Lipid nanoparticles and method for measuring optimal density range of target binding sites on surfaces of lipid nanoparticles

Abstract

The present invention addresses the problem of providing: lipid nanoparticles that enable selective delivery of a nucleic acid to a target cell; or a method for measuring the optimal density for selective delivery of a nucleic acid to a target cell. Provided are lipid nanoparticles that include: a nucleic acid encapsulated in the lipid nanoparticles; a cationic lipid; a sterol or a sterol derivative; an antibody selected from a VHH antibody, an Fab antibody, and an scFv antibody; a targeting polyalkylene glycol-modified lipid; and a non-targeting polyalkylene glycol-modified lipid. The antibody targets an antigen on a T-cell. The targeting polyalkylene glycol-modified lipid is a lipid in which the antibody is linked to polyalkylene glycol. The non-targeting polyalkylene glycol-modified lipid is a lipid not linked to the antibody. The density of the VHH antibody existing on the surfaces of the lipid nanoparticles in a state capable of binding to the antigen is 0.004-2.300 per 100 nm2 of the nanoparticle surfaces. The density of the Fab antibody existing on the surfaces of the lipid nanoparticles in a state capable of binding to the antigen is 0.003-1.113 per 100 nm2 of the nanoparticle surfaces. The density of the scFv antibody existing on the surfaces of the lipid nanoparticles in a state capable of binding to the antigen is 0.003-1.113 per 100 nm2 of the nanoparticle surfaces.

Description

Method for measuring the optimal density range of lipid nanoparticles and target binding sites on the surface of lipid nanoparticles

[0001] This disclosure relates to lipid nanoparticles. This disclosure also relates to a method for measuring the optimal density range of target binding sites on the surface of lipid nanoparticles.

[0002] Lipid nanoparticles (LNPs) are used as carriers to encapsulate nucleic acids such as siRNA (small interfering RNA) and mRNA and deliver them to cells. For example, Patent Document 1 reports lipid nanoparticles that serve as carriers for efficiently delivering nucleic acids into cells, and which contain cationic lipids as constituent lipids that are electrically neutral at physiological pH and change to cationic under weakly acidic pH environments such as endosomes.

[0003] As an attempt to selectively deliver antibodies to target cells, lipid nanoparticles conjugated with antibodies have been developed. For example, Patent Documents 2 and 3 disclose LNPs linked to single-stranded variable fragments (scFv) that target antigens present on the cell surface.

[0004] In the analysis of antibody content in antibody-conjugated lipid nanoparticles, the use of SDS-PAGE, Western blotting, and HPLC has been reported (for example, Non-Patent Document 1).

[0005] International Publication No. 2022 / 071582, International Publication No. 2023287861, International Publication No. 2024102772

[0006] J Control Release. 2022 Sep:349:379-387.

[0007] The use of antibody-conjugated lipid nanoparticles is one method for selectively delivering nucleic acids to target cells. However, the method for reproducibly achieving such selective delivery remains unknown.

[0008] The inventors focused on the density of VHH antibodies present on the surface of lipid nanoparticles linked with VHH antibodies, in a state where they can bind to antigens. Measuring this density using analytical instruments such as SDS-PAGE, Western blotting, and HPLC was technically difficult, but they found that it could be measured using a nanoflow cytometer. The inventors then discovered that there is an optimal density range for the antibodies necessary for reproducibly and selectively delivering the nucleic acids contained in the lipid nanoparticles to target cells.

[0009] Furthermore, the inventors have discovered that there is an optimal density range for Fab antibodies on lipid nanoparticles necessary for reproducibly and selectively delivering nucleic acids contained in the lipid nanoparticles to target cells.

[0010] Furthermore, the inventors have discovered that there is an optimal density range for scFv antibodies on lipid nanoparticles necessary for reproducibly and selectively delivering nucleic acids contained in the lipid nanoparticles to target cells.

[0011] Furthermore, the inventors have discovered that by using a nanoflow cytometer, it is possible to measure the optimal density range of target binding sites necessary for selectively delivering nucleic acids contained in lipid nanoparticles with linked target binding sites to target cells.

[0012] Furthermore, the inventors have found that, in lipid nanoparticles to which antibodies are linked, by utilizing the correlation between the density of target binding sites of the lipid nanoparticles measured using a nanoflow cytometer and the weight ratio of the targeted polyalkylene glycol-modified lipid to the nucleic acid in the lipid nanoparticles, it is possible to determine whether the density of target binding sites on the surface of lipid nanoparticles is within the optimal density range by measuring the weight ratio of the targeted polyalkylene glycol-modified lipid to the nucleic acid in lipid nanoparticles with an unknown antibody density.

[0013] This disclosure includes one or more embodiments described below: [1] A lipid nanoparticle comprising: a nucleic acid encapsulated in the lipid nanoparticle; a cationic lipid; a sterol or sterol derivative; a VHH antibody; a targeted polyalkylene glycol-modified lipid; and a non-targeted polyalkylene glycol-modified lipid, wherein the VHH antibody targets an antigen on a T cell; the targeted polyalkylene glycol-modified lipid is a lipid in which the VHH antibody is linked to a polyalkylene glycol; and the non-targeted polyalkylene glycol-modified lipid is a lipid in which the VHH antibody is not linked; and the density of the VHH antibody present on the surface of the lipid nanoparticle so as to be able to bind to the antigen is at a density of 100 nm on the surface of the nanoparticle. ２ Lipid nanoparticles having a density of 0.004 to 2,300 per nanoparticle. [2] The density of VHH antibodies capable of binding to the antigen on the surface of the lipid nanoparticles is such that the density ２ Lipid nanoparticles as described in [1], having a density of 0.008 to 2,300 particles per nanoparticle. [3] The density of VHH antibodies capable of binding to the antigen on the surface of the lipid nanoparticles is such that the density ２ Lipid nanoparticles according to [1] or [2], having 0.010 to 1.500 particles per unit. [4] Lipid nanoparticles according to any one of [1] to [3], wherein the VHH antibody binds to CD2, CD3, CD4, CD5, CD6, CD7, or CD8. [5] Lipid nanoparticles according to any one of [1] to [4], wherein the VHH antibody binds to CD4 or CD8. [6] Lipid nanoparticles according to any one of [1] to [5], wherein the VHH antibody binds to CD8. [7] Lipid nanoparticles according to any one of [1] to [5], wherein the VHH antibody binds to CD4.

[0014] [8] The density of VHH antibodies that are capable of binding to the antigen on the surface of the lipid nanoparticles is, at 100 nm on the surface of the nanoparticles. ２ Lipid nanoparticles as described in [6], having a density of 0.004 to 2,200 particles per nanoparticle. [9] The density of VHH antibodies capable of binding to the antigen on the surface of the lipid nanoparticles is such that the density ２The lipid nanoparticles according to [6], which are from 0.008 to 1.800 per unit.

[10] The density of the VHH antibody capable of binding to an antigen on the surface of the lipid nanoparticles is 100 nm on the nanoparticle surface ２ The lipid nanoparticles according to [6], which are from 0.010 to 1.500 per unit.

[11] The density of the VHH antibody capable of binding to an antigen on the surface of the lipid nanoparticles is 100 nm on the nanoparticle surface ２ The lipid nanoparticles according to [7], which are from 0.030 to 2.300 per unit.

[12] The density of the VHH antibody capable of binding to an antigen on the surface of the lipid nanoparticles is 100 nm on the nanoparticle surface ２ The lipid nanoparticles according to [7], which are from 0.035 to 2.300 per unit.

[13] The density of the VHH antibody capable of binding to an antigen on the surface of the lipid nanoparticles is 100 nm on the nanoparticle surface ２ The lipid nanoparticles according to [7], which are from 0.040 to 1.500 per unit.

[14] The VHH antibody is linked to the polyalkylene glycol of the targeted polyalkylene glycol-modified lipid via a linker, and the lipid nanoparticles according to any one of [1] to

[13] .

[15] The linker contains a peptide of 1 to 50 monomers, and the lipid nanoparticles according to

[14] .

[16] The VHH antibody contains a peptide represented by SEQ ID NO: 22 or SEQ ID NO: 23, and the lipid nanoparticles according to any one of [1] to

[15] .

[17] The sterol or sterol derivative is cholesterol, and the lipid nanoparticles according to any one of [1] to

[16] .

[18] The targeted polyalkylene glycol-modified lipid contains DSPE-PEG, and the lipid nanoparticles according to any one of [1] to

[17] .

[19] The non-targeted polyalkylene glycol-modified lipid contains a polyalkylene glycol-modified lipid in which the polyalkylene glycol is modified with a reactive group and a polyalkylene glycol-modified lipid in which the polyalkylene glycol is unmodified, and the lipid nanoparticles according to any one of [1] to

[18] .

[0015]

[20] The lipid nanoparticle according to

[19] , wherein the reactive group comprises a group selected from the group consisting of acetylene, transcyclooctene, cyclooctin, diarylcyclooctin, oxime, ketone, aldehyde, thiol, free cysteine, amine, maleimide, NHS (N-hydroxysuccinimide), NHS ester (N-hydroxysuccinimide ester), isocyanate, isothiocyanate, methyl ester phosphine, norbornene, tetrazine, methylcyclopropene, azetine, cyanide, azide, and dibenzocyclooctin.

[21] The lipid nanoparticle according to

[19] or

[20] , wherein the reactive group comprises maleimide.

[22] The lipid nanoparticle according to any one of

[19] to

[21] , wherein the polyalkylene glycol-modified lipid, in which polyalkylene glycol is modified with a reactive group, comprises DSPE-PEG, and the polyalkylene glycol-modified lipid, in which polyalkylene glycol is not modified, comprises DMG-PEG.

[23] The lipid nanoparticle according to any one of [1] to

[18] , wherein the untargeted polyalkylene glycol-modified lipid comprises a polyalkylene glycol-modified lipid in which polyalkylene glycol is modified at the untargeted site, and a polyalkylene glycol-modified lipid in which polyalkylene glycol is not modified.

[24] The lipid nanoparticle according to

[23] , wherein the polyalkylene glycol-modified lipid in which polyalkylene glycol is modified at the untargeted site comprises DSPE-PEG, and the polyalkylene glycol-modified lipid in which polyalkylene glycol is not modified is DMG-PEG.

[25] The lipid nanoparticle according to any one of [1] to

[24] , further comprising a phospholipid.

[26] The lipid nanoparticle according to

[25] , wherein the phospholipid is DSPC, DOPC, DOPE, or a mixture thereof.

[27] The lipid nanoparticle according to any one of [1] to

[26] , wherein the average particle size is 40 nm to 300 nm.

[28] Lipid nanoparticles according to any one of [1] to

[27] , wherein the nucleic acid is siRNA, antisense nucleic acid, heteroduplex nucleic acid, miRNA, gRNA, or mRNA.

[29] A lipid nanoparticle according to any one of [1] to

[28] , which is produced by a method comprising the steps of mixing a cationic lipid, a sterol or sterol derivative, a non-targeted polyalkylene glycol-modified lipid and the nucleic acid to produce non-targeted lipid nanoparticles encapsulating the nucleic acid, and linking the VHH antibody to the non-targeted lipid nanoparticles.

[30] The lipid nanoparticle according to

[29] , wherein the non-targeted polyalkylene glycol-modified lipid comprises a polyalkylene glycol-modified lipid in which polyalkylene glycol is modified with a first reactive group, the VHH antibody before linking comprises a second reactive group, and the linking step comprises the reaction of the first reactive group and the second reactive group to form the targeted polyalkylene glycol-modified lipid.

[0016]

[31] A method for measuring the optimal density range of target binding sites on the surface of lipid nanoparticles, wherein the lipid nanoparticles comprise nucleic acids encapsulated in the lipid nanoparticles, cationic lipids, sterols or sterol derivatives, target binding sites, targeted polyalkylene glycol-modified lipids, and untargeted polyalkylene glycol-modified lipids, wherein the target binding sites target target sites on target cells, the targeted polyalkylene glycol-modified lipids are lipids in which the target binding sites are linked to polyalkylene glycol, and the untargeted polyalkylene glycol-modified lipids are lipids in which the target binding sites are not linked, and the measurement method comprises gradually increasing the amount of target binding sites and using a nanoflow cytometer to measure the density of target binding sites that are capable of binding to target sites on the surface of lipid nanoparticles, and A measurement method comprising: measuring the expression level of nucleic acid delivered at the density of the target binding site within a target cell; and obtaining an optimal density range by setting the density range of target binding sites that achieves an expression level of 40% or more, with the maximum expression level being set as 100%.

[32] The method according to

[31] , comprising: setting the density range of target binding sites that achieves an expression level of 50% or more, with the maximum expression level being set as 100%, as the optimal density range;

[33] The method according to

[31] or

[32] , comprising: setting the density range of target binding sites that achieves an expression level of 60% or more, with the maximum expression level being set as 100%, as the optimal density range;

[34] The method according to any one of

[31] to

[33] , wherein the target cell is a T cell or a hematopoietic stem cell;

[35] The method according to any one of

[31] to

[34] , wherein the target binding site is a VHH antibody, a Fab antibody, or an scFv.

[36] The method according to any one of

[31] to

[35] , wherein the nucleic acid is mRNA.

[37] A method for producing lipid nanoparticles, comprising obtaining an optimal density range by the measurement method according to any one of

[31] to

[36] , and producing lipid nanoparticles having the optimal density.

[0017]

[38] A lipid nanoparticle comprising: a nucleic acid encapsulated in the lipid nanoparticle; a cationic lipid; a sterol or sterol derivative; a Fab antibody; a targeted polyalkylene glycol-modified lipid; and a non-targeted polyalkylene glycol-modified lipid, wherein the Fab antibody targets an antigen on a T cell; the targeted polyalkylene glycol-modified lipid is a lipid in which the Fab antibody is linked to a polyalkylene glycol; and the non-targeted polyalkylene glycol-modified lipid is a lipid in which the Fab antibody is not linked; and the density of Fab antibodies capable of binding to the antigen on the surface of the lipid nanoparticle is at 100 nm on the surface of the nanoparticle. ２ Lipid nanoparticles having 0.003 to 1.113 particles per nanoparticle.

[39] The density of Fab antibodies capable of binding to the antigen on the surface of the lipid nanoparticles is 100 nm on the surface of the nanoparticles. ２ Lipid nanoparticles as described in

[38] , wherein the number of nanoparticles per nanoparticle ranges from 0.004 to 1.113.

[40] The density of Fab antibodies capable of binding to an antigen on the surface of the lipid nanoparticles is such that the density ２Lipid nanoparticles according to

[38] or

[39] , having 0.006 to 1.000 particles per unit.

[41] Lipid nanoparticles according to any one of

[38] to

[40] , wherein the Fab antibody is linked to the polyalkylene glycol of the targeted polyalkylene glycol-modified lipid via a linker.

[42] Lipid nanoparticles according to

[41] , wherein the linker comprises a peptide of 1 to 50 units.

[43] Lipid nanoparticles according to any one of

[38] to

[42] , wherein the Fab antibody binds to CD2, CD3, CD4, CD5, CD6, CD7, or CD8.

[44] Lipid nanoparticles according to any one of

[38] to

[43] , wherein the Fab antibody binds to CD4 or CD8.

[45] Lipid nanoparticles according to any one of

[38] to

[44] , wherein the Fab antibody binds to CD8.

[46] The lipid nanoparticle according to any one of

[38] to

[45] , wherein the sterol or sterol derivative is cholesterol.

[47] The lipid nanoparticle according to any one of

[38] to

[46] , wherein the targeted polyalkylene glycol-modified lipid comprises DSPE-PEG.

[48] The lipid nanoparticle according to any one of

[38] to

[47] , wherein the untargeted polyalkylene glycol-modified lipid comprises a polyalkylene glycol-modified lipid in which polyalkylene glycol is modified with a reactive group, and a polyalkylene glycol-modified lipid in which polyalkylene glycol is unmodified.

[0018]

[49] The lipid nanoparticle according to

[48] , wherein the reactive group comprises a group selected from the group consisting of acetylene, transcyclooctene, cyclooctin, diarylcyclooctin, oxime, ketone, aldehyde, thiol, free cysteine, amine, maleimide, NHS (N-hydroxysuccinimide), NHS ester (N-hydroxysuccinimide ester), isocyanate, isothiocyanate, methyl ester phosphine, norbornene, tetrazine, methylcyclopropene, azetine, cyanide, azide, and dibenzocyclooctin.

[50] The lipid nanoparticle according to

[48] or

[49] , wherein the reactive group comprises maleimide.

[51] The lipid nanoparticle according to any one of

[48] to

[50] , wherein the polyalkylene glycol-modified lipid, in which polyalkylene glycol is modified with a reactive group, comprises DSPE-PEG, and the polyalkylene glycol-modified lipid, in which polyalkylene glycol is not modified, comprises DMG-PEG.

[52] The lipid nanoparticle according to any one of

[48] to

[51] , wherein the untargeted polyalkylene glycol-modified lipid comprises a polyalkylene glycol-modified lipid in which polyalkylene glycol is modified at the untargeted site, and a polyalkylene glycol-modified lipid in which polyalkylene glycol is not modified.

[53] The lipid nanoparticle according to

[52] , wherein the polyalkylene glycol-modified lipid in which polyalkylene glycol is modified at the untargeted site comprises DSPE-PEG, and the polyalkylene glycol-modified lipid in which polyalkylene glycol is not modified is DMG-PEG.

[54] The lipid nanoparticle according to any one of

[38] to

[53] , further comprising a phospholipid.

[55] The lipid nanoparticle according to

[54] , wherein the phospholipid is DSPC, DOPC, DOPE, or a mixture thereof.

[56] The lipid nanoparticle according to any one of

[38] to

[55] , wherein the average particle size is 40 nm to 300 nm.

[57] The lipid nanoparticle according to any one of

[38] to

[56] , wherein the nucleic acid is siRNA, antisense nucleic acid, heteroduplex nucleic acid, miRNA, gRNA, or mRNA.

[58] A lipid nanoparticle according to any one of

[38] to

[57] , produced by a method comprising the steps of: mixing a cationic lipid, a sterol or sterol derivative, a non-targeted polyalkylene glycol-modified lipid and the nucleic acid to produce non-targeted lipid nanoparticles encapsulating the nucleic acid; and linking the antibody to the non-targeted lipid nanoparticles.

[59] The lipid nanoparticle according to

[58] , wherein the non-targeted polyalkylene glycol-modified lipid comprises a polyalkylene glycol-modified lipid in which polyalkylene glycol is modified with a first reactive group, the antibody before linking comprises a second reactive group, and the linking step comprises the reaction of the first reactive group and the second reactive group to form the targeted polyalkylene glycol-modified lipid.

[0019]

[60] A lipid nanoparticle comprising: a nucleic acid encapsulated in the lipid nanoparticle; a cationic lipid; a sterol or sterol derivative; an scFv antibody; a targeted polyalkylene glycol-modified lipid; and a non-targeted polyalkylene glycol-modified lipid, wherein the scFv antibody targets an antigen on a T cell; the targeted polyalkylene glycol-modified lipid is a lipid in which the scFv antibody is linked to a polyalkylene glycol; and the non-targeted polyalkylene glycol-modified lipid is a lipid in which the scFv antibody is not linked; and the density of scFv antibodies present on the surface of the lipid nanoparticle so as to be able to bind to the antigen is at a density of 100 nm on the surface of the nanoparticle. ２ Lipid nanoparticles having a density of 0.001 to 3,400 per nanoparticle.

[61] The density of scFv antibodies capable of binding to the antigen on the surface of the lipid nanoparticles is such that the density ２ Lipid nanoparticles as described in

[60] , having a density of 0.002 to 3.100 particles per nanoparticle.

[62] The density of scFv antibodies capable of binding to an antigen on the surface of the lipid nanoparticles is such that the density ２Lipid nanoparticles according to

[60] or

[61] , having 0.005 to 2.800 particles per nanoparticle.

[63] Lipid nanoparticles according to any one of

[60] to

[62] , wherein the scFv antibody is linked to the polyalkylene glycol of the targeted polyalkylene glycol-modified lipid via a linker.

[64] Lipid nanoparticles according to

[63] , wherein the linker comprises a peptide of 1 to 50 units.

[65] Lipid nanoparticles according to any one of

[60] to

[64] , wherein the scFv antibody binds to CD2, CD3, CD4, CD5, CD6, CD7, or CD8.

[66] Lipid nanoparticles according to any one of

[60] to

[65] , wherein the scFv antibody binds to CD4 or CD8.

[67] Lipid nanoparticles according to any one of

[60] to

[66] , wherein the scFv antibody binds to CD8.

[68] The scFv antibody is a lipid nanoparticle according to any one of

[60] to

[66] that binds to CD4.

[0020]

[69] The density of scFv antibodies present on the surface of the lipid nanoparticles that are capable of binding to the antigen is such that the density ２ Lipid nanoparticles as described in

[67] , having a density of 0.001 to 1.900 particles per nanoparticle.

[70] The density of scFv antibodies capable of binding to an antigen on the surface of the lipid nanoparticles is such that the density ２ Lipid nanoparticles as described in

[67] , having a density of 0.002 to 1.400 particles per nanoparticle.

[71] The density of scFv antibodies capable of binding to an antigen on the surface of the lipid nanoparticles is such that the density ２ Lipid nanoparticles as described in

[67] , having a density of 0.005 to 1.100 particles per nanoparticle.

[72] The density of scFv antibodies capable of binding to an antigen on the surface of the lipid nanoparticles is such that the density ２ Lipid nanoparticles as described in

[68] , having a density of 0.020 to 3,400 particles per nanoparticle.

[73] The density of scFv antibodies capable of binding to an antigen on the surface of the lipid nanoparticles is such that the density ２Lipid nanoparticles as described in

[68] , having a density of 0.020 to 3.100 particles per nanoparticle.

[74] The density of scFv antibodies capable of binding to an antigen on the surface of the lipid nanoparticles is such that the density ２ Lipid nanoparticles according to

[68] , having 0.030 to 2.800 particles per nanoparticle.

[75] Lipid nanoparticles according to any one of

[60] to

[74] , wherein the sterol or sterol derivative is cholesterol.

[76] Lipid nanoparticles according to any one of

[60] to

[75] , wherein the targeted polyalkylene glycol-modified lipid comprises DSPE-PEG.

[77] Lipid nanoparticles according to any one of

[60] to

[76] , wherein the untargeted polyalkylene glycol-modified lipid comprises a polyalkylene glycol-modified lipid in which polyalkylene glycol is modified with a reactive group, and a polyalkylene glycol-modified lipid in which polyalkylene glycol is unmodified.

[0021]

[78] The lipid nanoparticle according to

[77] , wherein the reactive group comprises a group selected from the group consisting of acetylene, transcyclooctene, cyclooctin, diarylcyclooctin, oxime, ketone, aldehyde, thiol, free cysteine, amine, maleimide, NHS (N-hydroxysuccinimide), NHS ester (N-hydroxysuccinimide ester), isocyanate, isothiocyanate, methyl ester phosphine, norbornene, tetrazine, methylcyclopropene, azetine, cyanide, azide, and dibenzocyclooctin.

[79] The lipid nanoparticle according to

[77] or

[78] , wherein the reactive group comprises maleimide.

[80] The lipid nanoparticle according to any one of

[77] to

[79] , wherein the polyalkylene glycol-modified lipid, in which polyalkylene glycol is modified with a reactive group, comprises DSPE-PEG, and the polyalkylene glycol-modified lipid, in which polyalkylene glycol is not modified, comprises DMG-PEG.

[81] The lipid nanoparticle according to any one of

[60] to

[76] , wherein the untargeted polyalkylene glycol-modified lipid comprises a polyalkylene glycol-modified lipid in which polyalkylene glycol is modified at the untargeted site, and a polyalkylene glycol-modified lipid in which polyalkylene glycol is not modified.

[82] The lipid nanoparticle according to

[81] , wherein the polyalkylene glycol-modified lipid in which polyalkylene glycol is modified at the untargeted site comprises DSPE-PEG, and the polyalkylene glycol-modified lipid in which polyalkylene glycol is not modified is DMG-PEG.

[83] The lipid nanoparticle according to any one of

[60] to

[82] , further comprising a phospholipid.

[84] The lipid nanoparticle according to any one of

[60] to

[83] , wherein the phospholipid is DSPC, DOPC, DOPE, or a mixture thereof.

[85] The lipid nanoparticle according to any one of

[60] to

[84] , wherein the average particle size is 40 nm to 300 nm.

[86] The lipid nanoparticle according to any one of

[60] to

[85] , wherein the nucleic acid is siRNA, antisense nucleic acid, heteroduplex nucleic acid, miRNA, gRNA, or mRNA.

[87] A lipid nanoparticle according to any one of

[60] to

[86] , produced by a method comprising the steps of mixing a cationic lipid, a sterol or sterol derivative, a non-targeted polyalkylene glycol-modified lipid and the nucleic acid to produce non-targeted lipid nanoparticles encapsulating the nucleic acid, and linking the antibody to the non-targeted lipid nanoparticles.

[88] The lipid nanoparticle according to

[87] , wherein the non-targeted polyalkylene glycol-modified lipid comprises a polyalkylene glycol-modified lipid in which polyalkylene glycol is modified with a first reactive group, the antibody before linking comprises a second reactive group, and the linking step comprises the reaction of the first reactive group and the second reactive group to form the targeted polyalkylene glycol-modified lipid.

[0022]

[89] A method for determining whether the density of target binding sites on the surface of lipid nanoparticles is within the optimal density range, wherein the lipid nanoparticles comprise nucleic acids encapsulated in the lipid nanoparticles, cationic lipids, sterols or sterol derivatives, target binding sites, targeted polyalkylene glycol-modified lipids, and untargeted polyalkylene glycol-modified lipids, wherein the target binding sites target target sites on target cells, the targeted polyalkylene glycol-modified lipids are lipids in which the target binding sites are linked to polyalkylene glycol, and the untargeted polyalkylene glycol-modified lipids are lipids in which the target binding sites are not linked, and the method comprises the following steps (1) and (2): (1) obtaining the weight ratio of the targeted polyalkylene glycol-modified lipids to the nucleic acids in the lipid nanoparticles; (2) determining that the density of target binding sites on the surface of the lipid nanoparticles is within the optimal density range if the weight ratio obtained in step (1) is within the optimal weight ratio range; Here, the optimal weight ratio range is expressed by the following regression equation (A), which is based on the correlation between the density of target binding sites of lipid nanoparticles measured using a nanoflow cytometer and the weight ratio of the targeted polyalkylene glycol-modified lipid to the nucleic acid in the lipid nanoparticles: Y = aX + b (A) [wherein Y is the density of target binding sites of lipid nanoparticles measured using a nanoflow cytometer (100 nm of the nanoparticle surface)] ２ The method is a numerical range in which (Y1-b) / a, obtained by substituting the lower limit Y1 of the optimal density range into [the number of target binding sites per unit], X represents the weight ratio of the targeted polyalkylene glycol-modified lipid to the nucleic acid in the lipid nanoparticles, a represents the slope of the regression line, a > 0, and b represents the intercept of the regression line, has as its lower limit (Y1-b) / a and as its upper limit (Y2-b) / a, obtained by substituting the upper limit Y2 of the optimal density range, and the optimal density range is a numerical range obtained by any one of the methods in

[31] to

[36] .

[0023]

[90] A method for measuring the optimal weight ratio range of targeted polyalkylene glycol-modified lipids to nucleic acids in lipid nanoparticles, wherein the lipid nanoparticles comprise nucleic acids encapsulated in the lipid nanoparticles, a cationic lipid, a sterol or sterol derivative, a target binding site, a targeted polyalkylene glycol-modified lipid, and a non-targeted polyalkylene glycol-modified lipid, wherein the target binding site targets a target site on a target cell, the targeted polyalkylene glycol-modified lipid is a lipid in which the target binding site is linked to polyalkylene glycol, and the non-targeted polyalkylene glycol-modified lipid is a lipid in which the target binding site is not linked, and the method comprises a regression equation represented by the following equation (A) based on the correlation between the density of target binding sites of lipid nanoparticles measured using a nanoflow cytometer and the weight ratio of the targeted polyalkylene glycol-modified lipids to nucleic acids in the lipid nanoparticles: Y = aX + b (A) [wherein of equation (A), Y is the density of target binding sites of lipid nanoparticles measured using a nanoflow cytometer (100 nm on the nanoparticle surface). ２ The method comprises the step of obtaining an optimal weight ratio range, wherein the lower limit is (Y1-b) / a obtained by substituting the lower limit of the optimal density range Y1 into [the number of target binding sites per unit], X represents the weight ratio of the targeted polyalkylene glycol-modified lipid to the nucleic acid in the lipid nanoparticles, a represents the slope of the regression line, a>0, and b represents the intercept of the regression line, and the upper limit is (Y2-b) / a obtained by substituting the upper limit of the optimal density range Y2 into [the number of target binding sites per unit], X represents the weight ratio of the targeted polyalkylene glycol-modified lipid to the nucleic acid in the lipid nanoparticles, a represents the slope of the regression line, a>0, and b represents the intercept of the regression line, and the lower limit is (Y1, and the upper limit is (Y2-b) / a obtained by substituting the upper limit of the optimal density range Y2, and the optimal density range is a numerical range obtained by the method of any one of

[31] to

[36] .

[91] The method of

[89] or

[90] , wherein the target cell is a T cell or a hematopoietic stem cell.

[92] The method of any one of

[93] The method according to any one of

[89] to

[92] , wherein the nucleic acid is mRNA.

[0024]

[94] Lipid nanoparticles comprising a nucleic acid encapsulated in the lipid nanoparticles, a cationic lipid, a sterol or sterol derivative, a VHH antibody, a targeted polyalkylene glycol-modified lipid, and a non-targeted polyalkylene glycol-modified lipid, wherein the VHH antibody targets an antigen on a T cell, the targeted polyalkylene glycol-modified lipid is a lipid in which the VHH antibody is linked to polyalkylene glycol, and the non-targeted polyalkylene glycol-modified lipid is a lipid in which the VHH antibody is not linked, and the weight ratio of the targeted polyalkylene glycol-modified lipid to the nucleic acid in the lipid nanoparticles is 0.047 to 3.931.

[95] The lipid nanoparticles according to

[94] , wherein the weight ratio of the targeted polyalkylene glycol-modified lipid to the nucleic acid in the lipid nanoparticles is 0.054 to 3.931.

[96] The lipid nanoparticle according to

[94] or

[95] , wherein the weight ratio of targeted polyalkylene glycol-modified lipid to nucleic acid in the lipid nanoparticle is 0.057 to 2.578.

[97] The lipid nanoparticle according to any one of

[94] to

[96] , wherein the VHH antibody binds to CD2, CD3, CD4, CD5, CD6, CD7, or CD8.

[98] The lipid nanoparticle according to any one of

[94] to

[97] , wherein the VHH antibody binds to CD4 or CD8.

[99] The lipid nanoparticle according to any one of

[94] to

[98] , wherein the VHH antibody binds to CD8.

[100] The lipid nanoparticle according to any one of

[94] to

[98] , wherein the VHH antibody binds to CD4.

[0025]

[101] The lipid nanoparticle according to

[99] , wherein the weight ratio range of the targeted polyalkylene glycol-modified lipid to nucleic acid in the lipid nanoparticle is 0.047 to 3.762.

[102] The lipid nanoparticle according to

[99] , wherein the weight ratio range of the targeted polyalkylene glycol-modified lipid to nucleic acid in the lipid nanoparticle is 0.054 to 3.085.

[103] The lipid nanoparticle according to

[99] , wherein the weight ratio range of the targeted polyalkylene glycol-modified lipid to nucleic acid in the lipid nanoparticle is 0.057 to 2.578.

[104] The lipid nanoparticle according to

[100] , wherein the weight ratio range of the targeted polyalkylene glycol-modified lipid to nucleic acid in the lipid nanoparticle is 0.091 to 3.931.

[105] The lipid nanoparticle according to

[100] , wherein the weight ratio range of the targeted polyalkylene glycol-modified lipid to nucleic acid in the lipid nanoparticle is 0.099 to 3.931.

[106] The lipid nanoparticle according to

[100] , wherein the weight ratio range of the targeted polyalkylene glycol-modified lipid to the nucleic acid in the lipid nanoparticle is 0.108 to 2.578.

[107] The lipid nanoparticle according to any one of

[94] to

[106] , wherein the VHH antibody is linked to the polyalkylene glycol of the targeted polyalkylene glycol-modified lipid via a linker.

[108] The lipid nanoparticle according to

[107] , wherein the linker comprises a peptide of 1 to 50 units.

[109] The lipid nanoparticle according to any one of

[94] to

[108] , wherein the VHH antibody comprises a peptide represented by SEQ ID NO: 22 or SEQ ID NO: 23.

[110] The lipid nanoparticle according to any one of

[94] to

[109] , wherein the sterol or sterol derivative is cholesterol.

[111] The lipid nanoparticle according to any one of

[94] to

[110] , wherein the targeted polyalkylene glycol-modified lipid comprises DSPE-PEG.

[112] The non-targeted polyalkylene glycol-modified lipid comprises a polyalkylene glycol-modified lipid in which polyalkylene glycol is modified with a reactive group, and a polyalkylene glycol-modified lipid in which polyalkylene glycol is unmodified, as described in any one of

[94] to

[111] .

[0026]

[113] The lipid nanoparticle according to

[112] wherein the reactive group comprises a group selected from the group consisting of acetylene, transcyclooctene, cyclooctin, diarylcyclooctin, oxime, ketone, aldehyde, thiol, free cysteine, amine, maleimide, NHS (N-hydroxysuccinimide), NHS ester (N-hydroxysuccinimide ester), isocyanate, isothiocyanate, methyl ester phosphine, norbornene, tetrazine, methylcyclopropene, azetine, cyanide, azide, and dibenzocyclooctin.

[114] The lipid nanoparticle according to

[112] or

[113] wherein the reactive group comprises maleimide.

[115] The lipid nanoparticle according to any one of

[112] to

[114] , wherein the polyalkylene glycol-modified lipid, which is modified with a reactive group, comprises DSPE-PEG, and the polyalkylene glycol-modified lipid, which is not modified with polyalkylene glycol, is DMG-PEG.

[116] The lipid nanoparticle according to any one of

[94] to

[111] , wherein the untargeted polyalkylene glycol-modified lipid comprises a polyalkylene glycol-modified lipid, which is modified with polyalkylene glycol at an untargeted site, and a polyalkylene glycol-modified lipid, which is not modified with polyalkylene glycol.

[117] The lipid nanoparticle according to

[116] , wherein the polyalkylene glycol-modified lipid, which is modified with polyalkylene glycol at an untargeted site, comprises DSPE-PEG, and the polyalkylene glycol-modified lipid, which is not modified with polyalkylene glycol, is DMG-PEG.

[118] A lipid nanoparticle according to any one of

[94] to

[117] , further comprising a phospholipid.

[119] A lipid nanoparticle according to

[118] , wherein the phospholipid is DSPC, DOPC, DOPE, or a mixture thereof.

[120] A lipid nanoparticle according to any one of

[94] to

[119] , wherein the average particle size is 40 nm to 300 nm.

[121] A lipid nanoparticle according to any one of

[94] to

[120] , wherein the nucleic acid is siRNA, antisense nucleic acid, heterodouble-stranded nucleic acid, miRNA, gRNA, or mRNA.

[122] A lipid nanoparticle according to any one of

[94] to

[121] , comprising the steps of: mixing a cationic lipid, a sterol or sterol derivative, a non-targeted polyalkylene glycol-modified lipid, and the nucleic acid to produce non-targeted lipid nanoparticles encapsulating the nucleic acid; and linking the VHH antibody to the non-targeted lipid nanoparticles.

[123] The lipid nanoparticle according to

[122] , wherein the non-targeted polyalkylene glycol-modified lipid comprises a polyalkylene glycol-modified lipid in which polyalkylene glycol is modified with a first reactive group, the VHH antibody before linking comprises a second reactive group, and the linking step comprises the reaction of the first reactive group and the second reactive group to form the targeted polyalkylene glycol-modified lipid.

[0027] The lipid nanoparticles of the present invention can selectively deliver nucleic acids to target cells with high reproducibility by employing a specific density as the density of antibodies capable of binding to antigens. Furthermore, the measurement method of the present invention can measure the optimal density range of antibodies capable of binding to antigens for excellent selective delivery of nucleic acids to target cells. In addition, the determination method of the present invention can determine whether the density of target binding sites on the surface of lipid nanoparticles is within the optimal density range by measuring the weight ratio of targeted polyalkylene glycol-modified lipids to nucleic acids in lipid nanoparticles with an unknown antibody density.

[0028] Figure 1 shows the density of VHH antibodies present in an antigen-binding state for antibody-conjugated LNPs (lipid P) with different amounts of antibody 1 [mol%] in Example 1 [ / 100 nm]. 2 Figure 2 shows the amount of antibody 1 [mol%] in the antibody conjugate LNP (lipid P) in Example 1, and the density of VHH antibody present in a state capable of binding to the antigen [ / 100 nm]. 2 This shows the correlation with the median value of ]. Figure 3 shows the density of VHH antibody present in a state capable of binding to the antigen of antibody-conjugated LNP (lipid P) in Example 1 [ / 100 nm 2Figure 4 shows the correlation between the median of [ / 100 nm] and the mCherry expression rate in CD8-positive T cells when each LNP was transfected. Error bars indicate the standard deviation (n=3). Figure 4 shows the density of VHH antibody present in an antigen-binding state for antibody-conjugated LNP (lipid P) in Example 1. 2 Figure 5 shows the correlation between the median of [ ] and the mean mCherry luminescence intensity (MFI) in CD8-positive T cells when each LNP was transfected. Error bars indicate the standard deviation (n=3). Figure 5 shows that an antibody-lipid conjugate is generated after conjugation in the antibody-conjugated LNP in Example 1. Figure 6 shows the density of VHH antibodies present in an antigen-binding state for antibody-conjugated LNPs (lipid P) with different amounts of antibody 2 [mol%] in Example 2 [ / 100 nm]. 2 Figure 7 shows the amount of antibody 2 [mol%] in the antibody conjugate LNP (lipid P) preparation in Example 2, and the density of VHH antibody present in a state capable of binding to the antigen [ / 100 nm]. 2 This shows the correlation with the median value of ]. Figure 8 shows the density of VHH antibody present in a state capable of binding to the antigen of antibody-conjugated LNP (lipid P) in Example 2 [ / 100 nm 2 Figure 9 shows the correlation between the median of [ / 100 nm] and the mCherry expression rate in CD8-positive T cells when each LNP was transfected. Error bars indicate the standard deviation (n=3). Figure 9 shows the density of VHH antibody present in an antigen-binding state for antibody-conjugated LNP (lipid P) in Example 2 [ / 100 nm]. 2 The median of [ ] and the correlation with the mean mCherry luminescence intensity (MFI) in CD8-positive T cells transfected with each LNP are shown. Error bars indicate the standard deviation (n=3). Figure 10 shows that in the antibody-conjugated LNP in Example 2, an antibody-lipid conjugate is generated after conjugation.

[0029] Figure 11 shows the density of VHH antibodies present in an antigen-binding state for antibody-conjugated LNPs (lipid Q) with different amounts of antibody 1 [mol%] in Example 3 [ / 100 nm].2 Figure 12 shows the amount of antibody 1 [mol%] in the antibody conjugate LNP (lipid Q) preparation in Example 3, and the density of VHH antibody present in a state capable of binding to the antigen [ / 100 nm]. 2 This shows the correlation with the median value of ]. Figure 13 shows the density of VHH antibody present in a state capable of binding to the antigen of antibody-conjugated LNP (lipid Q) in Example 3 [ / 100 nm 2 Figure 14 shows the correlation between the median of [ / 100 nm] and the mCherry expression rate in CD8-positive T cells when each LNP was transfected. Error bars indicate the standard deviation (n=3). Figure 14 shows the density of VHH antibody present in an antigen-binding state for antibody-conjugated LNP (lipid Q) in Example 3. 2 Figure 15 shows the correlation between the median of [ ] and the mean mCherry luminescence intensity (MFI) in CD8-positive T cells when each LNP was transfected. Error bars indicate the standard deviation (n=3). Figure 15 shows that an antibody-lipid conjugate is generated after conjugation in the antibody-conjugated LNP in Example 3. Figure 16 shows the density of VHH antibodies present in an antigen-binding state for antibody-conjugated LNPs (lipid Q) with different amounts of antibody 2 [mol%] in Example 4 [ / 100 nm]. 2 Figure 17 shows the amount of antibody 2 [mol%] in the antibody conjugate LNP (lipid Q) preparation in Example 4, and the density of VHH antibody present in a state capable of binding to the antigen [ / 100 nm]. 2 This shows the correlation with the median value of ]. Figure 18 shows the density of VHH antibody present in an antigen-binding state of antibody-conjugated LNP (lipid Q) in Example 4 [ / 100 nm 2 Figure 19 shows the correlation between the median of [ / 100 nm] and the mCherry expression rate in CD8-positive T cells when each LNP was transfected. Error bars indicate the standard deviation (n=3). Figure 19 shows the density of VHH antibody present in an antigen-binding state for antibody-conjugated LNP (lipid Q) in Example 4. 2The median of [ ] and the correlation with the mean mCherry luminescence intensity (MFI) in CD8-positive T cells transfected with each LNP are shown. Error bars indicate the standard deviation (n=3). Figure 20 shows that in the antibody-conjugated LNP in Example 4, an antibody-lipid conjugate is generated after conjugation.

[0030] Figure 21 shows the density of VHH antibodies present in an antigen-binding state for antibody-conjugated LNPs (lipid R) with different amounts of antibody 1 [mol%] in Example 5 [ / 100 nm]. 2 Figure 22 shows the amount of antibody 1 [mol%] in the preparation of antibody conjugate LNP (lipid R) in Example 5, and the density of VHH antibody present in a state capable of binding to the antigen [ / 100 nm]. 2 This shows the correlation with the median value of ]. Figure 23 shows the density of VHH antibody present in an antigen-binding state in antibody-conjugated LNP (lipid R) in Example 5 [ / 100 nm 2 Figure 24 shows the correlation between the median of [ / 100 nm] and the mCherry expression rate in CD8-positive T cells when each LNP was transfected. Error bars indicate the standard deviation (n=3). Figure 24 shows the density of VHH antibody present in an antigen-binding state for antibody-conjugated LNP (lipid R) in Example 5. 2 Figure 25 shows the correlation between the median of [ ] and the mean mCherry luminescence intensity (MFI) in CD8-positive T cells when each LNP was transfected. Error bars indicate the standard deviation (n=3). Figure 25 shows that an antibody-lipid conjugate is generated after conjugation in the antibody-conjugated LNP in Example 5. Figure 26 shows the density of VHH antibodies present in an antigen-binding state for antibody-conjugated LNPs (lipid R) with different amounts of antibody 2 [mol%] in Example 6 [ / 100 nm]. 2 Figure 27 shows the amount of antibody 2 [mol%] in the antibody conjugate LNP (lipid R) preparation in Example 6, and the density of VHH antibody present in a state capable of binding to the antigen [ / 100 nm]. 2This shows the correlation with the median value of ]. Figure 28 shows the density of VHH antibody present in an antigen-binding state in antibody-conjugated LNP (lipid R) in Example 6 [ / 100 nm 2 Figure 29 shows the correlation between the median of [ / 100 nm] and the mCherry expression rate in CD8-positive T cells when each LNP was transfected. Error bars indicate the standard deviation (n=3). Figure 29 shows the density of VHH antibody present in an antigen-binding state for antibody-conjugated LNP (lipid R) in Example 6. 2 The median of [ ] and the correlation with the mean mCherry luminescence intensity (MFI) in CD8-positive T cells transfected with each LNP are shown. Error bars indicate the standard deviation (n=3). Figure 30 shows that in the antibody-conjugated LNP in Example 6, an antibody-lipid conjugate is generated after conjugation.

[0031] Figure 31 shows the density of VHH antibodies present in an antigen-binding state for antibody-conjugated LNPs (lipid S) with different amounts of antibody 1 [mol%] in Example 7 [ / 100 nm]. 2 Figure 32 shows the amount of antibody 1 [mol%] in the antibody conjugate LNP (lipid S) in Example 7 and the density of VHH antibody present in a state capable of binding to the antigen [ / 100 nm]. 2 This shows the correlation with the median value of ]. Figure 33 shows the density of VHH antibody present in an antigen-binding state in antibody-conjugated LNP (lipid S) in Example 7 [ / 100 nm 2 Figure 34 shows the correlation between the median of [ / 100 nm] and the mCherry expression rate in CD8-positive T cells when each LNP was transfected. Error bars indicate the standard deviation (n=3). Figure 34 shows the density of VHH antibody present in an antigen-binding state for antibody-conjugated LNP (lipid S) in Example 7 [ / 100 nm]. 2Figure 35 shows the correlation between the median of [ ] and the mean mCherry luminescence intensity (MFI) in CD8-positive T cells when each LNP was transfected. Error bars indicate the standard deviation (n=3). Figure 35 shows that an antibody-lipid conjugate is generated after conjugation in the antibody-conjugated LNP in Example 7. Figure 36 shows the density of VHH antibodies present in an antigen-binding state for antibody-conjugated LNPs (lipid S) with different amounts of antibody 2 [mol%] in Example 8 [ / 100 nm]. 2 Figure 37 shows the amount of antibody 2 [mol%] in the preparation of antibody conjugate LNP (lipid S) in Example 8, and the density of VHH antibody present in a state capable of binding to the antigen [ / 100 nm]. 2 This shows the correlation with the median value of ]. Figure 38 shows the density of VHH antibody present in an antigen-binding state in antibody-conjugated LNP (lipid S) in Example 8 [ / 100 nm 2 Figure 39 shows the correlation between the median of [ / 100 nm] and the mCherry expression rate in CD8-positive T cells when each LNP was transfected. Error bars indicate the standard deviation (n=3). Figure 39 shows the density of VHH antibody present in an antigen-binding state for antibody-conjugated LNP (lipid S) in Example 8. 2 The median of [ ] and the correlation with the mean mCherry luminescence intensity (MFI) in CD8-positive T cells transfected with each LNP are shown. Error bars indicate the standard deviation (n=3). Figure 40 shows that in the antibody-conjugated LNP in Example 8, an antibody-lipid conjugate is generated after conjugation.

[0032] Figure 41 is a schematic diagram of a method for measuring the density of VHH antibodies present on the surface of lipid nanoparticles that are capable of binding to an antigen. Figure 42 is a schematic diagram showing one embodiment of the method for producing lipid nanoparticles according to the present invention. Figure 43 shows the density of antibodies present in a state capable of binding to an antigen [ / 100 nm] for antibody conjugate LNPs (lipid S) with different amounts [mol%] of Fab antibody used in Example 10. 2Figure 44 shows the amount of Fab antibody [mol%] in the preparation of antibody conjugate LNP (lipid S) in Example 10, and the density of Fab antibody present in a state capable of binding to the antigen [ / 100 nm]. 2 This shows the correlation with the median value of ]. Figure 45 shows the density of Fab antibodies present in an antigen-binding state in antibody-conjugated LNP (lipid S) in Example 10 [ / 100 nm 2 Figure 46 shows the correlation between the median value of [ ] and the mean GFP emission intensity (MFI) in CD8-positive T cells when each LNP was transfected. Figure 46 shows the amount of VHH antibody [mol%] used to prepare the antibody-conjugated LNP in Example 9 and the density of VHH antibody present in a state where it can bind to the antigen [ / 100 nm]. 2 This shows the correlation with the median value of ]. Figure 47 shows the density of VHH antibody present in an antigen-binding state on antibody-conjugated LNP in Example 9 [ / 100 nm 2 Figure 48 shows the correlation between the median value of ] and the mCherry expression intensity in CD4-positive T cells when each LNP was transfected. Figure 49 shows that an antibody-lipid conjugate was generated after conjugation in the antibody-conjugated LNP in Example 9. Figure 49 shows the amount of scFv antibody [mol%] used in the preparation of the antibody-conjugated LNP in Example 11 and the density of scFv antibody present in a state that can bind to the antigen [ / 100 nm]. 2 This shows the correlation with the median value of ]. Figure 50 shows the density of scFv antibody present in an antigen-binding state on antibody-conjugated LNP in Example 11 [ / 100 nm 2 This shows the correlation between the median value of [ ] and the mCherry expression intensity in CD8-positive T cells when each LNP was transfected. Figure 51 shows that in the antibody-conjugated LNP in Example 11, an antibody-lipid conjugate was generated after conjugation.

[0033] Figure 52 shows the amount of scFv antibody [mol%] used in the preparation of the antibody-conjugated LNP in Example 12, and the density of the scFv antibody present in a state capable of binding to the antigen [ / 100 nm]. 2This shows the correlation with the median value of ]. Figure 53 shows the density of scFv antibody present in an antigen-binding state on antibody-conjugated LNP in Example 12 [ / 100 nm 2 Figure 54 shows the correlation between the median value of ] and the mCherry expression intensity in CD8-positive T cells when each LNP was transfected. Figure 54 shows that an antibody-lipid conjugate is generated after conjugation in the antibody-conjugated LNP in Example 12. Figure 55 shows the amount of scFv antibody [mol%] used in the preparation of the antibody-conjugated LNP in Example 13 and the density of scFv antibody present in a state that can bind to the antigen [ / 100 nm]. 2 This shows the correlation with the median value of ]. Figure 56 shows the density of scFv antibody present in an antigen-binding state on antibody-conjugated LNP in Example 13 [ / 100 nm 2 Figure 57 shows the correlation between the median value of ] and the mCherry expression intensity in CD8-positive T cells when each LNP was transfected. Figure 57 shows that in the antibody-conjugated LNP in Example 13, an antibody-lipid conjugate is generated after conjugation. Figure 58 shows the amount of scFv antibody [mol%] used in the preparation of the antibody-conjugated LNP in Example 14 and the density of scFv antibody present in a state that can bind to the antigen [ / 100 nm]. 2 This shows the correlation with the median value of ]. Figure 59 shows the density of scFv antibody present in an antigen-binding state on antibody-conjugated LNP in Example 14 [ / 100 nm 2 Figure 60 shows the correlation between the median value of [ ] and the mCherry expression intensity in CD8-positive T cells when each LNP was transfected. Figure 60 shows that an antibody-lipid conjugate was generated after conjugation in the antibody-conjugated LNP in Example 14. Figure 61 shows the correlation between the VHH antibody density and the VHH-Lipid / mRNA weight ratio in the VHH antibody-conjugated LNP in Example 15.

[0034] 1. Terminology

[0035] In this disclosure, the term “lipid nanoparticles (LNPs)” means particles comprising multiple lipid molecules physically bound to one another by intermolecular forces. Lipid nanoparticles may be, for example, microspheres (including monolayer and multilayer vesicles, e.g., liposomes), dispersed phases in emulsions, micelles, or internal phases in suspensions.

[0036] As used herein, “cationic lipid” includes ionizable cationic lipids and permanently cationic lipids. As used herein, the term “ionizable cationic lipid” refers to a lipid containing an ionizable moiety that can become positively charged under specific conditions (e.g., a specific pH range, e.g., physiological conditions), and is synonymous with “pH-sensitive cationic lipid.” The ionizable moiety may consist of an amine. In addition to the ionizable moiety, ionizable cationic lipids may contain alkyl or alkenyl groups. As used herein, the term “permanently cationic lipid” refers to a lipid consisting of a cationic moiety that is positively charged over any pH range. The permanently cationic moiety may consist of a quaternary amine. In addition to the cationic moiety, permanently cationic lipids may contain alkyl or alkenyl groups.

[0037] In this disclosure, the terms “nucleic acid,” “nucleic acid molecule,” “oligonucleotide,” “polynucleotide,” and “nucleotide” may be used interchangeably. These terms refer to polymers of deoxyribonucleotides (DNA), ribonucleotides (RNA), and their modified forms, which are used as distinct fragments or components of a larger structure, linear or branched, single-stranded, double-stranded, triple-stranded, or hybrids thereof. The term also includes RNA / DNA hybrids. Polynucleotides may include sense and antisense oligonucleotides or polynucleotide sequences of DNA or RNA. DNA or RNA molecules may be, but are not limited to, complementary DNA (cDNA), genomic DNA, synthetic DNA, recombinant DNA, or hybrids thereof, mRNA (including linear and circular mRNA), gRNA, shRNA, siRNA, miRNA, antisense RNA, and other RNA molecules. Furthermore, these terms may include oligonucleotides composed of native bases, sugars, and nucleoside-to-nucleoside covalent bonds, as well as oligonucleotides having non-native parts that function similarly to their respective native parts.

[0038] In this disclosure, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to polymers of amino acid residues. These terms may include natural amino acid polymers and non-natural amino acid polymers in which one or more amino acid residues are artificial chemical analogs of corresponding natural amino acids.

[0039] In this disclosure, the term “target binding site” refers to any type of molecule or part thereof that is limited to a specific cell or can specifically recognize and interact with / bind to a target molecule (e.g., a cell surface antigen) on the cell surface enriched in a specific cell. In some embodiments, the target binding site is selected from, but is not limited to, antibodies, antigen-binding fragments, peptides, nucleotides, ligands, ligand mimes, agonists and / or antagonists. In some embodiments, the target binding site may be any type of antibody or its antigen-binding fragment. In some embodiments, the target binding site includes antibodies, Fab', F(ab')2, Fab, Fv, rIgG, scFv, hcAbs (heavy chain antibodies), single-domain antibodies, VHH, VNAR, sdAbs, nanobodies, receptor ectodomains or their ligand-binding portions, or ligands (e.g., cytokines, chemokines), cell marker ligands, receptor ligands, peptides (artificial polypeptides), peptide aptamers, nucleic acids, nucleic acid aptamers, spiegelmers, or combinations thereof.

[0040] In this disclosure, "VHH antibody" (variable domain of heavy chain of heavy chain antibody) refers to a domain containing the variable region of a heavy chain antibody without a light chain, found in the serum of camelid animals (such as Bactrian camels, dromedary camels, llamas, and alpacas). VHH antibodies are known as the smallest unit of immunoglobulin fragments that can bind to an antigen.

[0041] In this disclosure, the term “target site” refers to a biomolecule including a specific protein, peptide, glycan, nucleic acid, or a complex thereof. A target site may be an antigen, cell surface receptor, enzyme active site, or intracellularly localized molecule.

[0042] In this disclosure, the term “antigen” refers to a molecule or part of a molecule to which an antibody can specifically bind. An antigen may have one or more epitopes. In some embodiments, the antigen is a protein specifically expressed by a particular cell. In some embodiments, the antigen is a membrane protein. In some embodiments, the antigen is expressed on the outer membrane of a cell. In some embodiments, the antigen is a cell surface protein. In some embodiments, the antigen is a cell surface receptor.

[0043] 2-1. Lipid Nanoparticles Containing VHH Antibody One aspect of the present invention is a lipid nanoparticle comprising: a nucleic acid encapsulated in the lipid nanoparticle; a cationic lipid; a sterol or sterol derivative; a VHH antibody; a targeted polyalkylene glycol-modified lipid; and a non-targeted polyalkylene glycol-modified lipid, wherein the VHH antibody targets an antigen on a T cell; the targeted polyalkylene glycol-modified lipid is a lipid in which the VHH antibody is linked to a polyalkylene glycol; and the non-targeted polyalkylene glycol-modified lipid is a lipid in which the VHH antibody is not linked; and the density of the VHH antibody present on the surface of the lipid nanoparticle so as to be able to bind to the antigen is at a density of 100 nm on the surface of the nanoparticle. ２ This relates to lipid nanoparticles, with a quantity ranging from 0.004 to 2,300 per nanoparticle.

[0044] Examples of the cationic lipids contained in the lipid nanoparticles of this aspect are not particularly limited, and for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), bis(3-pentyloctyl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate, 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), 9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), [(4-Hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315) , 2-(bis(2-(tetradecanoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-2-oxoethan-aminium bromide (HEDC),((2-((2-(dimethylamino)ethyl)thio)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate (S104), ((2-(3,4-dihydroxypyrrolidin-1-yl)acetyl)azanediyl)bis(ethane-2,1-diyl) (9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate), ((2-((3S,4R)-3,4-dihydroxypyrrolidin-1-yl)acetyl)azanediyl)bis(ethane-2,1-diyl) (9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate), or mixtures thereof. Other lipids are disclosed, for example, in US Pat. Nos. 9,011,903, 9,308,267, 10,167,253, and US Pat. Pub. No. 2024 / 0024252, which are incorporated herein by reference.

[0045] Examples of sterols or sterol derivatives contained in the lipid nanoparticles of this embodiment include animal-derived sterols such as cholesterol, cholesterol succinate, lanosterol, dihydrolanosterol, desmosterol, and dihydrocholesterol; plant-derived sterols (phytosterols) such as stigmasterol, sitosterol, β-sitosterol, campesterol, and brassicasterol; and microbial-derived sterols such as thymosterol and ergosterol. Examples of glycolipids include glyceroglycolipids such as sulfoxyribosylglyceride, diglycosyldiglyceride, digalactosyldiglyceride, galactosyldiglyceride, and glycosyldiglyceride; and sphingoglycolipids such as galactosylcerebroside, lactosylcerebroside, and ganglioside. Examples of saturated or unsaturated fatty acids include palmitic acid, oleic acid, stearic acid, arachidonic acid, and myristic acid, which have 12 to 20 carbon atoms.

[0046] The lipid nanoparticles of this embodiment include targeted polyalkylene glycol-modified lipids in which a VHH antibody is linked to polyalkylene glycol, and untargeted polyalkylene glycol-modified lipids in which the VHH antibody is not linked. Polyalkylene glycol is a hydrophilic polymer, and by constructing lipid nanoparticles using polyalkylene glycol-modified lipids as lipid membrane constituent lipids, the surface of the lipid nanoparticles can be modified with polyalkylene glycol. Surface modification with polyalkylene glycol may enhance the stability of the lipid nanoparticles, such as their retention in the bloodstream.

[0047] Examples of polyalkylene glycols that can be used to constitute the polyalkylene glycol-modified lipids include polyethylene glycol, polypropylene glycol, polytetramethylene glycol, and polyhexamethylene glycol. The molecular weight of the polyalkylene glycol is, for example, about 200 to 10,000, for example, about 300 to 10,000, preferably about 500 to 10,000, and more preferably about 1,000 to 5,000. In one embodiment of the present invention, the molecular weight of the polyalkylene glycol is about 200, about 300, about 350, about 400, about 500, about 550, about 750, about 1,000, about 1,500, about 2,000, about 3,000, about 3,500, about 4,000, about 5,000, or about 10,000 Da.

[0048] For example, stearylated polyethylene glycol (e.g., PEG-45 stearate (STR-PEG45)) can be used for modifying lipids with polyethylene glycol. Other options include N-[carbonyl-methoxypolyethylene glycol]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG), N-[carbonyl-methoxypolyethylene glycol]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol. Polyethylene glycol derivatives such as N-[carbonyl-methoxypolyethylene glycol]-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG) may be used.For example, N-[carbonyl-methoxypolyethylene glycol-2000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG2000), n-[carbonyl-methoxypolyethylene glycol-5000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG5000), N-[carbonyl-methoxypolyethylene glycol-750]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG750), N-[carbonyl-methoxypolyethylene glycol-2000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG2000) N-[carbonyl-methoxypolyethylene glycol-5000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG5000), 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-750 (DMG-PEG750), 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-5000 Polyethylene glycol derivatives such as (DMG-PEG5000), N-[carbonyl-methoxypolyethylene glycol-750]-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG750), N-[carbonyl-methoxypolyethylene glycol-2000]-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG2000), and N-[carbonyl-methoxypolyethylene glycol-5000]-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG5000) can also be used, but polyalkylene glycol-modified lipids are not limited to these.

[0049] In this embodiment, the targeted polyalkylene glycol-modified lipid constituting the lipid nanoparticles preferably has a VHH antibody linked to the polyalkylene glycol moiety of the polyalkylene glycol-modified lipid via a linker. In this disclosure, "linking" refers to linking by covalent bonds.

[0050] The linker contains or consists of, for example, peptides. The linker contains or consists of, for example, peptides of 1 to 50-mers, 5 to 45-mers, 5 to 35-mers, 5 to 30-mers, 5 to 25-mers, and 10 to 25-mers. In a preferred embodiment of the present invention, the linker is (Ser-Ser-Ser-Gly) m (Sequence ID 1) [wherein m is an integer from 1 to 5], (Glu-Ala-Ala-Ala-Lys) n (Sequence No. 2) [wherein n is an integer from 1 to 4], (Ala-Pro) ｐ [In the formula, p is an integer between 1 and 8], (His) ｑ [wherein the formula q is an integer from 1 to 10], includes at least one of the following:

[0051] In a preferred embodiment of the present invention, the linker is (OCH ２ CH ２ ) ｒ The formula includes the expression [where r is an integer between 1 and 10].

[0052] In a preferred embodiment of the present invention, the linker is of the following formula: The compound includes the site indicated by [wherein the formula, the left arrow indicates linkage with the peptide, the right arrow indicates linkage with the polyalkylene glycol, X is N or O, and r is an integer from 1 to 15].

[0053] In a preferred embodiment of the present invention, the linker is represented as -Z1-Z2-, where Z1 is linked to the VHH antibody, Z2 is linked to the polyalkylene glycol, and Z1 is It is represented as, and Z2 is, [In the formula, the left arrow indicates linkage to the adjacent Lys side chain amino group, the right arrow indicates linkage to the polyalkylene glycol, X is N or O, and r is an integer from 1 to 15]. In a preferred embodiment of the present invention, the linker is represented as -Z1-Z2-, where Z1 is linked to the VHH antibody, Z2 is linked to the polyalkylene glycol, and Z1 is It is represented as, and Z2 is, [In the formula, the left arrow indicates a linkage to the adjacent Lys side chain amino group, the right arrow indicates a linkage to the polyalkylene glycol, X is N or O, and r is an integer from 1 to 15.]

[0054] In a preferred embodiment of the present invention, the linker is represented as -Z1-Z2-, where Z1 is linked to the VHH antibody, Z2 is linked to the polyalkylene glycol, and Z1 is as follows: Z2 is one of the polypeptides shown, and Z2 is [In the formula, the left arrow indicates a linkage to the adjacent Cys side chain SH group, the right arrow indicates a linkage to the polyalkylene glycol, and X is N or O].

[0055] The VHH antibody linked to the targeted polyalkylene glycol-modified lipid constituting the lipid nanoparticles of this embodiment preferably binds to CD2, CD3, CD4, CD5, CD6, CD7, or CD8, more preferably to CD4 or CD8, and particularly preferably to CD8.

[0056] In one embodiment of the present invention, the antibody has at least one, preferably all, CDR sequences (based on the Kabat numbering scheme) selected from the group consisting of DYAIG (SEQ ID NO: 16), CIRIFDRHTYSADSVKG (SEQ ID NO: 17), and GSFWACTRPEGAMDY (SEQ ID NO: 18).

[0057] In one embodiment of the present invention, the antibody has at least one, preferably all, CDR sequences (based on the IMGT numbering scheme) selected from the group consisting of GSTFSDYG (SEQ ID NO: 19), IDWNGEHT (SEQ ID NO: 20), and AADALPYTVRKYNY (SEQ ID NO: 21).

[0058] In one embodiment of the present invention, the VHH antibody is antibody 1 represented by the following sequence. The underlined portion indicates the CDR sequence (based on the Kabat numbering scheme). EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGKGREGVLCIRIFDRHTYSADSVKGRFTISSDNAQNTVYLHMNSLKPEDTAVYYCAAGSFWACTRPEGAMDYWGKGTQVTVSS (Sequence ID 22)

[0059] In one embodiment of the present invention, the VHH antibody is antibody 2 represented by the following sequence. The underlined portion indicates the CDR sequence (based on the IMGT numbering scheme). EVQLVESGGGLVQAGGSLRLSCAASGSTFSDYGVGWFRQAPGKGREFVADIDWNGEHTSYADSVKGRFATSRDNAKNTAYLQMNSLKPEDTAVYYCAADALPYTVRKYNYWGQGTQVTVSS (Sequence ID 23)

[0060] In one embodiment of the present invention, the VHH antibody is antibody 4 represented by the following sequence. The underlined portion indicates the CDR sequence (based on the IMGT numbering scheme). EVQLVESGGGSVQPGGSLTLSCGTSGRTFNVMGWFRQAPGKEREFVAAVRWSSTGIYYTQYADSVKSRFTISRDNAKNTVYLEMNSLKPEDTAVYYCAADTYNSNPARWDGYDFRGQGTLVTVSS (Sequence ID 59)

[0061] In one embodiment, the targeted polyalkylene glycol-modified lipid includes DSPE-PEG.

[0062] In one embodiment, the untargeted polyalkylene glycol-modified lipid constituting the lipid nanoparticles of this embodiment includes polyalkylene glycol-modified lipids in which the polyalkylene glycol is modified with a reactive group. In one embodiment, the untargeted polyalkylene glycol-modified lipid constituting the lipid nanoparticles of this embodiment includes polyalkylene glycol-modified lipids in which the untargeted site is linked to the polyalkylene glycol site. Although not particularly limited, lipids formed by the reaction of polyalkylene glycol-modified lipids in which the polyalkylene glycol is modified with a reactive group (e.g., maleimide) with an untargeted molecule (e.g., L-Cysteine ​​when maleimide is the reactive group) are included in polyalkylene glycol-modified lipids in which the untargeted site is linked to the polyalkylene glycol site. In one embodiment, the untargeted polyalkylene glycol-modified lipid constituting the lipid nanoparticles of this embodiment includes polyalkylene glycol-modified lipids in which the polyalkylene glycol is unmodified.

[0063] In this disclosure, “reactive group” collectively refers to groups involved in the coupling reaction of two molecules, such as Michael addition reactions and click chemistry reactions. Examples of Michael addition reactions include, but are not limited to, nucleophilic addition reactions of thiol groups to maleimides. Examples of click chemistry reactions include, but are not limited to, [3+2] cycloaddition, thiol-ene reactions, Diels-Alder reactions, inverse electron-demand Diels-Alder reactions, and [4+1] cycloaddition. The reactive group preferably includes a group selected from the group consisting of acetylene, transcyclooctene, cyclooctin, diarylcyclooctin, oxime, ketone, aldehyde, thiol, free cysteine, amine, maleimide, NHS (N-hydroxysuccinimide), NHS ester (N-hydroxysuccinimide ester), isocyanate, isothiocyanate, methyl ester phosphine, norbornene, tetrazine, methylcyclopropene, azetine, cyanide, azide, and dibenzocyclooctin. The reactive group most preferably includes maleimide.

[0064] In one embodiment, the untargeted polyalkylene glycol-modified lipid, in which polyalkylene glycol is modified with a reactive group, includes DSPE-PEG.

[0065] In one embodiment, the untargeted polyalkylene glycol-modified lipid, in which the polyalkylene glycol is unmodified, includes DMG-PEG.

[0066] The lipid nanoparticles of this embodiment may further contain phospholipids as constituent lipids. Examples of phospholipids include glycerophospholipids such as phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, cardiolipin, plasmalogen, ceramidephosphorylglycerol phosphate, and phosphatidic acid; and sphingophospholipids such as sphingomyelin, ceramidephosphorylglycerol, and ceramidephosphorylethanolamine. Phospholipids derived from natural products such as egg yolk lecithin and soy lecithin can also be used. The fatty acid residues in glycerophospholipids and sphingophospholipids are not particularly limited, but examples include saturated or unsaturated fatty acid residues having 12 to 24 carbon atoms, with saturated or unsaturated fatty acid residues having 14 to 20 carbon atoms being preferred. Specifically, examples include acyl groups derived from fatty acids such as lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid, arachidonic acid, behenic acid, and lignoceric acid. When these glycerolipids or sphingolipids have two or more fatty acid residues, all fatty acid residues may be the same group or they may be different groups.

[0067] Examples of phospholipids include diphytanoyl phosphatidyl ethanolamine (DPhPE), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2- It contains dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0068] In this embodiment, the "density of VHH antibodies capable of binding to the antigen on the surface of lipid nanoparticles" is the value obtained by dividing the number of VHH antibodies capable of binding to the antigen on the surface of one lipid nanoparticle by the surface area of ​​one lipid nanoparticle. Here, the number of VHH antibodies capable of binding to the antigen on the surface of one lipid nanoparticle and the surface area of ​​one lipid nanoparticle are obtained by mixing antibody-conjugated lipid nanoparticles with a fluorescently labeled target antigen, removing the unreacted fluorescently labeled target antigen, and subjecting the sample to a nanoflow cytometer, simultaneously detecting the fluorescence intensity and lateral scattered light, respectively, and calculating the values. A schematic diagram is shown in Figure 41. In reality, as shown in Figures 1, 6, 11, 16, 21, 26, 31, and 36, the lipid nanoparticle group shows a distribution of particles with different antibody densities. In this case, the "density of VHH antibodies capable of binding to the antigen on the surface of lipid nanoparticles" is the median value of the antibody density of the particle group.

[0069] In this embodiment, the density of VHH antibodies present on lipid nanoparticles in a state capable of binding to the antigen is 100 nm. ２ The number of antibodies per unit area ranges from 0.004 to 2,300. The density of the VHH antibody is, for example, at 100 nm. ２Percentages of 0.005 or more, 0.006 or more, 0.007 or more, 0.008 or more, 0.009 or more, 0.010 or more, 0.015 or more, 0.020 or more, 0.025 or more, 0.030 or more, 0.035 or more, 0.040 or more, 0.045 or more, 0.050 or more, 0.055 or more, 0.060 or more, 0.065 or more, 0.070 or more, 0.075 or more, 0.080 or more, 0.085 or more, 0.090 or more, 0.095 or more, 0.100 or more, 0.105 or more, 0.110 or more , 0.115 or more, 0.120 or more, 0.125 or more, 0.130 or more, 0.135 or more, 0.140 or more, 0.145 or more, 0.150 or more, 0.155 or more, 0.160 or more, 0.165 or more, 0.170 or more, 0.175 or more, 0.180 or more, 0.185 or more, 0.190 or more, 0.195 or more, 0.200 or more, 0.205 or more, 0.210 or more, 0.215 or more, 0.220 or more, 0.225 or more, 0.230 or more, 0.235 or more, 0.240 or more, 0 .. 245 or more, 0.250 or more, 0.255 or more, 0.260 or more, 0.265 or more, 0.270 or more, 0.275 or more, 0.280 or more, 0.285 or more, 0.290 or more, 0.295 or more, 0.300 or more, 0.305 or more, 0. 310 or more, 0.315 or more, 0.320 or more, 0.325 or more, 0.330 or more, 0.335 or more, 0.340 or more, 0.345 or more, 0.350 or more, 0.355 or more, 0.360 or more, 0.365 or more, 0.370 or more, 0.3 75 or more, 0.380 or more, 0.385 or more, 0.390 or more, 0.395 or more, 0.400 or more, 0.405 or more, 0.410 or more, 0.415 or more, 0.420 or more, 0.425 or more, 0.430 or more, 0.435 or more, 0.440 or more, 0.445 or more, 0.450 or more, 0.455 or more, 0.460 or more, 0.465 or more, 0.470 or more, 0.475 or more, 0.480 or more, 0.485 or more, 0.490 or more, 0.495 or more, or 0.500 or more,2.300 or less, 2.250 or less, 2.200 or less, 2.150 or less, 2.100 or less, 2.050 or less, 2.000 or less, 1.950 or less, 1.900 or less, 1.850 or less, 1.800 or less , 1.750 or less, 1.700 or less, 1.650 or less, 1.600 or less, 1.550 or less, 1.500 or less, 1.450 or less, 1.400 or less, 1.350 or less, 1.300 or less, 1.250 or less Bottom, 1.200 or less, 1.150 or less, 1.100 or less, 1.050 or less, 1.000 or less, 0.950 or less, 0.900 or less, 0.850 or less, 0.800 or less, 0.750 or less, 0.700 The following are 0.650 or less, 0.600 or less, 0.550 or less, 0.500 or less, 0.450 or less, 0.400 or less, 0.350 or less, 0.300 or less, 0.250 or less, or 0.200 or less.

[0070] Furthermore, the density of the VHH antibody is, for example, 100 nm. ２Per 100 units: 0.004 to 2.300, 0.004 to 2.200, 0.004 to 2.100, 0.004 to 2.000, 0.004 to 1.900, 0.004 to 1.800, 0.004 to 1.700, 0.004 to 1.600, 0.004 to 1.500, 0.004 to 1.400, 0.004 to 1.300 pieces, 0.004 pieces to 1.200 pieces, 0.004 pieces to 1.100 pieces, 0.004 pieces to 1.000 pieces, 0.004 pieces to 0.900 pieces, 0.004 pieces to 0.800 pieces, 0.004 pieces ~0.700 pieces, 0.004 pieces to 0.600 pieces, 0.004 pieces to 0.500 pieces, 0.004 pieces to 0.400 pieces, 0.004 pieces to 0.300 pieces, 0.004 pieces to 0.200 pieces, 0.005 pieces to 2.300 pieces, 0.005 pieces to 2.200 pieces, 0.005 pieces to 2.000 pieces, 0.005 pieces to 1.900 pieces, 0.005 pieces to 1.800 pieces, 0.0 05 pieces to 1.700 pieces, 0.005 pieces to 1.600 pieces, 0.005 pieces to 1.500 pieces, 0.005 pieces to 1.400 pieces, 0.005 pieces to 1.300 pieces, 0.005 pieces to 1.200 pieces, 0.005 pieces to 1.100 pieces, 0.005 pieces to 1.000 pieces, 0.005 pieces to 0.900 pieces, 0.005 pieces to 0.800 pieces, 0.005 pieces to 0.7 00 pieces, 0.005 pieces to 0.600 pieces, 0.005 pieces to 0.500 pieces, 0.005 pieces to 0.400 pieces, 0.005 pieces to 0.300 pieces, 0.005 pieces to 0.200 pieces, 0.006 pieces to 2.300 pieces, 0.006 pieces to 2.200 pieces, 0.006 pieces to 2.000 pieces, 0.006 pieces to 1.900 pieces, 0.006 pieces to 1.800 pieces, 0.0 06 pieces to 1.700 pieces, 0.006 pieces to 1.600 pieces, 0.006 pieces to 1.500 pieces, 0.006 pieces to 1.400 pieces, 0.006 pieces to 1.300 pieces, 0.006 pieces to 1.200 pieces, 0.006 pieces to 1.100 pieces, 0.006 pieces to 1.000 pieces, 0.006 pieces to 0.900 pieces, 0.006 pieces to 0.800 pieces, 0.006 pieces to 0.7 00 pieces, 0.006 pieces to 0.600 pieces, 0.006 pieces to 0.500 pieces, 0.006 pieces to 0.400 pieces, 0.006 pieces to 0.300 pieces, 0.006 pieces to 0.200 pieces,0.007 to 2.300, 0.007 to 2.200, 0.007 to 2.000, 0.007 to 1.900, 0.007 to 1.800, 0.007 to 1.700, 0.007 to 1.600, 0.007 to 1.500, 0.007 to 1.400, 0.007 to 1.300, 0.007 to 1.200, 0.007 to 1.100, 0.007 to 1.000, 0.007 to 0.900, 0.007 to 0.800, 0.007 to 0.700, 0.007 to 0.600, 0.007 to 0.500, 0.007 to 0.400, 0.007 to 0.300, 0.007 to 0.200 0.008 to 2.300, 0.008 to 2.200, 0.008 to 2.000, 0.008 to 1.900, 0.008 to 1.800, 0.008 to 1.700, 0.008 to 1.600, 0.008 to 1.500, 0.008 to 1.400, 0.008 to 1.300, 0.008 to 1.200, 0.008 to 1.100, 0.008 to 1.000, 0.008 to 0.900, 0.008 to 0.800, 0.008 to 0.700, 0.008 to 0.600, 0.008 to 0.500, 0.008 to 0.400, 0.008 to 0.300, 0.008 to 0.200 0.009 to 2.300, 0.009 to 2.200, 0.009 to 2.000, 0.009 to 1.900, 0.009 to 1.800, 0.009 to 1.700, 0.009 to 1.600, 0.009 to 1.500, 0.009 to 1.400, 0.009 to 1.300, 0.009 to 1.200, 0.009 to 1.100, 0.009 to 1.000, 0.009 to 0.900, 0.009 to 0.800, 0.009 to 0.700, 0.009 to 0.600, 0.009 to 0.500, 0.009 to 0.400, 0.009 to 0.300, 0.009 to 0.2000.010 to 2.300, 0.010 to 2.200, 0.010 to 2.000, 0.010 to 1.900, 0.010 to 1.800, 0.010 to 1.700, 0.010 to 1.600, 0.010 to 1.500, 0.010 to 1.400, 0.010 to 1.300, 0.010 to 1.200, 0.010 to 1.100, 0.010 to 1.000, 0.010 to 0.900, 0.010 to 0.800, 0.010 to 0.700, 0.010 to 0.600, 0.010 to 0.500, 0.010 to 0.400, 0.010 to 0.300, 0.010 to 0.200 0.020 to 2.300, 0.020 to 2.200, 0.020 to 2.000, 0.020 to 1.900, 0.020 to 1.800, 0.020 to 1.700, 0.020 to 1.600, 0.020 to 1.500, 0.020 to 1.400, 0.020 to 1.300, 0.020 to 1.200, 0.020-1.100, 0.020-1.000, 0.020-0.900, 0.020-0.800, 0.020-0.700, 0.020-0.600, 0.020-0.500, 0.020-0.400, 0.020-0.300, 0.020-0.200 0.030 to 2.300, 0.030 to 2.200, 0.030 to 2.000, 0.030 to 1.900, 0.030 to 1.800, 0.030 to 1.700, 0.030 to 1.600, 0.030 to 1.500, 0.030 to 1.400, 0.030 to 1.300, 0.030 to 1.200, 0.030-1.100, 0.030-1.000, 0.030-0.900, 0.030-0.800, 0.030-0.700, 0.030-0.600, 0.030-0.500, 0.030-0.400, 0.030-0.300, 0.030-0.2000.035 to 2.300, 0.035 to 2.200, 0.035 to 2.000, 0.035 to 1.900, 0.035 to 1.800, 0.035 to 1.700, 0.035 to 1.600, 0.035 to 1.500, 0.035 to 1.400, 0.035 to 1.300, 0.035 to 1.200, 0.035-1.100, 0.035-1.000, 0.035-0.900, 0.035-0.800, 0.035-0.700, 0.035-0.600, 0.035-0.500, 0.035-0.400, 0.035-0.300, 0.035-0.200 0.040 to 2.300, 0.040 to 2.200, 0.040 to 2.000, 0.040 to 1.900, 0.040 to 1.800, 0.040 to 1.700, 0.040 to 1.600, 0.040 to 1.500, 0.040 to 1.400, 0.040 to 1.300, 0.040 to 1.200, 0.040-1.100, 0.040-1.000, 0.040-0.900, 0.040-0.800, 0.040-0.700, 0.040-0.600, 0.040-0.500, 0.040-0.400, 0.040-0.300, 0.040-0.200 0.050 to 2.300, 0.050 to 2.200, 0.050 to 2.000, 0.050 to 1.900, 0.050 to 1.800, 0.050 to 1.700, 0.050 to 1.600, 0.050 to 1.500, 0.050 to 1.400, 0.050 to 1.300, 0.050 to 1.200, 0.050-1.100, 0.050-1.000, 0.050-0.900, 0.050-0.800, 0.050-0.700, 0.050-0.600, 0.050-0.500, 0.050-0.400, 0.050-0.300, 0.050-0.2000.060 to 2.300, 0.060 to 2.200, 0.060 to 2.000, 0.060 to 1.900, 0.060 to 1.800, 0.060 to 1.700, 0.060 to 1.600, 0.060 to 1.500, 0.060 to 1.400, 0.060 to 1.300, 0.060 to 1.200, 0.060-1.100, 0.060-1.000, 0.060-0.900, 0.060-0.800, 0.060-0.700, 0.060-0.600, 0.060-0.500, 0.060-0.400, 0.060-0.300, 0.060-0.200 0.070 to 2.300, 0.070 to 2.200, 0.070 to 2.000, 0.070 to 1.900, 0.070 to 1.800, 0.070 to 1.700, 0.070 to 1.600, 0.070 to 1.500, 0.070 to 1.400, 0.070 to 1.300, 0.070 to 1.200, 0.070-1.100, 0.070-1.000, 0.070-0.900, 0.070-0.800, 0.070-0.700, 0.070-0.600, 0.070-0.500, 0.070-0.400, 0.070-0.300, 0.070-0.200 0.080 to 2.300, 0.080 to 2.200, 0.080 to 2.000, 0.080 to 1.900, 0.080 to 1.800, 0.080 to 1.700, 0.080 to 1.600, 0.080 to 1.500, 0.080 to 1.400, 0.080 to 1.300, 0.080 to 1.200, 0.080-1.100, 0.080-1.000, 0.080-0.900, 0.080-0.800, 0.080-0.700, 0.080-0.600, 0.080-0.500, 0.080-0.400, 0.080-0.300, 0.080-0.2000.090 pieces to 2.300 pieces, 0.090 pieces to 2.200 pieces, 0.090 pieces to 2.000 pieces, 0.090 pieces to 1.900 pieces, 0.090 pieces to 1.800 pieces, 0.0 90 pieces to 1.700 pieces, 0.090 pieces to 1.600 pieces, 0.090 pieces to 1.500 pieces, 0.090 pieces to 1.400 pieces, 0.090 pieces to 1.300 pieces, 0.090 pieces to 1.200 pieces, 0.090 pieces to 1.100 pieces, 0.090 pieces to 1.000 pieces, 0.090 pieces to 0.900 pieces, 0.090 pieces to 0.800 pieces, 0.090 pieces to 0.7 00 pieces, 0.090 pieces to 0.600 pieces, 0.090 pieces to 0.500 pieces, 0.090 pieces to 0.400 pieces, 0.090 pieces to 0.300 pieces, 0.090 pieces to 0.200 pieces, 0.100 pieces to 2.300 pieces, 0.100 pieces to 2.200 pieces, 0.100 pieces to 2.000 pieces, 0.100 pieces to 1.900 pieces, 0.100 pieces to 1.800 pieces, 0.10 0 to 1.700 pieces, 0.100 to 1.600 pieces, 0.100 to 1.500 pieces, 0.100 to 1.400 pieces, 0.100 to 1.300 pieces, 0.100 to 1. The possible values ​​are 200, 0.100 to 1.100, 0.100 to 1.000, 0.100 to 0.900, 0.100 to 0.800, 0.100 to 0.700, 0.100 to 0.600, 0.100 to 0.500, 0.100 to 0.400, 0.100 to 0.300, or 0.100 to 0.200.

[0071] In this embodiment, the density of VHH antibody present on lipid nanoparticles in a state capable of binding to the antigen is preferably 100 nm, from the viewpoint of achieving excellent target cell delivery. ２ The number ranges from 0.008 to 2,300 per unit, and more preferably from 0.010 to 1,500.

[0072] In one embodiment of this design, the VHH antibody binds to CD8. In this case, the density of the VHH antibody present on the lipid nanoparticles in a state capable of binding to the CD8 antigen is preferably 100 nm on the surface of the nanoparticles. ２ The number of nanoparticles is between 0.004 and 2,200 per nanoparticle, and more preferably on the surface of 100 nm of the nanoparticles. ２ The number of nanoparticles is between 0.008 and 1,800 per nanoparticle, and is particularly preferably on the surface of 100 nm of the nanoparticles. ２The number ranges from 0.010 to 1.500 per unit.

[0073] In one embodiment of this design, the VHH antibody binds to CD4. In this case, the density of the VHH antibody present on the lipid nanoparticles in a state capable of binding to the CD4 antigen is preferably 100 nm on the surface of the nanoparticles. ２ The number of nanoparticles is between 0.030 and 2,300 per nanoparticle, and more preferably on the surface of 100 nm of the nanoparticles. ２ The number of nanoparticles is between 0.035 and 2,300 per nanoparticle, and is particularly preferably 100 nm on the surface of the nanoparticles. ２ The number ranges from 0.040 to 1.500 per unit.

[0074] The nucleic acid encapsulated in the lipid nanoparticles of this embodiment is preferably a functional nucleic acid that controls the expression of a target gene present in the target cell. Examples of such functional nucleic acids include antisense nucleic acids (antisense oligonucleotides, antisense DNA, antisense RNA), heteroduplex nucleic acids, siRNA, microRNA (miRNA), mRNA, guide RNA (gRNA), etc. Alternatively, it may be plasmid DNA (pDNA) that serves as an siRNA expression vector for expressing siRNA in cells. The siRNA expression vector can be prepared from a commercially available siRNA expression vector, or it may be modified as appropriate.

[0075] The gene expression vector to be encapsulated in the lipid nanoparticles according to the present invention is not particularly limited, and vectors commonly used in gene therapy and the like can be used. Preferably, the gene expression vector to be encapsulated in the lipid nanoparticles according to the present invention is a nucleic acid vector such as a plasmid vector. The plasmid vector may remain circular, or it may be pre-cut into a linear shape before being encapsulated in the lipid nanoparticles according to the present invention. The gene expression vector can be designed by conventional methods using commonly used molecular biological tools based on the base sequence information of the gene to be expressed, and can be manufactured by various known methods.

[0076] When the nucleic acid to be encapsulated is mRNA, in one embodiment, the mRNA includes a miRNA binding site. miRNA (microRNA) is typically a small, non-coding single-stranded RNA molecule about 20–25 nucleotides long, produced from hairpin RNA precursors (pre-miRNA), and can form functional complexes with proteins. Typically, miRNA further binds to the UTR region of target mRNA as a functional complex with a protein, and can regulate the target gene by, but not limited to, mRNA degradation or translation inhibition or repression. One embodiment of the miRNA binding site is the miR 122-5p binding site, which can regulate protein expression in hepatocytes.

[0077] The average particle size of the lipid nanoparticles in this embodiment is, for example, 400 nm or less, 350 nm or less, 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less, or 100 nm or less, and 30 nm or more, 40 nm or more, 50 nm or more, or 60 nm or more. In one embodiment, the average particle size of the lipid nanoparticles in this embodiment is 40 nm to 300 nm, 40 nm to 250 nm, 40 nm to 200 nm, 40 nm to 150 nm, 40 nm to 100 nm, 50 nm to 300 nm, 50 nm to 250 nm, 50 nm to 200 nm, 50 nm to 150 nm, 50 nm to 100 nm, 60 nm to 300 nm, 60 nm to 250 nm, 60 nm to 200 nm, 60 nm to 150 nm, or 60 nm to 100 nm.

[0078] In this embodiment, the average particle diameter of lipid nanoparticles refers to the number-average particle diameter measured by a device capable of measuring particle diameter on a number basis, such as dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), or a nanoflow cytometer.

[0079] In one embodiment, the polydispersity index (PDI) of the lipid nanoparticles of this embodiment is 0.01 to 0.7, preferably 0.01 to 0.6, and more preferably 0.03 to 0.3. The zeta potential at pH 7.4 can be in the range of -50 mV to 5 mV, preferably -45 mV to 5 mV.

[0080] The morphology of the lipid nanoparticles in this embodiment is not particularly limited, but examples of morphologies when dispersed in an aqueous solvent include single-layer liposomes, multilayer liposomes, spherical micelles, or amorphous layered structures. The lipid nanoparticles according to the present invention are preferably single-layer liposomes or multilayer liposomes.

[0081] The animals to which the lipid nanoparticles of this embodiment are administered are not particularly limited and may be humans or other animals. Examples of non-human animals include mammals such as cattle, pigs, horses, sheep, goats, monkeys, dogs, cats, rabbits, mice, rats, hamsters, and guinea pigs, as well as birds such as chickens, quail, and ducks.

[0082] In one embodiment, the lipid nanoparticles of this embodiment are produced by a method comprising the steps of producing untargeted lipid nanoparticles containing nucleic acids and not containing VHH antibodies, and linking the VHH antibodies to the untargeted lipid nanoparticles. In another embodiment, the lipid nanoparticles of this embodiment are produced by a method comprising the steps of producing untargeted lipid nanoparticles containing nucleic acids and not containing VHH antibodies, and introducing targeted polyalkylene glycol-modified lipids linked to VHH antibodies into the untargeted lipid nanoparticles. The above-mentioned method for producing untargeted lipid nanoparticles is not particularly limited, and any method available to those skilled in the art can be employed. For example, they can be produced by an alcohol dilution method using a channel. This method involves introducing a solution in which lipid components are dissolved in an alcohol solvent and a solution in which water-soluble components to be encapsulated in lipid nanoparticles are dissolved in an aqueous solvent from separate channels and combining them to produce lipid nanoparticles. Examples of aqueous solvents used in the alcohol dilution method include buffers such as phosphate buffer, citrate buffer, and phosphate-buffered saline, physiological saline, and cell culture media. The above-mentioned non-targeted lipid nanoparticles may be prepared by suspending lipid nanoparticles in an aqueous solution.

[0083] In another embodiment, the lipid nanoparticles of this embodiment are produced by a method comprising the step of mixing nucleic acids, cationic lipids, sterols or sterol derivatives, targeted polyalkylene glycol-modified lipids linked to VHH antibodies, and untargeted polyalkylene glycol-modified lipids not linked to VHH antibodies. The production method is not particularly limited, and any method available to those skilled in the art can be used. For example, the lipid nanoparticles according to the present invention can be produced by the alcohol dilution method using the flow channel described above. Alternatively, for example, all lipid components can be dissolved in an organic solvent such as chloroform, and a lipid film can be formed by drying under reduced pressure using an evaporator or by spray drying using a spray dryer. Then, an aqueous solvent containing components to be encapsulated in the lipid nanoparticles, such as nucleic acids, can be added to the dried mixture, and the mixture can be further emulsified using an emulsifier such as a homogenizer, an ultrasonic emulsifier, or a high-pressure spray emulsifier. They can also be produced by methods well known for producing liposomes, such as reverse-phase evaporation. To control the size of lipid nanoparticles, you can use a membrane filter with uniform pore sizes and perform extrusion filtration under high pressure.

[0084] The composition of the aqueous solvent (dispersion medium) is not particularly limited, but examples include buffers such as phosphate buffer, citrate buffer, and phosphate-buffered saline, physiological saline, and cell culture media. These aqueous solvents (dispersion mediums) can stably disperse lipid nanoparticles, but further additions such as sugars (aqueous solutions) such as monosaccharides like glucose, galactose, mannose, fructose, inositol, ribose, and xylose sugars, disaccharides such as lactose, sucrose, cellobiose, trehalose, and maltose, trisaccharides such as raffinose and melesinose, polysaccharides such as cyclodextrin, and sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, and maltitol, or polyhydric alcohols (aqueous solutions) such as glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol monoalkyl ether, and 1,3-butylene glycol may also be added. To stably store lipid nanoparticles dispersed in this aqueous solvent for a long period, it is desirable to remove as much electrolyte as possible from the aqueous solvent in terms of physical stability, such as suppressing aggregation. Furthermore, in terms of the chemical stability of the lipids, it is desirable to set the pH of the aqueous solvent to slightly acidic to near neutral (pH 3.0 to 8.0) and / or remove dissolved oxygen by nitrogen bubbling or the like.

[0085] When freeze-drying or spray-drying the aqueous dispersion of lipid nanoparticles according to this embodiment, stability may be improved by using sugars (aqueous solutions) such as monosaccharides including glucose, galactose, mannose, fructose, inositol, ribose, and xylose sugar; disaccharides including lactose, sucrose, cellobiose, trehalose, and maltose; trisaccharides including raffinose and melesinose; polysaccharides including cyclodextrin; and sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, and maltitol. Furthermore, when freezing the aqueous dispersion, stability may be improved by using the aforementioned sugars or polyhydric alcohols (aqueous solutions) such as glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol monoalkyl ether, and 1,3-butylene glycol. In one embodiment, the lipid nanoparticles according to this embodiment are freeze-dried.

[0086] In one aspect, the present invention relates to a pharmaceutical formulation containing lipid nanoparticles according to this embodiment. The pharmaceutical formulation may contain a buffering agent. Examples of buffering agents include HEPES buffering agents, phosphate buffering agents, and Tris buffering agents. The pharmaceutical formulation may also contain a disaccharide. Examples of disaccharides include lactose, sucrose, cellobiose, trehalose, and maltose, with sucrose being preferred.

[0087] 2-2. Lipid Nanoparticles Containing Fab Antibody One aspect of the present invention is a lipid nanoparticle comprising: a nucleic acid encapsulated in the lipid nanoparticle; a cationic lipid; a sterol or sterol derivative; a Fab antibody; a targeted polyalkylene glycol-modified lipid; and a non-targeted polyalkylene glycol-modified lipid, wherein the Fab antibody targets an antigen on a T cell; the targeted polyalkylene glycol-modified lipid is a lipid in which the Fab antibody is linked to a polyalkylene glycol; and the non-targeted polyalkylene glycol-modified lipid is a lipid in which the Fab antibody is not linked; and the density of Fab antibodies present on the surface of the lipid nanoparticles that are capable of binding to the antigen is such that the density is 100 nm on the surface of the nanoparticle. ２ This concerns lipid nanoparticles, which number between 0.003 and 1.113 per nanoparticle.

[0088] The density of Fab antibodies present on lipid nanoparticles in a state capable of binding to the antigen in this embodiment is, for example, 100 nm. ２Percentages of 0.004 or more, 0.005 or more, 0.006 or more, 0.007 or more, 0.008 or more, 0.009 or more, 0.010 or more, 0.015 or more, 0.020 or more, 0.025 or more, 0.030 or more, 0.035 or more, 0.040 or more, 0.045 or more, 0.050 or more, 0.055 or more, 0.060 or more, 0.065 or more, 0.070 or more, 0.075 or more, 0.080 or more, 0.085 or more, 0.090 or more, 0.095 or more, 0.100 or more, 0.105 or more, 0 .. 110 or more, 0.115 or more, 0.120 or more, 0.125 or more, 0.130 or more, 0.135 or more, 0.140 or more, 0.145 or more, 0.150 or more, 0.155 or more, 0.160 or more, 0.165 or more, 0.170 or more, 0.17 5 or more, 0.180 or more, 0.185 or more, 0.190 or more, 0.195 or more, 0.200 or more, 0.205 or more, 0.210 or more, 0.215 or more, 0.220 or more, 0.225 or more, 0.230 or more, 0.235 or more, 0.240 0.245 or more, 0.250 or more, 0.255 or more, 0.260 or more, 0.265 or more, 0.270 or more, 0.275 or more, 0.280 or more, 0.285 or more, 0.290 or more, 0.295 or more, 0.300 or more, 0.305 or more , 0.310 or more, 0.315 or more, 0.320 or more, 0.325 or more, 0.330 or more, 0.335 or more, 0.340 or more, 0.345 or more, 0.350 or more, 0.355 or more, 0.360 or more, 0.365 or more, 0.370 or more, 0 . 375 or more, 0.380 or more, 0.385 or more, 0.390 or more, 0.395 or more, 0.400 or more, 0.405 or more, 0.410 or more, 0.415 or more, 0.420 or more, 0.425 or more, 0.430 or more, 0.435 or more, 0.440 or more, 0.445 or more, 0.450 or more, 0.455 or more, 0.460 or more, 0.465 or more, 0.470 or more, 0.475 or more, 0.480 or more, 0.485 or more, 0.490 or more, 0.495 or more, or 0.500 or more,1.110 or less, 1.100 or less, 1.050 or less, 1.000 or less, 0.950 or less, 0.900 or less, 0.850 or less, 0.800 or less, 0.750 or less, 0.700 or less, 0.650 or less, 0 .600 or less, 0.550 or less, 0.500 or less, 0.450 or less, 0.400 or less, 0.350 or less, 0.300 or less, 0.250 or less, 0.200 or less, 0.150 or less, 0.100 or less

[0089] Furthermore, the density of the Fab antibody is, for example, 100 nm. ２Percentage: 0.003 to 1.110, 0.003 to 1.100, 0.003 to 1.050, 0.003 to 1.000, 0.003 to 0.950, 0.003 to 0.900, 0.003 to 0.850, 0.003 to 0.800, 0.003 to 0.750, 0.003 to 0.700 0.003 pieces to 0.650 pieces, 0.003 pieces to 0.600 pieces, 0.003 pieces to 0.550 pieces, 0.003 pieces to 0.500 pieces, 0.003 pieces to 0.450 pieces, 0.003 pieces to 0.400 pieces, 0.003 pieces to 0.350 pieces, 0.003 pieces to 0.300 pieces, 0.003 pieces to 0.250 pieces, 0.003 pieces to 0.200 pieces, 0.004 pieces to 1.113 pieces, 0.004 pieces to 1.110 pieces, 0.004 pieces to 1.100 pieces, 0.004 pieces to 1.050 pieces, 0.004 pieces to 1.000 pieces, 0.0 04 pieces to 0.950 pieces, 0.004 pieces to 0.900 pieces, 0.004 pieces to 0.850 pieces, 0.004 pieces to 0.800 pieces, 0.004 pieces to 0.750 pieces, 0.004 pieces -0.700 pieces, 0.004 pieces - 0.650 pieces, 0.004 pieces - 0.600 pieces, 0.004 pieces - 0.550 pieces, 0.004 pieces - 0.500 pieces, 0.004 pieces - 0. 450 pieces, 0.004 pieces to 0.400 pieces, 0.004 pieces to 0.350 pieces, 0.004 pieces to 0.300 pieces, 0.004 pieces to 0.250 pieces, 0.004 pieces to 0.200 pieces 0.005 pieces to 1.113 pieces, 0.005 pieces to 1.110 pieces, 0.005 pieces to 1.100 pieces, 0.005 pieces to 1.050 pieces, 0.005 pieces to 1.000 pieces, 0.0 05 pieces to 0.950 pieces, 0.005 pieces to 0.900 pieces, 0.005 pieces to 0.850 pieces, 0.005 pieces to 0.800 pieces, 0.005 pieces to 0.750 pieces, 0.005 pieces ~0.700 pieces, 0.005 pieces~0.650 pieces, 0.005 pieces~0.600 pieces, 0.005 pieces~0.550 pieces, 0.005 pieces~0.500 pieces, 0.005 pieces~0. 450 pieces, 0.005 pieces to 0.400 pieces, 0.005 pieces to 0.350 pieces, 0.005 pieces to 0.300 pieces, 0.005 pieces to 0.250 pieces, 0.005 pieces to 0.200 pieces0.006 to 1.113, 0.006 to 1.110, 0.006 to 1.100, 0.006 to 1.050, 0.006 to 1.000, 0.006 to 0.950, 0.006 to 0.900, 0.006 to 0.850, 0.006 to 0.800, 0.006 to 0.750, 0.006 to 0.700, 0.006 to 0.650, 0.006 to 0.600, 0.006 to 0.550, 0.006 to 0.500, 0.006 to 0.450, 0.006 to 0.400, 0.006 to 0.350, 0.006 to 0.300, 0.006 to 0.250, 0.006 to 0.200 0.008 to 1.113, 0.008 to 1.110, 0.008 to 1.100, 0.008 to 1.050, 0.008 to 1.000, 0.008 to 0.950, 0.008 to 0.900, 0.008 to 0.850, 0.008 to 0.800, 0.008 to 0.750, 0.008 to 0.700, 0.008 to 0.650, 0.008 to 0.600, 0.008 to 0.550, 0.008 to 0.500, 0.008 to 0.450, 0.008 to 0.400, 0.008 to 0.350, 0.008 to 0.300, 0.008 to 0.250, 0.008 to 0.200 0.010 to 1.113, 0.010 to 1.110, 0.010 to 1.100, 0.010 to 1.050, 0.010 to 1.000, 0.010 to 0.950, 0.010 to 0.900, 0.010 to 0.850, 0.010 to 0.800, 0.010 to 0.750, 0.010 to 0.700, 0.010 to 0.650, 0.010 to 0.600, 0.010 to 0.550, 0.010 to 0.500, 0.010 to 0.450, 0.010 to 0.400, 0.010 to 0.350, 0.010 to 0.300, 0.010 to 0.250, 0.010 to 0.200.

[0090] From the viewpoint of achieving excellent target cell delivery property, the density of Fab antibodies present in a state capable of binding to an antigen on the lipid nanoparticles of the present aspect is preferably 100 nm ２The number of items per unit ranges from 0.004 to 1.113, and more preferably from 0.006 to 1.000.

[0091] In one embodiment of this present invention, the Fab antibody is antibody 3, comprising the following heavy chain variable region (VH) and light chain variable region (VL). <Heavy chain variable region> QVQLVESGGGVVQPGRSLRLSCAASGFTFSDFGMNWVRQAPGKGLEWVALIYYDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPHYDGYYHFFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC (SEQ ID NO: 57) <Light chain variable region> DIQMTQSPSSLSASVGDRVTITCKGSQDINNYLAWYQQKPGKAPKLLIYNTDILHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCYQYNNGYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 58)

[0092] Except for differences in the antibody used and the range of antibody densities, everything described in 2-1. Lipid nanoparticles containing VHH antibody applies to lipid nanoparticles containing Fab antibody in this embodiment. Any part of 2-1. describing VHH antibody should be interpreted as describing Fab antibody.

[0093] 2-3. Lipid Nanoparticles Containing ScFv Antibody One aspect of the present invention is a lipid nanoparticle comprising: a nucleic acid encapsulated in the lipid nanoparticle; a cationic lipid; a sterol or sterol derivative; an scFv antibody; a targeted polyalkylene glycol-modified lipid; and a non-targeted polyalkylene glycol-modified lipid, wherein the scFv antibody targets an antigen on a T cell; the targeted polyalkylene glycol-modified lipid is a lipid in which the scFv antibody is linked to a polyalkylene glycol; and the non-targeted polyalkylene glycol-modified lipid is a lipid in which the scFv antibody is not linked; and the density of scFv antibodies present on the surface of the lipid nanoparticles that are capable of binding to the antigen is such that the density ２ This relates to lipid nanoparticles, with a quantity ranging from 0.001 to 3,400 per nanoparticle.

[0094] In this embodiment, the density of scFv antibody present on lipid nanoparticles in a state capable of binding to the antigen is, for example, 100 nm. ２0.001 or more, 0.002 or more, 0.003 or more, 0.004 or more, 0.0005 or more, 0.006 or more, 0.007 or more, 0.008 or more, 0.009 or more, 0.010 or more, 0.015 or more, 0.020 or more, 0.025 or more, 0 .030 or more, 0.035 or more, 0.040 or more, 0.045 or more, 0.050 or more, 0.055 or more, 0.060 or more, 0.065 or more, 0.070 or more, 0.075 or more, 0.080 or more, 0.085 or more, 0.090 or more, 0.095 or more Above, 0.100 or more, 0.105 or more, 0.110 or more, 0.115 or more, 0.120 or more, 0.125 or more, 0.130 or more, 0.135 or more, 0.140 or more, 0.145 or more, 0.150 or more, 0.155 or more, 0.160 or more, 0.16 5 or more, 0.170 or more, 0.175 or more, 0.180 or more, 0.185 or more, 0.190 or more, 0.195 or more, 0.200 or more, 0.205 or more, 0.210 or more, 0.215 or more, 0.220 or more, 0.225 or more, 0.230 or more, 0 .. 235 or more, 0.240 or more, 0.245 or more, 0.250 or more, 0.255 or more, 0.260 or more, 0.265 or more, 0.270 or more, 0.275 or more, 0.280 or more, 0.285 or more, 0.290 or more, 0.295 or more, 0.300 or more Above, 0.305 or more, 0.310 or more, 0.315 or more, 0.320 or more, 0.325 or more, 0.330 or more, 0.335 or more, 0.340 or more, 0.345 or more, 0.350 or more, 0.355 or more, 0.360 or more, 0.365 or more, 0.3 70 or more, 0.375 or more, 0.380 or more, 0.385 or more, 0.390 or more, 0.395 or more, 0.400 or more, 0.405 or more, 0.410 or more, 0.415 or more, 0.420 or more, 0.425 or more, 0.430 or more, 0.435 or more, 0.440 or more, 0.445 or more, 0.450 or more, 0.455 or more, 0.460 or more, 0.465 or more, 0.470 or more, 0.475 or more, 0.480 or more, 0.485 or more, 0.490 or more, 0.495 or more, or 0.500 or more.3.400 or less, 3.300 or less, 3.200 or less, 3.100 or less, 3.000 or less, 2.900 or less, 2.800 or less, 2.700 or less, 2 .600 or less, 2.500 or less, 2.300 or less, 2.200 or less, 2.100 or less, 2.000 or less, 1.900 or less. 800 or less, 1.700 or less, 1.600 or less, 1.500 or less, 1.400 or less, 1.300 or less, 1.200 or less, 1.100 or less, 1.0 00 or less, 0.900 or less, 0.800 or less, 0.900 or less, 0.800 or less, 0.700 or less, 0.600 or less, 0.500 or less.

[0095] Furthermore, the density of the scFv antibody is, for example, 100 nm. ２Per unit: 0.001 to 3.4000, 0.001 to 3.300, 0.001 to 3.200, 0.001 to 3.100, 0.001 to 3.000, 0.001 to 2.900, 0.001 to 2.800, 0.001 to 2.700, 0.001 to 2.600, 0.001 to 2.500, 0.001 to 2.400, 0.001 to 2.300, 0.001 to 2.200, 0.001 to 2.100, 0.001 to 2.000, 0.001 to 1.900 , 0.001 to 1.800 pieces, 0.001 to 1.700 pieces, 0.001 to 1.600 pieces, 0.001 to 1.500 pieces, 0.001 to 1.400 pieces, 0.001 to 1.300 pieces, 0.001 to 1.200 pieces, 0.001 to 1.100 pieces , 0.001 pieces to 1.000 pieces, 0.001 pieces to 0.900 pieces, 0.001 pieces to 0.800 pieces, 0.001 pieces to 0.900 pieces, 0.001 pieces to 0.800 pieces, 0.001 pieces to 0.700 pieces, 0.001 pieces to 0.600 pieces, 0.001 pieces to 0.500 pieces, 0.002 to 3.4000 pieces, 0.002 to 3.300 pieces, 0.002 to 3.200 pieces, 0.002 to 3.100 pieces, 0.002 to 3.000 pieces, 0.002 to 2.900 pieces, 0.002 to 2.800 pieces, 0.002 to 2.700 pieces , 0.002 to 2.600 pieces, 0.002 to 2.500 pieces, 0.002 to 2.400 pieces, 0.002 to 2.300 pieces, 0.002 to 2.200 pieces, 0.002 to 2.100 pieces, 0.002 to 2.000 pieces, 0.002 to 1.900 pieces , 0.002 to 1.800 pieces, 0.002 to 1.700 pieces, 0.002 to 1.600 pieces, 0.002 to 1.500 pieces, 0.002 to 1.400 pieces, 0.002 to 1.300 pieces, 0.002 to 1.200 pieces, 0.002 to 1.100 pieces , 0.002 pieces to 1.000 pieces, 0.002 pieces to 0.900 pieces, 0.002 pieces to 0.800 pieces, 0.002 pieces to 0.900 pieces, 0.002 pieces to 0.800 pieces, 0.002 pieces to 0.700 pieces, 0.002 pieces to 0.600 pieces, 0.002 pieces to 0.500 pieces,0.003 to 3.4000, 0.003 to 3.300, 0.003 to 3.200, 0.003 to 3.100, 0.003 to 3.000, 0.003 to 2.900, 0.003 to 2.800, 0.003 to 2.700, 0.003 to 2.600, 0.003 to 2.500, 0.003 to 2.400, 0.003 to 2.300, 0.003 to 2.200, 0.003 to 2.100, 0.003 to 2.000, 0.003 to 1.900 0.003 to 1.800, 0.003 to 1.700, 0.003 to 1.600, 0.003 to 1.500, 0.003 to 1.400, 0.003 to 1.300, 0.003 to 1.200, 0.003 to 1.100, 0.003 to 1.000, 0.003 to 0.900, 0.003 to 0.800, 0.003 to 0.900, 0.003 to 0.800, 0.003 to 0.700, 0.003 to 0.600, 0.003 to 0.500 0.004 to 3.4000, 0.004 to 3.300, 0.004 to 3.200, 0.004 to 3.100, 0.004 to 3.000, 0.004 to 2.900, 0.004 to 2.800, 0.004 to 2.700, 0.004 to 2.600, 0.004 to 2.500, 0.004 to 2.400, 0.004 to 2.300, 0.004 to 2.200, 0.004 to 2.100, 0.004 to 2.000, 0.004 to 1.900 0.004 to 1.800, 0.004 to 1.700, 0.004 to 1.600, 0.004 to 1.500, 0.004 to 1.400, 0.004 to 1.300, 0.004 to 1.200, 0.004 to 1.100, 0.004 to 1.000, 0.004 to 0.900, 0.004 to 0.800, 0.004 to 0.900, 0.004 to 0.800, 0.004 to 0.700, 0.004 to 0.600, 0.004 to 0.5000.005 to 3.4000, 0.005 to 3.300, 0.005 to 3.200, 0.005 to 3.100, 0.005 to 3.000, 0.005 to 2.900, 0.005 to 2.800, 0.005 to 2.700, 0.005 to 2.600, 0.005 to 2.500, 0.005 to 2.400, 0.005 to 2.300, 0.005 to 2.200, 0.005 to 2.100, 0.005 to 2.000, 0.005 to 1.900 0.005 to 1.800, 0.005 to 1.700, 0.005 to 1.600, 0.005 to 1.500, 0.005 to 1.400, 0.005 to 1.300, 0.005 to 1.200, 0.005 to 1.100, 0.005 to 1.000, 0.005 to 0.900, 0.005 to 0.800, 0.005 to 0.900, 0.005 to 0.800, 0.005 to 0.700, 0.005 to 0.600, 0.005 to 0.500 0.006 to 3.4000, 0.006 to 3.300, 0.006 to 3.200, 0.006 to 3.100, 0.006 to 3.000, 0.006 to 2.900, 0.006 to 2.800, 0.006 to 2.700, 0.006 to 2.600, 0.006 to 2.500, 0.006 to 2.400, 0.006 to 2.300, 0.006 to 2.200, 0.006 to 2.100, 0.006 to 2.000, 0.006 to 1.900 0.006 to 1.800, 0.006 to 1.700, 0.006 to 1.600, 0.006 to 1.500, 0.006 to 1.400, 0.006 to 1.300, 0.006 to 1.200, 0.006 to 1.100, 0.006 to 1.000, 0.006 to 0.900, 0.006 to 0.800, 0.006 to 0.900, 0.006 to 0.800, 0.006 to 0.700, 0.006 to 0.600, 0.006 to 0.5000.008 to 3.4000 pieces, 0.008 to 3.300 pieces, 0.008 to 3.200 pieces, 0.008 to 3.100 pieces, 0.008 to 3.000 pieces, 0.008 to 2.900 pieces, 0.008 to 2.800 pieces, 0.008 to 2.700 pieces , 0.008 to 2.600 pieces, 0.008 to 2.500 pieces, 0.008 to 2.400 pieces, 0.008 to 2.300 pieces, 0.008 to 2.200 pieces, 0.008 to 2.100 pieces, 0.008 to 2.000 pieces, 0.008 to 1.900 pieces , 0.008 to 1.800 pieces, 0.008 to 1.700 pieces, 0.008 to 1.600 pieces, 0.008 to 1.500 pieces, 0.008 to 1.400 pieces, 0.008 to 1.300 pieces, 0.008 to 1.200 pieces, 0.008 to 1.100 pieces , 0.008 pieces to 1.000 pieces, 0.008 pieces to 0.900 pieces, 0.008 pieces to 0.800 pieces, 0.008 pieces to 0.900 pieces, 0.008 pieces to 0.800 pieces, 0.008 pieces to 0.700 pieces, 0.008 pieces to 0.600 pieces, 0.008 pieces to 0.500 pieces, 0.010 to 3.4000 pieces, 0.010 to 3.300 pieces, 0.010 to 3.200 pieces, 0.010 to 3.100 pieces, 0.010 to 3.000 pieces, 0.010 to 2.900 pieces, 0.010 to 2.800 pieces, 0.010 to 2.700 pieces , 0.010 to 2.600 pieces, 0.010 to 2.500 pieces, 0.010 to 2.400 pieces, 0.010 to 2.300 pieces, 0.010 to 2.200 pieces, 0.010 to 2.100 pieces, 0.010 to 2.000 pieces, 0.010 to 1.900 pieces The quantities are 0.010 to 1.800, 0.010 to 1.700, 0.010 to 1.600, 0.010 to 1.500, 0.010 to 1.400, 0.010 to 1.300, 0.010 to 1.200, 0.010 to 1.100, 0.010 to 1.000, 0.010 to 0.900, 0.010 to 0.800, 0.010 to 0.900, 0.010 to 0.800, 0.010 to 0.700, 0.010 to 0.600, and 0.010 to 0.500.

[0096] The density of the scFv antibody present in a state capable of binding to an antigen on the lipid nanoparticles of the present aspect is preferably 0.002 to 3.100 per 100 nm ２ from the viewpoint of achieving excellent target cell delivery property, and more preferably 0.005 to 2.800.

[0097] In one embodiment of the present aspect, the scFv antibody binds to CD8. In this case, the density of the scFv antibody present in a state capable of binding to the CD8 antigen on the lipid nanoparticles is preferably 0.001 to 1.900 per 100 nm ２ on the nanoparticle surface, more preferably 0.002 to 1.400 per 100 nm ２ on the nanoparticle surface, and particularly preferably 0.005 to 1.100 per 100 nm ２ on the nanoparticle surface.

[0098] In one embodiment of the present aspect, the scFv antibody binds to CD4. In this case, the density of the scFv antibody present in a state capable of binding to the CD4 antigen on the lipid nanoparticles is 0.020 to 3.400 per 100 nm ２ on the nanoparticle surface, preferably 0.020 to 3.100 per 100 nm ２ on the nanoparticle surface, and more preferably 0.030 to 2.800 per 100 nm ２ on the nanoparticle surface.

[0099] In one embodiment of this design, the scFv antibody is antibody 5 represented by the following sequence. The underlined portion indicates the CDR sequence (based on the Kabat numbering scheme). QVQLVESGGGVVQPGRSLRLSCAASGFTFSDFGMNWVRQAPGKGLEWVALIYYDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPHYDGYYHFFDSWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKGSQDINNYLAWYQQKPGKAPKLLIYNTDILHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCYQYNNGYTFGQGTKVEIK (Sequence ID 60)

[0100] In one embodiment of this design, the scFv antibody is antibody 6 represented by the following sequence. The underlined portion indicates the CDR sequence (based on the IMGT numbering scheme). QVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIK (Sequence ID 61)

[0101] Except for differences in the antibody used and the range of antibody densities, everything described in 2-1. Lipid nanoparticles containing VHH antibody applies to the scFv antibody-containing lipid nanoparticles of this embodiment. The parts of 2-1. that describe VHH antibody should be understood as describing scFv antibody.

[0102] 3. Method for Measuring the Optimal Density Range of Antibodies on the Surface of Lipid Nanoparticles In one embodiment, the present invention provides a method for measuring the optimal density range of antibodies on the surface of lipid nanoparticles, wherein the lipid nanoparticles comprise nucleic acids encapsulated within the lipid nanoparticles, cationic lipids, sterols or sterol derivatives, target binding sites, targeted polyalkylene glycol-modified lipids, and untargeted polyalkylene glycol-modified lipids, wherein the target binding sites target target sites on target cells, the targeted polyalkylene glycol-modified lipids are lipids in which the target binding sites are linked to polyalkylene glycol, and the untargeted polyalkylene glycol-modified lipids are lipids in which the target binding sites are not linked, and the measurement method comprises gradually increasing the amount of target binding sites and using a nanoflow cytometer to measure the density of target binding sites that are capable of binding to target sites on the surface of the lipid nanoparticles, and The present invention relates to a measurement method comprising: measuring the expression level of nucleic acids delivered at the aforementioned target binding site density within target cells; and obtaining an optimal density range by setting a target binding site density range that achieves an expression level of 40% or more, 50% or more, or 60% or more, when the maximum expression level is set to 100%.

[0103] In this disclosure, "nanoflow cytometer" is a device capable of analyzing minute particles or cells approximately 10 to 999 nanometers in size with high precision. Nanoflow cytometers can analyze minute particles or cells with higher precision than conventional flow cytometry. Nanoflow cytometers can analyze the size, shape, surface properties, or internal components of particles by irradiating a sample passing through a microchannel with a laser and detecting fluorescence or scattered light. While not particularly limited, examples of nanoflow cytometers include "nanoFCM" manufactured by NanoFCM Co., Ltd. and "CytoFLEX nano" manufactured by Beckman Coulter.

[0104] The composition and embodiments of the lipid nanoparticles to be measured in the measurement method of this embodiment are the same as the composition and embodiments of the lipid nanoparticles described in "2-1. Lipid nanoparticles containing VHH antibody," "2-2. Lipid nanoparticles containing Fab antibody," and "2-3. Lipid nanoparticles containing scFv antibody." However, the "targeted polyalkylene glycol-modified lipid" is not limited to polyalkylene glycol-modified lipids to which VHH antibody, Fab antibody, or scFv antibody is linked, and may also be polyalkylene glycol-modified lipids to which target binding sites other than VHH antibody, Fab antibody, and scFv antibody are linked to polyalkylene glycol.

[0105] According to the measurement method of this embodiment, the antibody density per unit surface area of ​​lipid nanoparticles can be quantified on a particle-by-particle basis using a nanoflow cytometer. This allows for the clear definition and control of antibody density, a critical quality characteristic (CQA), thereby enabling the production of lipid nanoparticles that selectively deliver nucleic acids to target cells with high reproducibility. Such particle-level quality evaluation is extremely useful from the viewpoint of ensuring the uniformity and functionality of the formulation.

[0106] 4. Method for Determining Whether the Density of Target Binding Sites on the Surface of Lipid Nanoparticles is Within an Optimal Density Range In one embodiment, the present invention is a method for determining whether the density of target binding sites on the surface of lipid nanoparticles is within an optimal density range, wherein the lipid nanoparticles comprise nucleic acids encapsulated within the lipid nanoparticles, cationic lipids, sterols or sterol derivatives, target binding sites, targeted polyalkylene glycol-modified lipids, and untargeted polyalkylene glycol-modified lipids, wherein the target binding sites target target sites on target cells, the targeted polyalkylene glycol-modified lipids are lipids in which the target binding sites are linked to polyalkylene glycol, and the untargeted polyalkylene glycol-modified lipids are lipids in which the target binding sites are not linked, and the method comprises the following steps (1) and (2): (1) obtaining the weight ratio of the targeted polyalkylene glycol-modified lipids to the nucleic acids in the lipid nanoparticles; (2) determining that the density of target binding sites on the surface of the lipid nanoparticles is within an optimal density range if the weight ratio obtained in step (1) is within an optimal weight ratio range; Here, the optimal weight ratio range is expressed by the following regression equation (A), which is based on the correlation between the density of target binding sites of lipid nanoparticles measured using a nanoflow cytometer and the weight ratio of the targeted polyalkylene glycol-modified lipid to the nucleic acid in the lipid nanoparticles: Y = aX + b (A) [wherein Y is the density of target binding sites of lipid nanoparticles measured using a nanoflow cytometer (100 nm of the nanoparticle surface)] ２ The present invention relates to a numerical range in which (Y1-b) / a, obtained by substituting the lower limit of the optimal density range Y1 into (Y1-b) / a, obtained by substituting the upper limit of the optimal density range Y2 into (Y2-b) / a, and the numerical range in which (Y2-b) / a, obtained by substituting the upper limit of the optimal density range Y2, is the lower limit of the numerical range, and the optimal density range is the numerical range obtained by the method described in "3. Method for measuring the optimal density range of antibodies on the surface of lipid nanoparticles" above.

[0107] The inventors have found that, in antibody-linked lipid nanoparticles, by utilizing the correlation between the density of target binding sites of the lipid nanoparticles, measured using a nanoflow cytometer, and the weight ratio of the targeted polyalkylene glycol-modified lipid to the nucleic acid in the lipid nanoparticles, it is possible to determine whether the density of target binding sites on the surface of lipid nanoparticles is within the optimal density range by measuring the weight ratio of the targeted polyalkylene glycol-modified lipid to the nucleic acid in lipid nanoparticles with an unknown antibody density. This method is extremely useful, for example, under conditions where a nanoflow cytometer cannot be used (e.g., under GxP standards).

[0108] The composition and embodiments of the lipid nanoparticles to be measured in the method of this embodiment are the same as those described in "2-1. Lipid nanoparticles containing VHH antibody," "2-2. Lipid nanoparticles containing Fab antibody," and "2-3. Lipid nanoparticles containing scFv antibody." However, the "targeted polyalkylene glycol-modified lipid" is not limited to polyalkylene glycol-modified lipids to which VHH antibody, Fab antibody, or scFv antibody is linked, and may also be polyalkylene glycol-modified lipids to which target binding sites other than VHH antibody, Fab antibody, and scFv antibody are linked to polyalkylene glycol.

[0109] The method for obtaining the weight ratio of targeted polyalkylene glycol-modified lipids to nucleic acids in lipid nanoparticles is not particularly limited, but can be calculated, for example, by dividing the concentration of targeted polyalkylene glycol-modified lipids by the concentration of nucleic acids in the lipid nanoparticles. The concentration of nucleic acids in lipid nanoparticles is not particularly limited, but can be obtained, for example, by a fluorescence quantification method using a fluorescent reagent such as RiboGreen reagent. The concentration of targeted polyalkylene glycol-modified lipids in lipid nanoparticles is not particularly limited, but can be obtained, for example, using HPLC, SDS-PAGE, SDS-CGE, etc.

[0110] 5. Method for Measuring the Optimal Weight Ratio Range of Targeted Polyalkylene Glycol Modified Lipids to Nucleic Acids in Lipid Nanoparticles In one embodiment, the present invention is a method for measuring the optimal weight ratio range of targeted polyalkylene glycol modified lipids to nucleic acids in lipid nanoparticles, wherein the lipid nanoparticles comprise nucleic acids encapsulated in the lipid nanoparticles, a cationic lipid, a sterol or sterol derivative, a target binding site, a targeted polyalkylene glycol modified lipid, and a non-targeted polyalkylene glycol modified lipid, wherein the target binding site targets a target site on a target cell, the targeted polyalkylene glycol modified lipid is a lipid in which the target binding site is linked to polyalkylene glycol, and the non-targeted polyalkylene glycol modified lipid is a lipid in which the target binding site is not linked, and the method comprises a regression equation represented by the following equation (A) based on the correlation between the density of target binding sites of lipid nanoparticles measured using a nanoflow cytometer and the weight ratio of the targeted polyalkylene glycol modified lipids to nucleic acids in the lipid nanoparticles: Y = aX + b (A) [wherein of equation (A), Y is the density of target binding sites of lipid nanoparticles measured using a nanoflow cytometer (100 nm on the nanoparticle surface). ２ The present invention relates to a method that includes the step of obtaining an optimal weight ratio range, wherein the lower limit is (Y1-b) / a obtained by substituting the lower limit of the optimal density range Y1 into the equation [where Y1 represents the number of target binding sites per unit, X represents the weight ratio of the targeted polyalkylene glycol-modified lipid to the nucleic acid in the lipid nanoparticles, a represents the slope of the regression line, a > 0, and b represents the intercept of the regression line], and the upper limit is (Y2-b) / a obtained by substituting the upper limit of the optimal density range Y2 into the equation, and the optimal density range is a numerical range obtained by the method described in "3. Method for measuring the optimal density range of antibodies on the surface of lipid nanoparticles" above.

[0111] The composition and embodiments of the lipid nanoparticles to be measured in the method of this embodiment are the same as those described in "2-1. Lipid nanoparticles containing VHH antibody," "2-2. Lipid nanoparticles containing Fab antibody," and "2-3. Lipid nanoparticles containing scFv antibody." However, the "targeted polyalkylene glycol-modified lipid" is not limited to polyalkylene glycol-modified lipids to which VHH antibody, Fab antibody, or scFv antibody is linked, and may also be polyalkylene glycol-modified lipids to which target binding sites other than VHH antibody, Fab antibody, and scFv antibody are linked to polyalkylene glycol.

[0112] The method for obtaining the weight ratio of targeted polyalkylene glycol-modified lipids to nucleic acids in lipid nanoparticles is as described in "4. Method for determining whether the density of target binding sites on the surface of lipid nanoparticles is within the optimal density range."

[0113] 6. Lipid nanoparticles containing VHH antibody within an optimal weight ratio range. In one embodiment, the present invention relates to lipid nanoparticles comprising: nucleic acid encapsulated in the lipid nanoparticle; a cationic lipid; a sterol or sterol derivative; a VHH antibody; a targeted polyalkylene glycol-modified lipid; and a non-targeted polyalkylene glycol-modified lipid, wherein the VHH antibody targets an antigen on a T cell; the targeted polyalkylene glycol-modified lipid is a lipid in which the VHH antibody is linked to polyalkylene glycol; the non-targeted polyalkylene glycol-modified lipid is a lipid in which the VHH antibody is not linked; and the weight ratio range of the targeted polyalkylene glycol-modified lipid to the nucleic acid in the lipid nanoparticle is 0.047 to 3.931.

[0114] In this embodiment, the weight ratio range of targeted polyalkylene glycol-modified lipids to nucleic acids in lipid nanoparticles is such that the optimal density range of VHH antibodies on the surface of the lipid nanoparticles is 100 nm on the nanoparticle surface. ２ When the number of particles per nanoparticle ranges from 0.004 to 2,300, this is the optimal weight ratio range measured according to the method described in "5. Method for measuring the optimal weight ratio range of targeted polyalkylene glycol-modified lipids to nucleic acids in lipid nanoparticles".

[0115] In this embodiment, the weight ratio range of targeted polyalkylene glycol-modified lipids to nucleic acids in lipid nanoparticles is preferably 0.054 to 3.931. This weight ratio range is such that the optimal density range of VHH antibodies on the surface of the lipid nanoparticles is 100 nm from the nanoparticle surface. ２ When the number of particles per nanoparticle ranges from 0.008 to 2,300, this is the optimal weight ratio range measured according to the method described in "5. Method for measuring the optimal weight ratio range of targeted polyalkylene glycol-modified lipids to nucleic acids in lipid nanoparticles".

[0116] In this embodiment, the weight ratio range of targeted polyalkylene glycol-modified lipids to nucleic acids in lipid nanoparticles is more preferably 0.057 to 2.578. This weight ratio range is such that the optimal density range of VHH antibodies on the surface of the lipid nanoparticles is 100 nm from the nanoparticle surface. ２ When the number of particles per nanoparticle ranges from 0.010 to 1.500, this is the optimal weight ratio range measured according to the method described in "5. Method for measuring the optimal weight ratio range of targeted polyalkylene glycol-modified lipids to nucleic acids in lipid nanoparticles".

[0117] In one embodiment of this specification, the VHH antibody binds to CD8. In this case, the weight ratio range of the targeted polyalkylene glycol-modified lipid to the nucleic acid in the lipid nanoparticles is preferably 0.047 to 3.762. This weight ratio range is such that the optimal density range of the VHH antibody on the surface of the lipid nanoparticles is 100 nm from the nanoparticle surface. ２ When the number of particles per nanoparticle ranges from 0.004 to 2.200, this is the optimal weight ratio range measured according to the method described in "5. Method for measuring the optimal weight ratio range of targeted polyalkylene glycol-modified lipids to nucleic acids in lipid nanoparticles".

[0118] In one embodiment of this specification, the VHH antibody binds to CD8. In this case, the weight ratio range of the targeted polyalkylene glycol-modified lipid to the nucleic acid in the lipid nanoparticles is more preferably 0.054 to 3.085. This weight ratio range is such that the optimal density range of the VHH antibody on the surface of the lipid nanoparticles is 100 nm from the nanoparticle surface. ２When the number of particles per nanoparticle ranges from 0.008 to 1.800, this is the optimal weight ratio range measured according to the method described in "5. Method for measuring the optimal weight ratio range of targeted polyalkylene glycol-modified lipids to nucleic acids in lipid nanoparticles".

[0119] In one embodiment of this specification, the VHH antibody binds to CD8. In this case, the weight ratio range of the targeted polyalkylene glycol-modified lipid to the nucleic acid in the lipid nanoparticles is particularly preferably 0.057 to 2.578. This weight ratio range is such that the optimal density range of the VHH antibody on the surface of the lipid nanoparticles is 100 nm from the nanoparticle surface. ２ When the number of particles per nanoparticle ranges from 0.010 to 1.500, this is the optimal weight ratio range measured according to the method described in "5. Method for measuring the optimal weight ratio range of targeted polyalkylene glycol-modified lipids to nucleic acids in lipid nanoparticles".

[0120] In one embodiment of this specification, the VHH antibody binds to CD4. In this case, the weight ratio range of the targeted polyalkylene glycol-modified lipid to the nucleic acid in the lipid nanoparticles is preferably 0.091 to 3.931. This weight ratio range is such that the optimal density range of the VHH antibody on the surface of the lipid nanoparticles is 100 nm from the nanoparticle surface. ２ When the number of particles per nanoparticle ranges from 0.030 to 2.300, this is the optimal weight ratio range measured according to the method described in "5. Method for measuring the optimal weight ratio range of targeted polyalkylene glycol-modified lipids to nucleic acids in lipid nanoparticles".

[0121] In one embodiment of this specification, the VHH antibody binds to CD4. In this case, the weight ratio range of the targeted polyalkylene glycol-modified lipid to the nucleic acid in the lipid nanoparticles is more preferably 0.099 to 3.931. This weight ratio range is such that the optimal density range of the VHH antibody on the surface of the lipid nanoparticles is 100 nm from the nanoparticle surface. ２ When the number of particles per nanoparticle ranges from 0.035 to 2.300, this is the optimal weight ratio range measured according to the method described in "5. Method for measuring the optimal weight ratio range of targeted polyalkylene glycol-modified lipids to nucleic acids in lipid nanoparticles."

[0122] In one embodiment of this specification, the VHH antibody binds to CD4. In this case, the weight ratio range of the targeted polyalkylene glycol-modified lipid to the nucleic acid in the lipid nanoparticles is particularly preferably 0.108 to 2.578. This weight ratio range is such that the optimal density range of the VHH antibody on the surface of the lipid nanoparticles is 100 nm from the nanoparticle surface. ２ When the number of particles is between 0.040 and 1.500, this is the optimal weight ratio range measured according to the method described in "5. Method for measuring the optimal weight ratio range of targeted polyalkylene glycol-modified lipids to nucleic acids in lipid nanoparticles".

[0123] Everything described in "2-1. Lipid Nanoparticles Containing VHH Antibodies" applies to the lipid nanoparticles of this embodiment.

[0124] 1. General Manufacturing Procedure for Antibody-Conjugated Lipid Nanoparticles 1-1. Plasmid Preparation for Antibody Expression The kozak sequence, Ig K leader sequence, antibody-coding sequence, and linker sequence were cloned between sequence A at terminal 5 and sequence B at terminal 3 of the mammalian expression vector pcDNA3.4. The antibody-coding sequence underwent codon optimization for Cricetulus griseus (CHO) (GenScript Biotech Corporation, Piscataway, US).

[0125]

[0126]

[0127]

[0128] 1-2. Transient expression of VHH antibody: 30 mL of ExpiCHO Expression Medium (#A2910001, Thermo Fisher Scientific) and 1 mL of ExpiCHO-S cells thawed at 37°C (1 x 10⁶). 7Cells / mL (Thermo Fisher Scientific) were added to a 125 mL flask (#4115-0500, Thermo Fisher Scientific) and cultured at 37°C, 8% CO2, and 125 rpm for 3–4 days (HERAcell CO2 incubator 150i, electromagnetic orbital shaker COSH6). The cell density was 1.0 x 10⁶. 6 Confirm that the concentration is 0.3–0.5 x 10 6 The cells were diluted to a concentration of cells / mL and cultured again at 37°C, 8% CO2, and 125 rpm for 3–4 days. This subculturing was repeated at least three times, and the cells were transferred to a 100 mL scale of medium in a 500 mL flask before plasmid transfection. Specifically, a solution of 80 μg of plasmid diluted in 4 mL of OptiPRO SFM (#12309019, Thermo Fisher Scientific) and a solution of 320 μL of ExpiFectamine CHO Reagent (#100033021, Thermo Fisher Scientific) diluted in 3.7 mL of OptiPRO SFM were mixed together, gently inverted, and the mixture was allowed to stand at room temperature for 1–5 minutes before being transfected to 6.0 x 10⁶ cells. 6 The cells were added to a flask containing ExpiCHO-S cells diluted to cells / mL and cultured at 37°C, 8% CO2, and 125 rpm for 18–22 hours. Subsequently, a mixture of 600 μL of ExpiFectamine CHO Enhancer (#100033018, Thermo Fisher Scientific) and 24 mL of ExpiCHO Feed (#A29101-02, Thermo Fisher Scientific) was added to the ExpiCHO-S cells and cultured at 32°C, 5% CO2, and 125 rpm for 10–12 days. The cell culture medium was then transferred to a 50 mL tube, centrifuged at 4000 rpm, 4°C for 5 minutes, and the supernatant was filtered through a 0.22 μm or 0.45 μm syringe filter and stored frozen at -80°C.

[0129] 1-3. IMAC Purification of VHH Antibody A HisTrap column (HisTrap excel 5 mL, #17371206, Cytiva) was set on an AKTA go (Cytiva, Marlborough, MA, USA) and washed and equilibrated by delivering 25 mL of Wash Buffer (50 mM phosphate buffer pH 7.4, 300 mM NaCl) at a flow rate of 5.0 mL / min and a pressure resistance of 0.5 MPa. After injecting the supernatant of the ExpiCHO-S cell culture medium after antibody expression into the column, it was washed with 25 mL of Wash Buffer to remove unwanted components. A concentration gradient was applied to a mixture of Wash Buffer and Elution Buffer (50 mM phosphate buffer pH 7.4, 300 mM NaCl, 500 mM imidazole) so that the proportion of Elution Buffer changed linearly from 0% to 50% during the delivery of 50 mL, and the antibody bound to the column was eluted. Each fraction was collected, the target fraction was identified by SDS-PAGE, and the fraction was transferred to an ultrafiltration filter unit (Vivaspin Turbo 15 3,000 MWCO, #VS15T91, Sartorius). The fraction was then concentrated by centrifugation at 4,000 × g at 4°C until the antibody concentration reached approximately 5–10 mg / mL.

[0130] 1-4. Affinity Purification of VHH Antibody with Protein A Affinity 10 mL of Pierce Centrifuge Columns (Thermo Fisher Scientific, #89898) were set in a Protein LoBind 50 mL tube (Eppendorf, #0030122240). 0.5 times the volume of the ExpiCHO-S cell culture supernatant to be purified was added to the column, and the mixture was centrifuged at 3,000 × g at 4°C for 1 minute. The filtrate was discarded. The column was washed twice with an equal volume of DDW to the culture supernatant, and the filtrate was discarded. The column was then equilibrated twice with an equal volume of PBS to the culture supernatant, and the filtrate was discarded. The culture supernatant was added to the column, and the mixture was centrifuged at 3,000 × g at 4°C for 1 minute. The filtrate was added back to the same column and centrifuged again in the same manner, and the filtrate was discarded. Furthermore, the column was washed three times with an equal volume of PBS as the culture supernatant, and the filtrate was discarded. A new Protein LoBind 50 mL tube was filled with 0.025 times the volume of 1.0 M Tris-HCl pH9.0 (for neutralization) as the culture supernatant, and the column was transferred to this tube. 0.5 times the volume of Elution Buffer (0.1 M Glycine, pH2.5) as the culture supernatant was added to the column, and after standing for 1-2 minutes, the column was centrifuged at 3,000×g at 4°C for 1 minute to elute the antibodies bound to the resin. This elution procedure was repeated twice. The resulting filtrate was transferred to an ultrafiltration filter unit (Vivaspin Turbo 15 3,000 MWCO) and concentrated by centrifugation at 4,000×g at 4°C until the antibody concentration reached approximately 5-10 mg / mL.

[0131] 1-5. SEC Purification of VHH Antibody A Superdex75 Increase 10 / 300 GL, #29148721, Cytiva SEC column (Superdex75 Increase 10 / 300 GL, #29148721, Cytiva) was set on an AKTA go (Cytiva, Marlborough, MA, USA) and washed and equilibrated by delivering 30 mL of PBS at a flow rate of 0.8 mL / min and a pressure resistance of 3.0 MPa. 500 μL of the purified sample using a HisTrap column or Protein A resin was injected, and after delivering 24 mL of PBS, each fraction was collected and the target fraction was identified by SDS-PAGE. The obtained samples were transferred to an ultrafiltration filter unit (Vivaspin Turbo 15 3,000 MWCO) and concentrated by centrifugation at 4,000 × g at 4°C until the antibody concentration reached approximately 3–5 mg / mL. The concentration was determined by BCA assay and stored at -80°C.

[0132] 1-6. Preparation of GGGYPYDVPDYAK-(PEG)4-S-Acetyl Peptide The GGGYPYDVPDYAK peptide was synthesized using Fmoc solid-phase peptide synthesis. The N-terminal primary amino group was protected with a Boc group in an On resin peptide. Next, the protecting group (ivDde group) of the lysine side chain contained in the above sequence was selectively deprotected with 5% hydrazine / DMF. Subsequently, commercially available SAT(PEG)4 (PEGylated N-succinimidyl S-acetylthioacetate) was bonded to the deprotected primary amino group of the lysine side chain on the solid phase to synthesize GGGYPYDVPDYAK-(PEG)4-S-Acetyl. After washing the obtained On resin peptide with DCM, it was cleaved from the resin and deprotected using a TFA / water / TIS / DODT (92.5 / 2.5 / 2.5 / 2.5) cleavage cocktail. After deprotection, the obtained crude peptide was collected, precipitated with ether, and dried overnight. Subsequently, the peptide was purified by reverse-phase chromatography (C18 column, mobile phase (Solution A: H2O (containing 0.1% TFA), Solution B: Acetonitrile (containing 0.1% TFA))). After removing acetonitrile from the recovered purified solution using an evaporator, purified GGGYPYDVPDYAK-(PEG)4-S-Acetyl powder was obtained using a freeze-dryer (purity of 90% or higher).

[0133] 1-7. Pretreatment of antibodies for conjugate (in the case of VHH antibody-Sortase linker adduct) The purified Sortase linker adduct antibody was conjugated with the GGGYPYDVPDYAK-(PEG)4-S-Acetyl peptide via the Sortase reaction. Specifically, the final concentrations were 50 μM antibody, 20 μM Sortase A (Protein Express Co., Ltd., 17.9 kDa), 500 μM GGGYPYDVPDYAK-(PEG)4-S-Acetyl peptide, 1 mM CaCl2, 50 mM Tris-HCl (pH 7.9), and 150 mM NaCl. The mixture was diluted in DDW, prepared in 1 mL / tube, and incubated at 37°C for 16 hours. Subsequently, the Sortase reaction solution was purified using the His tag. Specifically, a 10 mL Pierce Centrifuge Column (Thermo Fisher Scientific, #89965) equal to the amount of reaction solution to be purified was added to a 50 mL Protein LoBind tube with a Pierce Centrifuge Column (10 mL) (Thermo Fisher Scientific, #89898). The mixture was centrifuged at 700 × g, 4°C, for 1 minute, and the filtrate was discarded. 2.5 times the volume of PBS(-) was added to the reaction solution, and the mixture was centrifuged at 700 × g, 4°C, for 1 minute. This equilibration procedure was repeated a total of two times. The Sortase A reaction solution was added to the column, centrifuged at 700 × g, 4°C, for 1 minute, and the filtrate was added back to the same column. The mixture was centrifuged again at 700 × g, 4°C, for 1 minute, and the filtrate was collected. The column was then washed three times with 0.5 times the volume of PBS(-) and the filtrate was collected. The obtained filtrate was centrifuged at 4000 g and 4°C using an ultrafiltration filter unit (Vivaspin Turbo 15 3,000 MWCO) to concentrate it to approximately 3.2 mg / mL, and then SEC purification was performed. The Elution fraction was also obtained by adding 0.5 times the reaction solution volume of Elution Buffer (50 mM phosphate buffer pH 7.4, 300 mM NaCl, 500 mM imidazole), letting it stand for 2 minutes, and then centrifuging at 700 × g and 4°C for 1 minute.An SEC column (Superdex75 Increase 10 / 300 GL) was set on an AKTA go (Cytiva, Marlborough, MA, USA), washed and equilibrated, and then SEC purification of 500 μL of His-tagged samples was performed under conditions of 1x PBS, 0.8 mL / min, and a pressure of 3.0 MPa. Each fraction was collected, the target fraction was identified by SDS-PAGE, concentrated at 4000×g, 4°C on a Vivaspin Turbo 15 (3,000 MWCO), the concentration was determined by BCA assay, diluted to 2 mg / mL with PBS, and stored at -80°C. Deprotection of the S-Acetyl group was performed immediately before conjugation with LNP. An acetyl deprotection solution (Hydroxyamine 0.5 M (Pierce® Hydroxylamine-HCl, 26103, Thermo Scientific), EDTA 25 mM (Nacalai Tesque, 06894-14), pH 7.2-7.5, PBS(-)) was prepared immediately before use. The prepared acetyl deprotection solution was mixed with 2 mg / mL of acetyl-protected antibody to adjust the final concentration to 1 mg / mL antibody, 0.05 M Hydroxylamine, and 2.5 mM EDTA. The mixture was stirred at 25°C and 200 rpm for 2 hours to obtain SH-free antibody. The conjugate antibody prepared in this section was used in the production of antibody-conjugated lipid nanoparticles used in Examples 1-9 and 11-14 below.

[0134] 1-8. Pretreatment of Conjugate Antibodies (In the case of VHH antibody-reduced linker adducts) The artificially introduced cysteine ​​contained in antibody-reduced linker adducts forms disulfide bonds with glutathione and cysteine ​​in the culture medium, so it needs to be reduced before conjugation with LNPs. Specifically, a reduced solution was prepared by adding 0.5 M TCEP solution (Fujifilm Wako Pure Chemical Industries, Ltd., 207-20151) to EDTA 25 mM buffer to a final TCEP concentration of 10 mM. 15 μL of the reduced solution and 15 μL of 2 mg / mL antibody solution were mixed and shaken at 200 rpm for 5 minutes. The reaction mixture was added to a Zeba™ Spin Desalting Column (Thermo Scientific™, 89882) that had been purged twice with EDTA 25 mM buffer, centrifuged at 1500 × g, 20°C for 4 minutes, and the filtrate was collected. The protein concentration in the filtrate was measured using NanoDrop Lite (Thermo Scientific®). Based on the measurement results, the filtrate was diluted with 25 mM EDTA buffer to prepare an SH-free antibody solution with a protein concentration of 0.6 mg / mL.

[0135] 1-9. Preparation of Fab Antibodies Using known gene engineering techniques, the DNA sequence encoding antibody 3 was introduced into CHO cells using a mammalian cell expression vector to express the protein. The obtained antibodies were reduced with a reducing agent, the reducing agent was removed by dialysis, and the antibodies were dissolved in 20 mM histidine acetate and 0.15 M NaCl (pH 5.5) to prepare an antibody solution with an antibody concentration of 1-8 mg / mL.

[0136] 1-10. Preparation of scFv antibodies The scFv antibodies were prepared using the same procedure as described in 1-2, 1-3, 1-5, and 1-7 of "1. General manufacturing procedure for antibody-conjugated lipid nanoparticles". 1-11. Preparation of untargeted LNPs Figure 42 is a schematic diagram of the method for producing lipid nanoparticles according to the present invention. A lipid mixture (cationic lipids: Cholesterol: DSPC: DMG-PEG2000: DSPE-PEG(2000)-maleimide = 50:38.5:10:0.75:0.75, molar ratio) was dissolved in 100% EtOH to prepare a lipid solution (Cholesterol: Nacalai Tesque, 08722-81) (DSPC (1,2-Distearoyl-sn-glycero-3-phosphocholine): COATSOME MC-8080, NOF Corporation) (DMG-PEG2000 (1,2-Dimyristoyl-rac-glycero-3-methylpolyoxyethylene): SUNBRIGHT GM-020, NOF Corporation) (DSPE-PEG(2000)-maleimide (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (ammonium) (Salt): Avanti Polar Lipids, 880126P. The lipid concentration was adjusted so that the final lipid concentration after mixing with the nucleic acid solution was 2–3.5 mM. The nucleic acid solution was prepared by diluting CleanCap mCherry mRNA (5 moU) (TriLink BioTechnologies, Inc., L-7203-5) with 50 mM citrate buffer solution (Citric Acid: FUJIFILM Wako Pure Chemical Corporation, 030-5525, Sodium citrate dihydrate: Sigma-Aldrich, W302600). The ratio of lipids to nucleic acids (NP ratio) was set to 10, and the volume was adjusted to lipid:nucleic acid = 25:75.The obtained lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr instrument (NanoAssemblr® Ignite®, Precision Nanosystems) at a rate of 3 ml / min for the lipid solution and 9 ml / min for the nucleic acid solution, and the solution containing the composition was collected. The obtained solution was diluted by the same volume with 9% sucrose, 20 mM HEPES buffer solution (1 mM HEPES Buffer, Nacalai Tesque, 17557-94; sucrose: Fujifilm Wako Pure Chemical Industries, 196-00015), and dialyzed against the 9% sucrose, 20 mM HEPES buffer solution at 4°C for 16 to 24 hours using a SpectraPor® 4 Dialysis Membrane (12-14 kDa) (REPLIGEN, 132703). Next, the solution was concentrated by ultrafiltration using Amicon Ultra-15 100kDa (Merck Millipore, UFC910024) and sterilized using a sterile syringe filter (φ13mm / 0.2μm) (Pall Corporation, 4602).

[0137] 1-12. Conjugation of untargeted LNPs and antibodies SH-free antibodies were added to an untargeted LNP solution containing maleimide in an arbitrary ratio of 0.0078 mol% to 0.5 mol% relative to the total lipid moles multiplied by the mRNA yield. The antibody and LNPs were conjugated by stirring at 25°C and 200 rpm for 2 hours. Subsequently, five times the amount of L-Cysteine・HCl・HO (44889, Thermo Fisher Scientific) relative to the moles of DSPE-PEG(2000)-maleimide was added to quench the unreacted maleimide. Subsequently, for VHH antibody-conjugated LNPs, the antibody-conjugated LNPs were added to Spectra / Por® Float-A-Lyzer® G2 (MWCO: 1000kDa) (REPLIGEN, G235037) and dialyzed three times at 4°C for 2–12 hours in 9% sucrose, 20 mM HEPES buffer. The samples were sterilized using a sterile syringe filter (φ13mm / 0.2μm) (Pall Corporation, 4602), and the resulting antibody-conjugated LNPs were stored at -80°C. For Fab antibody-conjugated LNPs, size exclusion chromatography was performed using Sepharose CL6B (Cytiva) as the resin. The resins were separated using a column packed with Eco-pac Chromatography Columns (Bio-Rad). A 20 mM Arginine 9% Sucrose solution was used as the running buffer. A column packed with 15 mL of Sepharose CL6B Dispersion was equilibrated by delivering buffer for more than one hour, after which a sample containing antibody-conjugated LNPs was added from the top of the column. The solution discharged from the bottom of the column was collected, and the UV absorption at 280 nm was measured using Synergy 2 (Biotek) to confirm the fraction containing protein-bound lipid nanoparticles. The collected fractions were pooled and mixed by inversion to obtain the antibody-conjugated LNP solution.

[0138] 1-13. Measurement of physicochemical properties of antibody-conjugated LNPs The average particle size and PDI of LNPs were measured using a Zetasizer Nano ZSP instrument (Malvern Instruments, Malvern, UK) by adding 5 μL of LNP to 500 μL of D-PBS(-). 1-14. Measurement of mRNA inclusion rate in antibody-conjugated LNPs The mRNA inclusion rate in antibody-conjugated LNPs was measured using Ribogreen reagent (Invitrogen, #R114491). Specifically, measurement solution A was prepared by diluting the LNP solution with TE buffer to approximately 8 μg mRNA / mL. Measurement solution B was also prepared by diluting the LNP solution with TE buffer to approximately 1.2 μg mRNA / mL and containing 1% (w / w) X-triton100 (Sigma-Aldrich, T8787-50ML). On a 96-well microplate, 100 μL of each measurement solution was mixed with 100 μL of Ribogreen reagent diluted 200-fold in TE buffer. The mixture was incubated at room temperature for 5 minutes, and the fluorescence intensity was measured at an excitation wavelength of 485 nm and a measurement wavelength of 528 nm. Nucleic acid concentrations were calculated using a calibration curve created for nucleic acid concentrations ranging from 0 to 2.5 μg / ml. The mRNA inclusion rate in antibody-conjugated LNPs was calculated using the following formula: Inclusion rate % = ((Nucleic acid concentration of measurement solution B (μg / mL) - Nucleic acid concentration of measurement solution A (μg / mL)) ÷ Nucleic acid concentration of measurement solution B (μg / mL) × 100)

[0139] 1-15. Antibody Quantification in LNP Protein quantification was performed according to the protocol of the Pierce BCA Protein Assay Kit. Specifically, the 2 mg / mL Albumin standard included in the kit was diluted with PBS to create a calibration curve from 0 to 1000 μg / mL in 25 μL / wells. In addition, 25 μL / well of 100 μg mRNA / mL LNP was added. The kit's included solutions A and B were mixed in a 50:1 ratio and 200 μL was added to each well. After sealing and incubation at 37°C for 30 minutes at 200 rpm, the absorbance at 562 nm was measured with a plate reader to quantify the protein concentration. 1-16. Analysis of Antibody-Lipid Conjugate Sample Buffer was obtained by adding 200 μL of 1M DTT solution (Sigma, 43816) to 1000 μL of 2x Laemmli Sample Buffer (Bio-Rad, #161-0737). Equal volumes of Sample Buffer were added to LNPs with a concentration of 100 μg mRNA / mL, and the mixture was heated at 95°C for 5 minutes. An equal mixture of Precision Plus Protein All Blue Standards and Precision Plus Protein Unstained Standards was used as the molecular weight marker. Any kD Mini-Protean TGX Stain-Free Gels (Bio-Rad, Hercules, CA, USA) were placed in an electrophoresis apparatus (Bio-Rad, Mini-Protean Tetra cell), 1xTris / Glycine / SDS buffer was added, and 10 μL of sample was loaded. Electrophoresis was then performed at 200 V for 30 minutes. Images were taken after UV irradiation with ChemiDoc XRS+ (Bio-Rad). The gels were transferred to PVDF membranes (Trans-Blot Turbo Mini PVDF Transfer Packs, Bio-Rad) using the Trans-Blot Turbo Blotting system (Bio-Rad).The membrane was placed in a container and immersed in 15 mL of Bullet Blocking One for Western Blotting (Nacalai tesque, 13779-14), and shaken at room temperature for 5 minutes. The membrane was immersed in 10 mL of PBS-T, pH 7.4 (x1), and shaken at room temperature for 3 minutes, repeating this three times. 2.5 μL of anti-hemagglutinin monoclonal antibody peroxidase conjugate and 1 μL of Precision Protein StrepTactin-HRP Conjugate were added to 10 mL of PBS-T (x1) and mixed, the membrane was immersed, and shaken at room temperature for 30 minutes under light protection. The membrane was immersed in 10 mL of PBS-T (x1), and shaken at room temperature for 3 minutes, repeating this three times. Immediately before use, equal volumes of two solutions of Clarity Western ECL Substrate (Bio-Rad) were mixed, and the membrane was immersed in 6 mL of this solution and shaken at room temperature for 5 minutes. We imaged the membrane using ChemiDoc XRS Plus (Bio-Rad) and confirmed the formation of antibody-lipid conjugates.

[0140] 2. General Procedure for In vitro Protein Expression Evaluation T cells were isolated from PBMC (Lonza) using EasySep Human T cell Enrichment Cocktail (STEMCELL TECHNOLOGIES). ImmunoCult Human CD3 / CD28 / CD2 T cell Activator (STEMCELL TECHNOLOGIES) was added to the isolated T cells, and the T cells were cultured at 37°C under 5% CO2 conditions for 4 to 6 days. The culture medium used for the T cells was RPMI-1640, which contains 10% FBS, 100 IU / mL IL-2, 100 U / mL and 100 mg / mL Penicillin and Streptomycin. Cells cultured in a 96-well plate were measured at 0.1 × 10⁶ 6Cells were seeded at 100 μL / well, treated with a formulation, and cultured in a CO2 incubator for 24 hours. After 24 hours, cells were harvested and the cell surface antigens were labeled using BB515-labeled CD4 antibody, BV421-labeled CD3 antibody, and APC-labeled CD8 antibody. Dead cells were stained with FVS780 (BD Horizon®). Intracellular mCherry expression was measured using FACSymphony A1 (BD Biosciences).

[0141] 3. Measurement of Antibody Density on LNP Surfaces The density of VHH antibodies capable of binding to antigens on the surface of lipid nanoparticles was measured. A schematic diagram of the measurement method is shown in Figure 41. Specifically, fluorescently labeled antigens were added to VHH conjugate lipid nanoparticles, and the particle surface area and fluorescence intensity of each particle were measured individually using a nanoflow cytometer to quantify the VHH density capable of binding to antigens per unit surface area. Furthermore, the nucleic acid delivery efficiency of VHH conjugate lipid nanoparticles with different VHH densities was determined as the amount of protein expressed after mRNA encapsulated in the lipid nanoparticles was introduced into target cells. The density of Fab antibodies capable of binding to antigens on the surface of lipid nanoparticles was measured in the same manner.

[0142] The antibody density on the LNP surface was calculated based on its interaction with the fluorescently labeled antigen. Specifically, the reagent and antigen were mixed using AF 488 NHS ester (Lumiprobe #41820) according to the product protocol, and the mixture was fluorescently labeled with Recombinant Human CD8 alpha (hCD8a, Nippon Sinobiological Co., Ltd. #10980-H02H) by stirring for 60 minutes at 800 rpm, room temperature, and in the dark. Subsequently, the entire reaction mixture was added to an ultrafiltration unit (Vivaspin turbo15 10k #VS15T02), and unreacted fluorescent molecules were removed by washing three times with 5 mL of PBS at 4000 × g. The removal of unreacted fluorescent molecules was confirmed by electrophoresis. Specifically, 10 μL of fluorescently labeled antigen was mixed with 10 μL of 2x Laemmli Sample Buffer (Bio-Rad, #161-0737) and 2 μL of 1M DTT solution (Sigma, #43816), and heated at 95°C for 5 minutes. The molecular weight marker was an equal mixture of Precision Plus Protein All Blue Standards and Precision Plus Protein Unstained Standards. Any kD Mini-Protean TGX Stain-Free Gels (Bio-Rad, Hercules, CA, USA) were set up in an electrophoresis apparatus (Bio-Rad, Mini-Protean Tetra cell), 1x Tris / Glycine / SDS buffer was added, and 10 μL of the sample was loaded. Electrophoresis was then performed at 200 V for 25 minutes. The gel was imaged using ChemiDoc XRS+ (Bio-rad), and AF488 and protein were detected to confirm that unreacted fluorescent molecules were removed and the antigen was fluorescently labeled. Subsequently, the average number of fluorescent molecules bound to the antigen (DOL, Degree of Labeling) was calculated. Specifically, the antigen concentration was calculated using a BCA assay with BSA as the calibration curve, and the absorbance at 494 nm and the molar extinction coefficient (AF488, ε: 71000 cm⁻¹) were measured using Nanodrop. -1 M -1DOL was calculated by determining the fluorescence concentration from the given values. Throughout this experiment, AF488-labeled Human CD8 alpha with a DOL of 0.38 was consistently used. The reaction between antibody-conjugated LNP and the fluorescently labeled antigen was carried out as follows: 2 μL of antibody-conjugated LNP (40 μg mRNA / mL) and 3 μL of hCD8a-AF488 (4 μM) were mixed and stirred for 60 min at 700 rpm, 25°C, and in the dark. Subsequently, size exclusion chromatography (SEC) was performed to remove unreacted hCD8a-AF488. Specifically, the mixture was placed in an SEC column (Gen2 qEV single / 35, Izon Science, Medford, MA, USA), eluted with PBS(-), and each fraction was recovered. The AF488 fluorescence intensity (Ex 490 nm / Em 525 nm) was measured for each fraction. Subsequently, each fraction was diluted 6.67 times with 1.18% Triton-containing TE buffer, and then an equal volume of Ribogreen reagent diluted 200 times with TE buffer was added. The fluorescence intensity (Ex 485 nm / Em 528 nm) was measured to identify fractions containing LNPs. After that, the antibody-conjugate LNPs bound to the fluorescently labeled antigen were quantified using nanoflow cytometry (NanoFCM Flare, NanoFCM inc.) to determine the side scatter (SS) and AF488 signals. For calibration curve creation, NanoFCM Silica Nanospheres Cocktail #S16M-Exo and #FL23-MESF 488 were used, and FlowJo_v10.8.1 was used for analysis. Based on the quantitative values ​​of the number of AF488 molecules and particle size in each particle, the LNP surface area of ​​100 nm in each particle was calculated. 2 The number of antibodies per unit area was calculated, and the median value was defined as the antibody density.

[0143] In the examples, the following lipids (P) to (T) were used.

[0144] Lipid (P) (ALC-0315) was purchased from MedChemExpress (product number #HY-13817). Lipid (R) is a lipid disclosed in International Publication No. 2022 / 071582 (Patent Document 1), and was manufactured according to the method described in said document.

[0145] Synthesis of lipid (Q) (1) (2-Phenyl-1,3-dioxane-5,5-diyl)dimethanol (1.12 g), 2-hexyldecanoic acid (2.69 g), and DMAP (61.1 mg) were dissolved in DCM (6.0 ml), and EDCI (2.11 g) was added and the mixture was stirred overnight at room temperature. Saturated aqueous ammonium chloride solution was added to the reaction solution and extracted three times with DCM. The organic layer was washed with saturated brine, and then dehydrated with sodium sulfate. After filtration, the solvent was removed under reduced pressure to obtain the crude product. (2) Methanol (32 ml), DCM (8.0 ml), and HCl (4 mol / L in 1,4-Dioxane, 4.0 ml) were added to the obtained crude product (2.10 g) and the mixture was stirred at room temperature for 4 hours. An excess amount of sodium bicarbonate was added to the reaction solution and it was concentrated under reduced pressure. Water and ethyl acetate were added to the residue and extracted three times with ethyl acetate. The organic layer was washed with saturated brine, and then dehydrated with sodium sulfate. After filtration, the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography [eluent: hexane / ethyl acetate] to obtain the target 2,2-bis(hydroxymethyl)propane-1,3-diyl bis(2-hexyldecanoate) (1.46 g). (3) In a container equipped with a calcium chloride tube, 4,4-diethoxy-N,N-diethylbutan-1-amine, p-toluenesulfonic acid monohydrate, and toluene were added and stirred overnight at 85°C. An excess amount of saturated sodium bicarbonate aqueous solution was added to the reaction solution and extracted three times with ethyl acetate. The organic layer was washed with saturated brine, and then dehydrated with sodium sulfate. After filtration, the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography to obtain the target lipid (Q). 1H NMR (300 MHz, CDCl3) δ 4.48 (t, J = 5.1 Hz, 1H), 4.36 (s, 2H), 3.92 (d, J = 11.8 Hz, 2H), 3.83 (s, 2H), 3.60 (d, J = 11.8 Hz, 2H), 2.51 (q, J = 7.0 Hz, 4H), 2.44 (t, J = 6.9 Hz, 2H), 2.37-2.28 (m, 2H), 1.61-1.43 (m, 12H), 1.25 (brs, 40H), 1.04 (t, J = 7.1 Hz, 6H), 0.90-0.85 (m, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 738.7, measured value m / z = 738.7.

[0146] Synthesis of Lipids (S) (1) Synthesis of Compound a1 DL-10-camporsulfonic acid (CSA; 0.05 eq) was added to a mixture of 3,3-diethoxypropanenitrile (Combi-Blocks) (1.0 eq) and 1-heptanol (Combi-Blocks) (3 eq). The reaction mixture was heated at 100°C and stirred overnight. The reaction mixture was cooled to ambient temperature and purified on a silica gel short pad. The filtrate was concentrated under vacuum and dissolved in MeOH to a final concentration of 0.4 M. 8.0 M aqueous sodium hydroxide solution (1.5 eq) was added to the mixture. The mixture was stirred overnight at 60°C. The reaction mixture was diluted with ethyl acetate and saline solution. The aqueous layer was titrated to neutral pH with saturated aqueous ammonium chloride solution and extracted twice with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by column chromatography (ethyl acetate / hexane = 0-40%) to obtain compound a1.

[0147] (2) Synthesis of compound a2 The following compound a2 was synthesized according to the procedure described in WO2018 / 230710.

[0148] (3) Synthesis of lipid (S) Compound a2 (1.0 eq), compound a1 (2.3 eq), and 4-dimethylaminopyridine (DMAP) (Sigma-Aldrich, 0.2 eq) were dissolved in dichloromethane (DCM) (containing 0.2 M of compound a1) in a round-bottom flask, and 3-ethylcarbodiimide hydrochloride (EDCI) (TCI Chemicals, 2.8 eq) was added to the stirred solution. The resulting mixture was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, saturated aqueous NaCl solution was added, and the mixture was extracted three times with dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate, filtered, evaporated, and the residue was purified by column chromatography to obtain lipid (S) (1.1 g). ESI+: m / z = 957.2.

[0149] Synthesis of lipid (T) (1) Synthesis of compound b1: b1 was obtained in the same manner as compound a1, except that 4,4-dimethoxybutanenitrile (AK Scientific) was used instead of 3,3-diethoxypropanenitrile. (2) Synthesis of lipid (T): Lipid (T) was obtained in the same manner as lipid (S), except that compound b1 was used instead of compound a1. ESI+: m / z = 985.2.

[0150] Example 1 Each antibody-conjugate LNP with different VHH densities containing mCherry mRNA, as listed in Table 1 below (lipid composition before antibody conjugation: lipid (P):Cholesterol:DSPC:DMG-PEG2000:DSPE-PEG2000-maleimide=50:38.5:10:0.75:0.75), was transfected into activated T cells at 0.002 μg / mL or 0.004 μg / mL. After 24 hours, the mCherry expression rate in CD8-positive T cells or CD4-positive T cells was evaluated by flow cytometry. The results showed that antibody-conjugate lipid nanoparticles were carriers capable of selectively delivering mRNA to CD8-positive T cells compared to untargeted lipid nanoparticles. Among these, those with VHH densities capable of binding to the antigen ranged from 0.004 to 2.200 VHH / 100nm. 2The antibody-conjugate lipid nanoparticles were shown to be particularly excellent carriers capable of selectively delivering mRNA to CD8-positive T cells.

[0151]

[0152] Example 2 Each antibody-2 conjugate LNP with different VHH densities containing mCherry mRNA, as listed in Table 2 below (lipid composition before antibody conjugation: lipid (P):Cholesterol:DSPC:DMG-PEG2000:DSPE-PEG2000-maleimide=50:38.5:10:0.75:0.75), was transfected into activated T cells at 0.002 μg / mL or 0.004 μg / mL. After 24 hours, the mCherry expression rate in CD8-positive T cells or CD4-positive T cells was evaluated by flow cytometry. The results showed that antibody-2 conjugate lipid nanoparticles are carriers capable of selectively delivering mRNA to CD8-positive T cells compared to untargeted lipid nanoparticles. Among these, those with VHH densities capable of binding to the antigen ranging from 0.004 to 2.200 VHH / 100nm were particularly effective. 2 The antibody-2 conjugate lipid nanoparticles were shown to be particularly excellent carriers capable of selectively delivering mRNA to CD8-positive T cells.

[0153]

[0154] Example 3 Each antibody-conjugate LNP with different VHH densities containing mCherry mRNA, as listed in Table 3 below (lipid composition before antibody conjugation: lipid (Q):Cholesterol:DSPC:DMG-PEG2000:DSPE-PEG2000-maleimide=50:38.5:10:0.75:0.75), was transfected into activated T cells at 0.002 μg / mL or 0.004 μg / mL. After 24 hours, the mCherry expression rate in CD8-positive T cells or CD4-positive T cells was evaluated by flow cytometry. The results showed that antibody-conjugate lipid nanoparticles were carriers capable of selectively delivering mRNA to CD8-positive T cells compared to untargeted lipid nanoparticles. Among these, those with VHH densities capable of binding to the antigen ranging from 0.004 to 2.200 VHH / 100nm were particularly effective. 2 The antibody-conjugate lipid nanoparticles were shown to be particularly excellent carriers capable of selectively delivering mRNA to CD8-positive T cells.

[0155] Example 4 Each antibody-2 conjugate LNP with different VHH densities containing mCherry mRNA, as listed in Table 4 below (lipid composition before antibody conjugation: lipid (Q):Cholesterol:DSPC:DMG-PEG2000:DSPE-PEG2000-maleimide=50:38.5:10:0.75:0.75), was transfected into activated T cells at 0.002 μg / mL or 0.004 μg / mL. After 24 hours, the mCherry expression rate in CD8-positive T cells or CD4-positive T cells was evaluated by flow cytometry. The results showed that antibody-2 conjugate lipid nanoparticles are carriers capable of selectively delivering mRNA to CD8-positive T cells compared to untargeted lipid nanoparticles. Among these, those with VHH densities capable of binding to the antigen ranging from 0.004 to 2.200 VHH / 100nm were particularly effective. 2 The antibody-2 conjugate lipid nanoparticles were shown to be particularly excellent carriers capable of selectively delivering mRNA to CD8-positive T cells.

[0156]

[0157] Example 5 Each antibody-conjugate LNP with different VHH densities containing mCherry mRNA, as listed in Table 5 below (lipid composition before antibody conjugation: lipid(R):Cholesterol:DSPC:DMG-PEG2000:DSPE-PEG2000-maleimide=50:38.5:10:0.75:0.75), was transfected into activated T cells at 0.002 μg / mL or 0.004 μg / mL. After 24 hours, the mCherry expression rate in CD8-positive T cells or CD4-positive T cells was evaluated by flow cytometry. The results showed that antibody-conjugate lipid nanoparticles are carriers capable of selectively delivering mRNA to CD8-positive T cells compared to untargeted lipid nanoparticles. Among these, those with VHH densities capable of binding to the antigen ranged from 0.004 to 2.200 VHH / 100nm. 2 The antibody-conjugate lipid nanoparticles were shown to be particularly excellent carriers capable of selectively delivering mRNA to CD8-positive T cells.

[0158]

[0159] Example 6 Each antibody-2 conjugate LNP with different VHH densities containing mCherry mRNA, as listed in Table 6 below (lipid composition before antibody conjugation: lipid(R):Cholesterol:DSPC:DMG-PEG2000:DSPE-PEG2000-maleimide=50:38.5:10:0.75:0.75), was transfected into activated T cells at 0.002 μg / mL or 0.004 μg / mL. After 24 hours, the mCherry expression rate in CD8-positive T cells or CD4-positive T cells was evaluated by flow cytometry. The results showed that antibody-2 conjugate lipid nanoparticles are carriers capable of selectively delivering mRNA to CD8-positive T cells compared to untargeted lipid nanoparticles. Among these, those with a VHH density capable of binding to the antigen of 0.004–2.200 VHH / 100nm 2 The antibody-2 conjugate lipid nanoparticles were shown to be particularly excellent carriers capable of selectively delivering mRNA to CD8-positive T cells.

[0160]

[0161] Example 7 Each antibody-conjugate LNP with different VHH densities containing mCherry mRNA, as listed in Table 7 below (lipid composition before antibody conjugation: lipid (S):Cholesterol:DSPC:DMG-PEG2000:DSPE-PEG2000-maleimide=50:38.5:10:0.75:0.75), was transfected into activated T cells at 0.002 μg / mL or 0.004 μg / mL. After 24 hours, the mCherry expression rate in CD8-positive T cells or CD4-positive T cells was evaluated by flow cytometry. The results showed that antibody-conjugate lipid nanoparticles were carriers capable of selectively delivering mRNA to CD8-positive T cells compared to untargeted lipid nanoparticles. Among these, those with VHH densities capable of binding to the antigen ranging from 0.004 to 2.200 VHH / 100nm were particularly effective. 2 The antibody-conjugate lipid nanoparticles were shown to be particularly excellent carriers capable of selectively delivering mRNA to CD8-positive T cells.

[0162]

[0163] Example 8 Each antibody-2 conjugate LNP with different VHH densities containing mCherry mRNA, as listed in Table 8 below (lipid composition before antibody conjugation: lipid (S):Cholesterol:DSPC:DMG-PEG2000:DSPE-PEG2000-maleimide=50:38.5:10:0.75:0.75), was transfected into activated T cells at 0.002 μg / mL or 0.004 μg / mL. After 24 hours, the mCherry expression rate in CD8-positive T cells or CD4-positive T cells was evaluated by flow cytometry. The results showed that antibody-2 conjugate lipid nanoparticles are carriers capable of selectively delivering mRNA to CD8-positive T cells compared to untargeted lipid nanoparticles. Among these, those with a VHH density capable of binding to the antigen of 0.004–2.200 VHH / 100nm 2 The antibody-2 conjugate lipid nanoparticles were shown to be particularly excellent carriers capable of selectively delivering mRNA to CD8-positive T cells.

[0164]

[0165] Example 9 Two antibody-conjugate LNPs with different VHH densities containing mCherry mRNA, as listed in Table 9 below (lipid composition before antibody conjugation: lipid (P):Cholesterol:DSPC:DMG-PEG2000:DSPE-PEG2000-maleimide=50:38.5:10:0.75:0.75), were transfected into activated T cells at 0.03 μg / mL. After 24 hours, the mCherry expression rate in CD8-positive T cells or CD4-positive T cells was evaluated by flow cytometry. The results showed that, compared to untargeted lipid nanoparticles, the four antibody-conjugate lipid nanoparticles were carriers capable of selectively delivering mRNA to CD4-positive T cells. Among these, those with VHH densities capable of binding to the antigen ranging from 0.03 to 2,300 VHH / 100nm were particularly effective. 2 The antibody-2 conjugate lipid nanoparticles were shown to be particularly excellent carriers capable of selectively delivering mRNA to CD4-positive T cells.

[0166]

[0167] In Examples 1-9, LNPs showing an MFI of 40% or more of the maximum value (with the maximum MFI set to 100%) are indicated with a double underline. The antibody density of such LNPs ranges from 0.004 to 2,300 LNPs / 100nm. 2 It fell within the range.

[0168]

[0169] When the maximum MFI is set to 100%, LNPs showing an MFI of 50% or more of the maximum value are indicated with a double underline. The antibody density of such LNPs is 0.008 to 2,300 cells / 100nm. 2 It fell within the range.

[0170] When the maximum MFI is set to 100%, LNPs showing an MFI of 60% or more of the maximum value are indicated with a double underline. The antibody density of such LNPs is 0.01 to 1,500 cells / 100nm. 2 It fell within the range.

[0171]

[0172] Example 10 Each antibody-3 conjugate LNP (lipid composition before antibody conjugation: lipid (S):Cholesterol:DSPC:DMG-PEG2000:DSPE-PEG2000-maleimide=50:37.5:10:2:0.5) with different Fab antibody densities containing GFP mRNA, as listed in Table 13 below, was transfected into activated T cells at 1.39 μg / mL or 0.69 μg / mL. After 24 hours, the GFP expression rate in CD8-positive T cells or CD4-positive T cells was evaluated by flow cytometry. The results showed that antibody-3 conjugate lipid nanoparticles were carriers capable of selectively delivering mRNA to CD8-positive T cells compared to untargeted lipid nanoparticles. Among these, Fab antibody densities capable of binding to the antigen ranged from 0.003 to 1.113 molecules / 100nm. 2 The antibody-3 conjugate lipid nanoparticles were shown to be particularly excellent carriers capable of selectively delivering mRNA to CD8-positive T cells.

[0173]

[0174] In Example 10, when the maximum MFI is set to 100%, those showing an MFI of 40% or more of the maximum value are indicated by double underlines in the table below. The antibody density of such LNPs is 0.003 to 1.113 cells / 100nm. 2 The results fell within this range. Furthermore, when the maximum MFI is set to 100%, those showing an MFI of 50% or more of the maximum value are indicated with a double underline. The antibody density of such LNPs ranged from 0.004 to 1.113 cells / 100nm. 2 The results fell within this range. Furthermore, in the table below, LNPs showing an MFI of 60% or more of the maximum value (with the maximum MFI set to 100%) are indicated with a double underline. The antibody density of such LNPs ranged from 0.006 to 1,000 cells / 100nm. 2 It fell within the range.

[0175]

[0176] Example 11 Each antibody-5 conjugate LNP with different scFv densities containing mCherry mRNA, as listed in Table 15 below (lipid composition before antibody conjugation: lipid (P):Cholesterol:DSPC:DMG-PEG2000:DSPE-PEG2000-maleimide=50:38.5:10:0.75:0.75), was transfected into activated T cells at 0.03 μg / mL, and the mCherry expression rate in CD8-positive T cells or CD4-positive T cells was evaluated by flow cytometry after 24 hours. As a result, it was shown that antibody-5 conjugate lipid nanoparticles are carriers that can selectively deliver mRNA to CD8-positive T cells compared to untargeted lipid nanoparticles. Among them, those with an scFv density capable of binding to the antigen of 0.001 to 1.900 particles / 100 nm 2 The antibody-5 conjugate lipid nanoparticles were shown to be particularly excellent carriers capable of selectively delivering mRNA to CD8-positive T cells.

[0177]

[0178] Example 12 Each antibody-5 conjugate LNP with different scFv densities containing mCherry mRNA, as listed in Table 16 below (lipid composition before antibody conjugation: lipid (S):Cholesterol:DSPC:DMG-PEG2000:DSPE-PEG2000-maleimide=50:38.5:10:0.75:0.75), was transfected into activated T cells at 0.03 μg / mL, and the mCherry expression rate in CD8-positive T cells or CD4-positive T cells was evaluated by flow cytometry after 24 hours. As a result, it was shown that antibody-5 conjugate lipid nanoparticles are carriers that can selectively deliver mRNA to CD8-positive T cells compared to untargeted lipid nanoparticles. Among them, those with an scFv density capable of binding to the antigen of 0.001 to 1.900 particles / 100 nm 2 The antibody-5 conjugate lipid nanoparticles were shown to be particularly excellent carriers capable of selectively delivering mRNA to CD8-positive T cells.

[0179]

[0180] Example 13 Six antibody-conjugate LNPs with different scFv densities containing mCherry mRNA, as listed in Table 17 below (lipid composition before antibody conjugation: lipid (P):Cholesterol:DSPC:DMG-PEG2000:DSPE-PEG2000-maleimide=50:38.5:10:0.75:0.75), were transfected into activated T cells at 0.03 μg / mL. After 24 hours, the mCherry expression rate in CD8-positive T cells or CD4-positive T cells was evaluated by flow cytometry. The results showed that, compared to untargeted lipid nanoparticles, the six antibody-conjugate lipid nanoparticles were carriers capable of selectively delivering mRNA to CD4-positive T cells. Among these, those with an antigen-binding scFv density of 0.0030 to 3.400 particles / 100 nm were particularly effective. 2 The antibody-6 conjugate lipid nanoparticles were shown to be particularly excellent carriers capable of selectively delivering mRNA to CD4-positive T cells.

[0181] Example 14 Six antibody-conjugate LNPs with different scFv densities containing mCherry mRNA, as listed in Table 18 below (lipid composition before antibody conjugation: lipid (S):Cholesterol:DSPC:DMG-PEG2000:DSPE-PEG2000-maleimide=50:38.5:10:0.75:0.75), were transfected into activated T cells at 0.03 μg / mL. After 24 hours, the mCherry expression rate in CD8-positive T cells or CD4-positive T cells was evaluated by flow cytometry. The results showed that, compared to untargeted lipid nanoparticles, the six antibody-conjugate lipid nanoparticles were carriers capable of selectively delivering mRNA to CD4-positive T cells. Among these, those with an scFv density capable of binding to the antigen of 0.0030 to 3.400 particles / 100 nm were particularly effective. 2 The antibody-6 conjugate lipid nanoparticles were shown to be particularly excellent carriers capable of selectively delivering mRNA to CD4-positive T cells.

[0182] In Examples 11-14, samples showing an MFI of 40% or more of the maximum value (with the maximum MFI set to 100%) are indicated with a double underline. The antibody density of such LNPs ranges from 0.001 to 3,400 cells / 100nm. 2 It fell within the range.

[0183] In Examples 11-14, samples showing an MFI of 50% or more of the maximum value (MFI) is indicated by a double underline, with the maximum MFI set to 100%. The antibody density of such LNPs ranges from 0.002 to 3.100 LNPs / 100nm. 2 It fell within the range.

[0184] In Examples 11-14, LNPs showing an MFI of 60% or more of the maximum value (with the maximum MFI set to 100%) are indicated with a double underline. The antibody density of such LNPs ranges from 0.005 to 2,800 LNPs / 100nm. 2 It fell within the range.

[0185] Example 15 (1) Non-targeted LNP preparation lipid mixture (cationic lipid: Cholesterol: DSPC: DMG-PEG2000: DSPE-PEG(2000)-maleimide = 40:28.5:30:1.4:0.1, molar ratio) was dissolved in 100% EtOH to prepare a lipid solution (Cholesterol: Nacalai Tesque, 08722-81) (DSPC (1,2-Distearoyl-sn-glycero-3-phosphocholine): COATSOME MC-8080, NOF Corporation) (DMG-PEG2000 (1,2-Dimyristoyl-rac-glycero-3-methylpolyoxyethylene): SUNBRIGHT GM-020, NOF Corporation) (DSPE-PEG(2000)-maleimide(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene [glycol)-2000] (ammonium salt): Avanti Polar Lipids, 880126P). The lipid concentration was adjusted so that the final lipid concentration after mixing with the nucleic acid solution was 2-5 mM. The nucleic acid (mRNA) solution was adjusted by diluting it with 50 mM citrate buffer solution (Citric Acid: FUJIFILM Wako Pure Chemical Corporation, 030-5525, Sodium citrate dihydrate: Sigma-Aldrich, W302600). The ratio of lipid to nucleic acid (NP ratio) was set to 10, and the volume was adjusted to lipid:nucleic acid = 25:75. The obtained lipid solution and nucleic acid solution were mixed at room temperature using a Nanoassemblr (Precision Nanosystems) or an IJM Nanoscaler (KNAUER), and the solution containing the composition was recovered. The obtained solution was diluted by the same volume with a neutral pH buffer solution.Subsequently, the sample was dialyzed at 4°C for 16–24 hours using a SpectraPor® 4 Dialysis Membrane (12–14 kDa) (REPLIGEN, 132703) in a neutral pH buffer solution, and then concentrated by ultrafiltration using Amicon Ultra-15 100 kDa (Merck Millipore, UFC910024). Alternatively, buffer substitution and concentration were performed using a KrosFlo KR2i TFF system and RC flat-sheet (100 kDa) (Cytiva, DC100T02) in a neutral pH buffer solution. Finally, the sample was sterilized using a sterile syringe filter (φ13 mm / 0.2 μm) (Pall Corporation, 4602).

[0186] (2) Conjugation of untargeted LNPs and antibodies SH-free antibody (antibody 7-reduced linker 13 adduct) was added to an untargeted LNP solution containing maleimide in an arbitrary ratio of 0.00625 mol% to 0.2 mol% of the total lipid moles obtained by multiplying the mRNA yield, and the antibody and LNP were conjugated by reacting at 2-8°C for 1 hour. Subsequently, 5 times the amount of L-Cysteine・HCl・H2O (44889, Thermo Fisher Scientific) relative to the moles of DSPE-PEG(2000)-maleimide was added to quench the unreacted maleimide. Subsequently, antibody-conjugated LNPs were added to Spectra / Por® Float-A-Lyzer® G2 (MWCO: 1000kDa) (REPLIGEN, G235037), and dialysis was performed three times at 4°C for 2 to 12 hours in a neutral pH buffer solution. Alternatively, buffer replacement was performed using a KrosFlo KR2i TFF system and RC flat-sheet (100kDa) (Cytiva, DC100T02) with a neutral pH buffer solution. Finally, the samples were sterilized using a sterile syringe filter (φ13mm / 0.2μm) (Pall Corporation, 4602), and the obtained antibody-conjugated LNPs were stored at -80°C.

[0187] (3) Measurement of the physicochemical properties of antibody-conjugated LNPs The average particle size and PDI of LNPs were measured using a Zetasizer Nano ZSP instrument (Malvern Instruments, Malvern, UK) after adding 5 μL of LNPs to 500 μL of D-PBS(-).

[0188] (4) Measurement of mRNA inclusion rate in antibody-conjugated LNPs The mRNA inclusion rate in antibody-conjugated LNPs was measured using Ribogreen reagent (Invitrogen, #R114491). Specifically, the LNP solution was diluted with TE buffer to approximately 8 μg mRNA / mL to prepare measurement solution A. In addition, the LNP solution was diluted with TE buffer to approximately 1.2 μg mRNA / mL and contain 1% (w / w) X-triton 100 (Sigma-Aldrich, T8787-50ML) to prepare measurement solution B. 100 μL of each measurement solution and 100 μL of Ribogreen reagent diluted 200-fold with TE buffer were mixed on a 96-well microplate, incubated at room temperature for 5 minutes, and the fluorescence intensity at an excitation wavelength of 485 nm and a measurement wavelength of 528 nm was measured. Nucleic acid concentration was calculated using a calibration curve created for nucleic acid concentrations ranging from 0 to 2.5 μg / mL. The mRNA inclusion rate in antibody-conjugated LNPs was calculated using the following formula: Inclusion rate % = ((Nucleic acid concentration of measurement solution B (μg / mL) - Nucleic acid concentration of measurement solution A (μg / mL)) ÷ Nucleic acid concentration of measurement solution B (μg / mL) × 100)

[0189] (5) Physical properties of VHH antibody-conjugated LNPs The physical properties of the VHH antibody-conjugated LNPs used are shown below.

[0190] (6) Quantification of VHH antibody-lipid conjugates in VHH antibody-conjugate LNPs The VHH antibody-lipid conjugate (VHH-Lipid) in VHH antibody-conjugate LNPs was measured by HPLC. Specifically, a 1 mg / mL standard of prepared VHH-Lipid was diluted with SDS-containing water to prepare calibration curve samples ranging from 1 to 200 μg / mL (final SDS concentration: 0.25 w / v%). In addition, a 200 ug mRNA / mL antibody-conjugate LNP was diluted 2-fold with 0.5 w / v% SDS water and stirred to obtain a sample (final SDS concentration: 0.25 w / v%). The prepared calibration curve samples and sample were packed into vials and measured by HPLC. Detection was performed using a PDA detector (measurement wavelength: UV280 nm). The concentration of VHH-Lipid in the detected antibody-conjugate LNPs was calculated from the calibration curve samples. We confirmed that the VHH-Lipid concentration increased with increasing antibody preparation volume. From the obtained measurement results, we calculated the weight ratio of VHH-Lipid to mRNA.

[0191] (7) Correlation between VHH antibody density and VHH-Lipid / mRNA weight ratio in VHH antibody-conjugated LNPs The results for VHH antibody density and VHH-Lipid / mRNA weight ratio in VHH antibody-conjugated LNPs are shown in the table and Figure 61 below.

[0192] A strong correlation was observed between the VHH antibody density and the VHH-Lipid / mRNA weight ratio in VHH antibody-conjugated LNPs (correlation coefficient R). 2 (=0.8879). Based on these results, the optimal antibody density is at 100 nm on the LNP surface. ２ When the number of molecules per nanoparticle is between 0.004 and 2.300, the optimal weight ratio range for targeted polyalkylene glycol-modified lipids to nucleic acids in lipid nanoparticles is 0.047 to 3.931.

Claims

1. A lipid nanoparticle comprising: a nucleic acid encapsulated in the lipid nanoparticle; a cationic lipid; a sterol or sterol derivative; a VHH antibody; a targeted polyalkylene glycol-modified lipid; and a non-targeted polyalkylene glycol-modified lipid, wherein the VHH antibody targets an antigen on a T cell; the targeted polyalkylene glycol-modified lipid is a lipid in which the VHH antibody is linked to a polyalkylene glycol; and the non-targeted polyalkylene glycol-modified lipid is a lipid in which the VHH antibody is not linked; and the density of the VHH antibody present on the surface of the lipid nanoparticle in a manner that allows it to bind to the antigen is at a density of 100 nm on the surface of the nanoparticle. ２ Lipid nanoparticles, ranging in number from 0.004 to 2,300 per molecule.

2. The density of VHH antibodies capable of binding to the antigen on the surface of the lipid nanoparticles is at 100 nm on the nanoparticle surface. ２ Lipid nanoparticles according to claim 1, wherein the number of nanoparticles per nanoparticle ranges from 0.008 to 2,300.

3. The density of VHH antibodies capable of binding to the antigen on the surface of the lipid nanoparticles is at 100 nm on the nanoparticle surface. ２ Lipid nanoparticles according to claim 1, wherein the number of nanoparticles per nanoparticle ranges from 0.010 to 1.

500.

4. The lipid nanoparticle according to claim 1, wherein the VHH antibody binds to CD2, CD3, CD4, CD5, CD6, CD7, or CD8.

5. The lipid nanoparticle according to claim 1, wherein the VHH antibody binds to CD4 or CD8.

6. The lipid nanoparticles according to claim 1, wherein the VHH antibody binds to CD8.

7. The lipid nanoparticle according to claim 1, wherein the VHH antibody binds to CD4.

8. The density of VHH antibodies capable of binding to the antigen on the surface of the lipid nanoparticles is at 100 nm on the nanoparticle surface. ２ Lipid nanoparticles according to claim 6, wherein the number of nanoparticles per nanoparticle ranges from 0.004 to 2,200.

9. The density of VHH antibodies capable of binding to the antigen on the surface of the lipid nanoparticles is at 100 nm on the nanoparticle surface. ２ Lipid nanoparticles according to claim 6, wherein the number of nanoparticles is between 0.008 and 1,800 per nanoparticle.

10. The density of VHH antibodies capable of binding to the antigen on the surface of the lipid nanoparticles is at 100 nm on the nanoparticle surface. ２ Lipid nanoparticles according to claim 6, wherein the number of nanoparticles per nanoparticle ranges from 0.010 to 1.

500.

11. The density of VHH antibodies capable of binding to the antigen on the surface of the lipid nanoparticles is at 100 nm on the nanoparticle surface. ２ Lipid nanoparticles according to claim 7, wherein the number of nanoparticles per nanoparticle ranges from 0.030 to 2,300.

12. The density of VHH antibodies capable of binding to the antigen on the surface of the lipid nanoparticles is at 100 nm on the nanoparticle surface. ２ Lipid nanoparticles according to claim 7, wherein the number of nanoparticles per nanoparticle ranges from 0.035 to 2,300.

13. The density of the VHH antibody that can bind to the antigen on the surface of the lipid nanoparticle is from 0.040 to 1.500 per 100 nm of the nanoparticle surface ２ The lipid nanoparticle according to claim 7, wherein the density is from 0.040 to 1.500 per 100 nm of the nanoparticle surface 14. The lipid nanoparticle according to claim 1, wherein the VHH antibody is linked to the polyalkylene glycol of the targeted polyalkylene glycol-modified lipid via a linker.

15. The lipid nanoparticle according to claim 14, wherein the linker comprises a peptide of 1 to 50 units.

16. The lipid nanoparticle according to claim 1, wherein the VHH antibody comprises a peptide represented by SEQ ID NO: 22 or SEQ ID NO:

23.

17. The lipid nanoparticle according to claim 1, wherein the sterol or sterol derivative is cholesterol.

18. The lipid nanoparticles according to claim 1, wherein the targeted polyalkylene glycol-modified lipid comprises DSPE-PEG.

19. The lipid nanoparticle according to claim 1, wherein the untargeted polyalkylene glycol-modified lipid comprises a polyalkylene glycol-modified lipid in which polyalkylene glycol is modified with a reactive group, and a polyalkylene glycol-modified lipid in which polyalkylene glycol is unmodified.

20. The lipid nanoparticle according to claim 19, wherein the reactive group comprises a group selected from the group consisting of acetylene, transcyclooctene, cyclooctin, diarylcyclooctin, oxime, ketone, aldehyde, thiol, free cysteine, amine, maleimide, NHS (N-hydroxysuccinimide), NHS ester (N-hydroxysuccinimide ester), isocyanate, isothiocyanate, methyl ester phosphine, norbornene, tetrazine, methylcyclopropene, azetine, cyanide, azide, and dibenzocyclooctin.

21. The lipid nanoparticle according to claim 19, wherein the reactive group comprises maleimide.

22. The lipid nanoparticle according to claim 19, wherein the polyalkylene glycol-modified lipid, in which polyalkylene glycol is modified with a reactive group, includes DSPE-PEG, and the polyalkylene glycol-modified lipid, in which polyalkylene glycol is not modified, is DMG-PEG.

23. The lipid nanoparticle according to claim 1, wherein the untargeted polyalkylene glycol-modified lipid comprises a polyalkylene glycol-modified lipid in which polyalkylene glycol is modified at the untargeting site, and a polyalkylene glycol-modified lipid in which polyalkylene glycol is unmodified.

24. The lipid nanoparticle according to claim 23, wherein the polyalkylene glycol-modified lipid, in which polyalkylene glycol is modified at the non-targeting site, includes DSPE-PEG, and the polyalkylene glycol-modified lipid, in which polyalkylene glycol is not modified, is DMG-PEG.

25. Lipid nanoparticles according to claim 1, further comprising phospholipids.

26. The lipid nanoparticle according to claim 25, wherein the phospholipid is DSPC, DOPC, DOPE, or a mixture thereof.

27. Lipid nanoparticles according to claim 1, wherein the average particle size is 40 nm to 300 nm.

28. The lipid nanoparticle according to claim 1, wherein the nucleic acid is siRNA, antisense nucleic acid, heteroduplex nucleic acid, miRNA, gRNA, or mRNA.

29. Lipid nanoparticles according to claim 1, produced by a method comprising the steps of: mixing a cationic lipid, a sterol or sterol derivative, a non-targeted polyalkylene glycol-modified lipid, and the nucleic acid to produce non-targeted lipid nanoparticles encapsulating the nucleic acid; and linking the VHH antibody to the non-targeted lipid nanoparticles.

30. The lipid nanoparticle according to claim 29, wherein the untargeted polyalkylene glycol-modified lipid comprises a polyalkylene glycol-modified lipid in which polyalkylene glycol is modified with a first reactive group, the VHH antibody before linking comprises a second reactive group, and the linking step comprises the reaction of the first reactive group and the second reactive group to form the targeted polyalkylene glycol-modified lipid.

31. A method for measuring the optimal density range of target binding sites on the surface of lipid nanoparticles, wherein the lipid nanoparticles comprise nucleic acids encapsulated within the lipid nanoparticles, cationic lipids, sterols or sterol derivatives, target binding sites, targeted polyalkylene glycol-modified lipids, and untargeted polyalkylene glycol-modified lipids, wherein the target binding sites target target sites on target cells, the targeted polyalkylene glycol-modified lipids are lipids in which the target binding sites are linked to polyalkylene glycol, and the untargeted polyalkylene glycol-modified lipids are lipids in which the target binding sites are not linked, and the measurement method comprises gradually increasing the amount of target binding sites and using a nanoflow cytometer to measure the density of target binding sites that are capable of binding to target sites on the surface of the lipid nanoparticles, and A measurement method comprising: measuring the expression level of nucleic acids delivered at the density of target binding sites within target cells; and obtaining the optimal density range by setting the density range of target binding sites that achieves an expression level of 40% or more (with the maximum expression level being defined as 100%) as the optimal density range.

32. The method according to claim 31, comprising setting the density range of target binding sites that achieve an expression level of 50% or more, when the maximum expression level is set to 100%, as the optimal density range, and obtaining the optimal density range.

33. The method according to claim 31, comprising setting the density range of target binding sites that achieve an expression level of 60% or more, when the maximum expression level is set to 100%, as the optimal density range, and obtaining the optimal density range.

34. The method according to claim 31, wherein the target cell is a T cell or a hematopoietic stem cell.

35. The method according to claim 31, wherein the target binding site is a VHH antibody, a Fab antibody, or an scFv.

36. The method according to claim 31, wherein the nucleic acid is mRNA.

37. A method for producing lipid nanoparticles, comprising obtaining an optimal density range by the measurement method described in claim 31, and producing lipid nanoparticles having the optimal density.

38. A lipid nanoparticle comprising: a nucleic acid encapsulated in the lipid nanoparticle; a cationic lipid; a sterol or sterol derivative; a Fab antibody; a targeted polyalkylene glycol-modified lipid; and a non-targeted polyalkylene glycol-modified lipid, wherein the Fab antibody targets an antigen on a T cell; the targeted polyalkylene glycol-modified lipid is a lipid in which the Fab antibody is linked to a polyalkylene glycol; and the non-targeted polyalkylene glycol-modified lipid is a lipid in which the Fab antibody is not linked; and the density of Fab antibodies capable of binding to the antigen on the surface of the lipid nanoparticle is at 100 nm on the nanoparticle surface. ２ Lipid nanoparticles, numbering from 0.003 to 1.113 per molecule.

39. The density of Fab antibodies capable of binding to the antigen on the surface of the lipid nanoparticles is, at 100 nm on the nanoparticle surface. ２ Lipid nanoparticles according to claim 38, wherein the number of nanoparticles per nanoparticle ranges from 0.004 to 1.

113.

40. The density of Fab antibodies capable of binding to the antigen on the surface of the lipid nanoparticles is such that the density ２ Lipid nanoparticles according to claim 38, wherein the number of nanoparticles is between 0.006 and 1,000 per nanoparticle.

41. The lipid nanoparticle according to claim 38, wherein the Fab antibody is linked to the polyalkylene glycol of the targeted polyalkylene glycol-modified lipid via a linker.

42. The lipid nanoparticle according to claim 41, wherein the linker comprises a peptide of 1 to 50 units.

43. The lipid nanoparticle according to claim 38, wherein the Fab antibody binds to CD2, CD3, CD4, CD5, CD6, CD7, or CD8.

44. The lipid nanoparticle according to claim 38, wherein the Fab antibody binds to CD4 or CD8.

45. The lipid nanoparticle according to claim 38, wherein the Fab antibody binds to CD8.

46. ​​The lipid nanoparticle according to claim 38, wherein the sterol or sterol derivative is cholesterol.

47. The lipid nanoparticle according to claim 38, wherein the targeted polyalkylene glycol-modified lipid comprises DSPE-PEG.

48. The lipid nanoparticle according to claim 38, wherein the untargeted polyalkylene glycol-modified lipid comprises a polyalkylene glycol-modified lipid in which polyalkylene glycol is modified with a reactive group, and a polyalkylene glycol-modified lipid in which polyalkylene glycol is unmodified.

49. The lipid nanoparticle according to claim 48, wherein the reactive group comprises a group selected from the group consisting of acetylene, transcyclooctene, cyclooctin, diarylcyclooctin, oxime, ketone, aldehyde, thiol, free cysteine, amine, maleimide, NHS (N-hydroxysuccinimide), NHS ester (N-hydroxysuccinimide ester), isocyanate, isothiocyanate, methyl ester phosphine, norbornene, tetrazine, methylcyclopropene, azetine, cyanide, azide, and dibenzocyclooctin.

50. The lipid nanoparticle according to claim 48, wherein the reactive group comprises maleimide.

51. The lipid nanoparticle according to claim 48, wherein the polyalkylene glycol-modified lipid, in which polyalkylene glycol is modified with a reactive group, includes DSPE-PEG, and the polyalkylene glycol-modified lipid, in which polyalkylene glycol is not modified, is DMG-PEG.

52. The lipid nanoparticle according to claim 48, wherein the untargeted polyalkylene glycol-modified lipid comprises a polyalkylene glycol-modified lipid in which polyalkylene glycol is modified at the untargeting site, and a polyalkylene glycol-modified lipid in which polyalkylene glycol is unmodified.

53. The lipid nanoparticle according to claim 52, wherein the polyalkylene glycol-modified lipid, in which polyalkylene glycol is modified at the non-targeting site, includes DSPE-PEG, and the polyalkylene glycol-modified lipid, in which polyalkylene glycol is not modified, is DMG-PEG.

54. Lipid nanoparticles according to claim 38, further comprising phospholipids.

55. The lipid nanoparticle according to claim 54, wherein the phospholipid is DSPC, DOPC, DOPE, or a mixture thereof.

56. Lipid nanoparticles according to claim 38, wherein the average particle size is 40 nm to 300 nm.

57. The lipid nanoparticle according to claim 38, wherein the nucleic acid is siRNA, antisense nucleic acid, heteroduplex nucleic acid, miRNA, gRNA, or mRNA.

58. Lipid nanoparticles according to claim 38, produced by a method comprising the steps of: mixing a cationic lipid, a sterol or sterol derivative, a non-targeted polyalkylene glycol-modified lipid, and the nucleic acid to produce non-targeted lipid nanoparticles encapsulating the nucleic acid; and linking the antibody to the non-targeted lipid nanoparticles.

59. The lipid nanoparticle according to claim 58, wherein the untargeted polyalkylene glycol-modified lipid comprises a polyalkylene glycol-modified lipid in which polyalkylene glycol is modified with a first reactive group, the antibody before linking comprises a second reactive group, and the linking step comprises the reaction of the first reactive group and the second reactive group to form the targeted polyalkylene glycol-modified lipid.

60. A lipid nanoparticle comprising a nucleic acid encapsulated in the lipid nanoparticle, a cationic lipid, a sterol or sterol derivative, an scFv antibody, a targeted polyalkylene glycol-modified lipid, and a non-targeted polyalkylene glycol-modified lipid, wherein the scFv antibody targets an antigen on a T cell, the targeted polyalkylene glycol-modified lipid is a lipid in which the scFv antibody is linked to a polyalkylene glycol, and the non-targeted polyalkylene glycol-modified lipid is a lipid in which the scFv antibody is not linked, and the density of scFv antibodies present on the surface of the lipid nanoparticle capable of binding to the antigen is at 100 nm on the surface of the nanoparticle. ２ Lipid nanoparticles, ranging in number from 0.001 to 3,400 per molecule.

61. The density of scFv antibodies capable of binding to the antigen on the surface of the lipid nanoparticles is, at 100 nm on the nanoparticle surface. ２ Lipid nanoparticles according to claim 60, wherein the number of nanoparticles per nanoparticle ranges from 0.002 to 3.

100.

62. The density of scFv antibodies capable of binding to the antigen on the surface of the lipid nanoparticles is, at 100 nm on the nanoparticle surface. ２ Lipid nanoparticles according to claim 60, wherein the number of nanoparticles per nanoparticle ranges from 0.005 to 2,800.

63. The lipid nanoparticle according to claim 60, wherein the scFv antibody is linked to the polyalkylene glycol of the targeted polyalkylene glycol-modified lipid via a linker.

64. The lipid nanoparticle according to claim 63, wherein the linker comprises a peptide of 1 to 50 units.

65. The lipid nanoparticle according to claim 60, wherein the scFv antibody binds to CD2, CD3, CD4, CD5, CD6, CD7, or CD8.

66. The lipid nanoparticle according to claim 60, wherein the scFv antibody binds to CD4 or CD8.

67. The lipid nanoparticle according to claim 60, wherein the scFv antibody binds to CD8.

68. The lipid nanoparticle according to claim 60, wherein the scFv antibody binds to CD4.

69. The density of scFv antibodies capable of binding to the antigen on the surface of the lipid nanoparticles is, at 100 nm on the nanoparticle surface. ２ Lipid nanoparticles according to claim 67, wherein the number of nanoparticles is between 0.001 and 1.900 per nanoparticle.

70. The density of scFv antibodies capable of binding to the antigen on the surface of the lipid nanoparticles is, at 100 nm on the nanoparticle surface. ２ Lipid nanoparticles according to claim 67, wherein the number of nanoparticles is between 0.002 and 1,400 per nanoparticle.

71. The density of scFv antibodies capable of binding to the antigen on the surface of the lipid nanoparticles is, at 100 nm on the nanoparticle surface. ２ Lipid nanoparticles according to claim 67, wherein the number of nanoparticles is between 0.005 and 1.100 per nanoparticle.

72. The density of scFv antibodies capable of binding to the antigen on the surface of the lipid nanoparticles is, at 100 nm on the nanoparticle surface. ２ Lipid nanoparticles according to claim 68, wherein the number of nanoparticles is between 0.020 and 3,400 per nanoparticle.

73. The density of scFv antibodies capable of binding to the antigen on the surface of the lipid nanoparticles is, at 100 nm on the nanoparticle surface. ２ Lipid nanoparticles according to claim 68, wherein the number of nanoparticles per unit ranges from 0.020 to 3.

100.

74. The density of scFv antibodies capable of binding to the antigen on the surface of the lipid nanoparticles is, at 100 nm on the nanoparticle surface. ２ Lipid nanoparticles according to claim 68, wherein the number of nanoparticles is between 0.030 and 2,800 per nanoparticle.

75. The lipid nanoparticle according to claim 60, wherein the sterol or sterol derivative is cholesterol.

76. The lipid nanoparticle according to claim 60, wherein the targeted polyalkylene glycol-modified lipid comprises DSPE-PEG.

77. The lipid nanoparticle according to claim 60, wherein the untargeted polyalkylene glycol-modified lipid comprises a polyalkylene glycol-modified lipid in which polyalkylene glycol is modified with a reactive group, and a polyalkylene glycol-modified lipid in which polyalkylene glycol is unmodified.

78. The lipid nanoparticle according to claim 77, wherein the reactive group comprises a group selected from the group consisting of acetylene, transcyclooctene, cyclooctin, diarylcyclooctin, oxime, ketone, aldehyde, thiol, free cysteine, amine, maleimide, NHS (N-hydroxysuccinimide), NHS ester (N-hydroxysuccinimide ester), isocyanate, isothiocyanate, methyl ester phosphine, norbornene, tetrazine, methylcyclopropene, azetine, cyanide, azide, and dibenzocyclooctin.

79. The lipid nanoparticle according to claim 77, wherein the reactive group comprises maleimide.

80. The lipid nanoparticle according to claim 77, wherein the polyalkylene glycol-modified lipid, in which polyalkylene glycol is modified with a reactive group, includes DSPE-PEG, and the polyalkylene glycol-modified lipid, in which polyalkylene glycol is not modified, is DMG-PEG.

81. The lipid nanoparticle according to claim 60, wherein the untargeted polyalkylene glycol-modified lipid comprises a polyalkylene glycol-modified lipid in which polyalkylene glycol is modified at the untargeting site, and a polyalkylene glycol-modified lipid in which polyalkylene glycol is not modified.

82. The lipid nanoparticle according to claim 81, wherein the polyalkylene glycol-modified lipid, in which polyalkylene glycol is modified at the non-targeting site, includes DSPE-PEG, and the polyalkylene glycol-modified lipid, in which polyalkylene glycol is not modified, is DMG-PEG.

83. Lipid nanoparticles according to claim 60, further comprising phospholipids.

84. The lipid nanoparticle according to claim 60, wherein the phospholipid is DSPC, DOPC, DOPE, or a mixture thereof.

85. Lipid nanoparticles according to claim 60, wherein the average particle size is 40 nm to 300 nm.

86. The lipid nanoparticle according to claim 60, wherein the nucleic acid is siRNA, antisense nucleic acid, heteroduplex nucleic acid, miRNA, gRNA, or mRNA.

87. Lipid nanoparticles according to claim 60, produced by a method comprising the steps of: mixing a cationic lipid, a sterol or sterol derivative, a non-targeted polyalkylene glycol-modified lipid, and the nucleic acid to produce non-targeted lipid nanoparticles encapsulating the nucleic acid; and linking the antibody to the non-targeted lipid nanoparticles.

88. The lipid nanoparticle according to claim 87, wherein the untargeted polyalkylene glycol-modified lipid comprises a polyalkylene glycol-modified lipid in which polyalkylene glycol is modified with a first reactive group, the antibody before linking comprises a second reactive group, and the linking step comprises the reaction of the first reactive group and the second reactive group to form the targeted polyalkylene glycol-modified lipid.

89. A method for determining whether the density of target binding sites on the surface of lipid nanoparticles is within the optimal density range, wherein the lipid nanoparticles comprise nucleic acids encapsulated within the lipid nanoparticles, cationic lipids, sterols or sterol derivatives, target binding sites, targeted polyalkylene glycol-modified lipids, and untargeted polyalkylene glycol-modified lipids, wherein the target binding sites target target sites on target cells, the targeted polyalkylene glycol-modified lipids are lipids in which the target binding sites are linked to polyalkylene glycol, and the untargeted polyalkylene glycol-modified lipids are lipids in which the target binding sites are not linked, and the method comprises the following steps (1) and (2): (1) obtaining the weight ratio of the targeted polyalkylene glycol-modified lipids to the nucleic acids in the lipid nanoparticles; (2) determining that the density of target binding sites on the surface of the lipid nanoparticles is within the optimal density range if the weight ratio obtained in step (1) is within the optimal weight ratio range; Here, the optimal weight ratio range is expressed by the following regression equation (A), which is based on the correlation between the density of target binding sites of lipid nanoparticles measured using a nanoflow cytometer and the weight ratio of the targeted polyalkylene glycol-modified lipid to the nucleic acid in the lipid nanoparticles: Y = aX + b (A) [wherein Y is the density of target binding sites of lipid nanoparticles measured using a nanoflow cytometer (100 nm of the nanoparticle surface)] ２ The method is a numerical range in which (Y1-b) / a, obtained by substituting the lower limit Y1 of the optimal density range into (Y1-b) / a, obtained by substituting the upper limit Y2 of the optimal density range into (Y2-b) / a, and the numerical range in which (Y2-b) / a, obtained by substituting the upper limit Y2 of the optimal density range into (Y1-b) / a, which represents the number of target binding sites per unit, X represents the weight ratio of the targeted polyalkylene glycol-modified lipid to the nucleic acid in the lipid nanoparticle, a represents the slope of the regression line, a > 0, and b represents the intercept of the regression line. The optimal density range is a numerical range obtained by the method of claim 31.

90. A method for measuring the optimal weight ratio range of targeted polyalkylene glycol-modified lipids to nucleic acids in lipid nanoparticles, wherein the lipid nanoparticles comprise nucleic acids encapsulated in the lipid nanoparticles, a cationic lipid, a sterol or sterol derivative, a target binding site, a targeted polyalkylene glycol-modified lipid, and a non-targeted polyalkylene glycol-modified lipid, wherein the target binding site targets a target site on a target cell, the targeted polyalkylene glycol-modified lipid is a lipid in which the target binding site is linked to polyalkylene glycol, and the non-targeted polyalkylene glycol-modified lipid is a lipid in which the target binding site is not linked, and the method comprises a regression equation represented by the following equation (A) based on the correlation between the density of target binding sites of lipid nanoparticles measured using a nanoflow cytometer and the weight ratio of the targeted polyalkylene glycol-modified lipids to nucleic acids in the lipid nanoparticles: Y = aX + b (A) [wherein of equation (A), Y is the density of target binding sites of lipid nanoparticles measured using a nanoflow cytometer (100 nm on the nanoparticle surface). ２ The method includes the step of obtaining an optimal weight ratio range, wherein the lower limit is (Y1-b) / a obtained by substituting the lower limit of the optimal density range Y1 into [the number of target binding sites per unit], X represents the weight ratio of the targeted polyalkylene glycol-modified lipid to the nucleic acid in the lipid nanoparticles, a represents the slope of the regression line, a > 0, and b represents the intercept of the regression line], and the upper limit is (Y2-b) / a obtained by substituting the upper limit of the optimal density range Y2 into the value, and the optimal density range is a numerical range obtained by the method of claim 31.

91. The method according to claim 89 or 90, wherein the target cell is a T cell or a hematopoietic stem cell.

92. The method according to claim 89 or 90, wherein the target binding site is a VHH antibody, a Fab antibody, or an scFv.

93. The method according to claim 89 or 90, wherein the nucleic acid is mRNA.

94. Lipid nanoparticles comprising: nucleic acid encapsulated in the lipid nanoparticles; a cationic lipid; a sterol or sterol derivative; a VHH antibody; a targeted polyalkylene glycol-modified lipid; and a non-targeted polyalkylene glycol-modified lipid, wherein the VHH antibody targets an antigen on a T cell; the targeted polyalkylene glycol-modified lipid is a lipid in which the VHH antibody is linked to polyalkylene glycol; the non-targeted polyalkylene glycol-modified lipid is a lipid in which the VHH antibody is not linked; and the weight ratio of the targeted polyalkylene glycol-modified lipid to the nucleic acid in the lipid nanoparticles is 0.047 to 3.

931.

95. The lipid nanoparticle according to claim 94, wherein the weight ratio of targeted polyalkylene glycol-modified lipid to nucleic acid in the lipid nanoparticle is 0.054 to 3.

931.

96. The lipid nanoparticle according to claim 94, wherein the weight ratio of targeted polyalkylene glycol-modified lipid to nucleic acid in the lipid nanoparticle is 0.057 to 2.

578.

97. The lipid nanoparticle according to claim 94, wherein the VHH antibody binds to CD2, CD3, CD4, CD5, CD6, CD7, or CD8.

98. The lipid nanoparticle according to claim 94, wherein the VHH antibody binds to CD4 or CD8.

99. The lipid nanoparticle according to claim 94, wherein the VHH antibody binds to CD8.

100. The lipid nanoparticle according to claim 94, wherein the VHH antibody binds to CD4.

101. The lipid nanoparticle according to claim 99, wherein the weight ratio range of the targeted polyalkylene glycol-modified lipid to nucleic acid in the lipid nanoparticle is 0.047 to 3.

762.

102. The lipid nanoparticle according to claim 99, wherein the weight ratio range of the targeted polyalkylene glycol-modified lipid to nucleic acid in the lipid nanoparticle is 0.054 to 3.

085.

103. The lipid nanoparticle according to claim 99, wherein the weight ratio range of the targeted polyalkylene glycol-modified lipid to nucleic acid in the lipid nanoparticle is 0.057 to 2.

578.

104. The lipid nanoparticle according to claim 100, wherein the weight ratio range of the targeted polyalkylene glycol-modified lipid to the nucleic acid in the lipid nanoparticle is 0.091 to 3.

931.

105. The lipid nanoparticle according to claim 100, wherein the weight ratio range of the targeted polyalkylene glycol-modified lipid to the nucleic acid in the lipid nanoparticle is 0.099 to 3.

931.

106. The lipid nanoparticle according to claim 100, wherein the weight ratio range of the targeted polyalkylene glycol-modified lipid to the nucleic acid in the lipid nanoparticle is 0.108 to 2.

578.

107. The lipid nanoparticle according to claim 94, wherein the VHH antibody is linked to the polyalkylene glycol of the targeted polyalkylene glycol-modified lipid via a linker.

108. The lipid nanoparticle according to claim 107, wherein the linker comprises a peptide of 1 to 50 units.

109. The lipid nanoparticle according to claim 94, wherein the VHH antibody comprises a peptide represented by SEQ ID NO: 22 or SEQ ID NO:

23.

110. The lipid nanoparticle according to claim 94, wherein the sterol or sterol derivative is cholesterol.

111. The lipid nanoparticle according to claim 94, wherein the targeted polyalkylene glycol-modified lipid comprises DSPE-PEG.

112. The lipid nanoparticle according to claim 94, wherein the untargeted polyalkylene glycol-modified lipid comprises a polyalkylene glycol-modified lipid in which polyalkylene glycol is modified with a reactive group, and a polyalkylene glycol-modified lipid in which polyalkylene glycol is unmodified.

113. The lipid nanoparticle according to claim 112, wherein the reactive group comprises a group selected from the group consisting of acetylene, transcyclooctene, cyclooctin, diarylcyclooctin, oxime, ketone, aldehyde, thiol, free cysteine, amine, maleimide, NHS (N-hydroxysuccinimide), NHS ester (N-hydroxysuccinimide ester), isocyanate, isothiocyanate, methyl ester phosphine, norbornene, tetrazine, methylcyclopropene, azetine, cyanide, azide, and dibenzocyclooctin.

114. The lipid nanoparticle according to claim 112, wherein the reactive group comprises maleimide.

115. The lipid nanoparticle according to claim 112, wherein the polyalkylene glycol-modified lipid, in which polyalkylene glycol is modified with a reactive group, includes DSPE-PEG, and the polyalkylene glycol-modified lipid, in which polyalkylene glycol is not modified, is DMG-PEG.

116. The lipid nanoparticle according to claim 94, wherein the untargeted polyalkylene glycol-modified lipid comprises a polyalkylene glycol-modified lipid in which polyalkylene glycol is modified at the untargeting site, and a polyalkylene glycol-modified lipid in which polyalkylene glycol is unmodified.

117. The lipid nanoparticle according to claim 116, wherein the polyalkylene glycol-modified lipid, in which polyalkylene glycol is modified at a non-targeting site, includes DSPE-PEG, and the polyalkylene glycol-modified lipid, in which polyalkylene glycol is not modified, is DMG-PEG.

118. Lipid nanoparticles according to claim 94, further comprising phospholipids.

119. The lipid nanoparticle according to claim 117, wherein the phospholipid is DSPC, DOPC, DOPE, or a mixture thereof.

120. Lipid nanoparticles according to claim 94, wherein the average particle size is 40 nm to 300 nm.

121. The lipid nanoparticle according to claim 94, wherein the nucleic acid is siRNA, antisense nucleic acid, heterodouble-stranded nucleic acid, miRNA, gRNA, or mRNA.

122. Lipid nanoparticles according to claim 94, produced by a method comprising the steps of: mixing a cationic lipid, a sterol or sterol derivative, a non-targeted polyalkylene glycol-modified lipid, and the nucleic acid to produce non-targeted lipid nanoparticles encapsulating the nucleic acid; and linking the VHH antibody to the non-targeted lipid nanoparticles.

123. The lipid nanoparticle according to claim 122, wherein the untargeted polyalkylene glycol-modified lipid comprises a polyalkylene glycol-modified lipid in which polyalkylene glycol is modified with a first reactive group, the VHH antibody before linking comprises a second reactive group, and the linking step comprises the reaction of the first reactive group and the second reactive group to form the targeted polyalkylene glycol-modified lipid.

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