Lipid nanoparticle

Abstract

The purpose of the present invention is to provide: a lipid nanoparticle that enables efficient introduction of a drug into a cell; and constituent lipids of the lipid nanoparticle. Provided is a lipid nanoparticle comprising a nucleic acid encapsulated in the lipid nanoparticle, a cationic lipid, and a targeted polyalkylene glycol-modified lipid linked to a target-binding site, wherein the target-binding site targets a target site on a target cell.

Description

Lipid nanoparticles

[0001] This disclosure relates to 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, lipid nanoparticles that serve as carriers for efficiently delivering nucleic acids into cells have been reported, and these LNPs 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 (for example, Patent Document 1).

[0003] Attempts have been made to enable the selective delivery of nucleic acids to target cells. For example, Patent Documents 2 and 3 disclose LNPs formed by linking single-stranded variable fragments (scFv) that target antigens present on the cell surface.

[0004] International Publication No. 2022 / 071582, International Publication No. 2023 / 287861, International Publication No. 2024 / 102772

[0005] Although LNPs have been shown to be effective for selective delivery of nucleic acids, this remains challenging. Therefore, there is a need for novel lipid nanoparticles that enable selective delivery of nucleic acids to target cells by targeting specific sites on those cells.

[0006] The inventors have completed the present invention by finding that the cationic lipids in this disclosure can be constituent lipids of lipid nanoparticles that enable selective delivery of nucleic acids to target cells. This disclosure includes one or more embodiments described below: [1] Lipid nanoparticles comprising: nucleic acids encapsulated in the lipid nanoparticles; a cationic lipid; and a targeted polyalkylene glycol-modified lipid linked to a target binding site, wherein the target binding site targets a target site on a target cell, and the cationic lipid is a compound represented by the following general formula (I), or a pharmaceutically acceptable salt thereof: [In formula (I), a represents an integer from 3 to 5; b represents 0 or 1; R １ and R ２Each independently represents the following general formula (A): (In formula (A), R １１ and R １２ each independently represents a linear or branched C ５－１５ alkyl group; c represents 0 or 1; v represents an integer from 4 to 12) and represents a group represented by the formula; X represents the following general formula (B): (In formula (B), d represents an integer from 0 to 3; R ３ and R ４ each independently represents a C １－４ alkyl group or a C ２－４ alkenyl group (the C １－４ alkyl group or C ２－４ alkenyl group may have one or two hydrogen atoms substituted with a phenyl group), but R ３ and R ４ may be bonded to each other to form a 5- to 7-member non-aromatic heterocyclic ring (one or two hydrogen atoms of the ring may be substituted with a C １－４ alkyl group or a C ２－４ alkenyl group)) or a 5- to 7-member non-aromatic heterocyclic group (however, the group is bonded to (O-CO)b- by a carbon atom, and one or two hydrogen atoms of the ring may be substituted with a C １－４ alkyl group or a C ２－４ alkenyl group)) [2] The lipid nanoparticle according to [1], wherein the cationic lipid is a compound represented by any one of the following formulas, or a pharmaceutically acceptable salt thereof [3] The lipid nanoparticle according to [1] or [2], wherein the target binding site binds to an antigen on a T cell. [4] The lipid nanoparticle according to any one of [1] to [3], wherein the target binding site binds to CD2, CD3, CD4, CD5, CD6, CD7, or CD8. [5] The lipid nanoparticle according to any one of [1] to [4], wherein the target binding site binds to an antigen on a hematopoietic stem cell (HSC). [6] The lipid nanoparticle according to any one of [1] to [5], wherein the target binding site binds to CD90 or CD117. [7] The lipid nanoparticle according to any one of [1] to [6], wherein the target binding site is an antibody or an antigen-binding fragment thereof. [8] The lipid nanoparticle according to any one of [1] to [7], wherein the target binding site is an antibody, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an scFv, or a VHH antibody. [9] The lipid nanoparticle according to any one of [1] to [8], wherein the target binding site is linked to the polyalkylene glycol of the targeted polyalkylene glycol-modified lipid via a linker.

[10] The lipid nanoparticle according to [9], wherein the linker comprises a peptide of 1 to 50 units.

[11] 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 Lipid nanoparticles according to claim 9, comprising at least one of the following: [wherein q is an integer from 1 to 10]

[12] The linker is (OCH ２ CH ２ ) ｒ Lipid nanoparticles according to any one of [9] to

[11] , comprising [wherein r is an integer from 1 to 10].

[13] The linker is defined by the following formula: Lipid nanoparticles according to any one of [9] to

[12] , comprising a site represented by [wherein the formula, the left arrow indicates linkage with the peptide, the right arrow indicates linkage with polyalkylene glycol, X is N or O, and r is an integer from 1 to 15].

[14] The linker is represented as -Z1-Z2-, where Z1 is linked to the target binding site, Z2 is linked to the polyalkylene glycol, and Z1 is It is represented as, and Z2 is, A lipid nanoparticle according to any one of [9] to

[12] , represented by the formula [wherein 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].

[15] The linker is represented as -Z1-Z2-, where Z1 is linked to the target binding site, Z2 is linked to the polyalkylene glycol, and Z1 is as follows: Z2 is one of the polypeptides shown, and Z2 is A lipid nanoparticle according to any one of [9] to

[14] , represented by the formula [wherein the left arrow indicates linkage to the adjacent Cys side chain SH group, the right arrow indicates linkage to polyalkylene glycol, and X is N or O].

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

[15] , further comprising a sterol or sterol derivative.

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

[16] , further comprising a phospholipid.

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

[17] , further comprising an untargeted polyalkylene glycol-modified lipid in which the target binding site is not linked.

[19] The lipid nanoparticle according to

[18] , wherein the untargeted polyalkylene glycol-modified lipid comprises 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.

[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

[18] , 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.

[24] The lipid nanoparticles according to

[23] , 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.

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

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

[0007] The lipid nanoparticles of the present invention can selectively deliver nucleic acids to target cells.

[0008] Figure 1 shows that antibody 12 conjugate LNPs 1a-7a in Example 1 are carriers capable of delivering mRNA to CD3-positive T cells. Figure 2 shows that antibody 12 conjugate LNPs 1b-6b in Example 2 are carriers capable of delivering mRNA to CD8-positive T cells. Figure 3 shows that antibody 12 conjugate LNPs 1b-6b in Example 2 are carriers capable of delivering mRNA to CD4-positive T cells. Figure 4 shows that all antibody conjugate LNPs in Example 3 are carriers capable of selectively delivering mRNA to CD8-positive T cells. Error bars indicate standard deviation (n=3). Figure 5 shows that all antibody conjugate LNPs in Example 3 are carriers capable of selectively delivering mRNA to CD8-positive T cells. Error bars indicate standard deviation (n=3). Figure 6 shows that all antibody 1 conjugate LNPs in Example 4 are carriers capable of selectively delivering mRNA to CD8-positive T cells. Figure 7 shows that all antibody-1 conjugate LNPs in Example 4 are carriers capable of selectively delivering mRNA to CD8-positive T cells. Figure 8 shows that antibody-lipid conjugates are generated after conjugation in antibody-1 conjugate LNPs 1d to 7d in Example 4. Figure 9 shows that all antibody-2 conjugate LNPs in Example 5 are carriers capable of selectively delivering mRNA to CD8-positive T cells. Figure 10 shows that all antibody-2 conjugate LNPs in Example 5 are carriers capable of selectively delivering mRNA to CD8-positive T cells. Figure 11 shows that antibody-lipid conjugates are generated after conjugation in antibody-2 conjugate LNPs 1e to 7e in Example 5. Figure 12 shows that anti-CD3 antibody conjugate LNP 1f in Example 6 is a superior carrier capable of efficiently delivering to CD4-positive T cells and CD8-positive T cells compared to untargeted LNP R1f. Error bars indicate the standard deviation (n=3). Figure 13 shows that the anti-CD4 antibody conjugate LNP 2f in Example 6 is a carrier capable of selectively delivering mRNA to CD4-positive T cells. Error bars indicate the standard deviation (n=3).Figure 14 shows that anti-CD5 antibody conjugate LNP 3f in Example 6 is a superior carrier that can efficiently deliver to CD4-positive T cells and CD8-positive T cells compared to untargeted LNP R1f. Error bars indicate the standard deviation (n=3). Figure 15 shows that anti-CD5 antibody conjugate LNP 4f in Example 6 is a superior carrier that can efficiently deliver to CD4-positive T cells and CD8-positive T cells compared to untargeted LNP R1f. Error bars indicate the standard deviation (n=3). Figure 16 shows that anti-CD8 antibody conjugate LNP 5f in Example 6 is a carrier that can selectively deliver mRNA to CD8-positive T cells. Error bars indicate the standard deviation (n=3). Figure 17 shows that antibody-lipid conjugates are generated after conjugation in antibody conjugate LNPs 1f to 5f in Example 6. Figure 18 shows that 1 g of antibody-1 conjugate LNP in Example 7 is a carrier capable of selectively delivering mRNA to CD8-positive T cells. In the graph, "Lipofectamine MessengerMAX" is used instead of LNP as a control for mRNA carrier. Error bars indicate the standard deviation (n=3). Figure 19 shows that 1 g of antibody-1 conjugate LNP in Example 7 is a carrier capable of selectively delivering mRNA to CD8-positive T cells. In the graph, "Lipofectamine MessengerMAX" is used instead of LNP as a control for mRNA carrier. Error bars indicate the standard deviation (n=3). Figure 20 shows that 1 h of antibody-1 conjugate LNP in Example 8 is a carrier capable of selectively delivering mRNA to CD8-positive T cells. Error bars indicate the standard deviation (n=3). Figure 21 is a schematic diagram showing one embodiment of the method for producing lipid nanoparticles of the present invention.

[0009] The lipid nanoparticles of the present invention include nucleic acids, cationic lipids, and targeted polyalkylene glycol-modified lipids. In this disclosure, the term "lipid nanoparticle (LNP)" means a particle containing 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), a dispersed phase in an emulsion, a micelle, or an internal phase in a suspension. 1. Nucleic Acids It is preferable that the lipid nanoparticles of the present invention encapsulate a component intended for delivery into a target cell within a lipid membrane-covered particle. The component encapsulated within the lipid nanoparticles of the present invention is not particularly limited as long as it is of a size that can be encapsulated. Any substance such as nucleic acids, sugars, peptides, small molecule compounds, and metal compounds can be encapsulated in the lipid nanoparticles of the present invention. It is also preferable that the nucleic acid encapsulated in the lipid nanoparticles of the present invention is a functional nucleic acid that controls the expression of a target gene present in the target cell. Examples of 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, plasmid DNA (pDNA) may be used as an siRNA expression vector for expressing siRNA within cells. The siRNA expression vector can be prepared from commercially available siRNA expression vectors, or it may be modified as appropriate. In one embodiment of the present invention, the lipid nanoparticles according to the present invention comprise a pH-sensitive cationic lipid of the present invention, its salt or stereoisomer, and a nucleic acid, where the nucleic acid is siRNA, mRNA, or plasmid DNA.

[0010] 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.

[0011] 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 a hairpin RNA precursor (pre-miRNA), and can form functional complexes with proteins. Typically, miRNA further binds to the UTR region of a target mRNA as a functional complex with a protein and can regulate the target gene by mRNA degradation or translation inhibition or repression, but is not limited to these. One embodiment of the miRNA binding site described above is the miR 122-5p binding site, which can regulate protein expression in hepatocytes. 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. This 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 covalent bonds, as well as oligonucleotides having non-native parts that function similarly to their respective native parts.

[0012] 2. Cationic Lipids As used herein, “cationic lipids” include ionizable cationic lipids and permanently cationic lipids. As used herein, the term “ionizable cationic lipids” refers to lipids 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 lipids.” 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 lipids” refers to lipids 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.

[0013] The cationic lipid that constitutes the lipid nanoparticles of the present invention is defined by the following formula (I): [wherein a is an integer from 3 to 5; b is 0 or 1; R １ and R ２ Each of these independently gives the following general formula (A): (wherein formula (A), R １１ and R １２ Each of these is independently a linear or branched C ５－１５ X represents an alkyl group; c represents 0 or 1; v represents an integer from 4 to 12) and X is represented by the following general formula (B): (wherein formula (B), d represents an integer from 0 to 3; R ３ and R ４ Each is independently C １－４ Alkyl alkyl group or C ２－４ Alkenyl group (the C １－４ Alkyl alkyl group or C ２－４ The alkenyl group may have one or two hydrogen atoms substituted for a phenyl group, but R ３ and R ４ These are bonded to each other to form a 5-7 member non-aromatic heteroring (one or two hydrogen atoms of the ring are C １－４ Alkyl alkyl group or C ２－４A group represented by (which may be substituted with an alkenyl group) or a 5-7 membered non-aromatic heterocyclic group (where the group is bonded to a carbon atom by (O-CO)b-, and one or two hydrogen atoms of the ring are C １－４ Alkyl alkyl group or C ２－４ A pH-sensitive cationic lipid (hereinafter sometimes referred to as "the pH-sensitive cationic lipid of the present invention") is a compound represented by [which may be substituted with an alkenyl group] or a pharmaceutically acceptable salt thereof.

[0014] In general formula (I), a is an integer from 3 to 5, but is preferably 4. b is 0 or 1. When b is 0, it means that there is no -O-CO- group and it is a single bond. In general formula (I), R １ and R ２ Each of these independently represents a group represented by the following general formula (A). In general formula (A), R １１ and R １２ Each of these is independently a linear or branched C ２－１５ It represents an alkyl group (an alkyl group having 2 to 15 carbon atoms); c represents 0 or 1; and v represents an integer from 4 to 12.

[0015] Linear or branched C ２－１５Examples of alkyl groups include n-ethyl group; n-propyl group, 1-methylethyl group; n-butyl group, 1-methylpropyl group, 2-methylpropyl group, 1,1-dimethylethyl group; n-pentyl group, 1-methylbutyl group, 2-methylbutyl group, 3-methylbutyl group, 1-ethylpropyl group, 1,1-dimethylpropyl group, 2,2-dimethylpropyl group; n-hexyl group, 1-methylpentyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, 1-ethylbutyl group, 1,1-dimethylbutyl group, 2,2-dimethylbutyl group, 3,3-dimethylbutyl group, 1,2-dimethylbutyl group, 1-methyl-2,2-dimethylbutyl group; n-heptyl group, 1-methylhexyl group, 2-methylhexyl group, 3-methylhexyl group, 4-methylhexyl group, 5-methylhexyl group, 1-ethylpentyl group, 1,1-dimethylpentyl group, 2,2-dimethylpentyl group, 3,3-dimethylpentyl group, 4,4-dimethylpentyl group, 1-methyl-3,3-dimethylbutyl group, 2-methyl-3,3-dimethylbutyl group;

[0016] n-octyl group, 1-methylheptyl group, 2-methylheptyl group, 3-methylheptyl group, 4-methylheptyl group, 5-methylheptyl group, 6-methylheptyl group, 1-ethylhexyl group, 1,1-dimethylhexyl group, 2,2-dimethylhexyl group, 3,3-dimethylhexyl group, 4,4-dimethylhexyl group, 5,5-dimethylhexyl group, 1-methyl-4,4-dimethylpentyl group, 2-methyl-4,4-dimethylpentyl group, 3-methyl-4,4-dimethylpentyl group; n-nonyl group, 1-methyloctyl group, 2-methyloctyl group, 3-methyloctyl group, 4-methyloctyl group, 5-methyloctyl group, 6-methyloctyl group, 7-methyloctyl group, 1-ethylheptyl group, 1,1-dimethylheptyl group, 2,2-dimethylheptyl group, 3,3-dimethylheptyl group, 4,4-dimethylheptyl group, 5,5-dimethylheptyl group, 6,6-dimethylheptyl group, 1-methyl-5,5-dimethylhexyl group, 2-methyl-5,5-dimethylhexyl group, 3-methyl-5,5-dimethylhexyl group, 4-methyl-5,5-dimethylhexyl group; n-decyl group, 1-methylnonyl group, 2-methylnonyl group, 3-methylnonyl group, 4-methylnonyl group, 5-methylnonyl group, 6-methylnonyl group, 7-methylnonyl group, 8-methylnonyl group, 1-ethyloctyl group, 1,1-dimethyloctyl group, 2,2-dimethyloctyl group, 3,3-dimethyloctyl group, 4,4-dimethyloctyl group, 5,5-dimethyloctyl group, 6,6-dimethyloctyl group, 7,7-dimethyloctyl group, 1-methyl-6,6-dimethylheptyl group, 2-methyl-6,6-dimethylheptyl group, 3-methyl-6,6-dimethylheptyl group, 4-methyl-6,6-dimethylheptyl group, 5-methyl-6,6-dimethylheptyl group;

[0017] n-undecyl group, 1-methyldecyl group, 2-methyldecyl group, 3-methyldecyl group, 4-methyldecyl group, 5-methyldecyl group, 6-methyldecyl group, 7-methyldecyl group, 8-methyldecyl group, 9-methyldecyl group, 1-ethylnonyl group, 1,1-dimethylnonyl group, 2,2-dimethylnonyl group, 3,3-dimethylnonyl group, 4,4-dimethylnonyl group, 5,5-dimethylnonyl group, 6,6-dimethylnonyl group, 7,7-dimethylnonyl group, 8,8-dimethylnonyl group, 1-methyl-7,7-dimethyloctyl group, 2-methyl-7,7-dimethyloctyl group, 3-methyl-7,7-dimethyloctyl group, 4-methyl-7,7-dimethyloctyl group, 5-methyl-7,7-dimethyloctyl group, 6-methyl-7,7-dimethyloctyl group; n-dodecyl group, 1-methylundecyl group, 2-methylundecyl group, 3-methylundecyl group, 4-methylundecyl group, 5-methylundecyl group, 6-methylundecyl group, 7-methylundecyl group, 8-methylundecyl group, 9-methylundecyl group, 10-methylundecyl group, 1-ethyldecyl group, 1,1-dimethyldecyl group, 2,2-dimethyldecyl group, 3,3-dimethyldecyl group, 4,4-dimethyldecyl group, 5,5 -dimethyldecyl group, 6,6-dimethyldecyl group, 7,7-dimethyldecyl group, 8,8-dimethyldecyl group, 9,9-dimethyldecyl group, 1-methyl-8,8-dimethylnonyl group, 2-methyl-8,8-dimethylnonyl group, 3-methyl-8,8-dimethylnonyl group, 4-methyl-8,8-dimethylnonyl group, 5-methyl-8,8-dimethylnonyl group, 6-methyl-8,8-dimethylnonyl group, 7-methyl-8,8-dimethylnonyl group;

[0018] n-tridecyl group, 1-methyldodecyl group, 2-methyldodecyl group, 3-methyldodecyl group, 4-methyldodecyl group, 5-methyldodecyl group, 6-methyldodecyl group, 7-methyldodecyl group, 8-methyldodecyl group, 9-methyldodecyl group, 10-methyldodecyl group, 11-methyldodecyl group, 1-ethylundecyl group, 1,1-dimethylundecyl group, 2,2-dimethylundecyl group, 3,3-dimethylundecyl group, 4,4-dimethylundecyl group, 5,5-dimethylundecyl group, 6,6-di Methyl undecyl group, 7,7-dimethyl undecyl group, 8,8-dimethyl undecyl group, 9,9-dimethyl undecyl group, 10,10-dimethyl undecyl group, 1-methyl-9,9-dimethyldecyl group, 2-methyl-9,9-dimethyldecyl group, 3-methyl-9,9-dimethyldecyl group, 4-methyl-9,9-dimethyldecyl group, 5-methyl-9,9-dimethyldecyl group, 6-methyl-9,9-dimethyldecyl group, 7-methyl-9,9-dimethyldecyl group, 8-methyl-9,9-dimethyldecyl group; n-tetradecyl group, 1-methyltridecyl group, 2-methyltridecyl group, 3-methyltridecyl group, 4-methyltridecyl group, 5-methyltridecyl group, 6-methyltridecyl group, 7-methyltridecyl group, 8-methyltridecyl group, 9-methyltridecyl group, 10-methyltridecyl group, 11-methyltridecyl group, 12-methyltridecyl group, 1-ethyldodecyl group, 1,1-dimethyldodecyl group, 2,2-dimethyldodecyl group, 3,3-dimethyldodecyl group, 4,4-dimethyldodecyl group, 5,5-dimethyldodecyl group, 6,6-dimethyldodecyl group, 7,7-dimethyldodecyl group, 8,8-dimethyldodecyl group, 9,9-dimethyldodecyl group, 10,10-dimethyldodecyl group, 11,11-dimethyldodecyl group, 1-methyl-10,10-dimethylundecyl group, 2-methyl-10,10-dimethylundecyl group, 3-methyl-10,10-dimethylundecyl group, 4-methyl-10,10-dimethylundecyl group, 5-methyl-10,10-dimethylundecyl group, 6-methyl-10,10-dimethylundecyl group, 7-methyl-10,10-dimethylundecyl group, 8-methyl-10,10-dimethylundecyl group, 9-methyl-10,10-dimethylundecyl group;

[0019] n-pentadecyl group, 1-methyltetradecyl group, 2-methyltetradecyl group, 3-methyltetradecyl group, 4-methyltetradecyl group, 5-methyltetradecyl group, 6-methyltetradecyl group, 7-methyltetradecyl group, 8-methyltetradecyl group, 9-methyltetradecyl group, 10-methyltetradecyl group, 11-methyltetradecyl group, 12-methyltetradecyl group, 13-methyltetradecyl group, 1-ethyltridecyl group, 1,1-dimethyltridecyl group, 2,2-dimethyltridecyl group, 3,3-dimethyltridecyl group, 4,4-dimethyltridecyl group, 5,5-dimethyltridecyl group, 6,6-dimethyltridecyl group, 7,7-dimethyltridecyl group, 8,8- Examples include dimethyltridecyl group, 9,9-dimethyltridecyl group, 10,10-dimethyltridecyl group, 11,11-dimethyltridecyl group, 12,12-dimethyltridecyl group, 1-methyl-11,11-dimethyldodecyl group, 2-methyl-11,11-dimethyldodecyl group, 3-methyl-11,11-dimethyldodecyl group, 4-methyl-11,11-dimethyldodecyl group, 5-methyl-11,11-dimethyldodecyl group, 6-methyl-11,11-dimethyldodecyl group, 7-methyl-11,11-dimethyldodecyl group, 8-methyl-11,11-dimethyldodecyl group, 9-methyl-11,11-dimethyldodecyl group, and 10-methyl-11,11-dimethyldodecyl group.

[0020] In general formula (A), R １１ and R １２ Each of these is independently linear or branched C ２－１２ Preferably, it is an alkyl group (an alkyl group having 2 to 12 carbon atoms), and is linear or branched. ５－１２ It is more preferably an alkyl group (an alkyl group having 5 to 12 carbon atoms), and is linear or branched C ５－１０ It is more preferably an alkyl group (an alkyl group having 5 to 10 carbon atoms), and is linear or branched in shape. ６－９ It is most preferable that the alkyl group be an alkyl group having 6 to 9 carbon atoms. In addition, in the pH-sensitive cationic lipid of the present invention, R １ and R ２X represents any group represented by general formula (A), and may be the same group or different groups. In general formula (I), X represents a group represented by the following general formula (B) or a 5- to 7-membered non-aromatic heterocyclic group. The 5- to 7-membered non-aromatic heterocyclic group represented by X is bonded to a carbon atom by (O-CO)b-. In general formula (B), d represents an integer from 0 to 3. When d is 0, -(CH ２ )- means that there is no group and it is a single bond.

[0021] In general formula (B), R ３ and R ４ Each is independently C １－４ Alkyl alkyl groups (alkyl groups with 1 to 4 carbon atoms) or C ２－４ This indicates an alkenyl group (an alkenyl group with 1 to 4 carbon atoms). ３ and R ４ C, which is shown １－４ Alkyl alkyl group or C ２－４ The alkenyl group may have one or two hydrogen atoms substituted with phenyl groups. ３ and R ４ C １－４ Alkyl alkyl group or C ２－４ Any alkenyl group will suffice; they may be the same group or different groups. １－４ Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, and tert-butyl groups. ２－４ Examples of alkenyl groups include vinyl group, 1-propenyl group, 2-propenyl group, 1-methylvinyl group, 2-methyl-1-propenyl group, 1-butenyl group, 2-butenyl group, and 3-butenyl group. In general formula (B), R ３ and R ４ These may be bonded to each other to form a 5-7 member non-aromatic heterocycle. ３ and R ４ Examples of 5- to 7-membered non-aromatic heterorings formed by the bonding of these groups include 1-pyrrolidinyl groups, 1-piperidinyl groups, 1-morpholinyl groups, and 1-piperazinyl groups. ３ and R ４The 5- to 7-membered non-aromatic heterocycle formed by bonding with each other has one or two hydrogen atoms in the ring replaced by C １－４ alkyl group or C ２－４ alkenyl group may be substituted. When two hydrogen atoms in the ring are replaced by C １－４ alkyl group or C ２－４ alkenyl group, they may be substituted by the same group or different groups from each other. In general formula (I), when X is a 5- to 7-membered non-aromatic heterocyclic group, examples of the heteroatom contained in the heterocyclic group include a nitrogen atom, an oxygen atom, or a sulfur atom. The heteroatom constituting the heterocycle in the heterocyclic group may be one, or two or more identical or different heteroatoms may be present. The heterocycle in the heterocyclic group may be a saturated heterocycle, or may contain one or two or more double bonds, but the heterocycle does not become an aromatic ring.

[0022] As the pH-sensitive cationic lipid of the present invention, in general formula (I), R １ and R ２ are each independently, among general formula (A), R １１ and R １２ are each independently a linear or branched C ２－１２ alkyl group, c is 1, v is an integer of 6 to 10, a is an integer of 3 to 5, b is 1, X is a 5- to 7-membered non-aromatic heterocyclic group (however, the carbon atom in the heterocyclic group is bonded to (O-CO)b-), preferably a 1-pyrrolidinyl group, a 1-piperidinyl group, a 1-morpholinyl group, or a 1-piperazinyl group (bonded to (O-CO)b- by the carbon atom in the ring, and one hydrogen atom may be replaced by C １－４ alkyl group or C ２－４ alkenyl group).) and, in general formula (I), R １ and R ２ are each independently, among general formula (A), R １１ and R １２ are each independently a linear or branched C ２－１２It is an alkyl group, c is 1, v is an integer from 6 to 10, a is an integer from 3 to 5, b is 0, and among the general formula (B), X is such that d is 0, R ３ and R ４ are each independently C １－４ alkyl group or C ２－４ alkenyl group (the C ３ alkyl group or C ４ alkenyl group represented by R １－４ and R ２－４ may have one or two hydrogen atoms substituted with a phenyl group). Also, in the general formula (I), R １ and R ２ are each independently, among the general formula (A), R １１ and R １２ are each independently a linear or branched C ５－１２ alkyl group, c is 1, v is an integer from 6 to 10, a is an integer from 3 to 5, b is 1, X is a 5- to 7-member non-aromatic heterocyclic group (however, it is bonded to (O-CO)b- by a carbon atom in the heterocyclic group), preferably a 1-pyrrolidinyl group, a 1-piperidinyl group, a 1-morpholinyl group, or a 1-piperazinyl group (bonded to (O-CO)b- by a carbon atom in the ring, and one hydrogen atom may be substituted with a C １－４ alkyl group or C ２－４ alkenyl group).), or in the general formula (I), R １ and R ２ are each independently, among the general formula (A), R １１ and R １２ are each independently a linear or branched C ５－１２ alkyl group, c is 1, v is an integer from 6 to 10, a is an integer from 3 to 5, b is 0, and among the general formula (B), X is such that d is 0, R ３ and R ４ are each independently C １－４ alkyl group or C ２－４ alkenyl group (the C ３ alkyl group or C ４ alkenyl group represented by R １－４ and R ２－４Compounds in which the alkenyl group may have one or two hydrogen atoms substituted with phenyl groups are preferred. In particular, as the pH-sensitive cationic lipid of the present invention, R in general formula (I) １ and R ２ Each of them independently represents R in the general formula (A). １１ and R １２ Each of these is independently a linear or branched C ６－９ It is an alkyl group in which c is 1, v is an integer from 6 to 10, a is an integer from 3 to 5, b is 1, and X is a 1-pyrrolidinyl group, a 1-piperidinyl group, a 1-morpholinyl group, or a 1-piperazinyl group (bonded to (O-CO)b- by a carbon atom in the ring, with one hydrogen atom being C １－４ Alkyl or C ２－４ Compounds that may be substituted with an alkenyl group, or in general formula (I), R １ and R ２ Each of them independently represents R in the general formula (A). １１ and R １２ Each of these is independently a linear or branched C ６－９ It is an alkyl group where c is 1, v is an integer from 6 to 10, a is an integer from 3 to 5, b is 0, and X is the general formula (B) where d is 0, R ３ and R ４ Each of them independently C １－４ Compounds that are alkyl groups are preferred.

[0023] The pH-sensitive cationic lipid of the present invention is more preferably R in general formula (I). １ and R ２ Each of them independently represents R in the general formula (A). １１ and R １２ Each of them is independently a linear C ６－９ It is an alkyl group in which c is 1, v is an integer from 6 to 10, a is an integer from 3 to 5, b is 1, and X is a 1-pyrrolidinyl group, a 1-piperidinyl group, a 1-morpholinyl group, or a 1-piperazinyl group (bonded to (O-CO)b- by a carbon atom in the ring, with one hydrogen atom being C １－４ Alkyl or C２－４ Compounds that may be substituted with an alkenyl group; in general formula (I), R １ and R ２ Each of them independently represents R in the general formula (A). １１ and R １２ Each of these is independently branched in a chain-like manner. ６－９ It is an alkyl group in which c is 1, v is an integer from 6 to 10, a is an integer from 3 to 5, b is 1, and X is a 1-pyrrolidinyl group, a 1-piperidinyl group, a 1-morpholinyl group, or a 1-piperazinyl group (bonded to (O-CO)b- by a carbon atom in the ring, with one hydrogen atom being C １－４ Alkyl or C ２－４ Compounds that may be substituted with an alkenyl group; in general formula (I), R １ and R ２ Each of them independently represents R in the general formula (A). １１ and R １２ Each of them is independently a linear C ６－９ It is an alkyl group where c is 1, v is an integer from 6 to 10, a is an integer from 3 to 5, b is 0, and X is the general formula (B) where d is 0, R ３ and R ４ Each of them independently C １－４ Compounds that are alkyl groups; in general formula (I), R １ and R ２ Each of them independently represents R in the general formula (A). １１ and R １２ Each of these is independently branched in a chain-like manner. ６－９ It is an alkyl group where c is 1, v is an integer from 6 to 10, a is an integer from 3 to 5, b is 0, and X is the general formula (B) where d is 0, R ３ and R ４ Each of them independently C １－４ A compound that is an alkyl group;

[0024] The pH-sensitive cationic lipid of the present invention is R in general formula (I). １ and R ２ The same group, and of the general formula (A), R １１ and R１２ Each of them is independently a linear C ６－９ It is an alkyl group in which c is 1, v is an integer from 6 to 10, a is an integer from 3 to 5, b is 1, and X is a 1-pyrrolidinyl group, a 1-piperidinyl group, a 1-morpholinyl group, or a 1-piperazinyl group (bonded to (O-CO)b- by a carbon atom in the ring, with one hydrogen atom being C １－４ Alkyl or C ２－４ Compounds that may be substituted with an alkenyl group; in general formula (I), R １ and R ２ The same group, and of the general formula (A), R １１ and R １２ Each of these is independently branched in a chain-like manner. ６－９ It is an alkyl group in which c is 1, v is an integer from 6 to 10, a is an integer from 3 to 5, b is 1, and X is a 1-pyrrolidinyl group, a 1-piperidinyl group, a 1-morpholinyl group, or a 1-piperazinyl group (bonded to (O-CO)b- by a carbon atom in the ring, with one hydrogen atom being C １－４ Alkyl or C ２－４ Compounds that may be substituted with an alkenyl group; in general formula (I), R １ and R ２ The same group, and of the general formula (A), R １１ and R １２ Each of them is independently a linear C ６－９ It is an alkyl group where c is 1, v is an integer from 6 to 10, a is an integer from 3 to 5, b is 0, and X is the general formula (B) where d is 0, R ３ and R ４ Each of them independently C １－４ Compounds that are alkyl groups; in general formula (I), R １ and R ２ The same group, and of the general formula (A), R １１ and R １２ Each of these is independently branched in a chain-like manner. ６－９ It is an alkyl group where c is 1, v is an integer from 6 to 10, a is an integer from 3 to 5, b is 0, and X is the general formula (B) where d is 0, R３ and R ４ Each of them independently C １－４ Compounds that are alkyl groups are particularly preferred.

[0025] As used herein, the term “pharmaceutically acceptable” means suitable for use in a subject, such as a mammal, including humans.

[0026] The pharmaceutically acceptable salts of the present disclosure include, but are not limited to, salts containing chlorides, bromides, fluorides, iodides, nitrates, sulfates, methylsulfates, phosphates, acetates, benzoates, citrates, glutamates, and / or lactates. The pharmaceutically acceptable salts of the compounds of general formula (I) of the present disclosure can be synthesized by conventional chemical methods. For example, the pharmaceutically acceptable salts of the compounds of general formula (I) of the present disclosure can be prepared by ion-exchange chromatography or by reacting a free base in the compound with a stoichiometric or excess amount of a desired salt-forming inorganic or organic acid in a suitable solvent or a variety of solvent combinations. Generally, the compounds of general formula (I) of the present disclosure may contain one or more chiral centers. Compounds containing one or more chiral centers may include those described as "isomers," "stereoisomers," "diastereomers," "enantiomers," "optical isomers," or "racemic mixtures."

[0027] The pH-sensitive cationic lipid of the present invention is disclosed in International Publication No. 2022 / 071582 (Patent Document 1), and can be produced according to the method described in said document. All of the disclosures in said document are incorporated herein by reference.

[0028] The pH-sensitive cationic lipid constituting the lipid nanoparticles according to the present invention may be one type or two or more types. When there are two or more pH-sensitive cationic lipids constituting the lipid nanoparticles according to the present invention, the amount of the pH-sensitive cationic lipids according to the present invention means the total amount of lipid molecules corresponding to the pH-sensitive cationic lipids according to the present invention among the lipid molecules constituting the lipid nanoparticles.

[0029] 3. Target Binding Sites 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 may be an antibody, Fab', F(ab') ２This includes 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. 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, a cell surface receptor, an enzyme active site, or an intracellularly localized molecule. 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. In this disclosure, target cells are, for example, immune cells or hematopoietic stem cells. In this disclosure, target cells are preferably immune cells. In some embodiments of this disclosure, immune cells that are target cells include T cells, NK cells, monocytes, neutrophils, macrophages, and dendritic cells. In this disclosure, immune cells that are target cells are preferably T cells or NK cells. In this disclosure, immune cells that are target cells are most preferably T cells. In some embodiments of this disclosure, antigens are, for example, T cell antigens or hematopoietic stem cell antigens. In preferred embodiments of this disclosure, antigens are CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD90, or CD117. In this disclosure, the term “antibody” is used in its broadest sense and includes monoclonal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen-binding fragments of sufficient length to exhibit desired binding / recognition activity.An "antigen-binding fragment" is composed of a portion of a complete antibody, typically containing the antigen-binding site of the complete antibody and thus retaining the ability to bind to an antigen. In some embodiments, the antigen-binding fragment includes, but is not limited to, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an scFv, or a VHH antibody. Examples of antibodies or antigen-binding fragments include all or part of polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, bispecific antibodies, minibodies, and linear antibodies. In a preferred embodiment of the present invention, the target binding site is a single-chain antibody (e.g., scFv or VHH). In a more preferred embodiment of the present invention, from the viewpoint of manufacturing reproducibility and / or scale-up, the target binding site is scFv or VHH.

[0030] In one embodiment of the present invention, the antibody has at least one, preferably all, CDR sequences selected from the group consisting of DYAIG (SEQ ID NO: 3), CIRIFDRHTYSADSVKG (SEQ ID NO: 4), and GSFWACTRPEGAMDY (SEQ ID NO: 5).

[0031] In one embodiment of the present invention, the antibody has at least one, preferably all, CDR sequences selected from the group consisting of GSTFSDYG (SEQ ID NO: 6), IDWNGEHT (SEQ ID NO: 7), and AADALPYTVRKYNY (SEQ ID NO: 8).

[0032] In one embodiment of the present invention, the antibody has at least one, preferably all, CDR sequences selected from the group consisting of DFGMN (SEQ ID NO: 9), LIYYDGSNKFYADSVKG (SEQ ID NO: 10), PHYDGYYHFFDS (SEQ ID NO: 11), KGSQDINNYLA (SEQ ID NO: 12), NTDILHT (SEQ ID NO: 13), and YQYNNGYT (SEQ ID NO: 14).

[0033] In one embodiment of the present invention, the antibody has at least one, preferably all, CDR sequences selected from the group consisting of GFIFSNYG (SEQ ID NO: 15), IWYDGSNK (SEQ ID NO: 16), ARSYDMLTGSGDYYGL (SEQ ID NO: 17), QDITNY (SEQ ID NO: 18), GAS (SEQ ID NO: 19), and QQYNNYPLT (SEQ ID NO: 20).

[0034] In one embodiment of the present invention, the antibody has at least one, preferably all, CDR sequences selected from the group consisting of GYTFTNYG (SEQ ID NO: 21), INTHTGEP (SEQ ID NO: 22), TRRGYDWYFDV (SEQ ID NO: 23), QDINSY (SEQ ID NO: 24), RAN (SEQ ID NO: 25), and QQYDESPWT (SEQ ID NO: 26).

[0035] In one embodiment of the present invention, the antibody has at least one, preferably all, CDR sequences selected from the group consisting of GDTICISA (SEQ ID NO: 27), ITSGGST (SEQ ID NO: 28), and NADIAGHNCSGYLKEY (SEQ ID NO: 29).

[0036] In one embodiment of the present invention, the antibody has at least one, preferably all, CDR sequences selected from the group consisting of GFTFNKYA (SEQ ID NO: 30), IRSKYNNYAT (SEQ ID NO: 31), VRHGNFGNSYISYWAY (SEQ ID NO: 32), TGAVTSGNY (SEQ ID NO: 33), GTK (SEQ ID NO: 34), and VLWYSNRWV (SEQ ID NO: 35).

[0037] In one embodiment of the present invention, the antibody has at least one sequence selected from the group consisting of GYTFTSYV (SEQ ID NO: 36), INPYNDGT (SEQ ID NO: 37), AREKDNYATGAWFAY (SEQ ID NO: 38), QSLLYSTNQKNY (SEQ ID NO: 39), WAS (SEQ ID NO: 40), and QQYYSYRT (SEQ ID NO: 41).

[0038] In one embodiment of the present invention, the antibody has at least one, preferably all, CDR sequences selected from the group consisting of GFTFSTFP (SEQ ID NO: 42), LSPSGDST (SEQ ID NO: 43), TKVGFTTFYFDS (SEQ ID NO: 44), QNINKY (SEQ ID NO: 45), NIN (SEQ ID NO: 46), and LQHRTGWT (SEQ ID NO: 47).

[0039] In one embodiment of the present invention, the antibody is antibody 1 represented by the following sequence. The underlined portion indicates the CDR sequence. EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGKGREGVLCIRIFDRHTYSADSVKGRFTISSDNAQNTVYLHMNSLKPEDTAVYYCAAGSFWACTRPEGAMDYWGKGTQVTVSS (Sequence ID 48)

[0040] In one embodiment of the present invention, the antibody is antibody 2 represented by the following sequence. The underlined portion indicates the CDR sequence. EVQLVESGGGLVQAGGSLRLSCAASGSTFSDYGVGWFRQAPGKGREFVADIDWNGEHTSYADSVKGRFATSRDNAKNTAYLQMNSLKPEDTAVYYCAADALPYTVRKYNYWGQGTQVTVSS (Sequence ID 49)

[0041] In one embodiment of the present invention, the antibody is antibody 3 represented by the following sequence. The underlined portion indicates the CDR sequence. QVQLVESGGGVVQPGRSLRLSCAASGFTFSDFGMNWVRQAPGKGLEWVALIYYDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPHYDGYYHFFDSWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKGSQDINNYLAWYQQKPGKAPKLLIYNTDILHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCYQYNNGYTFGQGTKVEIK (Sequence ID 50)

[0042] In one embodiment of the present invention, the antibody is antibody 4 represented by the following sequence. The underlined portion indicates the CDR sequence. QVQLVESGGGVVQPGRSLRLSCAASGFTFSDFGMNWVRQAPGKGLEWVALIYYDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPHYDGYYHFFDSWGQGTLVTVSSSPNSASHSGSAPQTSSAPGSQDIQMTQSPSSLSASVGDRVTITCKGSQDINNYLAWYQQKPGKAPKLLIYNTDILHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCYQYNNGYTFGQGTKVEIK (Sequence ID 51)

[0043] In one embodiment of the present invention, the antibody is antibody 5 represented by the following sequence. The underlined portion indicates the CDR sequence. QVQLVESGGGVVQPGRSLRLSCAASGFTFSDFGMNWVRQAPGKGLEWVALIYYDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPHYDGYYHFFDSWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKGSQDINNYLAWYQQKPGKAPKLLIYNTDILHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCYQYNNGYTFGQGTKVEIK (Sequence ID 52)

[0044] In one embodiment of the present invention, the antibody is antibody 6 represented by the following sequence. The underlined portion indicates the CDR sequence. QVQLVESGGGVVQPGRSLRLSCAASGFTFSDFGMNWVRQAPGKCLEWVALIYYDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPHYDGYYHFFDSWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKGSQDINNYLAWYQQKPGKAPKLLIYNTDILHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCYQYNNGYTFGCGTKVEIK (Sequence ID 53)

[0045] In one embodiment of the present invention, the antibody is antibody 7 represented by the following sequence. The underlined portion indicates the CDR sequence. QVQLVESGGGVDQPGRSLRLSCAASGFIFSNYGIHWVRQAPGKGLEWVAVIWYDGSNKYFEDSVKGRFNISRDNSKNIVYLQMNSLRAEDTAVYFCARSYDMLTGSGDYYGLDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDITNYLAWFQQKPGKAPKSLIYGASSLQSGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPLTFGGGTKVEIK (Sequence ID 54)

[0046] In one embodiment of the present invention, the antibody is antibody 8 represented by the following sequence. The underlined portion indicates the CDR sequence. NIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCTRRGYDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFHHKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDMGIYYCQQYDESPWTFGGGTKLEMK (Sequence ID 55)

[0047] In one embodiment of the present invention, the antibody is antibody 9 represented by the following sequence. The underlined portion indicates the CDR sequence. EVQVVESGGGLVQAGGSLRLSCAASGDTICISAMYWYRQAPGKAREMVAAITSGGSTYYEDSVMGRFTIFRENAKNTVYLQMNSLKPEDTAVYYCNADIAGHNCSGYLKEYWGQGTQVTVSA (Sequence ID 56)

[0048] In one embodiment of the present invention, the antibody is antibody 10 represented by the following sequence. The underlined portion indicates the CDR sequence. EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGQPKS (Sequence ID 57)

[0049] In one embodiment of the present invention, the antibody is antibody 11 represented by the following sequence. The underlined portion indicates the CDR sequence. QVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIK (Sequence ID 58)

[0050] In one embodiment of the present invention, the antibody is antibody 12 represented by the following sequence. The underlined portion indicates the CDR sequence. EVYLVESGGGLVQPGGSVKLSCSASGFTFSTFPMAWVRQAPTQGLQWVATLSPSGDSTYYRDSVKGRFTISRDNVLNTLYLHMDILRSEDTATYYCTKVGFTTFYFDSWGQGVMVAVSSAGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTINCKASQNINKYLDWYQQKFGETPKLLIYNINNLHSGVPSRFSGSGSGPDFTLTISSLQPEDVATYFCLQHRTGWTFGGGTKVELRRA (Sequence ID 59)

[0051] In a preferred embodiment of the present invention, the target binding site is a single-domain antibody. In a preferred embodiment of the present invention, the single-domain antibody is a VHH antibody or a VNAR antibody. In a certain embodiment of the present invention, the target binding site is an artificial polypeptide. In a certain embodiment of the present invention, the artificial polypeptide is DARPin, adonectin, ancarin, and an aphibody. In this disclosure, “VHH antibody” (variable domain of heavy chain of heavy chain antibody) is a domain containing a variable region of a heavy chain antibody without a light chain, found in the serum of artiodactyla camelids (such as Bactrian camels, dromedary camels, llamas, and alpacas). VHH antibodies are known as the smallest unit of immunoglobulin fragments capable of binding to an antigen. In a preferred embodiment of the present invention, the target binding site binds to an antigen on a T cell. T cells (T lymphocytes) are the main cells of the immune system, for example, produced in the bone marrow and matured in the thymus. T cells may recognize antigens presented by antigen-presenting cells (APCs) via T cell receptors (TCRs) and induce a specific immune response. T cells have subtypes such as cytotoxic T cells (CD8-positive T cells), helper T cells (CD4-positive T cells), and regulatory T cells (Treg). Cytotoxic T cells may directly attack and destroy infected cells or tumor cells. Helper T cells may activate other immune cells and modulate the immune response. Helper T cells can activate B cells to promote antibody production and assist in infection control. Regulatory T cells (Treg) can suppress excessive immune responses and contribute to the prevention of autoimmune diseases. T cells may be used in cancer immunotherapy, such as CAR-T cell therapy. In CAR-T cell therapy, patient-derived T cells are genetically modified to express CARs (chimeric antigen receptors) that target specific antigens, and then reinjected into the patient to attack cells with specific antigens (ex-vivo CAR-T therapy). Furthermore, by genetically modifying a patient's T cells within their body and making them express CARs that target specific antigens, it is possible to attack cells possessing those specific antigens (in-vivo CAR-T therapy).

[0052] In a preferred embodiment of the present invention, the target binding site binds to CD2, CD3, CD4, CD5, CD6, CD7, or CD8. CD2, CD3, CD4, CD5, CD6, CD7, and CD8 are membrane proteins expressed on the surface of immune cells, and may be used as markers indicating the function or characteristics of specific immune cells. CD2, CD3, CD4, CD5, CD6, CD7, and CD8 play important roles in the regulation or signal transduction of immune responses. CD2 may be expressed on T cells and innate lymphoid cells, for example, and may be involved in cell adhesion and assisting T cell activation. CD3 may constitute part of the T cell receptor (TCR) complex and be responsible for signal transduction after antigen recognition. CD4 may be specifically expressed on helper T cells and may be an auxiliary molecule necessary for antigen recognition from antigen-presenting cells via MHC class II molecules. CD4 may be used in the evaluation of immune function and in cell fractionation in cell therapy. CD5 may be mainly expressed on T cells and some B cells and may contribute to the suppression of autoreactivity by regulating TCR signaling. CD6 may interact with CD5 and play a role in regulating T cell activation and immune synapse formation. CD7 may be expressed on T cells or NK cells and be involved in cell development and differentiation, as well as assisting in intercellular interactions. CD8 may be specifically expressed on cytotoxic T cells and function to recognize endogenous antigens (e.g., virus-derived antigens) via MHC class I molecules.

[0053] In a preferred embodiment of the present invention, the target binding site relates to lipid nanoparticles that bind to an antigen on hematopoietic stem cells (HSCs). Hematopoietic stem cells refer to pluripotent stem cells that can differentiate into any cell of the blood and immune system, such as red blood cells, white blood cells (lymphocytes, neutrophils, etc.), and platelets. In a preferred embodiment of the present invention, the target binding site binds to CD90 or CD117. CD90 (Thy-1) and CD117 (c-Kit) are membrane proteins expressed on the surface of cells and may be used as markers to identify specific cell populations. CD90 is expressed, for example, in stem cells, nerve cells, and fibroblasts and may be involved in intercellular interactions and signal transduction. CD117 is expressed, for example, in hematopoietic stem cells or mast cells and may regulate cell proliferation and differentiation through binding to stem cell factors (SCFs). In one embodiment, the target binding site binds to an antigen on a T cell, for example, one of CD2, CD3, CD4, CD5, CD6, CD7, or CD8, preferably CD5 or CD8, and more preferably CD8. In one embodiment, the target binding site is an antibody, Fab fragment, Fab' fragment, F(ab')2 fragment, scFv, or VHH antibody that binds to one of CD2, CD3, CD4, CD5, CD6, CD7, or CD8. In one embodiment, the target binding site binds to an antigen on a hematopoietic stem cell (HSC), for example, CD90 or CD117. In one embodiment, the target binding site is an antibody, Fab fragment, Fab' fragment, F(ab')2 fragment, scFv, or VHH antibody that binds to CD90 or CD117.

[0054] In one embodiment, when the target molecule is CD8, the target binding site is a VHH antibody represented by SEQ ID NO: 92 (Antibody 1) or SEQ ID NO: 93 (Antibody 2).

[0055] 4. Polyalkylene glycol-modified lipids: Polyalkylene glycols are hydrophilic polymers, and by using polyalkylene glycol-modified lipids as lipid membrane constituents to construct lipid nanoparticles, the surface of the lipid nanoparticles can be modified with polyalkylene glycol. Surface modification with polyalkylene glycol may improve the stability of lipid nanoparticles, such as their retention in the bloodstream.

[0056] Examples of polyalkylene glycols 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.

[0057] 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 glycolated lipids are not limited to these.

[0058] The lipid nanoparticles of this disclosure include targeted polyalkylene glycol-modified lipids as constituent lipids. In this disclosure, “targeted polyalkylene glycol-modified lipid” means a polyalkylene glycol-modified lipid to which a target binding site is linked. In one embodiment, the target binding site is linked to the polyalkylene glycol, preferably via a linker to the polyalkylene glycol-modified lipid. In this disclosure, “linking” means linking by covalent bond. The ratio of targeted polyalkylene glycol-modified lipid to the total amount of lipids constituting the lipid nanoparticles according to the present invention is not particularly limited as long as it does not impair the gene expression activity when the lipid nanoparticles according to the present invention are used as a gene carrier. 5. Linker In a preferred embodiment of the present invention, the target binding site is linked to the polyalkylene glycol of the targeted polyalkylene glycol-modified lipid via a linker. In a certain embodiment of the present invention, the linker refers to a group that links the target binding site and the polyalkylene glycol. The linker includes, for example, a peptide. The linker comprises or consists of peptides of, for example, 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:

[0059] 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].

[0060] 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].

[0061] In a preferred embodiment of the present invention, the linker is represented as -Z1-Z2-, where Z1 is linked to the target binding site, 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 target binding site, 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.]

[0062] In a preferred embodiment of the present invention, the linker is represented as -Z1-Z2-, where Z1 is linked to the target site, 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].

[0063] In a preferred embodiment of the present invention, the linker is represented as -Z1-Z2-, where Z1 is linked to the target binding site, 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 linkage to the adjacent Cys side chain SH group, the right arrow indicates linkage to polyalkylene glycol, and X is N or O]. 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.

[0064] 6. Further Lipids Among the constituent lipids of the lipid nanoparticles according to the present invention, lipids other than cationic lipids and targeted polyalkylene glycol-modified lipids can be those commonly used when forming liposomes. Examples of such lipids include phospholipids, sterols or sterol derivatives, glycolipids, or saturated or unsaturated fatty acids. These can be used individually or in combination of two or more.

[0065] Examples of phospholipids include glycerophospholipids such as phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, phospharidylcholine, 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.

[0066] 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).

[0067] Examples of sterols or sterol derivatives 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 saturated or unsaturated fatty acids with 12 to 20 carbon atoms such as palmitic acid, oleic acid, stearic acid, arachidonic acid, and myristic acid.

[0068] The constituent lipids of the lipid nanoparticles according to the present invention preferably include neutral lipids in addition to cationic lipids and targeted polyalkylene glycol-modified lipids, more preferably phospholipids or sterols, even more preferably sterols, and even more preferably cholesterol.

[0069] 7. Size of Lipid Nanoparticles The size of the lipid nanoparticles according to the present invention is preferably such that the average particle diameter is 400 nm or less, more preferably 300 nm or less, even more preferably 200 nm or less, and even more preferably 150 nm or less, in order to obtain high delivery efficiency to cells in living organisms. The average particle diameter of the lipid nanoparticles refers to the Z-mean particle diameter measured by dynamic light scattering (DLS). Measurement by dynamic light scattering can be performed by a conventional method using a commercially available DLS device, etc.

[0070] The morphology of the lipid nanoparticles according to the present invention 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.

[0071] 8. Non-targeted polyalkylene glycol modified lipids In one embodiment, the present invention relates to lipid nanoparticles further comprising non-targeted polyalkylene glycol modified lipids in which a target binding site is not linked. In this disclosure, "non-targeted polyalkylene glycol modified lipid" means a polyalkylene glycol modified lipid in which a target binding site is not linked. In one embodiment, the non-targeted polyalkylene glycol modified lipid includes a polyalkylene glycol modification in which the polyalkylene glycol is modified with a reactive group. In one embodiment, the non-targeted polyalkylene glycol modified lipid includes a polyalkylene glycol modified lipid in which a non-targeting site is linked to a polyalkylene glycol site. Although not particularly limited, lipids formed by the reaction of a polyalkylene glycol modification in which the polyalkylene glycol is modified with a reactive group (e.g., maleimide) with a non-targeting molecule (e.g., L-Cysteine ​​when maleimide is the reactive group) are included in polyalkylene glycol modified lipids in which a non-targeting site is linked to a polyalkylene glycol site. In one embodiment, the untargeted polyalkylene glycol-modified lipid includes a polyalkylene glycol-modified lipid in which the polyalkylene glycol is unmodified. 9. Reactive Groups In a preferred embodiment of the present invention, the untargeted polyalkylene glycol-modified lipid includes 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. Reactive groups collectively refer to groups that are 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 between a thiol group and maleimide. 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. In one embodiment of the present invention, the polyalkylene glycol-modified lipid, in which polyalkylene glycol is modified with a reactive group, includes DSPE-PEG. In one embodiment, the polyalkylene glycol-modified lipid, in which polyalkylene glycol is modified with a reactive group, is DSPE-PEG, in which polyethylene glycol is modified with maleimide. In one embodiment of the present invention, the polyalkylene glycol-modified lipid, in which polyalkylene glycol is unmodified, is DMG-PEG.

[0072] 10. Animals to be administered The animals to which the lipid nanoparticles according to the present invention 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. 11. Manufacturing method In one embodiment, the lipid nanoparticles of the present disclosure containing a target binding site are manufactured by a method comprising the steps of manufacturing untargeted lipid nanoparticles that do not contain a target binding site encapsulating nucleic acids, and linking the target binding site to the untargeted lipid nanoparticles. In another embodiment, the lipid nanoparticles of the present disclosure containing a target binding site are manufactured by a method comprising the steps of manufacturing untargeted lipid nanoparticles that do not contain a target binding site encapsulating nucleic acids, and introducing a targeted polyalkylene glycol-modified lipid linked to a target binding site into the untargeted lipid nanoparticles. The above methods for manufacturing untargeted lipid nanoparticles are 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 an alcohol dilution method using a channel. This method involves introducing a solution in which a lipid component is dissolved in an alcohol solvent and a solution in which a water-soluble component to be encapsulated in the lipid nanoparticles is dissolved in an aqueous solvent from separate channels and then combining them to produce lipid nanoparticles. Examples of aqueous solvents used in the alcohol dilution method include buffer solutions such as phosphate buffer, citrate buffer, and phosphate-buffered saline, as well as physiological saline and cell culture media. The above-mentioned untargeted lipid nanoparticles may also be prepared by suspending the lipid nanoparticles in an aqueous solution.

[0073] In another embodiment, lipid nanoparticles of the present disclosure containing a target binding site are produced by a method comprising the step of mixing nucleic acids, cationic lipids, and targeted polyalkylene glycol-modified lipids. The method for producing lipid nanoparticles according to the present invention is not particularly limited, and any method available to those skilled in the art can be employed. For example, 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. Liposomes can also be produced by methods well known for producing liposomes, such as reverse-phase evaporation. If it is desired to control the size of the lipid nanoparticles, extrusion (extrusion filtration) can be performed under high pressure using a membrane filter with uniform pore sizes.

[0074] 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.

[0075] When freeze-drying or spray-drying an aqueous dispersion of lipid nanoparticles according to the present invention, stability may be improved by using sugars (aqueous solutions) such as monosaccharides including glucose, galactose, mannose, fructose, inositol, ribose, and xylose; 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 above 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 of the present invention, the lipid nanoparticles according to the present invention are freeze-dried.

[0076] In one aspect, the present invention relates to a pharmaceutical formulation containing lipid nanoparticles according to the present invention. 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.

[0077] In the examples, the following lipids (P) to (X) were used as cationic lipids. Lipid (P) was ALC-0315, purchased from MedChemExpress (product number #HY-13817). The other lipids were those disclosed in International Publication No. 2022 / 071582 (Patent Document 1), and were manufactured according to the method described in that document.

[0078] 1. General Procedure 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). 1-2. Transient antibody expression: 30 mL of ExpiCHO Expression Medium (#A2910001, Thermo Fisher Scientific) and 1 mL of ExpiCHO-S cells thawed at 37°C (1 x 10⁶). 7 Cells / 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 6The 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. 6The 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. Then, 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. 1–3. For IMAC purification of antibodies, 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 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 antibodies bound to the column were 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.1-4. Affinity Purification of Antibodies with Protein A A 10 mL 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 Protein A resin suspension (Bipo Resin Protein A, #AAR-025, Protein Express Co., Ltd.), and the column 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 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) compared to 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) compared to 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 antibody 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. 1-5. For antibody SEC purification, an SEC column (Superdex75 Increase 10 / 300 GL, #29148721, Cytiva) was set up 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 a sample purified with HisTrap column or Protein A resin was injected, 24 mL of PBS was delivered, and each fraction was collected. The target fraction was identified by SDS-PAGE. The obtained sample was transferred to an ultrafiltration filter unit (Vivaspin Turbo 15 3,000 MWCO), concentrated by centrifugation at 4,000 × g, 4°C until the antibody concentration reached approximately 3–5 mg / mL, and the concentration was determined by BCA assay and stored at -80°C. 1–6. Preparation of GGGYPYDVPDYAK-(PEG)4-S-Acetyl peptide The GGGYPYDVPDYAK peptide was synthesized in solid phase 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 primary amino group of the deprotected lysine side chain on a solid phase to synthesize GGGYPYDVPDYAK-(PEG)4-S-Acetyl. The resulting peptide from On resin was washed with DCM, and then cleaved and deprotected using a TFA / water / TIS / DODT (92.5 / 2.5 / 2.5 / 2.5) cleavage cocktail. After deprotection, the resulting crude peptide was recovered, 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 more). 1-7. Pretreatment of antibodies for conjugate (in the case of antibody-Sortase linker adducts) The GGGYPYDVPDYAK-(PEG)4-S-Acetyl peptide was added to the purified antibody-Sortase linker adduct by a 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. These were diluted in DDW, prepared at 1 mL / tube, and incubated at 37°C for 16 hours. Subsequently, His-tagged purification of the Sortase reaction mixture was performed. Specifically, the same volume of HisPur Cobalt Resin (Thermo Fisher Scientific, #89965) as the reaction mixture to be purified was added to Pierce Centrifuge Columns (10 mL) (Thermo Fisher Scientific, #89898) set in a Protein LoBind 50 mL tube, centrifuged at 700 × g, 4°C for 1 minute, and the filtrate was discarded. Add 2.5 times the amount of PBS(-) to the reaction mixture and centrifuge at 700×g, 4°C, for 1 minute. This equilibration procedure was repeated a total of two times. Add the Sortase A reaction mixture to the column and centrifuge at 700×g, 4°C, for 1 minute. Add the filtrate back to the same column and centrifuge again at 700×g, 4°C, for 1 minute, then collect the filtrate. Furthermore, wash the column three times with 0.5 times the amount of PBS(-) to the reaction mixture and collect the filtrate. The obtained filtrate was centrifuged at 4000g, 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. Furthermore, the Elution fraction can be obtained by adding 0.5 times the amount of Elution Buffer (50 mM phosphate buffer, pH 7.4, 300 mM NaCl, 500 mM imidazole) to the reaction mixture, letting it stand for 2 minutes, and then centrifugating at 700×g, 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 purified sample 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℃ in Vivaspin Turbo 15 (3,000 MWCO), the concentration was determined by BCA assay, diluted to 2 mg / mL in PBS, and stored at -80℃. 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 hydroxyamine, 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-conjugate lipid nanoparticles used in Examples 1-8 below. 1-8. Pretreatment of Conjugate Antibodies (In the case of 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 0.5 M TCEP solution (Fujifilm Wako Pure Chemical Industries, Ltd., 207-20151) was added to 25 mM EDTA buffer to prepare a reduced solution with a final TCEP concentration of 10 mM. 15 μL of reducing 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 twice replaced with EDTA 25 mM buffer, and centrifuged at 1500 × g, 20°C for 4 minutes. 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 EDTA 25 mM buffer to prepare an SH-free antibody solution with a protein concentration of 0.6 mg / mL.1-9. Lipid solutions were prepared by dissolving a lipid mixture (cationic lipids: Cholesterol: DSPC: DMG-PEG2000: DSPE-PEG(2000)-maleimide = 50:38.5:10:0.75:0.75, molar ratio) in 100% EtOH (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 16–24 hours using a SpectraPor® 4 Dialysis Membrane (12–14 kDa) (REPLIGEN, 132703). Subsequently, the solution was concentrated by ultrafiltration using Amicon Ultra-15 100 kDa (Merck Millipore, UFC910024) and sterilized using a sterile syringe filter (φ13 mm / 0.2 μm) (Pall Corporation, 4602). 1–10. Conjugation of untargeted LNPs with antibodies: SH-free antibody was 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. Then, the antibody-conjugated LNPs were added to Spectra / Por® Float-A-Lyzer® G2 (MWCO: 1000kDa) (REPLIGEN, G235037) and dialyzed three times in 9% sucrose, 20 mM HEPES buffer at 4°C for 2 to 12 hours. The antibody-conjugated LNPs were sterilized using a sterile syringe filter (φ13mm / 0.2μm) (Pall Corporation, 4602) and stored at -80°C. 1-11. Measurement of the physicochemical properties of antibody-conjugated LNPs The average particle size and PDI of the 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(-).1-12. 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-triton 100 (Sigma-Aldrich, T8787-50ML). 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 1-13. 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, and a calibration curve from 0 to 1000 μg / mL was prepared at 25 μL / well. In addition, 25 μL / well of 100 μg mRNA / mL LNP was added. Solutions A and B included in the kit 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, and the protein concentration was quantified. 1-14. Analysis of antibody-lipid conjugates 2x Laemmli Sample Buffer (Bio-Rad, (#161-0737) 200 μL of 1M DTT solution (Sigma, 43816) was added to 1000 μL of solution to obtain Sample Buffer. An equal volume of Sample Buffer was added to LNPs with a concentration of 100 μg mRNA / mL, and the mixture was 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 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.2. General Procedure for Protein Expression Evaluation The nucleic acid delivery efficiency of antibody-conjugated lipid nanoparticles was determined as the amount of protein expressed after mRNA encapsulated in lipid nanoparticles was introduced into target cells. (1) 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 T cells was RPMI-1640 containing 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 seeded at 0.1 × 10⁶ cells / 100 μL / well, treated with the formulation, and cultured in a CO₂ incubator for 24 hours. After 24 hours, the cells were harvested and the antigens on the cell surface 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). (2) In vivo evaluation of protein expression Human peripheral blood mononuclear cells (Lonza) were intravenously transferred at a rate of 2 × 10⁷ cells / head to NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ), which are severely immunodeficient mice. Approximately two weeks after transplantation of PBMCs, each formulation was administered intravenously. The spleen was removed 24 hours after administration of the formulation. After spleen fragmentation and hemolysis, a cell suspension was prepared. Dead cells were stained with Fixable Vibility Stain 575V (BD Horizon®), and after Fc blocking, cells were labeled with various antibodies (see antibody list). Intracellular mcherry expression was measured using FACSymphony™ A1 (BD Biosciences).<List of antibodies> Fluorescent labeled antibodies BB5-5 anti-hCD4 Antibody PE-Cy5 anti-mCD45 Antibody PE-Cy7 anti-hCD3 Antibody APC anti-hCD8 Antibody BV711 anti-hCD45 Antibody.

[0079] Example 1 Each antibody-conjugate LNP particle containing EGFP mRNA, as listed in Table 1 below (lipid composition before antibody conjugation: cationic lipid:Cholesterol:DSPC:DMG-PEG2000 / DSPE-PEG2000-maleimide=50:38.5:10:1.5:1.0), was intravenously administered to mice (B6) at a dose of 1.0 mg / kg, and the spleen was collected 24 hours later. EGFP expression rates in CD3-positive T cells were evaluated using a flow cytometer, and the results showed that antibody-12 conjugate lipid nanoparticles 1a to 7a were carriers capable of delivering mRNA to CD3-positive T cells.

[0080]

[0081] Example 2 Antibodies and LNPs were conjugated in HEPES buffer (20 mM HEPES, 9% sucrose, pH 7.25) or PBS buffer (10 mM sodium phosphate, 2.68 mM KCl, 140 mM NaCl, pH 7.45) as described in Table 2 below to obtain antibody-conjugated LNP particles (lipid composition before antibody conjugation: cationic lipids: Cholesterol: DSPC: DMG-PEG2000 / DSPE-PEG2000-maleimide = 50: 38.5: 10: 1.5: 1.0). Each antibody-conjugated LNP containing Cre mRNA was intravenously administered to mice (Ai14) at a dose of 0.5 mg / kg, and the spleen was collected after 24 hours. Flow cytometry evaluation of tdTomato expression levels in CD8-positive and CD4-positive T cells demonstrated that antibody-12 conjugate lipid nanoparticles 1b-6b are carriers capable of delivering mRNA to CD8-positive and CD4-positive T cells.

[0082]

[0083] Example 3 Each antibody-conjugate lipid (R) lipid nanoparticle containing mCheery mRNA, as listed in Table 3 below (lipid composition before antibody conjugation: cationic lipid:Cholesterol:DSPC:DMG-PEG2000:DSPE-PEG2000-maleimide=50:38.5:10:1.5:1.0), was intravenously administered at 0.65 mg / kg to hPBMC-transplanted NSG mice, and the spleen was collected 24 hours later. The mCherry expression rate in CD8-positive T cells, CD4-positive T cells, CD19-positive B cells, and Treg cells was evaluated by flow cytometry. The results showed that antibody-conjugate lipid nanoparticles 1c to 7c were carriers capable of selectively delivering mRNA to CD8-positive T cells compared to untargeted LNP R1c.

[0084]

[0085] Example 4 Each antibody-conjugate LNP containing mCheery mRNA, as listed in Table 4 below (lipid composition before antibody conjugation: cationic lipid:Cholesterol:DSPC:DMG-PEG2000:DSPE-PEG2000-maleimide=50:38.5:10:0.75:0.75), was transfected into activated T cells at 0.02 μg / mL or 0.2 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 all antibody-conjugate lipid nanoparticles were carriers capable of selectively delivering mRNA to CD8-positive T cells compared to untargeted lipid nanoparticles.

[0086]

[0087] Example 5 Each antibody-2 conjugate LNP containing mCheery mRNA, as described in Table 5 below (lipid composition before antibody conjugation: cationic lipid:Cholesterol:DSPC:DMG-PEG2000:DSPE-PEG2000-maleimide=50:38.5:10:0.75:0.75), was transfected into activated T cells at 0.02 μg / mL or 0.2 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 all antibody-2 conjugate lipid nanoparticles were carriers capable of selectively delivering mRNA to CD8-positive T cells compared to untargeted lipid nanoparticles.

[0088]

[0089] Example 6 Each antibody-conjugated LNP containing mCheery mRNA, as described in Table 6 below (lipid composition before antibody conjugation: cationic lipid:Cholesterol:DSPC:DMG-PEG2000:DSPE-PEG2000-maleimide=50:38.5:10:0.75:0.75), was transfected into activated T cells at 0.02 μg / mL or 0.2 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 anti-CD3 antibody-conjugated LNP 1f and anti-CD5 antibody-conjugated LNPs 3f and 4f were superior carriers that could efficiently deliver to CD4-positive T cells and CD8-positive T cells compared to untargeted LNP R1f (note that both CD3 and CD5 are expressed in CD4-positive T cells and CD8-positive T cells). Furthermore, it was shown that LNP 2f conjugated with an anti-CD4 antibody can selectively deliver mRNA to CD4-positive T cells, and LNP 5f conjugated with an anti-CD8 antibody can selectively deliver mRNA to CD8-positive T cells.

[0090]

[0091] Example 7: Antibody-1 conjugate lipid nanoparticles containing mCheery mRNA (lipid composition before antibody conjugation: cationic lipid:Cholesterol:DSPC:DMG-PEG2000 / DSPE-PEG2000-maleimide=50:38.5:10:0.75:0.75) were transfected into activated T cells at 0.02 μg / mL or 0.2 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 the antibody-1 conjugate lipid nanoparticles are carriers capable of selectively delivering mRNA to CD8-positive T cells.

[0092]

[0093] Example 8: Antibody-1 conjugate lipid nanoparticles containing mCheery mRNA (lipid composition before antibody conjugation: cationic lipid:Cholesterol:DSPC:DMG-PEG2000:DSPE-PEG2000-maleimide=50:38.5:10:1.5:1.0) as shown in Table 8 below were intravenously administered at 0.4 mg / kg to hPBMC-transplanted NSG mice, and the spleens were collected 24 hours later. The mCherry expression rates in CD8-positive T cells, CD4-positive T cells, CD19-positive B cells, and Treg cells were evaluated by flow cytometry, and the results showed that the antibody-1 conjugate LNPs are carriers capable of selectively delivering mRNA to CD8-positive T cells.

[0094]

Claims

1. A lipid nanoparticle comprising a nucleic acid encapsulated in the lipid nanoparticle, a cationic lipid, and a targeting polyalkylene glycol-modified lipid linked to a target binding site, wherein the target binding site targets a target site on a target cell, and the cationic lipid is a compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof: Lipid nanoparticle: [In formula (I), a represents an integer of 3 to 5; b represents 0 or 1; R １ and R ２ each independently represent a group represented by the following general formula (A): (In formula (A), R １１ and R １２ each independently represent a linear or branched C ５－１５ alkyl group; c represents 0 or 1; v represents an integer of 4 to 12); X represents a group represented by the following general formula (B): (In formula (B), d represents an integer of 0 to 3; R ３ and R ４ each independently represent a C １－４ alkyl group or a C ２－４ alkenyl group (the C １－４ alkyl group or C ２－４ alkenyl group may have one or two hydrogen atoms substituted with a phenyl group), but R ３ and R ４ may be bonded to each other to form a 5- to 7-member non-aromatic heterocycle (one or two hydrogen atoms of the ring may be substituted with a C １－４ alkyl group or a C ２－４ alkenyl group) or a 5- to 7-member non-aromatic heterocyclic group (however, the group is bonded to (O-CO)b- by a carbon atom, and one or two hydrogen atoms of the ring may be substituted with a C １－４ alkyl group or a C ２－４ alkenyl group)] 2. The lipid nanoparticle according to claim 1, wherein the cationic lipid is a compound represented by any one of the following formulas, or a pharmaceutically acceptable salt thereof.

3. The lipid nanoparticle according to claim 1, wherein the target binding site binds to an antigen on a T cell.

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

5. The lipid nanoparticle according to claim 1, wherein the target binding site binds to an antigen on a hematopoietic stem cell (HSC).

6. The lipid nanoparticle according to claim 1, wherein the target binding site binds to CD90 or CD117.

7. The lipid nanoparticle according to claim 1, wherein the target binding site is an antibody or an antigen-binding fragment thereof.

8. The lipid nanoparticle according to claim 1, wherein the target binding site is an antibody, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an scFv, or a VHH antibody.

9. The lipid nanoparticle according to claim 1, wherein the target binding site is linked to the polyalkylene glycol of the targeted polyalkylene glycol-modified lipid via a linker.

10. The lipid nanoparticle according to claim 9, wherein the linker comprises a peptide of 1 to 50 units.

11. The linker is (Ser-Ser-Ser-Gly) (Sequence ID 1) m [In the formula, m is an integer from 1 to 5], (Glu-Ala-Ala-Ala-Lys) (Sequence ID 2) n [In the formula, n is an integer between 1 and 4], (Ala-Pro) ｐ [In the formula, p is an integer between 1 and 8], (His) ｑ Lipid nanoparticles according to claim 9, comprising at least one of the following: [wherein q is an integer from 1 to 10].

12. The linker is (OCH ２ CH ２ ) ｒ Lipid nanoparticles according to claim 9, comprising [wherein r is an integer from 1 to 10].

13. The linker is given by the following formula: Lipid nanoparticles according to claim 10, comprising a site indicated by [wherein the formula, the left arrow indicates linkage with the peptide, the right arrow indicates linkage with polyalkylene glycol, X is N or O, and r is an integer from 1 to 15].

14. The linker is represented as -Z1-Z2-, where Z1 is linked to the target binding site, Z2 is linked to the polyalkylene glycol, and Z1 is It is represented as, and Z2 is, Lipid nanoparticles according to claim 9, represented by the formula [wherein the left arrow indicates linkage to an adjacent Lys side chain amino group, the right arrow indicates linkage to a polyalkylene glycol, X is N or O, and r is an integer from 1 to 15].

15. The linker is represented as -Z1-Z2-, where Z1 is linked to the V target binding site, Z2 is linked to the polyalkylene glycol, and Z1 is as follows: Z2 is one of the polypeptides shown, and Z2 is Lipid nanoparticles according to claim 9, represented by the formula [wherein the left arrow indicates linkage to the adjacent Cys side chain SH group, the right arrow indicates linkage to polyalkylene glycol, and X is N or O].

16. Lipid nanoparticles according to claim 1 or 2, further comprising sterols or sterol derivatives.

17. Lipid nanoparticles according to claim 1 or 2, further comprising phospholipids.

18. Lipid nanoparticles according to claim 1 or 2, further comprising untargeted polyalkylene glycol-modified lipids in which the target binding site is not linked.

19. The lipid nanoparticle according to claim 18, 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 18, 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 18, 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. The lipid nanoparticle according to claim 1, wherein the nucleic acid is siRNA, antisense nucleic acid, heteroduplex nucleic acid, miRNA, gRNA, or mRNA.

Images