Lipid particle conjugated with antibody or antigen-binding fragment thereof

Abstract

The present invention provides: a lipid particle which exhibits excellent effects in terms of drug efficacy manifestation, safety (low toxicity), and the like; and a production method therefor. A lipid particle provided by the present invention is, for example, a lipid particle conjugated with an antibody that specifically binds to a cell surface antigen, or an antigen-binding fragment thereof, wherein: the antibody or antigen-binding fragment thereof is covalently bonded to at least a portion of a lipid (reactive polyethylene glycol (PEG) lipid) having a reactive PEG chain contained in lipid components constituting the lipid particle; and the ratio of the reactive PEG lipid, which is not covalently bonded to the antibody or antigen-binding fragment thereof, with respect to all of the lipid components constituting the lipid particle is at most 0.1 mol%.

Description

Lipid particles conjugated with antibodies or their antigen-binding fragments

[0001] The present invention relates to lipid particles that enable the introduction of nucleic acids as active ingredients into various types of cells, tissues, or organs, and to compositions containing the lipid particles and nucleic acids.

[0002] [Background of the Invention] In recent years, research and development of nucleic acid drugs containing nucleic acids as active ingredients has been actively pursued. For example, numerous studies have been conducted on nucleic acid drugs containing nucleic acids such as siRNA, miRNA, miRNA mimic, or antisense nucleic acids that have the effect of degrading or inhibiting the function of target mRNA. Research is also being conducted on nucleic acid drugs containing mRNA encoding the target protein, etc., for expressing the target protein in cells. In connection with this research and development, technologies for efficiently introducing nucleic acids into cells, tissues, or organs are being developed as drug delivery system (DDS) technologies.

[0003] The above-mentioned DDS technology is conventionally known to involve mixing nucleic acids and lipids to form a complex, and then allowing the nucleic acids to be taken up by cells via this complex. Conventionally known lipids used for the formation of the above complex include cationic lipids, hydrophilic polymer lipids, and helper lipids. As for the cationic lipids, for example, compounds described in the following prior art documents are known.

[0004] Patent Document 1 describes a pharmaceutical composition comprising lipid nanoparticles (LNPs), therapeutic nucleic acids (TNAs), and at least one pharmaceutically acceptable excipient, wherein the LNPs include single-stranded variable region fragments (scFvs) linked to the LNPs, and the scFvs target antigens present on the surface of cells. Patent Document 1 also describes that scFv may be chemically conjugated to LNP via an incleavable linker, such as a maleimide-containing linker (Claim 5, etc.), that scFv may be chemically conjugated to or covalently linked to PEGylated lipids of LNP, such as DSPE [*DSPE = distearoylphosphoethanolamine]-PEG2000, DSPE-PEG5000, to form a PEGylated lipid conjugate (Claim 42, 44, 45, etc.), and that the PEGylated lipids may constitute 0-20% (mol) of the total lipids present in the LNP (Paragraph

[0384] , etc.). In Example 2 of Patent Document 1, α-HER2 scFv (SEQ ID NO: 2) derived from trastuzumab was prepared, with its C-terminus modified by a Myc tag, a His tag, and a cysteine ​​residue; the cysteine ​​residue of the modified scFv was reduced, and it was incubated with LNPs prepared separately using DSPE-PEG-maleimide in different molar percentages (0.1%, 0.5%, 0.75%, 1%, 1.25%) and PEG lengths (2k, 5k) from lipid A (scFv / maleimide ratio was 0.05). Modified scFv that did not react with the LNPs were removed by dialysis to obtain α-HER2. The document describes the production of LNPs (maleimide conjugate LNPs) in which scFv is conjugated by an inescapable covalent bond; and the evaluation of the inclusion efficiency (Figures 4A and 4B) and uptake into cells (Figures 8A and 8B) of the maleimide conjugate LNPs. Example 2 of Patent Document 1 also describes the production of scFv (SEQ ID NO: 3) for HER2 targeting, in which the N-terminus is modified with a His tag and the C-terminus is modified with an LLQGA polypeptide.

[0005] Non-patent document 1 describes anti-CD3F(ab') for introduction into T cells. ２By reducing the fragment to generate an SH group and incubating it with a mixed lipid containing 0.5% DSPE-PEG5k-maleimide and LNPs separately prepared from nucleic acids (mRNA), (F(ab') ２ Fragment: Maleimide (molar ratio = 1:1), F(ab') ２ The report describes the creation of conjugated LNPs (aCD3-LNPs) from the fragments, and the accumulation of aCD3-LNPs in the spleen after systemic administration.

[0006] Patent Document 2 describes PEG lipids (claims 1, 3, 6, 9, etc.) having a PEG chain having a predetermined functional group (maleimide, azide, dibenzocyclooctin (DBCO), etc.) and a hydrophobic carbon chain having a predetermined structure, and LNPs (claim 16, etc.) containing said PEG lipids. Example 44 of Patent Document 2 describes the preparation of LNPs containing a PEG lipid having a predetermined functional group such as maleimide (0.1% mol / mol) and several other types of lipids in a predetermined ratio; and the incubation of an anti-CD5 antibody modified with N-succinimidyl-S-acetylthioacetate (SATA) with the LNPs, and the conjugation of the antibody to the LNPs by a reaction between SATA and maleimide.

[0007] Non-patent document 2 describes how LNPs were prepared by mixing a lipid membrane containing predetermined lipids in predetermined ratios with an aqueous phase containing plasmids (CAR19, shIL6), and then mixing it with PSPE-PEG2000-anti-CD3 antibody (in a 1:10 ratio to the plasmid) to produce LNPs conjugated with the antibody.

[0008] Patent Document 3 describes a lipid nanoparticle (LNP) comprising a lipid blend for targeted delivery of nucleic acids to immune cells, wherein the LNP further comprises a lipid-immune cell targeting group conjugate containing a compound represented by a predetermined formula: [lipid]-[optional linker]-[immune cell targeting group], and an ionizable cationic lipid represented by a predetermined formula, the LNP further comprising a nucleic acid placed therein (Claim 1, etc.). Patent Document 3 also describes that the immune cell targeting group (e.g., an antibody) may be covalently bonded to the lipid in the lipid blend via a PEG-containing linker (Claim 6, 30, etc.), the lipid-immune cell targeting group conjugate may be present in the lipid blend in a range of 0.001 to 0.5 mole percent (Claim 9, etc.), and the lipid blend may contain free PEG-lipid (e.g., PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE)) (Claim 10, 16, etc.). In the example of Patent Document 3, LNPs were prepared using an mRNA solution and an ethanolic lipid solution (cationic lipids, cholesterol, DSPC, and DMG-PEG(2000)) (Example 2). DSPE-PEG(2000)-maleimide was conjugated with a Fab as a T cell targeting group such as an anti-CD3 Fab, resulting in DSPE-PEG-Fab:DSPE-PEG-maleimide(cysteine ​​terminus):DSPE-PEG-OCH ３ The document describes the preparation of a micelle composition consisting of a mixture in a molar ratio of 1:2.45:3.45 to 10.35 (Example 4), and the preparation of LNPs containing T cell targeting groups by combining (mixing) the LNPs and the conjugate (Example 5).

[0009] Special Publication No. 2024-529343 (corresponding to WO2023 / 287861 pamphlet), WO2023 / 196445 pamphlet, WO2022 / 120388 pamphlet

[0010] Kheirolomoom et al., In situ T-cell transfection by anti-CD3-ated lipid nanoparticles leads to T-cell activation, migration, and phenotypic shift, Bioconjugates, Volume 281, 2022, 121339, https: / / doi.org / 10.1016 / j.biomaterials.2021.121339.Zhou et al., Lipid nanoparticles produce Chimeric antigen receptor T cells with interleukin-6 knockdown in vivo, Journal of Controlled Release, Volume 350, October 2022, Pages 298-307, https: / / doi.org / 10.1016 / j.jconrel.2022.08.033.

[0011] Lipid particles that can efficiently deliver and introduce nucleic acids into target cells are expected to contribute to the creation of gene therapies and nucleic acid drugs that exhibit superior effects in terms of drug efficacy and safety (low toxicity). The aforementioned prior art lipid particles still had room for improvement in these areas.

[0012] The present invention aims to provide lipid particles and a method for producing the same that exhibit excellent effects in terms of drug efficacy and safety (low toxicity).

[0013] Firstly, the inventors have found that the above problems can be solved by controlling the ratio of lipids (PEG lipids) that are not covalently bonded to antibodies, etc., among lipid components conjugated by covalent bonds with antibodies, etc. for targeting specific cells, which have a hydrophobic portion containing a carbon chain that satisfies a predetermined condition regarding the number of carbon atoms and a hydrophilic portion containing a polyethylene glycol (PEG) chain, to a certain level or lower. In particular, the effect of the active ingredient delivered together with the lipid particles as a composition (for example, encapsulated) is improved, and the possibility of re-administration (redosability) is improved, that is, the effect of the active ingredient is maintained (less likely to decrease) even after multiple administrations. Secondly, the inventors have found that the above problems can also be solved by controlling the ratio of lipids that are not covalently bonded to antibodies, etc., among the lipid components constituting the lipid particles, which have a hydrophobic portion containing a carbon chain and a hydrophilic portion containing a PEG chain equipped with a reactive group for covalent bonding (reactive PEG lipids), for example, by setting it to a certain level or lower. In particular, they have found that the effect of the active ingredient delivered as a composition together with the lipid particles is improved, and the possibility of re-administration (redosability) is improved. Furthermore, the inventors have found that by setting the length of the PEG chain (average molecular weight) to a certain level or higher, the above effects can be achieved while also suppressing side effects caused by administering lipid particles (for example, the production of IFNγ). Furthermore, the inventors have found that lipid particles composed of lipid components satisfying the above conditions are preferably manufactured by preparing a reaction product of the above-mentioned PEG lipid or reactive PEG lipid with an antibody, etc., further purifying the above preparation as necessary, and incorporating the molecule in which the PEG lipid or reactive PEG lipid and the antibody, etc. are covalently bonded into the raw materials of all lipid components constituting the lipid particles, thereby controlling the ratio of PEG lipid or reactive PEG lipid that is not bound to the antibody, etc. to the lipid components in the lipid particles. (Conversely, they have found that in a process in which the PEG lipid or reactive PEG lipid contained therein is reacted with the antibody, etc. after the lipid particles are prepared to form a covalent bond, it is difficult to control the ratio of PEG lipid or reactive PEG lipid that is not bound to the antibody, etc. to the lipid components in the lipid particles.)

[0014] In other words, the present invention encompasses at least the following:

[01] Lipid particles conjugated with an antibody or an antigen-binding fragment thereof that specifically binds to a cell surface antigen, wherein the antibody or antigen-binding fragment is covalently bonded to at least a portion of a lipid having a polyethylene glycol (PEG) chain (hereinafter referred to as "PEG lipid") contained in the lipid components constituting the lipid particle, and the ratio of the PEG lipid, which is not covalently bonded to the antibody or antigen-binding fragment and has 30 or more carbon atoms in its hydrophobic carbon chain, to the total lipid components constituting the lipid particle is controlled to a certain range.

[02] The lipid particle according to claim 01, wherein the total number of carbon atoms in the hydrophobic carbon chain of the PEG lipid is 32 or more.

[03] The lipid particle according to claim 01, wherein the total number of carbon atoms in the hydrophobic carbon chain of the PEG lipid is 34 or more.

[04] The lipid particle according to claim 01, wherein the total number of carbon atoms in the hydrophobic carbon chain of the PEG lipid is 36 or more. [1] Lipid particles conjugated with an antibody or an antigen-binding fragment thereof that specifically binds to a cell surface antigen, wherein the antibody or antigen-binding fragment is covalently bonded to at least a portion of a lipid having a reactive polyethylene glycol (PEG) chain (hereinafter referred to as "reactive PEG lipid") contained in the lipid components constituting the lipid particle, and the ratio of the reactive PEG lipid not covalently bonded to the antibody or antigen-binding fragment to the total lipid components constituting the lipid particle is 0.1 mol% or less. [2] The lipid particle according to claim 1, wherein the total number of carbon atoms in the hydrophobic carbon chain of the reactive PEG lipid is 30 or more. [3] The lipid particle according to claim 1, wherein the total number of carbon atoms in the hydrophobic carbon chain of the reactive PEG lipid is 32 or more. [4] The lipid particle according to claim 1, wherein the total number of carbon atoms in the hydrophobic carbon chain of the reactive PEG lipid is 34 or more. [5] The lipid particle according to claim 1, wherein the total number of carbon atoms in the hydrophobic carbon chain of the reactive PEG lipid is 36 or more. [6] The lipid particle according to any one of items 1 to 5 or 01 to 04, wherein the cell surface antigen is a cell surface antigen of a T cell or an NK cell.[7] The lipid particle according to any one of claims 1 to 6 or 01 to 04, wherein the antibody or its antigen-binding fragment is an anti-CD3 antibody or an anti-CD7 antibody, or an antigen-binding fragment thereof. [8] The lipid particle according to any one of claims 1 to 7, wherein the reactive PEG lipid has an azide group or an alkynyl group which is a reactive group for a click reaction. [9] The lipid particle according to any one of claims 1 to 8 or 01 to 04, wherein the reactive PEG lipid or the PEG lipid comprises a reactive derivative of a PEG lipid having a PEG chain with an average molecular weight of 5000 or more, or a PEG lipid having a PEG chain with an average molecular weight of 5000 or more.

[10] Lipid particles according to any one of claims 1 to 9 or 01 to 04, wherein the reactive PEG lipid or the PEG lipid comprises a reactive derivative of PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG-DSPE) or a reactive derivative of PEG-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (PEG-DPPE).

[11] Lipid particles according to any one of claims 1 to 10 or 01 to 04, wherein the ratio of the reactive PEG lipid or the PEG lipid that is not covalently bound to the antibody or its antigen-binding fragment to the total lipid components constituting the lipid particle is 0.01 mol% or less.

[12] Lipid particles according to any one of claims 1 to 11 or 01 to 04, wherein the ratio of the reactive PEG lipid or the PEG lipid that is not covalently bound to the antibody or its antigen-binding fragment to the total lipid components constituting the lipid particle is substantially zero.

[013] A method for producing lipid particles conjugated with an antibody or an antigen-binding fragment thereof that specifically binds to a cell surface antigen, wherein the antibody or antigen-binding fragment is covalently bonded to at least a portion of the PEG lipid contained in the lipid components constituting the lipid particle, the ratio of the PEG lipid, which is not covalently bonded to the antibody or antigen-binding fragment and has 30 or more carbon atoms in its hydrophobic carbon chain, to the total lipid components constituting the lipid particle is controlled to a certain range, and the method comprises the step of blending a molecule in which the PEG lipid and the antibody or antigen-binding fragment are covalently bonded, prepared from the reaction product of the PEG lipid and the antibody or antigen-binding fragment, into the raw materials of the total lipid components constituting the lipid particle.

[014] The method according to item 013, wherein the total number of carbon atoms in the hydrophobic carbon chain of the PEG lipid is 32 or more.

[015] The method according to item 013, wherein the total number of carbon atoms in the hydrophobic carbon chain of the PEG lipid is 34 or more.

[016] The method for producing lipid particles according to item 013, wherein the total number of carbon atoms in the hydrophobic carbon chain of the PEG lipid is 36 or more.

[13] A method for producing lipid particles conjugated with an antibody or antigen-binding fragment that specifically binds to a cell surface antigen, wherein the antibody or antigen-binding fragment is covalently bonded to at least a portion of the reactive PEG lipid contained in the lipid components constituting the lipid particles, the ratio of the reactive PEG lipid that is not covalently bonded to the antibody or antigen-binding fragment to the total lipid components constituting the lipid particles is 0.1 mol% or less, and the method for producing lipid particles comprises the step of blending a molecule in which the reactive PEG lipid and the antibody or antigen-binding fragment are covalently bonded, prepared from the reaction product of the reactive PEG lipid and the antibody or antigen-binding fragment, into the raw materials of the total lipid components constituting the lipid particles.

[14] The method for producing lipid particles according to item 13, wherein the total number of carbon atoms in the hydrophobic carbon chain of the reactive PEG lipid is 30 or more.

[15] The method for producing lipid particles according to item 13, wherein the total number of carbon atoms in the hydrophobic carbon chain of the reactive PEG lipid is 32 or more.

[16] The method for producing the product according to item 13, wherein the total number of carbon atoms in the hydrophobic carbon chain of the reactive PEG lipid is 34 or more.

[17] The method for producing the reactive PEG lipid, wherein the total number of carbon atoms in the hydrophobic carbon chain of the reactive PEG lipid is 36 or more.

[18] The method for producing the reactive PEG lipid, wherein the cell surface antigen is a cell surface antigen of a T cell or an NK cell.

[19] The method for producing the reactive PEG lipid, wherein the antibody or its antigen-binding fragment is an anti-CD3 antibody or an anti-CD7 antibody, or an antigen-binding fragment thereof.

[20] The method for producing the reactive PEG lipid, wherein the reactive PEG lipid has an azide group or an alkynyl group which is a reactive group for a click reaction.

[21] The method for producing the reactive PEG lipid, wherein the reactive PEG lipid or the PEG lipid comprises a reactive derivative of a PEG lipid having a PEG chain with an average molecular weight of 5000 or more, or a PEG lipid having a PEG chain with an average molecular weight of 5000 or more.

[22] A method for producing a nucleic acid according to any one of claims 13 to 21 or 013 to 016, wherein the reactive PEG lipid or PEG lipid comprises a reactive derivative of PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG-DSPE) or a reactive derivative of PEG-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (PEG-DPPE).

[023] A nucleic acid delivery composition comprising the lipid particles and nucleic acid according to any one of claims 01 to 04.

[024] The nucleic acid delivery composition according to claim 023, wherein the total number of carbon atoms in the hydrophobic carbon chain of the PEG lipid is 32 or more.

[025] The nucleic acid delivery composition according to claim 023, wherein the total number of carbon atoms in the hydrophobic carbon chain of the PEG lipid is 34 or more.

[026] The nucleic acid delivery composition according to claim 023, wherein the total number of carbon atoms in the hydrophobic carbon chain of the PEG lipid is 36 or more.

[23] A nucleic acid delivery composition comprising lipid particles and nucleic acids as described in any one of items 1 to 12.

[24] The nucleic acid delivery composition according to item 23, wherein the total number of carbon atoms in the hydrophobic carbon chain of the PEG lipid is 30 or more.

[25] The nucleic acid delivery composition according to item 23, wherein the total number of carbon atoms in the hydrophobic carbon chain of the PEG lipid is 32 or more.

[26] The nucleic acid delivery composition according to claim 23, wherein the total number of carbon atoms in the hydrophobic carbon chain of the PEG lipid is 34 or more.

[27] The nucleic acid delivery composition according to claim 23, wherein the total number of carbon atoms in the hydrophobic carbon chain of the PEG lipid is 36 or more. [23A] The nucleic acid delivery composition comprising lipid particles and nucleic acids according to any one of claims 1 to 14.

[28] The nucleic acid delivery composition according to any one of claims 23 to 27, 23A, or 023 to 026, wherein at least a portion of the nucleic acid is encapsulated in the lipid particles.

[29] The nucleic acid delivery composition according to claim 28, wherein the nucleic acid is DNA or RNA.

[30] A nucleic acid delivery method comprising the step of contacting the nucleic acid delivery composition according to any one of claims 23 to 27, 23A, or 023 to 026 with a target cell having a cell surface antigen to which an antibody or antigen-binding fragment conjugated to the lipid particle specifically binds.

[31] The nucleic acid delivery method according to claim 30, wherein the target cell is a T cell or an NK cell.

[32] A method for introducing nucleic acids in vivo, comprising the step of administering a nucleic acid introduction composition according to any one of items 023 to 27, 23A, or 023 to 026.

[33] A method for improving redosability by controlling the ratio of PEG lipids, which are not covalently bonded to the antibody or its antigen-binding fragment and have 30 or more carbon atoms in their hydrophobic carbon chain, to the total lipid components constituting the lipid particles, in lipid particles conjugated with an antibody or its antigen-binding fragment that specifically binds to a cell surface antigen covalently bonded to at least a portion of a lipid having a polyethylene glycol (PEG) chain contained in the lipid components constituting the lipid particles (hereinafter referred to as "PEG lipid").

[34] The method according to claim 33, wherein the total number of carbon atoms in the hydrophobic carbon chain of the PEG lipid is 32 or more.

[35] The method according to claim 33, wherein the total number of carbon atoms in the hydrophobic carbon chain of the PEG lipid is 34 or more.

[36] The method according to claim 33, wherein the total number of carbon atoms in the hydrophobic carbon chain of the PEG lipid is 36 or more.

[0015] The present invention improves the predetermined therapeutic effect obtained by efficiently delivering the active ingredient, typically nucleic acid (mRNA, etc.) encapsulated in the lipid particles, as part of a composition along with lipid particles, to cells, tissues, or organs in the body, and improves the redosability, meaning that the therapeutic effect can be maintained even after multiple administrations. This is a remarkable effect not observed with conventional lipid particles, and preferably, it can also suppress side effects caused by the administration of lipid particles, such as the production of IFNγ.

[0016] —Terminology— In this specification, "lipids having polyethylene glycol chains" are referred to as "PEG lipids." PEG lipids are derivatives in which PEG chains are attached to general lipid molecules. They have hydrophobic carbon chains and hydrophilic polyethylene glycol chains, but the molecule (polymer) as a whole is hydrophilic.

[0017] In this specification, PEG lipids are sometimes referred to as the abbreviation of the original lipid molecule - PEG, or PEG - the abbreviation of the original lipid molecule, to indicate that they are molecules formed by the bonding of a PEG chain to a pre-existing lipid molecule. For example, a PEG lipid formed by the bonding of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine to a PEG chain may be referred to as "DSPE-PEG" or "PEG-DSPE," using the abbreviation "DSPE" for the former and "PEG" for the latter. In such notations, the number in parentheses after PEG represents the average molecular weight (usually the number-average molecular weight) of the PEG chain. For example, "DSPE-PEG (5000)" represents a compound formed by the bonding of DSPE to a PEG chain with an average molecular weight of 5000.

[0018] In this specification, "lipids having a reactive PEG chain" are referred to as "reactive PEG lipids," and "lipids having a non-reactive PEG chain" are referred to as "non-reactive PEG lipids." A "reactive PEG chain" is a PEG chain that has a site (functional group, etc.) capable of reacting with an antibody or its antigen-binding fragment to form a covalent bond, while a "lipid having a non-reactive PEG chain" is a PEG chain that does not have such a site (functional group, etc.). Reactive PEG lipids can also be described as compounds obtained by chemical synthesis or other means from PEG lipids so that the PEG chain of the PEG lipid becomes reactive, that is, has a site (functional group, etc.) capable of reacting with an antibody or its antigen-binding fragment to form a covalent bond; therefore, they are also called "reactive derivatives" of PEG lipids. Reactive PEG lipids may be expressed by combining the notation of the original PEG lipid with the notation (abbreviation) of the introduced site. For example, "DSPE-PEG(2000)Azide" represents a compound in which an azide group for a click reaction has been introduced into DSPE-PEG(2000).

[0019] In this specification, "a molecule in which a PEG lipid and an antibody or antigen-binding fragment are covalently bonded" is referred to as "targeted PEG lipid."

[0020] In this specification, "approximately" refers to any number that falls within ±10%, ±5%, or ±1% of the number to which it is attached. Numbers described herein may be marked with "approximately" as needed.

[0021] —Lipid Particles— In the present invention, lipid particles are lipid particles conjugated with an antibody or an antigen-binding fragment that specifically binds to a cell surface antigen. Regardless of its form, embodiment, or method of manufacture, the conjugation refers to the presence of an antibody or an antigen-binding fragment that specifically binds to a cell surface antigen in order to enable the lipid particle to be introduced into a target cell.

[0022] "Lipid particles" can encapsulate (enclose) various substances according to their uses or form complexes through interactions. For example, when the lipid component constituting the lipid particles contains ionized lipids (e.g., cationized lipids), it can form complexes with various substances (e.g., negatively charged nucleic acids) that exhibit electrostatic interactions with it. The shape of the lipid particles is not particularly limited, and includes, for example, complexes formed by aggregating such that the lipid components form a substantially spherical shape, complexes formed by aggregating without forming a specific shape, complexes dissolved in a solvent, and complexes uniformly or non-uniformly dispersed in a dispersion medium. Conventionally, "lipid particles" in various embodiments have been known or common in this technical field, and in the present invention as well, embodiments of lipid particles similar to those in the prior art can be used, except for introducing configurations necessary to achieve the effects of the present invention.

[0023] - Lipid component Lipid particles are usually composed of a lipid component containing two or more types of lipids. The composition of the lipid component constituting the lipid particles (the types of lipids and their blending amounts) is not particularly limited, and various lipid components known or common in this technical field can be employed, except for introducing configurations necessary to achieve the effects of the present invention (meeting the specific conditions described in this specification).

[0024] As the lipid for constituting the lipid particles, for example, at least one selected from the group consisting of sterols, phospholipids, lipids having a polyethylene glycol chain (PEG lipids), and ionized lipids can be used, and it is preferable to use all four of these types.

[0025] Examples of sterols include cholesterol, cholesterol ester, cholesterol hemisuccinate, etc. As one embodiment of the present invention, it is preferable that the lipid component contains cholesterol as a sterol.

[0026] Phospholipids include, for example, phosphatidylcholine (e.g., dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), lysophosphatidylcholine, dioleoylphosphatidylcholine (DOPC), palmitoyloleoylphosphatidylcholine (POPC), dilinolenoylphosphatidylcholine (DLPC), diecoylphosphatidylcholine (DEPC), MC-1010 (NOF CORPORATION), MC-2020 (NOF CORPORATION), MC-4040 (NOF CORPORATION), MC-6060 (NOF CORPORATION), MC-8080 (NOF CORPORATION), etc.). Examples include phosphatidylserine (e.g., dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), dioleoylphosphatidylserine (DOPS), palmitoyloleoylphosphatidylserine (POPS)), phosphatidylethanolamine (e.g., dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine, lysophosphatidylethanolamine), phosphatidylinositol, phosphatidic acid, etc. In one embodiment of the present invention, the lipid component preferably contains DSPC, DPPC or other phosphatidylcholine as a phospholipid.

[0027] Examples of lipids having polyethylene glycol chains (PEG lipids) include PEG-dialkyloxyalkyl (PEG-DAA) (e.g., PEG-dilauryloxypropyl [C12 x 2], PEG-dimyristyloxypropyl [C14 x 2], PEG-dipalmityloxypropyl [C16 x 2], PEG-distearyloxypropyl [C18 x 2]), PEG-diacylglycerol (PEG-DAG) (e.g., PEG-dilauroylglycerol (PEG-DLG) [C12 x 2], PEG-dimyristoylglycerol (PEG-DMG) [C14 x 2], PEG-dipalmitoylglycerol (PEG-DPG) [C16 x 2], PEG-distearoylglycerol (PEG-DSG) [C18 x 2]), and SUNBRIGHT GM-020 (NOF CORPORATION), SUNBRIGHT GS-050 (NOF CORPORATION), PEG-phospholipids (e.g., PEG-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (PEG-DMPE) [C14 x 2] such as N-(carbonyl-methoxypolyethylene glycol)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, PEG-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (PEG-DPPE) [C16 x 2] such as N-(carbonyl-methoxypolyethylene glycol)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N-(carbonyl-methoxypolyethylene glycol)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine) PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG-DSPE) [C18 x 2], PEG-1,2-dimyristyl-sn-glycero-3-phosphoethanolamine [C14 x 2], PEG-1,2-dipalmityl-sn-glycero-3-phosphoethanolamine [C16 x 2], N-(carbonyl-methoxypolyethylene glycol)-1,2-dipalmityl-sn-glycero-3-phosphoethanolaminePEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine such as 2-distearoyl-sn-glycero-3-phosphoethanolamine [C18×2]), PEG-ceramide, PEG-cholesterol, PEG-C-DOMG, 2KPEG-CMG, etc. can be mentioned.,

[0028] As one embodiment of the present invention, the lipid component is a reactive PEG lipid (the reactive PEG lipid in (I) in the condition (1B) of the first embodiment of lipid particles described later, or the reactive PEG lipid in the conditions (2A) and (2B) of the second embodiment of lipid particles), and preferably includes a reactive derivative of PEG-DSPE, a reactive derivative of PEG-DPPE, or a reactive derivative of other PEG-phospholipids.,

[0029] As one embodiment of the present invention, the lipid component is a PEG lipid (a PEG lipid that may be optionally used in addition to the specific reactive PEG lipid and non-reactive PEG lipid according to conditions (1A) and (1B) in the first embodiment of lipid particles described later, or a PEG lipid that may be optionally used in addition to the specific reactive PEG lipid according to conditions (1A) and (1B) in the second embodiment of lipid particles), and preferably includes PEG-DMG.,

[0030] <Ionizable lipid> An ionizable lipid is a lipid having a site that ionizes in a solvent. Although ionizable lipids having various structures are known as ionizable lipids that can be incorporated into the lipid components constituting lipid particles, for example, the ionizable lipids (cationic lipids, etc.) described in WO2016 / 021683, WO2019 / 131770, WO2019 / 131839, WO2020 / 032184, WO2023 / 085299, etc. can be used in the present invention.,

[0031] The ionizable lipid used as a raw material for producing lipid particles may form a salt with an inorganic base, an organic base, an inorganic acid, an organic acid, a basic or acidic amino acid, preferably a pharmacologically acceptable salt.,

[0032] The proportions of each lipid included in the lipid component can be appropriately adjusted depending on the type of lipid used, its chemical structure, and the intended use of the resulting lipid particles. For example, the ratio (mol%) of sterols:phospholipids:PEG lipids:ionized lipids in the total lipids present in the lipid particles is typically 10-60%:0-50%:0.1-10%:10-80%, preferably 15-55%:5-40%:0.2-5%:20-70%, and more preferably 20-50%:5-30%:0.5-2%:30-60%.

[0033] • First Embodiment of Lipid Particles In one aspect of the present invention, lipid particles (first embodiment of lipid particles) satisfy two conditions: (1A) an antibody or its antigen-binding fragment is covalently bonded to at least a portion of the PEG lipids contained in the lipid components constituting the lipid particles, and (1B) the ratio of PEG lipids that are not covalently bonded to the antibody or its antigen-binding fragment, and have 30 or more carbon atoms in their hydrophobic carbon chains, to the total lipid components constituting the lipid particles is within a certain range.

[0034] In conditions (1A) and (1B), the "PEG lipids" can be PEG-dialkyloxyalkyl (PEG-DAA), PEG-diacylglycerol (PEG-DAG), and PEG-phospholipids, preferably those containing two hydrophobic carbon chains, as described above. In the case of reactive PEG lipids, the average molecular weight of the PEG chains contained in the PEG lipid is preferably 2000 or more (average number of repeating -C2H4-O- units of 44 or more), more preferably 3400 or more (average number of repeating -C2H4-O- units of 77 or more), and even more preferably 5000 or more (average number of repeating -C2H4-O- units of 113 or more). In the case of non-reactive PEG lipids, the average molecular weight of the PEG chains contained in the PEG lipid is preferably 1000 or more (average number of repeating -C2H4-O- units of 22 or more), more preferably 2000 or more (average number of repeating -C2H4-O- units of 44 or more).

[0035] In condition (1A), the form of covalent bonding between the antibody or its antigen-binding fragment and the PEG lipid is not particularly limited, and various forms of covalent bonding known or common in the art can be employed. For example, the antibody or its antigen-binding fragment may form a covalent bond with a lipid (reactive PEG lipid) having a PEG chain equipped with a maleimide group, a thiol group, an azide group, a DBCO or other alkyne group, a saltase recognition sequence, an amino group, glycine, etc., as described later in relation to the second embodiment of the lipid particles of the present invention.

[0036] In condition (1B), "PEG lipids that are not covalently bonded to an antibody or its antigen-binding fragment, and have 30 or more carbon atoms in their hydrophobic carbon chain" includes both (I) PEG lipids having a reactive PEG chain (reactive PEG lipid) but not covalently bonded to an antibody or its antigen-binding fragment (unreacted), and having 30 or more carbon atoms in their hydrophobic carbon chain, and (II) PEG lipids having a non-reactive PEG chain (unreactive PEG lipid) and having 30 or more carbon atoms in their hydrophobic carbon chain. The number of carbon atoms in the hydrophobic carbon chains of reactive and unreactive PEG lipids is preferably 32 or more, more preferably 34 or more, and even more preferably 36 or more.

[0037] "The number of carbon atoms in the hydrophobic carbon chain is 30 or more / 32 or more / 34 or more / 36 or more" means that the total number of carbon atoms in the carbon chains contained in the hydrophobic portion of PEG lipids, for example, PEG-dialkyloxyalkyl (PEG-DAA), PEG-diacylglycerol (PEG-DAG), and PEG-phospholipids (two carbon chains each) is 30 or more / 32 or more / 34 or more / 36 or more. As an example, PEG-DSPE has two stearoyl groups (-O-CO-(CH2) 16 PEG-phospholipids have two palmitoyl groups (-O-CO-(CH2)) and have 18 carbon atoms, meaning the hydrophobic carbon chain has 36 carbon atoms, and can be any of the PEG lipids with 30 or more carbon atoms in the hydrophobic carbon chain, 32 or more carbon atoms, 34 or more carbon atoms, or 36 or more carbon atoms. Furthermore, PEG-DPPE has two palmitoyl groups (-O-CO-(CH2)) 14It is a PEG-phospholipid having -CH3 (16 carbon atoms), that is, a hydrophobic carbon chain with 32 carbon atoms, and corresponds to a PEG-lipid with 30 or more or 32 or more carbon atoms in the hydrophobic carbon chain.

[0038] Under condition (1B), the ratio of PEG lipids that are not covalently bonded to the antibody or its antigen-binding fragment, and have 30 or more carbon atoms in their hydrophobic carbon chain, to the total lipid components constituting the lipid particles can be controlled to 0.5 mol% or less, 0.4 mol% or less, 0.3 mol% or less, 0.2 mol% or less, or 0.1 mol% or less, 0.01 mol% or less, or substantially zero (not included in a detectable amount). Furthermore, under condition (1B), the ratio of PEG lipids that are not covalently bonded to the antibody or its antigen-binding fragment, and have 30 or more carbon atoms in their hydrophobic carbon chain, to the total lipid components constituting the lipid particles can be controlled to 0.01 mol% to 0.5 mol%, 0.1 mol% to 0.5 mol%, 0.1 mol% to 0.4 mol%, 0.1 mol% to 0.3 mol%, approximately 0.5 mol%, approximately 0.4 mol%, approximately 0.3 mol%, approximately 0.2 mol%, approximately 0.1 mol%, or approximately 0.01 mol%.

[0039] When the first embodiment of the lipid particles of the present invention is produced by the production method of the present invention described herein, namely the production method (first embodiment of the production method, Pre method), which includes the step of purifying, if necessary, the reaction product of PEG lipid and antibody or its antigen-binding fragment, and incorporating a molecule in which PEG lipid and antibody or its antigen-binding fragment are covalently bonded (targeted PEG lipid) into the raw materials for all lipid components constituting the lipid particles, the non-reactive PEG lipid, even if the number of carbon atoms in the hydrophobic carbon chain is 30 or more, can be considered to satisfy condition (1B) as long as the ratio to the total lipid components constituting the lipid particles is within a certain range.

[0040] Purification of targeted PEG lipids from reaction products of PEG lipids with antibodies or their antigen-binding fragments can be performed by extracting the targeted PEG lipids from the reaction product or by removing molecules other than the targeted PEG lipids (i.e., PEG lipids that did not covalently bind to antibodies, antibodies that did not covalently bind to PEG lipids, etc.) from the reaction product. For such purification of targeted PEG lipids, methods such as dialysis filtration, ultrafiltration, and chromatography can be used. Examples of ultrafiltration include normal flow filtration and tangential flow filtration. Examples of chromatography include size exclusion chromatography (gel filtration chromatography), hydrophobic interaction chromatography, ion exchange chromatography, and affinity chromatography. As the mobile phase used in chromatography, for example, water, or an organic solvent described later, or a buffer described later, or a mixture thereof (for example, a mixed solvent containing 0-50% alcohols and water or a buffer) can be used.

[0041] On the other hand, when the first embodiment of the lipid particles of the present invention is manufactured by a manufacturing method (Post method) that includes a step of obtaining a lipid particle composition (dispersion) using a lipid component containing a reactive PEG lipid having 30 or more carbon atoms in its hydrophobic carbon chain as a raw material, and then reacting the reactive PEG lipid in the lipid particle preparation with an antibody or its antigen-binding fragment to form a covalent bond, the following conditions (1B) can be considered to be satisfied: (i) The sum of the amount of reactive PEG lipid having 30 or more carbon atoms in its hydrophobic carbon chain (a) and the amount of unreactive PEG lipid having 30 or more carbon atoms in its hydrophobic carbon chain (b) (a + b) is within a certain range, for example, 0.1 mol% or less, relative to the total lipid component used as a raw material. This is because even if all of a is not covalently bonded with the antibody or its antigen-binding fragment (unreacted), the total amount will still be within that certain range, for example, 0.1 mol% or less. (ii) The sum of the amount of reactive PEG lipids having 30 or more carbon atoms in their hydrophobic carbon chains that are not covalently bonded to the antibody or its antigen-binding fragment (a') and the amount of non-reactive PEG lipids having 30 or more carbon atoms in their hydrophobic carbon chains (b) (a' + b) is within a certain range relative to the total lipid components used as raw materials, for example, 0.1 mol% or less. Here, a' is the amount obtained by subtracting the amount of covalently bonded substances (a'') from a (a - a''), and the amount of a'' can be determined by multiplying the amount of the antigen or its antigen-binding fragment used in the reaction for covalent bonding by the reaction efficiency. The reaction efficiency can be measured, for example, by HPLC analysis of the unreacted antibody or its antigen-binding fragment.

[0042] ・Second Embodiment of Lipid Particles In one aspect of the present invention, the lipid particles of the present invention satisfy two conditions: (2A) an antibody or its antigen-binding fragment is covalently bound to at least a portion of a lipid having a reactive PEG chain (reactive PEG lipid) contained in the lipid components constituting the lipid particle, and (2B) the ratio of reactive PEG lipids not covalently bound to the antibody or its antigen-binding fragment to the total lipid components constituting the lipid particle is 0.1 mol% or less.

[0043] In conditions (2A) and (2B), the "reactive PEG lipid" and "unreactive PEG lipid" can be PEG-dialkyloxyalkyl (PEG-DAA), PEG-diacylglycerol (PEG-DAG), and PEG-phospholipid, respectively, preferably containing two hydrophobic carbon chains, and can be either reactively derivatized or underivatized. In the case of reactive PEG lipids, the average molecular weight of the PEG chains contained in the PEG lipid is preferably 2000 or more (average number of repeating -C2H4-O- units of 44 or more), more preferably 3400 or more (average number of repeating -C2H4-O- units of 77 or more), and even more preferably 5000 or more (average number of repeating -C2H4-O- units of 113 or more). In the case of unreactive PEG lipids, the average molecular weight of the PEG chains contained in the PEG lipid is preferably 1000 or more (average number of repeating -C2H4-O- units of 22 or more), more preferably 2000 or more (average number of repeating -C2H4-O- units of 44 or more).

[0044] In condition (2A), the form of covalent bonding between the antibody or its antigen-binding fragment and the reactive PEG lipid is not particularly limited, and various forms of covalent bonding known or common in the art can be employed. For example, lipids having a PEG chain equipped with a maleimide group, a thiol group, an azide group, a DBCO or other alkyne group, a saltase recognition sequence, an amino group, glycine, etc., can be used as the "reactive PEG lipid".

[0045] <Reaction between maleimide group and thiol group> Reactive PEG lipids having a maleimide group can react with antibodies or antigen-binding fragments having a thiol group, such as Fab' produced by treating F(ab')2 with a reducing agent to cleave the disulfide bond, or with antigens or antigen-binding fragments treated with sulfhydrylation reagents (e.g., SATA), to form a covalent bond (thioether bond). Bromomaleimide groups or bromoacetamide groups can also be used instead of maleimide groups.

[0046] <Click Chemistry: Reaction of Azide Groups with DBCO, etc.> Reactive PEG lipids having azide groups can react with antibodies or antigen-binding fragments having DBCO (dibenzocyclooctin), for example, derivatives obtained by treating antibodies, etc., with DBCO-NHS ester to bind NHS groups to amino groups (e.g., lysine residues) of the antibodies, etc., and form covalent bonds. Such reactions between azide groups and DBCO are preferable because they do not require a copper catalyst. In click chemistry, compounds having other alkyne groups (carbon-carbon triple bonds) can be used instead of DBCO, but a copper catalyst may be required. In addition, to introduce DBCO, etc., into antibodies, etc., a method using a transpeptidase such as saltase, as described below, can also be used. For example, a method in which an antibody, etc., to which a saltase recognition motif (LPXTG, etc.) has been introduced to the C-terminus beforehand is reacted with DBCO to which a glycine residue (GG, etc.) is bound, in the presence of saltase.

[0047] <Saltase Recognition Motif: Reaction of LPXTG etc. with Glycine Residues, etc.> Saltase is a transpeptidase known for protein modification, and saltase A (SrtA) and saltase B (SrtB) derived from Staphylococcus aureus can be used. Saltase A recognizes the Leu-Pro-Xxx-Thr-Gly motif (where Xxx is any amino acid; hereafter referred to as "LPXTG") near the carboxyl terminus (C-terminus) of one molecule, such as a protein or peptide, thereby mediating a transpeptidase reaction between a glycine residue (G) in the motif and a glycine residue at the amino terminus (N-terminus) of the other molecule. Therefore, an antibody or its antigen-binding fragment having the recognition motif of saltase A at its C-terminus can react with a reactive PEG lipid having a glycine residue at its telogen in the presence of saltase A, and can form a covalent bond more directly (without the need for specific reactive groups such as azide groups and DBCO as described above). The glycine residue may be one, two, three, or more consecutively, but one or two are preferred. The glycine residue may exist as a peptide having a glycine residue at its telogen (for example, in the molecular structure of Gly-Xxx-Xxx-Xxx-Xxx-Xxx-PEG lipid). The telogenous residue of the reactive PEG lipid is not limited to a glycine residue, but may be any other N-terminal amino acid residue or a functional group having an amino group. Saltase A may be a mutant (for example, one with an improved reaction rate than the wild type), and saltase B, which recognizes NPQTN, NPKTG, etc. as its recognition motif, can be used instead of saltase A.

[0048] In condition (2B), "reactive PEG lipids that are not covalently bonded to an antibody or its antigen-binding fragment" refers specifically to reactive PEG lipids that are not covalently bonded to an antibody or its antigen-binding fragment, and does not include the non-reactive PEG lipids described in condition (1B). The number of carbon atoms in the hydrophobic carbon chain of reactive PEG lipids that are not covalently bonded to an antibody or its antigen-binding fragment is preferably 30 or more, 32 or more, 34 or more, or 36 or more. The explanation for "the number of carbon atoms in the hydrophobic carbon chain is 30 or more / 32 or more / 34 or more / 36 or more" is the same as the explanation for condition (1B).

[0049] Under condition (2B), the ratio of reactive PEG lipids that are not covalently bound to the antibody or its antigen-binding fragment to the total lipid components constituting the lipid particles can be controlled to 0.5 mol% or less, 0.4 mol% or less, 0.3 mol% or less, 0.2 mol% or less, or 0.1 mol% or less, 0.01 mol% or less, or substantially zero (not included in a detectable amount). Furthermore, under condition (2B), the ratio of PEG lipids (similar to (I) and (II) mentioned above in relation to condition (1B)) that are not covalently bonded to an antibody or its antigen-binding fragment and have 30 or more carbon atoms in their hydrophobic carbon chains, to the total lipid components constituting the lipid particles can be controlled to 0.01 mol% to 0.5 mol%, 0.1 mol% to 0.5 mol%, 0.1 mol% to 0.4 mol%, 0.1 mol% to 0.3 mol%, approximately 0.5 mol%, approximately 0.4 mol%, approximately 0.3 mol%, approximately 0.2 mol%, approximately 0.1 mol%, or approximately 0.01 mol%.

[0050] In the production of the lipid particles of the present invention, a second embodiment is provided for, in this specification, by the production method of the present invention described herein, namely, a production method (second embodiment of the production method, Pre method) that includes the step of purifying, if necessary, the reaction product of reactive PEG lipid and an antibody or its antigen-binding fragment, and incorporating a molecule in which reactive PEG lipid and an antibody or its antigen-binding fragment are covalently bound (targeted PEG lipid) into the raw materials for all lipid components constituting the lipid particles. In this case, if the ratio of reactive PEG lipid that is not covalently bound to the antibody or its antigen-binding fragment (unreacted) to the total lipid components constituting the lipid particles is 0.1 mol% or less, then condition (2B) can be considered to be satisfied. This is because, in the purified product of targeted PEG lipid, the amount of unreacted material that is not covalently bound to the antibody or its antigen-binding fragment is at least 0.1 mol% or less, preferably 0.01 mol% or less, and can be considered substantially zero. The purification of targeted PEG lipid from the reaction product of reactive PEG lipid and an antibody or its antigen-binding fragment is the same as described for condition (1B).

[0051] When the second embodiment of the lipid particles of the present invention is manufactured by a manufacturing method (Post method) that includes a step of obtaining a lipid particle composition (dispersion) using a lipid component containing reactive PEG lipid as a raw material, and then reacting the reactive PEG lipid in the lipid particle preparation with an antibody or its antigen-binding fragment to form a covalent bond, the following conditions (2B) can be considered to be satisfied: (i) The amount of reactive PEG lipid (a) is 0.1 mol% or less relative to the total lipid component used as a raw material. This is because even if all of a is not covalently bonded to the antibody or its antigen-binding fragment (unreacted), the total amount will still be 0.1 mol% or less; (ii) The amount of reactive PEG lipid that is not covalently bonded to the antibody or its antigen-binding fragment (a') is 0.1 mol% or less relative to the total lipid component used as a raw material. Here, a' is the amount obtained by subtracting the amount of covalently bonded material (a'') from a (a - a''), and the amount of a'' can be determined by multiplying the amount of antigen or antigen-binding fragment used in the reaction for covalent bonding by the reaction efficiency. The reaction efficiency can be measured, for example, by HPLC analysis of the unreacted antibody or antigen-binding fragment.

[0052] • Antibodies or their antigen-binding fragments The antibodies or their antigen-binding fragments conjugated to the lipid particles of the present invention can be those that specifically bind to the target cell surface antigen, depending on the application. The target cell surface antigen is not particularly limited, but surface molecules that are specifically or highly expressed in the target cells are preferred.

[0053] Examples of antigen-binding fragments of antibodies include Fab, F(ab')2, Fab', Fv, reductive antibody (rIgG), disulfide-stabilized Fv (dsFv), single-chain Fv (single-chain antibody, scFv), dibody, tribody, HCAb, and VHH. When the lipid particles of the present invention target immune cells, as described below, Fab, Fab', and VHH are preferred as antibodies or their binding fragments.

[0054] In one embodiment of the present invention, the lipid particles of the present invention are used to introduce and express genes encoding CAR or exogenous TCR in immune cells, more specifically T cells responsible for cellular immunity among acquired immunity, NK cells responsible for innate immunity, monocytes, macrophages, dendritic cells, etc., and NK T cells which are T cells having the properties of NK cells, particularly in vivo. In such embodiments, the lipid particles of the present invention can be conjugated with antibodies or antigen-binding fragments that specifically bind to the cell surface antigens of immune cells, preferably T cells or NK cells. Examples of cell surface antigens of immune cells include CD3, CD4, CD5, CD7, CD8, CD16, CD28, CD56, etc. For example, when cytotoxic T cells are targeted, anti-CD3 antibodies, anti-CD7 antibodies, or anti-CD8 antibodies or their antigen-binding fragments are preferred, and when NK cells are targeted, anti-CD7 antibodies, anti-CD56 antibodies, or anti-CD16 antibodies or their antigen-binding fragments are preferred.

[0055] Antibodies or antigen-binding fragments that specifically bind to a desired target cell surface antigen can be prepared by known methods and used in the present invention. When introducing a functional group capable of forming a covalent bond with a functional group of a reactive PEG lipid, such as DBCO, into an antibody or its antigen-binding fragment using a transpeptidase for conjugation to lipid particles, the antibody or its antigen-binding fragment must first possess an oligopeptide corresponding to a saltase recognition motif. Such antibodies or antigen-binding fragments possessing a saltase recognition motif can also be prepared by known methods, such as genetic engineering.

[0056] In one aspect of the present invention, the lipid particles of the present invention can be used to encapsulate (encapsulate) nucleic acids or to form complexes through interactions, that is, to prepare the nucleic acid introduction composition of the present invention.

[0057] "Nucleic acid" refers to any molecule formed by the polymerization of nucleotides and molecules having equivalent functions to nucleotides. Examples include RNA, which is a polymer of ribonucleotides; DNA, which is a polymer of deoxyribonucleotides; polymers of a mixture of ribonucleotides and deoxyribonucleotides; and nucleotide polymers containing nucleotide analogs. Furthermore, nucleotide polymers containing nucleic acid derivatives may also be included. Nucleic acids may be single-stranded or double-stranded. Double-stranded nucleic acids also include double-stranded nucleic acids in which one strand hybridizes with the other under stringent conditions.

[0058] Nucleotide analogs can be any molecule that has been modified from ribonucleotides, deoxyribonucleotides, RNA, or DNA to improve nuclease resistance or stability compared to RNA or DNA, to increase affinity with complementary nucleic acids, to increase cell permeability, or to make them visible. Nucleotide analogs can be naturally occurring or unnatural molecules, such as sugar-modified nucleotide analogs or phosphate diester-modified nucleotide analogs.

[0059] As sugar-modified nucleotide analogs, any chemical structural substance can be added to or substituted for part or all of the chemical structure of the sugar of a nucleotide. Specific examples include nucleotide analogs substituted with 2'-O-methylribose, nucleotide analogs substituted with 2'-O-propylribose, nucleotide analogs substituted with 2'-methoxyethoxyribose, nucleotide analogs substituted with 2'-O-methoxyethylribose, nucleotide analogs substituted with 2'-O-[2-(guanidium)ethyl]ribose, nucleotide analogs substituted with 2'-fluororibose, nucleic acid analogs in which the sugar portion is replaced with a morpholino ring (morpholino nucleic acids), bridged nucleotides (BNA) having two cyclic structures by introducing a cross-linking structure to the sugar portion, more specifically, locked nucleotides (LNA) in which the oxygen atom at the 2' position and the carbon atom at the 4' position are cross-linked via methylene, and ethylene cross-linked nucleotides (Ethylene Examples include bridged nucleic acids (ENA) [Nucleic Acid Research, 32, e175 (2004)], amide-bridged nucleic acids (AmNA) in which the 2' and 4' carbon atoms are bridged via an amide bond, as well as peptide nucleic acids (PNA) [Acc. Chem. Res., 32, 624 (1999)], oxypeptide nucleic acids (OPNA) [J. Am. Chem. Soc., 123, 4653 (2001)], and peptide ribonucleic acid (PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)].

[0060] Phosphate diester bond-modified nucleotide analogs can be any nucleotide in which any chemical substance is added to or substituted for part or all of the phosphate diester bond in the chemical structure of the nucleotide. Specific examples include nucleotide analogs substituted with phosphorothioate bonds and nucleotide analogs substituted with N3'-P5' phosphoamide bonds [Cell Engineering, 16, 1463-1473 (1997)] [RNAi and Antisense Methods, Kodansha (2005)].

[0061] Nucleic acid derivatives can be any molecule to which another chemical substance has been added to the nucleic acid in order to improve nuclease resistance, stabilize it, increase affinity with complementary nucleic acid chains, increase cell permeability, or make it visible. Specific examples include 5'-polyamine-added derivatives, cholesterol-added derivatives, steroid-added derivatives, bile acid-added derivatives, vitamin-added derivatives, Cy5-added derivatives, Cy3-added derivatives, 6-FAM-added derivatives, and biotin-added derivatives.

[0062] The nucleic acids in the present invention are not particularly limited and may include, for example, nucleic acids intended for the improvement of diseases, symptoms, disorders, or pathological conditions, and for the alleviation or prevention of the onset of diseases, symptoms, disorders, or pathological conditions (which may be referred to as "treatment of diseases, etc." in this specification), or nucleic acids for regulating the expression of desired proteins that are useful for research purposes but do not contribute to the treatment of diseases, etc.

[0063] Information on disease-related genes or polynucleotides (hereinafter sometimes referred to as "disease-related genes") is available, for example, from the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and the National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.).

[0064] Specific examples of nucleic acids in the present invention include, for example, single-stranded DNA, double-stranded DNA, siRNA, miRNA, miRNA mimic, antisense nucleic acids, ribozymes, mRNA, circRNA, self-replicating RNA, gRNA, decoy nucleic acids, aptamers, etc., and may also be analogs or derivatives that have been artificially modified. Furthermore, nucleic acids may be linear or covalently closed circular. Preferred nucleic acids are DNA, RNA, such as single-stranded DNA, double-stranded DNA, siRNA, mRNA, circRNA, self-replicating RNA, and gRNA, or analogs or derivatives of these that have been artificially modified.

[0065] In the present invention, "siRNA" means a double-stranded RNA or its analogues having 10 to 30 bases, preferably 15 to 25 bases, and containing a complementary sequence. siRNA preferably has 1 to 3 overhanging bases, more preferably 2 bases, at its 3' end. The complementary sequence portion may be perfectly complementary or may contain non-complementary bases, but is preferably perfectly complementary.

[0066] The siRNA used in this invention is not particularly limited, and for example, siRNA for knocking down the expression of disease-related genes can be used. Disease-related genes refer to any genes or polynucleotides that produce transcription or translation products at abnormal levels or in abnormal forms in cells derived from affected tissue compared to non-disease control tissue or cells. In addition, the siRNA used in this invention can also be siRNA for regulating the expression of a desired protein useful for research.

[0067] In the present invention, "mRNA" means RNA containing a base sequence that can be translated into a protein. The mRNA in the present invention is not particularly limited as long as it is mRNA that can express a desired protein in a cell. Preferably, the mRNA is mRNA that is useful for pharmaceutical uses (e.g., disease treatment) and / or research purposes, and such mRNA is, for example, mRNA for expressing a marker protein such as luciferase in a cell.

[0068] In the present invention, "gRNA" means a guide RNA corresponding to the CRISPR system. The gRNA in the present invention may be in the form of a single RNA formed by the ligation of crRNA and tracrRNA, i.e., a chimeric RNA (sometimes called a single guide RNA, sgRNA, etc.), or it may be in the form of two unligated RNAs (a combination of two RNAs, or a combination of more than two RNAs).

[0069] In the present invention, "DNA" means DNA containing a base sequence that can be transcribed into mRNA. The DNA in the present invention is not particularly limited as long as it is DNA that can be transcribed into a desired mRNA within a cell. Preferably, the DNA is useful for pharmaceutical applications (e.g., gene therapy applications) and / or research purposes. Examples of such DNA include plasmid DNA (pDNA), single-stranded DNA (ssDNA), nanoplasmids, minicircle DNA (Minicircle), closed-end DNA (ceDNA), doggybone DNA (dbDNA), ministring DNA (msDNA), and linear DNA (linDNA). Examples of such DNA include DNA used to express marker proteins such as luciferase within a cell.

[0070] The DNA in this invention may include an enhancer or a promoter. The enhancer or promoter in this invention is not particularly limited as long as it can control the transcription to a desired mRNA within the cell. Examples of the above promoters or enhancers include the ApoE / hAAT enhancer or promoter, CAG promoter, CMV (Cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter or enhancer, SV40 promoter, DHFR (Dihydrofolate reducase) promoter, EF1α promoter, EF and CBA (Chicken β-Actin) promoter, PGK (Phosphoricerate Kinase) promoter, hSYN (human synapsin) promoter, MND promoter, RSV (Rous Sarcoma Virus LTR) promoter, and Chicken beta actin + intron promoter, TRE (tetracycline-responsive element) promoter, UBC (Ubiquitin C) promoter, MSCV U3 (Murine stem cell virus LTR) promoter, GALV U3 (Gibbon ape leukemia virus LTR) promoter, GUSB (Beta gluturonidase) promoter, MeCP2 promoter, GFAP (glial fibrillary acid protein) promoter, Human beta actin promoter, EBV (Epstein-Barr virus) promoter, SFFV (Spleen Focus) Examples include the Forming Virus LTR promoter. The CMV promoter or CAG promoter is preferred as the enhancer or promoter.

[0071] —Composition for introducing nucleic acids— In one aspect of the present invention, a composition for introducing nucleic acids is provided, which contains the lipid particles and nucleic acids of the present invention. In the composition for introducing nucleic acids, the nucleic acids are preferably encapsulated within substantially spherical lipid particles, but a portion of the nucleic acids may be complexed in other ways or present in the composition.

[0072] Nucleic acid introduction compositions may take the form of pharmaceutical compositions that have applications depending on the nucleic acid used, for example, for treating specific diseases. Diseases targeted for treatment or other procedures are not particularly limited, and examples include the diseases listed below (1) to (7). Unless otherwise specified, the information in parentheses indicates examples of disease-related genes. The nucleic acids used in this invention also include nucleic acids that regulate the expression levels of these disease-related genes (or the proteins they encode).

[0073] (1) Hematological disorders: Anemia (CDAN1, CDA1, RPS19, DBA, PKLR, PK1, NT5C3, UMPH1, PSN1, RHAG, RH50A, NRAMP2, SPTB, ALAS2, ANH1, ASB, ABCB7, ABC7, ASAT), lymphocyte insufficiency syndromes (TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11, MHC2TA, C2TA, RFX5), hemorrhagic disorders (TBXA2R, P2RX1, P2X1), H factor and Factor H-like factor 1 deficiency (HF1, CFH, HUS), factor V and factor VIII deficiency (MCFD2), factor VII deficiency (F7), factor X deficiency (F10), factor XI deficiency (F11), factor XII deficiency (F12, HAF), factor XIIIA deficiency (F13A1, F13A), factor XIIIB deficiency (F13B), Fanconi anemia (FANCA, FACA, FA1, FA, FAA, FAAP95, FAAP90, FLJ34064, FANCB, FAN CC, FACC, BRCA2, FANCD1, FANCD2, FANCD, FACD, FAD, FACE, FACE, FANCF, XRCC9, FANCG, BRIP1, BACH1, FANCJ, PHF9, FANCL, FAN CM, KIAA1596), hemophagocytic lymphohistiocytosis (PRF1, HPLH2, UNC13D, MUNC13-4, HPLH3, HLH3, FHL3), hemophilia A (F8, F8C, HEMA), hemophilia B (F9, HEMB), Blood disorders (PI, ATT, F5), leukocyte deficiencies (ITGB2, CD18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, EIF2B3, EIF2B5, LVWM, CACH, CLE, EIF2B4), sickle cell anemia (HBB), thalassemia (HBA2, HBB, HBD, LCRB, HBA1), von Willebrand disease (VWF), hypoalbuminemia, hypovolemia, severe congenital protein C deficiency, prothrombin deficiency, etc.

[0074] (2) Inflammatory and immune diseases: AIDS (KIR3DL1, NKAT3, NKB1, AMB11, KIR3DS1, IFNG, CXCL12, SDF1), autoimmune lymphoproliferative syndrome (TNFRSF6, APT1, FAS, CD95, ALPS1A), combined immunodeficiency (IL2RG, SCIDX1, SCIDX, IMD4), HIV infection (CCL5, SCYA5, D17S135E, TCP228, IL10, CSIF, CMKBR2, CCR2, DMKBR5, CCCKR5, CCR5), immunodeficiency (CD3E, CD3G, AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSF5, CD40LG, HIGM1, IGM, FOXP3, IPE Inflammation (IL10, IL-1, IL-13, IL-17, IL-23, CTLA4), severe combined immunodeficiency (JAK3, JAKL, DCLRE1C, ATREMI) S, SCIDA, RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG, SCIDX1, SCIDX, IMD4), primary immunodeficiency, secondary immunodeficiency, multifocal motor neuropathy, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, rheumatoid arthritis, psoriasis, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis, etc.), Sjögren's syndrome, Behçet's disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis pus nephritis, discoid lupus erythematosus, Castleman's disease, ankylosing spondylitis, polymyositis, dermatomyositis, polyarteritis nodosa, mixed connective tissue disease, scleroderma, deep lupus erythematosus, chronic thyroiditis, Graves' disease, autoimmune gastritis, type I and type II sugars. Urinary disease, autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia, atopic dermatitis, chronic active hepatitis, myasthenia gravis, graft-versus-host disease, Addison's disease, abnormal immune response, arthritis, dermatitis, radiation dermatitis, primary biliary cirrhosis, etc.

[0075] (3) Metabolic, hepatic, and renal diseases: Amyloid neuropathy (TTR, PALB), amyloidosis (APOA1, APP, AAA, CVAP, AD1, GSN, FGA, LYZ, TTR, PALB), non-alcoholic steatohepatitis and hepatic fibrosis (COL1A1), cirrhosis (KRT18, KRT8, CIRH1A, NAIC, TEX292, KI AA1988), cystic fibrosis (CFTR, ABCC7, CF, MRP7), glycogen storage disorders (SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1, GYS2, PYGL, PFKM), hepatocellular adenoma (TCF1, HFN1A, MODY3), liver failure (SCOD1, S CO1), hepatic lipase deficiency (LIPC), hepatoblastoma (CTNNNB1, PDGFRL, PDGRL, PRLTS, AXIN1, AXIN, TP53, P53, LFS1, IGF2R, MPRI, MET, CASP8, MCH5), medullary polycystic kidney disease (UMOD, HNFJ, FJHN, MCKD2, ADMKD2), phenylketonuria (PAH) PKU1, QDPR, DHPR, PTS), polycystic kidney and liver diseases (FCYT, PKHD1, APKD, PDK1, PDK2, PDK4, PDKTS, PRKCSH, G19P1, PCLD, SEC63), Hunter syndrome, lysosomal storage diseases, Fabry disease, Pompe disease, Gaucher disease, mucopolysaccharidosis, hypoparathyroidism, Wilson's disease, etc.

[0076] (4) Neurological disorders: ALS (SOD1, ALS2, STEX, FUS, TARDBP, VEGF), Alzheimer's disease (APP, AAA, CVAP, AD1, APOE, AD2, PSEN2, AD4, STM2, APBB2, FE65L1, NOS3, PLAU, URK, ACE, DCP1, ACE1, MPO, PACIP1, PAXIP1L, PTIP, A2M, BLMH, BMH, PSEN1, AD3), Autism (BZRAP1, MDGA2, GLO1, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4, KIAA1260, AUTSX2), Fragile X syndrome ( FMR2, FXR1, FXR2, mGLUR5), Huntington's disease (HD, IT15, PRNP, PRIP, JPH3, JP3, HDL2, TBP, SCA17), Parkinson's disease (NR4A2, NURR1, NOT, TINUR, SNCAIP, TBP, SCA17, SNCA, NACP, PARK1, PARK4, DJ1, DBH, NDUFV2), Rett syndrome (MECP2, RTT, PPMX, MRX16, MRX79, CDKL5, STK9), schizophrenia (GSK3, 5-HTT, COMT, DRD, SLC6A3, DAOA, DTNBP1), secretase-related disorders (APH-1), etc.

[0077] (5) Eye diseases: macular degeneration (Abcr, Ccl2, cp, Timp3, カテプシンD, Vld lr, Ccr2), cataract (CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, B FSP2, CP49, CP47, PAX6, AN2, MGDA, CRYBA1, CRYB1, CR YGC, CRYG3, CCL, LIM2, MP19, CRYGD, CRYG4, BSFP2, CP4 9. CP47, HSF4, CTM, MIP, AQP0, CRYAB, CRYA2, CTPP2, CRYBB1, CRYGD, CRYG4, CRYA1, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1), corneal turbidity (APOA1, TGFFB1, CSD2, CDGG1, CSD, BIGH3, CDG2, TASTD2, TROP2, M1S) 1. VSX1, RIX, PPCD, PPD, KTCN, COL8A2, FECD, PPCD2, PIP5K3, CFD), congenital hereditary flat cornea (KERA, CNA2), green cataract (MYOC, TIGR, GLC1A, JOAG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1B1, GLC3A, OPA1, NTG, NPG, CYP1B1, GLC3A), leaver Congenital agrarian syndrome (CRB1, RP12, CRX, CORD2, CRD, RPGRIP1, LCA6, C ORD9, RPE65, RP20, AIPL1, LCA4, GUCY2D, GUC2D, LCA1, CORD6、RDH12、LCA3)、Macula ジストロフCー(ELOVL4、ADMD、STGD2 , STGD3, RDS, RP7, PRPH2, PRPH, AVMD, AOFMD, VMD2)なI.

[0078] (6) Neoplastic diseases: malignant tumors, neovascular glaucoma, infantile hemangioma, hereditary angioedema, multiple myeloma, chronic sarcoma, metastatic melanoma, Kaposi's sarcoma, vascular proliferation, cachexia, metastasis of breast cancer, etc., cancer (e.g., colorectal cancer (e.g., familial colorectal cancer, hereditary nonpolyposis colorectal cancer, gastrointestinal stromal tumors, etc.), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, malignant mesothelioma, etc.), mesothelioma, pancreatic cancer (e.g., pancreatic ductal carcinoma, etc.), gastric cancer (e.g., papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous cell carcinoma, etc.), breast Cancer (e.g., invasive ductal carcinoma, non-invasive ductal carcinoma, inflammatory breast cancer, etc.), ovarian cancer (e.g., epithelial ovarian cancer, extragonadal germ cell tumor, ovarian germ cell tumor, low-grade ovarian tumor, etc.), prostate cancer (e.g., hormone-dependent prostate cancer, hormone-independent prostate cancer, etc.), liver cancer (e.g., primary liver cancer, extrahepatic cholangiocarcinoma, etc.), thyroid cancer (e.g., medullary thyroid carcinoma, etc.), kidney cancer (e.g., renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, etc.), uterine cancer, brain tumor (e.g., pineal astrocytic tumor) (Piliocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, etc.), melanoma, sarcoma, bladder cancer, hematological cancers including multiple myeloma, pituitary adenoma, glioma, acoustic neuroma, retinal sarcoma, pharyngeal cancer, laryngeal cancer, tongue cancer, thymoma, esophageal cancer, duodenal cancer, colon cancer, rectal cancer, hepatocellular carcinoma, pancreatic endocrine tumor, bile duct cancer, gallbladder cancer, penile cancer, ureteral cancer, testicular tumor, vulvar cancer, cervical cancer, uterine body cancer, uterine sarcoma, gestational trophoblastic disease, vaginal cancer, skin cancer, mycosis fungoides, basal cell tumor, soft tissue sarcoma, malignant lymphoma Hodgkin's disease, myelodysplastic syndrome, adult T-cell leukemia, chronic myeloproliferative disorders, pancreatic endocrine tumors, fibrous histiocytoma, leiomyosarcoma, rhabdomyosarcoma, cancer of unknown primary origin, etc.), leukemia (e.g., acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, etc.), chronic leukemia (e.g., chronic lymphoblastic leukemia, chronic myeloid leukemia, etc.), myeloplastic syndromes, etc.), uterine sarcoma (e.g., mixed mesodermal tumor, uterine leiomyosarcoma, endometrial stromal tumor, etc.), myelofibrosis, etc.

[0079] (7) Other diseases: IgA nephropathy, aplastic anemia, sarcoidosis, Williams syndrome, Marfan syndrome, muscular dystrophy, spinocerebellar degeneration, hypoparathyroidism, pemphigus, bullous pemphigoid, amyotrophic lateral sclerosis, spina bifida, hypertrophic cardiomyopathy, idiopathic thrombocytopenic purpura, ankylosing spondylitis, osteomalacia, dermatomyositis, IgG4-related disease, Usher syndrome, Apert syndrome, Alport syndrome, Angelman syndrome, West syndrome, spinal muscular atrophy, Werner syndrome, Osler disease, Crouzon syndrome, Creutzfeldt-Jakob disease, POEMS syndrome Group, prion disease, Shy-Drager syndrome, Charcot-Marie-Tooth disease, Sturge-Weber syndrome, Stevens-Johnson syndrome, SMON, Sotos syndrome, Dravet syndrome, Noonan syndrome, Buerger disease, Hirschsprung's disease, Pfeiffer syndrome, Tetralogy of Fallot, phenylketonuria, Prader-Willi syndrome, porphyria, mitochondrial disease, maple syrup urine disease, familial hypercholesterolemia, familial Mediterranean fever, Kabuki syndrome, fulminant hepatitis, tuberous sclerosis, polyarteritis nodosa, thrombotic thrombocytopenic purpura, microscopic Polyangiitis, primary sclerosing cholangitis, primary biliary cholangitis, eosinophilic sinusitis, Takayasu's arteritis, osteogenesis imperfecta, mixed connective tissue disease, neuromyelitis optica, autoimmune hepatitis, autoimmune hemolytic anemia, xeroderma pigmentosum, progressive supranuclear palsy, adult Still's disease, syringomyelia, congenital myopathy, systemic sclerosis, multiple system atrophy, aortitis syndrome, corticobasal degeneration, biliary atresia, fatal familial insomnia, toxic epidermal necrolysis, idiopathic interstitial pneumonia, achondroplasia, pustular psoriasis, pulmonary arterial hypertension, inclusion body myositis, chronic inflammatory demyelinating polyneuropathy, chronic active EB virus infection, Retinitis pigmentosa, Cushing's disease, familial chronic pyoderma, autosomal dominant polycystic kidney disease, 1p36 deletion syndrome, 22q11.2 deletion syndrome, HTLV-1 associated myelopathy, Aicardi syndrome, Weaver syndrome, granulomatosis with polyangiitis, Ehlers-Danlos syndrome, Emanuel syndrome, Klippel-Trenaunay-Weber syndrome, Cockayne syndrome, Costello syndrome, Coffin-Siris syndrome, Coffin-Lowry syndrome, Smith-Maginis syndrome, thanatophoric dysplasia, Tangier disease, CHARGE syndrome, Budd-Chiari syndrome, peroxisomal disease,Myoclonic absence epilepsy, Moebius syndrome, Menkes disease, lymphangioleiomyomatosis, Rubinstein-Taybe syndrome, Leber's hereditary optic neuropathy, subacute sclerosing panencephalitis, ossification of the ligamentum flavum, familial benign chronic pemphigus, oculocutaneous albinism, giant cell arteritis, ossification of the posterior longitudinal ligament, extensive spinal stenosis, hypertrichosis IgD syndrome, relapsing polychondritis, tricuspid atresia, congenital ichthyosis, polysplenia syndrome, pseudoxanthoma elastica, delayed endolymphatic hydrops, Nakajo-Nishimura syndrome, hypophosphatasia, idiopathic portal hypertension, Nasu-Hakola disease, refractory frequent partial seizure status epilepticus, urea cycle disorders, pulmonary alveolar proteinosis, paroxysmal nocturnal hemoglobinuria, hypertrophic dermatoperiosteopathy, bronchiolitis obliterans Arima syndrome, status epilepticus (biphasic) acute encephalopathy, Epstein syndrome, Fanconi anemia, 4p deletion syndrome, 5p deletion syndrome, Ulrich disease, Occipital-Horn syndrome, Carney complex, galactose-1-phosphate uridyltransferase deficiency, Galloway-Mowat syndrome, Mowat-Wilson syndrome, Young-Simpson syndrome, Landau-Kleffner syndrome, Rossmund-Thomson syndrome, suppurative aseptic arthritis, pyoderma gangrenosum, acne syndrome, interstitial cystitis, megalymphatic malformation, eosinophilic granulomatosis with polyangiitis, autoimmune hemorrhagic disease XIII, congenital erythrodysplasia anemia, septal optic nerve malformation, branchio-otorenephrosis, etc.

[0080] The nucleic acid delivery composition of the present invention, as a pharmaceutical composition, can be manufactured by methods known in the pharmaceutical technology field using a pharmaceutically acceptable carrier. Examples of dosage forms of the pharmaceutical composition include parenteral administration formulations such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, etc.) and topical formulations such as ointments, creams, solutions, and plasters. Parenteral administration formulations such as injections may contain conventional adjuvants such as buffers and / or stabilizers, and topical formulations may contain conventional pharmaceutical carriers.

[0081] The nucleic acid delivery composition of the present invention can be used to introduce active ingredients into a wide variety of cells, tissues, or organs. Examples of cells to which the composition of the present invention can be applied include spleen cells, nerve cells, glial cells, pancreatic B cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fibrous cells, muscle cells (e.g., skeletal muscle cells, cardiomyocytes, myoblasts, muscle satellite cells), adipocytes, immune cells (e.g., macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes, megakaryocytes), synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary gland cells, hepatocytes or stromal cells, egg cells, spermatocytes, or progenitor cells that can be differentiated into these cells, stem cells (e.g., induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells)), hematopoietic cells, oocytes, and fertilized eggs. Furthermore, tissues or organs to which the composition of the present invention can be applied include any tissue or organ in which the above-mentioned cells exist, such as the brain, various parts of the brain (e.g., olfactory bulb, amygdala, basal ganglia, hippocampus, thalamus, hypothalamus, subthalamic nucleus, cerebral cortex, medulla oblongata, cerebellum, occipital lobe, frontal lobe, temporal lobe, putamen, caudate nucleus, corpus callosum, substantia nigra), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonads, thyroid gland, gallbladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (e.g., large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cells, prostate, testes, ovaries, placenta, uterus, bone, joints, and skeletal muscle. These cells, tissues, or organs may also be cancerous cancer cells or cancerous tissue.

[0082] When the nucleic acid delivery composition of the present invention is used in vivo, typically as a pharmaceutical composition administered into the body, the target and dosage are not particularly limited and can be adjusted according to the application. The target may be a human or a non-human mammal (e.g., mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey). The dosage can be adjusted so that an effective amount of nucleic acid is delivered to the target cells and the desired effect is achieved.

[0083] In one embodiment of the present invention, the nucleic acid introduction composition of the present invention may be a composition comprising the lipid particles of the present invention and a nucleic acid encoding a chimeric antigen receptor (CAR) or a nucleic acid encoding an exogenous T cell receptor (TCR).

[0084] • Nucleic acid encoding CAR CAR is an artificially constructed hybrid protein containing an antigen-binding domain of an antibody (e.g., scFv) linked to a T cell signaling domain. Generally, CAR includes an antigen-binding domain of an antibody, an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain that can specifically recognize a surface antigen (e.g., cancer antigen peptide, surface receptors that are upregulated in cancer cells, etc.) that the target immune cells (e.g., T cells, NK cells) should recognize. The amino acid sequence of CAR and the base sequence of the nucleic acid encoding it are not particularly limited and can be adapted to the application of the nucleic acid delivery composition of the present invention.

[0085] Surface antigens specifically recognized by the antigen-binding domain of CAR include, for example, acute lymphoblastic carcinoma, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, intrahepatic bile duct cancer, joint cancer, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, oral cancer, vulvar cancer, chronic myeloid carcinoma, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), hypopharyngeal cancer, kidney cancer, laryngeal cancer, and leukemia (e.g., acute lymphoblastic leukemia, acute lymphoblastic leukemia). Examples of surface antigens that are upexpressed in various cancer cells include lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia), humoral neoplasms, liver cancer, lung cancer (e.g., non-small cell lung cancer), lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma), malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, cancers of the peritoneum, retinoplasm, and mesentery, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, gastric cancer, testicular cancer, thyroid cancer, and ureteral cancer. Specific examples of such surface antigens include CD19, EGF receptor, BCMA, CD30, Her2, ROR1, MUC16, CD20, mesothelin, B-cell mutation antigen (BCMA), CD123, CD3, prostate specific embrasure antigen (PSMA), CD33, MUC-1, CD138, CD22, GD2, PD- Examples include surface receptors such as L1, CEA, chondroitin sulfate protein glycycan-4, IL-13 receptor α chain, and IgG κ light chain; cancer antigen peptides derived from WT1, GPC3, MART-1, gp100, NY-ESO-1, MAGE-A4, etc.; and extracellular domains of transmembrane proteins such as Claudin (CLDN) 3, CLDN4, CLDN6, and CLDN18.2.

[0086] The antigen-binding domain of a CAR can be any antibody fragment capable of specifically recognizing a target antigen, and is not particularly limited. However, considering the ease of CAR production, it is preferable to use a single-chain antibody (scFv) in which the light chain variable region and the heavy chain variable region are linked via a linker peptide. The arrangement of the light chain variable region and the heavy chain variable region in a single-chain antibody is not particularly limited as long as both can reconstruct a functional antigen-binding domain, but it can usually be designed in the order of light chain variable region - linker peptide - heavy chain variable region from the N-terminus. It is preferable that a leader sequence is further added to the N-terminus of the antigen-binding domain to present the CAR on the surface of immune cells.

[0087] The base sequences of nucleic acids encoding the light chain variable region and heavy chain variable region can be obtained based on the amino acid sequence information of the light chain variable region and heavy chain variable region of an antibody or its antigen-binding fragment that specifically binds to a target cell surface antigen, or they can be obtained by cloning the light chain gene and heavy chain gene from the antibody-producing cell.

[0088] As the linker peptide, known linker peptides commonly used in the production of single-chain antibodies can be used. Based on the amino acid sequence of the linker peptide, the base sequence of the nucleic acid encoding it can also be designed.

[0089] For the extracellular hinge domain and transmembrane domain of CAR, domains derived from T cell surface molecules, which are common in CAR construction, can be used. Examples of such extracellular hinge domains and transmembrane domains include domains derived from CD8α or CD28.

[0090] As the intracellular signaling domain of a CAR, various domains commonly used in CAR construction can be appropriately combined and used. Examples of such intracellular signaling domains include those having a CD3ζ chain, those further having co-stimulus transmission motifs such as CD28, CD134, CD137, Lck, DAP10, ICOS, and 4-1BB between the transmembrane domain and the CD3ζ chain, and those having two or more co-stimulus transmission motifs.

[0091] The base sequences of nucleic acids encoding extracellular hinge domains, transmembrane domains, and intracellular signaling domains can be designed to correspond to the amino acid sequences of each domain, and the commonly known amino acid sequences of each domain and the nucleic acid sequences encoding them are publicly available.

[0092] The nucleic acid sequence encoding the entire CAR can be designed by concatenating the nucleic acid sequences encoding the antigen-binding domain (heavy chain variable region, light chain variable region, linker peptide, etc.), the extracellular hinge domain, the transmembrane domain, and the intracellular signal transduction domain.

[0093] Nucleic acids encoding exogenous TCRs T cell receptors (TCRs) are composed of dimers of TCR chains (α-chain and β-chain) that can specifically recognize surface antigens (e.g., cancer antigen peptides, etc.) that target T cells should recognize. They are receptors that recognize antigens or antigen-HLA (human leukocyte antigen) (MHC; major histocompatibility complex) complexes and transmit stimulating signals to T cells. Each TCR chain consists of a variable region and a constant region, and the variable region contains three complementarity-determining regions (CDR1, CDR2, CDR3). In this invention, TCRs include not only those in which the α-chain and β-chain constitute a heterodimer, but also those in which they constitute a homodimer. Furthermore, TCRs also include those with a partial or complete deletion of the constant region, those with rearranged amino acid sequences, and soluble TCRs.

[0094] Furthermore, "exogenous TCR" means that it is exogenous to T cells, which are the target cells of the lipid particles of the present invention. The amino acid sequence of the exogenous TCR may be the same as or different from the endogenous TCR expressed by T cells, which are the target cells of the lipid particles of the present invention.

[0095] The amino acid sequence of the exogenous TCR and the base sequence of the nucleic acid encoding it are not particularly limited and can be adapted to the application of the nucleic acid introduction composition of the present invention.

[0096] The base sequence of the nucleic acid encoding the exogenous TCR can be obtained based on the amino acid sequence information of the TCR chain (α and β chains), or it can be obtained by cloning the gene of the T cell expressing the target TCR.

[0097] Preparation of nucleic acids encoding CAR or exogenous TCR Nucleic acids encoding CAR or exogenous TCR can be prepared by general methods based on their base sequence, for example, by chemical synthesis as DNA or RNA strands, or by joining partially overlapping oligoDNA short chains using PCR or Gibson Assembly.

[0098] The nucleic acid encoding the CAR or exogenous TCR obtained in this manner may be used directly for the preparation of the nucleic acid delivery composition, or it may be converted into an expression vector (preferably a plasmid vector) before being used for the preparation of the nucleic acid delivery composition. When preparing an expression vector, the nucleic acid encoding the CAR or exogenous TCR is preferably DNA. When preparing the nucleic acid encoding the CAR or exogenous TCR as RNA, for example mRNA, it can also be obtained by first creating an expression vector using DNA as described above, and then using that as a template in an in vitro transcription system.

[0099] Functional expression vectors for T cells, NK cells, and other immune cells can be produced using common methods. For example, functional promoters in T cells can include the SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Morony's mouse leukemia virus) LTR, and HSV-TK (herpes simplex virus thymidine kinase) promoter, which are constitutive in mammalian cells, or gene promoters such as CD3, CD4, and CD8 that are specifically expressed in T cells.

[0100] In the case of nucleic acids encoding exogenous TCRs, the DNA encoding the α-chain and the DNA encoding the β-chain may be inserted into the same expression vector or into separate expression vectors. When inserted into the same expression vector, the expression vector may express both chains polycistronically (in this case, it is appropriate to insert an intervening sequence that allows polycistronic expression, such as IRES or FMV2A, between the DNAs encoding the two chains), or it may express them monocistronically.

[0101] —Method for Producing Lipid Particles— The lipid particles of the present invention can be produced by modifying the process, conditions, and other embodiments of various known methods for producing lipid particles, such that an antibody or antigen-binding fragment that specifically binds to a cell surface antigen is conjugated to the lipid particles of the first and second embodiments of the present invention, respectively, to satisfy the predetermined conditions.

[0102] Methods for producing lipid particles conjugated with antibodies or antigen-binding fragments can be broadly classified into (1) a production method in which lipid particles are prepared and then conjugated with antibodies or the like to the lipid particles (specific lipids contained therein), as described in Patent Documents 1 and 2 and Non-Patent Document 1 cited as prior art (referred to as the "Post method" in this specification), and (2) a production method in which lipid particles are prepared in which antibodies or the like are bound to specific lipids constituting the lipid particles, as described in Patent Document 3 and Non-Patent Document 2 cited as prior art, and then lipid particles are prepared using the specific lipids to which the antibodies or the like are bound and other lipids (referred to as the "Pre method" in this invention).

[0103] (1) Post method The Post method is a manufacturing method that includes the steps of preparing lipid particles and conjugating antibodies or the like to the lipid particles (specific lipids contained therein).

[0104] (1-1) Lipid Particle Preparation Process In the lipid particle preparation process of the Post method, an organic solvent solution containing the lipid components that make up the lipid particles (at this step, the antibody or its antigen-binding fragment is not yet covalently bonded to the reactive PEG lipid) is prepared and mixed with water or a buffer solution. This mixing process can be carried out by various emulsification methods or by using a microfluidic mixing system (e.g., Asia microfluidic system (Syrris) or Nanoassemblr (Precision Nanosystems)).

[0105] Examples of organic solvents include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butanol, acetone, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, or mixtures thereof. The organic solvent may contain 0-20% water or buffer solution.

[0106] Examples of buffer solutions include acidic buffers (e.g., acetate buffer, citrate buffer, 2-morpholinoethanesulfonic acid (MES) buffer, phosphate buffer) and neutral buffers (e.g., 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, tris(hydroxymethyl)aminomethane (Tris) buffer, phosphate buffer, phosphate-buffered saline (PBS)). Additives such as glycerol or salts such as NaCl may be added to the buffer solution.

[0107] When mixing using a microfluidic mixing system, it is preferable to mix 1 to 5 volumes of water or buffer solution with 1 volume of organic solvent solution. In this system, the flow rate of the mixture (mixture of organic solvent solution and water or buffer solution) is, for example, 0.01 to 115 mL / min, preferably 0.1 to 115 mL / min, and the temperature is, for example, 5 to 60°C, preferably 15 to 45°C.

[0108] The obtained lipid particles may be subjected to desalting, dialysis, and sterile filtration. For example, dialysis can replace the dispersion medium of the lipid particles with water or buffer solution. Dialysis should be performed using an ultrafiltration membrane with a molecular weight cutoff of 10 to 20 K at 4°C to room temperature. Dialysis may be repeated. Tangential flow filtration (TFF) may be used to replace the dispersion medium.

[0109] After replacing the dispersion medium, pH and osmotic pressure adjustments may be performed as needed. Examples of pH adjusting agents include sodium hydroxide, citric acid, acetic acid, triethanolamine, sodium hydrogen phosphate, sodium dihydrogen phosphate, and potassium dihydrogen phosphate. Examples of osmotic pressure adjusting agents include inorganic salts such as sodium chloride, potassium chloride, sodium hydrogen phosphate, potassium hydrogen phosphate, sodium dihydrogen phosphate, and potassium dihydrogen phosphate; polyols such as glycerol, mannitol, and sorbitol; and sugars such as glucose, fructose, lactose, and sucrose. The pH is usually adjusted to 6.5 to 8.0, preferably to 7.0 to 7.8. The osmotic pressure is preferably adjusted to 250 to 350 Osm / kg.

[0110] The average particle size of the lipid particles of the present invention is preferably 10 to 200 nm. The average particle size of the lipid particles can be calculated by performing cumulant analysis of the autocorrelation function using a particle size measuring device based on dynamic light scattering measurement technology, such as the Zetasiner Nano ZS (Malvern Instruments). Since the lipid particles of the present invention are usually on the order of nanometers, less than 1 μm, they can also be called lipid nanoparticles (LNPs).

[0111] (1-2) Conjugate step In the conjugate step of the Post method, an antibody or its antigen-binding fragment is reacted with a lipid having a reactive polyethylene glycol (PEG) chain (reactive PEG lipid) contained in the lipid component that makes up the lipid particle, and a covalent bond is formed. The embodiment of this conjugate step can be one that corresponds to the form of covalent bond between the reactive PEG and the antibody or its antigen-binding peptide as described above in relation to condition (2A), for example, the embodiment described below.

[0112] When conjugating lipid particles containing reactive PEG lipids having maleimide groups with an antibody or its antigen-binding fragment having a thiol group, an appropriate amount of the antibody, etc., should be added to a dispersion of the lipid particles prepared using an appropriate buffer, and the mixture should be reacted under appropriate conditions (e.g., pH 6.5-7.5, approximately 20-25°C (room temperature), several minutes to several hours).

[0113] When conjugating lipid particles containing reactive PEG lipids having azide groups with an antibody having DBCO or its antigen-binding fragment, an appropriate amount of the antibody, etc., should be added to a dispersion of the lipid particles prepared using an appropriate buffer, and the reaction should be carried out under appropriate conditions (e.g., pH 7.0-7.5, approximately 20-25°C (room temperature), several minutes to several hours).

[0114] When conjugating lipid particles containing reactive PEG lipids having glycine residues or amino groups with an antibody or antigen-binding fragment having a recognition sequence (LPXTG) for saltase A, the antibody etc. dissolved in a suitable buffer, a dispersion of the lipid particles prepared using a suitable buffer (organic solvent and / or buffer), and a dispersion of saltase A prepared using a suitable buffer are prepared under suitable conditions (e.g., pH 7.0-8.5, calcium (e.g., CaCl)). ２ The reaction should be carried out in the presence of the following: at 4 to 40°C, preferably about 20 to 25°C (room temperature), for several minutes to 24 hours.

[0115] (1-3) Other steps (purification steps, etc.) The method for producing lipid particles of the present invention may, if necessary, utilize steps other than the lipid particle preparation step and the conjugate step, such as a purification step, which may include chromatography (size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, affinity chromatography, etc.).

[0116] (2) Pre method The Pre method is a manufacturing method that includes the steps of preparing targeted PEG lipids by binding antibodies or the like to reactive PEG lipids, and preparing lipid particles using the obtained targeted PEG lipids.

[0117] (2-1) Targeted PEG Lipid Preparation Process In the targeted PEG lipid preparation process of the Pre method, a reactive PEG lipid is reacted with an antibody or its antigen-binding fragment to form a covalent bond. The embodiment of this targeted PEG lipid preparation process can be one that corresponds to the form of covalent bonding between the reactive PEG and the antibody or its antigen-binding peptide as described above in relation to condition (2A), for example, the embodiment described below.

[0118] As the reactive PEG lipid, a reactive PEG lipid as described herein in relation to the first or second embodiment of the lipid particles can be used. The concentration of the reactive PEG lipid in the solvent is preferably 0.5 to 100 mg / mL.

[0119] When covalently bonding an antibody or its antigen-binding fragment containing a thiol group to a reactive PEG lipid containing a maleimide group, an appropriate amount of the antibody, etc., should be added to a solution of the reactive PEG lipid prepared using an appropriate solvent (organic solvent and / or buffer), and the reaction should be carried out under appropriate conditions (e.g., pH 6.5 to 7.5, approximately 20 to 25°C (room temperature), several minutes to several hours).

[0120] When covalently bonding an antibody containing DBCO or its antigen-binding fragment to a reactive PEG lipid having an azide group, an appropriate amount of the antibody, etc., should be added to a solution of the reactive PEG lipid prepared using an appropriate solvent (organic solvent and / or buffer), and the reaction should be carried out under appropriate conditions (e.g., pH 7.0 to 8.5, approximately 20 to 25°C (room temperature), several minutes to several hours).

[0121] When covalently binding an antibody or its antigen-binding fragment having a recognition sequence for saltase A (LPXTG) to a reactive PEG lipid having a glycine residue or an amino group, the antibody, etc., dissolved in a suitable buffer, a suitable amount of the reactive PEG lipid prepared using a suitable solvent (organic solvent and / or buffer), and a suitable amount of saltase A prepared using a suitable buffer are prepared under suitable conditions (e.g., pH 7.0-8.5, calcium (e.g., CaCl)). ２ The reaction should be carried out in the presence of the following: at 4 to 40°C, preferably about 20 to 25°C (room temperature), for several minutes to 24 hours.

[0122] The organic solvent and buffer used in the targeted PEG lipid preparation step of the Pre method can be the same as those described in relation to the lipid particle preparation step of the Post method.

[0123] After reacting a reactive PEG lipid with an antibody or its antigen-binding fragment to form a covalent bond, it is desirable to further perform a purification process to remove molecules other than the reactive PEG lipid to which the antibody, etc., has covalently bonded (i.e., reactive PEG lipids that did not covalently bond with the antibody, etc., and antibodies, etc., that did not conjugate with the reactive PEG lipid). In such a purification process, as described above in relation to the first embodiment of the lipid particles of the present invention, for example, dialysis filtration, ultrafiltration (normal flow filtration, tangential flow filtration, etc.), and chromatography (size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, affinity chromatography, etc.) can be used.

[0124] (2-2) Lipid Particle Preparation Process In the lipid particle preparation process of the Pre method, the targeted PEG lipid prepared separately by the above process and other lipids contained in the lipid components constituting the lipid particles are used to perform a process to form lipid particles conjugated with antibodies, etc. Such a process may involve, for example, mixing an organic solvent solution in which lipids other than the reactive PEG lipid contained in the lipid components constituting the lipid particles are dissolved with water or a buffer solution using various emulsification methods, microfluidic mixing systems, etc., to prepare lipid particles containing the lipid components in advance, and then adding a solution of the targeted PEG lipid to the dispersion of these lipid particles and mixing. The Pre method is not particularly limited as long as at least a portion of the targeted PEG lipid is prepared before the formation of the lipid particles containing the targeted PEG lipid. One embodiment of the Pre method is, for example, a process of mixing (a) an organic solvent solution containing lipids other than reactive PEG lipids (organic solvent phase), (b) an aqueous solution or buffer solution containing an active ingredient such as nucleic acid (aqueous phase), and (c) an organic solvent solution (organic solvent phase) or aqueous solution or buffer solution (aqueous phase) containing targeted PEG lipids, preferably in a microfluidic mixing system, more preferably through a flow path designed to mix the organic solvent phase (a) and aqueous phase (b) in the system before mixing the organic solvent phase or aqueous phase (c), and after the mixing process, purifying the resulting mixture by removing the organic solvent by dialysis filtration or the like (see Example 4 below). Alternatively, lipid particles conjugated with antibodies can also be formed by preparing first lipid particles with the targeted PEG lipid and some of the other lipids contained in the lipid component, preparing second lipid particles with the remaining lipids contained in the lipid component, and mixing them. Water or an acidic buffer is preferred as the buffer for preparing the organic solvent phase or aqueous phase (c) (including when it is a mixed solvent) containing targeted PEG lipids.

[0125] The production method of the lipid particles of the first embodiment of the present invention by the Post method or Pre method can be adjusted in the Post method or Pre method described above to obtain lipid particles that satisfy the two conditions mentioned above in relation to the first embodiment of the lipid particles: (1A) the antibody or its antigen-binding fragment is covalently bonded to at least a portion of the PEG lipid contained in the lipid components constituting the lipid particle, and (1B) the ratio of PEG lipids that are not covalently bonded to the antibody or its antigen-binding fragment and have 30 or more carbon atoms in their hydrophobic carbon chain to the total lipid components constituting the lipid particle is within a certain range.

[0126] When producing the first embodiment of the lipid particles of the present invention by the Post method, (i) the sum of the amount of reactive PEG lipids having 30 or more carbon atoms in their hydrophobic carbon chains (a) and the amount of non-reactive PEG lipids having 30 or more carbon atoms in their hydrophobic carbon chains (b) (a + b) can be kept within a certain range relative to the total lipid components used as raw materials, for example, 0.1 mol% or less, or (ii) the sum of the amount of reactive PEG lipids having 30 or more carbon atoms in their hydrophobic carbon chains that are not covalently bonded to an antibody or its antigen-binding fragment (a') and non-reactive PEG lipids having 30 or more carbon atoms in their hydrophobic carbon chains (b) (a' + b) can be kept within a certain range relative to the total lipid components used as raw materials, for example, 0.1 mol% or less.

[0127] On the other hand, when producing the first embodiment of the lipid particles of the present invention by the Pre method, a molecule in which PEG lipid and antibody or antigen-binding fragment are covalently bonded (targeted PEG lipid), obtained by preparing a reaction product of PEG lipid and antibody or antigen-binding fragment and purifying it as necessary, can be incorporated into the raw materials for all lipid components constituting the lipid particles.

[0128] The production method of the second embodiment of the lipid particles of the present invention by the Post method or Pre method can be adjusted in the Post method or Pre method described above to obtain lipid particles that satisfy the two conditions mentioned above in relation to the second embodiment of the lipid particles, namely, (2A) the antibody or its antigen-binding fragment is covalently bound to at least a portion of the lipid having a reactive PEG chain (reactive PEG lipid) contained in the lipid components constituting the lipid particle, and (2B) the ratio of reactive PEG lipid that is not covalently bound to the antibody or its antigen-binding fragment to the total lipid components constituting the lipid particle is 0.1 mol% or less.

[0129] When producing the second embodiment of the lipid particles of the present invention by the Post method, (i) the amount of reactive PEG lipids (a) may be 0.1 mol% or less relative to the total lipid components used as raw materials, or (ii) the amount of reactive PEG lipids that are not covalently bound to antibodies or their antigen-binding fragments (a') may be 0.1 mol% or less relative to the total lipid components used as raw materials.

[0130] On the other hand, when producing the second embodiment of the lipid particles of the present invention by the Pre method, a reaction product of reactive PEG lipid and an antibody or its antigen-binding fragment can be prepared, purified as necessary, and the molecule in which the reactive PEG lipid and the antibody or its antigen-binding fragment are covalently bonded (targeted PEG lipid) can be incorporated into the raw materials for all lipid components constituting the lipid particles.

[0131] ―Method for Producing a Nucleic Acid-Introducing Composition― The nucleic acid-introducing composition of the present invention can be produced by adding nucleic acid to water or a buffer solution that is mixed with an organic solvent solution of lipid components in the step of preparing a dispersion of lipid particles, which is included in the method for producing lipid particles of the present invention as described above (i.e., a nucleic acid-introducing composition is obtained as equivalent to a dispersion of lipid particles). In such a production method (step), it is preferable to add nucleic acid in an amount such that the concentration in water or buffer solution is 0.05 to 2.0 mg / mL. An acidic buffer solution is preferred as the buffer solution for preparing the aqueous phase containing nucleic acid.

[0132] Further, the composition for nucleic acid introduction of the present invention can also be produced by mixing a dispersion of the lipid particles of the present invention obtained by the production method as described above and a nucleic acid by a known method.

[0133] In the composition for nucleic acid introduction of the present invention, the ratio (mass ratio) of the nucleic acid (regardless of whether it is encapsulated in the lipid particles or not) to the lipid particles of the present invention is preferably 1 to 20%.

[0134] The encapsulation rate of the nucleic acid in the composition for nucleic acid introduction of the present invention, that is, the ratio (weight ratio) of the nucleic acid encapsulated in the lipid particles rather than dissolved in the solvent to the total amount of the nucleic acid in the composition for nucleic acid introduction is preferably 90% or more. The encapsulation rate of the nucleic acid can be calculated based on, for example, the difference in fluorescence intensity depending on the presence or absence of addition of a surfactant (e.g., Triton-X100) that disintegrates the lipid particles after fluorescently labeling the nucleic acid with Quant-iT ＴＭ Ribogreen (registered trademark) (Invitrogen).

[0135] In the present specification, matters described with respect to a certain category of the present invention (for example, lipid particles) can be referred to while being appropriately rephrased as matters related to other categories (for example, a method for producing lipid particles, a composition for nucleic acid introduction, a nucleic acid introduction method, a method for improving the possibility of readministration).

[0136] The present invention will be further described in detail by the following examples, production examples and test examples, but these do not limit the present invention and may be changed without departing from the scope of the present invention.

[0137] In the following examples, "DSPE-PEG(2000) Azide" (AVANTI), "DSPE-PEG(3400) Azide" (Production Example 4), and "DSPE-PEG(5000) Azide" (AVANTI) are all compounds having a total of 36 carbon atoms in the hydrophobic carbon chain (2 stearoyl groups), and the average molecular weights of the PEG chains are 2000, 3400, and 5000, respectively, and each has an azide group as a reactive group for covalent bonding with antibodies, etc. "DPPE-PEG(2000) Azide" (AVANTI) and "DPPE-PEG(5000) Azide" (Production Example 5) are both compounds having a total of 32 carbon atoms in the hydrophobic carbon chain (2 palmitoyl groups), with average molecular weights of 2000 and 5000 respectively in the PEG chain, and possessing an azide group as a reactive group for covalent bonding with antibodies, etc. "DSPE-PEG(5000) Maleimide" (NOF CORPORATION) is a compound having a total of 36 carbon atoms in the hydrophobic carbon chain (2 stearoyl groups), with an average molecular weight of 5000 in the PEG chain, and possessing a maleimide group as a reactive group for covalent bonding with antibodies, etc. Gly-PEG(5000)-DSPE (Example 7) is a compound with a total of 36 carbon atoms in its hydrophobic carbon chain (2 stearoyl groups), an average molecular weight of 5000 in the PEG chain, and an N-terminal glycine group as a reactive group for covalent bonding with antibodies, etc. "SUNBRIGHT GM-020" (NOF CORPORATION) is a non-reactive PEG lipid with a total of 28 carbon atoms in its hydrophobic carbon chain and an average molecular weight of 2000 in the PEG chain.

[0138] [Preparation Example 1] Preparation of mCD3Fab-DBCO, hCD7Fab-DBCO, and hCD3Fab-DBCO Fab-DBCO was prepared by performing the following two steps. DBCO / Antibody ratio (DAR) and protein concentration were quantified by LC-MS and BCA methods, respectively. The results of the physical property evaluation are shown in Table 1.

[0139] Preparation process for Fabs having LPETGG-His6 at the C-terminus: Plasmid pMG2.2 vector encoding the mCD3Fab (clone: ​​145-2C11) gene, the hCD7Fab (Fab fragment of grisnilimap) gene, or hCD3 (clone: ​​OKT3) was introduced into CHOZN cells using an electroporation device (Maxcyte), and the cells were cultured for 6-8 days using EX-CELL Advanced CHO Feed 1 (with glucose). Subsequently, the samples were purified using a compacte Ni column and a Superdex 200 size exclusion column to prepare LPETGG-His-tagged Fab.

[0140] Preparation process for Fab-DBCO: LPETGG-His-tagged Fab is mixed with CaCl in a pH 7.4 HEPES buffer. ２ The mixture was then miscible with 5-(glycylglycyl-beta-alanyl)-11,12-didehydro-5,6-dihydrodibenzo[b,f]azosin and treated with Sortase A (P94S / D160N / D165A / K196T, Processings of the National Academy of Sciences of the United States of America. 2011;108:11399-11404) (Current Protocols in Protein Science, 89, 15.3.1-15.3.19). The treated product was purified by Ni column and dialysis to obtain Fab-DBCO.

[0141]

[0142] [Preparation Example 2] Preparation of mCD3Fab-PEG(5000)-DSPE mCD3Fab-DBCO solution (Preparation Example 1), an aqueous solution of DSPE-PEG(5000)Azide, and SUNBRIGHT GM-020 were mixed in amounts such that each compound was in a molar ratio of 1:1:10, and the mixture was reacted at 25°C for 24 hours to obtain mCD3Fab-PEG(5000)-DSPE.

[0143] [Preparation Example 3] Preparation of mCD3Fab' mCD3Fab' was obtained by adding 2.4 equivalents of TCEP Solution (Fujifilm) to mCD3F(ab') (BIO X Cell) and reacting at 25°C for 3 hours.

[0144] [Production Example 4] Production of DSPE-PEG (3400) Azide N ３ -PEG3.4K-CH ２ A mixture of COOH (250 mg), N-hydroxysuccinimide (16.5 mg), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (27 mg), and dichloromethane (5 mL) was stirred at 30°C for 18 hours and then concentrated under reduced pressure. The resulting residue, DSPE (78 mg), N,N-diisopropylethylamine (18 mg), and dichloromethane (5 mL) were stirred at 30°C for 18 hours. After concentrating the mixture under reduced pressure, it was purified by preparative HPLC (Boston Prime C18, water (ammonium carbonate) / acetonitrile:THF = 2:1) to obtain the title compound (170 mg). 1 H NMR (400 MHz, CDCl3) δ = 8.08 - 7.95 (m, 1H), 7.06 - 6.81 (m, 2H), 5.27 - 5.14 (m, 1H), 4.44 - 4.30 (m, 1H), 4.24 - 3.19 (m, 298H), 2.28 (q, J = 7.1 Hz, 4H), 1.58 (d, J = 1.9 Hz, 4H), 1.34 - 1.18 (m, 58H), 0.91 - 0.81 (m, 6H).

[0145] [Preparation Example 5] Preparation of mCD3Fab-PEG(2000)-DSPE mCD3Fab-DBCO solution (Preparation Example 1), an aqueous solution of DSPE-PEG(2000)Azide, and SUNBRIGHT GM-020 were mixed in amounts such that each compound was in a molar ratio of 1:1:10, and the mixture was reacted at 25°C for 24 hours to obtain mCD3Fab-PEG(2000)-DSPE.

[0146] [Preparation Example 6] Preparation of mCD3Fab-PEG(3400)-DSPE mCD3Fab-DBCO solution (Preparation Example 1), aqueous solution of DSPE-PEG(3400)Azide (Preparation Example 4), and SUNBRIGHT GM-020 were mixed in amounts such that each compound was in a molar ratio of 1:1:10, and the mixture was reacted at 25°C for 24 hours to obtain mCD3Fab-PEG(3400)-DSPE.

[0147] [Preparation Example 7] Preparation of Gly-PEG(5000)-DSPE A mixture of N-(tert-butoxycarbonyl)glycine (9 g), 4-nitrophenol (7.15 g), EDCI (12.8 g), DMAP (3.14 g), and DCM (45 mL) was stirred at room temperature for 12 hours, then poured into water and extracted with DCM. The organic layer was washed with saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether) and preparative HPLC (Welch Ultimate XB-CN, hexane / ethanol) to obtain 4-nitrophenyl N-(tert-butoxycarbonyl)glycinate (1 g). A mixture of DSPE-PEG(5000)Amine (600 mg), 4-nitrophenyl N-(tert-butoxycarbonyl)glycinate (93 mg), N,N-diisopropylethylamine (54 mg), and DCM (8 mL) was stirred at 30°C for 12 hours. The mixture was concentrated under reduced pressure, MeOH was added to the residue, and then dialyzed for 24 hours using an RC dialysis membrane (1000D) with MeOH / ion-exchanged water to obtain Boc-Gly-PEG(5000)-DSPE (540 mg). Trifluoroacetic acid (3 mL) was added to a mixture of Boc-Gly-PEG(5000)-DSPE (300 mg) and DCM (3 mL), and the mixture was stirred at 35°C for 2 hours. The mixture was concentrated under reduced pressure, the residue was neutralized with saturated sodium bicarbonate aqueous solution, and then acetonitrile was added. The obtained solution was dialyzed for 12 hours using an RC dialysis membrane (1000D) with deionized water, and then purified by preparative HPLC (Welch Xtimate C4, water (HCl) / acetonitrile) to obtain Gly-PEG(5000)-DSPE (115 mg). 1H NMR (400 MHz, CDCl3) δ 7.98 - 7.73 (m, 2H), 5.24-5.20 (m, 1H), 4.44 - 4.26 (m, 2H), 4.24 - 4.11 (m, 2H), 4.09 - 3.26 (m, 406H), 2.31 - 2.26 (m, 4H), 1.65 - 1.51 (m, 4H), 1.25-1.22 (m, 56H), 0.88 - 0.85 (m, 6H).

[0148] [Preparation Example 8] Preparation of hCD3Fab-PEG(5000)-DSPE A PBS solution (2.66 mL) of LPETGG-His-tagged hCD3Fab (clone: ​​OKT3, 15 mg) obtained in the first step of Preparation Example 1 was mixed with 5X conjugation buffer (250 mM HEPES, 750 mM NaCl, 50% glycerol (v / v), pH 7.4, 811 μL), N,N-dimethylacetamide solution (10 mM, 469 μL) of Gly-PEG(5000)-DSPE (Preparation Example 7), and CaCl ２A 15 μL aqueous solution, 20 mM Tris buffer (containing 150 mM NaCl and 10% Glycerol (v / v), pH 8.0, 6.7 μL) containing mutant saltase A (P94S / D160N / D165A / K196T, Proceedings of the National Academy of Sciences of the United States of America. 2011;108:11399-11404, 0.01 equivalent), and water (89 μL) were mixed at 22°C. After 18 hours, EDTA solution (0.2 M, 40.5 μL) was added, and the mixture was purified by preparative HPLC (HiTrap SP HP, mobile phase A: 20 mM acetate buffer at pH 5.0, mobile phase B: 20 ​​mM acetate buffer at pH 5.0 containing 50% ethanol (v / v) and 180 mM NaCl, gradient: 30-100% B). The resulting solution was concentrated with Amicon (10 K), dialyzed against PBS, and filtered through a 0.22 μm filter to obtain hCD3Fab-PEG(5000)-DSPE (5.83 mg). The MS value before the reaction was m / z 50916, while the representative MS value after the reaction was m / z 55623. No unreacted reactive PEG lipids were detected by HPLC analysis.

[0149] The HPLC analysis conditions are as follows: Mobile phase A: 0.05% trifluoroacetic acid (TFA) aqueous solution Mobile phase B: 0.05% TFA acetonitrile solution Detection method: Evaporative light scattering detector (ELSD) Column: Agilent PLRP-S

[0150] [Comparative Examples 1-2] Preparation of Control LNP1 and Control LNP2 (Post Method) Preparation of Azide-LNP mRNA encoding a CD19-targeted CAR having CD28 and CD3ζ as intracellular signaling domains (TriLink) was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.5) to obtain a 0.2 mg / ml nucleic acid solution.

[0151] mRNA encoding a CD19-targeting CAR, which has CD28 and CD3ζ as intracellular signaling domains, was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.5) to obtain a 0.2 mg / ml nucleic acid solution.

[0152] The lipid solutions for preparing control LNP1 and control LNP2, along with the nucleic acid solution, were heated at room temperature using a nanoassembly method. ＴＭ Ignite ＴＭ The mixture was prepared using a Precision Nanosystems at a flow rate ratio of 3 ml / min to 6 ml / min to obtain a dispersion containing the composition. The resulting dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4°C for 24 hours using Slide-A-Lyzer Dialesis (20K molecular weight cutoff, Thermo Fisher Scientific). Subsequently, the dispersion was concentrated by ultrafiltration using Amicon Ultra (30K molecular weight cutoff, Merck) and then filtered through a 0.2 μm syringe filter. The final mRNA concentration was adjusted to 350 μg / mL and stored at 4°C.

[0153] The binding reaction process between mCD3Fab-DBCO and Azide-LNP was performed by mixing the mCD3Fab-DBCO solution (Preparation Example 1) with the respective Azide-LNP dispersions for preparing Control LNP1 and Control LNP2, so that the molar concentration of Fab-DBCO relative to Azide was 3 / 100. The mixture was then reacted at 25°C for 24 hours. After that, the sucrose concentration was adjusted to 20%, and the mixture was stored at -80°C.

[0154] [Comparative Example 3] Preparation of Control LNP3 (Post Method) Preparation of Azide-LNP For the preparation of control LNP3, a lipid mixture (ionized lipid: DPPC: cholesterol: SUNBRIGHT GM-020: DSPE-PEG (5000) Azide = 60: 10.6: 27: 1.4: 1, mol%) was dissolved in 90% EtOH to obtain a lipid solution of 9.2 mg / ml. As the ionized lipid (cationic lipid), 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis(((3-pentyloctanoyl)oxy)methyl)propyl 3-pentyloctanoate, as described in WO2016 / 021683, was used.

[0155] mRNA (Elixirgen) encoding a GPC3 target CAR having CD28 and CD3ζ as intracellular signaling domains was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer at pH 5.5 to obtain a nucleic acid solution of 0.2 mg / ml.

[0156] The obtained lipid solution and nucleic acid solution were heated at room temperature using NanoAssemblr. ＴＭ Ignite ＴＭ The mixture was prepared using a Precision Nanosystems at a flow rate ratio of 3 ml / min to 6 ml / min to obtain a dispersion containing the composition. The resulting dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4°C for 24 hours using Slide-A-Lyzer Dialesis (20K molecular weight cutoff, Thermo Fisher Scientific). Subsequently, the dispersion was concentrated by ultrafiltration using Amicon Ultra (30K molecular weight cutoff, Merck) and then filtered through a 0.2 μm syringe filter. The final mRNA concentration was adjusted to 350 μg / mL and stored at 4°C.

[0157] The binding reaction step of hCD7Fab-DBCO and Azide-LNP was performed by mixing the hCD7Fab-DBCO solution (Preparation Example 1) with the Azide-LNP dispersion for the preparation of control LNP1 so that the molar concentration of Fab-DBCO relative to Azide was 1 / 100, and the mixture was reacted at 25°C for 24 hours. After that, the sucrose concentration was adjusted to 20%, and the mixture was stored at -80°C.

[0158] [Comparative Example 4] Preparation of Control LNP4 (Post Method) Preparation of Azide-LNP For the preparation of control LNP4, a lipid mixture (ionized lipid: DSPC: cholesterol: SUNBRIGHT GM-020: DPPE-PEG (2000) Azide = 60: 10.6: 27: 1.4: 1, mol%) was dissolved in 90% EtOH to obtain a lipid solution of 9.3 mg / ml. As the ionized lipid (cationic lipid), 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis(((3-pentyloctanoyl)oxy)methyl)propyl3-pentyloctanoate described in WO2016 / 021683 was used.

[0159] mRNA (Elixirgen) encoding a CD19-targeting CAR having CD28 and CD3ζ as intracellular signaling domains was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer at pH 5.5 to obtain a nucleic acid solution of 0.2 mg / ml.

[0160] The obtained lipid solution and nucleic acid solution were mixed at room temperature using a Nanoassembly™ Ignite™ instrument (Precision Nanosystems) at a flow rate ratio of 3 ml / min:6 ml / min to obtain a dispersion containing the composition. The obtained dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4°C for 24 hours using Slide-A-Lyzer Dialesis (20K molecular weight cutoff, Thermo Fisher Scientific). Subsequently, the dispersion was concentrated by ultrafiltration using Amicon Ultra (30K molecular weight cutoff, Merck) and then filtered through a 0.2 μm syringe filter. The final mRNA concentration was adjusted to 500 μg / mL and stored at 4°C.

[0161] The binding reaction process between mCD3Fab-DBCO and Azide-LNP was performed. The mCD3Fab-DBCO solution (Preparation Example 1) was mixed with the Azide-LNP dispersion for the preparation of control LNP4 so that the molar concentration of Fab-DBCO relative to Azide was 1 / 100, and the mixture was reacted at 25°C for 24 hours. After that, the sucrose concentration was adjusted to 20%, and the mixture was stored at -80°C.

[0162] [Comparative Example 5] Preparation of Control LNP5 (Post Method) Preparation of Maleimide-LNP For the preparation of Control LNP5, a lipid mixture (ionized lipid: DSPC: Cholesterol: SUNBRIGHT GM-020: DSPE-PEG(5000) Maleimide = 60:10.6:27:1.4:1, mol%) was dissolved in 90% EtOH to obtain a lipid solution of 9.3 mg / ml. As the ionized lipid (cationic lipid), 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis(((3-pentyloctanoyl)oxy)methyl)propyl3-pentyloctanoate described in WO2016 / 021683 was used.

[0163] mRNA (Elixirgen) encoding a CD19-targeting CAR having CD28 and CD3ζ as intracellular signaling domains was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer at pH 5.5 to obtain a nucleic acid solution of 0.2 mg / ml.

[0164] The obtained lipid solution and nucleic acid solution were mixed at room temperature using a Nanoassembly™ Ignite™ instrument (Precision Nanosystems) at a flow rate ratio of 3 ml / min:6 ml / min to obtain a dispersion containing the composition. The obtained dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4°C for 24 hours using Slide-A-Lyzer Dialesis (20K molecular weight cutoff, Thermo Fisher Scientific). Subsequently, the dispersion was concentrated by ultrafiltration using Amicon Ultra (30K molecular weight cutoff, Merck) and then filtered through a 0.2 μm syringe filter. The final mRNA concentration was adjusted to 500 μg / mL and stored at 4°C.

[0165] The binding reaction step between mCD3Fab' and Maleimide-LNP was performed. The mCD3Fab' solution (Preparation Example 3) was mixed with the Maleimide-LNP dispersion for the preparation of control LNP5 so that the molar concentration of Fab' relative to Maleimide was 1 / 100, and the mixture was reacted at 25°C for 24 hours. After that, the sucrose concentration was adjusted to 20%, and the mixture was stored at -80°C.

[0166] [Comparative Example 6] Preparation of Control LNP6 (Post Method) Preparation of Azide-LNP For the preparation of control LNP6, a lipid mixture (ionized lipid: DSPC: cholesterol: SUNBRIGHT GM-020: DSPE-PEG(5000) Azide = 60: 10.6: 27.7: 1.4: 0.3, mol%) was dissolved in 90% EtOH to obtain a lipid solution of 9.3 mg / ml. As the ionized lipid (cationic lipid), 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis(((3-pentyloctanoyl)oxy)methyl)propyl3-pentyloctanoate, as described in WO2016 / 021683, was used.

[0167] mRNA (Elixirgen) encoding a CD19-targeting CAR having CD28 and CD3ζ as intracellular signaling domains was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer at pH 5.5 to obtain a nucleic acid solution of 0.2 mg / ml.

[0168] The obtained lipid solution and nucleic acid solution were mixed at room temperature using a Nanoassembly™ Ignite™ instrument (Precision Nanosystems) at a flow rate ratio of 3 ml / min:6 ml / min to obtain a dispersion containing the composition. The obtained dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4°C for 24 hours using Slide-A-Lyzer Dialesis (20K molecular weight cutoff, Thermo Fisher Scientific). Subsequently, the dispersion was concentrated by ultrafiltration using Amicon Ultra (30K molecular weight cutoff, Merck) and then filtered through a 0.2 μm syringe filter. The final mRNA concentration was adjusted to 500 μg / mL and stored at 4°C.

[0169] The binding reaction process between mCD3Fab-DBCO and Azide-LNP was performed. The mCD3Fab-DBCO solution (Preparation Example 1) was mixed with the Azide-LNP dispersion for the preparation of control LNP6 so that the molar concentration of Fab-DBCO relative to Azide was 1 / 30, and the mixture was reacted at 25°C for 24 hours. After that, the sucrose concentration was adjusted to 20%, and the mixture was stored at -80°C.

[0170] [Examples 1-3] Preparation of LNP1, LNP2 and LNP3 (Post method) Preparation of Azide-LNP For the preparation of LNP1, a lipid mixture (ionized lipid: DPPC: cholesterol: SUNBRIGHT GM-020: DSPE-PEG (2000) Azide = 60: 10.6: 27.9: 1.4: 0.1, mol%) was dissolved in 90% EtOH to obtain a lipid solution of 9.3 mg / ml. For the preparation of LNP2 and LNP3, a lipid mixture (ionized lipid: DPPC: cholesterol: SUNBRIGHT GM-020: DSPE-PEG(5000) Azide = 60: 10.6: 27.9: 1.4: 0.1, mol%) was dissolved in 90% EtOH to obtain a lipid solution of 9.3 mg / ml. As the ionized lipid (cationic lipid), 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis(((3-pentyloctanoyl)oxy)methyl)propyl3-pentyloctanoate, as described in WO2016 / 021683, was used.

[0171] A 0.2 mg / ml nucleic acid solution was obtained by dissolving a CD19-targeting CAR mRNA (TriLink), which has CD28 and CD3ζ as intracellular signaling domains, in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.5).

[0172] The lipid solutions for LNP1 preparation, LNP2 preparation, and LNP3 preparation, along with the nucleic acid solution, were heated at room temperature using NanoAssemblr. ＴＭ Ignite ＴＭThe mixture was prepared using a Precision Nanosystems at a flow rate ratio of 3 ml / min to 6 ml / min to obtain a dispersion containing the composition. The resulting dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4°C for 24 hours using Slide-A-Lyzer Dialesis (20K molecular weight cutoff, Thermo Fisher Scientific). Subsequently, the dispersion was concentrated by ultrafiltration using Amicon Ultra (30K molecular weight cutoff, Merck) and then filtered through a 0.2 μm syringe filter. The final mRNA concentration was adjusted to 350 μg / mL and stored at 4°C.

[0173] The binding reaction process between mCD3Fab-DBCO and Azide-LNP was performed. The mCD3Fab-DBCO solution (Preparation Example 1) was mixed with the respective Azide-LNP dispersions for LNP1 and LNP2 preparation so that the molar concentration of Fab-DBCO relative to Azide was 3 / 10, and the mixture was reacted at 25°C for 24 hours. The mCD3Fab-DBCO solution (Preparation Example 1) was also mixed with the Azide-LNP dispersion for LNP3 preparation so that the molar concentration of Fab-DBCO relative to Azide was 1 / 10, and the mixture was reacted at 25°C for 24 hours. After that, the sucrose concentration was adjusted to 20%, and the mixture was stored at -80°C.

[0174] [Example 4] Preparation of LNP4 (Pre method) For the preparation of LNP4, a lipid mixture (ionized lipid: DSPC: cholesterol: SUNBRIGHT GM-020 = 60: 10.6: 27.99: 1.3, mol%) was dissolved in 90% EtOH to obtain a lipid solution of 9.3 mg / ml. As the ionized lipid (cationic lipid), 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis(((3-pentyloctanoyl)oxy)methyl)propyl3-pentyloctanoate, as described in WO2016 / 021683, was used.

[0175] An mRNA encoding a CD19-targeting CAR (TriLink), which has CD28 and CD3ζ as intracellular signaling domains, was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.5) to obtain a 0.2 mg / ml nucleic acid solution.

[0176] Furthermore, 0.01 mol% of mCD3Fab-PEG(5000)-DSPE (Preparation Example 2) relative to total lipids was diluted with water to obtain an antibody solution with a concentration of 0.02 mg / ml (Fab concentration).

[0177] The resulting lipid solution, nucleic acid solution, and antibody solution were heated at room temperature using Nanoassemblr. ＴＭ Ignite ＴＭ The components were mixed using a Precision Nanosystem at flow rates of 3 ml / min:6 ml / min:9 ml / min to obtain a dispersion containing the composition. At this time, the antibody solution was mixed through a channel for in-line dilution. The obtained dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4°C for 24 hours using Slide-A-Lyzer Dialesis (20 K molecular weight cutoff, Thermo Fisher Scientific). Subsequently, the dispersion was concentrated by ultrafiltration using Amicon Ultra (30 K molecular weight cutoff, Merck) and then filtered using a 0.2 μm syringe filter. The final mRNA concentration was adjusted to 200 μg / mL and the sucrose concentration to 20%, and the solution was stored at -80°C.

[0178] [Example 5] Preparation of LNP5 (Post method) Steps for preparing Azide-LNP For the preparation of LNP5, a lipid mixture (ionized lipid: DPPC: cholesterol: SUNBRIGHT GM-020: DSPE-PEG (5000) Azide = 60: 10.6: 27.9: 1.4: 0.1, mol%) was dissolved in 90% EtOH to obtain a lipid solution of 9.3 mg / ml. As the ionized lipid (cationic lipid), 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis(((3-pentyloctanoyl)oxy)methyl)propyl 3-pentyloctanoate, as described in WO2016 / 021683, was used.

[0179] mRNA (Elixirgen) encoding a GPC3 target CAR having CD28 and CD3ζ as intracellular signaling domains was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer at pH 5.5 to obtain a nucleic acid solution of 0.2 mg / ml.

[0180] The obtained lipid solution and nucleic acid solution were heated at room temperature using NanoAssemblr. ＴＭ Ignite ＴＭ The mixture was obtained by mixing the components at a flow rate ratio of 3 ml / min to 6 ml / min using a Precision Nanosystems. The resulting dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4°C for 24 hours using Slide-A-Lyzer Dialesis (20K molecular weight cutoff, Thermo Fisher Scientific). Subsequently, the dispersion was concentrated by ultrafiltration using Amicon Ultra (30K molecular weight cutoff, Merck) and then filtered through a 0.2 μm syringe filter. The final mRNA concentration was adjusted to 500 μg / mL and stored at 4°C.

[0181] The binding reaction process between hCD7Fab-DBCO and Azide-LNP was performed. The hCD7Fab-DBCO solution (Preparation Example 1) was mixed with the Azide-LNP dispersion for LNP5 preparation so that the molar concentration of Fab-DBCO relative to Azide was 1 / 10, and the mixture was reacted at 25°C for 24 hours. After that, the sucrose concentration was adjusted to 20%, and the mixture was stored at -80°C.

[0182] [Example 6] Preparation of LNP6 (Post method) Steps for preparing Azide-LNP For the preparation of LNP6, a lipid mixture (ionized lipid: DSPC: cholesterol: SUNBRIGHT GM-020: DPPE-PEG (2000) Azide = 60: 10.6: 27.9: 1.4: 0.1, mol%) was dissolved in 90% EtOH to obtain a lipid solution of 9.3 mg / ml. As the ionized lipid (cationic lipid), 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis(((3-pentyloctanoyl)oxy)methyl)propyl 3-pentyloctanoate, as described in WO2016 / 021683, was used.

[0183] mRNA (Elixirgen) encoding a CD19-targeting CAR having CD28 and CD3ζ as intracellular signaling domains was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer at pH 5.5 to obtain a nucleic acid solution of 0.2 mg / ml.

[0184] The obtained lipid solution and nucleic acid solution were mixed at room temperature using a Nanoassembly™ Ignite™ instrument (Precision Nanosystems) at a flow rate ratio of 3 ml / min:6 ml / min to obtain a dispersion containing the composition. The obtained dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4°C for 24 hours using Slide-A-Lyzer Dialesis (20K molecular weight cutoff, Thermo Fisher Scientific). Subsequently, the dispersion was concentrated by ultrafiltration using Amicon Ultra (30K molecular weight cutoff, Merck) and then filtered through a 0.2 μm syringe filter. The final mRNA concentration was adjusted to 500 μg / mL and stored at 4°C.

[0185] The binding reaction process between mCD3Fab-DBCO and Azide-LNP was performed by mixing an mCD3Fab-DBCO solution (Preparation Example 1) with an Azide-LNP dispersion for LNP6 preparation so that the molar concentration of Fab-DBCO relative to Azide was 1 / 10, and the mixture was reacted at 25°C for 24 hours. After that, the sucrose concentration was adjusted to 20%, and the mixture was stored at -80°C.

[0186] [Example 7] Preparation of LNP7 (Post method) Preparation of Maleimide-LNP For the preparation of LNP7, a lipid mixture (ionized lipid: DSPC: cholesterol: SUNBRIGHT GM-020: DSPE-PEG(5000) Maleimide = 60: 10.6: 27.9: 1.4: 0.1, mol%) was dissolved in 90% EtOH to obtain a lipid solution of 9.3 mg / ml. As the ionized lipid (cationic lipid), 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis(((3-pentyloctanoyl)oxy)methyl)propyl3-pentyloctanoate described in WO2016 / 021683 was used.

[0187] mRNA (Elixirgen) encoding a CD19-targeting CAR having CD28 and CD3ζ as intracellular signaling domains was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer at pH 5.5 to obtain a nucleic acid solution of 0.2 mg / ml.

[0188] The obtained lipid solution and nucleic acid solution were heated at room temperature using NanoAssemblr. ＴＭ Ignite ＴＭThe mixture was obtained by mixing the components at a flow rate ratio of 3 ml / min to 6 ml / min using a Precision Nanosystems. The resulting dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4°C for 24 hours using Slide-A-Lyzer Dialesis (20K molecular weight cutoff, Thermo Fisher Scientific). Subsequently, the dispersion was concentrated by ultrafiltration using Amicon Ultra (30K molecular weight cutoff, Merck) and then filtered through a 0.2 μm syringe filter. The final mRNA concentration was adjusted to 500 μg / mL and stored at 4°C.

[0189] The binding reaction step of mCD3Fab' and Maleimide-LNP was performed by mixing the mCD3Fab' solution (Preparation Example 3) with the Maleimide-LNP dispersion for LNP7 preparation so that the molar concentration of Fab' relative to Maleimide was 1 / 10, and the mixture was reacted at 25°C for 24 hours. After that, the sucrose concentration was adjusted to 20%, and the mixture was stored at -80°C.

[0190] [Example 8] Preparation of LNP8 (Pre method) For the preparation of LNP8, a lipid mixture (ionized lipid: DSPC: cholesterol: SUNBRIGHT GM-020 = 60:10.6:27.99:1.3, mol%) was dissolved in 90% EtOH to obtain a lipid solution of 9.3 mg / ml. As the ionized lipid (cationic lipid), 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis(((3-pentyloctanoyl)oxy)methyl)propyl3-pentyloctanoate, as described in WO2016 / 021683, was used.

[0191] An mRNA (Elixirgen) encoding a CD19-targeting CAR, which has CD28 and CD3ζ as intracellular signaling domains, was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.5) to obtain a nucleic acid solution of 0.2 mg / ml.

[0192] Furthermore, 0.01 mol% of mCD3Fab-PEG(2000)-DSPE (Preparation Example 5) relative to total lipids was diluted with water to obtain an antibody solution with a concentration of 0.02 mg / ml (Fab concentration).

[0193] The resulting lipid solution, nucleic acid solution, and antibody solution were heated at room temperature using Nanoassemblr. ＴＭ Ignite ＴＭ The components were mixed using a Precision Nanosystem at flow rates of 3 ml / min:6 ml / min:9 ml / min to obtain a dispersion containing the composition. At this time, the antibody solution was mixed through a channel for in-line dilution. The obtained dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4°C for 24 hours using Slide-A-Lyzer Dialesis (20 K molecular weight cutoff, Thermo Fisher Scientific). Subsequently, the dispersion was concentrated by ultrafiltration using Amicon Ultra (30 K molecular weight cutoff, Merck) and then filtered using a 0.2 μm syringe filter. The final mRNA concentration was adjusted to 200 μg / mL and the sucrose concentration to 20%, and the solution was stored at -80°C.

[0194] [Example 9] Preparation of LNP9 (Pre method) For the preparation of LNP9, a lipid mixture (ionized lipid: DSPC: cholesterol: SUNBRIGHT GM-020 = 60:10.6:27.99:1.3, mol%) was dissolved in 90% EtOH to obtain a lipid solution of 9.3 mg / ml. As the ionized lipid (cationic lipid), 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis(((3-pentyloctanoyl)oxy)methyl)propyl3-pentyloctanoate, as described in WO2016 / 021683, was used.

[0195] An mRNA (Elixirgen) encoding a CD19-targeting CAR, which has CD28 and CD3ζ as intracellular signaling domains, was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.5) to obtain a nucleic acid solution of 0.2 mg / ml.

[0196] Furthermore, 0.01 mol% of mCD3Fab-PEG(3400)-DSPE (Preparation Example 6) relative to total lipids was diluted with water to obtain an antibody solution with a concentration of 0.02 mg / ml (Fab concentration).

[0197] The resulting lipid solution, nucleic acid solution, and antibody solution were heated at room temperature using Nanoassemblr. ＴＭ Ignite ＴＭ The components were mixed using a Precision Nanosystem at flow rates of 3 ml / min:6 ml / min:9 ml / min to obtain a dispersion containing the composition. At this time, the antibody solution was mixed through a channel for in-line dilution. The obtained dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4°C for 24 hours using Slide-A-Lyzer Dialesis (20 K molecular weight cutoff, Thermo Fisher Scientific). Subsequently, the dispersion was concentrated by ultrafiltration using Amicon Ultra (30 K molecular weight cutoff, Merck) and then filtered using a 0.2 μm syringe filter. The final mRNA concentration was adjusted to 200 μg / mL and the sucrose concentration to 20%, and the solution was stored at -80°C.

[0198] [Example 10] Preparation of LNP10 (Pre method) For the preparation of LNP10, a lipid mixture (ionized lipid: DSPC: cholesterol: SUNBRIGHT GM-020 = 60: 10.6: 27.99: 1.3, mol%) was dissolved in 90% EtOH to obtain a lipid solution of 9.3 mg / ml. As the ionized lipid (cationic lipid), 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis(((3-pentyloctanoyl)oxy)methyl)propyl3-pentyloctanoate, as described in WO2016 / 021683, was used.

[0199] An mRNA (Elixirgen) encoding a CD19-targeting CAR, which has CD28 and CD3ζ as intracellular signaling domains, was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.5) to obtain a nucleic acid solution of 0.2 mg / ml.

[0200] Furthermore, 0.01 mol% of mCD3Fab-PEG(5000)-DSPE (Preparation Example 2) relative to total lipids was diluted with water to obtain an antibody solution with a concentration of 0.02 mg / ml (Fab concentration).

[0201] The resulting lipid solution, nucleic acid solution, and antibody solution were heated at room temperature using Nanoassemblr. ＴＭ Ignite ＴＭ The components were mixed using a Precision Nanosystem at flow rates of 3 ml / min:6 ml / min:9 ml / min to obtain a dispersion containing the composition. At this time, the antibody solution was mixed through a channel for in-line dilution. The obtained dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4°C for 24 hours using Slide-A-Lyzer Dialesis (20 K molecular weight cutoff, Thermo Fisher Scientific). Subsequently, the dispersion was concentrated by ultrafiltration using Amicon Ultra (30 K molecular weight cutoff, Merck) and then filtered using a 0.2 μm syringe filter. The final mRNA concentration was adjusted to 200 μg / mL and the sucrose concentration to 20%, and the solution was stored at -80°C.

[0202] [Example 11] Preparation of LNP11 (Pre method) For the preparation of LNP11, a lipid mixture (ionized lipid: DSPC: cholesterol: SUNBRIGHT GM-020 = 60:10.6:27.99:1.3, mol%) was dissolved in 90% EtOH to obtain a lipid solution of 7.7 mg / ml. MC3 (DLin-MC3-DMA) was used as the ionized lipid (cationic lipid).

[0203] An mRNA (Elixirgen) encoding a CD19-targeting CAR, which has CD28 and CD3ζ as intracellular signaling domains, was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.5) to obtain a nucleic acid solution of 0.2 mg / ml.

[0204] Furthermore, 0.01 mol% of mCD3Fab-PEG(5000)-DSPE (Preparation Example 2) relative to total lipids was diluted with water to obtain an antibody solution with a concentration of 0.02 mg / ml (Fab concentration).

[0205] The resulting lipid solution, nucleic acid solution, and antibody solution were heated at room temperature using Nanoassemblr. ＴＭ Ignite ＴＭ The components were mixed using a Precision Nanosystem at flow rates of 3 ml / min:6 ml / min:9 ml / min to obtain a dispersion containing the composition. At this time, the antibody solution was mixed through a channel for in-line dilution. The obtained dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4°C for 24 hours using Slide-A-Lyzer Dialesis (20 K molecular weight cutoff, Thermo Fisher Scientific). Subsequently, the dispersion was concentrated by ultrafiltration using Amicon Ultra (30 K molecular weight cutoff, Merck) and then filtered using a 0.2 μm syringe filter. The final mRNA concentration was adjusted to 200 μg / mL and the sucrose concentration to 20%, and the solution was stored at -80°C.

[0206] [Example 12] Preparation of LNP12 (Pre method) For the preparation of LNP12, a lipid mixture (ionized lipid: DSPC: cholesterol: SUNBRIGHT GM-020 = 60:10.6:27.99:1.4, mol%) was dissolved in 90% EtOH to obtain a lipid solution of 9.3 mg / ml. As the ionized lipid, 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis(((3-pentyloctanoyl)oxy)methyl)propyl3-pentyloctanoate, as described in WO2016 / 021683, was used.

[0207] The mRNA encoding the CD19 target CAR (Elixirgen) was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.5) to obtain a nucleic acid solution of 0.2 mg / ml.

[0208] The lipid solution for LNP preparation and the nucleic acid solution are mixed at room temperature using NanoAssemblr.ＴＭ Ignite ＴＭ The mixture was obtained by mixing the components at a flow rate ratio of 3 ml / min to 6 ml / min using a Precision Nanosystems. The obtained dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4°C for 24 hours using Slide-A-Lyzer Dialesis (20K molecular weight cutoff, Thermo Fisher Scientific). Subsequently, hCD3Fab-PEG(5000)-DSPE (Preparation Example 8) was mixed at 25°C to a concentration of 0.01 mol% of the total lipids and allowed to stand for 30 minutes. Then, the mixture was concentrated by ultrafiltration using Amicon Ultra (30K molecular weight cutoff, Merck) and filtered using a 0.2 μm syringe filter. The final mRNA concentration was adjusted to 200 μg / mL and the sucrose concentration to 20%, and the solution was stored at -80°C.

[0209] [Characterization of LNPs] The particle size, PDI, mRNA encapsulation rate, and ratio of uncovalently bonded reactive PEG lipids to the total lipid components of control LNPs 1-6 and LNPs 1-12 were measured by the following method. The results of these characterizations are shown in Table 2.

[0210] Particle size and PDI: The particle size and Polydispersity index (PDI) of LNPs were measured using a Zetasizer Nano ZS (Maivern Panalogical).

[0211] mRNA encapsulation rate: The mRNA encapsulation rate in LNPs is measured using Quant-it. TM Measurements were performed using the RiboGreen RNA Assay Kit (Thermo Fisher Scientific). The mRNA concentration measured after lysis of LNPs with 0.5% Triton X-100 was defined as the total mRNA concentration, and the mRNA concentration measured without the addition of Triton X-100 was defined as the mRNA concentration not encapsulated in the LNPs. The mRNA encapsulation rate in LNPs was then calculated.

[0212] The ratio of non-covalently bonded reactive PEG lipids to the total lipid components was calculated using the following formula: [Ratio of non-covalently bonded reactive PEG lipids] = [Total ratio of reactive PEG lipids] - [Total ratio of antibody or antigen-binding fragments] * [Reaction efficiency] The reaction efficiency was determined by analyzing unreacted antibody or antigen-binding fragments by HPLC. The HPLC analysis conditions are as follows: Mobile phase A: Aqueous solution of 0.2% trifluoroacetic acid (TFA) Mobile phase B: Acetonitrile solution of 0.2% TFA Detection method: UV 280 nm Column: Agilent PLRP-S

[0213] Since no unreacted reactive PEG lipids were detected in the sample of preparation example 8, the ratio of non-covalently bonded reactive PEG lipids to the total lipid components in LNP12 was set to 0%.

[0214]

[0215] [Test Example 1] In vivo CAR expression test in mouse T cells C57BL / 6NJcl mice were administered 0.1 mg / kg or 0.375 mg / kg of LNP (control LNP1-2, LNP1-4) via tail vein at a dose of 10 mL / kg once or twice at one-week intervals. The spleen and blood were collected 4 hours after the final dose. After isolating immune cells from the spleen, the cells were stained (immunostaining for CD45, CD90.2, CD4, CD8, and CD19 CAR), and the percentage of CD19 CAR-positive cells among CD90.2-positive T cells (Pan-T cells), CD4-positive T cells, and CD8-positive T cells was evaluated using an LSR Fortessa flow cytometer (BD Biosciences). Plasma was fractionated from the blood, and the concentration of IFN-γ was measured using the CUSTOMIZE U-PLEX ASSAY KIT.

[0216] Table 3 shows the results for control LNP1 and LNP1 (both with an average molecular weight of 2000 in the PEG chain). Control LNP1, which contained 0.973% reactive PEG lipids not covalently bonded to mCD3Fab, showed decreased CAR expression after the second administration compared to the first administration. On the other hand, LNP1, which contained 0.077% reactive PEG lipids not covalently bonded to mCD3Fab, maintained its CAR expression level even after the second administration. This comparative result demonstrates the effects of both the first and second embodiments of the lipid particles of the present invention.

[0217]

[0218] Table 4 shows the results for control LNP2 and LNP2 (both with an average molecular weight of 5000 in the PEG chain). Control LNP2, which contained 0.973% reactive PEG lipids not covalently bonded to mCD3Fab, showed decreased CAR expression after the second administration compared to the first administration. On the other hand, LNP2, which contained 0.072% reactive PEG lipids not covalently bonded to mCD3Fab, maintained its CAR expression level even after the second administration. Furthermore, LNP2 with 0.072% non-covalently bonded reactive PEG lipids showed a higher CAR expression level compared to control LNP2 with 0.973% non-covalently bonded reactive PEG lipids. This comparative result demonstrates the effects of both the first and second embodiments of the lipid particles of the present invention.

[0219]

[0220] As shown in Table 5, LNP2, which has an average molecular weight of 5000 in the PEG chain of the reactive PEG lipid, showed lower IFNγ production compared to LNP1, which has an average molecular weight of 2000 in the PEG chain of the reactive PEG lipid.

[0221]

[0222] As shown in Table 6, LNP4, which has a non-covalently bonded reactive PEG lipid content of 0.003%, showed higher CAR expression levels compared to LNP3, which has a non-covalently bonded reactive PEG lipid content of 0.095%.

[0223]

[0224] [Test Example 2] In vivo CAR expression test on mouse T cells and NK cells. Human CD7 gene-expressing hCD7KI mice were administered 0.125 mg / kg of LNP (control LNP3 and LNP5) via tail vein at a rate of 10 mL / kg, and the spleen was collected 4 hours after the final administration. After isolating immune cells from the spleen, the cells were stained (CD45, CD3, NKp46, G4S Linker), and the percentage of GPC3CAR-positive cells among CD3-positive T cells (Pan-T cells) and NKp46-positive NK cells was evaluated using an LSR Fortessa flow cytometer (BD Biosciences).

[0225] As shown in Table 7, LNP5, which contained 0.094% of non-covalently bonded reactive PEG lipids, showed a higher CAR expression level compared to control LNP3, which contained 0.995% of non-covalently bonded reactive PEG lipids. This comparative result demonstrates the effects of both the first and second embodiments of the lipid particles of the present invention.

[0226]

[0227] [Test Example 3] In vivo CAR expression test in mouse T cells C57BL / 6NJcl mice were administered 0.1 mg / kg of LNP (control LNPs 4-6, LNPs 6-11) via tail vein at a rate of 10 mL / kg, and the spleen was collected 4 hours after the final administration. After isolating immune cells from the spleen, the cells were stained (immunostaining for CD45, CD90.2, CD4, CD8, and CD19 CAR), and the percentage of CD19 CAR-positive cells among CD90.2-positive T cells (Pan-T cells), CD4-positive T cells, and CD8-positive T cells was evaluated using an LSR Fortessa flow cytometer (BD Biosciences). Plasma was fractionated from the blood, and the concentration of IFN-γ was measured using the CUSTOMIZE U-PLEX ASSAY KIT.

[0228] As shown in Table 8, LNP6, which contained 0.097% of non-covalently bonded reactive PEG lipids, showed a higher CAR expression level compared to control LNP4, which contained 0.996% of non-covalently bonded reactive PEG lipids. This comparative result demonstrates the effects of both the first and second embodiments of the lipid particles of the present invention.

[0229]

[0230] As shown in Table 9, LNP7, which contained 0.095% of non-covalently bonded reactive PEG lipids, showed a higher CAR expression level compared to control LNP5, which contained 0.993% of non-covalently bonded reactive PEG lipids. This comparative result demonstrates the effects of both the first and second embodiments of the lipid particles of the present invention.

[0231]

[0232] As shown in Table 10, LNPs 8-11, which contained 0.002%, 0.002%, 0.002%, and 0.001% of uncovalently bonded reactive PEG lipids, showed higher CAR expression levels compared to control LNP6, which contained 0.297% of uncovalently bonded reactive PEG lipids. This comparative result demonstrates the effects of both the first and second embodiments of the lipid particles of the present invention.

[0233]

[0234] As shown in Table 11, LNP9, which has an average molecular weight of 3400 in the PEG chain of the reactive PEG lipid, showed lower IFNγ production compared to LNP8, which has an average molecular weight of 2000. Furthermore, LNP10, which has an average molecular weight of 5000, showed even lower IFNγ production compared to LNP9, which has an average molecular weight of 3400.

[0235]

[0236] The lipid particles and nucleic acid delivery compositions of the present invention enable the efficient delivery of nucleic acids to various cells, tissues, or organs, and can therefore be used as a DDS (Drug Delivery System) technology in fields such as gene therapy and nucleic acid drugs, as well as as a nucleic acid delivery reagent for research purposes.

Claims

1. Lipid particles conjugated with an antibody or an antigen-binding fragment that specifically binds to a cell surface antigen, wherein the antibody or antigen-binding fragment is covalently bound to at least a portion of a lipid having a reactive polyethylene glycol (PEG) chain (hereinafter referred to as "reactive PEG lipid") contained in the lipid components constituting the lipid particle, and the ratio of the reactive PEG lipid that is not covalently bound to the antibody or antigen-binding fragment to the total lipid components constituting the lipid particle is 0.1 mol% or less.

2. The lipid particle according to claim 1, wherein the total number of carbon atoms in the hydrophobic carbon chain of the reactive PEG lipid is 30 or more.

3. The lipid particle according to claim 1, wherein the total number of carbon atoms in the hydrophobic carbon chain of the reactive PEG lipid is 32 or more.

4. The lipid particle according to claim 1, wherein the total number of carbon atoms in the hydrophobic carbon chain of the reactive PEG lipid is 34 or more.

5. The lipid particle according to claim 1, wherein the total number of carbon atoms in the hydrophobic carbon chain of the reactive PEG lipid is 36 or more.

6. The lipid particle according to claim 1, wherein the cell surface antigen is a cell surface antigen of a T cell or an NK cell.

7. The lipid particle according to claim 1, wherein the antibody or its antigen-binding fragment is an anti-CD3 antibody or an anti-CD7 antibody, or an antigen-binding fragment thereof.

8. The lipid particle according to claim 1, wherein the reactive PEG lipid has an azide group or an alkynyl group which is a reactive group for a click reaction.

9. The lipid particles according to claim 1, wherein the reactive PEG lipid comprises a reactive derivative of a PEG lipid having a PEG chain with an average molecular weight of 5000 or more.

10. The lipid particles according to claim 1, wherein the reactive PEG lipid comprises a reactive derivative of PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG-DSPE) or a reactive derivative of PEG-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (PEG-DPPE).

11. The lipid particle according to claim 1, wherein the ratio of the reactive PEG lipid that is not covalently bound to the antibody or its antigen-binding fragment to the total lipid components constituting the lipid particle is 0.01 mol% or less.

12. The lipid particle according to claim 1, wherein the ratio of the reactive PEG lipid that is not covalently bound to the antibody or its antigen-binding fragment to the total lipid components constituting the lipid particle is substantially zero.

13. A method for producing lipid particles conjugated with an antibody or an antigen-binding fragment thereof that specifically binds to a cell surface antigen, wherein the antibody or antigen-binding fragment is covalently bonded to at least a portion of a reactive PEG lipid contained in the lipid components constituting the lipid particle, the ratio of the reactive PEG lipid not covalently bonded to the antibody or antigen-binding fragment to the total lipid components constituting the lipid particle is 0.1 mol% or less, and the method includes the step of blending a molecule prepared from the reaction product of the reactive PEG lipid and the antibody or antigen-binding fragment, in which the reactive PEG lipid and the antibody or antigen-binding fragment are covalently bonded, into the raw materials for the total lipid components constituting the lipid particle.

14. The manufacturing method according to claim 13, wherein the total number of carbon atoms in the hydrophobic carbon chain of the reactive PEG lipid is 30 or more.

15. The manufacturing method according to claim 13, wherein the total number of carbon atoms in the hydrophobic carbon chain of the reactive PEG lipid is 32 or more.

16. The manufacturing method according to claim 13, wherein the total number of carbon atoms in the hydrophobic carbon chain of the reactive PEG lipid is 34 or more.

17. The manufacturing method according to claim 13, wherein the total number of carbon atoms in the hydrophobic carbon chain of the reactive PEG lipid is 36 or more.

18. The manufacturing method according to claim 13, wherein the cell surface antigen is a cell surface antigen of a T cell or an NK cell.

19. The method for producing an antibody or its antigen-binding fragment according to claim 13, wherein the antibody or its antigen-binding fragment is an anti-CD3 antibody or an anti-CD7 antibody, or an antigen-binding fragment thereof.

20. The manufacturing method according to claim 13, wherein the reactive PEG lipid has an azide group or an alkynyl group which is a reactive group for a click reaction.

21. The method for producing a PEG lipid according to claim 13, wherein the reactive PEG lipid includes a reactive derivative of a PEG lipid having a PEG chain with an average molecular weight of 5,000 or more.

22. The production method according to claim 13, wherein the reactive PEG lipid comprises a reactive derivative of PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG-DSPE) or a reactive derivative of PEG-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (PEG-DPPE).

23. A nucleic acid introduction composition comprising the lipid particles and nucleic acid described in claim 1.

24. The nucleic acid introduction composition according to claim 23, wherein the total number of carbon atoms in the hydrophobic carbon chain of the PEG lipid is 30 or more.

25. The nucleic acid introduction composition according to claim 23, wherein the total number of carbon atoms in the hydrophobic carbon chain of the PEG lipid is 32 or more.

26. The nucleic acid introduction composition according to claim 23, wherein the total number of carbon atoms in the hydrophobic carbon chain of the PEG lipid is 34 or more.

27. The nucleic acid introduction composition according to claim 23, wherein the total number of carbon atoms in the hydrophobic carbon chain of the PEG lipid is 36 or more.

28. The nucleic acid introduction composition according to claim 23, wherein at least a portion of the nucleic acid is encapsulated in the lipid particles.

29. The nucleic acid introduction composition according to claim 28, wherein the nucleic acid is DNA or RNA.

30. A method for introducing nucleic acids, comprising the step of contacting the nucleic acid introduction composition according to claim 23 with target cells having a cell surface antigen to which an antibody or antigen-binding fragment conjugated to the lipid particles specifically binds.

31. The nucleic acid delivery method according to claim 30, wherein the target cells are T cells or NK cells.

32. A method for introducing nucleic acids in vivo, comprising the step of administering the nucleic acid introduction composition described in claim 23.

33. A method for improving redosability by controlling the ratio of PEG lipids, which are not covalently bonded to the antibody or its antigen-binding fragment and have 30 or more carbon atoms in their hydrophobic carbon chain, to the total lipid components constituting the lipid particles, in lipid particles conjugated with an antibody or its antigen-binding fragment that specifically binds to a cell surface antigen covalently bonded to at least a portion of a lipid having a polyethylene glycol (PEG) chain contained in the lipid components constituting the lipid particles (hereinafter referred to as "PEG lipid").

34. The method according to claim 33, wherein the total number of carbon atoms in the hydrophobic carbon chain of the PEG lipid is 32 or more.

35. The method according to claim 33, wherein the total number of carbon atoms in the hydrophobic carbon chain of the PEG lipid is 34 or more.

36. The method according to claim 33, wherein the total number of carbon atoms in the hydrophobic carbon chain of the PEG lipid is 36 or more.

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