Method for producing antibody, and lipid composition for use in method for producing antibody

Abstract

The problem addressed by the present invention is to provide a method for producing an antibody using a lipid composition optimized for immunity without using mRNA having poor expression persistence of antigen protein, and to provide a lipid composition optimized for immunity without using mRNA having poor expression persistence of antigen protein. The present invention provides a method for producing an antibody, the method including administering to a non-human animal a lipid composition containing one or more compounds represented by formula (1) or formula (2) and DNA encoding an antigen protein.

Description

Method for producing antibodies and lipid compositions for use in the method for producing antibodies

[0001] The present invention relates to a method for producing an antibody, which comprises administering a lipid composition containing DNA encoding an antigen protein to a non-human animal. The present invention further relates to a lipid composition for use in the above-mentioned method for producing an antibody.

[0002] In order to produce antibodies, it is common to immunize animals by administering an antigen protein or a nucleic acid encoding the antigen protein. It is known that when administering an antigen protein or a nucleic acid encoding the antigen protein to an animal, the antigen protein or the nucleic acid encoding the antigen protein is administered using a cationic lipid.

[0003] For example, Patent Document 1 describes the production of antibodies by administering lipid nanoparticles prepared using mRNA and cationic lipids to non-human animals. Patent Document 2 describes a method of inducing an immune response by administering a complex of polynucleotides and cationic lipids to animals.

[0004] On the other hand, Patent Document 3 describes that lipid particles prepared using a compound having a specific structure exhibit a high nucleic acid encapsulation rate and excellent nucleic acid delivery.

[0005] Japanese Patent Application Laid-Open No. 2022-160674 Special Publication No. Hei 9-500013 International Publication No. WO2019 / 235635

[0006] The method described in Patent Document 1 uses mRNA, which has a short time from administration to translation, but has the problem of inferior antigen expression sustainability compared to DNA. Furthermore, mRNA is prone to degradation, making quality control difficult, and the need to synthesize mRNA from DNA makes the manufacturing process complicated. In the examples of Patent Document 2, a commercially available cationic lipid (lipofectamine) is used, but the cationic lipid is not optimized for antigen expression and immune induction by DNA.

[0007] In view of these circumstances, an object of the present invention is to provide a method for producing an antibody using a lipid composition optimized for immunity, without using mRNA that has poor sustained antigen protein expression.A further object of the present invention is to provide a lipid composition optimized for immunity, without using mRNA that has poor sustained antigen protein expression.

[0008] As a result of intensive research to solve the above-mentioned problems, the present inventors have found that antibodies can be produced in high yields by administering to a non-human animal a lipid composition containing one or more compounds represented by the following formula (1) or formula (2) and DNA encoding an antigen protein. The present invention was completed based on the above findings. According to the present invention, the following inventions are provided:

[0009] <1> A method for producing an antibody, comprising administering to a non-human animal a lipid composition containing one or more compounds represented by formula (1) or formula (2) and DNA encoding an antigen protein. <2> The method for producing an antibody according to <1>, wherein the lipid composition further comprises a phospholipid. <3> The method for producing an antibody according to <2>, wherein the phospholipid is at least one selected from the group consisting of dioleoylphosphatidylethanolamine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and stearoyloleoylphosphatidylcholine. <4> The method for producing an antibody according to <1>, wherein the compound is a compound represented by formula (1). <5> The method for producing an antibody according to any one of <1> to <4>, wherein the lipid composition further comprises a sterol. <6> The method for producing an antibody according to any one of <1> to <4>, wherein the lipid composition further comprises a lipid having a nonionic hydrophilic polymer chain. <7> The method for producing an antibody according to any one of <1> to <4>, wherein the administration method is topical administration. <8> A method for producing the antibody according to any one of <1> to <4>, further comprising fusing antibody-producing cells obtained from a non-human animal to which the lipid composition has been administered with proliferative cells to establish hybridomas, and culturing the hybridomas. <9> A lipid composition for use in the method for producing the antibody according to any one of <1> to <4>, comprising one or more compounds represented by formula (1) or formula (2) and DNA encoding an antigen protein.

[0010] According to the present invention, by administering a lipid composition containing a predetermined compound and DNA to an animal, the DNA is introduced into the cells, the antigen protein encoded by the DNA is expressed, and antibodies against the antigen protein can be produced.

[0011] FIG. 1 shows the results of measuring rabbit antibody production.

[0012] The present invention will be described in detail below. In this specification, the symbol "to" indicates a range that includes the numerical values ​​before and after it as the minimum and maximum values, respectively.

[0013] The method for producing an antibody according to the present invention comprises administering to a non-human animal a lipid composition comprising one or more compounds represented by formula (1) or formula (2) and DNA encoding an antigen protein.

[0014] <Configuration of lipid composition> The lipid composition contains one or more compounds represented by formula (1) or formula (2). The compound represented by formula (1) and the compound represented by formula (2) are compounds described in Example 25 and Example 125, respectively, of International Publication WO2019 / 235635.

[0015] As the one or more compounds represented by formula (1) or formula (2), it is preferable to use a compound represented by formula (1).

[0016] In the lipid composition, the amount of one or more compounds represented by formula (1) or formula (2) is preferably 20 mol% to 80 mol% relative to the total lipid amount, more preferably 35 mol% to 70 mol%, and even more preferably 40 mol% to 65 mol%.

[0017] The lipid composition contains DNA encoding an antigen protein. Any antigen protein can be used as the antigen protein, but a transmembrane protein is particularly preferred. Furthermore, the antigen protein is preferably a cancer antigen or a tumor antigen.

[0018] A list of cancer antigens and tumor antigens is shown below, but is not limited to these. The following antigens or partial peptides thereof can be used as antigenic proteins.

[0019] CD2, CD19, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD47, CD52, CD56, CD70, CD79, CD137, 4-IBB, 5T4, AGS-5, AGS-16, angiopoietin 2, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, carcinoembryonic antigen, CTLA4, Cripto, ED-B, ErbB1, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, FAP, fibronectin, folate receptor, ganglioside GM3, GD2 , glucocorticoid-inducible tumor necrosis factor receptor (GITR), gplOO, gpA33, GPNMB, ICOS, IGF1R, integrin av, integrin ανβ, LAG-3, Lewis Y, mesothelin, c-MET, MN carbonic anhydrase IX, MUC1, MUC16, nectin-4, NKGD2, NOTCH, OX40, OX40L, PD-1, PDL1, PSCA, PSMA, RANKL, ROR1, ROR2, SLC44A4, syndecan-1, TACI, TAG-72, tenascin, TIM3, TRAILR1, TRAILR2, VEGFR-1, VEGFR-2, VEGFR-3, and their mutants.

[0020] The form of DNA encoding an antigen protein is not particularly limited, but one example is a plasmid expression vector for expressing an antigen protein in eukaryotic cells. Nanoplasmids, which are smaller than conventional plasmids, can also be used as plasmid expression vectors. Expression vectors for eukaryotic cells generally contain four elements. The first element is a sequence encoding an origin of replication, which enables the plasmid to propagate within the host. The second element is an element that controls the initiation of transcription, specifically a promoter sequence and, optionally, an enhancer sequence. The third element is a sequence involved in transcript processing, such as a polyadenylation sequence. The fourth element is a sequence encoding the antigen protein. DNA encoding the antigen protein (which may be cDNA) can be operably linked to the expression vector. Two DNA sequences (a promoter sequence and a sequence encoding an antigenic protein) are said to be operably linked if the linkage between them does not (1) result in the introduction of a frameshift mutation, (2) interfere with the ability of the promoter sequence to direct the transcription of an immunogen-encoding gene sequence, or (3) interfere with the ability of the sequence encoding the antigenic protein to be transcribed by the promoter sequence.

[0021] The promoter may be derived from a virus (e.g., adenovirus, bovine papillomavirus, simian virus, etc.), a mammalian promoter such as an actin, collagen, or myosin promoter, or a prokaryotic promoter (e.g., bacteriophage T3 RNA polymerase promoter). Examples of promoters include the mouse metallothionein I gene promoter, herpes virus TK promoter, SV40 early promoter, Rous sarcoma virus promoter, and cytomegalovirus promoter.

[0022] In the lipid composition, the amount of DNA encoding the antigen protein is, for example, 0.01 to 50% by weight, preferably 0.1 to 30% by weight, more preferably 0.5 to 15% by weight, based on the total amount of lipids.

[0023] The lipid composition preferably further contains a phospholipid. Examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, etc., with phosphatidylcholine being preferred. The phospholipid may be a single phospholipid or a combination of multiple different phospholipids.

[0024] Examples of phosphatidylcholines include, but are not limited to, soybean lecithin (SPC), hydrogenated soybean lecithin (HSPC), egg yolk lecithin (EPC), hydrogenated egg yolk lecithin (HEPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), stearoyloleoylphosphatidylcholine (SOPC), and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC).

[0025] The phosphatidylethanolamine is not particularly limited, and examples thereof include dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylethanolamine (DLoPE), diphytanoylphosphatidylethanolamine (D(Phy)PE), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), ditetradecylphosphatidylethanolamine, dihexadecylphosphatidylethanolamine, dioctadecylphosphatidylethanolamine, and diphytanylphosphatidylethanolamine.

[0026] Examples of sphingomyelin include, but are not limited to, egg yolk-derived sphingomyelin and milk-derived sphingomyelin.

[0027] Among the above, the phospholipid is preferably at least one selected from the group consisting of dioleoylphosphatidylethanolamine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and stearoyloleoylphosphatidylcholine, with dioleoylphosphatidylethanolamine being particularly preferred.

[0028] In the lipid composition, the blending amount of the phospholipid is preferably 3 mol % or more and 55 mol % or less based on the total amount of the constituent lipid components.

[0029] The lipid composition preferably further contains a sterol. By including a sterol in the lipid composition, membrane fluidity can be reduced, and the lipid composition can be stabilized. Examples of sterols include, but are not limited to, cholesterol, phytosterols (sitosterol), stigmasterol, fucosterol, spinasterol, brassicasterol, ergosterol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, and cholesteryl-4'-hydroxybutyl ether. Among these, cholesterol is preferred.

[0030] In the lipid composition, the amount of sterol blended is preferably 10 mol% to 60 mol%, more preferably 20 mol% to 55 mol%, and even more preferably 25 mol% to 50 mol%, relative to the total amount of lipid.

[0031] The lipid composition preferably further contains a lipid having a nonionic hydrophilic polymer chain. By containing a lipid having a nonionic hydrophilic polymer chain in the lipid composition, a dispersion stabilizing effect of the lipid composition can be obtained.

[0032] Examples of nonionic hydrophilic polymers include, but are not limited to, nonionic vinyl polymers, nonionic polyamino acids, nonionic polyesters, nonionic polyethers, nonionic natural polymers, nonionic modified natural polymers, and block polymers or graft copolymers having two or more of these polymers as constituent units.

[0033] Of these nonionic hydrophilic polymers, nonionic polyethers, nonionic polyesters, nonionic polyamino acids, or nonionic synthetic polypeptides are preferred, nonionic polyethers or nonionic polyesters are more preferred, nonionic polyethers or nonionic monoalkoxy polyethers are even more preferred, and polyethylene glycol (polyethylene glycol will also be referred to as PEG hereinafter) is particularly preferred.

[0034] Lipids having a nonionic hydrophilic polymer include, but are not limited to, PEG-modified phosphoethanolamine, diacylglycerol PEG derivatives, dialkylglycerol PEG derivatives, cholesterol PEG derivatives, and ceramide PEG derivatives. Among these, diacylglycerol PEG is preferred. The weight-average molecular weight of the PEG chain of the nonionic hydrophilic polymer derivative is preferably 500 to 5,000, more preferably 750 to 3,000. The nonionic hydrophilic polymer chain may be branched or may have a substituent such as a hydroxymethyl group.

[0035] In the lipid composition, the amount of lipid having a nonionic hydrophilic polymer chain is preferably 0.25 mol% to 12 mol% of the total lipid amount, more preferably 0.5 mol% to 6 mol%, and even more preferably 1 mol% to 3 mol%.

[0036] A lipid composition comprising one or more compounds represented by formula (1) or formula (2) and DNA encoding an antigen protein can be used in the method for producing an antibody according to the present invention. That is, the present invention provides a lipid composition for use in the method for producing an antibody according to the present invention, comprising one or more compounds represented by formula (1) or formula (2) and DNA encoding an antigen protein.

[0037] <Method for producing lipid composition> A method for producing a lipid composition will be described. The method for producing a lipid composition is not limited, but the lipid composition can be produced by dissolving all or some of the oil-soluble components of the lipid composition in an organic solvent or the like to form an oil phase, dissolving water-soluble components such as DNA in water to form an aqueous phase, and mixing the oil phase and the aqueous phase. A micromixer may be used for mixing, or emulsification may be performed using an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure injection emulsifier, or the like.

[0038] Alternatively, the lipid-containing solution can be dried under reduced pressure using an evaporator or spray-dried using a spray dryer to prepare a dried mixture containing the lipid, and the mixture can be added to an aqueous solvent and further emulsified using the emulsifier described above.

[0039] An example of a method for producing a lipid composition containing DNA includes the following steps: (a) dissolving the components of the lipid composition in an organic solvent to obtain an oil phase; (b) mixing the oil phase obtained in step (a) with an aqueous phase containing DNA; (c) diluting the mixture containing the oil phase and aqueous phase obtained in step (b) to obtain a lipid particle dispersion; (d) removing the organic solvent from the lipid particle dispersion obtained in step (c); and (e) adjusting the concentration of the lipid particle dispersion obtained in step (d).

[0040] Step (a) involves dissolving components classified as lipids in an organic solvent (an alcohol such as ethanol, or an ester, etc.). The total lipid concentration after dissolution in the organic solvent is not particularly limited, but is generally 1 mmol / L to 100 mmol / L, preferably 5 mmol / L to 50 mmol / L, and more preferably 10 mmol / L to 30 mmol / L.

[0041] In step (b), the aqueous phase can be obtained by dissolving DNA in water or a buffer solution. The DNA concentration is not particularly limited, but is preferably 1 to 1,000 μg / mL, and more preferably 10 to 500 μg / mL. Components such as buffer components for pH adjustment and antioxidants can be added as needed. The pH of the aqueous phase is preferably 2.0 to 7.0, and more preferably 3.0 to 6.0. Buffer components such as acetic acid, citric acid, malic acid, phosphoric acid, MES, and HEPES are preferably used to adjust the pH to the above range. If necessary, salts such as sodium chloride and potassium chloride may be added to adjust the salt strength, and sugars or sugar alcohols such as sucrose, trehalose, and mannitol may be added to adjust the osmotic pressure.

[0042] In step (b), the oil phase and the aqueous phase may be mixed by any method, including a batch method and an in-line method using a flow channel device. For the in-line method, a micro-flow channel device is preferably used, and examples of the micro-flow channel device that can be used include a Y-mixer, a T-mixer, a herringbone mixer, a ring micromixer, and an impingement jet mixer. The mixing ratio (volume ratio) of the aqueous phase to the oil phase is preferably 5:1 to 1:1, and more preferably 4:1 to 2:1.

[0043] In step (c), the lipid particle dispersion is mixed with a diluent to reduce the content of organic solvent and stabilize the lipid particles. The diluent may be water, but may also include adjusting the pH or salt strength. The components contained in the diluent can be selected arbitrarily depending on the purpose. For example, a buffer solution (e.g., citrate buffer, citrate-buffered saline, acetate buffer, acetate-buffered saline, phosphate-buffered saline, Tris buffer, MES buffer, HEPES buffer, etc.) may be used to adjust the pH. In addition, sodium chloride, potassium chloride, sucrose, trehalose, fructose, mannitol, etc. may be included to adjust the salt strength or osmotic pressure, and the above buffer solutions may also be used with the addition of these additives.

[0044] The lipid particle dispersion and the diluted solution may be mixed by any method, including a batch method or an in-line method using a flow path device. The flow path device used during mixing may be a Y-mixer, a T-mixer, or the like. The ratio (volume ratio) of the lipid particle dispersion to the diluted solution is preferably 1:0.5 to 1:10, more preferably 1:1 to 1:5.

[0045] In some embodiments, in step (c), the lipid particle dispersion may be mixed with the dilution solution multiple times depending on the purpose.The dilution solutions used may be the same or different.In the lipid particle dispersion, the particle size of the lipid particles may change depending on the pH, so adjusting the pH of the dispersion is important.Therefore, for example, in order to adjust the pH of the lipid particle dispersion after mixing with the dilution solution, a buffer solution having an appropriate concentration and pH, or a buffer solution containing other components, may be used.

[0046] Furthermore, multiple dilution steps may be carried out continuously, and the interval between one dilution step and the next dilution step may be set arbitrarily, for example, 10 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, or 24 hours. Furthermore, the pH of the lipid particle dispersion after step (c) is preferably pH 3.0 to 10.0, more preferably pH 3.5 to pH 9.0, and particularly preferably pH 4.0 to pH 8.5.

[0047] The lipid particles can be subjected to sizing as needed. The sizing method is not particularly limited, but the particle size can be reduced using an extruder or the like. Furthermore, the dispersion containing the lipid particles can be subjected to freezing or freeze-drying by a general method.

[0048] In step (d), the method for removing the organic solvent from the lipid particle dispersion is not particularly limited and any common method can be used, for example, the organic solvent can be removed by dialysis using a pH buffer solution such as phosphate buffered saline or Tris buffer as the dialysis solution. If necessary, additives such as any salt or sugar can be added for the purpose of adjusting the osmotic pressure or protecting the dispersion from freezing.

[0049] In step (e), the concentration of the dispersion obtained in step (d) can be adjusted. When diluting, phosphate buffered saline, physiological saline, Tris-HCl buffer, or the like can be used as a diluent to dilute to an appropriate concentration. When concentrating, the dispersion obtained in step (d) can be concentrated by ultrafiltration using an ultrafiltration membrane, for example. The concentrated dispersion can be used as is, or it can be concentrated and then adjusted to a desired concentration using the diluent.

[0050] In some embodiments, the organic solvent removal step (step (d)) and the concentration adjustment step (step (e)) can be carried out continuously using tangential flow filtration (TFF). In this process, the organic solvent removal step and the concentration adjustment step may be carried out in any order. If necessary, the organic solvent removal step and the concentration adjustment step may each be carried out multiple times.

[0051] The solution that can be used for dialysis in step (d) or dilution in step (e) may contain an excipient, cryoprotectant, buffer, or antioxidant. Examples of excipients and cryoprotectants include, but are not limited to, sugars and sugar alcohols. Examples of sugars include sucrose, trehalose, maltose, glucose, lactose, and fructose, and examples of sugar alcohols include mannitol, sorbitol, inositol, and xylitol. Examples of buffers include, but are not limited to, ACES, BES, Bicine, CAPS, CHES, DIPSO, EPPS, HEPES, HEPPSO, MES, MOPS, MOPSO, TAPS, TAPSO, TES, Tricine, Tris, phosphoric acid, acetic acid, and citric acid. Examples of antioxidants include EDTA, ascorbic acid, and tocopherol.

[0052] In order to use the lipid particle dispersion as a cancer therapeutic agent, it is preferable to carry out sterile filtration.As a filtration method, hollow fiber membrane, reverse osmosis membrane, membrane filter, etc. can be used to remove unnecessary substances from the lipid particle dispersion.In the present invention, although not particularly limited, it is preferable to carry out filtration using a filter with a sterilizable pore size (preferably a 0.2 μm filtration sterilization filter).In addition, it is preferable to carry out sterile filtration after step (c), step (d), or step (e).Furthermore, if necessary, the lipid particle dispersion can be subjected to freeze-drying.

[0053] <Form of lipid composition> The lipid composition may be a lipid particle. Lipid particles refer to particles composed of lipids, and include compositions having any structure selected from lipid aggregates in which lipids are aggregated, micelles, and liposomes. However, the structure of the lipid particle is not limited to these, as long as the composition contains lipids. Liposomes include liposomes having a lipid bilayer structure and an aqueous phase inside, with a single bilayer membrane, and multi-phase liposomes with multiple layers stacked on top of each other. Either type of liposome may be included in the present invention.

[0054] The morphology of lipid particles can be confirmed by electron microscope observation or X-ray structural analysis.For example, by using a cryo-transmission electron microscope (cryo-TEM) method, it can be confirmed whether the lipid particles have a lipid bilayer structure (lamellar structure) and an inner water layer, like liposomes, or whether the particles have a core with high electron density inside and a structure packed with lipids and other components.Small-angle X-ray scattering (SAXS) measurement can also be used to confirm whether the lipid particles have a lipid bilayer structure (lamellar structure).

[0055] The particle size of the lipid particles is not particularly limited, but is preferably 10 to 1000 nm, more preferably 30 to 500 nm, and even more preferably 50 to 250 nm. The particle size of the lipid particles can be measured by a general method (e.g., dynamic light scattering method, laser diffraction method, etc.).

[0056] <Administration of lipid composition to non-human animals> In the present invention, a lipid composition containing one or more compounds represented by formula (1) or formula (2) and DNA encoding an antigen protein is administered to a non-human animal. Note that the lipid composition containing one or more compounds represented by formula (1) or formula (2) and DNA encoding an antigen protein may also be administered to a human.

[0057] The lipid composition can be administered to a living body alone or in admixture with an administration vehicle (eg, physiological saline or a buffer solution).

[0058] The non-human animal is preferably a non-human mammal, such as a rabbit, mouse, rat, or monkey, with rabbits and mice being preferred. Administration to the non-human animal can be by systemic administration or local administration, with local administration being preferred. Local administration can be by intramuscular injection, subcutaneous injection, or intradermal injection.

[0059] The dosage of the lipid composition to a non-human mammal is preferably such that the amount of DNA encapsulated therein is 1 to 1000 μg / head, more preferably 3 to 500 μg / head, and is preferably administered once every 1 to 4 weeks.

[0060] Antibodies against antigenic proteins are produced in the body of a non-human animal to which the lipid composition has been administered. Therefore, blood can be collected from the non-human animal to which the lipid composition has been administered, and the produced antibodies can be recovered.

[0061] Alternatively, antibody-producing cells obtained from a non-human animal to which a lipid composition has been administered may be fused with proliferative cells to establish hybridomas, and antibodies may be produced by culturing the hybridomas.

[0062] As used herein, the term "antibody" is used in the broadest sense to refer to an immunoglobulin molecule that recognizes and specifically binds to an antigen protein via at least one antigen recognition site in the variable region of the immunoglobulin molecule. Antibodies may be either polyclonal or monoclonal, and may be multi-chain, single-chain, or intact immunoglobulins. Intact antibodies are typically glycoproteins containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region is composed of one domain: CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0063] The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen protein, and the constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0064] The antibody can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0065] The present invention will now be described with reference to examples, but the present invention is not limited to these examples.

[0066] In the following test examples, the following compounds were used.

[0067] Test Example 1: Preparation of plasmid DNA-encapsulating lipid nanoparticles and measurement of antibody production in mice <Phospholipids used> DOPE (L-α-dioleoyl phosphatidylethanolamine) and SOPC (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine) were purchased from NOF Corporation.

[0068] <Preparation of Plasmid DNA-Encapsulating Lipid Nanoparticles> The compounds listed in Table 1, the phospholipids listed in Table 1, cholesterol (product name: Cholesterol HP; Nippon Fine Chemical Co., Ltd.), and 1,2-dimyristoyl-rac-glycero-3-(methylpolyoxyethylene 2000) (hereinafter, DMG-PEG2000) (product name: SUNBRIGHT® GM-020; NOF Corporation) were dissolved in ethanol in a molar ratio of ionized lipid:phospholipid:cholesterol:DMG-PEG2000=50:10:38.5:1.5 to a total lipid concentration of 12.5 mmol / L to obtain an oil phase.

[0069]

[0070] Plasmid DNA (Fujifilm Wako Pure Chemical Industries, Ltd.) encoding hEphA2 (Erythropoietin-producing hepatoma receptor-A2) was diluted with 50 mmol / L citrate buffer at pH 4.0 to obtain an aqueous phase at a weight ratio of total lipid to plasmid DNA of approximately 20:1. The aqueous and oil phases were then mixed using a NanoAssemblr (Precision NanoSystems) to obtain a volume ratio of aqueous to oil of 3:1. The mixture was then diluted two-fold with phosphate-buffered saline (PBS) or water to obtain a dispersion of plasmid DNA-lipid nanoparticles. This dispersion was dialyzed against 20 mM Tris buffer containing 8% sucrose using a dialysis cassette (Slide-A-Lyzer G2, MWCO: 10 kD, Thermo Fisher Scientific) to remove ethanol, and then concentrated using an ultrafiltration filter (Amicon Ultra 100 kDa, Merck) to obtain plasmid DNA-encapsulated lipid nanoparticles.

[0071] <Measurement of particle size and zeta potential> The particle size of the plasmid DNA-encapsulated lipid nanoparticles was measured using a particle size analyzer NanoSAQLA (Otsuka Electronics) after 20-fold dilution with phosphate-buffered saline (PBS). The zeta potential was measured using a zeta potential analyzer ELS-Z2 (Otsuka Electronics) after dilution with HEPES buffer (pH 7.5) or MES buffer (pH 5.5). The results are shown in Table 2.

[0072] <Evaluation of Plasmid DNA Encapsulation Rate> (Quantification of Total Plasmid DNA Concentration) Plasmid DNA was diluted with MilliQ water to prepare a 2-fold dilution series from 100 μg / mL to 3.1 μg / mL, and a calibration curve solution was prepared. 50 μL of the calibration curve solution or plasmid DNA lipid nanoparticles diluted 10-fold with MilliQ water was mixed with 450 μL of methanol to prepare a measurement solution.

[0073] The absorbance of each test solution was measured at 260 nm and 330 nm using a UV plate reader (Multiskan Go, Thermo Fisher Scientific), and the absorbance at 330 nm was subtracted from the absorbance at 260 nm to obtain the absorbance of each test solution. The total aqueous nucleic acid concentration was calculated from the calibration curve using the absorbance of each sample test solution.

[0074] (Quantification of Plasmid DNA Concentration in the External Aqueous Phase) The nucleic acid concentration in the external aqueous phase was quantified by the standard addition method using the Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher Scientific). First, the 20x TE buffer included in the kit was diluted with water to make 1x TE buffer. TE stands for Tris / EDTA (ethylenediaminetetraacetic acid). Plasmid DNA was diluted with TE buffer to final concentrations of 0 to 400 ng / mL, and a nucleic acid dilution series was prepared. 10 μL of plasmid DNA lipid nanoparticles diluted 25-fold with TE buffer and 90 μL of the nucleic acid dilution series were mixed in a 96-well plate, and then 100 μL of RiboGreen reagent diluted 200-fold with TE buffer was added to each well. Fluorescence (excitation wavelength: 485 nm, emission wavelength: 535 nm) was measured using a fluorescent plate reader (Infitite 200 Pro M nano +, TECAN). The nucleic acid concentration in the external aqueous phase of each measurement solution was calculated from the results obtained using the standard addition method.

[0075] (Calculation of nucleic acid encapsulation rate) Using the quantitative results of the total plasmid DNA concentration and the plasmid DNA concentration in the external aqueous phase obtained in the above steps, the nucleic acid (plasmid DNA) encapsulation rate of the plasmid DNA lipid nanoparticles was calculated according to the following formula. The results are shown in Table 2. Nucleic acid encapsulation rate (%) = (total plasmid DNA concentration - plasmid DNA concentration in the external aqueous phase) ÷ total plasmid DNA concentration × 100

[0076]

[0077] <Measurement of Mouse Antibody Production> After purchasing mice (Balb / c, female, 6 weeks old), they were allowed to acclimate for one week, and then the plasmid DNA lipid nanoparticle dispersion prepared in the above <Preparation of Plasmid DNA-Encapsulating Lipid Nanoparticles>, or CaLiVax (OZ biosciences, CV02000) (Comparative Example 1) mixed with plasmid DNA according to the recommended protocol, was administered intradermally to the mice at a plasmid DNA dose of 42 or 14 μg / head (week 0; hereinafter referred to as W00). Thereafter, similar administration was repeated every two weeks until W08. Blood was collected from the tail vein at W07 and 11, and serum was collected. The resulting serum was diluted 200-fold and added to CHO cells (MOCK) and CHO cells expressing hEphA2 protein. After 30 minutes of incubation, a fluorescently labeled secondary antibody (SouthernBiotech, 1030-09) was added. The addition of serum and secondary antibody to MOCK cells was used to quantify nonspecific adsorption. The median fluorescence intensity (MFI) per cell of the treated CHO cells was then quantified by flow cytometry (Beckman Coulter, CytoFlex). The MFI value for each example was calculated by subtracting the MFI of the mock cells from the MFI of the hEphA2 protein-expressing CHO cells, and the ratio of the MFIs to that of the comparative example, taken as 1, was used as the relative antibody production amount.

[0078] This test was conducted by dividing the test preparation into two parts, and the results of the first test (Comparative Example 1 and Example 1) are shown in Table 3, and the results of the second test (Examples 1 and 2, Comparative Example 2) are shown in Table 4.

[0079]

[0080]

[0081] <Measurement of Rabbit Antibody Production> After purchasing rabbits (Japanese White Rabbits, female, 12 weeks old), they were allowed to acclimate for one week, and then CNE-CPO (OZ biosciences, CNE-1000) prepared in Example 1 or Comparative Example 3 in the above <Preparation of Plasmid DNA-Encapsulating Lipid Nanoparticles> was administered intradermally to the back or intramuscularly to the thigh at a plasmid DNA dose of 200 μg / head (week 0; hereinafter referred to as W00). Thereafter, similar administration was repeated every two weeks until W10. Blood was collected from the ear vein at W03, 05, 07, 09, and 11, and serum was collected. The resulting serum was diluted 1000-fold and added to CHO cells (MOCK) and CHO cells expressing hEphA2 protein. After 30 minutes of incubation, a fluorescently labeled secondary antibody (SouthernBiotech, 4030-09) was added. The serum and secondary antibody were added to the MOCK cells to quantify nonspecific adsorption. The median fluorescence intensity (MFI) per cell of the treated CHO cells was then quantified by flow cytometry (Beckman Coulter, CytoFlex). The MFI value for each example was calculated by subtracting the MFI of the mock cells from the MFI of the hEphA2-expressing CHO cells, and this value was used to determine the amount of antibody production. The physical properties of Example 1 are shown in Table 5, and the results of antibody production are shown in Table 6 and FIG.

[0082]

[0083]

Claims

1. A method for producing an antibody, comprising administering to a non-human animal a lipid composition comprising one or more compounds represented by formula (1) or formula (2) and DNA encoding an antigen protein.

2. The method for producing an antibody according to claim 1, wherein the lipid composition further comprises a phospholipid.

3. The method for producing an antibody according to claim 2, wherein the phospholipid is at least one selected from the group consisting of dioleoylphosphatidylethanolamine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and stearoyloleoylphosphatidylcholine.

4. The method for producing an antibody according to claim 1, wherein the compound is a compound represented by formula (1).

5. The method for producing an antibody described in any one of claims 1 to 4, wherein the lipid composition further comprises a sterol.

6. The method for producing an antibody according to any one of claims 1 to 4, wherein the lipid composition further comprises a lipid having a non-ionic hydrophilic polymer chain.

7. A method for producing an antibody according to any one of claims 1 to 4, wherein the administration method is local administration.

8. A method for producing an antibody described in any one of claims 1 to 4, further comprising fusing antibody-producing cells obtained from the non-human animal to which the lipid composition has been administered with proliferative cells to establish hybridomas, and culturing the hybridomas.

9. A lipid composition for use in the method for producing an antibody described in any one of claims 1 to 4, comprising one or more compounds represented by formula (1) or formula (2) and DNA encoding an antigen protein.

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