Immunoconjugates

Abstract

To provide an antibody complex useful for improving antigen detection sensitivity in immunoassay using a low-molecular antibody.SOLUTION: An immune complex in which a low-molecular antibody to which lysine or a lysine tag is added is covalently bound to a polymer having a carboxy group.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an immunoconjugate comprising a low molecular weight antibody. [Background technology]

[0002] In the fields of biotechnology, medicine, and clinical testing, specific interactions between molecules, such as antigen-antibody reactions and enzyme reactions, are often utilized to detect and quantify biomolecules and their derivatives. For example, immunoassays such as enzyme-linked immunosorbent assay (ELISA) are used to analyze proteins such as physiologically active substances. In these immunoassays, the presence or absence of protein interaction with an antibody immobilized in a measurement area is measured indirectly by enzyme reaction or fluorescence detection using an enzyme-labeled or fluorescently labeled secondary antibody.

[0003] Immunoglobulins (IgG) are typically used as antibodies in immunoassays. However, because IgG has multiple domains, it cannot be artificially synthesized and must be obtained by immunizing animals. Therefore, research is being conducted on small molecules, including single-domain antibodies such as VHHs, as new antigen-binding molecules. VHHs are particularly useful because they have a molecular weight approximately one-tenth that of IgGs, are expected to bind to novel epitopes that cannot be bound by conventional antibodies, have a highly reversible protein structure, exhibit excellent resistance to heat and pressure, and can be produced using microorganisms such as yeast and bacteria.

[0004] On the other hand, it is known that VHHs have lower antigen affinity than IgGs, and that when VHH antibodies are directly physically adsorbed onto a substrate such as polystyrene, their intrinsic antigen-binding ability tends to decrease (Non-Patent Documents 1 to 3). Therefore, in order to maintain measurement sensitivity, immunoassays using small molecular weight antibodies require improved antigen affinity and maintenance of activity after physical adsorption onto a substrate. In response to this, known methods for enhancing the antigen affinity of single-domain antibodies such as VHH antibodies include expressing the single-domain antibody as a fusion protein by fusing it with a tag molecule, and then constructing an antibody multimer by self-associating via the tag molecule (Non-Patent Document 4), and fusing a peptide that specifically binds to a substrate to the end of a VHH antibody, and immobilizing the VHH while controlling its orientation via this substrate-specific binding peptide (Non-Patent Document 5). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] J. Virol. 2008, 82, 9753-9764 [Non-patent document 2] Mol. Immunol. 1994, 31, 219-226 [Non-patent document 3] Anal. Biochem. 2003, 312, 113-124 [Non-patent document 4] J. Immunol. Methods, 2007, 318, 88-94 [Non-Patent Document 5] Biotechnol. Prog., 2015, 31, 1563-1570 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention relates to providing an immune complex that is useful for improving the sensitivity of antigen detection in immunoassays using low-molecular-weight antibodies. [Means for solving the problem]

[0007] The present inventors conducted studies in light of the above-mentioned problems and found that when they constructed a conjugate in which lysine or a low-molecular-weight antibody tagged with a lysine tag was covalently bonded to a polymer having a carboxy group, the conjugate had improved affinity to a solid interface and improved antigen-binding activity, and was therefore useful for antigen detection in immunoassays.

[0008] That is, the present invention relates to the following 1) and 2). 1) An immune complex in which a low molecular weight antibody to which lysine or a lysine tag has been added is covalently bound to a polymer having a carboxy group. 2) An antigen detection method using the immune complex of 1). [Effects of the Invention]

[0009] According to the present invention, antigens can be detected with high sensitivity by immunoassay using low molecular weight antibodies. DETAILED DESCRIPTION OF THE INVENTION

[0010] In the present invention, the term "small molecule antibody" refers to an antibody fragment in which a portion of a full-length antibody (whole antibody) is deleted, which comprises at least the variable region of the heavy chain or light chain and has the ability to bind to a target molecule such as an antigen. Specific examples include single domain antibodies, single chain antibodies (scFv, single chain Fv), multivalent single chain antibodies (sc(Fv)n), constant region-fused single chain antibodies (scFv-Fc), Fab fragments, F(ab')2 fragments, immunoglobulin new antigen receptors (IgNAR), antibody fragments formed by linking these, or peptide aptamers, with single domain antibodies being preferred.

[0011] A single-domain antibody refers to an antibody that has the property of specifically binding to an antigen through a single variable region (antigen-binding domain). Single-domain antibodies include antibodies whose variable region consists only of the heavy chain variable region (heavy-chain single-domain antibodies) and antibodies whose variable region consists only of the light chain variable region (light-chain single-domain antibodies). As long as the single-domain antibody has a variable region, it may be an antibody or a fragment thereof in which part of the constant region has been deleted or substituted. VHH, a heavy-chain antibody identified in camelids (e.g., camels, llamas, alpacas, etc.), and VNAR, a heavy-chain antibody derived from cartilaginous fish (e.g., sharks), are known as types of single-domain antibodies, and VHH antibodies are preferred in the present invention. Useful VHH antibodies include VHH antibodies against SARS-CoV-2 (e.g., Japanese Patent No. 7414225, etc.) and VHH antibodies against norovirus (e.g., Japanese Patent Application No. 2023-056675, etc.).

[0012] An scFv antibody is an antibody in which only the variable regions of the heavy and light chains are linked together via a linker, and there are no particular limitations on its origin, and antibodies of any origin can be suitably used.

[0013] IgNAR antibodies are derived from cartilaginous fish (such as sharks and rays) and are heavy chain homodimers that bind to target molecules such as antigens using only the variable region of the heavy chain.

[0014] Such small molecule antibodies can be isolated and collected from the serum of animals bearing the antibodies, or they can be produced by a combination of solid-phase peptide synthesis and native chemical ligation (NCL) or by genetic engineering. Examples of genetic engineering methods include designing an artificial gene optimized for the expression of the antibody of interest in host cells through processes such as codon optimization of the nucleic acid encoding the antibody molecule, incorporating it into an appropriate vector, and then introducing it into host cells to produce the recombinant antibody.

[0015] Lysine or a lysine tag (Lys-Tag) is added to the small molecule antibody of the present invention. The lysine or lysine tag is added by introducing it into the N-terminal or C-terminal amino acid of the small molecule antibody, preferably the C-terminal amino acid. Techniques for adding a lysine or lysine tag to an antibody molecule are well known in the art, and can be produced, for example, by combining solid-phase peptide synthesis with native chemical ligation (NCL) or by genetic engineering. However, a preferred method involves designing an artificial gene optimized for expression of the target antibody in host cells by subjecting nucleic acids encoding the single domain antibody or multimers of the antibody of the present invention to steps such as codon optimization, incorporating the artificial gene into an appropriate vector, and introducing the vector into host cells to produce a recombinant antibody.

[0016] The lysine tag used in the present invention is a peptide containing two or more consecutive lysine residues, preferably 3 to 15 consecutive lysine residues, more preferably 6 to 12 consecutive lysine residues. As long as the lysine tag contains two or more consecutive lysine residues, it also includes those containing 1 to 30 amino acid residues other than lysine. A suitable lysine tag is a peptide consisting of six consecutive lysine residues (KKKKKK (SEQ ID NO: 4)). Furthermore, it is also possible to add a hinge sequence such as EPKTPKPQS (SEQ ID NO: 5) to the N-terminus of the lysine tag.

[0017] Furthermore, the low-molecular-weight antibodies of the present invention may further comprise a peptide tag consisting of a specific amino acid sequence in addition to a lysine or lysine tag. Examples of peptide tags include peptide tags for protein isolation / purification, such as a histidine tag (His tag (HHHHHH (SEQ ID NO: 7)), a FLAG tag (DYKDDDDK (SEQ ID NO: 8)), a Strep tag (WSHPQFEK (SEQ ID NO: 9)), and an HA tag (YPYDVPDYA (SEQ ID NO: 10)). The tag can be linked, for example, directly or via a peptide linker (e.g., GS), to the N-terminus or C-terminus of the single domain antibody or single domain antibody multimer of the present invention.

[0018] In the present invention, the polymer having a carboxy group that forms a complex with the above-mentioned low molecular weight antibody is preferably a polymer that has a carboxy group in the molecule, has high adhesiveness to solid surfaces, and does not adsorb the antigen to be detected. Examples of such polymers having a carboxy group include carboxy group-containing cellulose derivatives and carboxy group-containing acrylic polymers.

[0019] Among the above, examples of the carboxy group-containing cellulose derivatives include carboxymethyl cellulose, carboxyethyl cellulose, carboxymethylethyl cellulose, and salts thereof; alkyl glycidyl ether-modified carboxymethylated hydroxyethyl cellulose; and the like. Alkyl glycidyl ether-modified carboxymethylated hydroxyethyl cellulose is a modified HEC in which alkyl glycidyl ether groups have been introduced into hydroxyethyl cellulose (HEC) that has been hydrophobized with carboxymethyl. Examples of alkyl glycidyl ethers include butyl glycidyl ether, hexyl glycidyl ether, octyl glycidyl ether, 2-ethylhexyl glycidyl ether, dodecyl glycidyl ether (lauryl glycidyl ether), octadecyl glycidyl ether (stearyl glycidyl ether), tetracosyl glycidyl ether, 2-heptylnonyl glycidyl ether, 2-decyltetradecanyl glycidyl ether, phenyl glycidyl ether, orthocresyl glycidyl ether, and orthophenylphenol glycidyl ether. 4-25 alkyl glycidyl ether, more preferably C 12-24 Examples include alkyl glycidyl ethers, and more preferably stearyl glycidyl ether. That is, a more preferred alkyl glycidyl ether-modified carboxymethylated hydroxyethyl cellulose is stearyl glycidyl ether-modified carboxymethylated HEC.

[0020] Methods for introducing carboxy groups into cellulose or cellulose derivatives include, for example, a method of converting hydroxy groups of cellulose into carboxy groups by oxidation, and a method of reacting the hydroxy groups of cellulose with one or more compounds selected from the group consisting of compounds having carboxy groups, acid anhydrides of compounds having carboxy groups, and derivatives thereof.

[0021] Examples of the carboxy group-containing acrylic polymer include acrylic (co)polymers obtained by polymerizing a (meth)acrylic monomer (a) as the main component, a carboxy-containing monomer (b), and, if necessary, other polymerizable monomers (c). Here, "mainly as a component" means that the content is usually 40% by mass or more, preferably 50% by mass or more, more preferably 60% by mass or more, and even more preferably 70% by mass or more of the total polymer components.

[0022] Examples of the (meth)acrylic monomer (a) include derivatives of (meth)acrylic acid, (meth)acrylamide or derivatives thereof, etc. Here, (meth)acrylic acid means acrylic acid or methacrylic acid. Derivatives of (meth)acrylic acid include alkyl (meth)acrylates, cycloalkyl (meth)acrylates (e.g., cyclohexyl (meth)acrylate, cyclopentyl (meth)acrylate, etc.), aralkyl (meth)acrylates (e.g., benzyl (meth)acrylate, etc.), polycyclic (meth)acrylates (e.g., biphenyloxy (meth)acrylate (biphenyloxyethyl (meth)acrylate, etc.), 2-isobornyl (meth)acrylate, 2-norbornylmethyl (meth)acrylate, 5-norbornen-2-yl-methyl (meth)acrylate, 3-methyl-2-norbornylmethyl (meth)acrylate, dicyclopentenyl (meth)acrylate, dicyclopentenyloxyethyl (meth)acrylate, dicyclopentanyl (meth)acrylate, etc.), alkoxy or phenoxy (meth)acrylate (for example, 2-methoxyethyl (meth)acrylate, 2-ethoxyethyl (meth)acrylate, 2-methoxymethoxyethyl (meth)acrylate, 3-methoxybutyl (meth)acrylate, methoxydiethylene glycol (meth)acrylate, ethoxydiethylene ethylene glycol (meth)acrylate, methoxypolyethylene glycol (meth)acrylate, ethyl carbitol (meth)acrylate, phenoxyethyl (meth)acrylate, alkylphenoxypolyethylene glycol (meth)acrylate, etc.), hydroxyalkyl (meth)acrylates (e.g., 2-hydroxyethyl (meth)acrylate, 2-hydroxypropyl (meth)acrylate, 3-hydroxypropyl (meth)acrylate, 2-hydroxybutyl (meth)acrylate, 4-hydroxybutyl (meth)acrylate, 6 -hydroxyhexyl (meth)acrylate, 8-hydroxyoctyl (meth)acrylate, 10-hydroxydecyl (meth)acrylate, 12-hydroxylauryl (meth)acrylate, etc.), hydroxyl group-containing (meth)acrylates ([4-(hydroxymethyl)cyclohexyl]methyl acrylate, cyclohexanedimethanol mono(meth)acrylate, 2-hydroxy-3-phenoxypropyl (meth)acrylate, etc.), epoxy group-containing (for example, (meth)acrylates (4-hydroxybutyl (meth)acrylate glycidyl ether, etc.),Examples of the halogen-containing (meth)acrylate include 2,2,2-trifluoroethyl (meth)acrylate, 2,2,2-trifluoroethylethyl (meth)acrylate, tetrafluoropropyl (meth)acrylate, hexafluoropropyl (meth)acrylate, octafluoropentyl (meth)acrylate, heptadecafluorodecyl (meth)acrylate, and 3-chloro-2-hydroxypropyl (meth)acrylate, as well as alkylaminoalkyl (meth)acrylates such as dimethylaminoethyl (meth)acrylate.

[0023] Of these, alkyl(meth)acrylates are preferred, and alkyl(meth)acrylates in which the alkyl group has 1 to 24 carbon atoms, preferably 8 to 22 carbon atoms, are more preferred. Preferred alkyl (meth)acrylates include, for example, methyl (meth)acrylate, ethyl (meth)acrylate, n-propyl (meth)acrylate, isopropyl (meth)acrylate, 2-methyl-2-nitropropyl (meth)acrylate, n-butyl (meth)acrylate, isobutyl (meth)acrylate, s-butyl (meth)acrylate, t-butyl (meth)acrylate, n-pentyl (meth)acrylate, t-pentyl (meth)acrylate, 3-pentyl (meth)acrylate, 2,2-dimethylbutyl (meth)acrylate, n-hexyl (meth)acrylate, cetyl (meth)acrylate, stearyl (meth)acrylate, n-octyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, 4-methyl-2-propylpentyl (meth)acrylate, and n-octadecyl (meth)acrylate.

[0024] Examples of (meth)acrylamide derivatives include N-alkyl group-containing (meth)acrylamide derivatives such as N-methyl(meth)acrylamide, N,N-dimethyl(meth)acrylamide, N,N-diethyl(meth)acrylamide, N-isopropyl(meth)acrylamide, N-butyl(meth)acrylamide, and N-hexyl(meth)acrylamide.

[0025] Such (meth)acrylic monomers may be used alone or in combination of two or more.

[0026] Examples of the carboxy-containing monomer (b) include unsaturated carboxylic acids such as (meth)acrylic acid, crotonic acid, maleic acid, maleic anhydride, fumaric acid, citraconic acid, glutaconic acid, itaconic acid, acrylamido-N-glycolic acid, and cinnamic acid. These may be used alone or in combination of two or more. Among these, (meth)acrylic acid is preferred, and methacrylic acid is more preferred. The content of the carboxyl group-containing monomer is preferably 1 to 70% by mass, more preferably 1 to 50% by mass, and even more preferably 1 to 30% by mass, based on the total polymer component.

[0027] Examples of the other polymerizable monomers (c) include vinyl carboxylate ester monomers such as vinyl acetate, vinyl propionate, vinyl stearate, and vinyl benzoate; and aromatic ring-containing monomers such as styrene and α-methylstyrene.

[0028] The weight average molecular weight of such a carboxyl group-containing acrylic polymer determined by gel permeation chromatography is preferably 10,000 to 300,000, more preferably 50,000 to 200,000, from the viewpoints of adsorptivity and water dispersibility.

[0029] The carboxy group-containing acrylic polymer is preferably a (meth)acrylic acid / alkyl (meth)acrylate copolymer, and suitable examples thereof include a (meth)acrylic acid / methyl (meth)acrylate / stearyl (meth)acrylate copolymer, a (meth)acrylic acid / methyl (meth)acrylate / 2-ethylhexyl (meth)acrylate copolymer, and a (meth)acrylic acid / methyl (meth)acrylate / n-octadecyl (meth)acrylate copolymer.

[0030] In the present invention, examples of methods for covalently binding a low-molecular-weight antibody to a polymer having a carboxy group include a method in which the polymer having a carboxy group is amide-bonded to an amino group present on the low-molecular-weight antibody using a condensation reagent such as a carbodiimide reagent, and a method in which the carboxy group on the polymer is converted into a reactive derivative such as azide, chloride, or isocyanate and reacted with an amino group present on the low-molecular-weight antibody.

[0031] The immune complex of the present invention thus obtained is useful as a molecule for capturing or detecting an antigen in an antigen detection method. The antigen detection method of the present invention refers to a method for specifically detecting an antigen in a sample using the immune complex of the present invention, and a representative example is a sandwich immunoassay. A sandwich immunoassay is a method in which two types of antibodies (a capture antibody and a detection antibody) that recognize different epitopes on the antigen to be analyzed are prepared, and the antigen is detected by being sandwiched between the capture antibody and the detection antibody and captured. Sandwich immunoassays include a method in which an antigen captured by a capture antibody is detected with a labeled detection antibody, and a method in which an antigen captured by a capture antibody is detected by adding a labeled detection antibody and a substrate, but either method is acceptable. Specifically, sandwich immunoassays are carried out in the form of ELISA (Enzyme-linked immunosorbent assay) or immunochromatography (also called lateral flow assay), but in the present invention, they are preferably carried out as a lateral flow assay.

[0032] According to the antigen detection method of the present invention, by contacting a test sample that contains or may contain the target antigen, it is possible to confirm the presence or absence of the antigen in the sample. Specifically, in the antigen detection method of the present invention, for example, the immune complex of the present invention is brought into contact with a sample, the antigen in the sample is captured by the immune complex, a detection antibody is reacted with the complex, and the reaction product is measured, thereby detecting the presence of the antigen in the sample. The contact between the immune complex and the sample may be any as long as it allows a sufficient antigen-antibody reaction, and the concentration of the immune complex, the amount of contact, and the contact time may be appropriately set.

[0033] The antigen detection method of the present invention is preferably carried out in the form of a lateral flow assay, which is an immunoassay that operates along a single axial direction of a lateral flow strip. The strip used in lateral flow immunoassays consists of 1) a sample pad, 2) a conjugate or reagent pad, 3) a reaction membrane, and 4) an absorbent or waste reservoir. The components are immobilized on an inert backing material and are commercially available as a kit in a convenient dipstick format or in a plastic case. Here, 1) the sample pad is an adsorption pad onto which the sample is applied. 2) The conjugate pad or reagent pad contains an antibody (detection antibody) specific to the analyte antigen labeled with colored particles (usually colloidal gold nanoparticles, latex microparticles, etc.). 3) The reaction membrane is generally a nitrocellulose membrane or a cellulose acetate membrane, on which an immune complex (capture antibody) containing a low molecular weight antibody specific to the analyte antigen is immobilized in a line, and the control line contains an antibody that captures the antibody labeled with the particle. 4) The absorbent reservoir or waste liquid reservoir is an absorbent pad designed to absorb the sample that has migrated through the reaction membrane by capillary action. [Example]

[0034] Production Example 1 Synthesis of Polymers A1 to A3 (Carboxy Group-Containing Cellulose Derivatives) The following polymers A1 to A3 were synthesized for use in the immunoconjugate. A1: Stearyl glycidyl ether modified carboxymethylated HEC (carboxymethyl group substitution degree 0.5) A2: Stearyl glycidyl ether modified carboxymethylated HEC (carboxymethyl group substitution degree 0.3) A3: Stearyl glycidyl ether modified carboxymethylated HEC (carboxymethyl group substitution degree 0.1) Details of the raw materials and reagents used in the synthesis of each polymer are as follows: Hydroxyethyl cellulose (HEC): "Natrosol® 250 JR", manufactured by Ashland Corporation; weight average molecular weight 150,000 Isopropyl alcohol: Marubeni Chemix Corporation 48% by weight sodium hydroxide aqueous solution: manufactured by Nankai Chemical Industry Co., Ltd. Stearyl glycidyl ether: "S-EP(C)", manufactured by Yokkaichi Synthetic Co., Ltd. Sodium chloroacetate: Fujifilm Wako Pure Chemical Industries, Ltd.

[0035] [Production Example 1-1] Production of Polymer A1 70 g of HEC was placed in a 1 L separable flask, and under a nitrogen gas atmosphere, 331.4 g of an 85% isopropyl alcohol / water mixed solvent was added. After stirring with a stirring blade at 200 rpm for 5 minutes, 8.12 g of a 48% by weight aqueous sodium hydroxide solution was added and stirred at 25°C for 15 minutes. Next, 5.6 g of stearyl glycidyl ether was added and the mixture was stirred at 80°C for 13 hours to react, then cooled to 50°C. 8.12 g of a 48% by weight aqueous sodium hydroxide solution and 28.4 g of sodium chloroacetate were added and the mixture was stirred at 50°C for 5 hours to react. Next, 13.5 g of a 90% by weight aqueous acetic acid solution was added and stirred for 30 minutes to neutralize. The resulting suspension was evenly distributed into two 500 mL centrifuge tubes and centrifuged using a high-speed refrigerated centrifuge ("CR21GIII", Hitachi Koki Co., Ltd.; 1500 g, 40 seconds). The supernatant was recovered by decantation, and the same amount of 85% by mass isopropyl alcohol aqueous solution was added to redisperse the supernatant, followed by another centrifugation. The redispersion and centrifugation procedures were repeated in the same manner. After the third centrifugation, the precipitate was recovered, dried under reduced pressure at 80°C overnight in a vacuum dryer, and pulverized in a grinder to obtain powdered polymer A1. For use in VHH-polymer conjugates, the polymer was dispersed in PBS at 1 mg / mL and allowed to stand overnight to obtain a clear solution, which was then used.

[0036] [Production Example 1-2] Production of Polymer A2 A 1 mg / mL PBS solution of polymer A2 was obtained by the same procedure as in Production Example 1-1, except that the amount of sodium chloroacetate added was changed to 17.0 g.

[0037] [Production Example 1-3] Production of Polymer A3 A 1 mg / mL PBS solution of polymer A3 was obtained by the same procedure as in Production Example 1-1, except that the amount of sodium chloroacetate added was changed to 5.67 g.

[0038] Production Example 2: Synthesis of Polymers A5 to A7 (Carboxy Group-Containing Acrylic Polymers) The following polymers A5 to A7 were synthesized for use in the immunoconjugate. A5: Methacrylic acid / methyl methacrylate / stearyl methacrylate = 30 / 60 / 10wt%, 96kJ A6: Methacrylic acid / methyl methacrylate / stearyl methacrylate = 30 / 60 / 10 wt%, 120kJ A7: Methacrylic acid / methyl methacrylate / stearyl methacrylate = 10 / 80 / 10wt%, 98kJ [Production Example 2-1] Production of Polymer A5 A reaction vessel equipped with a stirrer, a reflux condenser, and a dropping tank was initially charged with 3 g (30% by mass) of methacrylic acid, 6 g (60% by mass) of methyl methacrylate, 1 g (10% by mass) of stearyl methacrylate, 7.4 g of methyl ethyl ketone (hereinafter referred to as "MEK"), and 0.8 g of water, and the mixture was stirred for 10 minutes while maintaining the temperature of the reaction vessel at 77°C. Next, a mixture of 27 g (30% by mass) of methacrylic acid, 54 g (60% by mass) of methyl methacrylate, 9 g (10% by mass) of stearyl methacrylate, 66.3 g of MEK, 7.4 g of water, and 0.2 g of a chain transfer agent (2-mercaptoethanol) was added continuously to the reaction vessel over a period of 5 hours. The polymerization reaction was continued for 1 hour, and then terminated by cooling to room temperature, yielding a polymer MEK solution (solids concentration: 45%). The mixture was then dried under reduced pressure overnight in a vacuum dryer at 80 °C and pulverized in a pulverizer to obtain powdered Polymer A5 (weight average molecular weight: 96,000). When used for VHH-polymer complexes, the polymer was dispersed in PBS to a concentration of 1 mg / mL, and a few drops of 1N sodium hydroxide solution was added. The resulting solution was heated and stirred at 80°C for 4 hours to obtain a clear solution (weight-average molecular weight: 96,000).

[0039] [Production Example 2-2] Production of Polymer A6 The same procedure as in Production Example 2-1 was carried out except that no chain transfer agent was added, to obtain a 1 mg / mL PBS solution of polymer A6 (weight average molecular weight: 120,000).

[0040] [Production Example 2-3] Production of Polymer A7 The same procedure as in Production Example 2-1 was carried out, except that no chain transfer agent was added and the monomers added were 10% by mass of methacrylic acid, 80% by mass of methyl methacrylate, and 10% by mass of stearyl methacrylate, to obtain a 1 mg / mL PBS solution of Polymer A7 (weight average molecular weight: 98,000).

[0041] In addition, the following polymers A4 and A8 were used. A4: Sodium carboxymethylcellulose (n = approx. 1,050), manufactured by Tokyo Chemical Industry Co., Ltd. A8: Demol EP (sodium maleate / styrene = 50 / 50 mol%, 20kJ), manufactured by Kao Corporation

[0042] The weight-average molecular weights of the polymers A4 to A8 were determined by gel permeation chromatography. The measurement sample was prepared by mixing 0.1 g of polymer with 10 mL of the eluent described below in a glass vial, stirring with a magnetic stirrer at 25°C for 10 hours, and filtering through a syringe filter (Advantec Co., Ltd.'s "DISMIC-13HP," pore size: 0.2 μm, material: PTFE). The measurement conditions are shown below. GPC equipment: Tosoh Corporation "HLC-8320GPC" Columns: "TSKgel SuperAWM-H", "TSKgel SuperAW3000", and "TSKgel guardcolum Super AW-H" manufactured by Tosoh Corporation Eluent: N,N-dimethylformamide dissolved with phosphoric acid and lithium bromide at concentrations of 60mmol / L and 50mmol / L, respectively. Flow rate: 0.5mL / min Standard material: Monodisperse polystyrene kits with known molecular weights, manufactured by Tosoh Corporation: "PStQuick B (F-550, F-80, F-10, F-1, A-1000)" and "PStQuick C (F-288, F-40, F-4, A-5000, A-500)"

[0043] [Production Example 3-1] (Production of VHH antibody B1) A VHH antibody against SARS-CoV-2 (VHH-COVE9, SEQ ID NO: 1) described in Japanese Patent Publication No. 7414225 was produced in accordance with the method described in the same publication (VHH antibody B1).

[0044] [Production Example 3-2] (Production of VHH antibody B2) A His-tagged VHH antibody (NoVHH53, SEQ ID NO: 2) against porcine norovirus described in Japanese Patent Application No. 2023-056675 was produced in accordance with the method described therein (VHH antibody B2).

[0045] [Production Example 3-3] (Production of Lys-tagged VHH antibody B1-L1) VHH antibody B1-L1 was synthesized in the same manner as in Production Example 3-1, except that a Lys tag (L1) sequence (KKKKKK (SEQ ID NO: 4)) was inserted before the His tag sequence (GSHHHHHH (SEQ ID NO: 3)).

[0046] [Production Example 3-4] (Production of Lys-tagged VHH antibody B1-L2) VHH antibody B1-L2 was synthesized using the same procedures as in Production Example 3-1, except that a Lys tag (L2) sequence (EPKTPKPQSKKKKKK (sequence number 6)) linked to EPKTPKPQS (sequence number 5) was inserted before the His tag sequence (GSHHHHHH (sequence number 3)).

[0047] [Production Example 3-5] (Production of Lys-tagged VHH antibody B2-L1) VHH antibody B2-L1 was synthesized in the same manner as in Production Example 3-2, except that a Lys tag (L1) sequence (KKKKKK (SEQ ID NO: 4)) was inserted before the His tag sequence (GSHHHHHH (SEQ ID NO: 3)).

[0048] [Production Example 3-6] (Production of Lys-tagged VHH antibody B2-L2) VHH antibody B2-L2 was synthesized using the same procedures as in Production Example 3-2, except that a Lys tag (L2) sequence (EPKTPKPQSKKKKKK (sequence number 6)) linked to EPKTPKPQS (sequence number 5) was inserted before the His tag sequence (GSHHHHHH (sequence number 3)).

[0049] Example 1 (Preparation of VHH polymer complex) In a 2 mL Eppendorf tube, 102 μL of a 1 mg / mL solution of polymer A1 in PBS and 100 μL of a 0.68 mg / mL solution of VHH antibody B1-L1 were mixed, and 20 μL of a condensation agent solution (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide as a condensation aid, each dissolved in MES buffer at pH 4.9 to a concentration of 50 mg / mL) was added. The mixture was stirred overnight at 25°C. The resulting VHH-polymer conjugate solution was further mixed with 356 μL of PBS to obtain a VHH-polymer conjugate reaction solution at 0.1 mg / mL (based on VHH antibody monomer). The resulting reaction solution was applied to an Amicon Ultra-0.5 (Merck Millipore, MWCO = 100,000). The buffer was exchanged three times with PBS to remove the condensation agent and unreacted VHH antibody, yielding a VHH-polymer conjugate solution.

[0050] [Example 2] A VHH-polymer complex solution was obtained by carrying out the same procedure as in Example 1, except that 204 μL of A2 was used as the polymer and the same parts by mass of B1-L2 was used as the VHH antibody.

[0051] [Example 3] A VHH-polymer complex solution was obtained by carrying out the same procedure as in Example 1, except that 204 μL of A2 was used as the polymer and the same parts by mass of B2-L1 was used as the VHH antibody.

[0052] [Example 4] A VHH-polymer complex solution was obtained by carrying out the same procedure as in Example 1, except that 204 μL of A2 was used as the polymer and the same parts by mass of B2-L2 was used as the VHH antibody.

[0053] [Example 5] A VHH-polymer complex solution was obtained by carrying out the same procedure as in Example 1, except that 204 μL of A4 was used as the polymer and the same parts by mass of B1-L1 was used as the VHH antibody.

[0054] [Example 6] A VHH-polymer complex solution was obtained by carrying out the same procedure as in Example 1, except that 204 μL of A4 was used as the polymer and the same parts by mass of B1-L2 were used as the VHH antibody.

[0055] [Example 7] A5 was used as the polymer and B1-L1 as the VHH antibody.

[0056] [Example 8] A6 was used as the polymer and B1-L2 as the VHH antibody.

[0057] [Example 9] A7 was used as the polymer and B1-L2 as the VHH antibody.

[0058] [Example 10] A8 was used as the polymer and B1-L2 as the VHH antibody.

[0059] [Comparative Example 1] Performance evaluation was carried out using B1 as the VHH antibody.

[0060] Comparative Example 2 A VHH-polymer complex solution was obtained by carrying out the same procedure as in Example 1, except that the same parts by mass of B1 were used as the VHH antibody.

[0061] Comparative Example 3 A VHH-polymer complex solution was obtained by carrying out the same procedure as in Example 2, except that the same parts by mass of B1 were used as the VHH antibody.

[0062] Comparative Example 4 Performance evaluation was carried out using B1-L1 as the VHH antibody.

[0063] Comparative Example 5 Performance evaluation was carried out using B1-L2 as the VHH antibody.

[0064] Comparative Example 6 A VHH-polymer complex solution was obtained by carrying out the same procedure as in Example 1, except that the same parts by mass of A3 were used as the polymer and the same parts by mass of B1 were used as the VHH antibody.

[0065] Comparative Example 7 A VHH-polymer complex solution was obtained by carrying out the same procedure as in Example 6, except that the same parts by mass of B1 were used as the VHH antibody.

[0066] [Comparative Example 8] A5 was used as the polymer and B1 as the VHH antibody.

[0067] Comparative Example 9 A6 was used as the polymer and B1 as the VHH antibody.

[0068] [Comparative Example 10] A7 was used as the polymer and B1 as the VHH antibody.

[0069] [Comparative Example 11] A8 was used as the polymer and B1 as the VHH antibody.

[0070] [evaluation] The VHHs and VHH-polymer conjugates obtained in the Examples and Comparative Examples were evaluated according to the following (1) to (4). The results are shown in Table 1.

[0071] (1) Measurement of the reaction rate of VHH-polymer complexes (Confirmed by SDS-PAGE) 65 μL of the VHH-polymer conjugate reaction mixture (0.1 mg / mL VHH monomer equivalent), 25 μL of NuPAGE™ LDS Sample Buffer (4x) (Thermo Fisher Scientific), and 10 μL of NuPAGE™ Sample Reducing Agent (10x) (Thermo Fisher Scientific) were mixed and heated at 100°C for 5 minutes. Then, 20 μL of the sample solution was applied to the wells of the gel. 5 μL of Novex™ Sharp Pre-stained Protein Standard (Thermo Fisher Scientific) was also applied to the wells as a molecular weight marker. The gel used was NuPAGE™ 10% Bis-Tris, 1.0 mm, Mini Protein Gel, 12-well (Thermo Fisher Scientific). The electrophoresis buffer was prepared by diluting NuPAGE™ MES SDS Running Buffer (20x) (Thermo Fisher Scientific) 20 times with water. Electrophoresis was performed for 40 minutes at 200 V using an XCell SureLock™ MiniCell Electrophoresis System (Thermo Fisher Scientific) connected to a PowerEase™ 90W Power Supply (Thermo Fisher Scientific). The gel was then stained with GelCode™ Blue Stain Reagent (Thermo Fisher Scientific) to confirm the presence or absence of a target protein band. A band was observed around 15 kDa in all samples, and the reaction rate of the VHH-polymer complex was estimated by comparing the band intensity with that of the starting VHH antibody monomer solution.

[0072] (2) Evaluation of the performance of each VHH and VHH polymer complex by sandwich ELISA (2-1) Evaluation of binding activity To each well of a 96-well plate ("Assay Plate (Medium Binding Capacity) 96-well (Clear)"; AGC TECHNO GLASS (IWAKI)), 100 μL of each VHH and VHH polymer conjugate solution, adjusted to 10 μg / mL in PBS, was added and incubated overnight at 4°C for immobilization. The VHH and VHH polymer conjugate solutions were then carefully removed using a pipette, followed by the addition of 200 μL of PBST (PBS containing 0.05% Tween 20) and careful removal with a pipette (washing) three times. Next, 200 μL of 5% skim milk / PBST was added to each well, and the plate was incubated at room temperature for 1 hour for blocking. The skim milk / PBST was carefully removed using a pipette, followed by the washing procedure three times. SARS-CoV-2 (2019-nCoV) Spike S1 (Fc-tagged S1 protein, Sino Biological Inc.) was diluted to 250 μg / mL in distilled water and then diluted to 0.1000 ng / mL in PBST (PBS containing 0.05% Tween 20). 100 μL of the prepared S1 protein was added to each well and incubated at room temperature for 1 hour. The target protein solution was then carefully removed using a pipette, and the plate was washed three times. 100 μL of Goat pAb to Hu IgG (HRP) (Abcam) diluted 1 / 5,000 in PBST was added to each well as the detection antibody. After incubation at room temperature for 1 hour, the antibody was carefully removed using a pipette, and the plate was washed four times. As a luminescent substrate, OPD tablets (Thermo Fisher Scientific) were dissolved in Stable Peroxide Substrate buffer (Thermo Fisher Scientific), and 100 μL of this solution was added to each well. Thereafter, the mixture was incubated at room temperature in the dark for 30 minutes, and the absorbance at 450 nm was measured using the GloMax (registered trademark) Explorer System (Promega).

[0073] (2-2) Evaluation of His activity A relative comparison of the immobilized amounts of solid-phase VHH antibody and VHH-polymer complex was performed using the same method as in (2-1), except that anti-His IgG-HRP (Abcam) diluted 1 / 1,000 with PBST was used as the detection antibody.

[0074] (2-3) Evaluation of anti-norovirus VHHs (B2, B2-L1, B2-L2) and VHH-polymer conjugates The binding activity of immobilized anti-norovirus VHH antibodies and VHH-polymer complexes was evaluated using the same method as in (2-1), except that Blocking Reagent for ELISA (Cosmo Bio) was used as the blocking solution, GII.19 VLP was used as the antigen, Rabbit anti-GII pAb was used as the primary detection antibody, and Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling Technology, Inc.) was used as the secondary detection antibody.

[0075] (3) Evaluation of the performance of VHH-polymer conjugates by on-plate conjugation using sandwich ELISA (3-1) Evaluation of binding activity To each well of a 96-well, medium-binding assay plate (clear, AGC TECHNO GLASS (IWAKI)), 100 μL of polymer solution (prepared at 10 μg / mL in PBS) was added and incubated overnight at 4°C for immobilization. After carefully removing the polymer solution with a pipette, 100 μL of a condensation agent solution (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide as a condensation aid, each dissolved in MES buffer at pH 4.9 to a concentration of 50 mg / mL) was added and incubated for 20 minutes at room temperature. After carefully removing the condensation agent solution with a pipette, 100 μL of polymer solution (prepared at 10 μg / mL in PBS) was added and incubated for 3 hours at room temperature, followed by overnight incubation at 4°C for immobilization. Next, the VHH antibody solution was carefully removed using a pipette, and 200 μL of PBST (PBS containing 0.05% Tween 20) was added and carefully removed using a pipette (washing) three times. Next, 200 μL of 5% skim milk / PBST was added to each well, and the wells were incubated at room temperature for 1 hour for blocking. The skim milk / PBST was carefully removed using a pipette, and the washing procedure was repeated three times. SARS-CoV-2 (2019-nCoV) Spike S1 (Fc-tagged S1 protein, Sino Biological Inc.) was diluted to 250 μg / mL in distilled water and then diluted to 0.1000 ng / mL in PBST (PBS containing 0.05% Tween 20). 100 μL of the prepared S1 protein was added to each well and incubated at room temperature for 1 hour. The target protein solution was then carefully removed using a pipette, and the plate was washed three times. 100 μL of Goat pAb to Hu IgG (HRP) (Abcam) diluted 1 / 5,000 in PBST was added to each well as the detection antibody. After incubation at room temperature for 1 hour, the antibody was carefully removed using a pipette, and the plate was washed four times. As a luminescent substrate, OPD tablets (Thermo Fisher Scientific) were dissolved in Stable Peroxide Substrate buffer (Thermo Fisher Scientific), and 100 μL of this solution was added to each well. Thereafter, the mixture was incubated at room temperature in the dark for 30 minutes, and the absorbance at 450 nm was measured using the GloMax (registered trademark) Explorer System (Promega).

[0076] (3-2) Evaluation of His activity A relative comparison of the amount of immobilized VHH antibody was performed in the same manner as in (3-1), except that anti-His IgG-HRP (Abcam) diluted 1 / 1,000 with PBST was used as the detection antibody.

[0077] (4) Evaluation of the performance of each VHH and VHH polymer complex as a capture antibody by lateral flow assay (4-1) Sensitization of labeled antibodies to dye particles NanoAct colored cellulose particles (chemically bonded, Asahi Kasei) were used as the dye particles. Sensitization of the labeled antibody was performed according to the attached protocol. The procedure is outlined below. 60 μL of NanoAct was dispensed into a 15 mL centrifuge tube, and 540 μL of 100 mM MES (pH 6), 7.5 μL of 4 wt% EDC, and 15 μL of 4 wt% NHS were added. The tube was left to stand at room temperature for 15 minutes to esterify the carboxyl groups. The tube was centrifuged at 13,000 × g for 20 minutes, the supernatant was removed, and 600 μL of 100 mM MES (pH 6) was added and sonicated. 60 μg of anti-hFc IgG antibody was added as the labeled antibody, followed by vortexing and incubation at 37°C for 120 minutes. Next, 7.2 mL of blocking buffer (1 wt% Casein, 100 mM Boric Acid, pH 8.5) was added, followed by vortexing and incubation at 37°C for 60 minutes. The mixture was centrifuged at 13,000 × g for 20 minutes, the supernatant was removed, 600 μL of 100 mM MES (pH 6) was added, and the mixture was sonicated. The mixture was centrifuged again at 13,000 × g for 15 minutes, the supernatant was removed, and 1.3 mL of storage solution (33 mM Boric Acid, 0.2 wt% Casein, 15 wt% Sucrose, pH 9.2) was added, the mixture was sonicated, and the mixture was stored at 4°C until use.

[0078] (4-2) Assembling the half strip FF High Performance FF120HP+ (Cytiva) cut to 5 mm x 40 mm was used as the nitrocellulose membrane. CM5 (Cytiva) cut to 5 mm x 20 mm was used as the absorbent pad. Half strips were assembled by laminating the nitrocellulose membrane and absorbent pad with a 10 mm overlap and adhering them with a backing sheet (GL-57888, Lohmann).

[0079] (4-3) Immobilization of capture antibodies onto nitrocellulose membrane The capture antibody, VHH antibody, and VHH-polymer complex were prepared in advance in PBS solution at 1 mg / mL. 1 μL of the antibody was spotted with a pipette at a point 2 cm from the bottom end of the strip assembled in (4-2) above. The antibody was then immobilized by drying at 37°C for 20 minutes. Hereinafter, the site where the capture antibody was immobilized is referred to as the test line.

[0080] (4-4) Preparation of developing solution 200 mM HEPES, pH 7 was prepared. NaCl and 0.5% Tween 20 were added to give final concentrations of 500 mM and 0.5% Tween 20, respectively, to prepare an HBST solution, which was used as a developing solution.

[0081] (4-5) Performance evaluation of the constructed immunochromatography Fc-tagged S1 protein was added to the developer solution to be evaluated, and serial dilutions of 10 ng / mL, 2 ng / mL, 0.4 ng / mL, and 0.08 ng / mL were used to prepare positive samples. 50 μL of the positive sample solution and 20 μL of the dye particles prepared in (4-1) were added to one well of a 96-well plate and mixed thoroughly by pipetting. The capture antibody-immobilized membrane prepared in (4-3) was then immersed in the well and allowed to stand until the solution had completely drained. As shown in Table 1, a clearly visible blue dot on the test line was designated "++," a visually visible dot was designated "+," a visible but very faint dot was designated "±," and an invisible dot was designated "-."

[0082] (Results of complex reaction rate) As shown in Table 1, the samples of Examples 1 to 6 were found to react with more VHH antibodies and form complexes with a high VHH antibody density compared to Comparative Examples 2, 3, and 7, which did not have a Lys tag, and Comparative Example 6, which had a low carboxylic acid content in the polymer.

[0083] (ELISA (1) results) As shown in Table 1, the samples of Examples 1 to 6 were found to immobilize a larger number of VHH antibodies on polystyrene plates and exhibit higher antigen binding activity than Comparative Examples 1 to 3 and 7, which did not have a Lys tag introduced, Comparative Examples 4 and 5, which were not conjugated to a polymer, and Comparative Example 6, which had a low carboxylic acid content in the polymer.

[0084] (ELISA (2) results) As shown in Table 1, the samples of Examples 1 to 6 were found to exhibit high antigen binding activity by forming well-oriented complexes with the carboxyl group-containing polymer on the polystyrene plate, compared to Comparative Examples 8 to 12, which did not have a Lys tag.

[0085] (Lateral flow assay results) As shown in Table 1, when the sample of Comparative Example 1 was used as the capture antibody, the lower detection limit was 2.0 ng. In contrast, when the sample of Example 1 was used as the capture antibody, detection was possible down to 0.4 ng. In other words, it was revealed that the detection sensitivity could be improved by more than five times by using a conjugate of a Lys-tagged VHH antibody and a polymer as the capture antibody.

[0086] [Table 1]

Claims

1. An immune complex comprising a lysine or a lysine-tagged low molecular weight antibody covalently bound to a polymer having a carboxy group.

2. The low molecular weight antibody may be a single domain antibody, a single chain antibody, a multivalent single chain antibody, a constant domain-fused single chain antibody, a Fab fragment, or a F(ab') ２ The conjugate of claim 1, which is a fragment, an immunoglobulin new antigen receptor or a peptide aptamer.

3. The complex of claim 1, wherein the low molecular weight antibody is a VHH antibody.

4. 3. The composite according to claim 1, wherein the polymer having a carboxy group is a carboxy group-containing cellulose derivative or a carboxy group-containing acrylic polymer.

5. 5. The composite according to claim 4, wherein the carboxy group-containing cellulose derivative is alkyl glycidyl ether-modified carboxymethylated hydroxyethyl cellulose.

6. 5. The composite according to claim 4, wherein the carboxy group-containing acrylic polymer is a (meth)acrylic acid / alkyl(meth)acrylate copolymer.

7. The conjugate according to claim 1 or 2, wherein the lysine tag is an oligopeptide containing six or more consecutive lysine residues.

8. The conjugate according to claim 1 or 2, wherein the lysine tag is an oligopeptide containing 6 to 12 consecutive lysine residues.

9. A method for detecting an antigen, which uses the immune complex according to claim 1.

10. The method according to claim 9, wherein the antigen is detected by ELISA or immunochromatography.

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