JP6732283B2 - Method for producing specific antibody against target antigen - Google Patents

Description

TECHNICAL FIELD The present invention relates to an antigen-binding carrier, a vector, a kit, and a method for producing a specific antibody against an antigen of interest.

Antibodies are widely used not only in basic life science research, but also as laboratory diagnostics or pharmaceuticals. An antibody is generally produced by immunizing an animal with a specific protein serving as an antigen, but it is not easy to obtain an antibody that can be used for basic life science research, laboratory diagnosis, or pharmaceuticals. In particular, it is often important in terms of function, and it is often difficult to produce an antibody against a biological substance having a functional structure that is evolutionarily highly conserved.
For the purpose of producing a site-specific antibody, a method has been developed in which a synthetic peptide or the like at a specific site of a protein is bound to various carriers and immunized (see, for example, Patent Document 1). In many cases, it is not possible to obtain an antibody, and a new technique for freely producing an antibody against a specific site of a protein is required.

In autoimmune diseases, an antibody against a biological substance having a highly conserved site, which is difficult to produce an antibody by a usual immunization method, is often produced.
In autoimmune disease, we have a common conserved C-terminal site in the large subunits P0, P1 and P2 of the P protein, an acidic phosphorylated protein in the ribosome that is often the target of autoantibodies. It was thought that the strong antigenicity of P protein originates from the higher-order structure and dynamic properties of P protein, and various analyzes have been performed. As a result, the P protein forms a unique complex, and the common C-terminal site (hereinafter, also referred to as “C-terminal antigen site”) serving as an antigen site flexibly moves, and the human P-protein complex. It was revealed that by administering the body to mice, a specific antibody against the common C-terminal antigenic site (hereinafter sometimes referred to as "anti-P antibody") is preferentially generated (for example, non-patent document). 1 and Patent Document 2.).

Japanese Patent Publication No. 2008-504219 JP, 2008-110943, A

Sato, H., et al., “Characterization of anti-P monoclonal antibodies directed against the ribosomal protein-RNA complex antigen and produced using Murphy Roths large autoimmune-prone mice”, Clin. Exp. Immunol., vol.179, p236. -244, 2014.

When an anti-P antibody is produced by using the human P protein complex as an antigen by the method described in Non-Patent Document 1 and Patent Document 2, three kinds of proteins, P0, P1, and P2, which compose the complex, are prepared. Had to be prepared and further complexed. Further, when preparing three kinds of proteins constituting the complex using an expression system using Escherichia coli, it was difficult to separate and purify from proteins derived from Escherichia coli.
Further, in the method for producing an antibody using the human P protein complex as an antigen, only an anti-P antibody can be produced, and an antibody specific to any protein cannot be produced.

The present invention has been made in view of the above circumstances, and is applicable to the production of an antibody specific to an arbitrary antigen by applying an antibody production technique using a human P protein complex as an antigen. It is intended to provide a carrier for easily binding an antigen.

As a result of intensive studies to solve the above problems, the present inventors have focused on archaebacterium aP1, which is an ortholog of human P protein. The aP1 forms a multimer even if the base aP0 does not exist, and the region sandwiched between the aP0 binding site of each aP1 and the C-terminal antigen site forms a tentacle-like structure having flexibility. Then, it was found that a specific antibody against the antigen protein can be produced by immunizing an animal with the aP1 having the C-terminal antigen site substituted with a specific antigen protein, and the present invention It came to completion.

The present invention includes the following aspects.
[1] A method for producing a specific antibody against an antigen of interest, comprising:
A binding step of binding an antigen of interest to an antigen-binding carrier to produce an antigen-binding carrier,
An immunization step of administering the antigen-binding carrier to a non-human mammal,
A collection step of collecting serum, spleen cells, lymph node cells, peripheral blood leukocytes, or B cells from the non-human mammal after the immunization step,
Equipped with
The antigen-binding carrier is
Core particles,
At least two flexible linkers linked to the core particles,
Equipped with
In the linker, Ri antigen binding sites der is opposite the tip to the connection portion between the core particles,
The core particle and the linker are any of the following proteins (a) to (c),
(A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1,
(B) a protein consisting of an amino acid sequence in which 1 or more and 5 or less amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1, and having a multimer-forming ability and a flexible region,
(C) a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1, and having a multimer-forming ability and a flexible region,
The production method, wherein the antigen has a highly conserved site and is a protein other than ribosomal protein.
[2] The production method according to [1], wherein the protein forms a multimer of dimers or more and 7 or less.
[3] In the binding step, the nucleic acid encoding the antigen is inserted into an expression vector containing the nucleic acid encoding the carrier for antigen binding to prepare an expression vector containing the nucleic acid encoding the antigen-binding carrier. The production method according to [1] or [2], wherein a host is transformed with an expression vector containing a nucleic acid encoding a binding carrier, and then the host is cultured to express the antigen-binding carrier.
[4] The production method according to any one of [1] to [3], wherein the antigen is Hbs1.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide an antigen-binding carrier that can be used for producing an antibody specific to an arbitrary antigen and is easy to handle.

It is a figure which shows typically one Embodiment of the carrier for antigen binding of this invention. It is a figure which shows typically the other embodiment of the carrier for antigen binding of this invention. 3 is an image showing the results of verifying the antigen specificity of the polyclonal antibody in Example 1. 5 is an image showing the result of verifying the antigen specificity of the polyclonal antibody in Example 2.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as necessary.

<< Carrier for antigen binding >>
In one embodiment, the present invention comprises a core particle and at least two flexible linkers connected to the core particle, wherein the tip of the linker on the side opposite to the connecting portion with the core particle is An antigen-binding carrier that is an antigen-binding site is provided.

The antigen-binding carrier of the present embodiment is easy to handle and can be used for producing a specific antibody against any antigen. As the antigen, it is also possible to use an antigen having a highly conserved site, which has been conventionally difficult to produce an antibody.

<Structure>
FIG. 1 is a diagram schematically showing an embodiment of the carrier for antigen binding of the present invention.
The core particle (11) in this embodiment is for supporting the linker (12).
The linker (12) in the present embodiment is for binding an antigen.
In the antigen-binding carrier (1A) of this embodiment, a plurality of flexible linkers (12) are linked to a core particle (11). The linker (12) has an antigen binding site (13) at the tip opposite to the connecting part with the core particle (11).
Although FIG. 1 illustrates an example in which four linkers (12) are connected, the present invention is not limited to this. Further, in FIG. 1, the core particles (11) are exemplified as spherical particles, but the present invention is not limited thereto.

In the antigen-binding carrier (1A) of the present embodiment, since the linker (12) has flexibility, the antigen bound to the tip on the side opposite to the connecting part with the core particle (11) can move flexibly. You can
The length of the linker (12) is preferably 100 Å or more and 200 Å or less, more preferably 100 Å or more and 180 Å or less, and further preferably 100 Å or more and 150 Å or less. When the length of the linker (12) is within the above range, structural damage is unlikely to occur between adjacent linkers, and the receptor on the surface of antibody-producing cells can be efficiently activated.

The shape of the core particle (11) is, for example, sphere, polyhedron (eg, tetrahedron, pentahedron, hexahedron (including cube), octahedron, dodecahedron, icosahedron, icosahedron, Kepler Poinsso solids, etc.), and aggregates thereof.
At least two linkers (12) are linked to the core particles (11), preferably 2 or more and 10 or less, more preferably 4 or more and 9 or less, and still more preferably 4 or more and 8 or less. By providing a plurality of linkers (12), the receptor existing on the surface of the antibody-producing cell (B cell) can be efficiently activated, and the antibody against the antigen can be preferentially produced.

The molecular weight of the antigen-binding carrier of the present embodiment may be, for example, 20,000 or more and 110,000 or less, for example, 40,000 or more and 70,000 or less, and for example, 40,000 or more and 50,000 or less.

The antigen-binding carrier of the present embodiment is preferably biocompatible. Here, “having biocompatibility” means avoiding attack by macrophages or T cells existing in the body of a non-human mammal after immunization to the non-human mammal, and preventing degradation. Means the ability to
Further, the antigen-binding carrier of the present embodiment preferably has heat resistance. By having heat resistance, for example, the antigen-binding carrier of the present embodiment is composed of a protein, and when produced using an Escherichia coli expression system, impurities such as Escherichia coli-derived protein can be denatured by heating. The carrier for antigen binding of the present embodiment can be efficiently removed by removing the carrier.

Moreover, the antigen-binding carrier of the present embodiment is not particularly limited to applicable antigens, and can also be applied to antigens having highly conserved sites, which have been difficult to produce antibodies in the past.
Applicable antigens include, for example, proteins such as histones, enzymes, ribosomal proteins, mitochondrial proteins, proliferating cell nuclear antigens (PCNA); RNA-protein complexes such as snRNPs; centromeres, sugar chains, glycoproteins; DNA, RNA, Examples include, but are not limited to, nucleic acids such as miRNA and siRNA (the nucleic acid may be single-stranded or double-stranded, and also include synthetic nucleic acids such as LNA).

The molecular weight of the antigen applicable to the carrier for antigen binding of the present embodiment is preferably 600 or more and 5000 or less, more preferably 1000 or more and 4000 or less, and further preferably 1500 or more and 3000 or less. preferable. When the molecular weight of the antigen is within the above range, structural damage due to the antigens is unlikely to occur, and the receptor on the surface of the antibody-producing cell can be efficiently activated.
More preferable antigens include, for example, peptides consisting of 10 or more and 30 or less amino acid residues.

The mode of binding with the antigen at the antigen-binding site of the carrier for antigen binding of the present embodiment is not particularly limited and includes, for example, covalent bond (eg, amide bond, disulfide bond, ester bond, etc.), electrostatic interaction. , Hydrogen bond, van der Waals bond, bond by hydrophobic effect, and the like.
Next, the material of the carrier for antigen binding of the present embodiment will be described in detail.

<Material>
The material forming the core particle and the linker is not particularly limited as long as it has biocompatibility. Examples of the material forming the core particles and the linker include proteins (for example, zein, casein, gelatin, gluten, serum albumin, collagen, and aP1 lacking the C-terminal antigen site (the amino acid sequence is shown in SEQ ID NO: 1)). And protein variants having substantially the same activity as those), polysaccharides (eg, alginate, cellulose, dextran, pullulane, polyhyaluronic acid, and their derivatives), chitin, poly(3- Hydroxyalkanoate) (particularly poly(β-hydroxybutyrate), poly(3-hydroxyoctanoate)), poly(3-hydroxy fatty acid), and other naturally-occurring polymer compounds; polyphosphazene, poly(vinyl alcohol) ), polyamide, polyesteramide, poly(amino acid), polyanhydride, polycarbonate, polyacrylate, polyalkylene (eg, polyethylene), polyacrylamide, polyalkylene glycol (eg, polyethylene glycol), polyalkylene oxide (eg, Polyethylene oxide etc.), polyalkylene terephthalate (eg polyethylene terephthalate etc.), polyorthoester, polyvinyl ether, polyvinyl ester, polyvinyl halide, polyvinylpyrrolidone, polyester, polysiloxane, polyurethane, polyhydroxy acid (eg polylactide, polyglycolide etc.) ), poly(hydroxybutyric acid), poly(hydroxyvaleric acid), poly[lactide-co-(ε-caprolactone)], poly[glycolide-co-(ε-caprolactone)], etc.), poly(hydroxyalkanoate), And synthetic polymer compounds such as copolymers thereof. The core particles may be composed of one kind of the above-mentioned materials, or may be composed of two or more kinds.

The cellulose includes synthetically modified ones, and examples thereof include cellulose derivatives (eg, alkyl cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester, nitrocellulose, and chitosan). As more specific cellulose derivatives, for example, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxymethyl cellulose, cellulose triacetate, Cellulose sulfate sodium salt and the like can be mentioned.

More specifically as the polyacrylate, for example, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(methacryl) Acid isodecyl), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and the like.

Among them, the material constituting the core particle and the linker is preferably a protein, and is archaebacterium-derived aP1 deficient in the C-terminal antigen site, or a protein variant having substantially the same activity as the aP1. Is more preferable.
Archaea-derived aP1 lacking the C-terminal antigen site, or a protein variant having substantially the same activity as the aP1 has heat resistance. Therefore, when the antigen-binding carrier of the present embodiment is produced using an Escherichia coli expression system, impurities such as Escherichia coli-derived proteins are removed by denaturing by heating to efficiently purify the antigen-binding carrier. You can

FIG. 2 is a view schematically showing another embodiment of the antigen-binding carrier of the present invention, which is a view schematically showing the tetramer of archaebacterium aP1 lacking the C-terminal antigen site.
The tetramer (1B) of archaeal aP1 lacking the C-terminal antigen site has an aP0 binding site (21), an antigen binding site (23), an aP0 binding site (21) and an antigen binding site (23). It contains aP1 (100) lacking the C-terminal antigenic site consisting of the sandwiched flexible region (22). Furthermore, the aP0 binding sites (21) in aP1 (100) lacking the C-terminal antigen site bind to each other to form a core particle-like structure, thereby forming a multimer.
In FIG. 2, aP1(100) lacking the C-terminal antigen site forms a tetramer, but the present invention is not limited to this.
The aP1 (100) lacking the C-terminal antigen site may form a multimer by mixing the P0 binding sites (21) with each other by mixing the aP1 (100), which does not include the base aP0. it can. In addition, by including aP0, it is possible to form a multimer of at most 7 mer. The aP1 lacking the C-terminal antigen site used in the carrier for antigen binding of the present embodiment preferably forms a multimer of dimer or more and 7 or less, more preferably tetramer or more and 7 or less. There is.

In addition to aP1 lacking the C-terminal antigen site used for the antigen-binding carrier in the present embodiment, it may contain aP0 or may not contain aP0. Above all, it is preferable that the carrier for antigen binding in the present embodiment does not contain aP0, since only one kind of protein is required.

SEQ ID NO: 1 is the amino acid sequence of archaeal aP1 lacking the C-terminal antigenic site.
In addition, the protein variant having substantially the same activity as aP1 lacking the C-terminal antigen site means, for example, that one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO:1. And a protein containing the amino acid sequence.
Here, the number of amino acids that may be deleted, substituted, or added is preferably 1 or more and 15 or less, more preferably 1 or more and 10 or less, still more preferably 1 or more and 5 or less.

Further, a protein variant having substantially the same activity as aP1 lacking the C-terminal antigenic site has, for example, 70% or more identity with the amino acid sequence shown in SEQ ID NO: 1, preferably 80% or more. , More preferably 90% or more, still more preferably 95% or more, and particularly preferably 99% or more of a protein containing an amino acid sequence.

Further, the protein variant has substantially the same activity as that of aP1 lacking the C-terminal antigen site, that is, the ability to form multimers and the flexible region.

The antigen-binding site in the linker may be, for example, a functional group capable of binding to the target antigen.
Examples of the functional groups include active ester structures (for example, p-nitrophenyl ester group, N-hydroxysuccinimide ester group, succinimide ester group, phthalic acid imide ester group, 5-norbornene-2,3-dicarboxyl group. Imide ester group), a carboxy group, an amino group, a thiol group, and the like, but are not limited thereto.

<Method for producing carrier for antigen binding of the present embodiment>
As the method for producing the carrier for antigen binding of the present embodiment, for example, a commercially available polymer compound having biocompatibility is used as the core particle and the linker, and a known method is appropriately selected to form the core particle and the linker. It may be manufactured by combining them. The bond mode between the core particle and the linker is not particularly limited, and examples thereof include covalent bond (eg, amide bond, disulfide bond, ester bond, etc.), electrostatic interaction, hydrogen bond, van der Waals bond, and hydrophobic effect. And the like.

As a method for producing the carrier for antigen binding of the present embodiment, more specifically, for example, a solution containing a linker is prepared, and after coating the core particle surface by a known method such as dipping or spraying, at room temperature or Chemical bonding can be carried out by drying under heating.
The solution concentration of the above linker is not particularly limited and may be, for example, 0.05% by weight or more, for example, 0.1% by weight or more and 30% by weight or less, for example, 0.1% by weight. The amount may be 20% by weight or less and, for example, 0.3% by weight or more and 10% by weight or less. When the linker concentration in the linker solution is within the above range, the number of linkers chemically bonded to the core particles can be fixed, and the linkers can be efficiently immobilized.

Further, the case where the antigen-binding carrier of the present embodiment is a protein will be described in detail in [Binding step] of <<Method for producing specific antibody against antigen of interest>> described below.

<<<vector>>>
In one embodiment, the present invention provides a vector containing a nucleic acid encoding an archaebacterium-derived aP1 deficient in the above-mentioned C-terminal antigen site, or a protein variant having substantially the same activity as the aP1.

According to the vector of the present embodiment, an antigen-binding carrier composed of archaebacterium-derived aP1 lacking the C-terminal antigenic site or a protein variant having substantially the same activity as the aP1 can be easily produced. it can.

Examples of the nucleic acid encoding the archaebacterium-derived aP1 deficient in the above C-terminal antigen site or a protein variant having substantially the same activity as the aP1 include, for example, a nucleic acid having the nucleotide sequence shown in SEQ ID NO: 2, Or a nucleic acid encoding a protein having 70% or more, for example 80% or more, for example 90% or more, for example 95% or more identity with the nucleotide sequence shown in SEQ ID NO: 2 and having a multimerizing ability and a flexible region. Nucleic acid having a base sequence and the like can be mentioned.

The vector of this embodiment is preferably an expression vector. The expression vector is not particularly limited, and examples thereof include plasmids derived from Escherichia coli such as pBR322, pBR325, pUC12 and pUC13; plasmids derived from Bacillus subtilis such as pUB110, pTP5 and pC194; plasmids derived from yeast such as pSH19 and pSH15; λ phage and the like. Bacteriophage; viruses such as adenovirus, adeno-associated virus, lentivirus, vaccinia virus, baculovirus; and vectors modified with these. When the antigen is a protein, these may be for producing an antigen-binding carrier expression vector which is a fusion protein of the antigen and an antigen-binding carrier.

In the above expression vector, the promoter for expressing an antigen-binding carrier is not particularly limited, and examples thereof include EF1α promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, HSV-tk promoter, and the like. For expression in plant cells, such as promoters for expression in host cells, cauliflower mosaic virus (CaMV) 35S promoters, REF (rubber elongation factor) promoters, insect cells such as polyhedrin promoter, p10 promoter It is possible to use a promoter or the like for expression using the above as a host. These promoters can be appropriately selected depending on the antigen-binding carrier or the host expressing the antigen-binding carrier.

The above-mentioned expression vector may further have a multicloning site, an enhancer, a splicing signal, a poly A addition signal, a selection marker, an origin of replication and the like.

<< kit >>
In one embodiment, the present invention provides a kit for producing a specific antibody against an antigen of interest, the kit comprising the above-mentioned antigen-binding carrier or the above-mentioned vector.

According to the kit of the present embodiment, a specific antibody against a target antigen can be produced with high efficiency in an immunized non-human mammal.

When the kit of the present embodiment is provided with the above-mentioned antigen-binding carrier, it may further be provided with a reagent, a reaction solvent, a catalyst and the like for binding the target antigen with the above-mentioned antigen-binding carrier. .. Known reagents, reaction solvents, catalysts, etc. can be appropriately selected depending on the target antigen and the type of the above-mentioned carrier for antigen binding.

Further, the kit of the present embodiment, in the case of including the above-mentioned vector, a primer for amplifying a nucleic acid encoding the above-mentioned carrier for antigen binding in the vector, a nucleic acid amplification reagent (for example, a nucleotide as a substrate) Triphosphate, nucleic acid synthase, amplification reaction buffer, etc.) and the like.
Nucleotide triphosphate is a substrate (dNTP, rNTP, etc.) depending on the nucleic acid synthase. The nucleic acid synthase is an enzyme depending on the nucleic acid amplification method used, and examples thereof include DNA polymerase, RNA polymerase, and reverse transcriptase. Examples of the buffer for amplification reaction include Tris buffer, phosphate buffer, veronal buffer, borate buffer, Good's buffer, and the like, and the pH is not particularly limited.
When the kit of the present embodiment is provided with the above vector, it may further be provided with a transfection reagent for introducing the above vector into a host cell.
The transfection reagent is selected from the group consisting of cationic polymers, cationic lipids, polyamine reagents, polyimine reagents, and calcium phosphate. Examples of such a transfection reagent include Effectene Transfection Reagent (cat. no. 301425, Qiagen, CA), TransFast™ Transfection Reagent (E2431, Promega, WI), TfxTM-20 Reagent (S2391, Profit, E2391, Prof). Transfection Reagent (301305, Qiagen, CA), PolyFect Transfection Reagent (301105, Qiagen, CA), LipofectAMINE 2000 Reagent (11668-019, Invitrogen corporation, CA), 4PEC. (101-30, Polyplus-transfection, France), ExGen 500 (R0511, Fermentas Inc., MD) and the like, but are not limited thereto.

Further, the kit of the present embodiment comprises the above-mentioned vector, when the target antigen is a protein, in order to insert a nucleic acid encoding the antigen protein into the vector, a restriction enzyme, a buffer for restriction enzyme treatment, It may further include a ligase, a ligase treatment buffer, and the like.

<<Method for producing specific antibody against target antigen>>
In one embodiment, the present invention is a method for producing a specific antibody against an antigen of interest, which comprises binding the antigen of interest to the above-described antigen-binding carrier to produce an antigen-binding carrier, and the antigen. An immunization step of administering the bound carrier to a non-human mammal, and a collection step of collecting serum, spleen cells, lymph node cells, peripheral blood leukocytes, or B cells from the non-human mammal after the immunization step. A manufacturing method is provided.

According to the production method of this embodiment, a specific antibody against a target antigen can be obtained with high efficiency.

In the present specification, the type of “antibody” may be a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment. In addition, "antibody" includes all classes and subclasses of immunoglobulin.
Also, as used herein, "polyclonal antibody" means an antibody preparation containing different antibodies against different epitopes.
Further, in the present specification, the “monoclonal antibody” means an antibody having substantially uniform specificity.
As used herein, the term “antigen-binding fragment” means a part (partial fragment) of an antibody that specifically recognizes a target protein. Specifically, Fab, Fab′, F(ab′)2, variable region fragment (Fv), disulfide bond Fv, single chain Fv (scFv), sc(Fv)2, diabody, multispecific antibody, And these polymers and the like.

As used herein, the term "specific binding" means that the antibody binds only to an antigen, for example, an in vitro assay, preferably a plasmon resonance assay using a purified wild type antigen (eg, BIAcore, GE). -Healthcare Uppsala, Sweden, etc.) can be quantified by binding to the epitope of the antigen of the antibody. Binding affinity can be defined by ka (rate constant for antibody binding from the antibody-antigen complex), kD (dissociation constant), and KD (kD/ka). The binding affinity (KD) when the antibody specifically binds to the antigen is preferably 10 ⁻⁸ mol/L or less, and more preferably 10 ⁻⁹ M to 10 ⁻¹³ mol/L. preferable.
Each step in the manufacturing method of this embodiment will be described in detail below.

[Joining process]
First, the target antigen is bound to the above-mentioned antigen-binding carrier to prepare an antigen-binding carrier.
Examples of the target antigen include the same as those exemplified in the above <<<Antigen binding carrier >>>.

As a method for binding the target antigen to the antigen-binding carrier, a known method may be appropriately selected according to the types of the target antigen and the antigen-binding carrier.
There is no particular limitation on the binding mode between the antigen-binding carrier and the antigen, and for example, a covalent bond using a functional group exemplified as the antigen-binding site of the linker in the <<antigen-binding carrier>> described above ( For example, amide bond, disulfide bond, ester bond, etc.), electrostatic interaction, hydrogen bond, van der Waals bond, bond by hydrophobic effect, etc.

More specifically, a method of binding the target antigen to the antigen-binding carrier includes a method of bringing the antigen-binding carrier into contact with a liquid in which the target antigen is dissolved or dispersed. The pH of the liquid in which the target antigen is dissolved or dispersed may be, for example, 4 or more and 11 or less, for example, 5 or more and 10 or less. When the pH is in the above range, the target antigen and the antigen-binding carrier can be bound efficiently, and the denaturation or decomposition of the target antigen can be suppressed.

Further, more specifically as a method of binding the target antigen to the antigen-binding carrier, when the antigen-binding site in the linker is a carboxy group and the target antigen has an amino group,
Examples thereof include a method of binding by a condensation reaction between a carboxy group at the antigen-binding site and an amino group of the antigen.

Further, as a method for binding the target antigen to the antigen-binding carrier, more specifically, when the antigen and the antigen-binding carrier are proteins, for example, they may be bound by the following method.
First, a nucleic acid encoding an antigen protein is inserted into an expression vector containing a nucleic acid encoding an antigen-binding carrier to prepare an expression vector containing a nucleic acid encoding an antigen-binding carrier. As an insertion method, for example, using a restriction enzyme, a method of inserting a nucleotide sequence of a nucleic acid encoding an antigen protein into the 3′ end of a nucleotide sequence of a nucleic acid encoding a carrier for antigen binding of the expression vector, and ligating, Alternatively, a cloning method by PCR using a primer to which a nucleotide sequence of a nucleic acid encoding an antigen protein is added may be used.

Then, the host is transformed with the expression vector containing the nucleic acid encoding the antigen-binding carrier. Examples of the host include bacteria such as Escherichia coli, animal cells, plant cells, insect cells and the like.
Then, the host is cultured to express the antigen-binding carrier. Those skilled in the art can determine the composition of the medium, the temperature of the culture, the time, the addition of the inducer, etc. according to known methods so that the transformant grows and the antigen-binding carrier is efficiently produced. Further, for example, when an antibiotic resistance gene is incorporated into an expression vector as a selection marker, transformants can be selected by adding an antibiotic to the medium.
Next, the antigen-binding carrier expressed by the host is purified by an appropriate method to obtain the antigen-binding carrier.
For example, when the antigen-binding carrier is aP1 or a protein variant having substantially the same activity as aP1, since it has heat resistance, it can be purified by removing impurities by heating.

[Immunization process]
Then, the antigen-binding carrier obtained in the binding step is administered to a non-human mammal.
Examples of the non-human mammal include, but are not limited to, mouse, rat, hamster, guinea pig, rabbit, dog, cat, horse, cow, sheep, pig, goat, marmoset, monkey and the like. The non-human mammal may be a healthy body or an autoimmune disease model animal. Among them, the non-human mammal is preferably an autoimmune disease model animal because it can efficiently produce an antibody.

The method of administering an antigen-binding carrier to a non-human mammal is not particularly limited, and for example, intraarterial injection, intravenous injection, subcutaneous injection, intranasal, transbronchial, intramuscular, transdermal, Orally, methods and the like known to those skilled in the art can be mentioned.

Further, the dose of the antigen-binding carrier to the non-human mammal is appropriately adjusted in consideration of the age, sex, weight of the non-human mammal, administration method, treatment time and the like.

[Collecting process]
Then, serum, spleen cells, lymph node cells, peripheral blood leukocytes, or B cells are collected from the non-human mammal after the immunization step.

When a polyclonal antibody is produced using the production method of the present embodiment, the polyclonal antibody is purified from the collected serum by a known method (for example, salting out, centrifugation, dialysis, column chromatography, etc.). Can be obtained.

When a monoclonal antibody is produced using the production method of the present embodiment, it can be produced by a hybridoma method or a recombinant DNA method.

Examples of the hybridoma method include Kohler and Milstein methods (see, for example, Kohler & Milstein, Nature, 256:495 (1975)). Examples of the antibody-producing cells used in the cell fusion step in this method include spleen cells, lymph node cells and peripheral blood leukocytes of the non-human mammal after the immunization step. In addition, antibody-producing cells (B cells) obtained by allowing an antigen to act in a medium on the above-mentioned cells previously isolated from non-immunized animals, or lymphocytes can also be used. .. As the myeloma cells, various known cell lines can be used. The antibody-producing cells and myeloma cells may be of different animal species origin as long as they can be fused, but are preferably of the same animal species origin.
More specifically, as a method for obtaining a hybridoma, for example, spleen cells obtained from the mouse after the immunization step, and produced by cell fusion between mouse myeloma cells, the subsequent screening, specific to the target protein Examples thereof include a method for obtaining a hybridoma that produces a monoclonal antibody.
Further, as a method for obtaining the monoclonal antibody produced by the hybridoma, for example, a method for obtaining a monoclonal antibody against the target protein by culturing the hybridoma, or from the ascites of a non-human mammal to which the hybridoma is administered, etc. may be mentioned. To be

As the recombinant DNA method, for example, DNA encoding the above-mentioned antibody is cloned from a hybridoma, B cell or the like and incorporated into an appropriate vector, and this is used as a host (eg, mammalian cell line, E. coli, yeast cell, insect cell, (For example, PJ Delves, Antibody Products: Essential Techniques, 1997 WILEY, P. Shepherd and C. Dean Monoclonal Ornamental Ornamental Ornamental Ornamental Ornamental Ornamental Ornamental Ornamental Ornamental Ornamental Ornamental Organisms), and the like. PRESS, Vandamme AM et al., Eur. J. Biochem. 192:767-775 (1990)).
In the expression of the DNA encoding the antibody, the DNA encoding the heavy chain or the light chain may be separately incorporated into an expression vector to transform the host, and the DNA encoding the heavy chain and the light chain may be expressed by a single expression. The host may be transformed by incorporating it into a vector (see, for example, International Patent Application No. 94/11523).
The antibody can be obtained by culturing the above-mentioned host, separating and purifying in the host or from the culture solution, and in a substantially pure and homogeneous form. For the separation and purification of the antibody, the method used in the purification of ordinary polypeptides can be used.
In the method using the transgenic animal production technology, for example, a transgenic animal (for example, cow, goat, sheep, pig, etc.) into which the antibody gene is incorporated is produced, and the antibody gene is derived from the milk of the transgenic animal. And a method for obtaining a large amount of monoclonal antibodies.

The binding activity of the antibody to the antigen obtained by the production method of the present embodiment can be determined by, for example, ELISA, Western blotting, Flow cytometry, Western blotting, Dot blotting, Radioimmunoassay, Immunoprecipitation, Immunoassay. It can be evaluated by a staining method or the like.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to the following examples.

[Example 1]
(1) Preparation of vector for expressing antigen-binding carrier (1-1) Archaea P. Construction of Plasmid Vector for Expression of Escherichia coli Inserting Full Length DNA of aP1 of Horikoshii. The full-length DNA of horikoshii aP1 was amplified by PCR using primers to which NdeI and BamHI sequences were added. Then, the Escherichia coli expression plasmid vector pET-3a (manufactured by Agilent Technologies) was treated with NdeI and BamHI to insert the PCR product. The obtained archaea P. SEQ ID NO: 3 shows the nucleotide sequence of a plasmid vector for expression in Escherichia coli in which the full length aP1 of horikoshii was inserted.

(1-2) Preparation of vector for expressing antigen-binding carrier Then, the archaeal P. cerevisiae obtained in (1-1) was used. Using the Escherichia coli expression plasmid vector into which the full length aP1 of horikoshii was inserted, the nucleic acid encoding the 18 amino acid residues corresponding to the C-terminal antigen site of aP1 was replaced with the nucleotide sequence encoding the antigen protein.
The C-terminal portion (18 amino acid residues) of mouse ribosomal protein S6 was used as an antigen protein. The nucleotide sequence of the nucleic acid encoding the C-terminal portion of mouse ribosomal protein S6 is shown in SEQ ID NO:4.
Specifically, a primer (SEQ ID NOS: 5 and 6) containing a part of the base sequence encoding the C-terminal portion of the mouse ribosomal protein S6 and a part of the base sequence of Escherichia coli expression plasmid vector pET-3a was used. The archaeal P. cerevisiae obtained in (1-1) was used. PCR was performed using the Escherichia coli expression plasmid vector, into which the full-length DNA of horikoshii aP1 was inserted, as a template in a circular form, and the obtained amplification product was used for E. coli transformation. The cloned plasmid was prepared from Escherichia coli to obtain an antigen-binding carrier expression vector.

(2) Expression and purification of antigen-binding carrier Next, the obtained antigen-binding carrier expression vector was transformed into Escherichia coli and cultured at 37°C for 12 hours to express the antigen-binding carrier. Then, the E. coli after culturing was recovered, crushed by ultrasonic treatment, and heated to 90° C. to denature unnecessary proteins such as those derived from E. coli. Then, the solution after the heat treatment was purified using column chromatography to obtain an antigen-bound carrier.

(3) Immunization of autoimmune disease model mouse and purification of antibody Next, the antigen-binding carrier obtained in (2) was mixed with an adjuvant and injected into the foot pad of a rabbit (New Zealand white) for immunization. .. Then, serum was collected from the rabbit 63 days after the immunization and purified using an affinity chromatograph filled with HiTrap Protein G (manufactured by GE Healthcare Japan) to obtain a rabbit polyclonal antibody (IgG).

(4) Verification of Antigen Specificity of Antibody Next, in order to verify the antigen specificity of the polyclonal antibody obtained in (3), the antigen-binding carrier prepared in (2) was used as a negative control. As a tetramer of aP1 of horikoshii and a positive control, the full-length protein of mouse ribosomal protein S6 was separated by SDS-PAGE, and then each protein was blotted on a protein-adsorbing membrane. Then, the membrane and the polyclonal antibody obtained in (3) were reacted at room temperature for 60 minutes. Then, immunostaining was performed using an anti-rabbit IgG antibody. Results are shown in FIG.
In FIG. 3, lane 1 shows a reaction of the polyclonal antibody with the antigen-binding carrier, and lane 2 shows the polyclonal antibody with an archaeal P. The one obtained by reacting the aP1 tetramer of horikoshii and lane 3 are obtained by reacting the polyclonal antibody with the full-length mouse ribosomal protein S6 protein.
In FIG. 3, the upper arrow indicates the position corresponding to the molecular weight of the full-length mouse ribosomal protein S6, and the lower arrow indicates the position corresponding to the molecular weight of the antigen-binding carrier.

From FIG. 3, it was shown that the obtained polyclonal antibody was an antibody specific to mouse ribosomal protein S6. In addition, when the polyclonal antibody is reacted with the antigen-binding carrier (lane 1 in FIG. 3), aP1 in which the C-terminal antigenic site is substituted with 18 amino acid residues of the C-terminal region of S6, which is an antigen, Usually, a tetramer is formed, but it is speculated that the band is decomposed into a trimer, a dimer, or a monomer by the reducing action of SDS, and the band extends to a position where the molecular weight is low.
Further, ribosomal proteins are proteins having highly conserved sites, and it is difficult to produce an antibody having excellent specificity by conventional methods, but it is easy to use the carrier for antigen binding of the present invention, It was shown that an antibody with excellent specificity can be produced.

[Example 2]
(1) Preparation of Vector for Expression of Antigen-Binding Carrier As the antigen protein, instead of the nucleic acid encoding the C-terminal portion (18 amino acid residues) of mouse ribosomal protein S6, the 140th to 15th 20 amino acid residues of yeast Hbs1 are used. Using the nucleic acid encoding SEQ ID NO: 7 (SEQ ID NO: 7), a part of the nucleotide sequence encoding the 20th to 140th amino acid residues of Hbs1 and a part of the nucleotide sequence of Escherichia coli expression plasmid vector pET-3a An antigen-binding carrier expression vector was produced in the same manner as in (1) of Example 1 except that the primers (SEQ ID NOS: 8 and 9) containing and were used.

(2) Expression and purification of antigen-binding carrier Next, using the same method as in (2) of Example 1, E. coli was transformed with the antigen-binding carrier expression vector to express the antigen-binding carrier and then purified. An antigen-bound carrier was obtained.

(3) Immunization of autoimmune disease model mouse and production of antibody Next, the antigen-binding carrier obtained in (2) was applied to rabbits (New Zealand white) using the same method as in (3) of Example 1. After administration, rabbit polyclonal antibody (IgG) was purified.

(4) Verification of Antigen Specificity of Antibody Next, in order to verify the antigen specificity of the polyclonal antibody obtained in (3), the antigen-binding carrier prepared in (2) was used as a negative control. The aP1 tetramer of horikoshii and, as a positive control, the yeast Hbs1 full-length protein was separated by SDS-PAGE, and then each protein was blotted on a protein-adsorbing membrane. Then, the membrane and the polyclonal antibody obtained in (3) were reacted at room temperature for 60 minutes. Then, immunostaining was performed using an anti-rabbit IgG antibody. The results are shown in Fig. 4.
In FIG. 4, lane 1 shows the above-mentioned polyclonal antibody and Archaea P. In lane 2, the polyclonal antibody and the antigen-binding carrier are reacted with horikoshii aP1 tetramer, and in lane 3, the polyclonal antibody and the full-length protein of yeast Hbs1 are expressed. It has been reacted.
Further, in FIG. 4, the upper arrow indicates the position corresponding to the molecular weight of the full-length protein of yeast Hbs1, and the lower arrow indicates the position corresponding to the molecular weight of the antigen-binding carrier.

From FIG. 4, it was shown that the obtained polyclonal antibody was an antibody specific to yeast Hbs1. In the reaction of the polyclonal antibody with the antigen-binding carrier (lane 2 in Fig. 4), the C-terminal antigenic site was replaced with the 20th amino acid residue at the 140th to 15th positions of yeast Hbs1 which is an antigen. AP1 normally forms a tetramer, but it is presumed that the band is extended to a position where the molecular weight is low by being decomposed into a trimer, a dimer, or a monomer by the reducing action of SDS. Was done.
In addition, Hbs1 is a protein having a highly conserved site, and it is difficult to produce an antibody having excellent specificity by the conventional method, but it is easy to obtain Hbs1 by using the antigen-binding carrier of the present invention. It was shown that an antibody having different specificity can be produced.

The antigen-binding carrier of the present invention is easy to handle and can be used for producing a specific antibody against any antigen.

1A... Antigen-binding carrier, 1B... Archaeon aP1 tetramer lacking C-terminal antigen site, 11... Core particle, 12... Linker, 13, 23... Antigen binding site, 21... aP0 binding site, 22... Movable Sex region, 100... aP1 lacking the C-terminal antigen site.

Claims (4)

A method for producing a specific antibody against an antigen of interest, comprising:
A binding step of binding an antigen of interest to an antigen-binding carrier to produce an antigen-binding carrier,
An immunization step of administering the antigen-binding carrier to a non-human mammal,
A collection step of collecting serum, spleen cells, lymph node cells, peripheral blood leukocytes, or B cells from the non-human mammal after the immunization step,
Equipped with
The antigen-binding carrier is
Core particles,
At least two flexible linkers linked to the core particles,
Equipped with
In the linker, Ri antigen binding sites der is opposite the tip to the connection portion between the core particles,
The core particle and the linker are any of the following proteins (a) to (c),
(A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1,
(B) a protein consisting of an amino acid sequence in which 1 or more and 5 or less amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1, and having a multimer-forming ability and a flexible region,
(C) a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1, and having a multimer-forming ability and a flexible region,
The production method, wherein the antigen has a highly conserved site and is a protein other than ribosomal protein. The production method according to claim 1 , wherein the protein forms a multimer of dimers or more and 7 or less. In the binding step, an expression vector containing a nucleic acid encoding the antigen-binding carrier, a nucleic acid encoding the antigen is inserted, to produce an expression vector containing a nucleic acid encoding the antigen-binding carrier, the antigen-binding carrier The method according to claim 1 or 2, wherein a host is transformed with an expression vector containing the encoding nucleic acid and then the host is cultured to express an antigen-binding carrier. The method according to any one of claims 1 to 3, wherein the antigen is Hbs1.