Heterodimeric protein production method, dimeric protein, monomeric protein, and target responsive heterodimeric protein screening method

Abstract

Provided is a heterodimeric protein production method for heterodimeric proteins such as bispecific antibodies, by which it is possible to produce a heterodimeric protein composed solely of domains comprising natural amino acid sequences.　This production method is a heterodimeric protein production method comprising a production step in which a dimeric protein including a reaction tag is reacted with a modifying protein for modifying the dimeric protein, to produce a heterodimeric protein. The dimeric protein including the reaction tag comprises a first monomeric protein and a second monomeric protein; the first monomeric protein comprises, in the following order, a first reaction tag and a first dimer formation domain capable of forming a dimer; the first reaction tag comprises a binding tag and a first C-intein; the second monomeric protein comprises, in the following order, a second reaction tag, the first dimer formation domain, and a second dimer formation domain capable of forming a dimer; the second reaction tag comprises a binding partner capable of binding to the binding tag, and a second C-intein; the modifying protein comprises a first modifying protein and a second modifying protein; the first modifying protein comprises a first N-intein capable of reacting with the first C-intein, and a first adduct added to the first monomeric protein; the second modifying protein comprises a second N-intein capable of reacting with the second C-intein, and a second adduct added to the second monomeric protein; the first adduct and the second adduct are different adducts; the first monomeric protein and the second monomeric protein form a dimer; and in the production step, the first N-intein of the first modifying protein reacts with the first C-intein of the first monomeric protein, the first adduct of the first modifying protein is joined to the first monomeric protein, the second N-intein of the second modifying protein reacts with the second C-intein of the second monomeric protein, and the second adduct of the second modifying protein is joined to the second monomeric protein.

Description

Method for producing heterodimeric proteins, dimeric proteins, monomeric proteins, and methods for screening heterodimeric proteins for target reactivity

[0001] The present invention relates to a method for producing a heterodimeric protein, a dimeric protein, a monomeric protein, and a method for screening for a target-reactive heterodimeric protein.

[0002] In multispecific antibodies, a single antibody molecule has antigen-binding domains that exhibit binding affinity to different antigens, and by changing the target antigen, development is underway for various medical applications such as anticancer agents and hemophilia treatment drugs.

[0003] However, multispecific antibodies have problems in that their production efficiency is extremely low and their production is difficult. For example, a bispecific antibody is composed of two types of heavy chains (H chains) and two types of light chains (L chains). Furthermore, when two types of H chains and two types of L chains are expressed to produce the bispecific antibody, there are 10 possible combinations of the two H chains and two L chains in the expressed antibody, which means that in addition to the desired bispecific antibody, as many as nine types of unnecessary antibodies are produced (Patent Document 1).

[0004] International Publication No. 2013 / 065708

[0005] Therefore, in the case of bispecific antibodies, a technique (knob-into-holes) has been developed in which mutations are introduced into the amino acid sequence of the Fc region of the antibody, allowing two different heavy chains to specifically associate with each other.

[0006] However, these techniques may result in the generation of antibodies against the Fc region, since non-natural amino acid sequences are present in these regions.

[0007] Therefore, an object of the present invention is to provide a method for producing heterodimeric proteins such as bispecific antibodies, which can also produce heterodimeric proteins composed only of domains consisting of natural amino acid sequences.

[0008] In order to achieve the above-mentioned object, the production method of the present invention (hereinafter also referred to as "production method") is a method for producing a heterodimeric protein, comprising a production step of reacting a dimeric protein comprising a reactive tag with a modifying protein that modifies the dimeric protein to produce a heterodimeric protein, wherein the dimeric protein comprising the reactive tag comprises a first monomeric protein and a second monomeric protein, wherein the first monomeric protein comprises, in this order, a first reactive tag and a first dimer-forming domain capable of forming a dimer, the first reactive tag comprises a binding tag and a first C intein, the second monomeric protein comprises, in this order, a second reactive tag and a second dimer-forming domain capable of forming a dimer with the first dimer-forming domain, the second reactive tag comprises a binding partner capable of binding to the binding tag and a second C intein, and the modified protein comprises the first modified protein and a second modified protein, the first modified protein comprises a first N intein capable of reacting with the first C intein and a first adduct added to the first monomeric protein; the second modified protein comprises a second N intein capable of reacting with the second C intein and a second adduct added to the second monomeric protein; the first adduct and the second adduct are different adducts; the first monomeric protein and the second monomeric protein form a dimer; and in the production process, the first N intein of the first modified protein reacts with the first C intein of the first monomeric protein, thereby linking the first adduct of the first modified protein to the first monomeric protein; and the second N intein of the second modified protein reacts with the second C intein of the second monomeric protein, thereby linking the second adduct of the second modified protein to the second monomeric protein.

[0009] The protein of the present invention (hereinafter also referred to as "dimeric protein") comprises a dimeric protein comprising a reactive tag, wherein the dimeric protein comprising the reactive tag comprises a first monomeric protein and a second monomeric protein, wherein the first monomeric protein comprises a first reactive tag and a first dimer-forming domain capable of forming a dimer, wherein the first reactive tag comprises a binding tag and a first C intein capable of reacting with a first N intein, wherein the second monomeric protein comprises, in this order, a second reactive tag and a second dimer-forming domain capable of forming a dimer with the first dimer-forming domain, wherein the second reactive tag comprises a binding partner capable of binding to the binding tag and a second C intein capable of reacting with a second N intein, and wherein the first monomeric protein and the second monomeric protein form a dimer.

[0010] The protein of the present invention (hereinafter also referred to as "first monomer protein") comprises a first monomer protein, wherein the first monomer protein comprises, in this order, a first reaction tag and a first dimerization domain capable of forming a dimer, the first reaction tag comprising a binding tag capable of binding to a binding partner and a first C intein capable of reacting with a first N intein, the first monomer protein capable of forming a dimer with a second monomer protein, the second monomer protein comprising a second reaction tag and a second dimerization domain capable of forming a dimer with the first dimerization domain, and the second reaction tag comprising a binding partner capable of binding to the binding tag and a second C intein capable of reacting with a second N intein.

[0011] The protein of the present invention (hereinafter also referred to as "second monomer protein") comprises a second monomer protein, wherein the second monomer protein comprises, in this order, a second reaction tag and a second dimerization domain capable of forming a dimer, the second reaction tag comprises a binding partner capable of binding to a binding tag and a second C intein capable of reacting with a second N intein, the second monomer protein is capable of forming a dimer with a first monomer protein, and the first monomer protein comprises, in this order, a first reaction tag and a first dimerization domain capable of forming a dimer with the second dimerization domain, and the first reaction tag comprises a binding tag capable of binding to the binding partner and a first C intein capable of reacting with a first N intein.

[0012] The nucleic acid of the present invention encodes the dimeric protein of the present invention, the first monomeric protein of the present invention, and / or the second monomeric protein of the present invention.

[0013] The expression vector of the present invention comprises the nucleic acid of the present invention.

[0014] The screening method for target-reactive heterodimeric proteins of the present invention (hereinafter also referred to as "screening method") comprises a production step of reacting a dimeric protein containing a reactive tag with a modifying protein that modifies the dimeric protein to produce a candidate heterodimeric protein; a detection step of contacting the candidate heterodimeric protein with a target and detecting a reaction between the candidate heterodimeric protein and the target; and a selection step of selecting the candidate heterodimeric protein whose reaction has been detected as a candidate substance that reacts with the target, wherein the production step is carried out by the production method of the present invention.

[0015] According to the present invention, heterodimeric proteins such as bispecific antibodies can also be produced that are composed only of domains consisting of natural amino acid sequences.

[0016] FIG. 1 is a schematic diagram showing an example of the components of a dimeric protein used in the production method of the present invention and each step of the production of a heterodimeric protein. FIG. 2 is a photograph showing each antibody fragment and the protein before and after PTS reaction in Example 1. FIG. 3 is a photograph showing the protein after PTS reaction and column purification in Example 1. FIG. 4 is a graph showing the elution pattern of the protein by gel filtration chromatography in Example 1. FIG. 5 is a graph showing the binding of the bispecific antibody to CD3-positive cells by flow cytometry in Example 1. FIG. 6 is a graph showing the binding of the bispecific antibody to HER2-positive cells by flow cytometry in Example 1. FIG. 7 is a graph showing the maintenance of binding of the bispecific antibody to HER2 by surface plasmon resonance in Example 1.

[0017] <Definition> As used herein, "protein" refers to a peptide polymer composed of unmodified amino acids (natural amino acids), modified amino acids, and / or artificial amino acids. The polymer may be, for example, linear, branched, or cyclic. The protein may also be referred to as a peptide or polypeptide.

[0018] As used herein, the term "monomer protein" refers to a protein that is not bound to or associated with other proteins.

[0019] As used herein, the term "dimeric protein" refers to a protein complex in which two proteins or protein subunits are bound or associated. When the two proteins are the same protein or subunit, the dimeric protein can also be called a homodimeric protein. When the two proteins are different proteins or subunits, the dimeric protein can also be called a heterodimeric protein.

[0020] As used herein, the term "fusion protein" refers to a protein in which two or more different or heterologous proteins are partially or entirely bound (linked) via peptide bonds. The fusion protein may be a natural fusion protein or an artificial fusion protein. Examples of the artificial fusion protein include proteins designed by genetic engineering techniques.

[0021] As used herein, "binding tag" refers to a polypeptide or substance that has specific binding properties for another molecule.

[0022] As used herein, the term "binding partner" refers to a polypeptide or substance that has specific binding affinity with the binding tag.

[0023] As used herein, the term "domain" refers to a structurally or functionally integrated region in a "protein," "polypeptide," and / or "peptide."

[0024] As used herein, the term "antibody" refers to a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Immunoglobulin genes include genes encoding constant regions such as κ, λ, α (including α1 and α2), γ (including γ1, γ2, γ3, and γ4), δ, ε, and μ, as well as genes capable of encoding numerous immunoglobulin variable regions such as V regions, D regions, and J regions. The antibody comprises, for example, a heavy chain and a light chain. The light chain comprises κ and λ, constituting the κ chain and the λ chain, respectively. The heavy chain comprises γ, μ, α, δ, or ε, constituting the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. The antibody may be a typical immunoglobulin (antibody) structural unit composed of a tetramer. In this case, the antibody is composed of two identical pairs of polypeptide chains, each pair consisting of one light chain (about 25 kDa) and one heavy chain (about 50-70 kDa), and the N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition.

[0025] As used herein, "antigen-binding fragment" refers to a portion or partial polypeptide comprising the antigen-binding site of an antibody. The antigen-binding fragment can be obtained by chemical or enzymatic treatment of an antibody. The antigen-binding fragment can also be obtained by recombinant means. The antigen-binding fragment can be, for example, Fab, Fab', F(ab') ２ , Fc and / or Fv fragments and derivatives thereof.

[0026] As used herein, the term "heavy chain antibody" refers to an antibody that is composed of only two heavy chains. The heavy chain antibody can also be referred to as an antibody that does not contain a light chain.

[0027] As used herein, the term "multispecific antibody" refers to an antibody or antibody derivative having specificity for more than one antigen and / or more than one epitope.

[0028] As used herein, "intein" refers to an autocatalytic enzyme that has both protein protease activity and protein ligase activity. For example, an intein is a protein that excises its own amino acid sequence from a protein by protein splicing and ligates the remaining amino acid sequence (extein) via a peptide bond. The "intein" can also be referred to as, for example, a protein intron.

[0029] As used herein, a "split intein" refers to an intein composed of two complementary half inteins, a C intein and an N intein, which form an active intein by tightly binding to the C intein and the N intein, and which exhibits enzymatic activity. The split intein is also called a split intein.

[0030] As used herein, the term "integrated intein" refers to a single intein that includes a C intein and an N intein, and the C intein and the N intein tightly bind to form an active intein that exhibits enzymatic activity.

[0031] As used herein, "complementary inteins" refers to a pair of inteins or split inteins with a C intein and an N intein.

[0032] As used herein, "C intein" refers to an intein polypeptide that shares homology with the C-terminal portion of an intein, which binds to a complementary N intein to form an active intein.

[0033] As used herein, "N intein" refers to an intein polypeptide that shares homology with the N-terminal portion of an intein, which binds to a complementary C intein to form an active intein.

[0034] As used herein, the term "salt" refers to an electrolyte that can be ionized in an aqueous solvent. The aqueous solvent refers to a solvent that contains water.

[0035] As used herein, a "solubilization domain" refers to a polypeptide that, when fused to a peptide, polypeptide, or protein, increases (e.g., increases or promotes) the expression level of the peptide, polypeptide, or protein expressed in a transformant compared to when the peptide, polypeptide, or protein is expressed without the solubilization domain.

[0036] As used herein, "purified" means identifying and separating, recovering from components in their natural state, being identified and separated, and / or recovered from components in their natural state. The "purification" can be carried out, for example, by obtaining at least one purification step. The purification can also be referred to as isolation.

[0037] As used herein, "separation" refers to the separation of an object from a substance that contains said object and / or the state of being separated. Said separation can also be referred to as liberation.

[0038] As used herein, "nucleic acid" refers to a polymer of deoxyribonucleotides (DNA), ribonucleotides (RNA), and / or modified nucleotides. When "nucleic acid" is used in combination with a specific protein, the "nucleic acid" refers to a polymer of nucleotides that encodes the amino acid sequence of the protein. Examples of the nucleic acid include genomic DNA, cDNA, and mRNA. The nucleic acid may be, for example, single-stranded or double-stranded. The nucleic acid can be interchangeably referred to as a "polynucleotide" or a "nucleic acid molecule."

[0039] As used herein, the term "host" refers to a cell and / or an individual into which exogenous nucleic acid is introduced. When the host is a cell, the host may also be referred to as a host cell.

[0040] As used herein, the terms "vector" and "expression vector" refer to a recombinant plasmid or virus containing a nucleic acid that is delivered to a host or host cell in vitro or in vivo. The term "vector" and "expression vector" includes viral vectors and non-viral vectors.

[0041] As used herein, the term "transformant" refers to a host into which exogenous nucleic acid has been introduced.

[0042] Herein, the origin of each protein, polypeptide, or peptide is not particularly limited and may be any animal. The animal may be, for example, a human or a non-human animal. Examples of the non-human animal include mammals such as mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, dolphins, and sea lions.

[0043] The present invention will be described below using examples, but the present invention is not limited to the following examples and can be practiced with any modifications. Furthermore, the descriptions in the present invention can be mutually incorporated unless otherwise specified. In this specification, when the expression "~" is used, it is used to mean that the numerical or physical values ​​before and after it are included. In addition, in this specification, the expression "A and / or B" includes "A only," "B only," and "both A and B."

[0044] <Method for producing heterodimeric proteins> In one aspect, the present invention provides a method for producing heterodimeric proteins such as bispecific antibodies, which can produce heterodimeric proteins composed only of domains consisting of natural amino acid sequences, or a method for producing heterodimeric proteins modified with different additions.The production method of the present invention is a method for producing a heterodimeric protein, and includes a production step of reacting a dimeric protein containing a reactive tag with a modifying protein that modifies the dimeric protein to produce a heterodimeric protein, wherein the dimeric protein containing the reactive tag includes a first monomeric protein and a second monomeric protein, the first monomeric protein includes, in this order, a first reactive tag and a first dimer-forming domain capable of forming a dimer, the first reactive tag includes a binding tag and a first C intein, the second monomeric protein includes, in this order, a second reactive tag and a second dimer-forming domain capable of forming a dimer with the first dimer-forming domain, the second reactive tag includes a binding partner that can bind to the binding tag and a second C intein, the modifying protein includes a first modifying protein and a second modifying protein, and the first modified protein The modified protein comprises a first N intein capable of reacting with the first C intein and a first adduct added to the first monomer protein, the second modified protein comprises a second N intein capable of reacting with the second C intein and a second adduct added to the second monomer protein, the first adduct and the second adduct being different adducts, and the first monomer protein and the second monomer protein form a dimer, and in the production process, the first N intein of the first modified protein reacts with the first C intein of the first monomer protein to link the first adduct of the first modified protein to the first monomer protein, and the second N intein of the second modified protein reacts with the second C intein of the second monomer protein to link the second adduct of the second modified protein to the second monomer protein.

[0045] When the first monomer protein and the second monomer protein are proteins that form homodimers, various combinations of dimers are produced by preparing a first monomer protein linked to the first adduct and a second monomer protein linked to the second adduct in advance and combining them. On the other hand, in the production method of the present invention, the first adduct is linked to the first monomer protein while the dimeric protein is formed by utilizing a reaction between the first N intein and the first C intein. Furthermore, in the production method of the present invention, the second adduct is linked to the second monomer protein while the dimeric protein is formed by utilizing a reaction between the second N intein and the second C intein. Therefore, even when the first monomer protein and the second monomer protein are proteins that form homodimers, the production method of the present invention can efficiently produce a heterodimeric protein consisting of a protein derived from the first monomer protein linked to the first adduct and a protein derived from the second monomer protein linked to the second adduct.

[0046] The present inventors conceived the idea that a heterodimeric protein composed of monomeric proteins to which different adducts are linked could be produced by inducing specific binding reactions between two monomeric proteins in a dimerized state and linking different adducts to each of the monomeric proteins. After extensive research, the present inventors discovered that this idea could be realized by using two types of monomeric proteins to which different inteins are linked, and two types of modified proteins to which inteins capable of reacting with the different inteins are linked, and an adduct to the monomeric protein is linked, thereby establishing the present invention. Specifically, the production method of the present invention is presumed to be capable of producing a heterodimeric protein composed of monomeric proteins to which different adducts are linked, as shown in Figure 1, by the mechanism described below. The present invention is not limited in any way to the mechanism described below.

[0047] As shown in FIG. 1(A), the production method of the present invention allows for the production of a heterodimeric protein using a first monomer protein 1, a second monomer protein 2, a first modified protein 3, and a second modified protein 4. The first monomer protein 1 comprises a first reactive tag 11 and a first dimer-forming domain 12, in this order from the N-terminus. The first reactive tag 11 also comprises a binding tag 13 and a first C intein 14, in this order from the N-terminus. The second monomer protein 2 comprises a second reactive tag 21 and a second dimer-forming domain 22, in this order from the N-terminus. The second reactive tag 21 also comprises a binding partner 23 and a second C intein 24, in this order from the N-terminus. The first modified protein 3 comprises a first N intein 31 and a first adduct 32, in this order from the N-terminus. The second modified protein 4 includes a second N intein 41 and a second adduct 42 in this order from the N-terminus.

[0048] First, a first monomer protein 1 and a second monomer protein 2 are contacted to prepare a dimer protein 10, in which the first monomer protein 1 and the second monomer protein 2 form a dimer, as shown in Figure 1(B). Next, when the dimer protein 10 is contacted with a first modified protein 3 and a second modified protein 4, the first N intein 31 of the first modified protein 3 reacts with the first C intein 14 of the first monomer protein 1 to form an active intein (arrow X), as shown in Figure 1(C). Furthermore, the second N intein 41 of the second modified protein 4 reacts with the second C intein 24 of the second monomer protein 2 to form an active intein (arrow Y). Then, peptide bond recombination occurs between the first C intein 14 and the first dimer-forming domain 12, and between the first adduct 32 and the first N intein 31, linking the first dimer-forming domain 12 to the C-terminus of the first adduct 32. Furthermore, peptide bond recombination occurs between the second C intein 24 and the second dimer-forming domain 22, and between the second adduct 42 and the second N intein 41, linking the second dimer-forming domain 22 to the C-terminus of the second adduct 42. As a result, as shown in FIG. 1(D), a heterodimeric protein 30 can be produced in which the first dimer-forming domain 12 and the second dimer-forming domain 22, which form dimers, are modified with different adducts 32, 42. Therefore, it is presumed that the production method of the present invention can produce heterodimeric proteins modified with any adduct.

[0049] In addition, in the production method of the present invention, the first adduct 32 and the second adduct 42 are added using an intein. Therefore, the monomer proteins constituting the resulting heterodimeric protein can be produced as a fusion protein linked via peptide bonds from the N-terminus to the C-terminus, similar to recombinant proteins. Therefore, in the production method of the present invention, the first dimer-forming domain 12, the second dimer-forming domain 22, the first adduct 32, and the second adduct 42 are natural amino acid sequences, i.e., amino acid sequences that do not contain amino acid mutations or artificial amino acid sequences, so that heterodimeric proteins in which multiple domains composed only of natural amino acid sequences are linked can also be produced. Therefore, as an example, by making the first adduct 32 and the second adduct 42 Fabs that bind to different antigens, and making the first dimer-forming domain 12 and the second dimer-forming domain 22 proteins composed of the hinge region of the antibody and the CH2 and CH3 regions of the Fc region, bispecific monoclonal antibodies composed of natural amino acid sequences can be suitably produced. Thus, according to the production method of the present invention, for example, even when a heterodimeric protein is produced using two types of monomer proteins that share the same amino acid sequence in the dimer-forming domain but have different amino acid sequences in other parts, the heterodimeric protein can be specifically produced without introducing a mutation in the amino acid sequence that would cause specific binding or association between the target monomer proteins.

[0050] 1(A) to 1(D) illustrate an example in which the first dimer-forming domain 12 and the second dimer-forming domain 22 are modified at their N-terminal ends with the first adduct 32 and the second adduct 42, respectively. However, the present invention is not limited to this example, and the first dimer-forming domain 12 and the second dimer-forming domain 22 may also be modified at their C-terminal ends with the first adduct 32 and the second adduct 42. In this case, the first monomer protein 1 includes the first reaction tag 11 and the first dimer-forming domain 12 in this order from the C-terminal end, and the first reaction tag 11 includes the binding tag 13 and the first N-intein 31 in this order from the C-terminal end. The second monomer protein 2 comprises a second reactive tag 21 and a second dimer-forming domain 22, in this order from the C-terminus, and the second reactive tag 21 comprises a binding partner 23 and a second N intein 41, in this order from the C-terminus. The first modified protein 3 comprises a first C intein 14 and a first adduct 32, in this order from the C-terminus. The second modified protein 4 comprises a second C intein 24 and a second adduct 42, in this order from the C-terminus. Except for these points, the production method of the present invention can modify the C-termini of the first dimer-forming domain 12 and the second dimer-forming domain 22 with the first adduct 32 and the second adduct 42, respectively. In the following description, the above-mentioned domains can be used interchangeably.

[0051] In the production method of the present invention, a dimeric protein containing a reactive tag is formed by dimerization of the first monomer protein and the second monomer protein. The first monomer protein and the second monomer protein can be prepared, for example, by genetic engineering techniques, as described below in the method for producing a monomer protein of the present invention. Therefore, the production method of the present invention may optionally include, prior to the production step, a formation step of reacting the first monomer protein with the second monomer protein to form a dimer. The production method of the present invention may also optionally include, prior to the production step, a preparation step of preparing the first monomer protein and / or the second monomer protein, and a formation step of reacting the first monomer protein with the second monomer protein to form a dimer. The preparation method in the preparation step can be based on the descriptions of the first monomer protein, second monomer protein, nucleic acid, expression vector, transformant, and method for producing a monomer protein of the present invention described below.

[0052] In the formation step, the first monomer protein and the second monomer protein are reacted. As a result, as shown in FIG. 1(B), in the formation step, the first monomer protein and the second monomer protein form a dimer, and a dimeric protein containing the reaction tag can be prepared or prepared. The first monomer protein and the second monomer protein can form a heterodimer via the binding tag and the binding partner. Therefore, the formation step may be performed in parallel with the manufacturing step described below.

[0053] The reaction in the formation step is preferably carried out in the presence of an aqueous solvent such as a buffer solution, physiological saline, or water. The first monomer protein and the second monomer protein may be solid or liquid, but are preferably liquid. When the first monomer protein and the second monomer protein are solids such as powders, the first monomer protein and the second monomer protein are preferably pre-dispersed in an aqueous solvent such as a buffer solution, physiological saline, or water. When the first monomer protein and the second monomer protein are liquids, the first monomer protein and the second monomer protein may be pre-diluted with an aqueous solvent such as a buffer solution, physiological saline, or water. As a specific example of the reaction, the reaction can be carried out by mixing a liquid containing the first monomer protein with a liquid containing the second monomer protein.

[0054] When the first monomer protein and the second monomer protein obtained in the preparation step are used as the first monomer protein and the second monomer protein, the first monomer protein and the second monomer protein may be purified or crudely purified proteins, or may be a transformant expressing the first monomer protein and / or the second monomer protein or a processed product of the transformant. Examples of the processed product include a lysate, extract, disrupted product, protein preparation, or crude or purified product thereof. The processed product may be, for example, a product that has been subjected to at least one purification treatment.

[0055] The first monomer protein includes, for example, the first reaction tag and a first dimerization domain capable of forming a dimer, in this order from the N-terminus. The first reaction tag includes, for example, the binding tag and the first C intein, in this order from the N-terminus. The second monomer protein includes, for example, the second reaction tag and a second dimerization domain capable of forming a dimer with the first dimerization domain, in this order from the N-terminus. The second reaction tag includes the binding partner and the second C intein. By arranging the first reaction tag on the N-terminus of the first dimerization domain in the first monomer protein, the first adduct can be introduced to the N-terminus of the first dimerization domain upon reaction between the first C intein and the first N intein. Furthermore, by arranging the second reactive tag on the N-terminus of the second dimer-forming domain of the second monomer protein, the second adduct can be introduced onto the N-terminus of the second dimer-forming domain upon reaction between the second C intein and the second N intein.

[0056] In the formation step, dimerization of the first monomer protein and the second monomer protein may occur, for example, by (1) binding between the first reactive tag and the second reactive tag, e.g., binding between the binding tag of the first reactive tag and the binding partner of the second reactive tag, or (2) binding between the first dimer-forming domain and the second dimer-forming domain, or by binding of both (1) and (2). However, binding of both (1) and (2) is preferred because it can improve dimer formation ability. The reactive tag can also be referred to as, for example, a domain in the first monomer protein and the second monomer protein that contributes to binding between the monomer protein and the modified protein and to the addition reaction of an adduct to the monomer protein. The reactive tag can also be referred to as, for example, a domain that disappears from the monomer protein after the addition reaction.

[0057] The dimer may be formed by a direct bond between the first monomer protein and the second monomer protein, by an indirect bond (association) between the first monomer protein and the second monomer protein, or by both a direct and an indirect bond between the first monomer protein and the second monomer protein. The direct bond is a covalent bond, specific examples of which include an amide bond (peptide bond) between amino acids and a disulfide bond between cysteines. The indirect bond is a non-covalent bond, specific examples of which include a hydrogen bond and a hydrophobic bond.

[0058] The first dimerization domain and the second dimerization domain can employ amino acid sequences capable of forming dimers, condition-dependent or condition-independent, when proteins containing the domains coexist. Specifically, the first dimerization domain and the second dimerization domain can utilize, for example, amino acid sequences of motif sequences forming each subunit of a protein dimer or its dimer. The first dimerization domain and the second dimerization domain can utilize amino acid sequences of motif sequences forming each subunit of a protein multimer or its dimer. The protein dimer may be a homodimer or a heterodimer. Examples of the protein dimer include immunoglobulins (antibodies) such as IgA, IgD, IgE, IgG, and IgM; leucine zipper-containing proteins such as myc family proteins such as AP-1 (c-fos and c-jun), myc, max, and mdx1; G protein-coupled receptors; kinesin; receptor tyrosine kinases such as the ErbB receptor family, platelet-derived growth factor receptor (PDGFR), neurotrophin (neurotrophic factor) receptor, insulin receptor, insulin-like growth factor receptor, vascular endothelial growth factor receptor (VEGFR), and stem cell factor receptor; and Toll-like receptors such as TLR1 to 11.

[0059] When the antibody dimerization motif sequence is used as the first dimerization domain and the second dimerization domain, the first dimerization domain and the second dimerization domain comprise, for example, an amino acid sequence comprising the CH2 region and CH3 region of the antibody Fc region, or the CH2 region, CH3 region and hinge region of the antibody Fc region. When the first dimerization domain and the second dimerization domain comprise the antibody Fc region, the first dimerization domain and the second dimerization domain comprise the amino acid sequence of all or a part of the antibody Fc region. When the first dimerization domain and the second dimerization domain comprise the CH2 region and CH3 region and hinge region of the antibody Fc region, the first dimerization domain and the second dimerization domain comprise all or a part of the amino acid sequence of the CH2 region and CH3 region of the antibody Fc region and all or a part of the amino acid sequence of the antibody hinge region, and preferably comprise all of the amino acid sequence of the CH2 region and CH3 region of the antibody Fc region and all or a part of the amino acid sequence of the antibody hinge region. When the first dimerization domain and the second dimerization domain comprise the CH2 region and CH3 region and hinge region of the antibody Fc region, the first dimerization domain and the second dimerization domain comprise, for example, contiguous amino acid sequences in the amino acid sequences of the CH2 region and CH3 region of the antibody Fc region and the hinge region. The amino acid sequence of the Fc region, or the CH2 region, CH3 region, and hinge region of the Fc region may be a naturally occurring amino acid sequence, i.e., an amino acid sequence containing no mutations, or may be a non-naturally occurring amino acid sequence, i.e., an amino acid sequence containing a mutation or artificial amino acid sequence.

[0060] The antibody is, for example, IgA, IgD, IgE, IgG, or IgM, and is preferably IgG. The IgG is, for example, IgG1, IgG2, IgG2a, IgG2b, IgG3, or IgG4. The antibody is, for example, an animal-derived antibody, and specific examples include a human antibody, a mouse antibody, a chicken antibody, a rat antibody, and a rabbit antibody. The first dimer-forming domain and the second dimer-forming domain are preferably human-derived antibodies, and more preferably human-derived IgG.

[0061] As the amino acid sequences of the human IgG1, IgG2, IgG3, and IgG4, reference can be made to, for example, the amino acid sequences registered in UniProt under accession numbers P01857, P01859, P01860, and P01861, respectively.

[0062] When the antibody dimerization motif sequence is used as the first dimerization domain and the second dimerization domain, the antibody may be an antibody with a modified Fc region. In this case, the first dimerization domain and the second dimerization domain may comprise an Fc region of a variant of the Fc region, or an amino acid sequence comprising the Fc region and hinge region of the variant of the Fc region. Examples of the modified Fc region include Fcab (Fc antigen binding, References 1 and 5), in which the amino acid sequence of the Fc region has been modified to confer target molecule binding ability, IgG hexamer (IgG hexamer, References 2 to 4), in which the amino acid sequence of the Fc region of an IgG antibody has been modified to confer hexamer-forming ability, DAF (Dual Action Fab, Reference 5), Charge pair (Amgen, Reference 5), SEEDbody (Reference 5), Knobs-in-holes (Reference 5), and DVI-IgG (Reference 5). Reference 1: G. Wozniak-Knopp et al., “Introducing antigen-binding sites in structural loops of immunoglobulin constant domains: Fc fragments with engineered HER2 / neu-binding sites and antibody properties”, Protein Engineering, Design and Selection, Volume 23, Issue 4, April 2010, Pages 289-297 Reference 2: Sopp, JM et al., “On-target IgG hexamerization driven by a C-terminal IgM tail-piece fusion variant confers augmented complement activation.”, Commun. Biol., 4, 1031 (2021). Reference 3: de Jong RN et al., “A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface.”, PLoS. Biol. (2016) 14(1): e1002344. Reference 4: Christoph A. Diebolder et al., “Complement Is Activated by IgG Hexamers Assembled at the Cell Surface”, Science, 343 (6176), pages 1260-1263 Reference 5: Christoph Spiess et al., “Alternative molecular formats and therapeutic applications for bispecific antibodies”, Molecular Immunology, Volume 67, Issue 2, Part A, 2015, Pages 95-106.

[0063] When the antibody dimerization motif sequence is used as the first dimerization domain and the second dimerization domain, the antibody may be a multispecific antibody. In this case, the first dimerization domain and the second dimerization domain may comprise, for example, an Fc region of the multispecific antibody, or an amino acid sequence comprising the CH2 region, CH3 region, and hinge region of the Fc region, and a domain added to the C-terminus of the Fc region. Examples of the multispecific antibody include IgG-scFv (Reference 5), IgG(L,H)-scFv (Reference 5), IgG(H)-V (Reference 5), KIH IgG-scFab (Reference 5), IgG-2scFv (Reference 5), Intrabody (Reference 5), and Tetravalent HCAb (Reference 5).

[0064] The binding tag and the binding partner are molecules that bind to each other condition-dependently or condition-independently when a protein containing the binding tag and a protein containing the binding partner coexist. The binding between the binding tag and the binding partner may be direct or indirect.

[0065] When the binding between the binding tag and the binding partner is a direct bond, the binding tag and the binding partner can be, for example, a peptide tag and peptide that can spontaneously form a covalent bond, or a peptide tag and peptide that can form a covalent bond through the modifying activity of another molecule.

[0066] When the peptide tag and peptide capable of spontaneously forming a covalent bond are used, examples of the binding tag and the binding partner include a Streptococcus pyogenes surface protein (SpyCatcher, SEQ ID NO: 1) and a peptide tag capable of binding to the SpyCatcher (SpyTag, SEQ ID NO: 2), or variants thereof. Examples of variants of the SpyCatcher and the SpyTag include SpyCatcher2 and SpyTag2 (Reference 6), SpyCatcher3 and SpyTag3 (Reference 7), and SnoopCatcher and SnoopTag (Reference 8). Reference 6: Anthony H. Keeble et al., “Evolving Accelerated Amidation by SpyTag / SpyCatcher to Analyze Membrane Dynamics”, Angew. Chem. Int. Ed., 2017, 56, pages 16521 - 16525 Reference 7: Anthony H. Keeble et al., “Approaching infinite affinity through engineering of Peptide-protein interaction”, PNAS, 2019, vol. 116, No. 52, pages 26523-26533 Reference 8: Veggiani G, Nakamura T, Brenner MD, Gayet RV, Yan J, Robinson CV, Howarth M. Programmable polyproteams built using twin peptide superglues. Proc Natl Acad Sci US A. 2016 Feb 2;113(5):1202-7. doi: 10.1073 / pnas.1519214113.

[0067] Amino acid sequence of Streptococcus pyogenes surface protein (SpyCatcher) (SEQ ID NO: 1) DSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVN Amino acid sequence of SpyTag (SEQ ID NO: 2) AHIVMVDAYKPTK

[0068] When a peptide tag and a peptide capable of forming a covalent bond by the modifying activity of another molecule are used, examples of the binding tag and the binding partner include a K tag and a Q tag, etc. The K tag and the Q tag can form a covalent bond by crosslinking the N-terminal lysine residue of the K tag with the N-terminal glutamic acid of the Q tag using, for example, bacterial transglutaminase.

[0069] When the binding between the binding tag and the binding partner is indirect, the binding tag and the binding partner can be, for example, an affinity tag and a molecule that binds to the affinity tag. The binding tag and the binding partner can be, for example, a peptide, polypeptide, or protein. Examples of the binding tag include a His-tag (Hisx6), His-Strep-tag, strep-tag, avidin tag, flag™-tag, HA (hemagglutinin)-tag, T7-tag, V5-peptide-tag, GST (glutathione-S-transferase)-tag, CBP (calmodulin-binding peptide)-tag, MBP (maltose-binding protein)-tag, and Myc-tag. When the binding partner is a molecule that exhibits specific binding to a target molecule, such as an antibody or an antigen-binding fragment thereof, or a derivative thereof, the binding tag can be a peptide consisting of any amino acid sequence to which the molecule exhibiting specific binding can bind.

[0070] The binding partner can be appropriately selected depending on the type of the binding tag. Specific examples of the binding partner include an antibody that recognizes the binding tag, an antigen-binding fragment thereof, or a derivative thereof; a nucleic acid molecule such as an aptamer; glutathione, calmodulin; a sugar chain such as mannose; a metal such as nickel, cobalt, or zinc, or an ion thereof; and the like.

[0071] The combination of the binding tag and the binding partner may be any combination that allows binding between the binding tag and the binding partner. Specifically, when the binding tag includes a His-tag, the binding partner may be, for example, nickel. When the binding tag includes a strep-tag or an avidin-tag, the binding partner may be, for example, biotin. When the binding tag includes an epitope tag such as a flag™-tag, HA-tag, T7-tag, V5-peptide-tag, and / or Myc-tag, the binding partner may be, for example, an antibody against each epitope tag, an antigen-binding fragment thereof, or a derivative thereof. When the binding tag includes a GST-tag, the binding partner may be, for example, glutathione. When the binding tag includes a CBP-tag, the binding partner may be, for example, calmodulin. When the binding tag includes an MBP-tag, the binding partner may be, for example, mannose.

[0072] The binding tag and the binding partner may be functional equivalents to the extent that they maintain the binding ability between the binding tag and the binding partner. When the binding tag or the binding partner is a peptide, polypeptide, or protein, the functional equivalent can be, for example, a polypeptide consisting of an amino acid sequence that has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the reference amino acid sequence of the binding tag or the binding partner, and has the ability to bind to the corresponding binding tag or binding partner. The functional equivalent can be, for example, an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, and / or added from the reference amino acid sequence of the binding tag or the binding partner, and has the ability to bind to the corresponding binding tag or binding partner. The one or several substitutions are, for example, 1 to 44, 1 to 33, 1 to 22, 1 to 11, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 or 2, or 1. The substitution is preferably, for example, a conservative substitution.

[0073] The first monomer protein may have one or more binding tags. In the latter case, the binding tags may be of one type or of multiple types. When the N-terminus of the first monomer protein is modified (added) with a first adduct, the binding tag is preferably located on the N-terminus side of the first dimerization domain.

[0074] The second monomer protein may have one or more binding partners. In the latter case, the binding partners may be of one type or of multiple types. When the N-terminus of the second monomer protein is modified (added) with a second adduct, the binding partner is preferably located on the N-terminal side of the second dimerization domain. The number of the binding partners and the number of the binding partners may be the same or different, but are preferably the same.

[0075] In the first monomer protein and the second monomer protein, the binding tag and the binding partner are interchangeable, and the combinations described above may be used interchangeably.

[0076] The first C intein and the first N intein are molecules that cause the binding tag to bind to the binding partner in a condition-dependent or condition-independent manner when a first monomer protein containing the first C intein and a first modified protein containing the first N intein coexist. The second C intein and the second N intein are molecules that cause the binding tag to bind to the binding partner in a condition-dependent or condition-independent manner when a second monomer protein containing the second C intein and a second modified protein containing the second N intein coexist. Inteins that can be used for the first C intein, the first N intein, the second C intein, and the second N intein are described below.

[0077] In the present invention, the C intein and N intein constituting the intein may be designed from, for example, an integrated intein, or a split intein may be used.

[0078] Examples of the integrated intein include gp41-1, gp41-8, NrdJ-1, SspGyrB, and Cfa. Examples of the split intein include DnaE (catalytic subunit α, dnaE-n, and dnaE-c, of cyanobacterial DNA polymerase III) and MCM2 (SEQ ID NO: 3, Hut MCM-2 intein from the archaea Halorhabdus utahensis DSM 12940, partial sequence of Uniprot: C7NUH7). In the amino acid sequence of SEQ ID NO: 3 below, the regions enclosed in brackets correspond, from the N-terminus, to the N intein, deleted region, and C intein (Reference 9). Reference 9: Ciragan A. et al., “Salt-inducible Protein Splicing in cis and trans by Inteins from Extremely Halophilic Archaea as a Novel Protein-Engineering Tool.” J Mol Biol. 2016 Nov 20;428(23):4573-4588. doi: 10.1016 / j.jmb.2016.10.006. Epub 2016 Oct 6. PMID: 27720988.

[0079] Amino acid sequence of MCM2 (SEQ ID NO: 3) [CVTGDTLVQAGDGRRRIRELAGETAEAGSIEELPNGRTIRDVDIDVWTMTDDETLTRRPVTAIHEYDAPETLYEVTLSTGEEVTVTPDHPFFIEQASGRVETPAEDLQPGDLVFVPEGSAMATDG][GIAQIDTSSDRLGPAESGL][GDIGLRTIENVESVPDHDYDSVYDLTVEGTHNFLANGMVVHN

[0080] When the intein is MCM2, examples of the C intein and N intein derived from MCM2 include a C intein consisting of the amino acid sequence of SEQ ID NO: 4 and an N intein consisting of the amino acid sequence of SEQ ID NO: 5 below.

[0081] Amino acid sequence of MCM2-derived C intein (SEQ ID NO: 4) GIAQIDTSSDRLGPAESGL / GDIGLRTIENVESVPDHDYDSVYDLTVEGTHNFLANGMVVHN

[0082] Amino acid sequence of MCM2-derived N intein (SEQ ID NO: 5): CVTGDTLVQAGDGRRRIRELAGETAEAGSIEELPNGRTIRDVDIDVWTMTDDETLTRRPVTAIHEYDAPETLYEVTLSTGEEVTVTPDHPFFIEQASGRVETPAEDLQPGDLVFVPEGSAMATD

[0083] C intein from gp41-1 (SEQ ID NO: 6) MMLKKILKIEELDERELIDIEVSGNHLFYANDILTHNSAG

[0084] N intein from gp41-1 (SEQ ID NO: 7) SGYCLDLKTQVQTPQGMKEISNIQVGDLVLSNTGYNEVLNVFPKSKKKSYKITLEDGKEIICSEEHLFPTQTGEMNISGGLKEGMCLYVKE

[0085] C intein from Cfa (SEQ ID NO: 8) VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASNC

[0086] Cfa-derived N intein (SEQ ID NO: 9) CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP

[0087] The intein may be an intein that undergoes a condition-dependent binding reaction (condition-dependent intein). The condition-dependent intein can also be, for example, an intein in which a binding reaction between the intein and / or the C intein and N intein occurs under certain conditions. Examples of the condition-dependent intein include salt-concentration-dependent inteins such as MCM2, a combination of HsaPolII and HsaCDC21 derived from Halobacterium salinarum NRC-1 (ATCC 700922) (see Reference 9), and temperature-dependent inteins (see Reference 10). The salt-concentration-dependent intein is, for example, an intein in which a binding reaction between the intein and / or the C intein and N intein occurs when the salt concentration is relatively high, i.e., the probability of a binding reaction occurring increases. The salt-concentration-dependent intein may be an intein in which a binding reaction occurs when the salt concentration reaches a certain level. The salt-concentration-dependent intein is presumed to undergo a binding reaction that is dependent on the ionic strength of the salt. Therefore, the salt used to induce the salt-concentration-dependent intein binding reaction is not particularly limited, and any salt can be used. Examples of the salt include chlorides such as sodium chloride, calcium chloride, potassium chloride, and magnesium chloride; sulfates such as sodium sulfate, calcium sulfate, potassium sulfate, and magnesium sulfate; nitrates such as sodium nitrate, calcium nitrate, potassium nitrate, and magnesium nitrate; carbonates such as sodium carbonate, calcium carbonate, potassium carbonate, and magnesium carbonate; and phosphates such as sodium phosphate, calcium phosphate, potassium phosphate, and magnesium phosphate. When the salt-concentration-dependent intein is MCM2, the salt is preferably sodium chloride. Reference 10: Zeidler, M. et al. "Temperature-sensitive control of protein activity by conditionally splicing inteins." Nat Biotechnol 22, 871-876 (2004). https: / / doi.org / 10.1038 / nbt979

[0088] The intein may be a functional equivalent to the extent that it maintains its protease activity and ligase activity. Examples of such functional equivalents include polypeptides having protease activity and ligase activity, which consist of an amino acid sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the reference amino acid sequence of the intein. Examples of such functional equivalents include polypeptides having protease activity and ligase activity, which consist of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the reference amino acid sequence of the intein. The one or several amino acids may be, for example, 1 to 44, 1 to 33, 1 to 22, 1 to 11, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 or 2, or 1. The substitution is preferably, for example, a conservative substitution.

[0089] The number of C inteins in the first monomer protein and the second monomer protein may be one or more, but is preferably one, as this allows the first monomer protein and the second monomer protein to be modified efficiently.

[0090] When a first adduct is added to the N-terminus of the first monomer protein, the first C intein is preferably positioned on the N-terminal side of the first dimerization domain. When a second adduct is added to the N-terminus of the second monomer protein, the binding partner is preferably positioned on the N-terminal side of the second dimerization domain.

[0091] The first C intein and the second C intein are configured to be capable of reacting with, for example, the first N intein and the second N intein, respectively. This allows the first modifying protein to be configured to modify (alter) the first monomer protein, and the second modifying protein to be configured to modify (alter) the second monomer protein. Therefore, the first C intein and the second C intein are preferably C inteins derived from different inteins. In this case, the first C intein and the second C intein comprise different C inteins. Furthermore, the first C intein and the first N intein are, for example, derived from the same intein. The second C intein and the second N intein are, for example, derived from the same intein.

[0092] The order of the first reactive tag and the first dimer-forming domain in the first monomer protein can be set, for example, depending on the addition position of the first adduct in the first modified protein. When the first adduct is added to the N-terminus of the first dimer-forming domain, the first reactive tag and the first dimer-forming domain are arranged in this order from the N-terminus in the first monomer protein. When the first adduct is added to the C-terminus of the first dimer-forming domain, the first dimer-forming domain and the first reactive tag are arranged in this order from the N-terminus in the first monomer protein.

[0093] In the first reaction tag, the order of the binding tag and the first C intein can be set, for example, depending on the addition position of the first adduct in the first modified protein. When the first adduct is added to the N-terminus of the first dimerization domain, the binding tag and the first C intein are arranged in this order from the N-terminus. In the second reaction tag, the order of the binding partner and the second C intein can be set, for example, depending on the addition position of the second adduct in the second modified protein. When the second adduct is added to the N-terminus of the second dimerization domain, the binding partner and the second C intein are arranged in this order from the N-terminus.

[0094] In the first monomer protein, the order of the binding tag, the first C intein, and the first dimerization domain can be set, for example, depending on the position of addition of the first adduct in the first modified protein. When the first adduct is added to the N-terminus of the first dimerization domain, the binding tag, the first C intein, and the first dimerization domain are arranged in this order from the N-terminus. Furthermore, in the second monomer protein, the order of the binding partner, the second C intein, and the second dimerization domain can be set, for example, depending on the position of addition of the second adduct in the second modified protein. When the second adduct is added to the N-terminus of the second dimerization domain, the binding partner, the second C intein, and the second dimerization domain are arranged in this order from the N-terminus.

[0095] In the first monomer protein, the binding tag, the first C intein, and the first dimerization domain are each directly or indirectly bound (linked). When producing a heterodimeric protein in which multiple domains composed only of natural amino acid sequences are linked, the first C intein and the first dimerization domain are preferably directly linked.

[0096] In the second monomer protein, the binding partner, the second C intein, and the second dimerization domain are each directly or indirectly bound (linked). When producing a heterodimeric protein in which multiple domains composed only of natural amino acid sequences are linked, it is preferable that the second C intein and the second dimerization domain are directly linked.

[0097] The direct bond means that the N- or C-terminal amino acid of a polypeptide or domain is bound to the C- or N-terminal amino acid of another polypeptide or domain via a peptide bond. On the other hand, the indirect bond means that the N- or C-terminal amino acid of a polypeptide or domain is bound to the C- or N-terminal amino acid of another polypeptide or domain via a linker peptide (peptide linker). That is, the N- or C-terminal amino acid of a polypeptide or domain is bound to the C- or N-terminal amino acid of the linker peptide via a peptide bond, and the amino acid at the other end of the linker peptide is bound to the N- or C-terminal amino acid of the other polypeptide or domain. The length of the linker peptide is, for example, 5 to 15 amino acids. The linker peptide may be a known linker peptide, and specific examples include a GS linker (GS, GGS, or GGGGS (SEQ ID NO: 10)), a linker peptide with repeated GS linkers ([GS] ｌ , [GGS] ｍ , or [GGGGS] ｎ (wherein l, m, and n are each an integer of 2 or more), GGGSGG (SEQ ID NO: 11), and the like.

[0098] In the present invention, when the heterodimeric protein is a multispecific antibody, the dimeric protein comprises, for example, the CH2 and CH3 regions of the Fc region of the immunoglobulin (antibody), or a portion or all of the hinge region of the immunoglobulin and the CH2 and CH3 regions of the Fc region of the immunoglobulin. In this case, the first dimer-forming domain of the first monomer protein and the second dimer-forming domain of the second monomer protein comprise, for example, the amino acid sequences of the CH2 and CH3 regions of the Fc region of the immunoglobulin (antibody), or a portion or all of the hinge region of the immunoglobulin and the CH2 and CH3 regions of the Fc region of the immunoglobulin. The immunoglobulin is, for example, a human antibody, preferably human IgG.

[0099] The first monomer protein may include, for example, another polypeptide, such as a solubility domain or a signal peptide, on the N-terminal side of the first reaction tag. The second monomer protein may also include, for example, another polypeptide, such as a solubility domain or a signal peptide, on the N-terminal side of the second reaction tag. The solubility domain is preferably a polypeptide that, when fused to the polypeptide or protein, increases the expression level of the first monomer protein or the second monomer protein expressed in the transformant described below by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 100% or more, compared to when expressed as a polypeptide or protein not containing the solubility domain. The solubility domain may be, for example, a protein or a partial polypeptide thereof. Specific examples of the soluble domain include GST, MBP, thioredoxin, antibodies, and modified antibodies such as single-chain antibodies.

[0100] When the first monomer protein includes the solubilization domain, the number of solubilization domains in the first monomer protein may be one or more. In the latter case, the number of solubilization domains may be one or more types.

[0101] When the second monomer protein includes the solubilization domain, the number of solubilization domains in the second monomer protein may be one or more. In the latter case, the number of solubilization domains may be one or more types.

[0102] The first monomer protein and the second monomer protein may include, for example, a purification tag used for purifying the first monomer protein, the second monomer protein, or the heterodimeric protein. The purification tag can be, for example, the affinity tag described above. The purification tag can be added, for example, to at least one of the N-terminus and C-terminus of the first dimerization domain and the second dimerization domain.

[0103] The reaction conditions in the formation step may be any conditions that allow the first monomer protein and the second monomer protein to form a dimer, and can be appropriately set taking into consideration the reaction conditions (binding conditions) between the binding tag and the binding partner, and / or the reaction conditions (binding conditions) between the first dimer-forming domain and the second dimer-forming domain. Specific examples of the reaction temperature in the formation step include 4 to 50°C or 10 to 45°C. The reaction time in the formation step is, for example, 0.1 to 24 hours or 0.5 to 12 hours. The reaction pH in the formation step is, for example, pH 4 to 10 or pH 5 to 9.

[0104] When the binding tag and the binding partner are directly bound, the reaction conditions for the formation step can be set to reaction conditions that allow sufficient formation of a bond between the binding tag and the binding partner. Specifically, when the binding tag and the binding partner are SpyTag and SpyCatcher, the formation step can be carried out, for example, at pH 5 to 8, at 4 to 37°C, in the presence of a buffer solution. When the bond between the binding tag and the binding partner is formed by an enzymatic reaction, the reaction conditions for the formation step can be set, for example, based on the activity conditions of the enzyme used in the enzymatic reaction.

[0105] When the first dimer-forming domain and the second dimer-forming domain are directly bound to each other, the reaction conditions for the formation step can be set to reaction conditions under which a bond between the first dimer-forming domain and the second dimer-forming domain is sufficiently formed. As a specific example, when the first dimer-forming domain and the second dimer-forming domain are bound to each other via a disulfide bond, the reaction conditions for the formation step can be set to, for example, reaction conditions under which the disulfide bond is not reduced.

[0106] When the heterodimeric protein is a multispecific antibody, the reaction conditions in the formation step may further be, for example, conditions under which the first monomeric protein and the second monomeric protein can form disulfides between the hinge regions of the immunoglobulin.

[0107] Next, in the production process, as shown in Figures 1(C) to 1(D), the dimeric protein is reacted with a modifying protein that modifies the dimeric protein to produce (produce) a heterodimeric protein. Therefore, the production process can also be considered a process of adding a polypeptide or protein derived from the modifying protein to the dimeric protein to produce the heterodimeric protein. Specifically, in the production process, the first N intein of the first modified protein reacts with the first C intein of the first monomer protein, and the first adduct of the first modified protein is linked to the first monomer protein, more specifically, to the N-terminal side of the first dimer-forming domain of the first monomer protein (hereinafter also referred to as the "first linking step"). Furthermore, in the production process, the second N intein of the second modified protein reacts with the second C intein of the second monomer protein, and the second adduct of the second modified protein is linked to the second monomer protein, more specifically, to the N-terminus of the first dimer-forming domain of the first monomer protein (hereinafter also referred to as the "second linking step"). This allows the dimeric protein to be converted into a heterodimeric protein composed of a protein containing the first adduct and the first dimer-forming domain in this order, and a protein containing the second adduct and the second dimer-forming domain in this order.

[0108] The reaction in the production process is preferably carried out in the presence of an aqueous solvent such as a buffer solution, physiological saline, or water. The dimeric protein, the first modified protein, and the second modified protein may be solid or liquid, but liquids are preferred. When the dimeric protein, the first modified protein, and the second modified protein are solids such as powders, the dimeric protein, the first modified protein, and the second modified protein are preferably pre-dispersed in an aqueous solvent such as a buffer solution, physiological saline, or water. When the dimeric protein, the first modified protein, and the second modified protein are liquids, the dimeric protein, the first modified protein, and the second modified protein may be pre-diluted with an aqueous solvent such as a buffer solution, physiological saline, or water. Specifically, the reaction can be carried out by mixing a liquid containing the dimeric protein with a liquid containing the first modified protein and / or a liquid containing the second modified protein.

[0109] In the manufacturing process, the first and second linking steps may be performed partly or entirely simultaneously, or may be performed separately. When the first and second linking steps are performed separately, the order of the first and second linking steps is not particularly limited, and the second linking step may be performed after the first linking step, or the first linking step may be performed after the second linking step.

[0110] The first modified protein comprises a first adduct added to the first monomer protein and a first N-intein capable of reacting with the first C-intein. The first modified protein comprises, for example, the first adduct and the first N-intein, in this order from the N-terminus. The second modified protein comprises a second adduct added to the second monomer protein and a second N-intein capable of reacting with the second C-intein. The second modified protein comprises, for example, the second adduct and the second N-intein, in this order from the N-terminus. By linking the first adduct to the first N-intein, the first modified protein can introduce the first adduct into the N-terminus of the first dimerization domain upon reaction between the first N-intein and the first C-intein. As a result, the first linking step can produce a fusion protein comprising the first dimerization domain and the first adduct. Furthermore, by linking the second adduct to the second N intein, the second modified protein can introduce the second adduct to the N-terminus of the second dimerization domain upon reaction between the second N intein and the second C intein. As a result, the second linking step can produce a fusion protein comprising the second dimerization domain and the second adduct.

[0111] The above description can be used to explain the first N intein in the first modified protein and the second N intein in the second modified protein. The first N intein and the second N intein are configured to be capable of reacting with, for example, the first C intein and the second C intein, respectively. This allows the first modified protein to be configured to modify (alter) the first monomer protein, and the second modified protein to be configured to modify (alter) the second monomer protein. For this reason, it is preferable that the first N intein and the second N intein are N inteins derived from different inteins. In this case, the first N intein and the second N intein contain different N inteins. Furthermore, the first C intein and the first N intein are derived from, for example, the same intein. The second C intein and the second N intein are derived from, for example, the same intein.

[0112] The first adduct and the second adduct refer to those added to the dimeric protein. The adducts can also be referred to as modifications, for example. The first adduct and the second adduct contain one or more desired peptides, polypeptides, or proteins, and the desired peptides, polypeptides, or proteins may be modified. The first adduct and the second adduct are different adducts. In the production method of the present invention, by making the first adduct and the second adduct different adducts, different adducts can be added to one dimer-forming domain and the other dimer-forming domain constituting the dimeric protein, thereby producing a heterodimeric protein. The adducts that can be used for the first adduct and the second adduct are also described below.

[0113] Examples of the adduct include proteins such as modified antibodies, heavy chain antibodies (VHH), receptors, cytokines, chemokines, toxins, enzymes, fluorescent proteins, and collagen-binding domains; peptides such as insulin; and the like.

[0114] The modified antibody is a protein comprising an antigen-binding fragment of the antibody, or a polypeptide in which the antigen-binding fragment is linked via a linker peptide. The modified antibody includes Fab, Fab', F(ab') ２ Examples of the modified antibody include a single-chain antibody (scFv), a tandem scFv, a BiTE, a diabody, a DART, a TandAb, a scDiabody, a heavy-chain antibody (VHH), and a variable domain of the VHH. The modified antibody may be a protein comprising an antigen-binding fragment of the antibody or a polypeptide in which the antigen-binding fragment is linked via a linker peptide, to which an antigen-binding fragment of another antibody or the like is further linked. In this case, the modified antibody can be configured as a protein comprising, for example, a domain other than the Fc region of DVD-IgG (Reference 5), scFv-(H)IgG (Reference 5), IgG(L)-scFv (Reference 5), scFv-(L)IgG (Reference 5), V(H)-IgG (Reference 5), IgG(L)-V (Reference 5), V(L)-IgG (Reference 5), 2scFv-IgG (Reference 5), scFv4-Ig (Reference 5), Zybody (Reference 5), scDiabody-CH3 (Reference 5), Diabody-CH3 (Reference 5), scDiabody-Fc (Reference 5), Diabody-Fc (Reference 5), or the like, more specifically, a part or all of the domain N-terminal to the Fc region.

[0115] The target antigen for the modified antibody or the heavy-chain antibody is not particularly limited and can be any antigen, including, for example, tumor antigens, viral antigens, bacterial antigens, parasitic antigens, antigens associated with autoimmune diseases, sugar chain antigens, and surface antigens of immune cells such as lymphocytes.

[0116] The surface antigen may be, for example, a CD (cluster of differentiation) antigen. The surface antigen may be an antigen that activates or inhibits activation of immune cells expressing the surface antigen when a modified antibody or a heavy chain antibody binds to the surface antigen. Examples of the immune cells include T cells, NK cells, NKT cells, B cells, and macrophages. Examples of antigens that activate T cells include CD2, CD3, CD4, CD5, CD8α, CD8β, CD27, CD28, CD134 (OX40), CD137 (4-1BB), CD154, GITR, and ICOS, with CD3 being preferred. Examples of the CD3 include CD3γ, CD3δ, CD3ε, CD3ζ, and CD3η, with CD3ε being preferred. Examples of the antigen that inhibits the activation of T cells include CTLA4, PD-1, LAG3, B7-H3, TIM3, and TIGIT. Examples of the antigen that activates NK cells include CD94 / NKG2C, CD94 / NKG2E, and NKG2D / NKG2D. Examples of the antigen that inhibits the activation of NK cells include KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, KIR2DL4, KIR2DL5, and KIR3DL3. Examples of the antigen that activates NKT cells include antigens that activate T cells and antigens that activate NK cells. Examples of the antigen that inhibits the activation of NKT cells include an antigen that inhibits the activation of T cells and an antigen that inhibits the activation of NK cells.

[0117] Examples of the receptor include cytokine receptors such as TNFα receptors; chemokine receptors; hormone receptors such as insulin receptors; and receptors for growth factors or proliferation factors such as EGF receptors.

[0118] Examples of the cytokines and chemokines include tumor necrosis factors (TNFs) such as TNF-α and TNF-β; lymphotoxins; interleukins such as IL-1 to IL-38 (e.g., IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-17, IL-18, IL-23, IL-33, IL-36, etc.); chemokines; hematopoietic factors; cell growth factors; adipokines; and growth factors such as VEGF.

[0119] Examples of the toxin include staphylococcal enterotoxin.

[0120] Examples of the enzyme include luciferase and phosphatase.

[0121] Examples of the fluorescent protein include GFP (Green Fluorescent Protein) and dsRED.

[0122] In the first modified protein, the first adduct and the first N-intein are directly or indirectly bound (linked). When producing a heterodimeric protein in which multiple domains composed only of natural amino acid sequences are linked, the first adduct and the first N-intein are preferably directly linked.

[0123] In the second modified protein, the second adduct and the second N-intein are directly or indirectly bound (linked). When a heterodimeric protein in which multiple domains composed only of natural amino acid sequences are linked is produced as the heterodimeric protein, the second adduct and the second N-intein are preferably directly linked.

[0124] In the first adduct and the second adduct, the above explanation can be applied to the direct or indirect bond.

[0125] In the present invention, when the heterodimeric protein is a multispecific antibody, the first adduct and the second adduct are, for example, polypeptides containing the antibody variant or the heavy chain antibody. Specifically, when the heterodimeric protein is a bispecific antibody, the first adduct is a polypeptide comprising an amino acid sequence containing the Fab region of a first antibody, or the Fab region and all or part of the hinge region of the antibody. Furthermore, the second adduct is a polypeptide comprising an amino acid sequence containing the Fab region of a second antibody, or the Fab region and all or part of the hinge region of the antibody. The first antibody and the second antibody are antibodies that bind to different antigens or different epitopes. When the first adduct and the second adduct comprise a portion of the hinge region of an antibody, the hinge regions comprised by the first adduct and the second adduct are configured to constitute the entire hinge region of the antibody when linked to the first dimerization domain and the second dimerization domain, respectively. The modified antibody is, for example, a modified human antibody, preferably a modified human IgG antibody.

[0126] The first modified protein may include, for example, the solubilization domain, the signal peptide, or another polypeptide at its N-terminus. The second modified protein may include, for example, the solubilization domain, the signal peptide, or another polypeptide at its N-terminus.

[0127] When the first modified protein includes the solubilization domain, the number of solubilization domains in the first modified protein may be one or more. In the latter case, the number of solubilization domains may be one type or multiple types.

[0128] When the second modified protein includes the solubilization domain, the number of solubilization domains in the second modified protein may be one or more. In the latter case, the number of solubilization domains may be one type or multiple types.

[0129] The first modified protein and the second modified protein may include, for example, a purification tag used for purifying the first modified protein, the second modified protein, or the heterodimeric protein. The purification tag can be, for example, the affinity tag described above. The purification tag can be added, for example, to at least one of the N-terminus and C-terminus of the first adduct and the second adduct.

[0130] In the production process, the reaction conditions for the first ligation step and the second ligation step may be the same or different. The reaction conditions for the first ligation step can be set, for example, depending on the types of the first C intein and the first N intein, more specifically, depending on the conditions for the enzymatic activity of the active intein formed by the binding of the first C intein and the first N intein. The reaction conditions for the second ligation step can be set, for example, depending on the types of the second C intein and the second N intein, more specifically, depending on the conditions for the enzymatic activity of the active intein formed by the binding of the second C intein and the second N intein. Specific examples of the reaction temperature for the production step include 0 to 40°C, 4 to 37°C, or 4 to 30°C. The reaction time for the production step is, for example, 1 minute to 48 hours, 30 minutes to 48 hours, or 1 to 48 hours. The reaction pH in the production step is, for example, pH 5 to 10, pH 6 to 9, or pH 6.5 to 9.

[0131] The production method of the present invention may include a purification step of purifying the heterodimeric protein after the production step. The purification method in the purification step can be, for example, a general protein purification method such as chromatography.

[0132] In this way, the production method of the present invention can produce a heterodimer protein from a dimer protein.

[0133] In another aspect, the present invention provides a dimeric protein suitable for use in producing a heterodimeric protein. The protein (dimeric protein) of the present invention includes a dimeric protein comprising a reactive tag, the dimeric protein comprising the reactive tag comprising a first monomeric protein and a second monomeric protein, the first monomeric protein comprising a first reactive tag and a first dimer-forming domain capable of forming a dimer, the first reactive tag comprising a binding tag and a first C intein capable of reacting with a first N intein, the second monomeric protein comprising a second reactive tag and a second dimer-forming domain capable of forming a dimer with the first dimer-forming domain, in this order, the second reactive tag comprising a binding partner capable of binding to the binding tag and a second C intein capable of reacting with a second N intein, and the first monomeric protein and the second monomeric protein forming a dimer. According to the dimeric protein of the present invention, a heterodimeric protein can be suitably produced by combining the first modified protein and the second modified protein.

[0134] In another aspect, the present invention provides a monomeric protein suitable for use in producing the dimeric protein. The protein of the present invention includes a first monomeric protein, the first monomeric protein comprising, in this order, a first reactive tag and a first dimerization domain capable of forming a dimer, the first reactive tag comprising a binding tag capable of binding to a binding partner and a first C intein capable of reacting with a first N intein, the first monomeric protein capable of forming a dimer with a second monomeric protein, the second monomeric protein comprising a second reactive tag and a second dimerization domain capable of forming a dimer with the first dimerization domain, and the second reactive tag comprising a binding partner capable of binding to the binding tag and a second C intein capable of reacting with a second N intein. The protein of the present invention also includes a second monomer protein, which includes, in this order, a second reactive tag and a second dimerization domain capable of forming a dimer; the second reactive tag includes a binding partner capable of binding to a binding tag and a second C intein capable of reacting with a second N intein; the second monomer protein is capable of forming a dimer with the first monomer protein; the first monomer protein includes, in this order, a first reactive tag and a first dimerization domain capable of forming a dimer with the second dimerization domain; and the first reactive tag includes a binding tag capable of binding to the binding partner and a first C intein capable of reacting with the first N intein.

[0135] <Nucleic Acid> In another aspect, the present invention provides a nucleic acid that can be used to synthesize a dimeric protein, a first monomeric protein, and / or a second monomeric protein. The nucleic acid of the present invention encodes the dimeric protein of the present invention, the first monomeric protein of the present invention, and / or the second monomeric protein of the present invention.

[0136] The nucleic acid of the present invention can be designed by substituting corresponding codons based on the amino acid sequence of the dimeric protein of the present invention, the first monomeric protein of the present invention, and / or the second monomeric protein of the present invention. The base sequence of the nucleic acid of the present invention may be, for example, codon-optimized.

[0137] The nucleic acid of the present invention may further encode the first modified protein and / or the second modified protein.

[0138] <Expression Vector> In another aspect, the present invention provides an expression vector that can be used to synthesize a dimeric protein, a first monomeric protein, and / or a second monomeric protein. The expression vector of the present invention comprises the nucleic acid of the present invention. The expression vector of the present invention allows the dimeric protein of the present invention, the first monomeric protein of the present invention, and / or the second monomeric protein of the present invention (hereinafter also referred to as "proteins of the present invention") to be suitably produced by genetic engineering techniques.

[0139] The expression vector of the present invention is, for example, an expression vector into which the nucleic acid of the present invention is inserted. The expression vector means, for example, a nucleic acid molecule that can transport an inserted gene into a target such as a cell.

[0140] The expression vector may contain, for example, a polynucleotide encoding the dimeric protein of the present invention, the first monomeric protein of the present invention, and / or the second monomeric protein of the present invention so as to be capable of expressing the protein of the present invention encoded by the polynucleotide of the nucleic acid of the present invention. The dimeric protein of the present invention, the first monomeric protein of the present invention, and / or the second monomeric protein of the present invention may be inserted, for example, partially or entirely, into the same expression vector, or may be inserted into separate expression vectors. When the dimeric protein of the present invention, the first monomeric protein of the present invention, and / or the second monomeric protein of the present invention are inserted into separate expression vectors, the expression vector of the present invention may be configured as an expression vector set including an expression vector containing a nucleic acid encoding the dimeric protein of the present invention, an expression vector containing a nucleic acid encoding the first monomeric protein of the present invention, and / or an expression vector containing a nucleic acid encoding the second monomeric protein of the present invention.

[0141] The expression vector may further include a nucleic acid encoding the first modified protein and / or the second modified protein. In this case, the nucleic acid encoding the first modified protein and / or the second modified protein may be inserted into an expression vector containing a nucleic acid encoding the dimeric protein of the present invention, the first monomeric protein of the present invention, and / or the second monomeric protein of the present invention, or may be inserted into another expression vector.

[0142] The expression vector can be prepared, for example, by inserting a polynucleotide encoding the protein of the present invention, i.e., the nucleic acid of the present invention, into a backbone vector (hereinafter also referred to as a "basic vector"). The type of the expression vector is not particularly limited and can be appropriately determined depending on, for example, the type of the host.

[0143] Examples of the host include non-human hosts such as microorganisms, animal cells, insect cells, or cultured cells thereof, isolated human cells or cultured cells thereof, and mammalian cells. Examples of the prokaryotic organism include bacteria such as Escherichia genus such as Escherichia coli and Pseudomonas genus such as Pseudomonas putida. Examples of the eukaryotic organism include yeast such as Saccharomyces cerevisiae. Examples of the animal cells include HEK293 cells, Expi293F cells, COS cells, and CHO cells, and examples of the insect cells include Sf9 and Sf21.

[0144] Examples of the expression vector (basic vector) include viral vectors and non-viral vectors. When transforming a host using the heat shock method as the introduction method, examples of the expression vector include binary vectors. Examples of the expression vector include pETDuet-1, pQE-80L, and pUCP26Km. When transforming bacteria such as Escherichia coli, examples of the expression vector include pETDuet-1 vector (Novagen), pQE-80L (QIAGEN), pBR322, pB325, pAT153, and pUC8. When transforming yeast, examples of the expression vector include pYepSec1, pMFa, and pYES2. When transforming insect cells, examples of the expression vector include pAc and pVL. When transforming mammalian cells, examples of the expression vector include pcDNA3.1, pcDNA3.4, pCAG, pCAGEN, pCDM8, and pMT2PC.

[0145] The expression vector preferably has a regulatory sequence that regulates the expression of the polynucleotide encoding the protein of the present invention and the expression of the protein of the present invention encoded by the polynucleotide encoding the protein of the present invention. Examples of the regulatory sequence include a promoter, a terminator, an enhancer, a polyadenylation signal sequence, and an origin of replication (ori). The location of the regulatory sequence in the expression vector is not particularly limited. In the expression vector, the regulatory sequence may be located, for example, so long as it is capable of functionally regulating the expression of the polynucleotide encoding the protein of the present invention and the expression of the protein of the present invention encoded thereby, and can be located based on known methods. For example, the regulatory sequence may utilize a sequence already contained in the basic vector, or the regulatory sequence may be further inserted into the basic vector, or the regulatory sequence contained in the basic vector may be replaced with another regulatory sequence.

[0146] The expression vector may further comprise, for example, a coding sequence for a selection marker, such as a drug resistance marker, a fluorescent protein marker, an enzyme marker, or a cell surface receptor marker.

[0147] Insertion of DNA, insertion of the regulatory sequence, and / or insertion of the coding sequence of the selection marker into the expression vector may be carried out, for example, by a method using restriction enzymes and ligase, or by using a commercially available kit, etc.

[0148] <Transformant and method for producing the transformant> In another aspect, a transformant capable of producing the protein of the present invention and a method for producing the same are provided. The transformant of the present invention contains a nucleic acid encoding the protein of the present invention. The transformant of the present invention can suitably produce the protein of the present invention.

[0149] Furthermore, a method for producing a transformant of the present invention includes the step of introducing the nucleic acid of the present invention into a host. According to the method for producing a transformant of the present invention, the transformant can be produced.

[0150] In the transformant of the present invention, the nucleic acid encoding the protein of the present invention can be the same as that described above for the nucleic acid encoding the protein of the present invention. The nucleic acid of the present invention may be the expression vector of the present invention.

[0151] In the transformant of the present invention, the nucleic acid of the present invention is present as an exogenous molecule. Therefore, the transformant of the present invention can be produced, for example, by introducing the nucleic acid of the present invention into the host.

[0152] The method for introducing the nucleic acid is not particularly limited and can be performed by a known method. The nucleic acid may be introduced, for example, via the expression vector. The introduction method can be appropriately selected depending on, for example, the type of host. Examples of the introduction method include introduction using a gene gun such as a particle gun, the calcium phosphate method, the polyethylene glycol method, lipofection using liposomes, electroporation, ultrasonic nucleic acid introduction, DEAE-dextran method, direct injection using microglass tubes, the hydrodynamic method, the cationic liposome method, methods using an introduction adjuvant, and agrobacterium-mediated methods. Examples of liposomes include lipofectamine and cationic liposomes, and examples of introduction adjuvants include atelocollagen, nanoparticles, and polymers. When the host is a microorganism, a method mediated by, for example, E. coli or Ps. putida is preferred. The polynucleotide encoding the protein of the present invention may be introduced into the host, for example, via the expression vector of the present invention.

[0153] <Method for Producing Proteins> In another aspect, the present invention provides a method for producing a dimeric protein that can be suitably used in producing a heterodimeric protein, or a monomeric protein that can be suitably used in producing the dimeric protein. The protein production method of the present invention includes an expression step of expressing the nucleic acid of the present invention, the expression vector of the present invention, and / or the expression vector set of the present invention. According to the protein production method of the present invention, the dimeric protein of the present invention, the first monomeric protein, and / or the second monomeric protein can be produced.

[0154] The protein of the present invention may be expressed, for example, using the expression vector of the present invention. The method for expressing the protein of the present invention is not particularly limited, and any known method can be used, for example, a host or a cell-free protein synthesis system.

[0155] In the former case, it is preferable to use a host into which the protein of the present invention or a nucleic acid encoding the protein of the present invention has been introduced, and to express the protein of the present invention in the host by culturing the host. In this way, for example, by introducing a nucleic acid encoding the protein of the present invention into a host, a transformant that synthesizes the protein of the present invention can be produced, and the protein of the present invention can be synthesized by culturing the transformant.

[0156] The method for culturing the host is not particularly limited and can be appropriately determined depending on the type of the host. The medium used for culturing is not particularly limited and can be appropriately determined depending on the type of the host.

[0157] In the latter case, it is preferable to express the polynucleotide of the protein of the present invention in a cell-free protein synthesis system. In this case, an expression vector may be used to express the polynucleotide of the protein of the present invention. The cell-free protein synthesis system can be carried out by a known method using, for example, a cell extract, a buffer containing various components, and an expression vector into which a polynucleotide encoding the protein of the present invention has been introduced, and for example, a commercially available reagent kit can be used.

[0158] The method for producing the protein of the present invention may include, for example, a recovery step of recovering the protein of the present invention. The protein of the present invention obtained in the recovery step may be, for example, a crude product or a purified protein.

[0159] When the protein is recovered from the culture medium, in the recovery step, insoluble matter is removed, for example, by filtering the culture supernatant, centrifuging it, etc. Then, in the recovery step, the culture supernatant from which the insoluble matter has been removed is separated and purified, for example, by an appropriate combination of concentration using an ultrafiltration membrane; salting out such as ammonium sulfate precipitation; dialysis; and chromatography using various columns such as an ion exchange column and a gel filtration column, thereby obtaining the protein of the present invention.

[0160] When the protein is recovered from the transformant, the recovery step involves disrupting the transformant by, for example, pressure treatment, ultrasonic treatment, etc. Then, the resulting disruption solution is subjected to removal of insoluble matter, separation, and purification as described above to obtain the protein of the present invention.

[0161] The protein of the present invention obtained by the production method of the present invention may be used, for example, as a crudely purified protein as is, or as a partially purified protein, or as a single purified protein.

[0162] In the production method of the present invention, the obtained protein of the present invention may be powdered by, for example, freeze-drying, vacuum drying, spray drying, etc. In this case, in the production method of the present invention, for example, the protein of the present invention may be dissolved in advance in a buffer such as acetate buffer, phosphate buffer, triethanolamine buffer, Tris-HCl buffer, or Good's buffer (e.g., HEPES, PIPES, MES, MOPS, etc.).

[0163] <Heterodimeric protein screening method> In another aspect, the present invention provides a method for screening heterodimeric proteins that react with a desired target. The target-reactive heterodimeric protein screening method of the present invention comprises a production step of reacting a dimeric protein containing a reactive tag with a modifying protein that modifies the dimeric protein to produce a candidate heterodimeric protein, a detection step of contacting the candidate heterodimeric protein with a target and detecting the reaction between the candidate heterodimeric protein and the target, and a selection step of selecting the candidate heterodimeric protein whose reaction is detected as a candidate substance that reacts with the target, and the production step is carried out by the production method of the present invention. According to the screening method of the present invention, heterodimeric proteins that react with a desired target can be screened.

[0164] In the production process, the candidate heterodimeric protein can be produced in the same manner as the production method of the present invention. The candidate heterodimeric protein is preferably an antibody such as a multispecific antibody or a bispecific antibody. When the candidate heterodimeric protein is an antibody, the antigen-binding domain of the antibody may be, for example, a candidate antigen-binding domain whose binding to a target as described below is evaluated.

[0165] The candidate heterodimeric protein may contain a label, for example, a fluorescent substance such as a fluorescent protein or a fluorescent dye; an enzyme such as luciferase or phosphatase; or the like.

[0166] Next, in the detection step, the candidate heterodimeric protein is contacted with a target, and the reaction between the candidate heterodimeric protein and the target is detected. In the detection step, the contact is carried out, for example, in the presence of the aqueous solvent. The contact conditions are not particularly limited and can be set, for example, depending on the type of target. The temperature in the detection step is, for example, 4 to 37°C, or 18 to 25°C. The time for the detection step is, for example, 0 to 120 minutes, or 30 to 60 minutes.

[0167] The target can be a desired target for screening the reactivity of the heterodimeric protein. The examples of the target antigen can be used as the target. During the contact, the target may be immobilized on a carrier or may be free. Examples of the carrier include a substrate, beads, and a container. Examples of the container include a microplate and a tube. When the target is a molecule that can be expressed in a cell, the target may be a cell that expresses the target.

[0168] Next, in the detection step, a reaction between the candidate heterodimeric protein and the target is detected. The reaction can be determined depending on the type of the candidate heterodimeric protein and the target. Specifically, when the candidate heterodimeric protein includes an antibody, such as a multispecific antibody, or an antigen-binding fragment thereof, the reaction is, for example, binding. When the candidate heterodimeric protein includes an enzyme, the reaction is, for example, a catalytic reaction. When the candidate heterodimeric protein includes a receptor, the reaction is, for example, binding.

[0169] Then, in the detection step, by detecting the presence or absence of reaction between the candidate heterodimeric protein and the target, for example, the presence or absence of reactivity of the candidate heterodimeric protein to the target can be detected or analyzed (qualitative analysis). In addition, in the detection step, by detecting the degree of reaction between the candidate heterodimeric protein and the target, for example, the strength of the reaction of the candidate heterodimeric protein to the target can be detected or analyzed (quantitative analysis).

[0170] If a reaction between the candidate heterodimeric protein and the target cannot be detected, the candidate heterodimeric protein can be evaluated as not reacting with the target, and if a reaction is detected, the candidate heterodimeric protein can be evaluated as reacting with the target.

[0171] The method for analyzing the reaction between the candidate heterodimeric protein and the target is not particularly limited and can be determined depending on the type of reaction. When the candidate heterodimeric protein contains a label and the reaction is binding, the reaction between the candidate heterodimeric protein and the target can be carried out, for example, by detecting the label of the complex formed by binding between the candidate heterodimeric protein and the target.

[0172] In the selection step, the candidate heterodimeric protein for which the reaction was detected is selected as a candidate substance that reacts with the target. Thus, according to the screening method of the present invention, candidate heterodimeric proteins that are presumed to have reactivity with the target can be screened.

[0173] The present invention will be described in detail below using examples, but the present invention is not limited to the embodiments described in the examples.

[0174] [Example 1] It was confirmed that different adducts can be introduced into the dimeric protein by the production method of the present invention.

[0175] Specifically, in Example 1, a protein containing a portion of the hinge region of a human IgG antibody and the CH2 and CH3 regions of the Fc region was used as a dimeric protein model, and a Fab of a HER2 antibody and a Fab of an OKT3 antibody were used as a first adduct model and a second adduct model, respectively. A first monomer protein, a second monomer protein, a first modified protein, and a second modified protein containing these as parts were then prepared, and it was confirmed that a bispecific antibody capable of binding to both HER2 and CD3 could be produced by introducing the Fab of a HER2 antibody (Herceptin) and the Fab of an OKT3 antibody into a protein containing an Fc region using the production method of the present invention.

[0176] (1) Preparation of Proteins of Each Antibody Fragment of Herceptin-OKT3 Bispecific Antibody To prepare the Herceptin-OKT3 bispecific antibody, the following monomer proteins and modified proteins were prepared. - First monomer protein: a protein comprising SpyTag (binding tag), gp41-1 C intein (first C intein), and the C-terminal region of the hinge region and Fc region of human IgG1 (SpyTag-VHH) - Second monomer protein: a protein comprising SpyCatcher (binding partner), cfa C intein (second C intein), and the C-terminal region of the hinge region and Fc region of human IgG1 (SpyCatcher-VHH) - First modified protein: (CD3-Fab) a complex of a protein comprising the VL and CL regions of hOKT3 antibody (CD3-Fab-L) and a protein comprising the VH and CH1 regions of hOKT3 antibody and the N-terminal region of the hinge region of human IgG1, and go41-1 N intein (first N intein) (CD3-Fab-H) Second modified protein (Her2-Fab): A complex of a protein (Her2-Fab-L) containing the VL and CL regions of Herceptin antibody and a protein (Her2-Fab-C) containing the VH and CH1 regions of Herceptin antibody, the N-terminal region of the hinge region of human IgG1, and a cfa N intein (second N intein).

[0177] A plasmid vector capable of expressing CD3-Fab-H was constructed as follows. First, a synthetic gene (Eurofins Genomics) containing a nucleotide sequence encoding a signal peptide, the OKT3 VH-CH1 domain (SEQ ID NO: 12), a partial sequence of the N-terminal side of the hinge region of human IgG1, the gp41-1 N intein (SEQ ID NO: 7), a G1 linker, and a His tag was amplified by PCR. The resulting full-length synthetic gene was ligated into an animal cell expression vector (pCAGEN) to construct an expression vector for the recombinant protein. The expression vector contains, from the N-terminus to the C-terminus, the signal peptide, the OKT3 VH-CH1 domain, a partial sequence of the N-terminal side of the hinge region, the gp41-1 N intein, the G1 linker, and the His tag, as shown in brackets.

[0178] CD3-Fab-H region (SEQ ID NO: 13) [MEFGLSWLFLVAILKGVQC][QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYSLDYWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC][DKT][SGYCLDLKTQVQTPQGMKEISNIQVGDLVLSNTGYNEVLNVFPKSKKKSYKITLEDGKEIICSEEHLFPTQTGEMNISGGLKEGMCLYVKE][GGSGG][HHHHHH]

[0179] Next, a plasmid vector capable of expressing CD3-Fab-L was constructed as follows. First, a synthetic gene (Eurofins Genomics) containing a nucleotide sequence encoding the signal peptide and the VL-CL domain of OKT3 (SEQ ID NO: 14) was amplified by PCR. The resulting full-length synthetic gene was ligated into an animal cell expression vector (pCAGEN) to construct an expression vector for the recombinant protein. In this expression vector, the signal peptide and the VL-CL domain of OKT3 are ligated in this order from the N-terminus to the C-terminus as the CD3-Fab-L region (SEQ ID NO: 15), as shown in parentheses.

[0180] CD3-Fab-L region (SEQ ID NO: 15) [MDFQVQIFSFLLISASVIISRG][DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTVA][APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC]

[0181] A plasmid vector capable of expressing Her2-Fab-H was constructed as follows. First, a synthetic gene (Eurofins Genomics) containing a nucleotide sequence encoding a signal peptide, the Herceptin VH-CH1 domain (SEQ ID NO: 16), the N-terminal partial sequence of the hinge region of human IgG1, the Cfa N intein (SEQ ID NO: 9), a G1 linker, and a His tag was amplified by PCR. The resulting full-length synthetic gene was ligated into an animal cell expression vector (pCAGEN) to construct an expression vector for the recombinant protein. The expression vector contains the Her2-Fab-H region (SEQ ID NO: 17), which contains, from the N-terminus to the C-terminus, the signal peptide, the Herceptin VH-CH1 domain, the N-terminal partial sequence of the hinge region, the Cfa N intein, the G1 linker, and the His tag, as shown in parentheses.

[0182] Her2-Fab-H region (SEQ ID NO: 17) [MEGFLSWLFLVAILKGVQC][EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC][DKT][CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP][GGSGG][HHHHHH]

[0183] Next, a plasmid vector capable of expressing Her2-Fab-L was constructed as follows. First, a synthetic gene (Eurofins Genomics) containing a nucleotide sequence encoding the signal peptide and the Herceptin VL-CL domain (SEQ ID NO: 18) was amplified in full length by PCR. The resulting full-length synthetic gene was ligated into an animal cell expression vector (pCAGEN) to construct an expression vector for the recombinant protein. In this expression vector, the signal peptide and the Herceptin VL-CL domain are ligated in this order from the N-terminus to the C-terminus as the Her2-Fab-L region (SEQ ID NO: 19), as shown in parentheses.

[0184] Her2-Fab-L region (SEQ ID NO: 19) [METPAQLLFLLLLWLPESTG][DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPT FGQGTKVEI][KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC]

[0185] A plasmid vector capable of expressing SpyCatcher-VHH was constructed as follows. First, a full-length synthetic gene (Eurofins Genomics) containing a nucleotide sequence encoding a signal peptide, Ia1 (single-chain antibody (soluble domain), SEQ ID NO: 20), G1 linker, SpyCatcher (SEQ ID NO: 1), G1 linker, Cfa C intein (SEQ ID NO: 8), a C-terminal partial sequence of the hinge region of human IgG1, and the CH2 and CH3 regions of the Fc region of human IgG (SEQ ID NO: 21) was amplified by PCR. The resulting full-length synthetic gene was ligated into an animal cell expression vector (pCDNA3.4) to construct a recombinant protein expression vector. In the expression vector, the SpyCatcher VHH region (SEQ ID NO: 22) has, from the N-terminus to the C-terminus, as shown in parentheses, a signal peptide, Ia1, a G1 linker, SpyCatcher, a G1 linker, a Cfa C intein, a C-terminal partial sequence of the hinge region of human IgG1, and the CH2 and CH3 regions of the Fc region of human IgG linked in this order.

[0186] Fc region (SEQ ID NO: 21) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0187] Ia1 (SEQ ID NO: 20) QVQLQESGGGLVQAGGSLLLSCAASGRTFSSYAMGWFRQAPGKEREFVAAINWSGGSTSYADSVKGRFTISRDNTKNTVYLQMNSLKPEDTAAFYCAATYNPYSRDHYFPRMTTEYDYWGQGTQVTVSS

[0188] SpyCatcher-VHH region (SEQ ID NO: 22) [MEFGLSWLFLVAILKGVQC][QVQLQESGGGLVQAGGSLLLSCAASGRTFSSYAMGWFRQAPGKEREFVAAINWSGGSTSYADSVKGRFTISRDNTKNTVYLQMNSLKPEDTAAFYCAATYNPYSRDHY FPRMTTEYDYWGQGTQVTVSS][GGSGG][DSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG][GGSGG][VKIISRKS LGTQNVYDIGVEKDHNFLLKNGLVASNC][FNASYTCPPCP][APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK]

[0189] A plasmid vector capable of expressing SpyTag-VHH was constructed as follows. First, a full-length synthetic gene (Eurofins Genomics) containing a signal peptide, cAbGFP4 (single-chain antibody (soluble domain), SEQ ID NO: 23), G1 linker, SpyTag (SEQ ID NO: 2), gp41-1 C intein (SEQ ID NO: 6), a partial sequence of the C-terminal side of the hinge region of human IgG1, and sequences encoding the CH2 and CH3 regions of the Fc region of human IgG was amplified by PCR. The resulting full-length synthetic gene was ligated into an animal cell expression vector (pCDNA3.4) to construct an expression vector for the recombinant protein. In the expression vector, a SpyTag-VHH region (SEQ ID NO: 24) is formed, from the N-terminus to the C-terminus, of a signal peptide, cAbGFP4, a G1 linker, SpyTag, gp41-1 C intein, a C-terminal partial sequence of the hinge region of human IgG1, and the CH2 and CH3 regions of the Fc region of human IgG, linked in this order as shown in brackets.

[0190] cAbGFP4 (SEQ ID NO: 23) QVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS

[0191] SpyTag-VHH region (SEQ ID NO: 24) [MEFGLSWLFLVAILKGVQC][QVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS][GGSGG][AHIVMVDAYKPTK][GGSGG][MMLKKILKIEELDERELIDIEVSGNHLFYANDILTHNSAG][ASYTCPPCP ][APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK]

[0192] Next, each of the recombinant protein plasmid expression vectors was transfected into Expi293F cells. The cells were transfected with a combination of the CD3-Fab-H expression vector and the CD3-Fab-L expression vector, a combination of the Her2-Fab-H expression vector and the Her2-Fab-L expression vector, or a combination of the SpyCatcher-VHH expression vector and the SpyTag-VHH expression vector. The resulting transformants were cultured in HE400 medium at 37°C in a CO2 incubator for 7 days, and the recombinant proteins (SpyTag-VHH + SpyCatcher-VHH (Fc dimer), CD3-Fab-L + CD3-Fab-H (CD3-Fab), and Her2-Fab-L + Her2-Fab-H (Her2-Fab)) were expressed in the culture supernatant.

[0193] Next, after the culture, the culture supernatant containing the recombinant protein was collected.

[0194] (2) Purification of CD3-Fab and Her2-Fab. The culture supernatant was centrifuged at 1500 rpm for 5 minutes. The supernatant was mixed with reagent solution to a final concentration of 500 mmol / L NaCl and 10 mmol / L imidazole. The mixture was then loaded onto a 3 ml Ni-NTA column (FUJIFILM) equilibrated with wash buffer (500 mmol / L NaCl, 50 mmol / L Tris-HCl, 10 mmol / L imidazole). After washing with 30 ml of wash buffer, the column was eluted with 15 ml of elute buffer (500 mmol / L NaCl, 50 mmol / L Tris-HCl, 500 mmol / L imidazole). The collected eluate fraction was dialyzed against dialysis buffer (150 mmol / L NaCl, 50 mmol / L HEPES).

[0195] (3) Purification of Fc dimers. The culture supernatant was centrifuged at 1500 rpm for 5 minutes. It was loaded onto a 1 ml Protein A column (SUPrA) equilibrated with Wash Buffer (50 mmol / L sodium phosphate buffer, pH 7.5). After washing with 10 ml of Wash Buffer, it was eluted with 5 ml of Elute Buffer (20 mmol / L sodium citrate, 100 mmol / L NaCl, pH 3.0). To neutralize the Elute Buffer, 5 ml of 100 mmol / L sodium phosphate buffer (pH 7.5) was added. The collected eluted fraction was dialyzed against dialysis buffer (150 mmol / L NaCl, 50 mmol / L HEPES).

[0196] Each of the obtained protein samples was diluted with ultrapure water, and the absorbance at 280 nm and 330 nm was measured using an ultraviolet-visible spectrophotometer (UV-1800, manufactured by Shimadzu Corporation). The molar concentration of the protein in each protein sample was calculated using the following formula (1): C = (A280 - A320) ÷ (l × ε) × (dilution factor) (1), where C is the molar concentration of the protein, A280 is the absorbance at 280 nm, A320 is the absorbance at 320 nm, l is the cell length, and ε is the molar extinction coefficient of the protein.

[0197] (4) Preparation of Bispecific Antibodies Herceptin-OKT3 bispecific antibodies were prepared using the SpyTag-VHH+SpyCatcher-VHH (Fc dimer), CD3-Fab, and Her2-Fab obtained in Example 1(1). Specifically, Herceptin-Okt3 bispecific antibodies were obtained by PTS reaction using SpyTag-VHH+SpyCatcher-VHH, CD3-Fab, and Her2-Fab. 2 μL of protein containing each antibody fragment obtained in Examples 1(1) and 1(2) (dissolved in 150 mmol / L NaCl, 50 mmol / L HEPES (pH 7.6), and 1 mmol / L TCEP) was added to a microtube to prepare a pre-PTS reaction sample. After preparation, the pre-PTS reaction sample was allowed to stand in an incubator at 37°C for 24 hours, allowing the PTS reaction to occur. After the PTS reaction, a post-PTS reaction sample was obtained.

[0198] (5) Examination of Bispecific Antibody Binding by Binding of Each Antibody Fragment The post-PTS reaction samples obtained in Example 1(4) were examined by SDS-PAGE to determine whether Herceptin-OKT3 bispecific antibodies had been produced. Specifically, 10 μl of 5×SDS buffer was added to 40 μl of the post-PTS reaction sample, Fc region (complex of SpyTag-VHH and SpyCatcher-VHH), Herceptin-Fab (Fab composed of Her2-Fab-L and Her2-Fab-H, Herceptin), CD3-Fab (Fab composed of CD3-Fab-L and CD3-Fab-H, hOKT3), or pre-PTS reaction sample, and the suspension was then heated at 95°C for 5 minutes. After the heat treatment, the mixture was applied to the wells of a 12.5% ​​polyacrylamide gel and electrophoresis was performed at 150-200 V. The Fc region, Herceptin, hOKT3, pre-PTS reaction samples, post-PTS reaction samples, and markers were applied to the 12.5% ​​polyacrylamide gel. After electrophoresis, the polyacrylamide gel was stained with CBB (Coomassie Brilliant Blue) staining solution for 5 minutes and then destained with a destaining solution. The results are shown in Figure 2.

[0199] Figure 2 is a photograph showing each antibody fragment and protein before and after the PTS reaction. In Figure 2, the top of the photograph indicates the type of sample, and the right side of the photograph indicates the molecular weight (kDa). In Figure 2, the lanes, from left to right, show the Fc region, Herceptin, hOKT3, the sample before the PTS reaction (0 hours of reaction), the sample after the PTS reaction (24 hours of reaction), and the marker. As shown in Figure 2, bands representing the four proteins that constitute each antibody were detected in the post-PTS reaction sample. These results indicated that the post-PTS reaction sample contained Herceptin-OKT3 bispecific antibodies produced by the PTS reaction.

[0200] (6) Purification of Bispecific Antibodies Next, the PTS reaction sample obtained in Example 1(4) above was purified. Specifically, it was purified using a column for purifying proteins having an Fc region, and then using a column for purifying proteins having an Fab region. First, the protein having an Fc region was purified using a KanCapA column. A KanCapA column (manufactured by KANEKA Corporation, column volume 500 μl) was equilibrated with TBS buffer (150 mmol / l NaCl and 50 mmol / l Tris-HCl (pH 7.5)), and the PTS reaction sample obtained in Example 1(4) above was loaded onto the column, and the flow-through fraction was collected. After the collection, 20 ml of TBS buffer was loaded, and the wash fraction was collected. Further, 2.8 ml of Elute buffer (100 mmol / L Glycine-HCl (pH 2.8)) was loaded, and the eluted fraction was collected. The eluted fraction was neutralized by pre-loading the collection tube with 1.2 ml of 1 mol / L Tris-HCl (pH 9.0). The collected eluted fraction was then placed in a transparent membrane (semipermeable membrane) and dialyzed using dialysis buffer (1x PBS). Next, proteins having Fab regions were purified from the dialyzed protein sample. A KanCapL column (manufactured by KANEKA, column volume 500 μl) was equilibrated with 1x PBS, and the dialyzed protein sample was loaded, and the flow-through fraction was collected. After the collection, 10 ml of 1x PBS was passed through, and the wash fraction was collected. Next, 7 ml of Elute buffer (100 mmol / L Glycine-HCl (pH 2.8)) was loaded, and the eluted fraction was collected. In order to neutralize the eluted fraction, 3 ml of 1 mol / L Tris-HCl (pH 9.0) was added to the collection tube beforehand. The collected eluted fraction was then placed in a transparent membrane (semipermeable membrane) and dialyzed using the dialysis buffer (1×PBS). After the dialysis, the fraction was concentrated using a concentration tube. A purified sample was obtained by the concentration.

[0201] (7) Investigation of Purified Bispecific Antibody The purified sample obtained in Example 1(6) above was investigated using SDS-PAGE to determine whether it contained Herceptin-Okt3 bispecific antibody. Specifically, 10 μl of 5×SDS buffer was added to the PTS reaction sample, the elution fraction, flow-through fraction, or wash fraction (40 μl each) purified using the KanCap A column, and the purified sample purified using the KanCap L column, and the suspension was then added. After suspension, the suspension was heated at 95°C for 5 minutes. After the heat treatment, the suspension was applied to the wells of a 12.5% ​​or 10% polyacrylamide gel and electrophoresed at 150-200 V. The marker, PTS reaction sample, flow-through fraction, wash fraction, and elution fraction were applied to the 12.5% ​​polyacrylamide gel. The purified sample was applied to the 10% acrylamide gel. After electrophoresis, the polyacrylamide gel was stained with Coomassie Brilliant Blue (CBB) staining solution for 5 minutes and then destained with a destaining solution. The results are shown in Figure 3.

[0202] 3A and 3B are photographs showing proteins after PTS reaction and column purification. In FIG. 3A, (A) shows photographs of proteins before and after purification using a KanCapA column, and FIG. 3B shows photographs of proteins in the sample after purification using a KanCapL column. In FIG. 3A, the top of the photograph indicates the type of sample, and the left side of the photograph indicates the molecular weight (kDa). In FIG. 3B, the top of the photograph indicates the type of sample, and the left side of the photograph indicates the molecular weight (kDa). In FIG. 3A, the lanes indicate, from left to right, a marker, a post-PTS reaction sample (24-hour reaction), a flow-through fraction (flow-through), a wash fraction (wash), and an elution fraction (elution). As shown in FIG. 3A, bands for each antibody fragment of interest were detected in the elution fraction, but bands for contaminating proteins were also detected. On the other hand, as shown in Figure 3(B), in the sample purified using the KanCapL column, bands corresponding to each antibody fragment of interest were detected, but no bands corresponding to contaminating proteins were detected. These results demonstrate that the desired Herceptin-Okt3 bispecific antibody can be purified by performing purification using two columns.

[0203] (8) Examination of the homogeneity of the purified bispecific antibody The Herceptin-Okt3 bispecific antibody purified in Example 1(6) above was examined using gel filtration chromatography to determine whether the Herceptin-Okt3 bispecific antibody was homogeneous. Specifically, the protein purified in Example 1(6) above was passed through a gel filtration chromatography column (Superdex 200 increase 30 / 100 GL, GE Healthcare) at 0.5 mL / min, and the absorbance at 212 nm was measured at room temperature (approximately 25°C). 1x PBS was used as the buffer in the measurement. The results are shown in Figure 4.

[0204] Figure 4 is a graph showing the elution pattern of a protein obtained by gel filtration chromatography. In Figure 4, the horizontal axis represents excluded volume (ml) and the vertical axis represents absorbance. As shown in Figure 4, the purified protein obtained by the method of the present disclosure exhibited a single peak at 15 ml at an absorption wavelength of 212 nm. These results demonstrate that the purified Herceptin-Okt3 bispecific antibody obtained by the method of the present disclosure has a single identity.

[0205] (9) Evaluation of Activity on CD3-Positive Cells The Herceptin-Okt3 bispecific antibody obtained in Example 1(6) was examined by flow cytometry for binding to cells expressing Her2 or CD3. Specifically, an HPB-ALL strain overexpressing CD3 on the cell membrane surface was used, and measurements were performed using a flow cytometer. Equal amounts of HPB-ALL cultured in a T75 flask were transferred to two 15 ml Falcon tubes and centrifuged at 1500 rpm for 5 minutes. After centrifugation, the supernatant was aspirated and removed, and the required amount of 1x PBS was added to suspend the cells. 1x10 ６A cell suspension of 1000 cells / ml was obtained. Subsequently, 1 ml of the suspension was dispensed into each of three microtubes (a) to (c) and diluted with 1x PBS. After dilution, two microtubes (a) and (b) were centrifuged at 2000 rpm, 25°C, and 7 minutes. After centrifugation, the supernatant was aspirated and removed using an aspirator, and 1 ml of 1x PBS was added. After the addition, two microtubes (a) and (b) were centrifuged again at 2000 rpm, 25°C, and 7 minutes. After centrifugation, the supernatant was aspirated and removed using an aspirator. 1x PBS and the Herceptin-Okt3 bispecific antibody purified in Example 1(6) were added to one microtube (b) so that the final concentration of the Herceptin-Okt3 bispecific antibody was 0.05 μmol / L, and the mixture was mixed by inversion. After mixing by inversion, the mixture was allowed to stand for 20 minutes. After standing, the mixture was centrifuged at 2000 rpm, 25°C, and 7 minutes. Then, 1 μl of OKT3-FITC (Cosmo Bio) and 1 ml of 1×PBS were added to microtube (a), and 1 μl of anti-Fc-FITC (AbCam) and 1 ml of 1×PBS were added to microtube (b), followed by inversion mixing. After inversion mixing, the mixture was left standing for 20 minutes. After standing, the mixture was centrifuged at 2000 rpm, 25°C, and 7 minutes. After centrifugation, the supernatant was aspirated and removed, and 1 ml of 1×PBS was added. After addition, the mixture was centrifuged again at 2000 rpm, 25°C, and 7 minutes. After centrifugation, the supernatant was aspirated and removed, and 1 ml of 1×PBS was added and suspended. After suspension, the mixture was sterilized using a mesh filter. After setting the measurement conditions, measurements were performed using a cell analyzer RF-500 (Sysmex Corporation) in the order of negative control (c), positive control (a), and sample (b). After the measurements, the measurement results were graphed using an FCSalyzer. These results are shown in Figure 5.

[0206] Figure 5 is a graph showing the binding of bispecific antibodies to CD3-positive cells by flow cytometry. In Figure 5, the horizontal axis represents fluorescence intensity, and the vertical axis represents cell count. As shown in Figure 5, when the Herceptin-Okt3 bispecific antibody obtained by the method of the present disclosure was added to CD3-positive cells, an increase in fluorescence intensity was observed compared to when the Herceptin-Okt3 bispecific antibody was not added to CD3-positive cells. These results demonstrate that the Herceptin-Okt3 bispecific antibody obtained by the production method of the present invention binds to CD3-positive cells.

[0207] (10) Evaluation of Activity on HER2-Positive Cells The Herceptin-Okt3 bispecific antibody obtained in Example 1(6) was examined by flow cytometry for its binding to breast cancer cells. Specifically, the SK-BR-3 cell line, in which HER2 is overexpressed on the cell membrane surface, was used, and measurements were performed using a flow cytometer. The supernatant of SK-BR-3 cells cultured in a T75 flask was aspirated and washed with 5 ml of 1xPBS / 5 mmol / L EDTA. After washing, the supernatant was aspirated and 5 ml of trypsin was added. The cells were then allowed to stand in an incubator at 37°C for 5 minutes. After standing, the flask was gently tapped. After tapping, DMEM medium was added to a total volume of 10 ml, and the cells were detached from the flask by suspension to obtain a cell suspension. Next, the cell suspension was transferred to a 15 ml Falcon tube and centrifuged at 1500 rpm for 5 minutes. After centrifugation, the supernatant was aspirated using an aspirator, and the required amount of 1x PBS was added and suspended to obtain a cell suspension. Then, 1x10 cells were added to three microtubes (d) to (f). ６1 ml of each cell suspension was taken and diluted with 1x PBS. After dilution, two microtubes (d) and (e) were centrifuged at 2000 rpm, 25°C, and 7 minutes. After centrifugation, the supernatant was aspirated and removed using an aspirator, and 1 ml of 1x PBS was added. After the addition, two microtubes (d) and (e) were centrifuged again at 2000 rpm, 25°C, and 7 minutes. After centrifugation, the supernatant was aspirated and removed using an aspirator. After the aspirated removal, 1 μl of 5 mg / ml Herceptin (Chugai Pharmaceutical Co., Ltd.) and 1 ml of 1x PBS were added to one microtube (d) and mixed by inversion. In parallel, 1 ml of 1x PBS and the Herceptin-Okt3 bispecific antibody purified in Example 1(6) were added to one microtube (e) so that the final concentration of the Herceptin-Okt3 bispecific antibody was 0.1 μmol / L, and the mixture was mixed by inversion. After mixing by inversion, the two microtubes (d) and (e) were allowed to stand for 20 minutes. After standing, the two microtubes (d) and (e) were centrifuged at 2000 rpm, 25°C, and 7 minutes. After centrifugation, the supernatant was aspirated and 1 ml of 1x PBS was added. After the addition, the two microtubes (d) and (e) were centrifuged again at 2000 rpm, 25°C, and 7 minutes. After the centrifugation, the supernatant was aspirated and removed. 1 μl of anti-Fc-FITC (AbCam) and 1 ml of 1×PBS were added to two microtubes (d) and (e), and the mixture was mixed by inversion. After the inversion, the mixture was allowed to stand for 20 minutes. After the standing, the two microtubes were centrifuged at 2000 rpm, 25°C, and 7 minutes. After the centrifugation, the supernatant was aspirated and removed, and 1 ml of 1×PBS was added. After the addition, the two microtubes (d) and (e) were centrifuged again at 2000 rpm, 25°C, and 7 minutes. After the centrifugation, the supernatant was aspirated and removed, and 1 ml of 1×PBS was added and suspended. After the suspension, the mixture was sterilized using a mesh filter. After setting the measurement conditions, measurements were carried out in the order of negative control (f), positive control (d), and sample (e) using a cell analyzer RF-500 (manufactured by Sysmex Corporation).After the measurements, the results were graphed using FCSalyzer. These results are shown in Figure 6.

[0208] Figure 6 is a graph showing the binding of bispecific antibodies to HER2-positive cells by flow cytometry. In Figure 6, the horizontal axis represents fluorescence intensity, and the vertical axis represents cell number. As shown in Figure 6, when the Herceptin-Okt3 bispecific antibody obtained by the production method of the present invention was added to HER2-positive cells, an increase in fluorescence intensity was observed compared to when the Herceptin-Okt3 bispecific antibody was not added to HER2-positive cells. These results demonstrate that the Herceptin-Okt3 bispecific antibody obtained by the method of the present disclosure binds to Her2-positive breast cancer cells.

[0209] (11) Evaluation of Maintenance of Activity on HER2-Positive Cells by Surface Plasmon Resonance The Herceptin-Okt3 bispecific antibody obtained in Example 1(6) above was examined using surface plasmon resonance to determine whether it could maintain binding to Her2. Specifically, the Herceptin-Okt3 bispecific antibody was passed through HER2 immobilized on a sensor chip at a predetermined flow rate, and measurements were performed using a molecular interaction analyzer. The Herceptin-Okt3 bispecific antibody purified in Example 1(6) above was diluted with 1×PBS-Tween 20 to concentrations of 1 nmol / L, 2 nmol / L, 4 nmol / L, 8 nmol / L, and 16 nmol / L. After the dilution, the binding strength was measured by passing the diluted solution containing the Herceptin-Okt3 bispecific antibody over HER2-hFc immobilized on a CM5 sensor chip (GE Healthcare) using a surface plasmon resonance measurement device (Biacore T200, Cytiva). The results are shown in Figure 7.

[0210] 7 is a graph showing the maintenance of binding of bispecific antibodies to HER2 by surface plasmon resonance. In FIG. 7, the horizontal axis represents time (minutes) and the vertical axis represents response units (RU). As shown in FIG. 7, when the Herceptin-Okt3 bispecific antibody obtained by the method of the present disclosure was added to HER2, the dissociation equilibrium constant Kd value was 2.25×10 －９These results demonstrate that the Herceptin-Okt3 bispecific antibody obtained by the production method of the present invention maintains its binding to HER2.

[0211] Although the present invention has been described above with reference to the embodiments, the present invention is not limited to the above embodiments. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

[0212] The patents, patent applications, and publications cited herein are incorporated by reference into this specification in their entirety as if the contents were specifically set forth herein.

[0213] <Supplementary Notes> Some or all of the above embodiments and examples can be described as in the following supplementary notes, but are not limited to the following. <Method for Producing a Heterodimeric Protein> (Supplementary Note 1) A method for producing a heterodimeric protein, comprising: a production step of producing a heterodimeric protein by reacting a dimeric protein comprising a reactive tag with a modifying protein that modifies the dimeric protein, wherein the dimeric protein comprising the reactive tag comprises a first monomeric protein and a second monomeric protein, wherein the first monomeric protein comprises, in this order, a first reactive tag and a first dimer-forming domain capable of forming a dimer, wherein the first reactive tag comprises a binding tag and a first C intein, wherein the second monomeric protein comprises, in this order, a second reactive tag and a second dimer-forming domain capable of forming a dimer with the first dimer-forming domain, wherein the second reactive tag comprises a binding partner capable of binding to the binding tag and a second C intein, and wherein the modifying protein comprises a first modifying protein and a second modifying protein, the first modified protein comprises a first N intein capable of reacting with the first C intein and a first adduct added to the first monomeric protein; the second modified protein comprises a second N intein capable of reacting with the second C intein and a second adduct added to the second monomeric protein; the first adduct and the second adduct are different adducts; the first monomeric protein and the second monomeric protein form a dimer; and in the production process, the first N intein of the first modified protein reacts with the first C intein of the first monomeric protein to link the first adduct of the first modified protein to the first monomeric protein; and the second N intein of the second modified protein reacts with the second C intein of the second monomeric protein to link the second adduct of the second modified protein to the second monomeric protein.(Appendix 2) The production method according to Appendix 1, comprising a step of reacting the first monomeric protein with the second monomeric protein to form a dimer. (Appendix 3) The production method according to Appendix 1 or 2, wherein the binding tag and the binding partner are a peptide tag and peptide capable of spontaneously forming a covalent bond. (Appendix 4) The production method according to any of Appendixes 1 to 3, wherein the bond between the binding tag and the binding partner is a covalent bond. (Appendix 5) The production method according to any of Appendixes 1 to 4, wherein the binding tag and the binding partner are a Streptococcus pyogenes surface protein (SpyCatcher) and a peptide tag (SpyTag) capable of binding to the SpyCatcher, respectively, or the binding tag and the binding partner are the SpyTag and the SpyCatcher, respectively. (Appendix 6) The production method according to any of Appendixes 1 to 5, wherein the dimeric protein comprises a part or all of the Fc region of an immunoglobulin. (Supplementary Note 7) The method for producing according to Supplementary Note 6, wherein the immunoglobulin is IgG, IgA, IgE, IgD, or IgM. (Supplementary Note 8) The method for producing according to Supplementary Note 7, wherein the IgG is IgG1, IgG2, IgG2a, IgG2b, IgG3, or IgG4. (Supplementary Note 9) The method for producing according to any of Supplements 6 to 8, wherein the Fc region of the immunoglobulin does not contain a mutation. (Supplementary Note 10) The method for producing according to any of Supplements 1 to 9, wherein the first C intein and the first N intein are the C intein and N intein of an integrated intein or a split intein, respectively, and / or the second C intein and the second N intein are the C intein and N intein of an integrated intein or a split intein, respectively. (Appendix 11) The method of any one of Appendices 1 to 10, wherein the first adduct and / or the second adduct is a protein. (Appendix 12) The method of Appendices 11, wherein the protein is selected from the group consisting of a modified antibody, a heavy chain antibody (VHH), a receptor, a cytokine, a chemokine, a toxin, an enzyme, a fluorescent protein, and a collagen-binding domain.(Appendix 13) The method of production according to Appendix 12, wherein the antibody variant is selected from the group consisting of Fab, single-chain antibody (scFv), tandem scFv, heavy-chain antibody (VHH), and the variable domain of the VHH. (Appendix 14) The method of production according to any of Appendices 1 to 13, wherein the dimeric protein comprises a part or all of an immunoglobulin Fc region, the first adduct and the second adduct comprise an antibody variant and / or a heavy-chain antibody (VHH), and the heterodimeric protein is a multispecific antibody. (Appendix 15) The method of production according to any of Appendices 1 to 14, wherein in the dimeric protein, the first monomeric protein and the second monomeric protein are linked by a disulfide bond. <Dimeric Protein> (Appendix 16) A dimeric protein comprising a reactive tag, wherein the dimeric protein comprising the reactive tag comprises a first monomeric protein and a second monomeric protein, wherein the first monomeric protein comprises a first reactive tag and a first dimer-forming domain capable of forming a dimer, wherein the first reactive tag comprises a binding tag and a first C intein capable of reacting with a first N intein, wherein the second monomeric protein comprises, in this order, a second reactive tag and a second dimer-forming domain capable of forming a dimer with the first dimer-forming domain, wherein the second reactive tag comprises a binding partner capable of binding to the binding tag and a second C intein capable of reacting with a second N intein, wherein the first monomeric protein and the second monomeric protein form a dimer. (Appendix 17) The protein according to Appendix 16, wherein the binding tag and the binding partner are a peptide tag and a peptide capable of spontaneously forming a covalent bond. (Appendix 18) The protein of appendix 16 or 17, wherein the bond between the binding tag and the binding partner is a covalent bond.(Supplementary Note 19) The protein according to any one of Supplementary Notes 16 to 18, wherein the binding tag and the binding partner are a Streptococcus pyogenes surface protein (SpyCatcher) and a peptide tag (SpyTag) capable of binding to the SpyCatcher, respectively; or the binding tag and the binding partner are the SpyTag and the SpyCatcher, respectively. (Supplementary Note 20) The protein according to any one of Supplementary Notes 16 to 19, wherein the dimeric protein comprises a part or all of an Fc region of an immunoglobulin. (Supplementary Note 21) The protein according to Supplementary Note 20, wherein the immunoglobulin is IgG, IgA, IgE, IgD, or IgM. (Supplementary Note 22) The protein according to Supplementary Note 21, wherein the IgG is IgG1, IgG2, IgG2a, IgG2b, IgG3, or IgG4. (Supplementary Note 23) The protein of any of Supplementary Notes 20 to 22, wherein the Fc region of the immunoglobulin does not contain a mutation. (Supplementary Note 24) The protein of any of Supplementary Notes 16 to 23, wherein the first C intein and the first N intein are the C intein and N intein of an integrated intein or a split intein, respectively, and / or the second C intein and the second N intein are the C intein and N intein of an integrated intein or a split intein, respectively. <Monomer Protein> (Appendix 25) A protein comprising a first monomer protein, wherein the first monomer protein comprises, in this order, a first reactive tag and a first dimerization domain capable of forming a dimer, the first reactive tag comprising a binding tag capable of binding to a binding partner and a first C intein capable of reacting with a first N intein, the first monomer protein capable of forming a dimer with a second monomer protein, the second monomer protein comprising a second reactive tag and a second dimerization domain capable of forming a dimer with the first dimerization domain, and the second reactive tag comprising a binding partner capable of binding to the binding tag and a second C intein capable of reacting with a second N intein.(Supplementary Note 26) A protein comprising a second monomeric protein, wherein the second monomeric protein comprises, in this order, a second reactive tag and a second dimerization domain capable of forming a dimer, the second reactive tag comprising a binding partner capable of binding to a binding tag and a second C intein capable of reacting with a second N intein, the second monomeric protein capable of forming a dimer with a first monomeric protein, the first monomeric protein comprising, in this order, a first dimerization domain capable of forming a dimer with a first reactive tag and the second dimerization domain, and the first reactive tag comprising a binding tag capable of binding to the binding partner and a first C intein capable of reacting with a first N intein. (Supplementary Note 27) A protein according to Supplementary Note 25 or 26, wherein the binding tag and the binding partner are a peptide tag and peptide capable of spontaneously forming a covalent bond. (Appendix 28) The protein according to any one of Appendices 25 to 27, wherein the bond between the binding tag and the binding partner is a covalent bond. (Appendix 29) The protein according to any one of Appendices 25 to 28, wherein the binding tag and the binding partner are a Streptococcus pyogenes surface protein (SpyCatcher) and a peptide tag (SpyTag) capable of binding to the SpyCatcher, respectively, or the binding tag and the binding partner are the SpyTag and the SpyCatcher, respectively. (Appendix 30) The protein according to any one of Appendices 25 to 29, wherein the dimeric protein comprises part or all of an Fc region of an immunoglobulin. (Appendix 31) The protein according to Appendice 30, wherein the immunoglobulin is IgG, IgA, IgE, IgD, or IgM. (Appendix 32) The protein of appendix 31, wherein the IgG is IgG1, IgG2, IgG2a, IgG2b, IgG3, or IgG4. (Appendix 33) The protein of any of appendices 30 to 32, wherein part or all of the Fc region of the immunoglobulin does not contain a mutation.(Appendix 34) The protein according to any one of Appendices 25 to 33, wherein the first C intein and the first N intein are the C intein and N intein of an integrated intein or a split intein, respectively, and / or the second C intein and the second N intein are the C intein and N intein of an integrated intein or a split intein, respectively. <Nucleic Acid> (Appendix 35) A nucleic acid encoding a protein according to any one of Appendices 25 and 27 to 34. (Appendix 36) A nucleic acid encoding a protein according to any one of Appendices 26 to 34. <Expression Vector> (Appendix 37) An expression vector comprising the nucleic acid according to Appendix 35 and / or the nucleic acid according to Appendix 36. (Appendix 38) The expression vector according to Appendix 37, comprising a nucleic acid encoding a first modified protein and / or a nucleic acid encoding a second modified protein, wherein the first modified protein comprises a first N intein capable of reacting with the first C intein and a first adduct added to the first monomeric protein, and the second modified protein comprises a second N intein capable of reacting with the second C intein and a second adduct added to the second monomeric protein, and the first adduct and the second adduct are different adducts. (Appendix 39) The expression vector according to Appendix 38, wherein the first adduct and / or the second adduct is a protein. (Appendix 40) The expression vector according to Appendix 39, wherein the protein is selected from the group consisting of a modified antibody, a heavy-chain antibody (VHH), a receptor, a cytokine, a chemokine, a toxin, a fluorescent protein, and a collagen-binding domain. (Appendix 41) The expression vector according to Appendix 40, wherein the modified antibody is selected from the group consisting of Fab, single-chain antibody (scFv), tandem scFv, heavy-chain antibody (VHH), and variable domain of the VHH. (Appendix 42) An expression vector set comprising an expression vector comprising the nucleic acid according to Appendix 35 and an expression vector comprising the nucleic acid according to Appendix 36.(Appendix 43) The expression vector set according to Appendix 42, comprising an expression vector containing a nucleic acid encoding a first modified protein and / or an expression vector containing a nucleic acid encoding a second modified protein, wherein the first modified protein comprises a first N intein capable of reacting with the first C intein and a first adduct added to the first monomeric protein, and the second modified protein comprises a second N intein capable of reacting with the second C intein and a second adduct added to the second monomeric protein, and the first adduct and the second adduct are different adducts. (Appendix 44) The expression vector set according to Appendix 43, wherein the first adduct and / or the second adduct is a protein. (Appendix 45) The expression vector set according to Appendix 44, wherein the protein is selected from the group consisting of a modified antibody, a heavy-chain antibody (VHH), a receptor, a cytokine, a chemokine, a toxin, a fluorescent protein, and a collagen-binding domain. (Appendix 46) The expression vector set according to Appendix 45, wherein the modified antibody is selected from the group consisting of Fab, single-chain antibody (scFv), tandem scFv, heavy-chain antibody (VHH), and the variable domain of the VHH. <Transformant> (Appendix 47) A transformant comprising the nucleic acid according to Appendix 35, the nucleic acid according to Appendix 36, the expression vector according to any one of Appendices 37 to 41, and / or the expression vector set according to any one of Appendices 42 to 45. <Method for producing a protein> (Appendix 48) A method for producing a protein, comprising an expression step of expressing the nucleic acid according to Appendix 35, the nucleic acid according to Appendix 36, the expression vector according to any one of Appendices 37 to 41, and / or the expression vector set according to any one of Appendices 42 to 45. (Appendix 49) The production method according to Appendix 48, wherein the expression step comprises: a culturing step of culturing the transformant according to Appendix 47; and an isolation step of isolating the first monomer protein and / or the second monomer protein.<Screening Method> (Appendix 50) A method for screening for target-reactive heterodimeric proteins, comprising: a production step of reacting a dimeric protein containing a reactive tag with a modifying protein that modifies the dimeric protein to produce a candidate heterodimeric protein; a detection step of contacting the candidate heterodimeric protein with a target and detecting a reaction between the candidate heterodimeric protein and the target; and a selection step of selecting the candidate heterodimeric protein for which the reaction has been detected as a candidate substance reactive with the target, wherein the production step is performed by the production method described in any of Appendices 1 to 15. (Appendix 51) The screening method described in Appendices 50, wherein the reaction is binding. (Appendix 52) The screening method described in Appendices 50 or 51, wherein the target is selected from the group consisting of a tumor antigen and a CD (cluster of differentiation) antigen. (Appendix 53) The screening method described in any of Appendices 50 to 52, wherein the candidate heterodimeric protein contains a label, and the detection step involves detecting the label.

[0214] As described above, according to the present invention, heterodimeric proteins such as bispecific antibodies can be produced, even if they are composed only of domains consisting of natural amino acid sequences. Furthermore, according to the present invention, desired modified peptides can be loaded onto the dimeric proteins. Therefore, the present invention is extremely useful, for example, in the fields of pharmaceuticals and pharmaceutical manufacturing.

Claims

1. A method for producing a heterodimeric protein, comprising: a production step of reacting a dimeric protein containing a reactive tag with a modifying protein that modifies the dimeric protein to produce a heterodimeric protein, the dimeric protein containing the reactive tag includes a first monomeric protein and a second monomeric protein; the first monomer protein comprises, in this order, a first reactive tag and a first dimerization domain capable of forming a dimer; the first reactive tag comprises a binding tag and a first C intein; the second monomer protein comprises, in this order, a second reactive tag and a second dimerization domain capable of forming a dimer with the first dimerization domain; the second reactive tag comprises a binding partner capable of binding to the binding tag and a second C intein; the modified protein includes a first modified protein and a second modified protein; the first modified protein comprises a first N intein capable of reacting with the first C intein and a first adduct added to the first monomer protein; the second modified protein comprises a second N intein capable of reacting with the second C intein and a second adduct added to the second monomer protein; the first adduct and the second adduct are different adducts, the first monomer protein and the second monomer protein form a dimer; In the manufacturing process, the first N intein of the first modified protein reacts with the first C intein of the first monomeric protein to link a first adduct of the first modified protein to the first monomeric protein; the second N intein of the second modified protein reacts with the second C intein of the second monomeric protein to link the second adduct of the second modified protein to the second monomeric protein. Manufacturing method.

2. The method according to claim 1 , further comprising a step of reacting the first monomer protein with the second monomer protein to form a dimer.

3. The method of claim 1 or 2, wherein the binding tag and the binding partner are a peptide tag and a peptide capable of spontaneously forming a covalent bond.

4. The method of claim 1 or 2, wherein the bond between the binding tag and the binding partner is a covalent bond.

5. the binding tag and the binding partner are, respectively, a Streptococcus pyogenes surface protein (SpyCatcher) and a peptide tag capable of binding to the SpyCatcher (SpyTag); or The method of claim 1 or 2, wherein the binding tag and the binding partner are the SpyTag and the SpyCatcher, respectively.

6. The method according to claim 1 or 2, wherein the dimeric protein comprises a part or all of the Fc region of an immunoglobulin.

7. The method of claim 6 , wherein the immunoglobulin is IgG, IgA, IgE, IgD, or IgM.

8. The method of claim 7, wherein the IgG is IgG1, IgG2, IgG2a, IgG2b, IgG3, or IgG4.

9. The method of claim 6 , wherein the Fc region of the immunoglobulin does not contain any mutations.

10. the first C intein and the first N intein are the C intein and N intein of an integrated or split intein, respectively; and / or The method of claim 1 or 2, wherein the second C intein and the second N intein are the C intein and the N intein of an integrated intein or a split intein, respectively.

11. The method according to claim 1 or 2, wherein the first adduct and / or the second adduct is a protein.

12. The method of claim 11 , wherein the protein is selected from the group consisting of a modified antibody, a heavy chain antibody (VHH), a receptor, a cytokine, a chemokine, a toxin, an enzyme, a fluorescent protein, and a collagen-binding domain.

13. The method of claim 12, wherein the antibody variant is selected from the group consisting of Fab, single-chain antibody (scFv), tandem scFv, heavy-chain antibody (VHH), and the variable domain of the VHH.

14. The dimeric protein contains a part or all of an Fc region of an immunoglobulin, the first adduct and the second adduct comprise an antibody variant and / or a heavy chain antibody (VHH); The method of claim 1 or 2, wherein the heterodimeric protein is a multispecific antibody.

15. The method according to claim 1 or 2, wherein the first monomer protein and the second monomer protein in the dimeric protein are linked by a disulfide bond.

16. a dimeric protein containing a reactive tag, the dimeric protein containing the reactive tag includes a first monomeric protein and a second monomeric protein; the first monomer protein comprises a first reactive tag and a first dimerization domain capable of forming a dimer; the first reactive tag comprises a first C intein capable of reacting with a binding tag and a first N intein; the second monomer protein comprises, in this order, a second reactive tag and a second dimerization domain capable of forming a dimer with the first dimerization domain; the second reactive tag comprises a binding partner capable of binding to the binding tag and a second C intein capable of reacting with a second N intein; the first monomer protein and the second monomer protein form a dimer; protein.

17. a first monomeric protein; the first monomer protein comprises, in this order, a first reactive tag and a first dimerization domain capable of forming a dimer; the first reactive tag comprises a binding tag capable of binding to a binding partner and a first C intein capable of reacting with a first N intein; the first monomeric protein is capable of forming a dimer with a second monomeric protein; the second monomer protein comprises a second reactive tag and a second dimerization domain capable of forming a dimer with the first dimerization domain; The protein, wherein the second reactive tag comprises a binding partner capable of binding to the binding tag and a second C intein capable of reacting with a second N intein.

18. a second monomeric protein; the second monomer protein comprises, in this order, a second reactive tag and a second dimerization domain capable of forming a dimer; the second reactive tag comprises a binding partner capable of binding to a binding tag and a second C intein capable of reacting with a second N intein; the second monomer protein is capable of forming a dimer with the first monomer protein; the first monomer protein comprises, in this order, a first reactive tag and a first dimerization domain capable of forming a dimer with the second dimerization domain; The first reactive tag comprises a binding tag capable of binding to the binding partner and a first C intein capable of reacting with a first N intein.

19. A nucleic acid encoding the protein of claim 16.

20. A nucleic acid encoding the protein of claim 17.

21. A nucleic acid encoding the protein of claim 18.

22. 20. An expression vector comprising the nucleic acid of claim 19.

23. An expression vector set comprising an expression vector comprising the nucleic acid of claim 20 and the nucleic acid of claim 21.

24. A method for producing a protein, comprising an expression step of expressing an expression vector set comprising the nucleic acid described in any one of claims 19 to 21, the expression vector described in claim 22, and / or an expression vector comprising the nucleic acid described in claim 20 and the nucleic acid described in claim 21.

25. a production step of reacting a dimeric protein containing a reactive tag with a modifying protein that modifies the dimeric protein to produce a candidate heterodimeric protein; A detection step of contacting the candidate heterodimeric protein with a target and detecting a reaction between the candidate heterodimeric protein and the target; a selection step of selecting the candidate heterodimer protein for which the reaction has been detected as a candidate substance that reacts with the target; A method for screening for a target-reactive heterodimeric protein, wherein the production step is carried out by the production method according to claim 1 or 2.

26. The screening method according to claim 25, wherein the reaction is binding.

27. 26. The method of claim 25, wherein the target is selected from the group consisting of a tumor antigen and a CD (cluster of differentiation) antigen.

28. the candidate heteroprotein comprises a label; The screening method according to claim 25 , wherein the detection step detects the label.