Method for producing heterodimer protein, dimer protein, monomeric protein, and method for screening target-reactive heterodimer protein

Abstract

Provided is a method for producing a heterodimer protein, which is also capable of producing a heterodimer protein comprising only a domain comprising a natural amino acid sequence, for example, a heterodimer protein such as a bispecific antibody. This production method is a method for producing a heterodimer protein, comprising a production step for producing a heterodimer protein by reacting a dimer protein containing a reaction tag with a modified protein that modifies the dimer protein, the reaction tag-containing dimer protein comprises a first monomeric protein comprising, in order, a first reaction tag comprising a binding tag and a first C-intein and a first dimer-forming domain capable of forming a dimer, and a second monomeric protein comprising, in order, a second reaction tag comprising a binding tag and a second C-intein, and a second monomeric protein comprising, in order, a second reaction tag comprising a binding partner capable of binding to the binding tag and a second C-intein and a second dimer-forming domain capable of forming a dimer with the first dimer-forming domain, the modified proteins comprise a first modified protein comprising a first N-intein capable of reacting with the first C-intein and a first adduct attached to the first monomeric protein, and a second modified protein comprising a second N-intein capable of reacting with the second C-intein. And a second modified protein comprising a second N-intein capable of reacting with the second C-intein and a second adduct attached to the second monomeric protein, the first adduct and the second adduct being different adducts, the first monomeric protein and the second monomeric protein forming a dimer, in the production step, the first N-intein of the first modified protein reacts with the first C-intein of the first monomeric protein such that a first adduct of the first modified protein is linked to the first monomeric protein. The second N-intein of the second modified protein reacts with a second C-intein of the second monomeric protein such that a second adduct of the second modified protein is linked to the second monomeric protein.

Description

Technical Field

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

[0002] In multispecific antibodies, one antibody molecule has antigen-binding domains that bind to different antigens. By changing the target antigen, they have been developed for various medical uses, such as anticancer drugs and hemophilia treatment drugs.

[0003] However, multispecific antibodies have the problem of extremely low manufacturing efficiency and difficulty in manufacturing. For example, bispecific antibodies are composed of two heavy chains (H chains) and two light chains (L chains). In addition, when the bispecific antibody is manufactured by expressing two H chains and two L chains, there are 10 combinations of two H chains and two L chains in the expressed antibody, and in addition to the target bispecific antibody, 9 unnecessary antibodies are also manufactured (Patent Document 1).

[0004] Prior art literature:

[0005] Patent Literature

[0006] Patent Document 1: International Publication No. 2013 / 065708 Summary of the invention

[0007] Problems to be solved by the invention

[0008] Therefore, in bispecific antibodies, technologies such as knob-into-holes have been developed to introduce mutations into the amino acid sequence of the Fc region of the antibody to allow two different heavy chains to specifically bind to each other.

[0009] However, in these techniques, due to the presence of non-natural amino acid sequences in the Fc region of antibodies, antibodies against these regions may be generated.

[0010] Therefore, an object of the present invention is to provide a method for producing a heterodimeric protein, which can also produce a heterodimeric protein composed of a domain consisting only of a natural amino acid sequence, for example, a heterodimeric protein such as a bispecific antibody.

[0011] Solution to the problem

[0012] 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.

[0013] The method comprises the steps of reacting a dimeric protein containing a reactive tag with a modified protein for modifying the dimeric protein to produce a heterodimeric protein,

[0014] The dimer protein containing the reactive tag comprises a first monomer protein and a second monomer protein,

[0015] The first monomer protein sequentially comprises a first reactive tag and a first dimer-forming domain capable of forming a dimer,

[0016] The first reaction tag comprises a binding tag and a first C intein,

[0017] The second monomeric protein sequentially comprises a second reactive tag and a second dimer-forming domain capable of forming a dimer with the first dimer-forming domain,

[0018] The second reactive tag comprises a binding partner capable of binding to the binding tag and a second C-intein,

[0019] The modified protein comprises a first modified protein and a second modified protein,

[0020] The first modified protein comprises a first N intein capable of reacting with the first C intein and a first adduct attached to the first monomeric protein,

[0021] The second modified protein comprises a second N intein capable of reacting with the second C intein and a second adduct attached to the second monomeric protein,

[0022] the first adduct and the second adduct are different adducts,

[0023] The first monomer protein and the second monomer protein form a dimer,

[0024] In the manufacturing process

[0025] the first N intein of the first modified protein reacts with the first C intein of the first monomeric protein such that a first adduct of the first modified protein is attached to the first monomeric protein,

[0026] The second N intein of the second modified protein reacts with the second C intein of the second monomeric protein such that a second adduct of the second modified protein is attached to the second monomeric protein.

[0027] The protein of the present invention (hereinafter also referred to as "dimeric protein") includes a dimeric protein containing a reactive tag,

[0028] The dimer protein containing the reactive tag comprises a first monomer protein and a second monomer protein,

[0029] The first monomeric protein comprises a first reactive tag and a first dimer-forming domain capable of forming a dimer,

[0030] The first reactive tag comprises a binding tag and a first C intein capable of reacting with the first N intein,

[0031] The second monomeric protein sequentially comprises a second reactive tag and a second dimer-forming domain capable of forming a dimer with the first dimer-forming domain,

[0032] 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,

[0033] The first monomeric protein and the second monomeric protein form a dimer.

[0034] The protein of the present invention (hereinafter also referred to as "first monomer protein") comprises a first monomer protein,

[0035] The first monomer protein sequentially comprises a first reactive tag and a first dimer-forming domain capable of forming a dimer,

[0036] 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,

[0037] The first monomer protein is capable of forming a dimer with the second monomer protein,

[0038] The second monomeric protein comprises a second reactive tag and a second dimer-forming domain capable of forming a dimer with the first dimer-forming domain,

[0039] 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.

[0040] The protein of the present invention (hereinafter also referred to as "second monomer protein") comprises a second monomer protein,

[0041] The second monomer protein sequentially comprises a second reactive tag and a second dimer-forming domain capable of forming a dimer,

[0042] The second reactive tag comprises a binding partner capable of binding to the binding tag and a second C intein capable of reacting with the second N intein,

[0043] The second monomer protein is capable of forming a dimer with the first monomer protein,

[0044] The first monomeric protein sequentially comprises a first reactive tag and a first dimer-forming domain capable of forming a dimer with the second dimer-forming domain,

[0045] 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.

[0046] 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.

[0047] The expression vector of the present invention comprises the nucleic acid of the present invention.

[0048] The method for screening a target-reactive heterodimeric protein of the present invention (hereinafter also referred to as "screening method") comprises:

[0049] 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;

[0050] A detection step, contacting the candidate heterodimeric protein with a target, and detecting a reaction between the candidate heterodimeric protein and the target;

[0051] a selection step of selecting the candidate heterodimeric protein in which the reaction is detected as a candidate substance that reacts with the target,

[0052] The above-mentioned production process is carried out by the production method of the present invention.

[0053] Effects of the Invention

[0054] According to the present invention, in the case of heterodimeric proteins such as bispecific antibodies, heterodimeric proteins consisting only of domains consisting of natural amino acid sequences can be produced. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] [ Figure 1 ] Figure 1 This is a schematic diagram showing an example of the constituent elements of the dimeric protein and each step of producing the heterodimeric protein used in the production method of the present invention.

[0056] [ Figure 2 ] Figure 2 These are photographs showing proteins before and after the reaction of each antibody fragment with PTS in Example 1.

[0057] [ Figure 3 ] Figure 3 These are photographs showing the protein after the PTS reaction and during column purification in Example 1.

[0058] [ Figure 4 ] Figure 4 This is a diagram showing the elution pattern of the protein measured by gel filtration chromatography in Example 1.

[0059] [ Figure 5 ] Figure 5 This is a graph showing the binding of the bispecific antibody to CD3-positive cells measured by flow cytometry in Example 1.

[0060] [ Figure 6 ] Figure 6 This is a graph showing the binding of the bispecific antibody to HER2-positive cells measured by flow cytometry in Example 1.

[0061] [ Figure 7 ] Figure 7 This is a graph showing the binding maintenance between the bispecific antibody and HER2 obtained by the surface plasmon resonance method in Example 1. DETAILED DESCRIPTION

[0062] <Definition>

[0063] In this specification, "protein" refers to a polymer of peptides composed of unmodified amino acids (natural amino acids), modified amino acids and / or artificial amino acids. The shape of the polymer can be, for example, straight chain, branched chain, and cyclic. The protein can also be called a peptide or polypeptide.

[0064] In the present specification, "monomeric protein" refers to a protein that is not bound to or associated with other proteins.

[0065] In this specification, "dimeric protein" refers to a protein complex in a state of being bound or associated by two proteins or protein subunits. When the two proteins are the same protein or subunit, the dimeric protein may also be referred to as a homodimeric protein. In addition, when the two proteins are different proteins or subunits, the dimeric protein may also be referred to as a heterodimeric protein.

[0066] In this specification, "fusion protein" refers to a protein in which two or more different proteins or heterologous proteins are partially or completely bound (connected) by peptide bonds. The fusion protein can be a natural fusion protein or an artificial fusion protein. The artificial fusion protein can be, for example, a protein designed by genetic engineering methods.

[0067] In this specification, "binding tag" refers to a polypeptide or substance that has specific binding properties to other molecules.

[0068] In the present specification, "binding partner" refers to a polypeptide or substance that has specific binding properties with the binding tag.

[0069] In the present specification, "domain" refers to a region of three-dimensional structure or functional integration in a "protein", "polypeptide" and / or "peptide".

[0070] In this specification, "antibody" refers to a protein containing one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Immunoglobulin genes include, for example, genes encoding constant regions such as κ, λ, α (including α1, α2), γ (including γ1, γ2, γ3, γ4), δ, ε and μ, as well as genes capable of encoding numerous immunoglobulin variable regions such as V region, D region, J region, etc. The antibody, for example, includes a heavy chain and a light chain. The light chain includes κ and λ, which constitute the κ chain and the λ chain, respectively. The heavy chain includes γ, μ, α, δ, or ε, which constitute the immunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively. The antibody can be a structural unit of a typical immunoglobulin (antibody) composed of a tetramer. In this case, the antibody consists of two pairs of identical polypeptide chains, each pair consisting of a light chain (about 25 kDa) and a heavy chain (about 50 to 70 kDa). Additionally, the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily involved in antigen recognition.

[0071] In this specification, "antigen binding fragment" refers to a portion or a portion of a 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 methods. Examples of the antigen binding fragment include Fab, Fab', F(ab')2, Fc and / or Fv fragments, and their derivatives.

[0072] In the present specification, "heavy chain antibody" refers to an antibody consisting of only two heavy chains. The heavy chain antibody may also be referred to as an antibody without a light chain.

[0073] In the present specification, a "multispecific antibody" refers to an antibody or antibody derivative having specificity for two or more antigens and / or two or more epitopes.

[0074] In the present specification, "intein" refers to an autocatalytic enzyme having both protein protease activity and protein ligase activity. The intein is a protein formed by, for example, cutting out its own amino acid sequence from a protein by protein splicing in certain proteins, and linking the remaining amino acid sequence (intein) by peptide bonds. The "intein" may also be referred to as a "protein intron".

[0075] In the present specification, "split intein" refers to an intein composed of two complementary half inteins, namely, a C intein and an N intein, which tightly bind to form an active intein and exhibit enzyme activity. The split intein is also called an "isolated intein".

[0076] In the present specification, the term "intein" refers to an intein that includes a C intein and an N intein, and that forms an active intein by tightly binding the C intein and the N intein, and exhibits enzymatic activity.

[0077] In the present specification, "complementary intein" refers to an intein or a combination (pair) of a C intein and an N intein of a split intein.

[0078] In the present specification, "C intein" refers to an intein polypeptide having homology to the C-terminal portion of an intein. The C intein combines with a complementary N intein to form an active intein.

[0079] In the present specification, "N intein" refers to an intein polypeptide having homology to the N-terminal portion of an intein. The N intein combines with a complementary C intein to form an active intein.

[0080] In this specification, "salt" refers to an electrolyte that can be ionized in an aqueous solvent. The aqueous solvent refers to a solvent containing water.

[0081] In the present specification, a "solubilization domain" refers to a polypeptide that, when fused to a peptide, polypeptide or protein, increases (e.g., increases or promotes) the expression amount of the peptide, polypeptide or protein expressed in a transformant compared to when expressed as a peptide, polypeptide or protein without a solubilization domain.

[0082] In this specification, "purification" means identifying and separating, recovering from natural components, or refers to a state of being identified and separated, and / or recovered from natural components. The "purification" can be performed, for example, by at least one purification step. The purification can also be referred to as separation.

[0083] In this specification, "separation" refers to the separation of a target object from a substance containing the target object and / or the state after separation. The separation may also be referred to as release.

[0084] In this specification, "nucleic acid" refers to a polymer of deoxyribonucleotides (DNA), ribonucleotides (RNA) and / or modified nucleotides. In this specification, when "nucleic acid" is used in conjunction with a specific protein, the "nucleic acid" refers to a nucleotide polymer encoding the amino acid sequence of the protein. The nucleic acid includes, for example, genomic DNA, cDNA, mRNA, etc. The nucleic acid can be, for example, single-stranded or double-stranded. The nucleic acid can be referred to interchangeably with "polynucleotide" or "nucleic acid molecule".

[0085] In this specification, "host" refers to a cell and / or individual into which an exogenous nucleic acid is introduced. When the host is a cell, the host may also be referred to as a host cell.

[0086] In the present specification, "vector" and "expression vector" refer to a recombinant plasmid or virus containing a nucleic acid delivered to a host or host cell in vitro or in vivo. Examples of the "vector" and "expression vector" include viral vectors and non-viral vectors.

[0087] In the present specification, the "transformant" refers to a host into which a foreign nucleic acid has been introduced.

[0088] In the present specification, the sources of various proteins, polypeptides or peptides are not particularly limited and may be any animal. The animal may be, for example, a human or a non-human animal. The non-human animal may be, for example, a mammal such as a mouse, a rat, a rabbit, a dog, a cat, a cow, a horse, a pig, a monkey, a dolphin, a sea lion, etc.

[0089] The present invention will be described below by examples, but the present invention is not limited to the following examples and can be implemented by any changes. In addition, unless otherwise specified, each description in the present invention can be referenced to each other. In this specification, when the expression of "~" is used, it is used with the meaning of the numerical value or physical value before and after it. In addition, in this specification, the expression of "A and / or B" includes "only A", "only B", "both A and B".

[0090] <Method for producing heterodimeric protein>

[0091] In one embodiment, the present invention provides a method for producing a heterodimeric protein, such as a bispecific antibody, which can produce a heterodimeric protein composed of a domain consisting only of a natural amino acid sequence, or can produce a heterodimeric protein modified by different adducts. The production method of the present invention is a production method for a heterodimeric protein, comprising a production step of producing a heterodimeric protein by reacting a dimeric protein containing a reaction tag with a modified protein that modifies the dimeric protein, wherein the dimeric protein containing the reaction tag comprises a first monomer protein and a second monomer protein, wherein the first monomer protein sequentially comprises a first reaction tag and a first dimer-forming domain capable of forming a dimer, wherein the first reaction tag comprises a binding tag and a first C-intein, wherein the second monomer protein sequentially comprises a second reaction tag and a second dimer-forming domain capable of forming a dimer with the first dimer-forming domain, wherein the second reaction tag comprises a binding partner capable of binding to the binding tag and a second C-intein, wherein the modified protein comprises a first modified protein and a second modified protein, wherein the first modified protein The protein comprises a first N intein capable of reacting with the first C intein and a first adduct attached 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 attached 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 manufacturing process, the first N intein of the first modified protein reacts with the first C intein of the first monomeric protein so that the first adduct of the first modified protein is linked 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 so that the second adduct of the second modified protein is linked to the second monomeric protein.

[0092] When the first monomer protein and the second monomer protein are proteins that form homodimers, various combinations of dimers are generated by preparing in advance the first monomer protein to which the first adduct is linked and the second monomer protein to which the second adduct is linked 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 by utilizing the reaction of the first N intein and the first C intein in the state where the dimeric protein is formed. In addition, in the production method of the present invention, the second adduct is linked to the second monomer protein by utilizing the reaction of the second N intein and the second C intein in the state where the dimeric protein is formed. Therefore, according to the production method of the present invention, even if the first monomer protein and the second monomer protein are proteins that form homodimers, a heterodimeric protein composed of a protein derived from the first monomer protein to which the first adduct is linked and a protein derived from the second monomer protein to which the second adduct is linked can be efficiently produced.

[0093] The inventors of the present invention have obtained the concept that a heterodimeric protein composed of monomeric proteins connected to different adducts can be produced by making two monomeric proteins in a dimer state, causing each monomeric protein to undergo a specific binding reaction and connect to different adducts. After in-depth research, the inventors of the present invention have found that the above concept can be implemented by using two monomeric proteins to which binding tags or binding partners and different inteins are attached, and two modified proteins to which inteins capable of reacting with the different inteins and adducts attached to the monomeric proteins are connected, thereby establishing the present invention. Specifically, if Figure 1 As shown, it is speculated that the production method of the present invention can produce a heterodimeric protein composed of monomeric proteins to which different adducts are linked by the following mechanism. In addition, the present invention is not limited to the following mechanism.

[0094] like Figure 1As shown in (A), in the production method of the present invention, a heterodimeric protein can be produced 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 includes a first reaction tag 11 and a first dimer-forming domain 12 in order from the N-terminal side. In addition, the first reaction tag 11 includes a binding tag 13 and a first C intein 14 in order from the N-terminal side. The second monomer protein 2 includes a second reaction tag 21 and a second dimer-forming domain 22 in order from the N-terminal side. In addition, the second reaction tag 21 includes a binding partner 23 and a second C intein 24 in order from the N-terminal side. In addition, the first modified protein 3 includes a first N intein 31 and a first adduct 32 in order from the N-terminal side. The second modified protein 4 includes a second N intein 41 and a second adduct 42 in order from the N-terminal side.

[0095] First, by contacting a first monomer protein 1 with a second monomer protein 2, such as Figure 1 As shown in (B), a dimer protein 10 formed by a first monomer protein 1 and a second monomer protein 2 is prepared. Then, when the dimer protein 10 is brought into contact with a first modified protein 3 and a second modified protein 4, as shown in Figure 1 As shown in (C), the first N intein 31 of the first modified protein 3 forms an active intein with the first C intein 14 of the first monomeric protein 1 and reacts (arrow X). In addition, the second N intein 41 of the second modified protein 4 forms an active intein with the second C intein 24 of the second monomeric protein 2 and reacts (arrow Y). Subsequently, 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, and the first dimer-forming domain 12 is connected to the C-terminal side of the first adduct 32. In addition, 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, and the second dimer-forming domain 22 is connected to the C-terminal side of the second adduct 42. As a result, as shown in FIG. Figure 1 As shown in (D), a heterodimeric protein 30 can be produced in which the first dimer-forming domain 12 and the second dimer-forming domain 22 that form a dimer are modified with different adducts 32 and 42. Therefore, according to the production method of the present invention, it is estimated that a heterodimeric protein modified with an arbitrary adduct can be produced.

[0096] In addition, in the production method of the present invention, an intein is used to attach the first adduct 32 and the second adduct 42. Therefore, the monomeric protein constituting the obtained heterodimeric protein can be used as a fusion protein connected from the N-terminus to the C-terminus by a peptide bond, just like the recombinant protein, to produce a heterodimeric protein. Therefore, in the production method of the present invention, by making the first dimer-forming domain 12, the second dimer-forming domain 22, the first adduct 32 and the second adduct 42 a natural amino acid sequence, that is, an amino acid sequence that does not contain amino acid mutations or artificial amino acid sequences, a heterodimeric protein formed by connecting multiple domains consisting only of natural amino acid sequences can also be produced. Therefore, as an example, by setting the first adduct 32 and the second adduct 42 to Fabs that bind to different antigens, respectively, and setting the first dimer-forming domain 12 and the second dimer-forming domain 22 to proteins consisting of the CH2 region and the CH3 region of the antibody hinge region and the Fc region, a bispecific monoclonal antibody consisting of a natural amino acid sequence can be appropriately produced. Thus, according to the production method of the present invention, for example, even when two monomeric proteins having the same amino acid sequence in the dimer-forming domain and different amino acid sequences in other parts are used to produce a heterodimeric protein, the heterodimeric protein can be specifically produced without introducing amino acid sequence variations that cause specific binding or association between the target monomeric proteins.

[0097] In addition, Figure 1 In (A) to (D), the case where the first adduct 32 and the second adduct 42 are modified at the N-terminal side of the first dimer-forming domain 12 and the second dimer-forming domain 22 is described as an example, but the present invention is not limited thereto, and the first adduct 32 and the second adduct 42 may be modified at the C-terminal of the first dimer-forming domain 12 and the second dimer-forming domain 22. In this case, the first monomeric protein 1 includes the first reaction tag 11 and the first dimer-forming domain 12 in order from the C-terminal side, and the first reaction tag 11 includes the binding tag 13 and the first N intein 31 in order from the C-terminal side. The second monomeric protein 2 includes the second reaction tag 21 and the second dimer-forming domain 22 in order from the C-terminal side, and the second reaction tag 21 includes the binding partner 23 and the second N intein 41 in order from the C-terminal side. In addition, the first modified protein 3 includes the first C intein 14 and the first adduct 32 in order from the C-terminal side. The second modified protein 4 includes the second C intein 24 and the second adduct 42 in order from the C-terminal side. In addition, the production method of the present invention can modify the first adduct 32 and the second adduct 42 at the C-terminal side of the first dimer-forming domain 12 and the second dimer-forming domain 22. In the following description, each domain can be replaced as described above and the description thereof can be cited.

[0098] In the production method of the present invention, the dimeric protein containing the reaction tag is formed by dimerization of the first monomer protein and the second monomer protein. In addition, the first monomer protein and the second monomer protein can be prepared, for example, by the genetic engineering technology described in the production method of the monomer protein of the present invention described later. Therefore, the production method of the present invention can optionally include a formation step of reacting the first monomer protein and the second monomer protein to form a dimer before the production step. In addition, the production method of the present invention can optionally include a production step of preparing the first monomer protein and / or the second monomer protein, and a formation step of reacting the first monomer protein and the second monomer protein to form a dimer before the production step. The production method in the production step can refer to the description of the first monomer protein, the second monomer protein, the nucleic acid, the expression vector, the transformant and the production method of the monomer protein of the present invention described later.

[0099] In the forming step, the first monomer protein and the second monomer protein are reacted. Figure 1 As shown in (B), in the formation step, the first monomer protein and the second monomer protein form a dimer, and a dimer protein containing the reaction tag can be prepared or prepared. The first monomer protein and the second monomer protein can form a heterodimer through the binding tag and the binding partner. Therefore, the formation step can be carried out in parallel with the manufacturing step described later.

[0100] The reaction in the forming step is preferably carried out in the presence of an aqueous solvent such as a buffer, 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 solid such as powder, the first monomer protein and the second monomer protein are preferably dispersed in an aqueous solvent such as a buffer, saline, or water in advance. In addition, when the first monomer protein and the second monomer protein are liquid, the first monomer protein and the second monomer protein may be diluted in advance with an aqueous solvent such as a buffer, saline, or water. As a specific example of the reaction, the reaction may be carried out by mixing a liquid containing the first monomer protein and a liquid containing the second monomer protein.

[0101] When the first monomer protein and the second monomer protein obtained in the preparation process 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 roughly purified proteins, or may be transformants expressing the first monomer protein and / or the second monomer protein or processed products of the transformants. Examples of the processed products include lysates, extracts, fragments, protein preparations, or roughly purified or purified products thereof. The processed products may be, for example, substances that have been subjected to at least one purification treatment.

[0102] The first monomer protein, for example, comprises the first reaction tag and the first dimer-forming domain capable of forming a dimer in order from the N-terminal side. The first reaction tag, for example, comprises the binding tag and the first C intein in order from the N-terminal side. In addition, the second monomer protein, for example, comprises the second reaction tag and the second dimer-forming domain capable of forming a dimer with the first dimer-forming domain in order from the N-terminal side. The second reaction tag comprises the binding partner and the second C intein. The first monomer protein can introduce the first adduct to the N-terminal side of the first dimer-forming domain when the first C intein reacts with the first N intein by configuring the first reaction tag to the N-terminal side of the first dimer-forming domain. In addition, the second monomer protein can introduce the second adduct to the N-terminal side of the second dimer-forming domain when the second C intein reacts with the second N intein by configuring the second reaction tag to the N-terminal side of the second dimer-forming domain.

[0103] In the formation step, the dimerization of the first monomer protein and the second monomer protein can be generated, for example, by (1) the binding of the first reaction tag and the second reaction tag, for example, by the binding tag of the first reaction tag and the binding partner of the second reaction tag, or by (2) the binding of the first dimer-forming domain and the second dimer-forming domain, or by the binding of both (1) and (2). Since the dimer-forming ability can be improved, it is preferably generated by the binding of both (1) and (2). The reaction tag can be regarded as, for example, a domain in the first monomer protein and the second monomer protein that contributes to the binding of the monomer protein and the modified protein and the addition reaction of the adduct with the monomer protein. In addition, the reaction tag can also be regarded as, for example, a domain that disappears from the monomer protein after the addition reaction.

[0104] The dimer may be formed by direct binding of the first monomer protein to the second monomer protein, may be formed by indirect binding (association) of the first monomer protein to the second monomer protein, or may be formed by both direct binding and indirect binding of the first monomer protein to the second monomer protein. The direct binding is covalent bonding, and specifically, amide bonds (peptide bonds) between amino acids, disulfide bonds between cysteines, etc. may be cited. The indirect bonding is non-covalent bonding, and specifically, hydrogen bonds, hydrophobic bonds, etc. may be cited.

[0105] The first dimer-forming domain and the second dimer-forming domain may use amino acid sequences that can form dimers conditionally dependently or conditionally independent when proteins containing the domains coexist. As a specific example, the first dimer-forming domain and the second dimer-forming domain may use amino acid sequences of motif sequences of each subunit or dimer-forming motif sequence present in a protein dimer. The first dimer-forming domain and the second dimer-forming domain may use amino acid sequences of motif sequences of each subunit or dimer-forming motif sequence present in a protein multimer. The protein dimer may be a homodimer or a heterodimer. As the protein dimer, for example, there can be mentioned immunoglobulins (antibodies) such as IgA, IgD, IgE, IgG, and IgM; proteins containing leucine zippers such as AP-1 (c-fos and c-jun), myc, max, mdx1 and other myc family proteins; G protein-coupled receptors; kinesin; receptor tyrosine kinases, such as the ErbB receptor family such as the epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), neurotrophin receptor, insulin receptor, insulin-like growth factor receptor, vascular endothelial growth factor receptor (VEGFR), stem cell factor receptor, etc.; Toll-like receptors such as TLR1 to 11, etc.

[0106] When the dimer-forming motif sequence of the antibody is used as the first dimer-forming domain and the second dimer-forming domain, the first dimer-forming domain and the second dimer-forming domain, for example, contain the CH2 region and CH3 region of the antibody Fc region, or the CH2 region and CH3 region of the antibody Fc region and the amino acid sequence of the hinge region. When the first dimer-forming domain and the second dimer-forming domain contain the Fc region of the antibody, the first dimer-forming domain and the second dimer-forming domain contain all or part of the amino acid sequence of the Fc region of the antibody. When the first dimer-forming domain and the second dimer-forming domain contain the CH2 region, CH3 region and hinge region of the Fc region of the antibody, the first dimer-forming domain and the second dimer-forming domain contain all or part of the amino acid sequence of the CH2 region and CH3 region of the Fc region of the antibody, and all or part of the amino acid sequence of the hinge region of the antibody, preferably contain all the amino acid sequence of the CH2 region and CH3 region of the Fc region of the antibody, and all or part of the amino acid sequence of the hinge region of the antibody. When the first dimer-forming domain and the second dimer-forming domain comprise the CH2 region, CH3 region and hinge region of the antibody Fc region, the first dimer-forming domain and the second dimer-forming domain, for example, comprise a continuous amino acid sequence in the amino acid sequence of the CH2 region, CH3 region and hinge region of the antibody Fc 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 natural amino acid sequence, i.e., an amino acid sequence without variation, or a non-natural amino acid sequence, i.e., an amino acid sequence comprising a variation or an artificially synthesized amino acid sequence.

[0107] The antibody is, for example, IgA, IgD, IgE, IgG and IgM, preferably IgG. The IgG is, for example, IgG1, IgG2, IgG2a, IgG2b, IgG3 or IgG4. The antibody is, for example, an antibody derived from an animal, and specific examples thereof include human antibodies, mouse antibodies, bird antibodies, rat antibodies, rabbit antibodies, etc. The first dimer-forming domain and the second dimer-forming domain are preferably derived from human antibodies, more preferably from human IgG.

[0108] As the amino acid sequences of human IgG1, IgG2, IgG3, and IgG4, for example, the amino acid sequences registered as UniProt Accession Nos. P01857, P01859, P01860, and P01861, respectively, can be referred to.

[0109] When the dimer-forming motif sequence of the antibody is used as the first dimer-forming domain and the second dimer-forming domain, the antibody may be an antibody in which the Fc region is altered. In this case, the first dimer-forming domain and the second dimer-forming domain may comprise a variant Fc region containing the Fc region, or a variant Fc region of the Fc region and an amino acid sequence of a hinge region. Examples of variants of the Fc region include Fcabs (Fc antigen binding, references 1 and 5) in which the amino acid sequence of the Fc region is altered and imparted with the ability to bind to a target molecule, IgG hexamers (IgG Hexamers, references 2 to 4) in which the amino acid sequence of the Fc region of an IgG antibody is altered and imparted with the ability to form a hexamer, DAF (Dual Action Fab, reference 5), Charge pair (Amgen, reference 5), SEEDbody (reference 5), Knobs-in-holes (reference 5), DVI-IgG (reference 5), and the like.

[0110] Reference 1: G.Wozniak-Knopp et al., "Introducing antigen-binding sites in structural loops of immunoglobulin constant: Fc fragments with domains engineered HER2 / neu-binding sites and antibody properties", ProteinEngineering, Design and Selection, Volume 23, Issue 4, April 2010, Pages 289-297

[0111] Reference 2: Sopp, JMet al., "On-target IgG hexamer isation driven by aC-terminal IgM tail-piece fusion variant confers augmented complementactivation.", Commun. Biol., 4, 1031 (2021).

[0112] Reference 3: de Jong RN et al., "A Novel Platform for the Potentiation ofTherapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamersat the Cell Surface.", PLoS. Biol. (2016) 14(1):e1002344.

[0113] Reference 4: Christoph A.Diebolder et al., "Complement Is Activated byIgG Hexamers Assembled at the Cell Surface", Science, 343(6176), pages 1260-1263

[0114] 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

[0115] When the dimer-forming motif sequence of the antibody is used as the first dimer-forming domain and the second dimer-forming domain, the antibody can be a multispecific antibody. In this case, the first dimer-forming domain and the second dimer-forming domain can include, for example, an Fc region containing the multispecific antibody, or a CH2 region and a CH3 region and a hinge region containing the Fc region, and an amino acid sequence of a domain attached to the C-terminus of the Fc region. The multispecific antibody can include, for example, 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), Tetravalent HCab (reference 5), etc.

[0116] The binding tag and the binding partner are molecules that bind to the binding partner conditionally or 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.

[0117] When the binding between the binding tag and the binding partner is direct binding, the binding tag and the binding partner can utilize, for example, a peptide tag and a peptide that can spontaneously form a covalent bond, or a peptide tag and a peptide that form a covalent bond through the modification activity of other molecules.

[0118] When the peptide tag and peptide capable of spontaneously forming a covalent bond are used, the binding tag and the binding partner may be, for example, a surface protein of Streptococcus pyogenes (SpyCatcher, sequence number 1) and a peptide tag capable of binding to the SpyCatcher (SpyTag, sequence number 2) or a variant thereof, etc. The variants of the SpyCatcher and the SpyTag may be, for example, SpyCatcher2 and SpyTag2 (reference 6), SpyCatcher3 and SpyTag3 (reference 7), SnoopCatcher and SnoopTag (reference 8), etc.

[0119] Reference 6: Anthony H.Keeble et al., "Evolving Accelerated Amidation bySpyTag / SpyCatcher to Analyze Membrane Dynamics", Angew.Chem.Int.Ed., 2017, 56, pages 16521-16525

[0120] 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

[0121] Reference 8: Veggiani G, Nakamura T, Brenner MD, Gayet RV, Yan J, Robinson CV, Howarth M. Programmable polyproteams built using twin peptidesuperglues. Proc Natl Acad Sci US A. 2016 Feb 2;113(5):1202-7.doi:10.1073 / pnas.1519214113.

[0122] Amino acid sequence of Streptococcus pyogenes surface protein (SpyCatcher) (SEQ ID NO: 1)

[0123] DSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVN

[0124] Amino acid sequence of SpyTag (SEQ ID NO: 2)

[0125] AHIVMVDAYKPTK

[0126] When a peptide tag and a peptide capable of forming a covalent bond through the modification activity of other molecules are used, the binding tag and the binding partner can be, for example, a K tag and a Q tag, etc. The K tag and the Q tag can be cross-linked with the N-terminal lysine residue of the K tag and the N-terminal glutamic acid of the Q tag by using a bacterial transglutaminase, thereby forming a covalent bond.

[0127] When the binding between the binding tag and the binding partner is indirect binding, the binding tag and the binding partner can utilize, for example, an affinity tag and a molecule that binds to the affinity tag. The binding tag and the binding partner are, for example, a peptide, a polypeptide or a protein. The binding tag can be, for example, a His-tag (His×6), a His-Strep-tag, a strep-tag, an avidin tag, a Flag (trademark)-tag, a HA (hemagglutinin)-tag, a T7-tag, a V5-peptide-tag, a GST (glutathione-S-transferase)-tag, a CBP (calmodulin binding peptide)-tag, an MBP (maltose binding protein)-tag, a Myc-tag, and the like. If the binding partner is an antibody or an antigen-binding fragment thereof, or a derivative thereof, or the like, which shows specific binding to a target molecule, the binding tag can be a peptide formed by any amino acid sequence that can bind to a molecule that exhibits the specific binding.

[0128] The binding partner can be appropriately set according to the type of the binding tag. As specific examples, the binding partner can include antibodies or antigen-binding fragments thereof that recognize the binding tag, or derivatives thereof; nucleic acid molecules such as aptamers; glutathione, calmodulin; sugar chains such as mannose; metals such as nickel, cobalt, zinc, or ions thereof.

[0129] The combination of the binding tag and the binding partner can be any combination as long as the binding tag and the binding partner can bind. As a specific example, if the binding tag includes a His tag, the binding partner is, for example, nickel. In addition, if the binding tag includes a Strep-tag or an avidin tag, the binding partner includes, for example, biotin. If the binding tag includes an epitope tag such as a Flag (trademark)-tag, HA-tag, T7-tag, V5-peptide-tag and / or Myc-tag, the binding partner can include, for example, antibodies or antigen-binding fragments thereof against each epitope tag, or their derivatives, etc. If the binding tag includes a GST-tag, the binding partner can include, for example, glutathione. If the binding tag includes a CBP-tag, the binding partner can be, for example, calmodulin. If the binding tag includes an MBP-tag, the binding partner can include, for example, mannose.

[0130] The binding tag and the binding partner may be functional equivalents within the scope of maintaining the binding ability between the binding tag and the binding partner. If the binding tag or the binding partner is a peptide, polypeptide or protein, the functional equivalent may be, for example, an amino acid sequence having 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 relative to the reference amino acid sequence of the binding tag or binding partner, and a polypeptide having the binding ability to the corresponding binding tag or binding partner. The functional equivalent may be, for example, an amino acid sequence consisting of one or more amino acids deleted, substituted, inserted and / or added in the reference amino acid sequence of the binding tag or binding partner, and a polypeptide having the binding ability to the corresponding binding tag or binding partner. The “one or more” is, 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, 1; the substitution is, for example, preferably a conservative substitution.

[0131] In the first monomer protein, the binding tag may be one or more. In the latter case, the binding tag may be one or more. In the first monomer protein, when the first adduct is modified (attached) at the N-terminus of the first monomer protein, the binding tag is preferably arranged at the N-terminus side of the first dimer-forming domain.

[0132] In the second monomer protein, the binding partner may be one or more. In the latter case, the binding partner may be one or more. In the second monomer protein, when the second adduct is modified (attached) at the N-terminus of the second monomer protein, the binding partner is preferably arranged at the N-terminus side of the second dimer-forming domain. The number of the binding partner and the binding partner may be the same or different, but preferably the same.

[0133] In the first monomeric protein and the second monomeric protein, the binding tag and the binding partner can be interchanged, and the combinations described above can be used interchangeably.

[0134] When a first monomeric protein including the first C intein and a first modified protein including the first N intein coexist, the first C intein and the first N intein are molecules that allow the binding of the binding tag to the binding partner to occur conditionally dependently or conditionally independently. Furthermore, when a second monomeric protein including the second C intein and a second modified protein including the second N intein coexist, the second C intein and the second N intein are molecules that allow the binding of the binding tag to the binding partner to occur conditionally dependently or conditionally independently. Inteins that can be used for the first C intein, the first N intein, the second C intein, and the second N intein will be described below.

[0135] In the present invention, the C intein and the N intein constituting the intein can be designed from, for example, an integrated intein, or a split intein can be used.

[0136] 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 of DNA polymerase III from cyanobacteria, dnaE-n and dnaE-c), MCM2 (sequence number 3, Hut MCM-2 intein from the archaeon Halorhabdus utahensis DSM 12940, partial sequence of Uniprot: C7NUH7), etc. In the amino acid sequence of sequence number 3 below, the regions in brackets correspond to the N intein, the deletion region, and the C intein, respectively, from the N-terminal side (reference 9).

[0137] Reference 9: Ciragan A.et al., "Salt-inducible Protein Splicing in cisand trans by Inteins from Extremely Halophilic Archaea as a Novel Protein-Engineering Tool." J Mol Biol.2016Nov 20;428(23):4573-4588.doi:10.1016 / j.jmb.2016.10.006.Epub2016Oct 6.PMID:27720988.

[0138] Amino acid sequence of MCM2 (SEQ ID NO: 3)

[0139] [CVTGDTLVQAGDGRRRIRELAGETAEAGSIEELPNGRTIRDVDIDVWTMTDDETLTRRPVTAIHEYDAPETLYEVTLSTGEEVTVTPDHPFFIEQASGRVETPAEDLQPGDLVFVPEGSAMATDG][GIAQIDTSSDRLGPAESGL][GDIGLRTIENVESVPDHDYDSVYDLTVEGTHNFLANGMVVHN

[0140] When the intein is MCM2, examples of the C intein and N intein derived from the 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.

[0141] Amino acid sequence of the C intein derived from MCM2 (SEQ ID NO: 4)

[0142] GIAQIDTSSDRLGPAESGL / GDIGLRTIENVESVPDHDYDSVYDLTVEGTHNFLANGMVVHN

[0143] Amino acid sequence of the N-intein derived from MCM2 (SEQ ID NO: 5)

[0144] CVTGDTLVQAGDGRRRIRELAGETAEAGSIEELPNGRTIRDVDIDVWTMTDDETLTRRPVTAIHEYDAPETLYEVTLSTGEEVTVTPDHPFFIEQASGRVETPAEDLQPGDL

[0145] VFVPEGSAMATD

[0146] C intein derived from gp41-1 (SEQ ID NO: 6) MMLKKILKIEELDERELIDIEVSGNHLFYANDILTHNSAG

[0147] N intein derived from gp41-1 (SEQ ID NO: 7)

[0148] SGYCLDLKTQVQTPQGMKEISNIQVGDLVLSNTGYNEVLNVFPKSKKKSYKITLEDGKEIICSEEHLFPTQTGEMNISGGLKEGMCLYVKE

[0149] C intein derived from Cfa (SEQ ID NO: 8)

[0150] VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASNC

[0151] N-intein derived from Cfa (SEQ ID NO: 9)

[0152] CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP

[0153] The intein may be an intein that undergoes a binding reaction condition-dependently (condition-dependent intein). The condition-dependent intein may also be, for example, an intein in which the intein and / or the C intein undergoes a binding reaction with the N intein under certain conditions. The condition-dependent intein may include, for example, salt concentration-dependent inteins such as MCM2, a combination of HsaPol II and HsaCDC21 derived from Halobacteriumsalinarum NRC-1 (ATCC 700922) (shown in reference 9); and temperature-dependent intein (shown in reference 10). The salt concentration-dependent intein is, for example, an intein in which the intein and / or the C intein undergoes a binding reaction with the N intein when the salt concentration is relatively high, i.e., an intein in which the probability of the binding reaction increases. The salt concentration-dependent intein may be an intein in which a binding reaction occurs when the salt concentration reaches a certain concentration. It is speculated that the occurrence of the binding reaction of the salt concentration-dependent intein depends on the ionic strength of the salt. Therefore, the salt used for 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; phosphates such as sodium phosphate, calcium phosphate, potassium phosphate, and magnesium phosphate; and the like. If the salt concentration-dependent intein is MCM2, the salt is preferably sodium chloride.

[0154] 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

[0155] The intein may be a functional equivalent within the range of maintaining its protease activity and ligase activity. The functional equivalent may include, for example, a polypeptide having protease activity and ligase activity, which is composed of an amino acid sequence having 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 identity with respect to the reference amino acid sequence of the intein. The functional equivalent may include, for example, a polypeptide having protease activity and ligase activity, which is composed of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the reference amino acid sequence of the intein. The one or more 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, for example, preferably a conservative substitution.

[0156] The number of C inteins in the first monomeric protein and the second monomeric protein may be one or more, but is preferably one in order to efficiently modify the first monomeric protein and the second monomeric protein.

[0157] When the first adduct is modified (added) to the N-terminus of the first monomeric protein, the first C-intein is preferably arranged on the N-terminus side of the first dimer-forming domain. In the second monomeric protein, when the second adduct is modified (added) to the N-terminus of the second monomeric protein, the binding partner is preferably arranged on the N-terminus side of the second dimer-forming domain.

[0158] The first C intein and the second C intein are, for example, respectively configured to react with the first N intein and the second N intein. Thus, the first modified protein can be configured to modify (change) the first monomeric protein, and the second modified protein can be configured to modify (change) the second monomeric 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 contain different C inteins. In addition, 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.

[0159] In the first monomeric protein, the order of the first reaction tag and the first dimer-forming domain can be set, for example, according to the attachment position of the first adduct in the first modified protein. When the first adduct is attached to the N-terminal side of the first dimer-forming domain, in the first monomeric protein, the first reaction tag and the first dimer-forming domain are arranged in sequence from the N-terminal side. When the first adduct is attached to the C-terminal side of the first dimer-forming domain, in the first monomeric protein, the first dimer-forming domain and the first reaction tag are arranged in sequence from the N-terminal side.

[0160] In the first reaction tag, the order of the binding tag and the first C intein can be set, for example, according to the attachment position of the first adduct in the first modified protein. When the first adduct is attached to the N-terminal side of the first dimer-forming domain, the binding tag and the first C intein are sequentially arranged from the N-terminal side. In the second reaction tag, the order of the binding partner and the second C intein can be set, for example, according to the attachment position of the second adduct in the second modified protein. When the second adduct is attached to the N-terminal side of the second dimer-forming domain, the binding partner and the second C intein are sequentially arranged from the N-terminal side.

[0161] In the first monomeric protein, the order of the binding tag, the first C intein, and the first dimer-forming domain can be set, for example, according to the attachment position of the first adduct in the first modified protein. When the first adduct is attached to the N-terminal side of the first dimer-forming domain, the binding tag, the first C intein, and the first dimer-forming domain are sequentially arranged from the N-terminal side. In addition, in the second monomeric protein, the order of the binding partner, the second C intein, and the second dimer-forming domain can be set, for example, according to the attachment position of the second adduct in the second modified protein. When the second adduct is attached to the N-terminal side of the second dimer-forming domain, the binding partner, the second C intein, and the second dimer-forming domain are sequentially arranged from the N-terminal side.

[0162] In the first monomeric protein, the binding tag, the first C intein and the first dimer-forming domain are respectively directly or indirectly bound (connected). When a heterodimeric protein is produced in which a plurality of domains consisting only of natural amino acid sequences are connected, it is preferred that the first C intein and the first dimer-forming domain are directly connected.

[0163] In the second monomeric protein, the binding partner, the second C intein and the second dimer-forming domain are respectively directly or indirectly bound (connected). When a heterodimeric protein is produced in which a plurality of domains consisting only of natural amino acid sequences are connected, it is preferred that the second C intein and the second dimer-forming domain are directly connected.

[0164] The direct binding refers to the binding of the amino acid at the N-terminus or C-terminus of a certain polypeptide or domain to the amino acid at the C-terminus or N-terminus of another polypeptide or domain by forming a peptide bond. On the other hand, the indirect binding refers to the binding of the amino acid at the N-terminus or C-terminus of a certain polypeptide or domain to the amino acid at the C-terminus or N-terminus of another polypeptide or domain through a connecting peptide (peptide linker), that is, the amino acid at the N-terminus or C-terminus of a certain polypeptide or domain is bound to the amino acid at the C-terminus or N-terminus of the connecting peptide by forming a peptide bond, and the amino acid at the other end of the connecting peptide is bound to the amino acid at the N-terminus or C-terminus of another polypeptide or domain. The length of the connecting peptide is, for example, 5 to 15 amino acids. The connecting peptide can use a known connecting peptide, as a specific example, GS linker (GS, GGS or GGGGS (SEQ ID NO: 10)), a connecting peptide formed by repeating the GS linker ([GS] l , [GGS] m , or [GGGGS] n (l, m and n are each integers greater than 2)), GGGSGG (serial number 11), etc.

[0165] In the present invention, if the heterodimeric protein is a multispecific antibody, the dimeric protein, for example, includes the CH2 region and CH3 region of the Fc region of the immunoglobulin (antibody), or part or all of the hinge region of the immunoglobulin and the CH2 region and CH3 region 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, for example, include the CH2 region and CH3 region of the Fc region of the immunoglobulin (antibody), or part or all of the hinge region of the immunoglobulin and the amino acid sequence of the CH2 region and CH3 region of the Fc region of the immunoglobulin. The immunoglobulin is, for example, a human antibody, preferably human IgG.

[0166] The first monomer protein may contain other polypeptides, such as solubilization domains, signal peptides, etc., on the N-terminal side of the first reaction tag, for example. In addition, the second monomer protein may contain other polypeptides, such as solubilization domains, signal peptides, etc., on the N-terminal side of the second reaction tag, for example. The solubilization domain is preferably a polypeptide that, when the solubilization domain is fused with the polypeptide or protein, the expression amount of the first monomer protein or the second monomer protein expressed in the transformant described later is at least increased by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 100% or more compared to the case where the polypeptide or protein without the solubilization domain is expressed. The solubilization domain may be, for example, a protein or a partial polypeptide thereof. Specific examples of the solubilizing domain include GST, MBP, thioredoxin, antibodies, antibody variants such as single-chain antibodies, and the like.

[0167] If the first monomer protein comprises the solubilization domain, the number of solubilization domains in the first monomer protein may be one or more. In the latter case, the solubilization domain may be one or more.

[0168] If the second monomer protein comprises the solubilization domain, the number of the solubilization domains in the second monomer protein may be one or more. In the latter case, the solubilization domain may be one or more.

[0169] The first monomer protein and the second monomer protein may include, for example, a purification tag for purifying the first monomer protein, the second monomer protein or the heterodimer protein. The purification tag may, for example, use the affinity tag. The purification tag may, for example, be attached to at least one of the N-terminus (side) and the C-terminus (side) of the first dimer-forming domain and the second dimer-forming domain.

[0170] The reaction conditions in the formation process can be appropriately set as long as the first monomer protein and the second monomer protein can form a dimer, and the reaction conditions (binding conditions) of the binding tag and the binding partner and / or the reaction conditions (binding conditions) of the first dimer-forming domain and the second dimer-forming domain can be considered. As a specific example, the reaction temperature in the formation process is, for example, 4 to 50°C, or 10 to 45°C. The reaction time in the formation process is, for example, 0.1 to 24 hours or 0.5 to 12 hours. The reaction pH of the formation process is, for example, pH 4 to 10 or pH 5 to 9.

[0171] When the binding tag and the binding partner are directly bound, the reaction conditions of the formation process can be set to reaction conditions that allow sufficient binding between the binding tag and the binding partner. As a specific example, when the binding tag and the binding partner are the SpyTag and the SpyCatcher, the formation process can be performed, for example, at pH 5 to 8, 4 to 37° C., and in the presence of a buffer. When the binding between the binding tag and the binding partner is generated by an enzyme reaction, the reaction conditions of the formation process can be set, for example, based on the activity conditions of the enzyme used for the enzyme reaction.

[0172] When the first dimer-forming domain and the second dimer-forming domain are directly bound, the reaction conditions of the formation step can be set to reaction conditions that allow the first dimer-forming domain and the second dimer-forming domain to fully form a bond. As a specific example, if the first dimer-forming domain and the second dimer-forming domain are bound via a disulfide bond, in the formation step, for example, the reaction conditions can be set to be such that the disulfide bond is not reduced.

[0173] When the heterodimeric protein is a multispecific antibody, the reaction conditions of the forming step can be further set to conditions such that the first monomer protein and the second monomer protein can form a disulfide bond between the hinge regions of the immunoglobulin.

[0174] Next, in the manufacturing process, Figure 1As shown in (C) to (D), the heterodimeric protein is produced (generated) by reacting the dimeric protein with a modified protein that modifies the dimeric protein. Therefore, the production process can also be said to be a process of adding a polypeptide or protein derived from the modified protein to the dimeric protein to generate 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 monomeric protein, and the first adduct of the first modified protein is connected to the first monomeric protein, more specifically, to the N-terminal side of the first dimer-forming domain of the first monomeric protein (hereinafter also referred to as the "first connection process"). In addition, in the production process, the second N intein of the second modified protein reacts with the second C intein of the second monomeric protein, and the second adduct of the second modified protein is connected to the second monomeric protein, more specifically, to the N-terminal side of the first dimer-forming domain of the first monomeric protein (hereinafter also referred to as the "second connection process"). Thus, in the production step, the dimeric protein can be converted into a heterodimeric protein composed of a protein containing the first adduct and the first dimer-forming domain in sequence, and a protein containing the second adduct and the second dimer-forming domain in sequence.

[0175] The reaction in the manufacturing process is preferably carried out in the presence of an aqueous solvent such as a buffer, saline, water, etc. The dimeric protein, the first modified protein, and the second modified protein may be solid or liquid, but preferably liquid. When the dimeric protein, the first modified protein, and the second modified protein are solids such as powders, it is preferred that the dimeric protein, the first modified protein, and the second modified protein are dispersed in an aqueous solvent such as a buffer, saline, water, etc. in advance. In addition, 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 diluted in advance with an aqueous solvent such as a buffer, saline, water, etc. As a specific example of the reaction, it can be carried out by mixing a liquid containing the dimeric protein, a liquid containing the first modified protein, and / or a liquid containing the second modified protein.

[0176] In the manufacturing process, the first connecting process and the second connecting process may be partially or completely performed simultaneously, or may be performed separately. In the case where the first connecting process and the second connecting process are performed separately, there is no particular restriction on the order of the first connecting process and the second connecting process, and the second connecting process may be performed after the first connecting process is performed, or the first connecting process may be performed after the second connecting process is performed.

[0177] The first modified protein includes a first adduct attached to the first monomeric protein and a first N intein capable of reacting with the first C intein. The first modified protein includes, for example, the first adduct and the first N intein in order from the N-terminal side. In addition, the second modified protein includes a second adduct attached to the second monomeric protein and a second N intein capable of reacting with the second C intein. The second modified protein includes, for example, the second adduct and the second N intein in order from the N-terminal side. The first modified protein can introduce the first adduct to the N-terminal side of the first dimer-forming domain when the first N intein reacts with the first C intein by connecting the first adduct to the first N intein. As a result, in the first connection step, a fusion protein comprising the first dimer-forming domain and the first adduct can be generated. In addition, the second modified protein can introduce the second adduct to the N-terminal side of the second dimer-forming domain when the second N intein reacts with the second C intein by connecting the second adduct to the second N intein. As a result, in the second ligation step, a fusion protein including the second dimer-forming domain and the second adduct can be produced.

[0178] The description of the first N intein in the first modified protein and the second N intein in the second modified protein can refer to the above description. The first N intein and the second N intein are, for example, respectively configured to react with the first C intein and the second C intein. Thus, the first modified protein is configured to be able to modify (change) the first monomer protein, and the second modified protein is configured to be able to modify (change) the second monomer protein. Therefore, the first N intein and the second N intein are preferably N inteins derived from different inteins. In this case, the first N intein and the second N intein contain different N inteins. In addition, 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.

[0179] The first adduct and the second adduct refer to substances attached to the dimeric protein. For example, the adducts may also be referred to as modified substances. The first adduct and the second adduct include 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 setting the first adduct and the second adduct as different adducts, one dimer-forming domain and another dimer-forming domain constituting the dimeric protein may be attached with different adducts, thereby enabling the production of heterodimeric proteins. Next, the adducts that can be used for the first adduct and the second adduct are described.

[0180] Examples of the adducts include antibody variants, heavy chain antibodies (VHH), proteins such as receptors, cytokines, chemokines, toxins, enzymes, fluorescent proteins, and collagen binding domains; peptides such as insulin; and the like.

[0181] The antibody variant is a protein comprising an antigen-binding fragment of the antibody or a polypeptide formed by connecting the antigen-binding fragment through a connecting peptide. The antibody variant can include Fab, Fab', F(ab')2, single-chain antibody (scFv), tandem scFv, BiTE, Diabody, DART, TandAb, scDiabody, heavy chain antibody (VHH) and the variable domain of the VHH. The antibody variant can be a protein further connected to the antigen-binding fragment of another antibody relative to the protein comprising the antigen-binding fragment of the antibody or the polypeptide formed by connecting the antigen-binding fragment through a connecting peptide. In this case, the antibody variant can be constituted as a protein comprising 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), etc., and more specifically, it can be constituted as a protein comprising a part or all of the domain closer to the N-terminal side than the Fc region.

[0182] The target antigen in the antibody variant or the heavy chain antibody is not particularly limited and may be any antigen. Examples of the target antigen include tumor antigens, viral antigens, bacterial antigens, parasite antigens, antigens associated with autoimmune diseases, sugar chain antigens, and surface antigens of immune cells such as lymphocytes.

[0183] The surface antigen can, for example, include a cluster of differentiation (CD) antigen. The surface antigen can be an antigen that activates immune cells expressing the surface antigen when an antibody variant or a heavy chain antibody binds to the surface antigen, or an antigen that inhibits its activation. The immune cells can include, for example, T cells, NK cells, NKT cells, B cells, macrophages, etc. The antigens that activate the T cells are, for example, CD2, CD3, CD4, CD5, CD8α, CD8β, CD27, CD28, CD134 (OX40), CD137 (4-1BB), CD154, GITR, ICOS, etc., preferably CD3. The CD3 can include, for example, CD3γ, CD3δ, CD3ε, CD3ζ, CD3η, etc., preferably CD3ε. The antigens that inhibit the activation of the T cells can include, for example, CTLA4, PD-1, LAG3, B7-H3, TIM3, TIGIT, etc. Antigens that activate the NK cells include, for example, CD94 / NKG2C, CD94 / NKG2E, NKG2D / NKG2D, etc. Antigens that inhibit the activation of NK cells include, for example, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, KIR2DL4, KIR2DL5, KIR3DL3, etc. Antigens that activate the NKT cells include, for example, antigens that activate the T cells and antigens that activate the NK cells. Antigens that inhibit the activation of NKT cells include, for example, antigens that inhibit the activation of T cells and antigens that inhibit the activation of NK cells.

[0184] Examples of the receptor include cytokine receptors such as TNFα receptor, chemokine receptors, hormone receptors such as insulin receptor, and growth factor or proliferation factor receptors such as EGF receptor.

[0185] The cytokines and chemokines include, for example, tumor necrosis factors (TNF, Tumor Necrosis Factor) such as TNF-α and TNF-β; lymphotoxin; interleukins IL-1 to IL-38 (for example, 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 proliferation factors; adipokines; growth factors such as VEGF, etc.

[0186] Examples of the toxin include staphylococcal enterotoxin and the like.

[0187] Examples of the enzyme include luciferase and phosphatase.

[0188] Examples of the fluorescent protein include GFP (green fluorescent protein) and dsRED.

[0189] In the first modified protein, the first adduct and the first N-intein may be directly or indirectly bound (linked). When a heterodimeric protein is produced in which a plurality of domains consisting only of natural amino acid sequences are linked, it is preferred that the first adduct and the first N-intein are directly linked.

[0190] In the second modified protein, the second adduct and the second N intein may be directly or indirectly bound (linked). When a heterodimeric protein is produced in which a plurality of domains consisting only of natural amino acid sequences are linked, the second adduct and the second N intein are preferably directly linked.

[0191] In the first adduct and the second adduct, the direct or indirect connection may refer to the aforementioned description.

[0192] In the present invention, when the heterodimeric protein is a multispecific antibody, the first adduct and the second adduct are, for example, polypeptides comprising the antibody variant or the heavy chain antibody. As a specific example, when the heterodimeric protein is a bispecific antibody, the first adduct is a polypeptide comprising an amino acid sequence comprising the Fab region of the first antibody or the Fab region and all or part of the hinge region of the antibody. In addition, the second adduct is a polypeptide comprising an amino acid sequence comprising the Fab region of the 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 contain a part of the hinge region of the antibody, the hinge regions contained in the first adduct and the second adduct are respectively set to constitute the entire hinge region of the antibody when connected to the first dimer-forming domain and the second dimer-forming domain. The antibody variant is, for example, an antibody variant of a human antibody, preferably an antibody variant of human IgG.

[0193] The first modified protein may, for example, contain other polypeptides at its N-terminal side, such as the solubilization domain, the signal peptide, etc. In addition, the second modified protein may contain other polypeptides at its N-terminal side, such as the solubilization domain, the signal peptide, etc.

[0194] If the first modified protein comprises the solubilization domain, the number of solubilization domains in the first modified protein may be one or more. In the latter case, the solubilization domain may be one or more.

[0195] If the second modified protein comprises the solubilization domain, the number of solubilization domains in the second modified protein may be one or more. In the latter case, the solubilization domain may be one or more.

[0196] The first modified protein and the second modified protein may include, for example, a purification tag for purifying the first modified protein, the second modified protein or the heterodimeric protein. The purification tag may, for example, use the affinity tag. The purification tag may, for example, be attached to at least one of the N-terminus (side) and the C-terminus (side) of the first adduct and the second adduct.

[0197] In the manufacturing process, the reaction conditions of the first connection process and the second connection process may be the same or different. The reaction conditions of the first connection process may be set, for example, according to the types of the first C intein and the first N intein, more specifically, based on the enzyme activity conditions of the active intein formed by the combination of the first C intein and the first N intein. In addition, the reaction conditions of the second connection process may be set, for example, according to the types of the second C intein and the second N intein, more specifically, based on the enzyme activity conditions of the active intein formed by the combination of the second C intein and the second N intein. As a specific example, the reaction temperature of the manufacturing process is, for example, 0 to 40°C, 4 to 37°C, or 4 to 30°C. The reaction time of the manufacturing process is, for example, 1 minute to 48 hours, 30 minutes to 48 hours, or 1 to 48 hours. The reaction pH of the manufacturing process is, for example, pH 5 to 10, pH 6 to 9, or pH 6.5 to 9.

[0198] 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 may be a conventional protein purification method such as chromatography.

[0199] Thus, the production method of the present invention can produce a heterodimeric protein from a dimeric protein.

[0200] <Dimeric protein>

[0201] In another embodiment, the present invention provides a dimeric protein suitable for the manufacture of heterodimeric proteins. The protein (dimeric protein) of the present invention includes a dimeric protein containing a reaction tag, and the dimeric protein containing a reaction tag includes a first monomer protein and a second monomer protein, the first monomer protein includes a first reaction tag and a first dimer-forming domain capable of forming a dimer, the first reaction tag includes a binding tag and a first C intein capable of reacting with a first N intein, the second monomer protein includes a second reaction tag and a second dimer-forming domain capable of forming a dimer with the first dimer-forming domain in sequence, the second reaction tag includes 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 monomer protein and the second monomer protein form a dimer. According to the dimeric protein of the present invention, heterodimeric proteins can be appropriately manufactured by combining the first modified protein and the second modified protein.

[0202] <Monomeric protein>

[0203] In another embodiment, the present invention provides a monomer protein suitable for making the dimer protein. The protein of the present invention comprises a first monomer protein, the first monomer protein sequentially comprising a first reaction tag and a first dimer-forming 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 dimer-forming domain capable of forming a dimer with the first dimer-forming domain, 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. In addition, the protein of the present invention includes a second monomer protein, which in turn comprises a second reaction tag and a second dimer-forming 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 capable of reacting with the second N intein, the second monomer protein can form a dimer with the first monomer protein, the first monomer protein in turn comprises a first reaction tag and a first dimer-forming domain capable of forming a dimer with the second dimer-forming domain, the first reaction tag comprises a binding tag capable of binding to the binding partner and a first C intein capable of reacting with the first N intein.

[0204] <Nucleic Acid>

[0205] In another embodiment, 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.

[0206] The nucleic acid of the present invention can be designed based on the amino acid sequence of the dimer protein of the present invention, the first monomer protein of the present invention and / or the second monomer protein of the present invention by replacing the corresponding codons. The base sequence of the nucleic acid of the present invention can be codon-optimized, for example.

[0207] The nucleic acid of the present invention may also encode the first modified protein and / or the second modified protein.

[0208] <Expression vector>

[0209] In another embodiment, 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. According to the expression vector 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 (hereinafter also referred to as "protein of the present invention") can be appropriately produced by genetic engineering methods.

[0210] The expression vector of the present invention, for example, has the nucleic acid of the present invention inserted therein. The expression vector refers to, for example, a nucleic acid molecule capable of delivering the inserted gene to a target cell or the like.

[0211] The structure of the expression vector is not particularly limited as long as it contains 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 that the protein of the present invention encoded by the polynucleotide of the nucleic acid of the present invention can be expressed. 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 can be partially or completely inserted into the same expression vector, or can be inserted into different expression vectors respectively. If 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 different expression vectors, the expression vector of the present invention constitutes an expression vector set, including: an expression vector of a nucleic acid encoding the dimeric protein of the present invention, an expression vector of a nucleic acid encoding the first monomeric protein of the present invention, and / or an expression vector of a nucleic acid encoding the second monomeric protein of the present invention.

[0212] The expression vector may further comprise 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 comprising a nucleic acid encoding the dimer protein of the present invention, the first monomer protein of the present invention, and / or the second monomer protein of the present invention, or may be inserted into another expression vector.

[0213] 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 vector serving as a backbone (hereinafter also referred to as a "basic vector"). The type of the expression vector is not particularly limited and can be appropriately determined, for example, according to the type of the host.

[0214] The host may include, for example, non-human hosts such as microorganisms, animal cells, insect cells, or cultured cells thereof, isolated human cells or cultured cells thereof, mammalian cells, etc. The prokaryotes may include, for example, bacteria of the genus Escherichia such as Escherichia coli, and the genus Pseudomonas such as Pseudomonas putida. The eukaryotes may include, for example, yeasts such as Saccharomyces cerevisiae. The animal cells may include, for example, HEK293 cells, Expi293F cells, COS cells, CHO cells, etc. The insect cells may include, for example, Sf9, Sf21, etc.

[0215] The expression vector (basic vector) can include viral vectors and non-viral vectors. When the host is transformed by heat shock method as the introduction method, the expression vector can include binary vectors. The expression vector can include, for example, pETDuet-1, pQE-80L, pUCP26Km, etc. When transforming bacteria such as Escherichia coli, the expression vector can include, for example, pETDuet-1 vector (Novagen), pQE-80L (QIAGEN), pBR322, pB325, pAT153, pUC8, etc. When transforming the yeast, the expression vector can include, for example, pYepSec1, pMFa, pYES2, etc. When transforming the insect cells, the expression vector can include, for example, pAc, pVL, etc. When transforming the mammalian cells, the expression vector can include, for example, pcDNA3.1, pcDNA3.4, pCAG, pCAGEN, pCDM8, pMT2PC, etc.

[0216] Preferably, the expression vector has a regulatory sequence, for example, regulating 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 of the protein of the present invention. The regulatory sequence may, for example, include a promoter, a terminator, an enhancer, a polyadenylation signal sequence, a replication origin sequence (ori), etc. In the expression vector, the configuration of the regulatory sequence is not particularly limited. In the expression vector, the regulatory sequence may, for example, be configured to be able to functionally regulate the expression of the polynucleotide encoding the protein of the present invention and the expression of the protein of the present invention encoded by it, and may be configured according to known methods. The regulatory sequence may, for example, use a sequence previously present in the basic vector, or the regulatory sequence may be further inserted into the basic vector, or the regulatory sequence present in the basic vector may be replaced with another regulatory sequence.

[0217] The expression vector may further include a coding sequence for a selection marker, such as a drug resistance marker, a fluorescent protein marker, an enzyme marker, a cell surface receptor marker, and the like.

[0218] The method of inserting DNA, inserting the regulatory sequence and / or inserting the coding sequence of the selection marker into the expression vector can be performed, for example, by a method using restriction enzymes and ligases, or by using a commercially available kit or the like.

[0219] <Transformant and method for producing transformant>

[0220] In another embodiment, the present invention provides a transformant capable of producing the protein of the present invention and a method for producing the protein of the present invention. The transformant of the present invention contains a nucleic acid encoding the protein of the present invention. According to the transformant of the present invention, the protein of the present invention can be suitably produced.

[0221] In addition, the method for producing a transformant of the present invention comprises 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.

[0222] In the transformant of the present invention, the nucleic acid encoding the protein of the present invention can be referred to the description of the nucleic acid encoding the protein of the present invention. As the nucleic acid of the present invention, the expression vector of the present invention can be used.

[0223] 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.

[0224] The method for introducing the nucleic acid is not particularly limited and can be carried out by known methods. For example, the nucleic acid can be introduced through the expression vector. The introduction method can be appropriately set according to the type of the host. The introduction method can include, for example, an introduction method using a gene gun such as a particle gun, a calcium phosphate method, a polyethylene glycol method, a liposome transfection method using a liposome, an electroporation method, an ultrasonic nucleic acid introduction method, a DEAE-dextran method, a direct injection method using a micro glass tube, etc., a hydrodynamic method, a cationic liposome method, a method using an introduction aid, a method through Agrobacterium, etc. The liposome can include, for example, a lipofectamine and a cationic liposome, and the introduction aid can include, for example, exo-telopeptide collagen, nanoparticles, and polymers. When the host is a microorganism, for example, a method using Escherichia coli (E. coli) or Pseudomonas (Ps. putida) is preferably used. The polynucleotide encoding the protein of the present invention can be introduced into the host through the expression vector of the present invention, for example.

[0225] <Method for producing protein>

[0226] In another embodiment, the present invention provides a method for producing a dimeric protein suitable for producing a heterodimeric protein or a monomeric protein suitable for producing the dimeric protein. The method for producing a protein of the present invention comprises an expression step of expressing the nucleic acid of the present invention, the expression vector of the present invention and / or the expression vector group of the present invention. According to the method for producing a protein of the present invention, the dimeric protein of the present invention, the first monomeric protein and / or the second monomeric protein can be produced.

[0227] The protein of the present invention can be expressed, for example, by using the expression vector of the present invention. The method for expressing the protein of the present invention is not particularly limited, and a known method can be used, for example, using a host or a cell-free protein synthesis system.

[0228] In the former case, for example, it is preferred to use the host into which the protein of the present invention or a nucleic acid encoding the protein of the present invention is introduced, and to culture the host so as to express the protein of the present invention in the host. Thus, 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 by culturing the transformant, the protein of the present invention can be synthesized.

[0229] The host culture method is not particularly limited and can be appropriately set according to the type of the host. The culture medium used for culture is not particularly limited and can be appropriately determined according to the type of the host.

[0230] In the latter case, it is preferred to express the polynucleotide of the protein of the present invention in a cell-free protein synthesis system. In this case, the polynucleotide of the protein of the present invention can be expressed using an expression vector. The cell-free protein synthesis system can be performed by a known method using, for example, a cell extract, a buffer containing various components, and an expression vector introduced with a polynucleotide encoding the protein of the present invention, and can use a commercially available kit.

[0231] The method for producing a 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 purified product or a purified protein.

[0232] When recovering from the culture solution, in the recovery step, insoluble matter can be removed by filtering the culture supernatant, centrifuging, etc. In the recovery step, for example, the culture supernatant after the insoluble matter has been removed can be concentrated using an ultrafiltration membrane; salting out using ammonium sulfate precipitation or the like; dialysis; or chromatography using various columns such as ion exchange columns and gel filtration columns can be appropriately combined to separate and purify the protein of the present invention.

[0233] When recovering from the transformant, in the recovery step, the transformant is disrupted by, for example, pressure treatment, ultrasonic treatment, etc. The resulting disrupted liquid can be separated and purified by removing insoluble matter as described above to obtain the protein of the present invention.

[0234] The protein of the present invention obtained by the production method of the present invention can be used as a crude purified protein as it is, can be used as a partially purified protein that has been partially purified, or can be used as a purified protein that has been purified alone.

[0235] In addition, the production method of the present invention can also powder the obtained protein of the present invention by, for example, freeze drying, vacuum drying or spray drying. In this case, the production method of the present invention can, for example, pre-dissolve the protein of the present invention in a buffer solution such as acetate buffer, phosphate buffer, triethanolamine buffer, Tris-HCl buffer, GOOD's buffer (e.g., HEPES, PIPES, MES, MOPS, etc.).

[0236] <Screening method for heterodimeric proteins>

[0237] In another embodiment, the present invention provides a method for screening heterodimeric proteins that react with a desired target. The method for screening target-reactive heterodimeric proteins of the present invention includes: a manufacturing process, in which a dimeric protein containing a reaction tag is reacted with a modified protein that modifies the dimeric protein to manufacture a candidate heterodimeric protein; a detection process, in which the candidate heterodimeric protein is contacted with a target and the reaction between the candidate heterodimeric protein and the target is detected; a selection process, in which the candidate heterodimeric protein in which the reaction is detected is selected as a candidate substance that reacts with the target, and the manufacturing process is implemented by the manufacturing method of the present invention. By the screening method of the present invention, heterodimeric proteins that react with the desired target can be screened out.

[0238] In the production process, the candidate heterodimeric protein can be implemented 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 can be used as a candidate antigen-binding domain for evaluating the binding property to the target described below.

[0239] The candidate heterodimeric protein may include a label, such as a fluorescent substance such as a fluorescent protein or a fluorescent dye, or an enzyme such as a luciferase or a phosphatase.

[0240] Next, in the detection process, the candidate heterodimeric protein is contacted with the target, and the reaction between the candidate heterodimeric protein and the target is detected. In the detection process, the contact can be performed, for example, in the presence of the aqueous solvent. The contact conditions are not particularly limited, and can be set, for example, according to the type of target. The temperature of the detection process is, for example, 4 to 37°C, or 18 to 25°C. The time of the detection process is, for example, 0 to 120 minutes, 30 to 60 minutes.

[0241] The target may be a desired target for screening the reactivity of the heterodimeric protein. The target may be an example of the target antigen. In the contacting, the target may be fixed on a carrier or may be free. Examples of the carrier include substrates, beads, containers, etc. Examples of the container include microtiter plates, tubes, etc. When the target is a molecule that can be expressed in a cell, the target may be a cell that expresses the target.

[0242] Next, in the detection step, the reaction between the candidate heterodimeric protein and the target is detected. The reaction can be determined according to the types of the candidate heterodimeric protein and the target. As a specific example, when the candidate heterodimeric protein comprises an antibody such as a multispecific antibody or an antigen-binding fragment thereof, the reaction is, for example, binding. In addition, when the candidate heterodimeric protein comprises an enzyme, the reaction is, for example, a catalytic reaction. When the candidate heterodimeric protein comprises a receptor, the reaction is, for example, binding.

[0243] Then, in the detection process, by detecting the presence or absence of a 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 process, by detecting the degree of reaction between the candidate heterodimeric protein and the target, for example, the intensity of the reaction of the candidate heterodimeric protein to the target can be detected or analyzed (quantitative analysis).

[0244] If no reaction between the candidate heterodimeric protein and the target is detected, it can be evaluated that the candidate heterodimeric protein does not react with the target; if the reaction is detected, it can be evaluated that the candidate heterodimeric protein reacts with the target.

[0245] The analysis method of the reaction of the candidate heterodimeric protein with the target is not particularly limited and can be determined according to the type of the reaction. When the candidate heterodimeric protein comprises a label and the reaction is binding, the reaction of the candidate heterodimeric protein with the target, for example, the complex produced by the binding of the candidate heterodimeric protein with the target, can be performed by detecting the label.

[0246] Then, in the selection step, the candidate heterodimeric protein in which the reaction is detected is selected as a candidate substance that reacts with the target. Thus, according to the screening method of the present invention, it is possible to screen for a candidate heterodimeric protein that is presumed to be reactive with the target.

[0247] [Example]

[0248] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the embodiments described in the examples.

[0249] [Example 1]

[0250] It was confirmed that various additions can be introduced into the dimeric protein by the production method of the present invention.

[0251] Specifically, in Example 1, a protein containing a portion of the hinge region of a human IgG antibody and the CH2 region and CH3 region of the Fc region was used as a model of a dimer protein, and the Fab of the HER2 antibody and the Fab of the OKT3 antibody were used as a first adduct model and a second adduct model, respectively. Then, a first monomer protein, a second monomer protein, a first modified protein, and a second modified protein containing these components were prepared, and by the preparation method of the present invention, the Fab of the HER2 antibody (Herceptin) and the Fab of the OKT3 antibody were introduced into a protein containing an Fc region, and it was confirmed that a bispecific antibody capable of binding to HER2 and CD3 can be prepared.

[0252] (1) Preparation of proteins of each antibody fragment of the Herceptin-OKT3 bispecific antibody

[0253] In order to prepare the Herceptin-OKT3 bispecific antibody, the following monomeric proteins and modified proteins were prepared.

[0254] First monomer protein: a protein comprising SpyTag (binding tag), gp41-1C intein (first C intein), the C-terminal region of the hinge region of human IgG1, and the Fc region (SpyTag-VHH)

[0255] Second monomer protein: a protein comprising SpyCatcher (binding partner), cfa C intein (second C intein), the C-terminal side region of the hinge region of human IgG1, and the Fc region (SpyCatcher-VHH)

[0256] ·First modified protein (CD3-Fab):

[0257] A protein comprising the VL and CL regions of the hOKT3 antibody (CD3-Fab-L),

[0258] A complex of a protein (CD3-Fab-H) comprising the VH and CH1 regions of the hOKT3 antibody and the N-terminal region of the hinge region of human IgG1, and the go41-1 N intein (first N intein)

[0259] · Second modified protein (Her2-Fab):

[0260] A protein comprising the VL and CL regions of the Herceptin antibody (Her2-Fab-L),

[0261] A complex of a protein (Her2-Fab-C) comprising the VH and CH1 regions of the Herceptin antibody and the N-terminal region of the hinge region of human IgG1, and the cfa N-intein (second N-intein)

[0262] The plasmid vector capable of expressing CD3-Fab-H is constructed by the following steps. First, a synthetic gene (Eurofins Genomics) containing a base sequence encoding a signal peptide, the VH-CH1 domain of OKT3 (SEQ ID NO: 12), a partial sequence on the N-terminal side of the hinge region of human IgG1, a gp41-1 N-intein (SEQ ID NO: 7), a G1 linker, and a His tag is amplified in full length by PCR. The obtained full-length synthetic gene is connected to an animal cell expression vector (pCAGEN) to construct an expression vector for the recombinant protein. As shown in brackets, the expression vector is sequentially connected to a signal peptide, a VH-CH1 domain of OKT3, a partial sequence on the N-terminal side of the hinge region, a gp41-1 N-intein, a G1 linker, and a His tag as the CD3-Fab-H region (SEQ ID NO: 13) from the N-terminus to the C-terminus.

[0263] CD3-Fab-H region (SEQ ID NO: 13)

[0264] [MEFGLSWLFLVAILKGVQC][QVQLVQSGGGVVQPGRSLRLSKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYSLDYWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC][DKT][SGYCLDLKTQVQTPQGMKEISNIQVGDLVLSNTGYNEVLNVFPKSKKKSYKITLEDGKEIICSEEHLFPTQTGEMNISGGLKEGMCLYVKE][GGSGG][HHHHHH]

[0265] Next, a plasmid vector capable of expressing CD3-Fab-L was constructed by the following steps. First, a synthetic gene (Eurofins Genomics) containing a base sequence encoding a signal peptide and the VL-CL domain of OKT3 (SEQ ID NO. 14) was amplified in full length by PCR. The obtained full-length synthetic gene was connected to an expression vector (pCAGEN) for animal cells to construct an expression vector for the recombinant protein. As shown in brackets, the expression vector is connected to the signal peptide and the VL-CL domain of OKT3 in sequence from the N-terminus to the C-terminus as the CD3-Fab-L region (SEQ ID NO. 15).

[0266] CD3-Fab-L region (SEQ ID NO: 15)

[0267] [MDFQVQIFSFLLISASVIISRG][DIQMTQSPSSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPF TFGQGTKLQITRTVA][APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC]

[0268] The plasmid vector capable of expressing Her2-Fab-H is constructed by the following steps. First, for a synthetic gene (Eurofins Genomics company) comprising a base sequence encoding a signal peptide, the VH-CH1 domain of Herceptin (sequence number 16), a partial sequence of the N-terminal side of the hinge region of human IgG1, a Cfa N intein (sequence number 9), a G1 linker and a His tag, the full length is amplified by PCR. The obtained full-length synthetic gene is connected to an animal cell expression vector (pCAGEN) to construct an expression vector for the recombinant protein. As shown in brackets, the expression vector is sequentially connected to a signal peptide, a VH-CH1 domain of Herceptin, a partial sequence of the N-terminal side of the hinge region, a Cfa N intein, a G1 linker and a His tag as the Her2-Fab-H region (sequence number 17) from the N-terminus to the C-terminus.

[0269] Her2-Fab-H region (SEQ ID NO: 17)

[0270] [MEFGLSWLFLVAILKGVQC][EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC][DKT][CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP][GGSGG][HHHHHH]

[0271] Next, a plasmid vector capable of expressing Her2-Fab-L was constructed by the following steps. First, a synthetic gene (Eurofins Genomics) containing a base sequence encoding a signal peptide and the VL-CL domain of Herceptin (SEQ ID NO: 18) was amplified in full length by PCR. The obtained full-length synthetic gene was connected to an expression vector (pCAGEN) for animal cells to construct an expression vector for the recombinant protein. As shown in brackets, the expression vector is connected to the signal peptide and the VL-CL domain of Herceptin in sequence from the N-terminus to the C-terminus as the Her2-Fab-L region (SEQ ID NO: 19).

[0272] Her2-Fab-L region (SEQ ID NO: 19)

[0273] [METPAQLLFLLLLWLPESTG][DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPT FGQGTKVEI][KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC]

[0274] The plasmid vector capable of expressing SpyCatcher-VHH was constructed by the following steps. First, a synthetic gene (Eurofins Genomics) containing a base sequence encoding a signal peptide, Ia1 (single-chain antibody (solubilization domain), sequence number 20), a G1 linker, SpyCatcher (sequence number 1), a G1 linker, a Cfa C intein (sequence number 8), a partial sequence on the C-terminal side of the hinge region of human IgG1, and the CH2 region and CH3 region (sequence number 21) of the Fc region of human IgG was amplified in full length by PCR. The obtained full-length synthetic gene was connected to an animal cell expression vector (pCDNA3.4) to construct an expression vector for the recombinant protein. As shown in brackets, the expression vector as the SpyCatcherVHH region (SEQ ID NO. 22) is connected in sequence from N-terminus to C-terminus to a signal peptide, Ia1, a G1 linker, SpyCatcher, a G1 linker, a CfaC intein, a partial sequence on the C-terminal side of the hinge region of human IgG1, and the CH2 region and CH3 region of the Fc region of human IgG.

[0275] Fc region (SEQ ID NO: 21)

[0276] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0277] Ia1 (Sequence number 20)

[0278] QVQLQESGGGLVQAGGSLLLSCAASGRTFSSYAMGWFRQAPGKEREFVAAINWSGGSTSYADSVKGRFTISRDNTKNTVYLQMNSLKPEDTAAFYCAATYNPYSRDHYFPRMTTEYDYWGQGTQVTVSS

[0279] SpyCatcher - VHH region (Sequence number 22)

[0280] [MEFGLSWLFLVAILKGVQC][QVQLQESGGGLVQAGGSLLLSCAASGRTFSSYAMGWFRQAPGKEREFVAAINWSGGSTSYADSVKGRFTISRDNTKNTVYLQMNSLKPEDTAAFYCAATYNPYSRDHYFPRMTTEYDYWGQGTQVTVSS][GGSGG][DSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG][GGSGG][VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASNC][FNASYTCPPCP][APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK]

[0281] The plasmid vector capable of expressing SpyTag-VHH is constructed by the following steps. First, for a synthetic gene (Eurofins Genomics company) comprising a partial sequence of the C-terminal side of the hinge region of human IgG1 and the base sequence of the CH2 region and CH3 region of the Fc region of human IgG, the full length is amplified by PCR. The obtained full-length synthetic gene is connected to an animal cell expression vector (pCDNA3.4) to construct an expression vector of the recombinant protein. As shown in brackets, the expression vector is sequentially connected to a signal peptide, cAb GFP4, G1 linker, SpyTag, gp41-1C intein, a partial sequence of the C-terminal side of the hinge region of human IgG1 and the CH2 region and CH3 region of the Fc region of human IgG as the SpyTag-VHH region (sequence number 24) from N-terminus to C-terminus.

[0282] cAb GFP4 (SEQ ID NO: 23)

[0283] QVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS

[0284] SpyTag-VHH region (sequence number 24)

[0285] [MEFGLSWLFLVAILKGVQC][QVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLK PEDTAVYYCNVNVGFEYWGQGTQVTVSS][GGSGG][AHIVMVDAYKPTK][GGSGG][MMLKKILKIEELDERELIDIEVSSGNHLFYANDILTHNSAG][ASYTCPPCP ][APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK]

[0286] Next, each plasmid expression vector of the recombinant protein was transfected into Expi293F cells respectively. During the transfection process, a combination of the expression vector of CD3-Fab-H and the expression vector of CD3-Fab-L, a combination of the expression vector of Her2-Fab-H and the expression vector of Her2-Fab-L, or a combination of the expression vector of SpyCatcher-VHH and the expression vector of SpyTag-VHH was introduced into the cells. For the obtained transformants, HE400 medium was used to culture at 37° C. in a CO2 incubator for 7 days, and recombinant proteins (SpyTag-VH+SpyCatcher-VHH (Fc dimer), CD3-Fab-L+CD3-Fab-H (CD3-Fab), Her2-Fab-L+Her2-Fab-H (Her2-Fab)) were expressed in the culture supernatant.

[0287] Next, the culture supernatant containing the recombinant protein after the culture is collected.

[0288] (2) Purification of CD3-Fab and Her2-Fab

[0289] The culture supernatant was centrifuged at 1500rpm for 5 minutes. The reagent solution was added to the supernatant after centrifugation to a final concentration of 500mmol / l NaCl and 10mmol / l imidazole, and then passed through a Ni-NTA column (manufactured by FUJIFILM) with a column volume of 3ml balanced with a wash buffer (WashBuffer) (500mmol / l NaCl, 50mmol / l Tris-HCl, 10mmol / l imidazole), washed with 30ml of wash buffer, and eluted with 15ml of elution buffer (Elute Buffer) (500mmol / l NaCl, 50mmol / l Tris-HCl, 500mmol / l imidazole). The eluted fraction recovered was dialyzed using a dialysis buffer (150mmol / l NaCl, 50mmol / l HEPES).

[0290] (3) Purification of Fc dimers

[0291] The culture supernatant was centrifuged at 1500rpm for 5 minutes. It was passed through a protein A column (SUPrA company system) balanced with a washing buffer (50mmol / l sodium phosphate buffer, pH 7.5) and a column volume of 1ml, washed with 10ml washing buffer, and eluted with 5ml elution buffer (20mmol / l sodium citrate, 100mmol / l NaCl, pH 3.0). When the elution buffer was passed, 5ml of 100mmol / l sodium phosphate buffer (pH 7.5) was added to the eluent to neutralize it. The eluted fraction recovered was dialyzed with a dialysis buffer (150mmol / l NaCl, 50mmol / l HEPES).

[0292] Each protein sample 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 according to the following formula (1).

[0293] C = (A280-A320) / (l×ε)×(dilution factor)…(1)

[0294] C: molar concentration of protein

[0295] A280: absorbance at 280nm

[0296] A320: absorbance at 320nm

[0297] l: cuvette length

[0298] ε: molar absorptivity of protein

[0299] (4) Preparation of bispecific antibodies

[0300] The Herceptin-OKT3 bispecific antibody was prepared using the SpyTag-VHH+SpyCatcher-VHH (Fc dimer), CD3-Fab, and Her2-Fab obtained in Example 1(1). Specifically, SpyTag-VHH+SpyCatcher-VHH, CD3-Fab, and Her2-Fab were reacted by the PTS reaction to obtain the Herceptin-Okt3 bispecific antibody. A protein containing 2 μl of each antibody fragment obtained in Example 1(1) and (2) (dissolved in 150 mmol / l NaCl, 50 mmol / l HEPES (pH7.6), and 1 mmol / l TCEP) was added to a microtube to prepare a pre-PTS reaction sample. After the preparation, the pre-PTS reaction sample was placed in an incubator at 37°C for 24 hours to perform a PTS reaction. After the PTS reaction, a sample after the PTS reaction was obtained.

[0301] (5) Detection of the binding of bispecific antibodies formed by binding of respective antibody fragments

[0302] For the post-PTS reaction sample obtained in Example 1 (4), SDS-PAGE was used to detect whether the Herceptin-OKT3 bispecific antibody was prepared. Specifically, 10 μl of 5×SDS buffer was added to 40 μl of the post-PTS reaction sample, Fc region (SpyTag-VHH and SpyCatcher-VHH complex), 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 suspended. After the suspension, heat treatment was performed at 95°C for 5 minutes. After the heat treatment, it was applied to the wells of a 12.5% ​​polyacrylamide gel and electrophoresed at 150-200V. The Fc region, Herceptin, hOKT3, the sample before PTS reaction, the sample after PTS reaction and the marker were applied to the 12.5% ​​polyacrylamide gel. After the electrophoresis, the polyacrylamide gel was stained with CBB (Coomassie Brilliant Blue) staining solution for 5 minutes and destained with a destaining solution. The results are shown in FIG. Figure 2 shown.

[0303] Figure 2 These are photographs showing proteins before and after the reaction of each antibody fragment with PTS. Figure 2In the figure, the type of sample is shown on the top of the photo, and the molecular weight (kDa) is shown on the right side of the photo. Figure 2 The columns in FIG. 1 show, from the left, the Fc region, Herceptin, hOKT3, the sample before PTS reaction (0 hours of reaction), the sample after PTS reaction (24 hours of reaction), and the marker. Figure 2 As shown, bands representing four proteins constituting each antibody were detected in the sample after the PTS reaction. These results show that the Herceptin-OKT3 bispecific antibody was produced by the PTS reaction in the sample after the PTS reaction.

[0304] (6) Purification of bispecific antibodies

[0305] Next, the PTS reaction sample obtained in Example 1 (4) was purified. Specifically, after purifying the protein having the Fc region using a purification column, the protein having the Fab region was purified using a purification column. First, the protein having the Fc region was purified using a KanCap A column. After equilibrating a KanCap A column (manufactured by KANEKA, column volume 500 μl) with TBS buffer (150 mmol / l NaCl and 50 mmol / l Tris-HCl (pH 7.5)), the PTS reaction sample obtained in Example 1 (4) was loaded onto the column, and the fraction directly passing through the column was recovered. After the recovery, 20 ml of TBS buffer was loaded and the washing fraction was recovered. 2.8 ml of elution buffer (100 mmol / l glycine-HCl (pH 2.8)) was then loaded and the eluted fraction was recovered. When recovering the eluted fraction, in order to neutralize the eluted fraction, 1.2 ml of 1 mol / l Tris-HCl (pH 9.0) was added to the recovery tube in advance for recovery. Next, the recovered elution fraction was placed in a transparent membrane (semipermeable membrane) and dialyzed using a dialysis buffer (1×PBS). Next, the protein sample after the dialysis was purified to have a protein with a Fab region. After balancing the KanCapL column (manufactured by KANEKA, column volume 500μl) with 1×PBS, the dialyzed protein sample was loaded, and the fraction that passed directly through the column was recovered. After the recovery, 10ml of 1×PBS was added to recover the washing fraction. Next, 7ml of elution buffer (100mmol / l glycine-HCl (pH2.8)) was added, and the elution fraction was recovered. When the elution fraction was recovered, in order to neutralize the elution fraction, 3ml of 1mol / l Tris-HCl (pH9.0) was added to the recovery tube in advance for recovery. Next, the recovered elution fraction was placed in a transparent membrane (semipermeable membrane) and dialyzed using the dialysis buffer (1×PBS). After the dialysis, it was concentrated using a concentrator. The purified sample was obtained by the concentration.

[0306] (7) Detection of purified bispecific antibodies

[0307] SDS-PAGE was used to detect whether the purified sample obtained in Example 1 (6) contained the Herceptin-Okt3 bispecific antibody. Specifically, 10 μl of 5×SDS buffer was added to the PTS reaction sample, the elution fraction purified by KanCap A column, the fraction directly passing through the column or the washing fraction (40 μl each), and the purified sample purified by KanCap L column, and suspended. After the suspension, heat treatment was performed at 95°C for 5 minutes. After the heat treatment, it was applied to the wells of a 12.5% ​​or 10% polyacrylamide gel, and electrophoresis was performed at 150-200V. The marker, PTS reaction sample, the fraction directly passing through the column, the washing fraction and the elution fraction were applied to the 12.5% ​​polyacrylamide gel. In addition, the purified sample was applied to a 10% acrylamide gel. After the electrophoresis, the polyacrylamide gel was stained with CBB staining solution for 5 minutes and decolorized with a decolorizing solution. These results are as shown in FIG. Figure 3 shown.

[0308] Figure 3 is a photograph showing the protein after PTS reaction and during column purification. Figure 3 In FIG. 1 , (A) shows a photograph of the protein before and after purification by a KanCap A column. Figure 3 (B) shows a photograph of the protein of the purified sample purified by a KanCap L column. Figure 3 In (A), the sample type is shown at the top of the photo, and the molecular weight (kDa) is shown on the left side of the photo. Figure 3 In (B), the type of sample is shown at the top of the photo, and the molecular weight (kDa) is shown on the left side of the photo. Figure 3 In (A), the columns show the marker, the sample after the PTS reaction (reaction for 24 hours), the fraction directly passed through the column (directly passed through the column), the washing fraction (washing), and the elution fraction (elution) from the left. Figure 3 As shown in (A), bands of each target antibody fragment were detected in the eluted fraction, and bands of the included proteins were also detected. Figure 3 As shown in (B), in the purified sample purified by KanCap L column, bands of each target antibody fragment were detected, and bands of contaminant proteins were not detected. These results indicate that only the target Herceptin-Okt3 bispecific antibody can be purified by using a two-step column for purification.

[0309] (8) Detection of the Uniqueness of Purified Bispecific Antibodies

[0310] The Herceptin-Okt3 bispecific antibody purified in Example 1 (6) was tested for singleness using gel filtration chromatography. Specifically, the protein purified in Example 1 (6) was passed through a gel filtration chromatography column (Superdex200increase 30 / 100GL, manufactured by GE Healthcare) at a flow rate of 0.5 mL / min, and the absorbance at 212 nm was measured at room temperature (about 25°C). 1×PBS was used as a buffer in the measurement. These results are shown in Table 1. Figure 4 shown.

[0311] Figure 4 is a diagram showing the elution pattern of proteins measured by gel filtration chromatography. Figure 4 In the figure, the horizontal axis represents the excluded volume (ml) and the vertical axis represents the absorbance. Figure 4 As shown, the purified protein obtained by the method of the present invention exhibits a single peak at 15 mL at an absorption wavelength of 212 nm. These results indicate that the purified Herceptin-Okt3 bispecific antibody obtained by the method of the present invention has a single property.

[0312] (9) Evaluation of the activity of CD3 positive cells

[0313] Whether the Herceptin-Okt3 bispecific antibody obtained in Example 1 (6) binds to cells expressing Her2 or CD3 was detected by flow cytometry. Specifically, an HPB-ALL cell line in which CD3 is overexpressed on the cell membrane surface was used and measured by flow cytometry. An equal amount of HPB-ALL cultured in a T75 flask was transferred to two 15 ml Falcon tubes and centrifuged at 1500 rpm for 5 minutes. After the centrifugation, the supernatant was removed by aspiration using an aspirator, and the necessary amount of 1×PBS was added for suspension to obtain 1×10 6cells / ml of cell suspension. Then, 1 ml of the suspension was injected into each of the three microtubes (a) to (c) and diluted with 1×PBS. After the dilution, the two microtubes (a) and (b) were centrifuged at 2000 rpm, 25°C, and 7 minutes. After the centrifugation, the supernatant was removed by suction using an aspirator, and 1 ml of 1×PBS was added. After the addition, the two microtubes (a) and (b) were centrifuged again at 2000 rpm, 25°C, and 7 minutes. After the centrifugation, the supernatant was removed by suction using an aspirator, and 1×PBS and the purified Herceptin-Okt3 bispecific antibody in Example 1 (6) were added to one microtube (b) until the final concentration of the Herceptin-Okt3 bispecific antibody was 0.05 μmol / l, and the mixture was inverted and mixed. After the inversion and mixing, it was allowed to stand for 20 minutes. After the standing, centrifuge at 2000rpm, 25℃, and 7 minutes. Then, add 1μl of OKT3-FITC (manufactured by Cosmo Bio) and 1ml of 1×PBS to tube (a), and add 1μl of anti-Fc-FITC (manufactured by AbCam) and 1ml of 1×PBS to tube (b), and mix them separately by inversion. After the inversion mixing, stand for 20 minutes. After the standing, centrifuge at 2000rpm, 25℃, and 7 minutes. After the centrifugation, use an aspirator to remove the supernatant, and add 1ml of 1×PBS. After the addition, centrifuge again at 2000rpm, 25℃, and 7 minutes. After the centrifugation, use an aspirator to remove the supernatant, and add 1ml of 1×PBS to suspend. After the suspension, sterilize with a mesh filter. After setting the measurement conditions, the negative control (c), positive control (a), and sample (b) were measured using the cell analyzer RF-500 (Sysmex). After the measurement, the measurement results were plotted into a graph using FC Salyzer. These results are shown in Figure 1. Figure 5 shown.

[0314] Figure 5 This is a graph showing the binding of the bispecific antibody to CD3-positive cells detected by flow cytometry. Figure 5 In the figure, the horizontal axis represents the fluorescence intensity and the vertical axis represents the number of cells. Figure 5 As shown, when the Herceptin-Okt3 bispecific antibody obtained by the method of the present invention is added to CD3-positive cells, the fluorescence intensity increases compared to when it is not added to CD3-positive cells. These results indicate that the Herceptin-Okt3 bispecific antibody obtained by the production method of the present invention binds to CD3-positive cells.

[0315] (10) Evaluation of activity on HER2-positive cells

[0316] The Herceptin-Okt3 bispecific antibody obtained in Example 1 (6) was detected by flow cytometry to determine whether it binds to breast cancer cells. Specifically, the SK-BR-3 cell line in which HER2 is overexpressed on the cell membrane surface was used and measured by flow cytometry. The supernatant of the SK-BR-3 cultured in the T75 flask was removed by suction using an aspirator and washed with 5 ml of 1×PBS / 5mmol / l EDTA. After the washing, the supernatant was removed by suction using an aspirator and 5 ml of trypsin was added. Then, it was allowed to stand in an incubator at 37°C for 5 minutes. After the standing, the flask was tapped gently. After the tapping, DMEM culture medium was added to make the total amount 10 ml, and the cells were separated from the flask by suspension to obtain a cell suspension. Next, the cell suspension was transferred to a 15 ml Falcon tube and the cell suspension was centrifuged at 1500 rpm for 5 minutes. After centrifugation, the supernatant was removed by suction using an aspirator, and a necessary amount of 1×PBS was added for suspension to obtain a cell suspension. Then, 1 ml of 1×10 6The suspension of cells was diluted with 1×PBS. After the dilution, two microtubes (d) and (e) were centrifuged at 2000rpm, 25°C, and 7 minutes. After the centrifugation, the supernatant was removed by suction using an aspirator, and 1 ml of 1×PBS was added. After the addition, the two microtubes (d) and (e) were centrifuged again at 2000rpm, 25°C, and 7 minutes. After the centrifugation, the supernatant was removed by suction using an aspirator. After the removal by suction, 1 μl of 5 mg / ml Herceptin (manufactured by Chugai Pharmaceutical Co., Ltd.) and 1 ml of 1×PBS were added to one microtube (d), and the mixture was inverted. At the same time, 1 ml of 1×PBS and the purified Herceptin-Okt3 bispecific antibody in Example 1 (6) were added to one microtube (e) until the final concentration of the Herceptin-Okt3 bispecific antibody was 0.1 μmol / l, and the mixture was inverted. After the inversion mixing, the two microtubes (d) and (e) are allowed to stand for 20 minutes. After the standing, the two microtubes (d) and (e) are centrifuged at 2000rpm, 25°C, and 7 minutes. After the centrifugation, the supernatant is removed by suction using an aspirator, and 1 ml of 1×PBS is added. After the addition, the two microtubes (d) and (e) are centrifuged again at 2000rpm, 25°C, and 7 minutes. After the centrifugation, the supernatant is removed by suction using an aspirator, and 1μl of anti-Fc-FITC (manufactured by AbCam) and 1 ml of 1×PBS are added to the two microtubes (d) and (e), and the mixture is inverted. After the inversion mixing, the mixture is allowed to stand for 20 minutes. After the standing, the two microtubes are centrifuged at 2000rpm, 25°C, and 7 minutes. After the centrifugation, the supernatant is removed by suction using an aspirator, and 1 ml of 1×PBS is added. After the addition, the two microtubes (d) and (e) are centrifuged again at 2000 rpm, 25°C, and 7 minutes. After the centrifugation, the supernatant is removed by suction using an aspirator, and 1 ml of 1×PBS is added for suspension. After the suspension, sterilization is performed using a mesh filter. After setting the measurement conditions, the cell analyzer RF-500 (manufactured by Sysmex) is used to perform measurements in the order of negative control (f), positive control (d), and sample (e). After the measurement, the measurement results are plotted into a graph using FCSalyzer. These results are shown in FIG. Figure 6 shown.

[0317] Figure 6 is a graph showing the binding of the bispecific antibody to HER2-positive cells detected by flow cytometry. Figure 6 In the figure, the horizontal axis represents the fluorescence intensity and the vertical axis represents the number of cells. Figure 6As shown, when the Herceptin-Okt3 bispecific antibody obtained by the production method of the present invention is added to HER2-positive cells, the fluorescence intensity increases compared to when it is not added to HER2-positive cells. These results indicate that the Herceptin-Okt3 bispecific antibody obtained by the method of the present invention binds to HER2-positive breast cancer cells.

[0318] (11) Evaluation of the maintenance of activity against HER2-positive cells by surface plasmon resonance

[0319] The surface plasmon resonance method was used to examine whether the Herceptin-Okt3 bispecific antibody obtained in Example 1 (6) could maintain binding to HER2. Specifically, the Herceptin-Okt3 bispecific antibody was allowed to flow through the HER2 fixed on the sensor chip at a predetermined flow rate, and was measured using an intermolecular interaction analyzer. The purified Herceptin-Okt3 bispecific antibody in Example 1 (6) was diluted to 1 nmol / l, 2 nmol / l, 4 nmol / l, 8 nmol / l and 16 nmol / l with 1×PBS-Tween20. After the dilution, a surface plasmon resonance measuring device (BiacoreT200, manufactured by Cytiva) was used to allow the diluted solution containing the Herceptin-Okt3 bispecific antibody to flow through the HER2-hFc fixed on the CM5 sensor chip (manufactured by GE Healthcare) to measure the binding strength. The results are as follows. Figure 7 shown.

[0320] Figure 7 It is a graph showing the maintenance of the binding of the bispecific antibody to HER2 detected by surface plasmon resonance. Figure 7 In the figure, the horizontal axis represents time (minutes) and the vertical axis represents response units (RU). Figure 7 As shown in Figure 2, when the Herceptin-Okt3 bispecific antibody obtained by the method of the present invention is added to HER2, the dissociation equilibrium constant Kd value is 2.25×10 -9 (M) These results indicate that the Herceptin-Okt3 bispecific antibody obtained by the production method of the present invention maintains binding to HER2.

[0321] Although the present invention has been described above with reference to the embodiments, the present invention is not limited to the above embodiments. A person skilled in the art can make various changes to the structure and details of the present invention that he or she can understand within the scope of the present invention.

[0322] Patents, patent applications and documents cited in this specification are cited as references to this specification to the extent that their contents themselves are the same as those specifically described in this specification.

[0323] <Note>

[0324] A part or all of the above-mentioned embodiments and examples may be described as the following supplementary notes, but are not limited to the following contents.

[0325] <Method for producing heterodimeric protein>

[0326] (Note 1)

[0327] A method for producing a heterodimeric protein.

[0328] The method comprises the steps of reacting a dimeric protein containing a reactive tag with a modified protein for modifying the dimeric protein to produce a heterodimeric protein,

[0329] The dimer protein containing the reactive tag comprises a first monomer protein and a second monomer protein,

[0330] The first monomer protein sequentially comprises a first reactive tag and a first dimer-forming domain capable of forming a dimer,

[0331] The first reaction tag comprises a binding tag and a first C intein,

[0332] The second monomeric protein sequentially comprises a second reactive tag and a second dimer-forming domain capable of forming a dimer with the first dimer-forming domain,

[0333] The second reactive tag comprises a binding partner capable of binding to the binding tag and a second C-intein,

[0334] The modified protein comprises a first modified protein and a second modified protein,

[0335] The first modified protein comprises a first N intein capable of reacting with the first C intein and a first adduct attached to the first monomeric protein,

[0336] The second modified protein comprises a second N intein capable of reacting with the second C intein and a second adduct attached to the second monomeric protein,

[0337] the first adduct and the second adduct are different adducts,

[0338] The first monomer protein and the second monomer protein form a dimer,

[0339] In the manufacturing process,

[0340] the first N intein of the first modified protein reacts with the first C intein of the first monomeric protein such that a first adduct of the first modified protein is attached to the first monomeric protein,

[0341] The second N intein of the second modified protein reacts with the second C intein of the second monomeric protein such that a second adduct of the second modified protein is attached to the second monomeric protein.

[0342] (Note 2)

[0343] The production method according to Supplementary Note 1, comprising a formation step of reacting the first monomer protein and the second monomer protein to form a dimer.

[0344] (Note 3)

[0345] The production method according to Supplementary Note 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.

[0346] (Note 4)

[0347] According to any one of Supplementary Notes 1 to 3, the binding between the binding tag and the binding partner is a covalent bond.

[0348] (Note 5)

[0349] The method according to any one of Notes 1 to 4, wherein the binding tag and the binding partner are a surface protein of Streptococcus pyogenes (SpyCatcher) and a peptide tag (SpyTag) capable of binding to the SpyCatcher, respectively; or

[0350] The binding tag and the binding partner are the SpyTag and the SpyCatcher, respectively.

[0351] (Note 6)

[0352] The production method according to any one of Supplementary Notes 1 to 5, wherein the dimeric protein comprises a part or all of the Fc region of an immunoglobulin.

[0353] (Note 7)

[0354] The production method according to Appendix 6, wherein the immunoglobulin is IgG, IgA, IgE, IgD or IgM.

[0355] (Note 8)

[0356] The production method according to Supplementary Note 7, wherein the IgG is IgG1, IgG2, IgG2a, IgG2b, IgG3 or IgG4.

[0357] (Note 9)

[0358] The production method according to any one of Supplementary Notes 6 to 8, wherein the Fc region of the immunoglobulin does not contain a mutation.

[0359] (Note 10)

[0360] According to any one of the manufacturing methods of Notes 1 to 9, the first C intein and the first N intein are respectively the C intein and the N intein of an integrated intein or a split intein, and / or the second C intein and the second N intein are respectively the C intein and the N intein of an integrated intein or a split intein.

[0361] (Note 11)

[0362] The production method according to any one of Supplementary Notes 1 to 10, wherein the first adduct and / or the second adduct is a protein.

[0363] (Note 12)

[0364] According to the production method described in Supplementary Note 11, the protein is selected from the group consisting of antibody variants, heavy chain antibodies (VHH), receptors, cytokines, chemokines, toxins, enzymes, fluorescent proteins and collagen binding domains.

[0365] (Note 13)

[0366] The production method according to Supplementary Note 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.

[0367] (Note 14)

[0368] The manufacturing method according to any one of Supplementary Notes 1 to 13,

[0369] The dimeric protein comprises part or all of the Fc region of an immunoglobulin,

[0370] The first adduct and the second adduct comprise antibody variants and / or heavy chain antibodies (VHH),

[0371] The heterodimeric protein is a multispecific antibody.

[0372] (Note 15)

[0373] The production method according to any one of Supplementary Notes 1 to 14, wherein in the dimeric protein, the first monomer protein and the second monomer protein are linked via a disulfide bond.

[0374] <Dimeric protein>

[0375] (Note 16)

[0376] A protein comprising a dimeric protein containing a reactive tag,

[0377] The dimer protein containing the reactive tag comprises a first monomer protein and a second monomer protein,

[0378] The first monomeric protein comprises a first reactive tag and a first dimer-forming domain capable of forming a dimer,

[0379] The first reactive tag comprises a binding tag and a first C intein capable of reacting with the first N intein,

[0380] The second monomeric protein sequentially comprises a second reactive tag and a second dimer-forming domain capable of forming a dimer with the first dimer-forming domain,

[0381] 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,

[0382] The first monomeric protein and the second monomeric protein form a dimer.

[0383] (Note 17)

[0384] The protein according to Supplementary Note 16, wherein the binding tag and the binding partner are a peptide tag and a peptide capable of spontaneously forming a covalent bond.

[0385] (Note 18)

[0386] According to the protein of Appendix 16 or 17, the binding between the binding tag and the binding partner is a covalent bond.

[0387] (Note 19)

[0388] The protein according to any one of Supplementary Notes 16 to 18, wherein the binding tag and the binding partner are a surface protein of Streptococcus pyogenes (SpyCatcher) and a peptide tag (SpyTag) capable of binding to the SpyCatcher, respectively; or

[0389] The binding tag and the binding partner are the SpyTag and the SpyCatcher, respectively.

[0390] (Note 20)

[0391] The protein according to any one of Supplementary Notes 16 to 19, wherein the dimeric protein comprises a part or the whole of the Fc region of an immunoglobulin.

[0392] (Note 21)

[0393] The protein according to Appendix 20, wherein the immunoglobulin is IgG, IgA, IgE, IgD or IgM.

[0394] (Note 22)

[0395] The protein according to Supplementary Note 21, wherein the IgG is IgG1, IgG2, IgG2a, IgG2b, IgG3 or IgG4.

[0396] (Note 23)

[0397] The protein according to any one of Appendix 20 to 22, wherein the Fc region of the immunoglobulin does not contain a mutation.

[0398] (Note 24)

[0399] According to the protein described in any one of Notes 16 to 23, the first C intein and the first N intein are respectively the C intein and the N intein of an integrated intein or a split intein, and / or the second C intein and the second N intein are respectively the C intein and the N intein of an integrated intein or a split intein.

[0400] <Monomeric protein>

[0401] (Note 25)

[0402] A protein comprising a first monomeric protein,

[0403] The first monomer protein sequentially comprises a first reactive tag and a first dimer-forming domain capable of forming a dimer,

[0404] 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,

[0405] The first monomer protein is capable of forming a dimer with the second monomer protein,

[0406] The second monomeric protein comprises a second reactive tag and a second dimer-forming domain capable of forming a dimer with the first dimer-forming domain,

[0407] 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.

[0408] (Note 26)

[0409] A protein comprising a second monomeric protein,

[0410] The second monomer protein sequentially comprises a second reactive tag and a second dimer-forming domain capable of forming a dimer,

[0411] The second reactive tag comprises a binding partner capable of binding to the binding tag and a second C intein capable of reacting with the second N intein,

[0412] The second monomer protein is capable of forming a dimer with the first monomer protein,

[0413] The first monomeric protein sequentially comprises a first reactive tag and a first dimer-forming domain capable of forming a dimer with the second dimer-forming domain,

[0414] 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.

[0415] (Note 27)

[0416] The protein according to Appendix 25 or 26, wherein the binding tag and the binding partner are a peptide tag and a peptide capable of spontaneously forming a covalent bond.

[0417] (Note 28)

[0418] According to any one of Appendix 25 to 27, the binding between the binding tag and the binding partner is a covalent bond.

[0419] (Note 29)

[0420] The protein according to any one of Notes 25 to 28, wherein the binding tag and the binding partner are a surface protein of Streptococcus pyogenes (SpyCatcher) and a peptide tag (SpyTag) capable of binding to the SpyCatcher, respectively; or

[0421] The binding tag and the binding partner are the SpyTag and the SpyCatcher, respectively.

[0422] (Note 30)

[0423] The protein according to any one of Supplementary Notes 25 to 29, wherein the dimeric protein comprises a part or the whole of the Fc region of an immunoglobulin.

[0424] (Note 31)

[0425] The protein according to Appendix 30, wherein the immunoglobulin is IgG, IgA, IgE, IgD or IgM.

[0426] (Note 32)

[0427] The protein according to Supplementary Note 31, wherein the IgG is IgG1, IgG2, IgG2a, IgG2b, IgG3 or IgG4.

[0428] (Note 33)

[0429] The protein according to any one of Supplementary Notes 30 to 32, wherein a part or all of the Fc region of the immunoglobulin does not contain mutations.

[0430] (Note 34)

[0431] According to the protein described in any one of Notes 25 to 33, the first C intein and the first N intein are respectively the C intein and the N intein of an integrated intein or a split intein, and / or the second C intein and the second N intein are respectively the C intein and the N intein of an integrated intein or a split intein.

[0432] <Nucleic Acid>

[0433] (Note 35)

[0434] A nucleic acid encoding the protein described in any one of Notes 25 and 27 to 34.

[0435] (Note 36)

[0436] A nucleic acid encoding the protein according to any one of Appendix 26 to 34.

[0437] <Expression vector>

[0438] (Note 37)

[0439] An expression vector comprising the nucleic acid described in Appendix 35 and / or the nucleic acid described in Appendix 36.

[0440] (Note 38)

[0441] An expression vector according to Supplement 37, comprising a nucleic acid encoding a first modified protein and / or a nucleic acid encoding a second modified protein,

[0442] The first modified protein comprises a first N intein capable of reacting with the first C intein and a first adduct attached to the first monomeric protein,

[0443] The second modified protein comprises a second N intein capable of reacting with the second C intein and a second adduct attached to the second monomeric protein,

[0444] The first adduct and the second adduct are different adducts.

[0445] (Note 39)

[0446] According to the expression vector of Supplementary Note 38, the first adduct and / or the second adduct is a protein.

[0447] (Note 40)

[0448] According to the expression vector described in Appendix 39, the protein is selected from the group consisting of antibody variants, heavy chain antibodies (VHH), receptors, cytokines, chemokines, toxins, fluorescent proteins and collagen binding domains.

[0449] (Note 41)

[0450] According to the expression vector described in Appendix 40, 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.

[0451] (Note 42)

[0452] An expression vector set comprising: an expression vector containing the nucleic acid described in Appendix 35; and an expression vector containing the nucleic acid described in Appendix 36.

[0453] (Note 43)

[0454] The expression vector set according to Supplement 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,

[0455] The first modified protein comprises a first N intein capable of reacting with the first C intein and a first adduct attached to the first monomeric protein,

[0456] The second modified protein comprises a second N intein capable of reacting with the second C intein and a second adduct attached to the second monomeric protein,

[0457] The first adduct and the second adduct are different adducts.

[0458] (Note 44)

[0459] According to the expression vector set of Supplementary Note 43, the first adduct and / or the second adduct is a protein.

[0460] (Note 45)

[0461] According to the expression vector group described in Appendix 44, the protein is selected from the group consisting of antibody variants, heavy chain antibodies (VHH), receptors, cytokines, chemokines, toxins, fluorescent proteins and collagen binding domains.

[0462] (Note 46)

[0463] According to the expression vector set described in Appendix 45, 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.

[0464] <Transformants>

[0465] (Note 47)

[0466] A transformant comprising the nucleic acid of Appendix 35, the nucleic acid of Appendix 36, the expression vector of any one of Appendixes 37 to 41 and / or the expression vector set of any one of Appendixes 42 to 45.

[0467] <Method for producing protein>

[0468] (Note 48)

[0469] A method for producing a protein, comprising the step of expressing the nucleic acid described in Appendix 35, the nucleic acid described in Appendix 36, the expression vector described in any one of Appendixes 37 to 41, and / or the expression vector group described in any one of Appendixes 42 to 45.

[0470] (Note 49)

[0471] According to the manufacturing method described in Supplementary Note 48, the expression step comprises:

[0472] A step of culturing the transformant described in Supplementary Note 47;

[0473] A separation step of isolating the first monomeric protein and / or the second monomeric protein.

[0474] <Screening method>

[0475] (Note 50)

[0476] A method for screening target-reactive heterodimeric proteins, comprising:

[0477] 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;

[0478] A detection step, contacting the candidate heterodimeric protein with a target, and detecting a reaction between the candidate heterodimeric protein and the target;

[0479] a selection step of selecting the candidate heterodimeric protein in which the reaction is detected as a candidate substance that reacts with the target,

[0480] The manufacturing process is carried out by the manufacturing method described in any one of Supplementary Notes 1 to 15.

[0481] (Note 51)

[0482] The screening method according to Note 50, wherein the reaction is binding.

[0483] (Note 52)

[0484] The screening method according to Appendix 50 or 51, wherein the target is selected from the group consisting of tumor antigens and cluster of differentiation (CD) antigens.

[0485] (Note 53)

[0486] The screening method according to any one of Supplementary Notes 50 to 52, wherein

[0487] The candidate foreign protein includes a marker,

[0488] In the detection step, the marker is detected.

[0489]

Industrial Applicability

[0490] As described above, according to the present invention, for heterodimeric proteins such as bispecific antibodies, heterodimeric proteins consisting of domains consisting only of natural amino acid sequences can also be manufactured. In addition, according to the present invention, the desired modified peptide can be loaded on the dimeric protein. Therefore, the present invention is extremely useful in, for example, the medical field, the pharmaceutical manufacturing field, etc.

Claims

1. A method for producing a heterodimeric protein, The method comprises the steps of reacting a dimeric protein containing a reactive tag with a modified protein for modifying the dimeric protein to produce a heterodimeric protein, The dimer protein containing the reactive tag comprises a first monomer protein and a second monomer protein, The first monomer protein sequentially comprises a first reactive tag and a first dimer-forming domain capable of forming a dimer, The first reaction tag comprises a binding tag and a first C intein, The second monomeric protein sequentially comprises 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, The modified protein comprises 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 attached 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 attached to the second monomeric 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 such that a first adduct of the first modified protein is attached 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 such that a second adduct of the second modified protein is attached to the second monomeric protein. 2 . The production method according to claim 1 , comprising a step of reacting the first monomer protein and the second monomer protein to form a dimer. 3 . 3 . The production method according to claim 1 , wherein the binding tag and the binding partner are a peptide tag and a peptide capable of spontaneously forming a covalent bond. The manufacturing method according to claim 1 or 2, wherein the binding between the binding tag and the binding partner is a covalent bond.

5. according to the manufacture method described in claim 1 or 2, wherein said binding label and said binding partner are respectively Streptococcus pyogenes surface protein (SpyCatcher) and can be combined with said SpyCatcher peptide label (SpyTag), or The binding tag and the binding partner are the SpyTag and the SpyCatcher, respectively. The production method according to claim 1 or 2, wherein the dimeric protein comprises part or all of the Fc region of an immunoglobulin. The production method according to claim 6 , wherein the immunoglobulin is IgG, IgA, IgE, IgD or IgM. The production method according to claim 7, wherein the IgG is IgG1, IgG2, IgG2a, IgG2b, IgG3 or IgG4.

9. The production method according to claim 6 or 7, wherein the Fc region of the immunoglobulin does not contain mutations.

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

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

12. The manufacturing method according to claim 11, wherein the protein is selected from the group consisting of antibody variants, heavy chain antibodies (VHH), receptors, cytokines, chemokines, toxins, enzymes, fluorescent proteins and collagen binding domains.

13. The manufacturing method according to 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 manufacturing method according to claim 1 or 2, wherein The dimeric protein comprises part or all of the Fc region of an immunoglobulin, The first adduct and the second adduct comprise antibody variants and / or heavy chain antibodies (VHH), The heterodimeric protein is a multispecific antibody. 15 . The production method according to claim 1 or 2 , wherein in the dimeric protein, the first monomer protein and the second monomer protein are linked by a disulfide bond.

16. A protein comprising a dimeric protein containing a reactive tag, The dimer protein containing the reactive tag comprises a first monomer protein and a second monomer protein, The first monomeric protein comprises 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 capable of reacting with the first N intein, The second monomeric protein sequentially comprises 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 capable of reacting with a second N intein, The first monomeric protein and the second monomeric protein form a dimer.

17. A protein comprising a first monomeric protein, The first monomer protein sequentially comprises a first reactive tag and a first dimer-forming 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 monomer protein is capable of forming a dimer with the second monomer protein, The second monomeric protein comprises 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 capable of reacting with a second N intein.

18. A protein comprising a second monomeric protein, The second monomer protein sequentially comprises a second reactive tag and a second dimer-forming domain capable of forming a dimer, The second reactive tag comprises a binding partner capable of binding to the binding tag and a second C intein capable of reacting with the second N intein, The second monomer protein is capable of forming a dimer with the first monomer protein, The first monomeric protein sequentially comprises a first reactive tag and a first dimer-forming domain capable of forming a dimer with the second dimer-forming 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. A nucleic acid encoding the protein of claim 16 .

20. A nucleic acid encoding the protein of claim 17. A nucleic acid encoding the protein of claim 18 . An expression vector comprising the nucleic acid of claim 19 .

23. An expression vector set comprising an expression vector containing the nucleic acid according to claim 20 and the nucleic acid according to claim 21.

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

25. A method for screening a target-reactive 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 candidate heterodimeric protein; a detection step, 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 heterodimeric protein in which the reaction is detected as a candidate substance that reacts with the target, The manufacturing process is carried out by the manufacturing method according to claim 1 or 2.

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

27. The screening method according to claim 25 or 26, wherein the target is selected from the group consisting of tumor antigens and cluster of differentiation (CD) antigens.

28. The screening method according to claim 25 or 27, wherein: The candidate foreign protein comprises a tag, In the detection step, the marker is detected.

Images