Method for producing heteromultimeric protein, protein, nucleic acid, expression vector, transformant, and method for producing protein

Abstract

Provided is a novel production method with which it is possible to produce a heteromultimeric protein such as a bispecific antibody. A method for producing a heteromultimeric protein according to the present invention comprises: a complex formation step for forming a first complex of the two proteins by bringing the two proteins into contact with each other, the two proteins comprising a first protein and a second protein, the first protein comprises a first binding tag, a first cleavage domain, and a first domain in this order from the N-terminal to the C-terminal, and the second protein comprises a first binding partner capable of binding to the first binding tag, a second cleavage domain, and a second domain in this order from the N-terminal to the C-terminal. The first protein and the second protein are capable of forming a dimer by means of the binding between the first domain and the second domain, the first protein and the second protein are bound to the first binding partner by means of the first binding tag, and the first domain is bound to the second domain to form the first complex; and a generation step in which the first cleaved domain and the second cleaved domain in the first complex are cleaved to generate a heterodimer of the first domain and the second domain.

Description

Technical Field

[0001] The present invention relates to a method for producing a heteromeric protein, a protein, a nucleic acid, an expression vector, a transformant, and a method for producing a protein. Background Art

[0002] In multispecific antibodies, one antibody molecule has antigen-binding domains that exhibit binding properties to different antigens, and various medical applications such as anticancer drugs and hemophilia treatment drugs have been developed by changing the target antigens.

[0003] However, there are problems in that the production efficiency of multispecific antibodies is extremely low and production is difficult. For example, a bispecific antibody consists of two heavy chains (H chains) and two light chains (L chains). In addition, when producing the bispecific antibody by expressing two H chains and two L chains, there are 10 combinations of two H chains and two L chains in the expressed antibodies, and in addition to the target bispecific antibody, nine unnecessary antibodies are produced (Patent Document 1).

[0004] Prior Art Documents:

[0005] Patent Documents

[0006] Patent Document 1: WO 2013 / 065708 Summary of the Invention

[0007] Problems to be Solved by the Invention

[0008] Therefore, an object of the present invention is to provide a novel method for producing a heteromeric protein such as a bispecific antibody.

[0009] Means for Solving the Problems

[0010] To achieve the above object, the production method of the present invention (hereinafter also referred to as "production method") is a method for producing a heteromeric protein, comprising:

[0011] a complex formation step of bringing two proteins into contact to form a first complex of the two proteins,

[0012] the two proteins include a first protein and a second protein,

[0013] the first protein sequentially includes a first binding tag, a first cleavage domain, and a first domain from the N-terminus to the C-terminus,

[0014] the second protein sequentially includes a first binding partner capable of binding to the first binding tag, a second cleavage domain, and a second domain from the N-terminus to the C-terminus,

[0015] The first protein and the second protein can form a dimer through the binding between the first domain and the second domain.

[0016] The first binding tag binds to the first binding partner, and the first domain binds to the second domain to form the first complex.

[0017] A production process that cleaves the first cleavage domain and the second cleavage domain in the first complex to generate a heterodimer of the first domain and the second domain.

[0018] The protein of the present invention sequentially includes a first binding tag capable of binding to a first binding partner, a first cleavage domain, and a first domain from the N-terminus to the C-terminus.

[0019] The protein of the present invention sequentially includes a first binding partner capable of binding to the first binding tag, a second cleavage domain, and a second domain from the N-terminus to the C-terminus.

[0020] The protein of the present invention sequentially includes a second binding tag capable of binding to a second binding partner, a third cleavage domain, and a third domain from the N-terminus to the C-terminus.

[0021] The protein of the present invention sequentially includes a first binding partner capable of binding to the second binding tag, a fourth cleavage domain, and a fourth domain from the N-terminus to the C-terminus.

[0022] The protein of the present invention contains two proteins.

[0023] The two proteins include a first protein and a second protein.

[0024] The first protein sequentially includes a first binding tag, a first cleavage domain, and a first domain from the N-terminus to the C-terminus.

[0025] The second protein sequentially includes a first binding partner capable of binding to the first binding tag, a second cleavage domain, and a second domain from the N-terminus to the C-terminus.

[0026] The first protein and the second protein form a dimer through the binding between the first domain and the second domain.

[0027] The first binding tag binds to the first binding partner.

[0028] The nucleic acid of the present invention encodes the protein of the present invention.

[0029] The vector of the present invention contains the nucleic acid of the present invention.

[0030] The transformant of the present invention contains the nucleic acid and / or vector of the present invention.

[0031] The method for producing the protein of the present invention includes an expression step of expressing the nucleic acid and / or vector of the present invention.

[0032] Effects of the Invention

[0033] According to the present invention, a novel production method capable of producing hetero - multimeric proteins such as bispecific antibodies can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Figure 1 It is a schematic diagram showing an example of the constituent elements of the protein used in the production method of the present invention and each step of producing a heterodimeric protein.

[0035] Figure 2 Figure 2 It is a schematic diagram showing an example of the constituent elements of the protein used in the production method of the present invention and each step of producing a heterotetrameric protein.

[0036] Figure 3 Figure 3 It is a schematic diagram showing an example of the constituent elements of the protein used in the production method of the present invention and each step of producing a heterotetrameric protein.

[0037] Figure 4 Figure 4 It is a photograph showing the result of SDS - PAGE.

[0038] Figure 5 Figure 5 It is a photograph showing the result of SDS - PAGE.

[0039] Figure 6 Figure 6 It is a graph showing the elution pattern of Herceptin obtained by size - exclusion chromatography.

[0040] Figure 7 Figure 7 It is a graph showing the binding of the light chain of the CD3 antibody and the heavy chain of the CD3 antibody to CD3 - positive cells measured by flow cytometry.

[0041] Figure 8 Figure 8 It is a graph showing the binding of the light chain of the Herceptin antibody and the heavy chain of the Herceptin antibody to HER2 - positive cells measured by flow cytometry.

[0042] Figure 9 Figure 9 ​​​​​​​​​​​​​​​​​​It is a photograph showing the results of SDS-PAGE.

[0043] Figure 10 Figure 10 It is a photograph showing the results of SDS-PAGE. Detailed implementation mode

[0044] <Definition>

[0045] 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. Examples of the shape of the polymer include straight chain, branched chain, and cyclic. The protein may also be referred to as a peptide or polypeptide.

[0046] In this specification, "monomeric protein" refers to a protein in a state where it is not bound or associated with other proteins.

[0047] In this specification, "dimeric protein" refers to a protein complex in a state where two proteins or protein subunits are bound or associated. 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 or more proteins are different proteins or subunits, the dimeric protein may also be referred to as a heterodimeric protein.

[0048] In this specification, "tetrameric protein" refers to a protein complex in a state where four proteins or protein subunits are bound or associated. When the four proteins are the same protein or subunit, the tetrameric protein may also be referred to as a homotetrameric protein. In addition, when one or more of the four proteins or subunits are different from other proteins or subunits, the tetrameric protein may also be referred to as a heterotetrameric protein.

[0049] In this specification, "multimeric protein" refers to a protein complex in a state where two or more proteins or protein subunits are bound or associated.

[0050] In this specification, "binding tag" refers to a polypeptide or substance that has specific binding properties with other molecules.

[0051] In this specification, "binding partner" refers to a polypeptide or substance that has specific binding properties with the binding tag.

[0052] In this specification, "domain" refers to a region of steric structure or functional integration in "protein", "polypeptide", and / or "peptide".

[0053] ​​In the present specification, the "cleavage domain" refers to a domain composed of a peptide, which is cleaved or decomposed by a substance having self-cleavage activity or other cleavage activity and is cleaved into two or more domains. Examples of substances having other cleavage activities include proteases, peptidases, modified inteins, and the like.

[0054] In the present specification, an "antibody" refers to a protein comprising one or more polypeptides encoded substantially or in part 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, and genes capable of encoding numerous immunoglobulin variable regions such as V regions, D regions, and J regions. The antibody includes, for example, heavy chains and light chains. The light chains include κ and λ, which constitute κ chains and λ chains, respectively. The heavy chains include γ, μ, α, δ, or ε, which constitute immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. The antibody may be a structural unit of a typical immunoglobulin (antibody) composed of a tetramer. In this case, the antibody is composed of two pairs of identical polypeptide chains, each pair consisting of a light chain (about 25 kDa) and a heavy chain (about 50-70 kDa). In addition, the N-terminus of each chain also defines a variable region composed of about 100-110 or more amino acids mainly involved in antigen recognition.

[0055] In the present specification, an "antigen-binding fragment" refers to a partial or partial polypeptide containing the antigen-binding site of an antibody. The antigen-binding fragment can be obtained by chemically or enzymatically treating the 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 derivatives thereof.

[0056] In the present specification, "purification" refers to identification and separation, recovery from components in a natural state, or a state of being identified and separated, and / or a state of recovery from components in a natural state. The "purification" can be carried out, for example, by obtaining at least one purification step. The purification can also be referred to as separation.

[0057] In the present specification, "separation" refers to separating the target from the substance containing the target and / or the state after separation. The separation can also be referred to as release.

[0058] 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 combination 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, etc. The nucleic acid can be referred to interchangeably with "polynucleotide" or "nucleic acid molecule".

[0059] In this specification, "host" refers to a cell and / or an individual into which an exogenous nucleic acid has been introduced. When the host is a cell, the host can also be referred to as a host cell.

[0060] In this specification, "vector" and "expression vector" refer to a recombinant plasmid or virus containing in vitro or in vivo a nucleic acid that is delivered to a host or a host cell. Examples of the "vector" and "expression vector" include viral vectors and non-viral vectors.

[0061] In this specification, "transformant" refers to a host into which an exogenous nucleic acid has been introduced.

[0062] In this specification, the source of various proteins, polypeptides, or peptides is not particularly limited and can be any animal. The animal can be, for example, a human or a non-human animal. Examples of the non-human animal include mammals such as mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, dolphins, sea lions, etc.

[0063] The present invention will be described below by way of examples, but the present invention is not limited to the following examples, etc., and can be implemented with any modifications. In addition, unless otherwise specified, the descriptions in the present invention and each embodiment can be cited with each other. In this specification, when using the expression "~", it is used in the sense of including the numerical values or physical values before and after it. In addition, in this specification, the expression "A and / or B" includes "only A", "only B", and "both A and B".

[0064] <Method for producing heteromultimeric protein>

[0065] In one embodiment, the present invention provides a method for producing a heteromultimeric protein. The production method of the present invention includes:

[0066] A complex formation step of bringing two proteins into contact to form a first complex of the two proteins,

[0067] The two proteins include a first protein and a second protein,

[0068] The first protein sequentially includes a first binding tag, a first cleavage domain, and a first domain from the N-terminus to the C-terminus.

[0069] The second protein sequentially includes a first binding partner capable of binding to the first binding tag, a second cleavage domain, and a second domain from the N-terminus to the C-terminus.

[0070] The first protein and the second protein can form a dimer through the binding between the first domain and the second domain.

[0071] By binding the first binding tag to the first binding partner and binding the first domain to the second domain, the first complex is formed.

[0072] A production step of cleaving the first cleavage domain and the second cleavage domain in the first complex to generate a heterodimer of the first domain and the second domain.

[0073] The inventors envisioned that for proteins that form dimers, by attaching specific binding tags or binding partners to each protein and forming a complex through the binding between the binding tag and the binding partner, it might be possible to efficiently produce dimers of two proteins. Through in-depth research, the inventors found that by using proteins with cleavage domains and the binding tag or the binding partner attached to two proteins, after forming the complex, a heterodimeric protein is obtained by cleaving the cleavage domain, thus establishing the present invention. In addition, regarding the presumed reaction mechanism of the production method of the present invention, an example of producing a heterodimeric protein will be given for illustration. However, as described later, the production method of the present invention can also be applied to other productions other than heterodimeric proteins. Therefore, according to the production method of the present invention, heteromultimeric proteins can be produced.

[0074] (Embodiment 1)

[0075] In the production method of the present embodiment, an example of producing a heterodimeric protein from two proteins will be described.

[0076] Taking the case of producing a heterodimeric protein 20 using a first protein 1 and a second protein 2 as an example for description. As Figure 1 (A) shows, the first protein 1 sequentially includes a first binding tag 11, a first cleavage domain 12, and a first domain 13 from the N-terminus to the C-terminus. In addition, the second protein 2 sequentially includes a first binding partner 21 capable of binding to the first binding tag 11, a second cleavage domain 22, and a second domain 23 from the N-terminus to the C-terminus.

[0077] First, as Figure 1As shown in (B), the first protein 1 and the second protein 2 are brought into contact. As a result, as shown by X, a bond is formed between the first domain 13 and the second domain 23. In addition, as shown by arrow Y, a bond is formed between the first binding tag 11 and the first binding partner 21. Thus, the first protein 1 and the second protein 2 form the first complex 10. Next, as Figure 1 shown in (C), the first cleavage domain 12 and the second cleavage domain 22 of the first complex 10 are cleaved. As a result, the first binding tag 11 and the first binding partner 21 disposed on the N-terminal sides of the first cleavage domain 12 and the second cleavage domain 22 are detached from the first complex 10. As a result, as Figure 1 shown in (C), a heterodimer protein 20 containing the first domain 13 and the second domain 23 can be produced. Therefore, according to the production method of the present invention, it is presumed that a desired heterodimer protein can be produced by arranging monomer proteins constituting the desired heterodimer in the first domain 13 and the second domain 23.

[0078] In the production method of the present embodiment, the first complex is formed by the binding of the first binding tag to the first binding partner and the binding of the first domain to the second domain. In addition, the first protein and the second protein can be prepared, for example, by genetic engineering techniques described in the method for producing a protein of the present invention described later. Therefore, the production method of the present invention may optionally include a first expression step of expressing the first protein and the second protein in a host cell before the complex formation step. The expression method in the expression step can refer to the description of the method for producing a protein, nucleic acid, expression vector, transformant, and protein of the present invention described later.

[0079] In the complex formation step, the first protein and the second protein are reacted. As a result, as Figure 1 shown in (B), in the complex formation step, the first complex of the first protein and the second protein is formed by the binding of the first binding tag to the first binding partner and the binding of the first domain to the second domain.

[0080] In the complex formation step, the complex of the first protein and the second protein can be formed, for example, by (1) the binding between the first binding tag and the first binding partner, or (2) the binding between the first domain and the second domain, or the binding of both (1) and (2). Since the ability to form dimers can be improved, it is preferable to form the complex by the binding of both (1) and (2).

[0081] The binding between the first domain and the second domain may be direct binding, indirect binding (association), or formed by both direct binding and indirect binding between the first domain and the second domain, preferably direct binding. The direct binding is a covalent bond, and specific examples include amide bonds (peptide bonds, isopeptide bonds, etc.) between amino acids, disulfide bonds between cysteines, etc. The indirect binding is a non-covalent bond, and specific examples include hydrogen bonds, hydrophobic bonds, etc.

[0082] The first domain and the second domain may adopt amino acid sequences that can form dimers conditionally or unconditionally when proteins containing each domain coexist. As specific examples, the first domain and the second domain can utilize the amino acid sequences of each subunit present in a protein dimer or the motif sequence that forms a dimer. The first domain and the second domain can utilize the amino acid sequences of each subunit present in a protein multimer or the motif sequence that forms a dimer. The protein dimer can be a homodimer or a heterodimer. As the protein dimer, for example, dimers of heavy and light chains of immunoglobulins (antibodies) such as IgA, IgD, IgE, IgG, IgM, etc.; proteins containing leucine zippers such as AP-1 (c-fos and c-jun), myc, max, mdx1, etc. in the myc family; G protein-coupled receptors; kinesin; receptor tyrosine kinases such as ErbB receptor family including epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), neurotrophic factor receptor, insulin receptor, insulin-like growth factor receptor, vascular endothelial growth factor receptor (VEGFR), stem cell factor receptor, etc.; Toll-like receptors such as TLR1-11, etc.

[0083] When the dimer formation motif sequence of the antibody is used as the first domain and the second domain, the first domain and the second domain respectively contain, for example, the light chain and the heavy chain of the antibody that binds to the first target antigen.

[0084] 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 include human antibodies, mouse antibodies, avian antibodies, rat antibodies, rabbit antibodies, etc. The first domain and the second domain are preferably derived from human antibodies, more preferably from human IgG.

[0085] As the amino acid sequences of the human IgG1, IgG2, IgG3, and IgG4, the amino acid sequences registered under UniProt accession numbers P01857, P01859, P01860, and P01861, respectively, can be referred to, for example.

[0086] When the dimer formation motif sequence of the antibody is used as the first domain and the second domain, the antibody can be an antibody with an altered constant region. In this case, the first domain and the second domain may include the amino acid sequences of the light chain or the heavy chain containing the constant region variant. Examples of the variant of the constant region include Fcab (Fc antigen binding, references 1 and 5) in which the amino acid sequence of the constant region is altered and binding ability to a target molecule is conferred, IgG hexamer (IgG Hexamer, references 2 to 4) in which the amino acid sequence of the constant region of an IgG antibody is altered and hexamer formation ability is conferred, DAF (Dual Action Fab, reference 5), Charge pair (Amgen, reference 5), SEEDbody (reference 5), Knobs-in-holes (reference 5), DVI-IgG (reference 5), etc.

[0087] Reference 1: G. Wozniak-Knopp et al., “Introducing antigen-binding sites in structural loops of immunoglobulin constant domains: Fc fragments with engineered HER2 / neu-binding sites and antibody properties”, Protein Engineering, Design and Selection, Volume 23, Issue 4, April 2010, Pages 289 - 297

[0088] Reference 2: Sopp, J.M. et al., “On-target IgG hexamerisation driven by a C-terminal IgM tail-piece fusion variant confers augmented complement activation.”, Commun. Biol., 4, 1031 (2021).

[0089] Reference 3: de Jong RN et al., “A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface.”, PLoS.Biol.(2016)14(1):e1002344.

[0090] Reference 4: Christoph A. Diebolder et al., “Complement Is Activated by IgG Hexamers Assembled at the Cell Surface”, Science, 343(6176), pages 1260-1263

[0091] 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

[0092] The first binding tag and the first binding partner are molecules in which the binding of the first binding tag to the first binding partner occurs conditionally or unconditionally when a protein containing the first binding tag and a protein containing the first binding partner coexist. The binding between the first binding tag and the first binding partner can be direct binding or indirect binding.

[0093] When the binding between the first binding tag and the first binding partner is direct binding, the first binding tag and the first binding partner can utilize, for example, peptide tags and peptides that can spontaneously form covalent bonds, or peptide tags and peptides that can form covalent bonds through the modification activity of other molecules.

[0094] When using the peptide tags and peptides capable of spontaneously forming covalent bonds, examples of the first binding tag and the first binding partner include Streptococcus pyogenes surface protein (SpyCatcher, SEQ ID NO: 1) and a peptide tag (SpyTag, SEQ ID NO: 2) or its variant capable of binding to the SpyCatcher; Streptococcus pneumoniae protein (SnoopCatcher, SEQ ID NO: 3) and a peptide tag (SnoopTag, SEQ ID NO: 4) or its variant capable of binding to the SnoopCatcher; modified Clostridium perfringens protein Cpe0147 439-563 (SEQ ID NO: 5) and a peptide tag Cpe0147 439-563 capable of binding to the Cpe0147 565-587 (SEQ ID NO: 6) or its variant, etc. Variants of the SpyCatcher and the SpyTag include, for example, SpyCatcher2 and SpyTag2 (Reference 6), SpyCatcher3 and SpyTag3 (Reference 7), SnoopCatcher and SnoopTag (Reference 8), etc. These peptide tags and peptides capable of spontaneously forming covalent bonds are bound, for example, by isopeptide bonds.

[0095] Reference 6: Anthony H. Keeble et al., “Evolving Accelerated Amidation by SpyTag / SpyCatcher to Analyze Membrane Dynamics”, Angew. Chem. Int. Ed., 2017, 56, pages 16521 - 16525

[0096] 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

[0097] Reference 8: Veggiani G, Nakamura T, Brenner MD, Gayet RV, Yan J, Robinson CV, Howarth M. Programmable polyproteams built using twin peptide superglues. Proc Natl Acad Sci U S A. 2016 Feb 2; 113(5): 1202-7. doi: 10.1073 / pnas.1519214113.

[0098] Amino acid sequence of Streptococcus pyogenes surface protein (SpyCatcher) (SEQ ID NO: 1)

[0099] DSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVE

[0100] TAAPDGYEVATAITFTVNEQGQVTVN

[0101] Amino acid sequence of SpyTag (SEQ ID NO: 2)

[0102] AHIVMVDAYKPTK

[0103] Amino acid sequence of Streptococcus pneumoniae protein (SnoopCatcher) (SEQ ID NO: 3)

[0104] KPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFEN

[0105] SEPAGYKPVQNKPIVAFQIVNGEVRDVTSIVPQDIPATYEFTNDKHYITNEPIPPK

[0106] Amino acid sequence of SnoopTag (SEQ ID NO: 4)

[0107] KLGDIEFIKVNK

[0108] Modified Clostridium perfringens protein Cpe0147 439-563 Amino acid sequence of (SEQ ID NO: 5)

[0109] MTLKTTVAADGVNGSSEKEALVSFENSKDGVDVKDTIDYKDLVANEKYNLTGKLMH

[0110] VKDDGSLEEVATKTTEVTAVENGSGQWELDFGNQKLQVGEKYVVFENAESVENLID

[0111] TDNNYELDTK

[0112] Peptide tag Cpe0147 565-587 The amino acid sequence of (SEQ ID NO: 6)

[0113] QVVKHEDKNDKAQTLIVEKPLE

[0114] When using the peptide tag and peptide that can form a covalent bond through the modification activity of other molecules, examples of the first binding tag and the first binding partner include a K tag and a Q tag. For example, the K tag and the Q tag can form a covalent bond by crosslinking the N-terminal lysine residue of the K tag with the N-terminal glutamate of the Q tag using a transglutaminase derived from bacteria.

[0115] When the binding between the first binding tag and the first binding partner is an indirect binding, the first binding tag and the first binding partner can utilize, for example, an affinity tag and a molecule that binds to the affinity tag. The first binding tag and the first binding partner are, for example, a peptide, a polypeptide, or a protein. Examples of the binding tag include a His-tag (His×6), a His-Strep-tag, a strep-tag, an avidin tag, a Flag (trademark)-tag, an 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, etc. If the first binding partner is a molecule that shows specific binding to a target molecule, such as an antibody or an antigen-binding fragment thereof, or a derivative thereof, the binding tag can be a peptide formed from any amino acid sequence that can bind to the molecule showing the specific binding.

[0116] The first binding partner can be appropriately set according to the type of the first binding tag. As specific examples, the first binding partner can be an antibody or an antigen-binding fragment thereof that recognizes the first binding tag, or a derivative thereof; a nucleic acid molecule such as an aptamer; glutathione, calmodulin; a sugar chain such as mannose; a metal such as nickel, cobalt, zinc, or an ion thereof.

[0117] The combination of the first binding tag and the first binding partner only needs to be a combination in which the first binding tag and the first binding partner can bind. As a specific example, if the first binding tag contains a His tag, the first binding partner is, for example, nickel. Additionally, if the first binding tag contains a Strep-tag or an avidin tag, the first binding partner, for example, contains biotin. If the first binding tag contains an epitope tag such as a Flag (trademark)-tag, an HA-tag, a T7-tag, a V5-peptide-tag, and / or a Myc-tag, the first binding partner can, for example, be an antibody against each epitope tag or its antigen-binding fragment, or their derivatives, etc. If the first binding tag contains a GST-tag, the first binding partner can, for example, be glutathione. If the first binding tag contains a CBP-tag, the first binding partner can, for example, be calmodulin. If the first binding tag contains an MBP-tag, the first binding partner can, for example, be mannose.

[0118] The first binding tag and the first binding partner can be functional equivalents within the range of maintaining the binding ability between the first binding tag and the first binding partner. If the first binding tag or the first binding partner is a peptide, polypeptide, or protein, the functional equivalents can, for example, be polypeptides consisting of amino acid sequences having 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the reference amino acid sequence of the first binding tag or the first binding partner and having the binding ability to the corresponding first binding tag or the first binding partner. The functional equivalents can, for example, be polypeptides consisting of amino acid sequences in which one or more amino acids are deleted, substituted, inserted, and / or added in the reference amino acid sequence of the first binding tag and the first binding partner and 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 preferably a conservative substitution.

[0119] In the first protein, the first binding tag can be one or more. In the latter case, the first binding tag can be one type or multiple types. In the first protein, the first binding tag is preferably located on the N-terminal side of the first protein.

[0120] In the second protein, the first binding partner may be one or more. In the latter case, the first binding partner may be one type or multiple types. In the second protein, the first binding partner is preferably located on the N-terminal side of the second protein. The number of the first binding tags and the first binding partners in each protein may be the same or different, but is preferably the same.

[0121] In the first protein and the second protein, the first binding tag and the first binding partner may be interchanged, and the combinations described above can be used interchangeably.

[0122] The first cleavage domain and the second cleavage domain are domains having an amino acid sequence that undergoes domain cleavage either conditionally or unconditionally. The cleavage is carried out in the subsequent production process after the formation of the first complex. Therefore, the first cleavage domain and the second cleavage domain are preferably cleavage domains that undergo cleavage conditionally.

[0123] The first cleavage domain and the second cleavage domain may be the same cleavage domain or different cleavage domains. Additionally, the first cleavage domain and the second cleavage domain may be one or more. In the latter case, the cleavage domains may be one type or multiple types. For example, in the subsequent production process, by making the first cleavage domain and the second cleavage domain the same cleavage domain, two cleavage domains can be cleaved in a single reaction, thus enabling efficient production of heterodimeric proteins.

[0124] The first cleavage domain and / or the second cleavage domain preferably contains a self-cleaving peptide and / or a cleavage sequence of a protease or peptidase. Examples of the self-cleaving peptide may include 2A self-cleaving peptide, intein, or its variants, etc. Examples of the cleavage sequence of the protease or peptidase may include the cleavage sequence of thrombin, Factor Xa recognition sequence, GST fusion protein cleavage enzyme (PreScission (trademark) protease) recognition sequence, furin protease-sensitive sequence, and carboxypeptidase-sensitive sequence, etc. The first cleavage domain and / or the second cleavage domain preferably contains a cleavage sequence of a protease or peptidase because this can inhibit non-specific cleavage.

[0125] In the first protein, the order of the first binding tag and the first cleavage domain can be set, for example, according to their positional relationship with the first domain. The first cleavage domain is disposed at a position closer to the first domain than the first binding tag. Thus, in the generation process described later, when the first cleavage tag is cleaved, the first binding tag can be detached from the first domain. Therefore, in the manufacturing method of the present embodiment, in the first protein, the first binding tag, the first cleavage domain, and the first domain are arranged in this order, for example, from the N-terminus to the C-terminus.

[0126] In the second protein, the order of the first binding partner and the second cleavage domain can be set, for example, according to their positional relationship with the second domain. The second cleavage domain is disposed at a position closer to the second domain than the first binding partner. Thus, in the generation process described later, when the second cleavage tag is cleaved, the first binding partner can be detached from the second domain. Therefore, in the manufacturing method of the present embodiment, in the second protein, the first binding partner, the second cleavage domain, and the second domain are arranged in this order, for example, from the N-terminus to the C-terminus.

[0127] In the first protein, the first binding tag, the first cleavage domain, and the first domain are directly or indirectly connected to each other. In addition, in the second protein, the first binding partner, the second cleavage domain, and the second domain are directly or indirectly connected to each other.

[0128] The direct binding means that the amino acid at the N-terminus or C-terminus of a certain polypeptide or domain binds 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 means that the amino acid at the N-terminus or C-terminus of a certain polypeptide or domain binds to the amino acid at the C-terminus or N-terminus of another polypeptide or domain through a linker peptide (peptide linker), that is, the amino acid at the N-terminus or C-terminus of a certain polypeptide or domain binds to the amino acid at the C-terminus or N-terminus of the linker peptide by forming a peptide bond, and the amino acid at the other end of the linker peptide binds to the amino acid at the N-terminus or C-terminus of another polypeptide or domain. The length of the linker peptide is, for example, 5 to 15 amino acids. Known linker peptides can be used as the linker peptide. As a specific example, a GS linker (GS, GGS, or GGGGS (SEQ ID NO: 7)), a linker peptide formed by repeating the GS linker ([GS]l, [GGS] m , or [GGGGS] n (l, m, and n are integers of 2 or more respectively)), GGGSGG (SEQ ID NO: 8), etc. can be cited.

[0129] The first protein may contain other polypeptides, such as solubilizing domains, signal peptides, etc., on the N-terminal side of the first binding tag. In addition, the second protein may contain other polypeptides, such as solubilizing domains, signal peptides, etc., on the N-terminal side of the first binding partner. The solubilizing domain is preferably such that when the solubilizing domain is fused to the polypeptide or protein, the expression level of the target protein, such as the first protein or the second protein expressed in the transformant described below, is increased by at least 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 100% or more, compared to the case where the polypeptide or protein is expressed without the solubilizing domain. The solubilizing domain may be, for example, a protein or a partial polypeptide thereof. As specific examples, the solubilizing domain may be GST, MBP, thioredoxin, an antibody, an antibody variant such as a single-chain antibody, etc.

[0130] If the first protein contains the solubilizing domain, the number of solubilizing domains in the first protein may be one or more. In the latter case, the solubilizing domain may be one or more types.

[0131] If the second protein contains the solubilizing domain, the number of solubilizing domains in the second protein may be one or more. In the latter case, the solubilizing domain may be one or more types.

[0132] The first protein and the second protein may include, for example, purification tags for purifying the first protein, the second protein, or the heterodimeric protein. The purification tag may be, for example, the affinity tag. The purification tag may be attached to at least one of the N-terminal (side) and C-terminal (side) of the first domain and the second domain.

[0133] The reaction conditions in the complex formation step may be any conditions under which the first protein and the second protein can form a dimer, and can be appropriately set by considering the reaction conditions (binding conditions) of the binding tag and the binding partner and / or the reaction conditions (binding conditions) of the first domain and the second domain. As a specific example, the reaction temperature in the complex formation step is, for example, 4 to 50 °C, or 10 to 45 °C. The reaction time in the complex formation step is, for example, 0.1 to 24 hours or 0.5 to 12 hours. The reaction pH in the complex formation step is, for example, pH 4 to 10 or pH 5 to 9.

[0134] When the binding tag and the binding partner bind directly, the reaction conditions of the complex formation step can be set to conditions under which binding between the binding tag and the binding partner is sufficiently formed. As a specific example, when the binding tag and the binding partner are the SpyTag and the SpyCatcher, the complex formation step can be carried out, for example, at pH 5 to 8, 4 to 37°C and in the presence of a buffer. When the binding tag and the binding partner are the SnoopCatcher and the SnoopTag, the complex formation step can be carried out, 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 enzymatic reaction, the reaction conditions of the complex formation step can be set, for example, based on the activity conditions of the enzyme used for the enzymatic reaction.

[0135] When the first domain and the second domain bind directly, the reaction conditions of the complex formation step can be set to conditions under which binding between the first domain and the second domain is sufficiently formed. As a specific example, if the first domain and the second domain are bound by a disulfide bond, in the complex formation step, the reaction conditions can be set, for example, to conditions under which the disulfide bond is not reduced. Additionally, as another specific example, if the first protein and the second protein are bound by an isopeptide bond, in the complex formation step, the reaction conditions can be set, for example, to conditions under which the isopeptide bond is not hydrolyzed.

[0136] When the heterodimeric protein is part of a multispecific antibody, the reaction conditions of the complex formation step can be further set to, for example, conditions under which the first protein and the second protein can form a disulfide bond between the heavy chain and the light chain.

[0137] The production method of the present embodiment may include a first purification step of purifying the first complex after the complex formation step. The purification method in the purification step can be a conventional protein purification method such as chromatography.

[0138] Next, in the generation step, the first cleavage domain and the second cleavage domain in the first complex are cleaved. As a result, as shown in Figure 1 (C), in the generation step, the bound first binding tag and the first binding partner dissociate, generating the heterodimeric protein of the first domain and the second domain.

[0139] In the generation step, the reaction conditions for the cleavage (e.g., reaction temperature, reaction time, reaction pH, etc.) can be set, for example, as the conditions under which the first cleavage domain and the second cleavage domain undergo a cleavage reaction. As a specific example, when the first cleavage domain and the second cleavage domain are thrombin cleavage sequences, the reaction temperature for the cleavage is, for example, 0 to 40°C, 4 to 37°C, or 4 to 30°C. The reaction time for the cleavage is, for example, 1 minute to 48 hours, 30 minutes to 48 hours, or 1 to 48 hours. The reaction pH for the cleavage is, for example, pH 5 to 10, pH 6 to 9, or pH 6.5 to 9.

[0140] In the generation step, if the first cleavage domain and / or the second cleavage domain is a domain cleaved by a protease or peptidase, the generation step can be carried out in the presence of the protease or peptidase. In this case, the generation step can be carried out under reaction conditions under which the protease or peptidase exhibits cleavage activity.

[0141] The production method of the present invention may include, after the generation step, a second purification step of purifying the heterodimeric protein. The purification method in the purification step can be, for example, a conventional protein purification method such as chromatography.

[0142] Thus, the production method of the present invention can produce a heterodimeric protein from two proteins.

[0143] In addition, in the production method of the present embodiment, an example of producing a heterodimeric protein using two isolated proteins is given and described, but the present invention is not limited thereto, and a first complex can also be produced from two proteins in a host cell described later. At this time, in the production method of the present embodiment, the first complex can be formed in the host cell by expressing the first protein and the second protein in the host cell. In the production method of the present embodiment, for example, a heterodimeric protein can be produced by purifying the first complex from the host cell and performing the generation step.

[0144] In addition, in the production method of the present embodiment, monomeric proteins are used for the two proteins, but the present invention is not limited thereto, and either one or both can be a dimeric or higher oligomeric protein, that is, the multimeric protein. As a specific example, when producing a heterotrimeric protein as the heteromultimeric protein, the first domain can be used as one protein in the trimeric protein, and the second domain can be used as a dimer of two proteins in the trimeric protein, thereby producing the trimeric protein. Therefore, the production method of the present invention can produce any multimeric protein.

[0145] (Embodiment 2)

[0146] Next, in the manufacturing method of the present embodiment, an example of manufacturing a heterotetrameric protein from four proteins will be given. In the manufacturing method of the present embodiment, as the proteins for manufacturing the heteromeric protein, in addition to the first protein 1 and the second protein 2 in the manufacturing method of Embodiment 1, the third protein and the fourth protein are also included, and these proteins are used to manufacture the heterotetrameric protein. Therefore, unless otherwise specified, the descriptions of various structures and processes in the manufacturing method of Embodiment 2 can refer to the descriptions of various structures and processes in the manufacturing method of Embodiment 1.

[0147] In the manufacturing method of the present embodiment, in addition to the two proteins used in the manufacturing method of Embodiment 1, the third protein 103 and the fourth protein 104 are also included. In the manufacturing method of the present embodiment, the case of manufacturing the heterotetrameric protein 120 using the first protein 101, the second protein 102, the third protein 103, and the fourth protein 104 will be described as an example. As Figure 2 (A) shows, the first protein 101 sequentially includes the first binding tag 111, the first cleavage domain 112, and the first domain 113 from the N-terminus to the C-terminus. The second protein 102 sequentially includes the first binding partner 121 capable of binding to the first binding tag 111, the second cleavage domain 122, and the second domain 123 from the N-terminus to the C-terminus. The third protein 103 sequentially includes the second binding tag 131, the third cleavage domain 132, and the third domain 133 from the N-terminus to the C-terminus. The fourth protein 104 sequentially includes the second binding partner 141 capable of binding to the second binding tag 131, the fourth cleavage domain 142, and the fourth domain 143 from the N-terminus to the C-terminus.

[0148] First, as Figure 2 (B) shows, in the complex formation step, the first protein 101, the second protein 102, the third protein 103, and the fourth protein 104 are brought into contact. Thereby, as shown by X1, a binding is formed between the first domain 113 and the second domain 123. In addition, as shown by X2, a binding is formed between the third domain 133 and the fourth domain 143. Furthermore, as shown by X3, a binding is formed between the second domain 123 and the fourth domain 143. Then, as shown by Y1, the binding between the first binding tag 111 and the first binding partner 121 is formed, and as shown by Y2, the binding between the second binding tag 131 and the second binding partner 141 is formed. Thereby, the first protein 101, the second protein 102, the third protein 103, and the fourth protein 104 form the second complex 110. Next, as Figure 2As shown in (C), the first cleavage domain 112, the second cleavage domain 122, the third cleavage domain 132, and the fourth cleavage domain 142 of the second complex 110 are cleaved. As a result, the first binding tag 111, the first binding partner 121, the second binding tag 131, and the second binding partner 141, which are arranged on the N-terminal side of the first cleavage domain 112, the second cleavage domain 122, the third cleavage domain 132, and the fourth cleavage domain 142, are detached from the second complex 110. Consequently, as Figure 2 shown in (C), a heterotetrameric protein 120 containing the first domain 113, the second domain 123, the third domain 133, and the fourth domain 143 can be produced. Therefore, according to the production method of the present invention, it is presumed that by arranging the monomeric proteins constituting the desired heterotetramer in the first domain 113, the second domain 123, the third domain 133, and the fourth domain 143, the desired heterotetrameric protein can be produced.

[0149] In the production method of the present embodiment, the second complex is formed by the binding of the first binding tag to the first binding partner, the binding of the second binding tag to the second binding partner, and the binding of the first domain to the second domain, the binding of the second domain to the fourth domain, and the binding of the third domain to the fourth domain. In addition, the first protein, the second protein, the third protein, and the fourth protein can be prepared, for example, by the genetic engineering techniques described in the method for producing the protein of the present invention described below. Therefore, the production method of the present invention may optionally include a first expression step of expressing the first protein, the second protein, the third protein, and the fourth protein in a host cell before the complex formation step. The expression method in the expression step may refer to the description of the method for producing the protein, nucleic acid, expression vector, transformant, and protein of the present invention described below.

[0150] In the complex formation step, the first protein, the second protein, the third protein, and the fourth protein are reacted. As a result, as Figure 2 shown in (B), in the complex formation step, the first protein, the second protein, the third protein, and the fourth protein form a second complex, and the second complex is formed by the binding of the first binding tag to the first binding partner, the binding of the second binding tag to the second binding partner, and the binding of the first domain to the second domain, the binding of the second domain to the fourth domain, and the binding of the third domain to the fourth domain.

[0151] In the complex formation step, the complex of the first to fourth proteins can be formed, for example, by (1) the binding between the binding tag and the binding partner, that is, the binding between the first binding tag and the first binding partner and / or the binding between the second binding tag and the second binding partner, (2) the binding between domains, that is, the binding between the first domain and the second domain, the binding between the second binding domain and the fourth domain, and / or the binding between the third domain and the fourth domain, or by the combination of both (1) and (2). Since the ability to form dimers can be improved, it is preferred to form the complex by the combination of both (1) and (2).

[0152] The binding between the first domain and the second domain, the binding between the second binding domain and the fourth domain, and / or the binding between the third domain and the fourth domain can be a direct binding, an indirect binding (association), or can be formed by both the direct binding and the indirect binding between the first domain and the second domain. The direct binding is a covalent bond binding. Specific examples include amide bonds (peptide bonds, isopeptide bonds, etc.) between amino acids, disulfide bonds between cysteines, etc. The indirect binding is a non-covalent bond binding. Specific examples include hydrogen bonds, hydrophobic bonds, etc.

[0153] The first to fourth domains can adopt amino acid sequences that can form a tetramer condition-dependently or non-condition-dependently when the proteins containing each domain coexist. As specific examples, the first to fourth domains can utilize the amino acid sequences of each subunit in a protein tetramer or the motif sequence that forms its dimer. The first to fourth domains can utilize the amino acid sequences of each subunit in a protein multimer or the motif sequence that forms its tetramer. The protein tetramer can be a heterotetramer containing partially identical subunits or a heterotetramer with all different subunits. Examples of the protein tetramer include immunoglobulins (antibodies) such as IgA, IgD, IgE, IgG, IgM; small bispecific antibodies diabody; etc. When using antibody subunits as the first to fourth domains, antibodies with altered constant regions can be used as the antibodies.

[0154] As the first to fourth domains, when using the motif sequence that forms the tetramer of the antibody, as an example, the first domain is the light chain of the antibody that binds to the first target, the second domain is the heavy chain of the antibody that binds to the first target, the third domain is the light chain of the antibody that binds to the second target, and the fourth domain is the heavy chain of the antibody that binds to the second target. The antibody that binds to the first target and the antibody that binds to the second target may recognize the same antigen or different antigens, but preferably recognize different antigens. In addition, the antibody that binds to the first target and the antibody that binds to the second target may recognize the same epitope or different epitopes, but preferably recognize different epitopes. By using the antibody that binds to the first target and the antibody that binds to the second target as antibodies that recognize different antigens or epitopes, the production method of the present invention can, for example, appropriately produce bispecific antibodies. As the first to fourth domains, when using the motif sequence that forms the tetramer of the antibody and using the first domain and the third domain as the light chains of the antibody and the second domain and the fourth domain as the heavy chains of the antibody, it is preferred that the second domain and the fourth domain use the heavy chains of antibodies in which the constant regions that specifically associate are altered. Examples of the antibodies in which the constant regions that specifically associate are altered include charge pairs, knobs-in-holes, and the like.

[0155] The first binding tag and the first binding partner are molecules in which the first binding tag and the first binding partner bind condition-dependently or non-condition-dependently when the protein containing the first binding tag and the protein containing the first binding partner coexist. The binding between the first binding tag and the first binding partner may be direct binding or indirect binding. The second binding tag and the second binding partner are molecules in which the first binding tag and the second binding partner bind condition-dependently or non-condition-dependently when the protein containing the second binding tag and the protein containing the second binding partner coexist. The binding between the second binding tag and the second binding partner may be direct binding or indirect binding. In the production method of the present embodiment, the binding between the first binding tag and the first binding partner and the binding between the second binding tag and the second binding partner are preferably direct binding. Thus, in the production method of the present embodiment, for example, the binding between non-specific binding tags and binding partners can be suppressed, and thus the desired heterotetramer can be efficiently produced.

[0156] Specific examples of the second binding tag and the second binding partner may cite the examples of the first binding tag and the first binding partner in Embodiment 1.

[0157] The first binding tag and the second binding tag are configured to be capable of binding to a first binding partner and the second binding partner, respectively. That is, the binding between the first binding tag and the second binding partner is configured to be less specific than the binding between the first binding tag and the first binding partner, and the binding between the second binding tag and the first binding partner is configured to be less specific than the binding between the second binding tag and the second binding partner. Thus, the first protein can specifically bind to the second protein, and the third protein can specifically bind to the fourth protein. As a specific example, the first binding tag and the first binding partner, and the second binding tag and the second binding partner are preferably combinations of different binding tags and binding partners.

[0158] In the third protein, the second binding tag may be one or more. In the latter case, the second binding tag may be one type or multiple types. In the third protein, the second binding tag is preferably located on the N-terminal side of the third protein.

[0159] In the fourth protein, the second binding partner may be one or more. In the latter case, the second binding partner may be one type or multiple types. In the fourth protein, the second binding partner is preferably located on the N-terminal side of the fourth protein. The number of the second binding tags in the third protein and the number of the second binding partners in the fourth protein may be the same or different, but are preferably the same.

[0160] In the third protein and the fourth protein, the second binding tag and the second binding partner may be interchanged, and the combinations described above may be used interchangeably.

[0161] The first to fourth cleavage domains are domains that undergo domain cleavage either conditionally or unconditionally. The cleavage is carried out in the subsequent generation process after the formation of the second complex. Therefore, the first to fourth cleavage domains are preferably cleavage domains that undergo cleavage conditionally.

[0162] Specific examples of the third cleavage domain and the fourth cleavage domain may refer to the descriptions of the first cleavage domain and the second cleavage domain in Embodiment 1. The first to fourth cleavage domains preferably contain cleavage sequences of the protease or peptidase, because this can inhibit non-specific cleavage.

[0163] The 1st to 4th cleavage domains may be partially or completely identical cleavage domains, or may be partially or completely different cleavage domains. Additionally, in the 1st to 4th proteins, the 1st to 4th cleavage domains may each be one or more. In the latter case, the cleavage domains may be of one type or multiple types. For example, in the production process described later, by making the 1st to 4th cleavage domains identical cleavage domains, the 1st to 4th cleavage domains can be cleaved in a single reaction, thus enabling efficient production of a tetrameric dimer protein.

[0164] In the 3rd protein, the order of the 2nd binding tag and the 3rd cleavage domain can be set, for example, according to their positional relationship with the 3rd domain. The 3rd cleavage domain is disposed at a position closer to the 3rd domain than the 2nd binding tag. Thereby, in the production process described later, when the 3rd cleavage tag is cleaved, the 2nd binding tag can be detached from the 3rd domain. Therefore, in the production method of the present embodiment, in the 3rd protein, the 2nd binding tag, the 3rd cleavage domain, and the 3rd domain are arranged in sequence from the N-terminus to the C-terminus, for example.

[0165] In the 4th protein, the order of the 2nd binding partner and the 4th cleavage domain can be set, for example, according to their positional relationship with the 4th domain. The 4th cleavage domain is disposed at a position closer to the 4th domain than the 2nd binding partner. Thereby, in the production process described later, when the 4th cleavage tag is cleaved, the 2nd binding partner can be detached from the 4th domain. Therefore, in the production method of the present embodiment, in the 4th protein, the 2nd binding partner, the 4th cleavage domain, and the 4th domain are arranged in sequence from the N-terminus to the C-terminus, for example.

[0166] In the 3rd protein, the 2nd binding tag, the 3rd cleavage domain, and the 3rd domain are each directly or indirectly connected. Additionally, in the 4th protein, the 2nd binding partner, the 4th cleavage domain, and the 4th domain are each directly or indirectly connected. The direct connection and the indirect connection can refer to the descriptions of the direct connection and the indirect connection in the explanations of the 1st protein and the 2nd protein in Embodiment 1.

[0167] The 3rd protein may, for example, contain other polypeptides on the N-terminal side of the 2nd binding tag, such as the solubilizing domain, the signal peptide, etc. In addition, the 4th protein may, for example, contain other polypeptides on the N-terminal side of the 2nd binding partner, such as the solubilizing domain, the signal peptide, etc.

[0168] If the third protein contains the solubilizing domain, the number of solubilizing domains in the third protein may be one or more. In the latter case, the solubilizing domain(s) may be one or more types.

[0169] If the fourth protein contains the solubilizing domain, the number of solubilizing domains in the fourth protein may be one or more. In the latter case, the solubilizing domain(s) may be one or more types.

[0170] The third protein and the fourth protein may contain, for example, a purification tag for purifying the third protein, the fourth protein, or the heterotetrameric protein. The purification tag may use, for example, the affinity tag. The purification tag may be attached to at least one of the N-terminus (side) and C-terminus (side) of the third domain and the fourth domain.

[0171] The reaction conditions in the complex formation step may be any conditions under which the first to fourth proteins can form a tetramer, and can be appropriately set in consideration of the reaction conditions (binding conditions) of the binding tag and the binding partner and / or the reaction conditions (binding conditions) of the first to fourth domains.

[0172] When the third domain and the fourth domain bind directly, the reaction conditions in the complex formation step may be set to conditions under which binding is sufficiently formed between the third domain and the fourth domain. As a specific example, if the third domain and the fourth domain are bound by a disulfide bond, in the complex formation step, for example, the reaction conditions may be set such that the disulfide bond is not reduced. Additionally, as another specific example, if the third protein and the fourth protein are bound by an isopeptide bond, in the complex formation step, for example, the reaction conditions may be set such that the isopeptide bond is not hydrolyzed. Also, when the second domain and the fourth domain bind directly, the reaction conditions in the complex formation step may be set to conditions under which binding is sufficiently formed between the second domain and the fourth domain. As a specific example, if the second domain and the fourth domain are bound by a disulfide bond, in the complex formation step, for example, the reaction conditions may be set such that the disulfide bond is not reduced. Additionally, as another specific example, if the second protein and the fourth protein are bound by an isopeptide bond, in the complex formation step, for example, the reaction conditions may be set such that the isopeptide bond is not hydrolyzed.

[0173] When the heterodimeric protein is a multispecific antibody, the reaction conditions of the complex formation step can be further set to, for example, conditions that enable the first to fourth proteins to form disulfide bonds between the heavy chain and the light chain and between the heavy chains.

[0174] The production method of the present embodiment may include a first purification step of purifying the second complex after the complex formation step. The purification method in the purification step may be, for example, a conventional protein purification method such as chromatography.

[0175] Next, in the generation step, the first cleavage domain, the second cleavage domain, the third cleavage domain, and the fourth cleavage domain in the second complex are cleaved. Thus, as Figure 2 (C) shows, in the generation step, the first binding tag and the first binding partner that are bound and the second binding tag and the second binding partner that are bound are detached, and a heterotetramer of the first domain, the second domain, the third domain, and the fourth domain is generated.

[0176] In the generation step, the reaction conditions for the cleavage (for example, reaction temperature, reaction time, reaction pH, etc.) can be set to, for example, conditions under which the first to fourth cleavage domains undergo a cleavage reaction.

[0177] In the generation step, if the first cleavage domain, the second cleavage domain, the third cleavage domain, and / or the fourth cleavage domain is a domain cleaved by a protease or a peptidase, the generation step can be carried out in the presence of the protease or the peptidase. In this case, the generation step can be carried out under reaction conditions under which the protease or the peptidase exhibits cleavage activity.

[0178] The production method of the present invention may include a second purification step of purifying the heterotetrameric protein after the generation step. The purification method in the purification step may be, for example, a conventional protein purification method such as chromatography.

[0179] Thus, the production method of the present invention can produce a heterotetrameric protein from four proteins.

[0180] In addition, in the manufacturing method of the present embodiment, an example of manufacturing a heterodimeric protein using four isolated proteins has been described, but the present invention is not limited thereto, and a second complex may also be manufactured from the four proteins in a host cell described later. At this time, in the manufacturing method of the present embodiment, the second complex may be formed in the host cell by expressing the first to fourth proteins in the host cell. In the manufacturing method of the present embodiment, for example, the heterotetrameric protein may be manufactured by purifying the second complex from the host cell and performing the generation step.

[0181] In addition, in the manufacturing method of the present embodiment, monomeric proteins are used for the four proteins, but the present invention is not limited thereto, and any one or both of them may be a dimeric or higher oligomeric protein, that is, the polymeric protein.

[0182] In the manufacturing method of the present embodiment, the second protein and the fourth protein are associated and bound only through the binding of the second domain and the fourth domain, but the second protein and the fourth protein may also be configured to be able to specifically bind through other domains. As a specific example, the second protein may further include a fifth cleavage domain and a third binding tag in sequence at the C-terminus. In addition, the fourth protein may further include a sixth cleavage domain and a third binding partner capable of binding to the third binding tag in sequence at the C-terminus. In this case, in the complex formation step, the third binding tag and the third binding partner are further bound. Moreover, in the generation step, for example, the fifth cleavage domain and the sixth cleavage domain in the second complex are further cleaved.

[0183] It will be described in Figure 3 more detail. First, as Figure 3As shown in (B), in the complex formation step, the first protein 201, the second protein 202, the third protein 203, and the fourth protein 204 are brought into contact. Thereby, binding occurs between the binding tag and the binding partner of each protein. Specifically, as shown in Y1, the first binding tag 211 and the first binding partner 221 bind. In addition, as shown in Y2, the second binding tag 231 and the second binding partner 241 bind. Furthermore, as shown in Y3, the third binding tag 251 and the third binding partner 261 bind. On the other hand, binding also occurs between the first domain 213, the second domain 223, the third domain 233, and the fourth domain 243 of each protein. Specifically, as shown in X1, the first domain 213 and the second domain 223 bind. In addition, as shown in X2, the third domain 233 and the fourth domain 243 bind. Furthermore, as shown in X3, the second domain 223 and the fourth domain 243 bind. As a result, in the complex formation step, the first protein 201, the second protein 202, the third protein 203, and the fourth protein 204 form the second complex 210.

[0184] Next, in the generation step, the first cleavage domain, the second cleavage domain, the third cleavage domain, the fourth cleavage domain, the fifth cleavage domain, and the sixth cleavage domain in the second complex are cleaved. Thereby, as Figure 3 shown in (C), in the generation step, the bound first binding tag 211 and the first binding partner 221, the bound second binding tag 231 and the second binding partner 241, and the bound third binding tag 251 and the third binding partner 261 dissociate, generating the heterotetramer 220 of the first domain 213, the second domain 223, the third domain 233, and the fourth domain 243.

[0185] Specific examples of the third binding tag and the third binding partner may refer to the examples of the first binding tag and the first binding partner in Embodiment 1. The first binding tag, the second binding tag, and the third binding tag are each configured to be capable of binding to the first binding partner, the second binding partner, and the third binding partner, respectively. Thereby, the first protein can bind to the second protein, and the third protein can bind to the fourth protein. Therefore, the first binding tag and the first binding partner, the second binding tag and the second binding partner, and the third binding tag and the third binding partner are preferably combinations of different binding tags and binding partners.

[0186] In addition, specific examples of the 5th cleavage domain and the 6th cleavage domain may refer to the examples of the 1st cleavage domain and the 1st cleavage domain in Embodiment 1. The 1st to 6th cleavage domains are preferably the same cleavage domain.

[0187] <Protein>

[0188] In another embodiment, the present invention provides a protein that can be suitably used for producing the heteromultimeric protein. The protein of the present invention is the following 1st protein, 2nd protein, 3rd protein, and / or 4th protein.

[0189] The protein of the present invention (1st protein) sequentially includes a 1st binding tag capable of binding to a 1st binding partner, a 1st cleavage domain, and a 1st domain from the N-terminus to the C-terminus.

[0190] The protein of the present invention (2nd protein) sequentially includes a 1st binding partner capable of binding to the 1st binding tag, a 2nd cleavage domain, and a 2nd domain from the N-terminus to the C-terminus.

[0191] The protein of the present invention (3rd protein) sequentially includes a 2nd binding tag capable of binding to a 2nd binding partner, a 3rd cleavage domain, and a 3rd domain from the N-terminus to the C-terminus.

[0192] The protein of the present invention (4th protein) sequentially includes a 1st binding partner capable of binding to the 2nd binding tag, a 4th cleavage domain, and a 4th domain from the N-terminus to the C-terminus.

[0193] The 2nd protein and / or 4th protein of the present invention may further sequentially include a cleavage domain and a binding tag or a binding partner on the C-terminal side.

[0194] The 1st domain of the 1st protein and / or the 3rd domain of the 3rd protein of the present invention are, for example, polypeptides containing the amino acid sequence of the light chain region of an immunoglobulin. In addition, the 2nd domain of the 2nd protein and / or the 4th domain of the 4th protein of the present invention are, for example, polypeptides containing the amino acid sequence of the heavy chain region of an immunoglobulin.

[0195] <Heteromultimeric protein>

[0196] In another embodiment, the present invention provides a heteromultimeric protein that can be suitably used for producing the heteromultimeric protein. The protein (heteromultimeric protein) of the present invention includes two proteins,

[0197] The two proteins include a 1st protein and a 2nd protein,

[0198] The first protein sequentially includes a first binding tag, a first cleavage domain, and a first domain from the N-terminus to the C-terminus.

[0199] The second protein sequentially includes a first binding partner capable of binding to the first binding tag, a second cleavage domain, and a second domain from the N-terminus to the C-terminus.

[0200] The first protein and the second protein form a dimer through the binding between the first domain and the second domain.

[0201] The first binding tag binds to the first binding partner.

[0202] Preferably, the heteromultimeric protein of the present invention further contains a third protein and a fourth protein.

[0203] The third protein sequentially includes a second binding tag, a third cleavage domain, and a third domain from the N-terminus to the C-terminus.

[0204] The fourth protein sequentially includes a second binding partner capable of binding to the second binding tag, a fourth cleavage domain, and a fourth domain from the N-terminus to the C-terminus.

[0205] The second protein and the fourth protein form a dimer through the binding between the second domain and the fourth domain.

[0206] The third protein and the fourth protein form a dimer through the binding between the third domain and the fourth domain.

[0207] The first binding tag binds to the first binding partner.

[0208] The second binding tag binds to the second binding partner.

[0209] When the heteromultimeric protein of the present invention is a bispecific antibody, the first domain of the first protein and the third domain of the third protein are polypeptides containing the amino acid sequence of the light chain of an immunoglobulin. Additionally, the second domain of the second protein and the fourth domain of the fourth protein are polypeptides containing the amino acid sequence of the heavy chain of an immunoglobulin.

[0210] <Nucleic Acid>

[0211] In another embodiment, the present invention provides a nucleic acid that can be used to synthesize a heteromultimeric protein. The nucleic acid of the present invention encodes the protein and / or heteromultimeric protein of the present invention.

[0212] The nucleic acid of the present invention can, for example, encode any one or more of the proteins and heteromultimeric proteins of the present invention, and can also encode multiple ones.

[0213] The nucleic acid of the present invention can be designed by replacing the corresponding codons based on the amino acid sequence of the heteromultimeric protein of the present invention. The base sequence of the nucleic acid of the present invention can, for example, be codon-optimized, preferably codon-optimized for the host cell described below.

[0214] <Expression vector>

[0215] In another embodiment, the present invention provides an expression vector that can be used for synthesizing proteins and / or heteromultimeric proteins. The expression vector of the present invention contains the nucleic acid of the present invention. According to the expression vector of the present invention, the proteins and / or heteromultimeric proteins of the present invention (hereinafter collectively referred to as "the material proteins of the present invention") can be appropriately produced by genetic engineering techniques.

[0216] The expression vector of the present invention only needs to contain, for example, a nucleic acid encoding any one or more of the proteins and heteromultimeric proteins of the present invention, and can also contain nucleic acids encoding multiple proteins and heteromultimeric proteins.

[0217] The nucleic acid of the present invention has been inserted into the expression vector of the present invention, for example. The expression vector refers to, for example, a nucleic acid molecule that can transport the inserted gene into a target such as a cell.

[0218] The expression vector only needs to contain, for example, a polynucleotide encoding the material 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, and its structure is not particularly limited. The material proteins of the present invention can, for example, be partially or entirely inserted into the same expression vector, or can be inserted into different expression vectors respectively. If a part of the material protein of the present invention is inserted into different expression vectors, the expression vector of the present invention constitutes an expression vector group including the expression vectors containing the nucleic acids encoding the material proteins of the present invention.

[0219] The expression vector can be prepared, for example, by inserting a polynucleotide encoding the material protein of the present invention, that is, the nucleic acid of the present invention, into a vector as a backbone (hereinafter also referred to as "basic vector"). The type of the expression vector is not particularly limited, and can be appropriately determined according to the type of the host, for example.

[0220] Examples of the host cell (host) include non-human hosts such as microorganisms, animal cells, insect cells, or their cultured cells, isolated human cells or their cultured cells, mammalian cells, etc. Examples of the prokaryote include Escherichia coli ( Escherichiacoli ) bacteria such as Escherichia and Pseudomonas Pseudomonas putida ) and other bacteria of the genus Pseudomonas. Examples of the eukaryote may include Saccharomyces cerevisiae Saccharomyces cerevisiae ) and other yeasts. Examples of the animal cells may include HEK293 cells, Expi293F cells, COS cells, CHO cells, etc. Examples of the insect cells may include Sf9, Sf21, etc.

[0221] Examples of the expression vector (basic vector) may include viral vectors and non-viral vectors. When transforming the host by the heat shock method as the introduction method, examples of the expression vector may include binary vectors. Examples of the expression vector may include pETDuet-1, pQE-80L, pUCP26Km, etc. When transforming bacteria such as Escherichia coli, examples of the expression vector may include pETDuet-1 vector (Novagen), pQE-80L (QIAGEN), pBR322, pB325, pAT153, pUC8, etc. When transforming the yeast, examples of the expression vector may include pYepSec1, pMFa, pYES2, etc. When transforming the insect cells, examples of the expression vector may include pAc, pVL, etc. When transforming the mammalian cells, examples of the expression vector may include pcDNA3.1, pcDNA3.4, pCAG, pCAGEN, pCDM8, pMT2PC, etc.

[0222] Preferably, the expression vector has regulatory sequences, such as regulating the expression of the polynucleotide encoding the material protein of the present invention and the expression of the material protein of the present invention encoded by the polynucleotide of the material protein of the present invention. Examples of the regulatory sequences may include promoters, terminators, enhancers, polyadenylation signal sequences, replication origin sequences (ori), etc. In the expression vector, the configuration of the regulatory sequences is not particularly limited. In the expression vector, the regulatory sequences may be configured as long as they can functionally regulate the expression of the polynucleotide encoding the material protein of the present invention and the expression of the material protein of the present invention encoded thereby, and can be configured according to known methods. The regulatory sequences may use the sequences pre-existing in the basic vector, or the regulatory sequences may be further inserted into the basic vector, or the regulatory sequences in the basic vector may be replaced with other regulatory sequences.

[0223] The expression vector may also have a coding sequence for a selection marker, for example. Examples of the selection marker may include drug resistance markers, fluorescent protein markers, enzyme markers, cell surface receptor markers, etc.

[0224] Insertion of DNA into the expression vector, insertion of the regulatory sequence, and / or insertion of the coding sequence of the selection marker can be carried out, for example, by methods using restriction enzymes and ligases, or by using commercially available kits, etc.

[0225] <Transformant>

[0226] In another embodiment, the present invention provides a transformant capable of producing the material protein of the present invention and a method for producing the same. The transformant of the present invention contains a nucleic acid encoding the material protein of the present invention. According to the transformant of the present invention, the material protein of the present invention can be appropriately produced.

[0227] In addition, the method for producing the transformant of the present invention includes the step of introducing the nucleic acid of the present invention into a host. According to the method for producing the transformant of the present invention, the transformant can be produced.

[0228] In the transformant of the present invention, the nucleic acid encoding the material protein of the present invention may refer to the description of the nucleic acid encoding the material protein of the present invention. As the nucleic acid of the present invention, the expression vector of the present invention can be used.

[0229] In the transformant of the present invention, the nucleic acid of the present invention exists 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.

[0230] 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, for example. Examples of the introduction method include the introduction method using a gene gun such as a particle gun, the calcium phosphate method, the polyethylene glycol method, the liposome transfection method using liposomes, the electroporation method, the ultrasonic nucleic acid introduction method, the DEAE-dextran method, the direct injection method using a micropipette, the hydrodynamic method, the cationic liposome method, the method using an introduction adjuvant, the method using Agrobacterium, etc. Examples of the liposome include liposome transfection reagent (lipofectamine) and cationic liposome, etc. Examples of the introduction adjuvant include terminal peptide collagen, nanoparticles, and polymers, etc. When the host is a microorganism, for example, a method using Escherichia coli (E. coli) or Pseudomonas putida (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.

[0231] <Method for producing protein>

[0232] In another embodiment, the present invention provides a method for producing a protein and / or a heteromultimeric protein suitable for use in the production of a protein and / or a heteromultimeric protein. The method for producing the protein of the present invention includes an expression step of expressing the nucleic acid of the present invention, the expression vector of the present invention, and / or the expression vector group of the present invention. According to the method for producing the protein of the present invention, the material protein of the present invention can be produced.

[0233] The expression of the material protein of the present invention can be carried out, for example, by using the expression vector of the present invention. The method for expressing the material protein of the present invention is not particularly limited, and known methods can be employed. For example, a host can be used, or a cell-free protein synthesis system can be used.

[0234] In the former case, for example, it is preferable to use the host into which the material protein of the present invention or the nucleic acid encoding the same has been introduced, and culture the host to express the material protein of the present invention in the host. Thus, for example, by introducing the nucleic acid encoding the material protein of the present invention into a host, a transformant capable of synthesizing the material protein of the present invention can be produced, and by culturing the transformant, the material protein of the present invention can be synthesized.

[0235] The method for culturing the host is not particularly limited and can be appropriately set according to the type of the host. The culture medium for culturing is not particularly limited and can be appropriately determined according to the type of the host.

[0236] In the latter case, it is preferable to express the polynucleotide of the material protein of the present invention in a cell-free protein synthesis system. In this case, an expression vector can be used to express the polynucleotide of the material protein of the present invention. The cell-free protein synthesis system can be carried out by known methods, for example, using a cell extract, a buffer containing various components, and an expression vector into which the polynucleotide encoding the material protein of the present invention has been introduced. For example, a commercially available kit can be used.

[0237] The method for producing the protein of the present invention can include, for example, a recovery step of recovering the material protein of the present invention. The material protein of the present invention obtained from the recovery step can be, for example, a crude purify or a purified protein.

[0238] When recovering from the culture solution, in the recovery step, for example, insolubles are removed by filtering the culture supernatant, centrifuging, etc. In the recovery step, for example, the culture supernatant after removing the insolubles can be concentrated by ultrafiltration membrane; salting out using ammonium sulfate precipitation, etc.; dialysis; chromatography using various columns such as ion exchange columns and gel filtration columns can be appropriately combined for separation and purification to obtain the material protein of the present invention.

[0239] When recovering from the transformant, in the recovery step, the transformant is disrupted, for example, by pressure treatment, ultrasonic treatment, etc. The resulting disrupted solution can be subjected to removal of insolubles, separation, and purification as described above to obtain the material protein of the present invention.

[0240] The material protein of the present invention obtained by the production method of the present invention can be used, for example, directly as a crudely purified protein, or as a partially purified protein that is partially purified, or as a purified protein that is singly purified.

[0241] In addition, the production method of the present invention can also powderize the obtained material protein of the present invention by, for example, freeze-drying, vacuum drying, or spray drying. In this case, in the production method of the present invention, for example, the protein of the present invention can be pre-dissolved in a buffer solution such as an acetate buffer, a phosphate buffer, a triethanolamine buffer, a Tris-HCl buffer, a GOOD's buffer (such as HEPES, PIPES, MES, MOPS, etc.).

[0242]

Examples

[0243] The present invention will be described in detail below by way of examples, but the present invention is not limited to the embodiments described in the examples.

[0244] [Example 1]

[0245] It was confirmed that a heteromultimeric protein could be produced by the production method of the present invention.

[0246] (1) Preparation of plasmid vector

[0247] First, it was confirmed that a first complex containing two proteins forming a heterodimer could be formed by the production method of the present invention. Specifically, a plasmid vector capable of expressing the following four proteins was constructed.

[0248] · First protein: A protein containing a solubilizing domain, SpyTag (binding tag), and the light chain of a CD3 antibody (VHH-SpyTag-CD3 L chain)

[0249] · Second protein: A protein containing a solubilizing domain, SpyCatcher (binding partner), the heavy chain and variable domain of a CD3 antibody (VHH-SpyCatcher-CD3 H chain)

[0250] · Third protein: The light chain of a Herceptin antibody

[0251] · Fourth protein: The heavy chain of a Herceptin antibody

[0252] The plasmid vector capable of expressing VHH-SpyTag-CD3 L chain was constructed through the following steps. First, for the synthetic gene (Eurofins Genomics) containing the base sequence encoding a signal peptide, aGFP4 (single-chain antibody (solubilizing domain), SEQ ID NO: 9), G1 linker, SpyTag (SEQ ID NO: 2), thrombin cleavage sequence, the light chain variable region of M291, and the light chain constant region of M291, the full length was amplified by PCR. The obtained full-length synthetic gene was ligated into an animal cell expression vector (pCDNA3.4) to construct an expression vector for the recombinant protein. As shown in the parentheses, the said expression vector, as the VHH-SpyTag-CD3 L chain region (SEQ ID NO: 10), sequentially ligates a signal peptide, aGFP4, G1 linker, SpyTag, thrombin cleavage sequence, the light chain variable region of M291, and the light chain constant region of M291 from the N-terminus to the C-terminus.

[0253] aGFP4 (SEQ ID NO: 9)

[0254] QVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDS

[0255] VKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS

[0256] VHH-SpyTag-CD3 L chain region (SEQ ID NO: 10)

[0257] [MEFGLSWLFLVAILKGVQC][QVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS][GGSGG][AHIVMVDAYKPTK][GGSGGGGSGG][LVPRGSHMHM][DIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYKQKSGTSPKRWTYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFGSGTKLEI][KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC]

[0258] The plasmid vector capable of expressing VHH-SpyCatcher-CD3 H chain was constructed through the following steps. First, for the synthetic gene (Eurofins Genomics) containing the base sequence encoding the signal peptide, Ia1 (single-chain antibody (solubilizing domain), SEQ ID NO: 11), G1 linker, SpyCatcher (SEQ ID NO: 1), G2 linker, thrombin cleavage sequence, heavy chain variable region of M291, heavy chain constant region of M291, hinge region, and Fc region, the full length was amplified by PCR. The obtained full-length synthetic gene was ligated into an animal cell expression vector (pCDNA3.4) to construct an expression vector for the recombinant protein. As shown in the brackets, the said expression vector sequentially ligates the signal peptide, Ia1, G1 linker, SpyCatcher, G2 linker, thrombin cleavage sequence, heavy chain variable region of M291, heavy chain constant region of M291, hinge region, and Fc region from the N-terminus to the C-terminus as the VHH-SpyCatcher-CD3 H chain region (SEQ ID NO: 12).

[0259] Ia1 (SEQ ID NO: 11)

[0260] QVQLQESGGGLVQAGGSLLLSCAASGRTFSSYAMGWFRQAPGKEREFVAAINWSGGSTSYADS

[0261] VKGRFTISRDNTKNTVYLQMNSLKPEDTAAFYCAATYNPYSRDHYFPRMTTEYDYWGQGTQVT

[0262] VSS

[0263] VHH-SpyCatcher-CD3H chain region (Sequence No. 12)

[0264] [MEFGLSWLFLVAILKGVQC][QVQLQESGGGLVQAGGSLLLSCAASGRTFSSYAMGWFRQAPGKEREFVAAINWSGGSTSYADSVKGRFTISRDNTKNTVYLQMNSLKPEDTAAFYCAATYNPYSRDHYFPRMTTEYDYWGQGTQVTVSS][GGSGG][DSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG][GGSGGGGSGG][LVPRGSHMHM][QVQLQQSGAELARPGASVKMSCKASGYTFISYTMHWVKQRPGQGLEWIGYINPRSGYTHYNQKLKDKATLTADKSSSSAYMQLSSLTSEDYAVYYCARSAYYDYDGFAYWGQGTLVTVSA][ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV][EPKSCDKTH][TCPPCP][APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK]

[0265] The plasmid vector capable of expressing the light chain of Herceptin antibody was constructed through the following steps. First, for the synthetic gene (Eurofins Genomics) containing the base sequence encoding the signal peptide, the variable region of the light chain of h4D5, and the constant region of the light chain of h4D5, the full length was amplified by PCR. The obtained full-length synthetic gene was ligated into the animal cell expression vector (pCAGGS) to construct the expression vector for the recombinant protein. As shown in the brackets, the said expression vector, as the light chain region of Herceptin antibody (SEQ ID NO: 13), sequentially ligates the signal peptide, the variable region of the light chain of h4D5, and the constant region of the light chain of h4D5 from the N-terminus to the C-terminus.

[0266] The light chain region of Herceptin antibody (SEQ ID NO: 13)

[0267] [METPAQLLFLLLLWLPESTG][DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEI][KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC]

[0268] The plasmid vector capable of expressing the heavy chain of Herceptin antibody was constructed through the following steps. First, for the synthetic gene (Eurofins Genomics) containing the base sequence encoding the signal peptide, the variable region of the heavy chain of h4D5, the constant region of the heavy chain of h4D5, the hinge region, and the Fc region of h4D5, the full length was amplified by PCR. The obtained full-length synthetic gene was ligated into the animal cell expression vector (pCAGGS) to construct the expression vector for the recombinant protein. As shown in the brackets, the said expression vector, as the heavy chain region of Herceptin antibody (SEQ ID NO: 14), sequentially ligates the signal peptide, the variable region of the heavy chain of h4D5, the constant region of the heavy chain of h4D5, the hinge region, and the Fc region of h4D5 from the N-terminus to the C-terminus.

[0269] The heavy chain region of Herceptin antibody (SEQ ID NO: 14)

[0270] [MEFGLSWLFLVAILKGVQC][EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS][ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV][EPKSCDKTH][TCPPCP][APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKALGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK]

[0271] Next, the four plasmid expression vectors of the recombinant protein were transfected into Expi293F cells. First, the frozen Expi293F cells were thawed and inoculated in HE400 medium. After inoculation, the cells were cultured with shaking at 37 °C, 8% CO2, and 125 rpm until the cell density reached 3 - 5×10 6 / ml and the viability rate was over 95%. After the culture, 25 ml of HE400 medium was added to make the cell density reach 75×10 6Cells. After the addition, the total amount of plasmid vector was added to Opti-MEM® such that the final concentration was 30,000 ng / ml. At the same time, PEI was added to Opti-MEM® different from the Opti-MEM® to which the plasmid vector was added such that the final concentration reached 40 μg / ml. After the addition, each Opti-MEM® was left standing at room temperature (hereinafter 24°C) for 5 minutes. After the standing, the respective Opti-MEM® were mixed and left standing at room temperature for 20 minutes. After the standing, the mixture was added to 25 ml of HE400 medium and cultured with shaking at 37°C, 8% CO2, and 125 rpm for 20 hours. After the culture, sodium valproate (final concentration 1.25 μmol / l), sodium propionate (final concentration 4 μmol / l), and 750 μl of 20 wt% tryptone were added. After the addition, the culture was continued with shaking at 37°C, 8% CO2, and 125 rpm for 6 days. After the culture, the culture supernatant was recovered.

[0272] (2) Purification of the protein

[0273] The culture supernatant was poured onto a protein A column (manufactured by SUPrA or BIO RAD) such that the column capacity corresponding to every 30 ml of the culture was 1 ml. Thereafter, the fraction passing through the column was recovered. Then, a washing buffer (50 mmol / l sodium phosphate buffer) was passed through the column, and the washing fraction was recovered. After the recovery, an elution buffer (20 mmol / l sodium citrate + 100 mmol / l sodium chloride (pH 3.0)) was passed through the column, and the elution fraction was recovered. The recovered elution fraction was placed in a dialysis membrane and dialyzed using a dialysis buffer (150 mmol / l NaCl, 50 mmol / l HEPES). After the dialysis, it was concentrated using a concentrator tube to obtain a purified sample.

[0274] (3) Detection of the purified protein

[0275] Use SDS-PAGE to detect whether a complex containing VHH-SpyTag-CD3 L chain and VHH-SpyCatcher-CD3 H chain is formed in the purified sample obtained in Example 1(2). Specifically, 10 μl of 5× SDS buffer was added to the purified sample (40 μl) and suspended. After the suspension, it was heat-treated at 95 °C for 5 minutes. After the heat treatment, it was applied to the wells of a 10% polyacrylamide gel and electrophoresed under the condition of 150 - 200 V. A marker and the purified sample were applied to the 10% polyacrylamide gel. After the electrophoresis, the polyacrylamide gel was stained with CBB (Coomassie Brilliant Blue) staining solution for 5 minutes and decolorized with a decolorizing solution. The results are as Figure 4 shown.

[0276] Figure 4 A photograph showing the SDS-PAGE results. In Figure 4 it, the left side of the photograph indicates the molecular weight (kDa). As Figure 4 shown, a band of the estimated molecular weight of the complex was detected in the purified sample. In addition, bands of the estimated molecular weights of the light chain of the Herceptin antibody and the heavy chain of the Herceptin antibody were also detected. These results indicate that a complex containing two proteins can be formed by the production method of the present invention.

[0277] (4) Detection of the complex cleaved with thrombin

[0278] Next, it was detected whether the band of the estimated molecular weight of the complex in Example 1(3) was a complex containing VHH-SpyTag-CD3 L chain and VHH-SpyCatcher-CD3 H chain. Specifically, protein purification was carried out in the same manner as in Example 1(2), and the purified elution fraction was added to thrombin buffer (2.5 mmol / l CaCl2, 150 mmol / l NaCl, 20 mmol / l Tris-HCl (pH 8.0)). After the addition, dialysis was carried out for 6 hours. Then, thrombin (cutting ability 2 unit / μl, manufactured by Wako Pure Chemical Industries, Ltd.) was added according to the protein yield. After the addition, it was allowed to stand overnight at 25 °C. After the standing, the sample after the cleavage reaction was purified using a protein A column. After the purification, a purified sample cleaved with thrombin was obtained. Then, SDS-PAGE was carried out. In the SDS-PAGE, the purified sample and the purified sample cleaved with thrombin were used as samples, and in addition, it was carried out in the same manner as in Example 1(3). The results are as Figure 5 shown.

[0279] Figure 5 It is a photograph showing the results of SDS-PAGE. Figure 5 In it, the types of samples are shown above the photograph, and the molecular weights (kDa) are shown on the left side of the photograph. As Figure 5 shown, no band of the estimated molecular weight of the complex was detected in the thrombin-cleaved purified sample. In addition, as Figure 5 shown, in the thrombin-cleaved purified sample, bands of the estimated molecular weights of the light chain of the Herceptin antibody, the heavy chain of the Herceptin antibody, the light chain of the CD3 antibody, the heavy chain of the CD3 antibody, and the bound VHH-SpyTag and VHH-SpyCatcher were detected. These results indicate that a complex containing two proteins can be formed by the production method of the present invention. In addition, it was found that the cleavage domain of the complex was cleaved by thrombin.

[0280] (5) Separation of Herceptin by chromatography

[0281] For the purified sample purified in the above-mentioned Example 1(3), Herceptin was separated using size exclusion chromatography. Specifically, the protein purified in the above-mentioned Example 1(3) was passed through a gel filtration chromatography column (Superdex 200 increase 30 / 100 GL, 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 the buffer in the measurement. The results are as Figure 6 shown.

[0282] Figure 6 It is a graph showing the elution pattern of Herceptin determined by size exclusion chromatography. In Figure 6 it, the horizontal axis represents the elution time (time), and the vertical axis represents the absorbance. As Figure 6 shown, the elution of Herceptin obtained by the production method of the present invention was confirmed ( Figure 6 the arrow in it).

[0283] (6) Activity evaluation of CD3-positive cells

[0284] Whether the light chain of the CD3 antibody and the heavy chain of the CD3 antibody obtained by the same method as in Example 1(5) bind to cells expressing CD3 was detected by flow cytometry. Specifically, the HPB-ALL cell line with overexpression of CD3 on the cell membrane surface was used and measured by flow cytometry. The HPB-ALL cultured in a T75 flask was transferred equally to two 15 ml Falcon tubes and centrifuged at 1500 rpm for 5 minutes. After the centrifugation, the supernatant was aspirated and removed using a pipette, and a necessary amount of 1×PBS was added for suspension to obtain a cell suspension of 1×10 6 cells / ml. 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, two microtubes (a) and (b) were centrifuged at 2000 rpm, 25 °C for 7 minutes. After the centrifugation, the supernatant was aspirated and removed using a pipette, and 1 ml of 1×PBS was added. After the addition, two microtubes (a) and (b) were centrifuged again at 2000 rpm, 25 °C for 7 minutes. After the centrifugation, the supernatant was aspirated and removed using a pipette, and 1×PBS and the CD3 antibody purified by the same method as in Example 1(5) were added to one microtube (b) until the final concentration of the CD3 antibody was 0.05 μmol / l, and then mixed by inversion. After the mixing by inversion, it was allowed to stand for 20 minutes. After the standing, it was centrifuged at 2000 rpm, 25 °C for 7 minutes. Then, 1 μl of OKT3-FITC (manufactured by Cosmo Bio Co., Ltd.) and 1 ml of 1×PBS were added to microtube (a), and 1 μl of anti-Fc-FITC (manufactured by AbCam) and 1 ml of 1×PBS were added to microtube (b), and they were mixed by inversion respectively. After the mixing by inversion, it was allowed to stand for 20 minutes. After the standing, it was centrifuged at 2000 rpm, 25 °C for 7 minutes. After the centrifugation, the supernatant was aspirated and removed using a pipette, and 1 ml of 1×PBS was added. After the addition, it was centrifuged again at 2000 rpm, 25 °C for 7 minutes. After the centrifugation, the supernatant was aspirated and removed using a pipette, and 1 ml of 1×PBS was added for suspension. After the suspension, it was sterilized using a mesh filter. After setting the measurement conditions, measurements were performed using a cell analyzer RF-500 (manufactured by Sysmex Corporation) in the order of negative control (c), positive control (a), and sample (b). After the measurement, the measurement results were plotted using FCSalyzer. These results are as Figure 7 shown.

[0285] Figure 7 A figure showing the binding of the light chain of the CD3 antibody and the heavy chain of the CD3 antibody to CD3-positive cells detected by flow cytometry.Figure 7 Among them, the horizontal axis represents fluorescence intensity and the vertical axis represents the number of cells. As Figure 7 shown, when the light chain of the CD3 antibody and the heavy chain of the CD3 antibody obtained by the production method of the present invention are added to CD3-positive cells, the fluorescence intensity increases compared to when not added to CD3-positive cells. These results indicate that the light chain of the CD3 antibody and the heavy chain of the CD3 antibody obtained by the production method of the present invention bind to CD3-positive cells.

[0286] (7) Evaluation of the activity against HER2-positive cells

[0287] It was detected by flow cytometry whether the light chain of the Herceptin antibody and the heavy chain of the Herceptin antibody obtained in Example 1(5) bind to breast cancer cells. Specifically, the SK-BR-3 cell line with overexpression of HER2 on the cell membrane surface was used and measured by flow cytometry. The supernatant of SK-BR-3 cultured in a T75 flask was aspirated and removed using an aspirator, and the necessary amount of 1×PBS was added for suspension to obtain a cell suspension. Then, 1 ml of 1×10 6The suspension of the cells was diluted with 1×PBS. After the dilution, two microtubes (d) and (e) were centrifuged at 2000 rpm, 25 °C for 7 minutes. After the centrifugation, the supernatant was aspirated and removed using a pipette, and 1 ml of 1×PBS was added. After the addition, the two microtubes (d) and (e) were centrifuged again at 2000 rpm, 25 °C for 7 minutes. After the centrifugation, the supernatant was aspirated and removed using a pipette. After the aspiration and removal, 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 it was mixed by inverting. At the same time, 1 ml of 1×PBS and the purified Herceptin antibody in Example 1(5) were added to one microtube (e) until the final concentration of the Herceptin antibody was 0.1 μmol / l, and it was mixed by inverting. After the mixing by inverting, the two microtubes (d) and (e) were allowed to stand for 20 minutes. After the standing, the two microtubes (d) and (e) were centrifuged at 2000 rpm, 25 °C for 7 minutes. After the centrifugation, the supernatant was aspirated and removed using a pipette, and 1 ml of 1×PBS was added. After the addition, the two microtubes (d) and (e) were centrifuged again at 2000 rpm, 25 °C for 7 minutes. After the centrifugation, the supernatant was aspirated and removed using a pipette, 1 μl of anti-Fc-FITC (manufactured by AbCam) and 1 ml of 1×PBS were added to the two microtubes (d) and (e), and it was mixed by inverting. After the mixing by inverting, it was allowed to stand for 20 minutes. After the standing, the two microtubes were centrifuged at 2000 rpm, 25 °C for 7 minutes. After the centrifugation, the supernatant was aspirated and removed using a pipette, and 1 ml of 1×PBS was added. After the addition, the two microtubes (d) and (e) were centrifuged again at 2000 rpm, 25 °C for 7 minutes. After the centrifugation, the supernatant was aspirated and removed using a pipette, and 1 ml of 1×PBS was added for resuspension. After the resuspension, it was sterilized using a mesh filter. After setting the measurement conditions, using a cell analyzer RF-500 (manufactured by Sysmex), measurements were performed in the order of negative control (f), positive control (d), and sample (e). After the measurement, the measurement results were plotted using FCSalyzer. These results are as Figure 8 shown.

[0288] Figure 8 is a graph showing the binding of the light chain of the Herceptin antibody and the heavy chain of the Herceptin antibody to HER2-positive cells detected by flow cytometry. Figure 8 In, the horizontal axis represents the fluorescence intensity and the vertical axis represents the number of cells. As Figure 8As shown, when the light chain of the Herceptin antibody obtained by the manufacturing method of the present invention and the heavy chain of the Herceptin antibody are added to HER2-positive cells, the fluorescence intensity increases compared to when they are not added to HER2-positive cells. These results indicate that the light chain of the Herceptin antibody and the heavy chain of the Herceptin antibody obtained by the method of the present invention bind to HER2-positive breast cancer cells.

[0289] (8) Detection of the formation of two complexes

[0290] Since it was found that a complex containing two proteins can be formed by the manufacturing method of the present invention, it was confirmed whether a complex of the light chain of the Herceptin antibody and the heavy chain of the Herceptin antibody can also be formed by the same method as in Example 1(1). Specifically, plasmid vectors capable of expressing the following four proteins constituting the heterotetramer were constructed. The plasmid vectors for the first protein and the second protein were constructed by the same method as in Example 1(1).

[0291] · First protein: A protein containing a variable domain, SpyTag (binding tag), and the light chain of the CD3 antibody (VHH-SpyTag-CD3 L chain)

[0292] · Second protein: A protein containing SpyCatcher (binding partner), the heavy chain of the CD3 antibody, and a variable domain (VHH-SpyCatcher-CD3 H chain)

[0293] · Fifth protein: A protein containing a variable domain, SnoopTag (binding partner), and the light chain of the Herceptin antibody (VHH-SnoopTag-Herceptin L chain)

[0294] · Sixth protein: A protein containing SnoopCatcher (binding partner), the heavy chain of the Herceptin antibody, and a variable domain (VHH-SnoopCatcher-Herceptin H chain)

[0295] The plasmid vector capable of expressing VHH-SnoopTag-Herceptin L chain was constructed through the following steps. First, for the synthetic gene (Eurofins Genomics) containing the base sequence encoding a signal peptide, aGFP4 (single-chain antibody (solubilizing domain), SEQ ID NO: 9), G1 linker, SnoopTag (SEQ ID NO: 4), G2 linker, thrombin cleavage sequence, light chain variable region of H4D5, and light chain constant region of H4D5, the full length was amplified by PCR. The obtained full-length synthetic gene was ligated into an animal cell expression vector (pCAGEN) to construct an expression vector for the recombinant protein. As shown in the brackets, the said expression vector successively ligates a signal peptide, aGFP4, G1 linker, SnoopTag, G2 linker, thrombin cleavage sequence, light chain variable region of H4D5, and light chain constant region of H4D5 from the N-terminus to the C-terminus as the VHH-SnoopTag-Herceptin L chain region (SEQ ID NO: 15).

[0296] VHH-SnoopTag-Herceptin L chain region (SEQ ID NO: 15)

[0297] [METPAQLLFLLLLWLPESTG][QVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS][GGSGG][KLGDIEFIKVNK][GGSGGGGSGG][LVPRGSHMHM][DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEI][KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC]

[0298] The plasmid vector capable of expressing VHH-SnoopCatcher-Herceptin H chain was constructed through the following steps. First, for the synthetic gene (Eurofins Genomics) containing the base sequence encoding the signal peptide, said Ia1 (SEQ ID NO: 11), G1 linker, SnoopCatcher (SEQ ID NO: 3), G2 linker, thrombin cleavage sequence, heavy chain variable region of H4D5, heavy chain constant region of H4D5, hinge region and Fc region, the full length was amplified by PCR. The obtained full-length synthetic gene was ligated into an animal cell expression vector (pCAGGS) to construct an expression vector for the recombinant protein. As shown in the brackets, the said expression vector, as the VHH-SnoopCatcher-Herceptin H chain region (SEQ ID NO: 16), sequentially ligates the signal peptide, Ia1, G1 linker, SnoopCatcher, G2 linker, thrombin cleavage sequence, heavy chain variable region of H4D5, heavy chain constant region of H4D5, hinge region and Fc region from the N-terminus to the C-terminus.

[0299] VHH-SnoopCatcher-Herceptin H chain region (SEQ ID NO: 16)

[0300] [MEFGLSWLFLVAILKGVQ][CQVQLQESGGGLVQAGGSLLLSCAASGRTFSSYAMGWFRQAPGKEREFVAAINWSGGSTSYADSVKGRFTISRDNTKNTVYLQMNSLKPEDTAAFYCAATYNPYSRDHYFPRMTTEYDYWGQGTQVTVSS][GGSGG][KPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKPIVAFQIVNGEVRDVTSIVPQDIPATYEFTNDKHYITNEPIPPK][GGSGGGGSGG][LVPRGSHMHM][EVQLVESGGGLVQPGGS

[0301] LRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS][ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV][EPKSCDKTH][TCPPCP][APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKALGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0303] Next, transfection of the 4 plasmid expression vectors of the recombinant protein, cell culture, and recovery of the culture supernatant were carried out in the same manner as in Example 1(1). After the recovery, protein purification was carried out in the same manner as in Example 1(2) to obtain a purified sample. SDS-PAGE was used to detect whether complexes containing VHH-SpyTag-CD3 L chain and VHH-SpyCatcher-CD3 H chain, and complexes containing VHH-SnoopTag-Herceptin L chain and VHH-SnoopCatcher-Herceptin H chain were formed in the purified sample. Specifically, 10 μl of 5× SDS buffer was added to the purified sample (40 μl) and suspended. After the suspension, heat treatment was carried out at 95 °C for 5 minutes. After the heat treatment, it was applied to the wells of a 10% polyacrylamide gel and electrophoresis was carried out under the conditions of 150 - 200 V. The marker and the purified sample were applied to the 10% polyacrylamide gel. After the electrophoresis, the polyacrylamide gel was stained with CBB (Coomassie Brilliant Blue) staining solution for 5 minutes and decolorized with a decolorizing solution. These results are as Figure 9 shown.

[0304] Figure 9 ​This is a photograph showing the results of SDS-PAGE. In Figure 9 , the left side of the photograph indicates the molecular weight (kDa). As Figure 9 shown, bands corresponding to the estimated molecular weights of the two complexes were detected in the purified sample. These results indicate that the two complexes can be formed by the production method of the present invention.

[0305] (9) Detection of the two complexes cleaved with thrombin

[0306] Next, it was examined whether the bands corresponding to the estimated molecular weights of the two complexes in Example 1(8) were complexes containing VHH-SpyTag-CD3 L chain and VHH-SpyCatcher-CD3 H chain, and complexes containing VHH-SnoopTag-Herceptin L chain and VHH-SnoopCatcher-Herceptin H chain. Samples containing the complex of VHH-SpyTag-CD3 L chain and VHH-SpyCatcher-CD3 H chain, and samples containing the complex of VHH-SnoopTag-Herceptin L chain and VHH-SnoopCatcher-Herceptin H chain were used, and otherwise, the same method as in Example 1(4) was carried out. These results are as Figure 10 shown.

[0307] Figure 10 This is a photograph showing the results of SDS-PAGE. Figure 10 , the molecular weight (kDa) is shown on the left side of the photograph. As Figure 10 shown, bands corresponding to the estimated molecular weights of the complexes containing VHH-SpyTag-CD3 L chain and VHH-SpyCatcher-CD3 H chain, and the complexes containing VHH-SnoopTag-Herceptin L chain and VHH-SnoopCatcher-Herceptin H chain were not detected in the purified sample cleaved with thrombin. Furthermore, as Figure 10As shown, in the purified sample cleaved by thrombin, bands of the estimated molecular weights of the light chain of the Herceptin antibody, the heavy chain of the Herceptin antibody, the light chain of the CD3 antibody, the heavy chain of the CD3 antibody, the bound VHH-SpyTag and VHH-SpyCatcher, and the bound VHH-SnoopTag and VHH-SnoopCatcher were detected. These results indicate that two complexes can be formed by the production method of the present invention. In addition, it was found that the cleavage domains of the two complexes were cleaved by thrombin.

[0308] As described above, the present invention has been described with reference to the embodiments, but the present invention is not limited to the above embodiments. Those skilled in the art can make various changes within the scope of the present invention that they can understand to the structure and details of the present invention.

[0309] This application claims priority based on Japanese Patent Application No. 2022-158540 filed on September 30, 2022, and incorporates all of its disclosure herein.

[0310] <Supplementary Note>

[0311] Part or all of the above embodiments and examples can be described as in the following supplementary notes, but are not limited to the following.

[0312] <Method for Producing Heteromultimeric Protein>

[0313] (Supplementary Note 1)

[0314] A method for producing a heteromultimeric protein, comprising:

[0315] a complex formation step of bringing two proteins into contact to form a first complex of the two proteins,

[0316] the two proteins include a first protein and a second protein,

[0317] the first protein sequentially includes a first binding tag, a first cleavage domain, and a first domain from the N-terminus to the C-terminus,

[0318] the second protein sequentially includes a first binding partner capable of binding to the first binding tag, a second cleavage domain, and a second domain from the N-terminus to the C-terminus,

[0319] the first protein and the second protein can form a dimer through the binding between the first domain and the second domain,

[0320] the first complex is formed by the binding of the first binding tag to the first binding partner and the binding of the first domain to the second domain;

[0321] A production step of cleaving the first cleavage domain and the second cleavage domain in the first complex to produce a heterodimer of the first domain and the second domain.

[0322] (Supplementary Note 2)

[0323] The manufacturing method according to Supplementary Note 1, wherein the first cleavage domain and the second cleavage domain are the same cleavage domain.

[0324] (Supplementary Note 3)

[0325] The manufacturing method according to Supplementary Note 1 or 2, wherein the first cleavage domain and / or the second cleavage domain contains a self-cleaving peptide and / or a cleavage sequence of a protease or peptidase.

[0326] (Supplementary Note 4)

[0327] The manufacturing method according to Supplementary Note 3, wherein the cleavage sequence of the protease is the cleavage sequence of thrombin.

[0328] (Supplementary Note 5)

[0329] The manufacturing method according to any one of Supplementary Notes 1 to 4, wherein the first binding tag and the first binding partner are a peptide tag and a peptide capable of spontaneously forming a covalent bond.

[0330] (Supplementary Note 6)

[0331] The manufacturing method according to any one of Supplementary Notes 1 to 5, wherein the first binding tag and the first binding partner are a combination of a binding tag and a binding partner selected from the following (1) to (3):

[0332] (1) Modified Streptococcus pyogenes surface protein (SpyCatcher) and a peptide tag (SpyTag) capable of binding to the SpyCatcher;

[0333] (2) Modified Streptococcus pneumoniae protein (SnoopCatcher) and a peptide tag (SnoopTag) capable of binding to the SnoopCatcher

[0334] (3) Modified Clostridium perfringens protein Cpe0147 439-563 and a peptide tag Cpe0147 439-563 capable of binding to the Cpe0147 565-587 .

[0335] (Supplementary Note 7)

[0336] The production method according to any one of Supplementary Notes 1 to 6, wherein the bond between the first domain and the second domain is a disulfide bond.

[0337] (Supplementary Note 8)

[0338] The production method according to any one of Supplementary Notes 1 to 7 further includes a first expression step of expressing the first protein and the second protein in a host cell before the complex formation step.

[0339] (Supplementary Note 9)

[0340] The production method according to any one of Supplementary Notes 1 to 8 further includes a first purification step of purifying the first complex after the complex formation step.

[0341] (Supplementary Note 10)

[0342] The production method according to any one of Supplementary Notes 1 to 9 further includes a second purification step of purifying the heterodimer after the production step.

[0343] (Supplementary Note 11)

[0344] The production method according to any one of Supplementary Notes 1 to 7

[0345] The complex formation step is a step of bringing four proteins into contact to form a second complex of the four proteins,

[0346] The four proteins include the first protein, the second protein, a third protein, and a fourth protein,

[0347] The third protein sequentially includes a second binding tag, a third cleavage domain, and a third domain from the N-terminus to the C-terminus,

[0348] The fourth protein sequentially includes a second binding partner capable of binding to the second binding tag, a fourth cleavage domain, and a fourth domain from the N-terminus to the C-terminus,

[0349] The second protein and the fourth protein can form a dimer through the binding between the second domain and the fourth domain,

[0350] The third protein and the fourth protein can form a dimer through the binding between the third domain and the fourth domain,

[0351] By binding the first binding tag to the first binding partner, the second binding tag to the second binding partner, and the first domain to the second domain, the second domain to the fourth domain, and the third domain to the fourth domain, the second complex is formed.

[0352] The generation step cleaves the first cleavage domain, the second cleavage domain, the third cleavage domain, and the fourth cleavage domain in the second complex to generate a heterotetramer of the first domain, the second domain, the third domain, and the fourth domain.

[0353] (Appendix 12)

[0354] The manufacturing method according to Appendix 11, wherein the third cleavage domain and the fourth cleavage domain are the same cleavage domain.

[0355] (Appendix 13)

[0356] The manufacturing method according to Appendix 11 or 12, wherein the third cleavage domain and / or the fourth cleavage domain comprises a self-cleaving peptide and / or a cleavage sequence of a protease or peptidase.

[0357] (Appendix 14)

[0358] The manufacturing method according to Appendix 13, wherein the cleavage sequence of the protease is the cleavage sequence of thrombin.

[0359] (Appendix 15)

[0360] The manufacturing method according to any one of Appendices 11 to 14, wherein the second binding tag and the second binding partner are a peptide tag and a peptide that can spontaneously form a covalent bond.

[0361] (Appendix 16)

[0362] The manufacturing method according to any one of Appendices 11 to 15, wherein the second binding tag and the second binding partner are a combination of a binding tag and a binding partner selected from the following (1) to (3):

[0363] (1) Streptococcus pyogenes surface protein (SpyCatcher) and a peptide tag (SpyTag) capable of binding to the SpyCatcher;

[0364] (2) Streptococcus pneumoniae protein (SnoopCatcher) and a peptide tag (SnoopTag) capable of binding to the SnoopCatcher

[0365] (3) Modified Clostridium perfringens protein Cpe0147 439-563 and a peptide tag Cpe0147 that can bind to the Cpe0147 439-563 . 565-587

[0366] (Supplementary Note 17)

[0367] The production method according to any one of Supplementary Notes 11 to 16, wherein the binding between the third domain and the fourth domain, and / or the binding between the second domain and the fourth domain is a disulfide bond binding.

[0368] (Supplementary Note 18)

[0369] The production method according to any one of Supplementary Notes 11 to 17, wherein

[0370] the first domain is the light chain of an antibody that binds to a first target,

[0371] the second domain is the heavy chain of an antibody that binds to the first target,

[0372] the third domain is the light chain of an antibody that binds to a second target,

[0373] the fourth domain is the heavy chain of an antibody that binds to the second target.

[0374] (Supplementary Note 19)

[0375] The production method according to Supplementary Note 18, wherein the antibody that binds to the first target and the antibody that binds to the second target recognize different epitopes.

[0376] (Supplementary Note 20)

[0377] The production method according to Supplementary Note 18 or 19, wherein the antibody that binds to the first target and the antibody that binds to the second target recognize different antigens.

[0378] (Supplementary Note 21)

[0379] The production method according to any one of Supplementary Notes 18 to 20, wherein the antibody that binds to the first target and the antibody that binds to the second target are IgG, IgA, IgE, IgD or IgM.

[0380] (Supplementary Note 22)

[0381] The production method according to Supplementary Note 21, wherein the IgG is IgG1, IgG2, IgG2a, IgG2b, IgG3 or IgG4.

[0382] (Supplementary Note 23) ​

[0383] The production method according to any one of Attachments 11 to 22, wherein the first cleavage domain, the second cleavage domain, the third cleavage domain, and the fourth cleavage domain are the same cleavage domain.

[0384] (Attachment 24)

[0385] The production method according to any one of Attachments 11 to 23, wherein the combination of the first binding tag and the first binding partner, and the combination of the second binding tag and the second binding partner are different combinations of binding tags and binding partners.

[0386] (Attachment 25)

[0387] The production method according to any one of Attachments 11 to 24, wherein

[0388] The second protein further sequentially includes a fifth cleavage domain and a third binding tag at the C-terminus,

[0389] The fourth protein further sequentially includes a sixth cleavage domain and a third binding partner capable of binding to the third binding tag at the C-terminus,

[0390] In the complex formation step, the third binding tag and the third binding partner further bind,

[0391] In the generation step, the fifth cleavage domain and the sixth cleavage domain in the second complex are cleaved.

[0392] (Attachment 26)

[0393] The production method according to Attachment 25, wherein the fifth cleavage domain and the sixth cleavage domain are the same cleavage domain.

[0394] (Attachment 27)

[0395] The production method according to Attachment 25 or 26, wherein the fifth cleavage domain and / or the sixth cleavage domain includes a self-cleaving peptide and / or a cleavage sequence of a protease or peptidase.

[0396] (Attachment 28)

[0397] The production method according to Attachment 27, wherein the cleavage sequence of the protease is the cleavage sequence of thrombin.

[0398] (Attachment 29)

[0399] The production method according to any one of Attachments 25 to 28, wherein the third binding tag and the third binding partner are a peptide tag and a peptide capable of spontaneously forming a covalent bond.

[0400] (Supplementary Note 30)

[0401] The manufacturing method according to any one of Supplementary Notes 25 to 29, wherein the third binding tag and the third binding partner are a combination of a binding tag and a binding partner selected from the following (1) to (3):

[0402] (1) Streptococcus pyogenes surface protein (SpyCatcher) and a peptide tag (SpyTag) capable of binding to the SpyCatcher;

[0403] (2) Streptococcus pneumoniae protein (SnoopCatcher) and a peptide tag (SnoopTag) capable of binding to the SnoopCatcher

[0404] (3) Modified Clostridium perfringens protein Cpe0147 439-563 and a peptide tag Cpe0147 capable of binding to the Cpe0147 439-563 565-587 .

[0405] (Supplementary Note 31)

[0406] The manufacturing method according to any one of Supplementary Notes 25 to 30, wherein the first cleavage domain, the second cleavage domain, the third cleavage domain, the fourth cleavage domain, the fifth cleavage domain, and the sixth cleavage domain are the same cleavage domain.

[0407] (Supplementary Note 32)

[0408] The manufacturing method according to any one of Supplementary Notes 25 to 31, wherein the first binding tag and the first binding partner, the second binding tag and the second binding partner, and the third binding tag and the third binding partner are combinations of different binding tags and binding partners.

[0409] (Supplementary Note 33)

[0410] The manufacturing method according to any one of Supplementary Notes 11 to 32 further includes a first expression step of expressing the first protein, the second protein, the third protein, and the fourth protein in a host cell before the complex formation step.

[0411] (Supplementary Note 34)

[0412] The manufacturing method according to any one of Supplementary Notes 11 to 33 further includes a first purification step of purifying the second complex after the complex formation step.

[0413] (Supplementary Note 35)​

[0414] The manufacturing method according to any one of Attachments 11 to 34 further includes a second purification step of purifying the heterodimer after the generation step.

[0415] <Protein>

[0416] (Attachment 36)

[0417] A protein that sequentially includes a first binding tag capable of binding to a first binding partner, a first cleavage domain, and a first domain from the N-terminus to the C-terminus.

[0418] (Attachment 37)

[0419] The protein according to Attachment 36, wherein the first cleavage domain includes a self-cleaving peptide and / or a cleavage sequence of a protease or peptidase.

[0420] (Attachment 38)

[0421] The protein according to Attachment 37, wherein the cleavage sequence of the protease is the cleavage sequence of thrombin.

[0422] (Attachment 39)

[0423] The protein according to any one of Attachments 36 to 38, wherein the first binding tag and the first binding partner are a peptide tag and a peptide capable of spontaneously forming a covalent bond.

[0424] (Attachment 40)

[0425] The protein according to any one of Attachments 36 to 39, wherein the first binding tag and the first binding partner are a combination of a binding tag and a binding partner selected from the following (1) to (3):

[0426] (1) Streptococcus pyogenes surface protein (SpyCatcher) and a peptide tag (SpyTag) capable of binding to the SpyCatcher;

[0427] (2) Streptococcus pneumoniae protein (SnoopCatcher) and a peptide tag (SnoopTag) capable of binding to the SnoopCatcher

[0428] (3) Modified Clostridium perfringens protein Cpe0147 439-563 and a peptide tag Cpe0147 capable of binding to the Cpe0147 439-563 565-587 .

[0429] (Attachment 41)

[0430] ​The protein according to any one of Attachments 36 to 40, wherein the first domain is the light chain of an antibody that binds to a first target.

[0431] (Attachment 42)

[0432] A protein that sequentially includes, from the N-terminus to the C-terminus, a first binding partner capable of binding to a first binding tag, a second cleavage domain, and a second domain.

[0433] (Attachment 43)

[0434] The protein according to Attachment 42, wherein the second cleavage domain includes a self-cleaving peptide and / or a cleavage sequence of a protease or peptidase.

[0435] (Attachment 44)

[0436] The protein according to Attachment 43, wherein the cleavage sequence of the protease is the cleavage sequence of thrombin.

[0437] (Attachment 45)

[0438] The protein according to any one of Attachments 42 to 44, wherein the first binding tag and the first binding partner are a peptide tag and a peptide that can spontaneously form a covalent bond.

[0439] (Attachment 46)

[0440] The protein according to any one of Attachments 42 to 45, wherein the first binding tag and the first binding partner are a combination of a binding tag and a binding partner selected from the following (1) to (3):

[0441] (1) Streptococcus pyogenes surface protein (SpyCatcher) and a peptide tag (SpyTag) capable of binding to the SpyCatcher;

[0442] (2) Streptococcus pneumoniae protein (SnoopCatcher) and a peptide tag (SnoopTag) capable of binding to the SnoopCatcher

[0443] (3) Modified Clostridium perfringens protein Cpe0147 439-563 and a peptide tag Cpe0147 capable of binding to the Cpe0147 439-563 binding 565-587 .

[0444] (Attachment 47)

[0445] The protein according to any one of Attachments 42 to 46, wherein the second domain is the heavy chain of an antibody that binds to a first target.

[0446] (Supplementary Note 48)

[0447] The protein according to any one of Supplementary Notes 42 to 47, further comprising, in sequence at the C-terminus, a fifth cleavage domain, a third binding tag, or a third binding partner capable of binding to the third binding tag.

[0448] (Supplementary Note 49)

[0449] The protein according to Supplementary Note 48, wherein the fifth cleavage domain comprises a self-cleaving peptide and / or a cleavage sequence of a protease or peptidase.

[0450] (Supplementary Note 50)

[0451] The protein according to Supplementary Note 49, wherein the cleavage sequence of the protease is the cleavage sequence of thrombin.

[0452] (Supplementary Note 51)

[0453] The protein according to any one of Supplementary Notes 48 to 50, wherein the third binding tag and the third binding partner are a peptide tag and a peptide capable of spontaneously forming a covalent bond.

[0454] (Supplementary Note 52)

[0455] The protein according to any one of Supplementary Notes 48 to 51, wherein the second cleavage domain and the fifth cleavage domain are the same cleavage domain.

[0456] (Supplementary Note 53)

[0457] The protein according to any one of Supplementary Notes 48 to 52, wherein the first binding tag and the first binding partner, and the third binding tag and the third binding partner are combinations of different binding tags and binding partners.

[0458] (Supplementary Note 54)

[0459] The protein according to any one of Supplementary Notes 48 to 53, wherein the third binding tag and the third binding partner are a combination of a binding tag and a binding partner selected from the following (1) to (3):

[0460] (1) Streptococcus pyogenes surface protein (SpyCatcher) and a peptide tag (SpyTag) capable of binding to the SpyCatcher;

[0461] (2) Streptococcus pneumoniae protein (SnoopCatcher) and a peptide tag (SnoopTag) capable of binding to the SnoopCatcher

[0462] (3) Modified Clostridium perfringens protein Cpe0147439-563 and a peptide tag Cpe0147 capable of binding to said Cpe0147 439-563 。 565-587 。

[0463] (Supplementary Note 55)

[0464] A protein comprising, in sequence from the N-terminus to the C-terminus, a second binding tag capable of binding to a second binding partner, a third cleavage domain, and a third domain.

[0465] (Supplementary Note 56)

[0466] The protein according to Supplementary Note 55, wherein the third cleavage domain comprises a self-cleaving peptide and / or a cleavage sequence of a protease or peptidase.

[0467] (Supplementary Note 57)

[0468] The protein according to Supplementary Note 56, wherein the cleavage sequence of the protease is the cleavage sequence of thrombin.

[0469] (Supplementary Note 58)

[0470] The protein according to any one of Supplementary Notes 55 to 57, wherein the second binding tag and the second binding partner are a peptide tag and a peptide capable of spontaneously forming a covalent bond.

[0471] (Supplementary Note 59)

[0472] The protein according to any one of Supplementary Notes 55 to 58, wherein the second binding tag and the second binding partner are a combination of a binding tag and a binding partner selected from the following (1) to (3):

[0473] (1) Streptococcus pyogenes surface protein (SpyCatcher) and a peptide tag (SpyTag) capable of binding to said SpyCatcher;

[0474] (2) Streptococcus pneumoniae protein (SnoopCatcher) and a peptide tag (SnoopTag) capable of binding to said SnoopCatcher

[0475] (3) Modified Clostridium perfringens protein Cpe0147 439-563 and a peptide tag Cpe0147 capable of binding to said Cpe0147 439-563 。 565-587 。

[0476] (Supplementary Note 60)

[0477] The protein according to any one of Supplementary Notes 55 to 59, wherein the third domain is the light chain of an antibody that binds to a second target.

[0478] (Supplementary Note 61)

[0479] A protein that sequentially includes, from the N-terminus to the C-terminus, a first binding partner capable of binding to a second binding tag, a fourth cleavage domain, and a fourth domain.

[0480] (Supplementary Note 62)

[0481] The protein according to Supplementary Note 61, wherein the fourth cleavage domain includes a self-cleaving peptide and / or a cleavage sequence of a protease or peptidase.

[0482] (Supplementary Note 63)

[0483] The protein according to Supplementary Note 62, wherein the cleavage sequence of the protease is a cleavage sequence of thrombin.

[0484] (Supplementary Note 64)

[0485] The protein according to any one of Supplementary Notes 61 to 63, wherein the second binding tag and the first binding partner are a peptide tag and a peptide capable of spontaneously forming a covalent bond.

[0486] (Supplementary Note 65)

[0487] The protein according to any one of Supplementary Notes 61 to 64, wherein the second binding tag and the first binding partner are a combination of a binding tag and a binding partner selected from the following (1) to (3):

[0488] (1) Streptococcus pyogenes surface protein (SpyCatcher) and a peptide tag (SpyTag) capable of binding to the SpyCatcher;

[0489] (2) Streptococcus pneumoniae protein (SnoopCatcher) and a peptide tag (SnoopTag) capable of binding to the SnoopCatcher

[0490] (3) Modified Clostridium perfringens protein Cpe0147 439-563 and a peptide tag Cpe0147 capable of binding to the Cpe0147 439-563 binding 565-587 .

[0491] (Supplementary Note 66)

[0492] The protein according to any one of Supplementary Notes 61 to 65, wherein the fourth domain is a heavy chain of an antibody that binds to a second target.

[0493] (Supplementary Note 67)

[0494] The protein according to any one of Appendices 61 to 66, further comprising, in sequence at the C-terminus, a sixth cleavage domain and a third binding partner capable of binding to the third binding tag.

[0495] (Appendix 68)

[0496] The protein according to Appendix 67, wherein the sixth cleavage domain comprises a self-cleaving peptide and / or a cleavage sequence of a protease or peptidase.

[0497] (Appendix 69)

[0498] The protein according to Appendix 68, wherein the cleavage sequence of the protease is the cleavage sequence of thrombin.

[0499] (Appendix 70)

[0500] The protein according to any one of Appendices 67 to 69, wherein the third binding tag and the third binding partner are a peptide tag and a peptide capable of spontaneously forming a covalent bond.

[0501] (Appendix 71)

[0502] The protein according to any one of Appendices 67 to 70, wherein the fourth cleavage domain and the sixth cleavage domain are the same cleavage domain.

[0503] (Appendix 72)

[0504] The protein according to any one of Appendices 67 to 71, wherein the second binding tag and the second binding partner, and the third binding tag and the third binding partner are combinations of different binding tags and binding partners.

[0505] (Appendix 73)

[0506] The protein according to any one of Appendices 67 to 72, wherein the third binding tag and the third binding partner are a combination of a binding tag and a binding partner selected from the following (1) to (3):

[0507] (1) Streptococcus pyogenes surface protein (SpyCatcher) and a peptide tag (SpyTag) capable of binding to the SpyCatcher;

[0508] (2) Streptococcus pneumoniae protein (SnoopCatcher) and a peptide tag (SnoopTag) capable of binding to the SnoopCatcher

[0509] (3) Modified Clostridium perfringens protein Cpe0147 439-563 and a peptide capable of binding to the Cpe0147 439-563The conjugated peptide tag Cpe0147 565-587 。

[0510] (Supplementary Note 74)

[0511] A protein according to any one of Supplementary Notes 36 to 73, which is used in the method for producing a heteromultimeric protein according to any one of Supplementary Notes 1 to 35.

[0512] <Heteromultimeric protein>

[0513] (Supplementary Note 75)

[0514] A protein,

[0515] which contains two proteins,

[0516] wherein the two proteins include a first protein and a second protein,

[0517] wherein the first protein sequentially includes a first binding tag, a first cleavage domain, and a first domain from the N-terminus to the C-terminus,

[0518] wherein the second protein sequentially includes a first binding partner capable of binding to the first binding tag, a second cleavage domain, and a second domain from the N-terminus to the C-terminus,

[0519] wherein the first protein and the second protein form a dimer through the binding between the first domain and the second domain,

[0520] wherein the first binding tag binds to the first binding partner.

[0521] (Supplementary Note 76)

[0522] The protein according to Supplementary Note 75, wherein the first cleavage domain and the second cleavage domain are the same cleavage domain.

[0523] (Supplementary Note 77)

[0524] The protein according to Supplementary Note 75 or 76, wherein the first cleavage domain and / or the second cleavage domain contains a self-cleaving peptide and / or a cleavage sequence of a protease or peptidase.

[0525] (Supplementary Note 78)

[0526] The protein according to any one of Supplementary Notes 75 to 77, wherein the first binding tag and the first binding partner are a peptide tag and a peptide capable of spontaneously forming a covalent bond.

[0527] (Supplementary Note 79)

[0528] The protein according to any one of Attachments 75 to 78, wherein the first binding tag and the first binding partner are a combination of a binding tag and a binding partner selected from the following (1) to (3):

[0529] (1) Streptococcus pyogenes surface protein (SpyCatcher) and a peptide tag (SpyTag) capable of binding to the SpyCatcher;

[0530] (2) Streptococcus pneumoniae protein (SnoopCatcher) and a peptide tag (SnoopTag) capable of binding to the SnoopCatcher

[0531] (3) Modified Clostridium perfringens protein Cpe0147 439-563 and a peptide tag Cpe0147 capable of binding to the Cpe0147 439-563 565-587

[0532] (Attachment 80)

[0533] The protein according to any one of Attachments 75 to 79, wherein the binding between the first domain and the second domain is a disulfide bond binding.

[0534] (Attachment 81)

[0535] The protein according to any one of Attachments 75 to 80,

[0536] further contains a third protein and a fourth protein,

[0537] the third protein sequentially contains a second binding tag, a third cleavage domain, and a third domain from the N-terminus to the C-terminus,

[0538] the fourth protein sequentially contains a second binding partner capable of binding to the second binding tag, a fourth cleavage domain, and a fourth domain from the N-terminus to the C-terminus,

[0539] the second protein and the fourth protein form a dimer through the binding between the second domain and the fourth domain,

[0540] the third protein and the fourth protein form a dimer through the binding between the third domain and the fourth domain,

[0541] the first binding tag binds to the first binding partner,

[0542] the second binding tag binds to the second binding partner.

[0543] (Attachment 82) ​​

[0544] The protein according to Note 81, wherein the third cleavage domain and the fourth cleavage domain are the same cleavage domain.

[0545] (Note 83)

[0546] The protein according to Note 81 or 82, wherein the third cleavage domain and / or the fourth cleavage domain comprises a self-cleaving peptide and / or a cleavage sequence of a protease or peptidase.

[0547] (Note 84)

[0548] The protein according to Note 83, wherein the cleavage sequence of the protease is the cleavage sequence of thrombin.

[0549] (Note 85)

[0550] The protein according to any one of Notes 81 to 84, wherein the second binding tag and the second binding partner are a peptide tag and a peptide capable of spontaneously forming a covalent bond.

[0551] (Note 86)

[0552] The protein according to any one of Notes 81 to 85, wherein the second binding tag and the second binding partner are a combination of a binding tag and a binding partner selected from the following (1) to (3):

[0553] (1) Streptococcus pyogenes surface protein (SpyCatcher) and a peptide tag (SpyTag) capable of binding to the SpyCatcher;

[0554] (2) Streptococcus pneumoniae protein (SnoopCatcher) and a peptide tag (SnoopTag) capable of binding to the SnoopCatcher

[0555] (3) Modified Clostridium perfringens protein Cpe0147 439-563 and a peptide tag Cpe0147 capable of binding to the Cpe0147 439-563 binding 565-587 .

[0556] (Note 87)

[0557] The protein according to any one of Notes 81 to 86, wherein the binding between the third domain and the fourth domain, and / or the binding between the second domain and the fourth domain is a disulfide bond binding.

[0558] (Note 88)

[0559] The protein according to any one of Notes 81 to 87, wherein,

[0560] The first domain is the light chain of an antibody that binds to a first target.

[0561] The second domain is the heavy chain of an antibody that binds to the first target.

[0562] The third domain is the light chain of an antibody that binds to a second target.

[0563] The fourth domain is the heavy chain of an antibody that binds to the second target.

[0564] (Supplementary Note 89)

[0565] The protein according to Supplementary Note 88, wherein the antibody that binds to the first target and the antibody that binds to the second target recognize different epitopes.

[0566] (Supplementary Note 90)

[0567] The protein according to Supplementary Note 88 or 89, wherein the antibody that binds to the first target and the antibody that binds to the second target recognize different antigens.

[0568] (Supplementary Note 91)

[0569] The protein according to any one of Supplementary Notes 88 to 90, wherein the antibody that binds to the first target and the antibody that binds to the second target are IgG, IgA, IgE, IgD or IgM.

[0570] (Supplementary Note 92)

[0571] The protein according to Supplementary Note 91, wherein the IgG is IgG1, IgG2, IgG2a, IgG2b, IgG3 or IgG4.

[0572] (Supplementary Note 93)

[0573] The protein according to any one of Supplementary Notes 81 to 92, wherein the first cleavage domain, the second cleavage domain, the third cleavage domain and the fourth cleavage domain are the same cleavage domain.

[0574] (Supplementary Note 94)

[0575] The protein according to any one of Supplementary Notes 81 to 93, wherein the first binding tag and the first binding partner, and the second binding tag and the second binding partner are combinations of different binding tags and binding partners.

[0576] (Supplementary Note 95)

[0577] The protein according to any one of Supplementary Notes 81 to 94, wherein

[0578] The second protein further sequentially includes a fifth cleavage domain and a third binding tag at its C-terminus.

[0579] The fourth protein further sequentially includes a sixth cleavage domain and a third binding partner capable of binding to the third binding tag at its C-terminus.

[0580] The third binding tag and the third binding partner bind to each other.

[0581] (Supplementary Note 96)

[0582] The protein according to Supplementary Note 95, wherein the fifth cleavage domain and the sixth cleavage domain are the same cleavage domain.

[0583] (Supplementary Note 97)

[0584] The protein according to Supplementary Note 95 or 96, wherein the fifth cleavage domain and / or the sixth cleavage domain includes a self-cleaving peptide and / or a cleavage sequence of a protease or peptidase.

[0585] (Supplementary Note 98)

[0586] The protein according to Supplementary Note 97, wherein the cleavage sequence of the protease is the cleavage sequence of thrombin.

[0587] (Supplementary Note 99)

[0588] The protein according to any one of Supplementary Notes 95 to 98, wherein the third binding tag and the third binding partner are a peptide tag and a peptide capable of spontaneously forming a covalent bond.

[0589] (Supplementary Note 100)

[0590] The protein according to any one of Supplementary Notes 95 to 99, wherein the third binding tag and the third binding partner are a combination of a binding tag and a binding partner selected from the following (1) to (3):

[0591] (1) Streptococcus pyogenes surface protein (SpyCatcher) and a peptide tag (SpyTag) capable of binding to the SpyCatcher;

[0592] (2) Streptococcus pneumoniae protein (SnoopCatcher) and a peptide tag (SnoopTag) capable of binding to the SnoopCatcher

[0593] (3) Modified Clostridium perfringens protein Cpe0147 439-563 and a peptide tag Cpe0147 capable of binding to the Cpe0147 439-563 565-587 .​

[0594] (Supplementary Note 101)

[0595] The protein according to any one of Supplementary Notes 95 to 100, wherein the first cleavage domain, the second cleavage domain, the third cleavage domain, the fourth cleavage domain, the fifth cleavage domain, and the sixth cleavage domain are the same cleavage domain.

[0596] (Supplementary Note 102)

[0597] The protein according to any one of Supplementary Notes 95 to 101, wherein the combination of the first binding tag and the first binding partner, the second binding tag and the second binding partner, and the third binding tag and the third binding partner are different combinations of binding tags and binding partners.

[0598] <Nucleic acid>

[0599] (Supplementary Note 103)

[0600] A nucleic acid encoding the protein according to any one of Supplementary Notes 36 to 102.

[0601] <Expression vector>

[0602] (Supplementary Note 104)

[0603] An expression vector comprising the nucleic acid according to Supplementary Note 103.

[0604] <Transformant>

[0605] (Supplementary Note 105)

[0606] A transformant comprising the nucleic acid according to Supplementary Note 103 and / or the expression vector according to Supplementary Note 104.

[0607] <Method for producing a protein>

[0608] (Supplementary Note 106)

[0609] A method for producing a protein, comprising an expression step of expressing the nucleic acid according to Supplementary Note 103 and / or the expression vector according to Supplementary Note 104.

[0610] (Supplementary Note 107)

[0611] According to the production method described in Supplementary Note 106, wherein the expression step includes:

[0612] A culturing step of culturing the transformant according to Supplementary Note 105;

[0613] A separating step of separating the protein according to any one of Supplementary Notes 36 to 102.

[0614]

Industrial Applicability

[0615] As described above, according to the present invention, hetero-multimeric proteins such as bispecific antibodies can be effectively produced. Therefore, the present invention is extremely useful in, for example, the pharmaceutical field, the field of drug manufacturing, and the like.

Claims

1. A method for manufacturing a heteromultimeric protein, comprising: a complex formation step of bringing two proteins into contact to form a first complex of the two proteins, wherein the two proteins include a first protein and a second protein, the first protein sequentially includes a first binding tag, a first cleavage domain, and a first domain from the N-terminus to the C-terminus, the second protein sequentially includes a first binding partner capable of binding to the first binding tag, a second cleavage domain, and a second domain from the N-terminus to the C-terminus, the first protein and the second protein can form a dimer through the binding between the first domain and the second domain, forming the first complex by binding the first binding tag to the first binding partner and binding the first domain to the second domain; a generation step of cleaving the first cleavage domain and the second cleavage domain in the first complex to generate a heterodimer of the first domain and the second domain.

2. The manufacturing method according to claim 1, wherein the first cleavage domain and the second cleavage domain are the same cleavage domain.

3. The manufacturing method according to claim 1 or 2, wherein the first cleavage domain and / or the second cleavage domain contains a self-cleaving peptide and / or a cleavage sequence of a protease or peptidase.

4. The manufacturing method according to claim 1 or 2, wherein the first binding tag and the first binding partner are a peptide tag and a peptide capable of spontaneously forming a covalent bond.

5. The manufacturing method according to claim 1 or 2, wherein the first binding tag and the first binding partner are a combination of a binding tag and a binding partner selected from the following (1) to (3): (1) Modified Streptococcus pyogenes surface protein (SpyCatcher) and a peptide tag (SpyTag) capable of binding to the SpyCatcher; (2) Modified Streptococcus pneumoniae protein (SnoopCatcher) and a peptide tag (SnoopTag) capable of binding to the SnoopCatcher (3) Modified Clostridium perfringens protein Cpe0147 439-563 and a peptide tag Cpe0147 capable of binding to said Cpe0147 439-563 565-587 .​ 6. The manufacturing method according to claim 1 or 2, wherein the binding between the first domain and the second domain is an isopeptide bond binding.

7. The manufacturing method according to claim 1 or 2, wherein, the complex formation step is a step of bringing four proteins into contact to form a second complex of the four proteins, the four proteins include the first protein, the second protein, a third protein, and a fourth protein, the third protein sequentially includes a second binding tag, a third cleavage domain, and a third domain from the N-terminus to the C-terminus, the fourth protein sequentially includes a second binding partner capable of binding to the second binding tag, a fourth cleavage domain, and a fourth domain from the N-terminus to the C-terminus, the second protein and the fourth protein can form a dimer through the binding between the second domain and the fourth domain, The 3rd protein and the 4th protein can form a dimer through the binding between the 3rd domain and the 4th domain. By binding the 1st binding tag to the 1st binding partner, the 2nd binding tag to the 2nd binding partner, and the 1st domain to the 2nd domain, the 2nd domain to the 4th domain, and the 3rd domain to the 4th domain, the 2nd complex is formed. The generation step cleaves the 1st cleavage domain, the 2nd cleavage domain, the 3rd cleavage domain, and the 4th cleavage domain in the 2nd complex to generate a heterotetramer of the 1st domain, the 2nd domain, the 3rd domain, and the 4th domain.

8. The manufacturing method according to claim 7, wherein the 3rd cleavage domain and the 4th cleavage domain are the same cleavage domain.

9. The manufacturing method according to claim 7, wherein the 3rd cleavage domain and / or the 4th cleavage domain contains a self-cleaving peptide and / or a cleavage sequence of a protease or peptidase.

10. The manufacturing method according to claim 7, wherein the 2nd binding tag and the 2nd binding partner are a peptide tag and a peptide that can spontaneously form a covalent bond.

11. The manufacturing method according to claim 7, wherein the 2nd binding tag and the 2nd binding partner are a combination of a binding tag and a binding partner selected from the following (1) to (3): (1) Streptococcus pyogenes surface protein (SpyCatcher) and a peptide tag (SpyTag) capable of binding to the SpyCatcher; (2) Streptococcus pneumoniae protein (SnoopCatcher) and a peptide tag (SnoopTag) capable of binding to the SnoopCatcher (3) Modified Clostridium perfringens protein Cpe0147 439-563 and a peptide tag Cpe0147 capable of binding to the Cpe0147 439-563 565-587 .​ 12. The manufacturing method according to claim 7, wherein the binding between the 3rd domain and the 4th domain, and / or the binding between the 2nd domain and the 4th domain is a disulfide bond binding.

13. The manufacturing method according to claim 7, wherein the 1st domain is the light chain of an antibody that binds to a 1st target, the 2nd domain is the heavy chain of an antibody that binds to the 1st target, the 3rd domain is the light chain of an antibody that binds to a 2nd target, the 4th domain is the heavy chain of an antibody that binds to the 2nd target.

14. The manufacturing method according to claim 13, wherein the antibody that binds to the 1st target and the antibody that binds to the 2nd target recognize different epitopes.

15. The manufacturing method according to claim 13 or 14, wherein the antibody that binds to the 1st target and the antibody that binds to the 2nd target recognize different antigens.

16. The manufacturing method according to claim 7, wherein the 1st cleavage domain, the 2nd cleavage domain, the 3rd cleavage domain, and the 4th cleavage domain are the same cleavage domain.

17. The manufacturing method according to claim 7, wherein the first binding tag and the first binding partner, and the second binding tag and the second binding partner are combinations of different binding tags and binding partners.

18. The manufacturing method according to claim 7, wherein the second protein further sequentially includes a fifth cleavage domain and a third binding tag at the C-terminus, the fourth protein further sequentially includes a sixth cleavage domain and a third binding partner capable of binding to the third binding tag at the C-terminus, in the complex formation step, the third binding tag and the third binding partner further bind to each other, in the generation step, the fifth cleavage domain and the sixth cleavage domain in the second complex are cleaved.

19. The manufacturing method according to claim 18, wherein the fifth cleavage domain and the sixth cleavage domain are the same cleavage domain.

20. The manufacturing method according to claim 18 or 19, wherein the fifth cleavage domain and / or the sixth cleavage domain includes a self-cleaving peptide and / or a cleavage sequence of a protease or peptidase.

21. The manufacturing method according to claim 18 or 19, wherein the third binding tag and the third binding partner are a peptide tag and a peptide capable of spontaneously forming a covalent bond.

22. The manufacturing method according to claim 18 or 19, wherein the third binding tag and the third binding partner are a combination of a binding tag and a binding partner selected from the following (1) to (3): (1) Streptococcus pyogenes surface protein (SpyCatcher) and a peptide tag (SpyTag) capable of binding to the SpyCatcher; (2) Streptococcus pneumoniae protein (SnoopCatcher) and a peptide tag (SnoopTag) capable of binding to the SnoopCatcher (3) Modified Clostridium perfringens protein Cpe0147 439-563 and a peptide tag Cpe0147 that can bind to said Cpe0147 439-563 565-587 .​ 23. The manufacturing method according to claim 18 or 19, wherein the first cleavage domain, the second cleavage domain, the third cleavage domain, the fourth cleavage domain, the fifth cleavage domain, and the sixth cleavage domain are the same cleavage domain.

24. The manufacturing method according to claim 18 or 19, wherein the first binding tag and the first binding partner, the second binding tag and the second binding partner, and the third binding tag and the third binding partner are combinations of different binding tags and binding partners.

25. A protein that sequentially includes a first binding tag capable of binding to a first binding partner, a first cleavage domain, and a first domain from the N-terminus to the C-terminus.

26. A protein that sequentially includes a first binding partner capable of binding to a first binding tag, a second cleavage domain, and a second domain from the N-terminus to the C-terminus.

27. A protein that sequentially includes a second binding tag capable of binding to a second binding partner, a third cleavage domain, and a third domain from the N-terminus to the C-terminus.

28. A protein that, from the N-terminus to the C-terminus, sequentially comprises a first binding partner capable of binding to a second binding tag, a fourth cleavage domain, and a fourth domain.

29. A protein, which contains two proteins, wherein the two proteins include a first protein and a second protein, the first protein sequentially comprises a first binding tag, a first cleavage domain, and a first domain from the N-terminus to the C-terminus, the second protein sequentially comprises a first binding partner capable of binding to the first binding tag, a second cleavage domain, and a second domain from the N-terminus to the C-terminus, the first protein and the second protein form a dimer through the binding between the first domain and the second domain, and the first binding tag binds to the first binding partner.

30. The protein according to claim 29, which further contains a third protein and a fourth protein, the third protein sequentially comprises a second binding tag, a third cleavage domain, and a third domain from the N-terminus to the C-terminus, the fourth protein sequentially comprises a second binding partner capable of binding to the second binding tag, a fourth cleavage domain, and a fourth domain from the N-terminus to the C-terminus, the second protein and the fourth protein form a dimer through the binding between the second domain and the fourth domain, the third protein and the fourth protein form a dimer through the binding between the third domain and the fourth domain, the first binding tag binds to the first binding partner, and the second binding tag binds to the second binding partner.

31. A nucleic acid encoding the protein according to any one of claims 25 to 29.

32. An expression vector comprising the nucleic acid according to claim 31.

33. A transformant comprising the nucleic acid according to claim 31.

34. A method for producing a protein, which comprises an expression step of expressing the nucleic acid according to claim 31.

Images