Antigen-binding molecules and combinations

Abstract

To provide an antibody modification technology for exerting the effector function of an antibody drug in a target tissue-specific manner, is useful for reducing antibody drug side effects, and can also be applied to various other protein engineering applications.SOLUTION: Provided are a first antigen-binding molecule, a second antigen-binding molecule, and a combination thereof. The second antigen-binding molecule binds to a complex of an antigen and antigen-binding molecule, which contains the first antigen and the first antigen-binding molecule, and increases the binding activity of the first antigen-binding molecule to the first antigen.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This invention relates to antigen-binding molecules and combinations. [Background technology]

[0002] Antibodies are proteins that bind specifically to antigens with high affinity. A wide variety of molecules, from small molecules to proteins, are known to act as antigens. Since the development of monoclonal antibody production techniques, antibody modification techniques have advanced, making it easier to obtain antibodies that recognize specific molecules. For example, a domino antibody that recognizes the light chain portion of a primary antibody and specifically recognizes the antigen-binding primary antibody is used in immunological assays such as ELISA (Patent Document 1). A junction epitope antibody that stabilizes the protein-protein interaction between IL-6 and gp80 modifies its downstream signaling (Scientific Reports (2017) 7, 1-15 Ralph, A. et al. (Non-Patent Document 9)).

[0003] Antibodies are attracting attention as pharmaceuticals due to their high stability in plasma and low incidence of side effects. Antibodies not only bind to antigens and exhibit agonist and antagonist activity, but also induce cytotoxic activity (also called effector function) by effector cells, such as ADCC (Antibody-Dependent Cytotoxicity), ADCP (Antibody-Dependent Cell phagocytosis), and CDC (Complement-Dependent Cytotoxicity). Pharmaceuticals for cancer, immunological diseases, chronic diseases, and infectious diseases have been developed utilizing these functions of antibodies (Paul J. Carter and Greg A. Lazar, Next generation antibody drugs: pursuit of the 'high-hanging fruit', [online], December 1, 2017, Nature Reviews Drug Discovery, [Retrieved January 22, 2018], Internet <https: / / www.nature.com / articles / nrd.2017.227> (Non-patent Literature 1)).

[0004] For example, drugs utilizing agonist antibodies against costimulatory molecules that promote the activation of cytotoxic T cells have been developed as anticancer agents (Clinical and Experimental Immunology (2009) 157, 9-19 Peggs, KSet al. (Non-Patent Literature 2)). In recent years, it has become clear that inhibitory antibodies against immune checkpoints that have antagonist activity against co-inhibitory molecules are useful as anticancer agents, and antibody drugs that inhibit the interaction of CTLA4 / CD80 and PD-1 / PD-L1, such as Ipilimumab, Nivolumab, Pembrolizumab, and Atezolizumab, have been successively launched (Non-Patent Literature 1).

[0005] Second-generation antibody drugs have been developed by artificially enhancing, adding, weakening, or deleting the function of natural IgG antibodies, thereby enhancing or adding, weakening or deleting, the function of which is tailored to the application of the antibody. Examples of second-generation antibody drugs include antibodies with enhanced or deleting effector function (Current Pharmaceutical Biotechnology (2016) 17, 1298-1314 Mimoto, F. et al. (Non-Patent Literature 3)), antibodies that bind to antigens in a pH-dependent manner (Nature Biotechnology (2010) 28, 1203-1208 Igawa, T. et al. (Non-Patent Literature 4)), and antibodies that bind to two or more antigens with a single molecule (antibodies that bind to two antigens are generally called "bispecific antibodies") (MAbs. (2012) Mar 1, 4(2) (Non-Patent Literature 5)).

[0006] Bispecific antibodies are expected to become more effective pharmaceuticals. For example, by using a protein expressed on the cell membrane of T cells as one antigen and a cancer antigen as the other antigen, antibodies with enhanced antitumor activity (in this specification, this antitumor activity is abbreviated as "TDCC activity" (T-cell Dependent Cytotoxicity) and is included in effector function) have been developed to crosslink cytotoxic T cells with cancer cells (Journal of Biomolecular Screening (2015) 20, 519-27 Nazarian, AA et al. (Non-patent document 10)). Examples of bispecific antibodies include molecules in which the two Fab regions of the antibody have different sequences (common light chain bispecific antibodies and hybrid hybridomas), molecules in which antigen-binding sites have been added to the N-terminus or C-terminus of the antibody (DVD-Ig and scFv-IgG), molecules in which one Fab region binds to two antigens (Two-in-one IgG), and molecules in which the loop region of the CH3 region has been used as a new antigen-binding site (Fcab) (Nature Review (2010), 10, 301-316 Chan, AC and Carter PJ (Non-Patent Literature 6), Peds (2010), 23(4), 289-297 Wozniak-Knopp, G. et al. (Non-Patent Literature 7)).

[0007] On the other hand, antibodies that utilize effector function tend to act on normal cells with low expression of the target antigen, making them prone to side effects. Therefore, attempts are being made to make the effector function of antibody drugs exerted specifically on target tissues. For example, antibodies whose binding ability changes upon binding to cellular metabolites (Patent Document 2), antibodies that exhibit antigen-binding ability after being cleaved by proteases (Patent Document 3), and a technology to control the crosslinking between antibody-mediated chimeric antigen receptor T cells (CAR-T cells) and cancer cells by adding a compound (ABT-737) (Nature Chemical Biology (2018) 14, 112-117 Hill ZB et al. (Non-Patent Document 8)) have been reported. [Prior art documents] [Chartered documents]

[0008]

Patent Document 1

Patent document 2

Patent document 3

Non-licensed literature

[0009]

Non-licensed literature 1

Non-licensed Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Summary of the Invention

Problems to be Solved by the Invention

[0010] Attempts to specifically exert the effector function of the above-mentioned antibody drugs on target tissues are still in development, and further attempts are expected. Therefore, the present invention aims to provide an antibody modification technique that can specifically exert the effector function of an antibody drug on a target tissue, is useful for reducing the side effects of the antibody drug, and can also be applied to various other protein engineering fields.

Means for Solving the Problems

[0011] As a result of intensive studies, the present inventors have found the inventions [1] to

[46] below. [1] A second antigen-binding molecule that binds to an antigen-antigen-binding molecule complex comprising a first antigen and a first antigen-binding molecule that binds to the first antigen, and increases the binding activity of the first antigen-binding molecule to the first antigen. [2] The second antigen-binding molecule according to [1], wherein its binding activity to the first antigen is higher in the presence of the first antigen-binding molecule compared to the absence of the first antigen-binding molecule. [3] The second antigen-binding molecule according to [1] or [2], wherein the first antigen is an immune-related molecule or a cellular metabolite. [4] The second antigen-binding molecule according to [3], wherein the immune-related molecule is a molecule present in the cell membrane of an immune cell. [5] The second antigen-binding molecule according to [4], wherein the immune cell is at least one selected from the group consisting of granulocytes, macrophages, dendritic cells, T cells, and B cells. [6] The second antigen-binding molecule according to any one of [3] to [5], wherein the immune-related molecule is CD3. [7] The second antigen-binding molecule according to [6], wherein the first antigen-binding molecule comprises a CD3-binding polypeptide whose combination of amino acid sequences of the heavy chain variable region and the light chain variable region is selected from any combination of SEQ ID NO: 1 and SEQ ID NO: 122, SEQ ID NO: 114 and SEQ ID NO: 115, SEQ ID NO: 116 and SEQ ID NO: 117, SEQ ID NO: 118 and SEQ ID NO: 119, and SEQ ID NO: 120 and SEQ ID NO: 121, respectively, or a first modified polypeptide modified from the CD3-binding polypeptide, wherein the CD3-binding activity of the first modified polypeptide is lower than that of the CD3-binding polypeptide. [8] The second antigen-binding molecule according to [3], wherein the cell metabolite is adenosine or a derivative thereof. [9] The second antigen-binding molecule according to [8], wherein the first antigen-binding molecule comprises an adenosine-binding polypeptide having a combination of amino acid sequences of the heavy chain variable region and the light chain variable region selected from any combination of SEQ ID NO: 106 and SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO: 109, SEQ ID NO: 110 and SEQ ID NO: 111, and SEQ ID NO: 112 and SEQ ID NO: 113, respectively, or a second modified polypeptide modified from the adenosine-binding polypeptide, wherein the adenosine-binding activity of the second modified polypeptide to adenosine is lower or higher than that of the adenosine-binding polypeptide.

[10] A second antigen-binding molecule according to any one of [1] to [9], wherein the first antigen-binding molecule has multiple antigen specificity and further binds to at least a second antigen.

[11] The second antigen-binding molecule according to

[10] , wherein the second antigen is a cancer antigen or an immune-related molecule.

[12] A second antigen-binding molecule according to any one of [1] to

[11] , having multiple antigen specificity and further binding to at least a third antigen.

[13] The second antigen-binding molecule according to

[12] , wherein the third antigen is a cancer antigen or an immune-related molecule.

[14] The second antigen-binding molecule according to any one of [1] to

[13] , wherein the first antigen-binding molecule has multiple antigen specificity and further binds to at least a second antigen, and the second antigen-binding molecule has multiple antigen specificity and further binds to at least a third antigen, and the combination of the first antigen, the second antigen and the third antigen is any of the following combinations (1) to (5). (1) A combination in which the first antigen is an immune-related molecule, the second antigen is the first cancer antigen, and the third antigen is the second cancer antigen. (2) A combination in which the first antigen is a cellular metabolite of a target cell, the second antigen is a cancer antigen, and the third antigen is an immune-related molecule. (3) A combination in which the first antigen is a cellular metabolite of a target cell, the second antigen is an immune-related molecule, and the third antigen is a cancer antigen. (4) A combination in which the first antigen is a first immune-related molecule, the second antigen is a cancer antigen, and the third antigen is a second immune-related molecule. (5) A combination in which the first antigen is a first immune-related molecule, the second antigen is a second immune-related molecule, and the third antigen is a cancer antigen. The combination of the first antigen-binding molecule and the second antigen-binding molecule described in

[15] [1].

[16] A first antigen-binding molecule that binds to a first antigen, wherein the binding activity of the first antigen-binding molecule to the first antigen is increased by a second antigen-binding molecule that binds to an antigen-antigen-binding molecule complex comprising the first antigen and the first antigen-binding molecule.

[17] The first antigen-binding molecule according to

[16] , wherein the binding activity of the second antigen-binding molecule to the first antigen is higher in the presence of the first antigen-binding molecule compared to the absence of the first antigen-binding molecule.

[18] The first antigen-binding molecule according to

[16] or

[17] , wherein the first antigen is an immune-related molecule or a cellular metabolite.

[19] The first antigen-binding molecule according to

[18] , wherein the immune-related molecule is a molecule present in the cell membrane of an immune cell.

[20] The first antigen-binding molecule according to

[19] , wherein the immune cell is at least one selected from the group consisting of granulocytes, macrophages, dendritic cells, T cells, and B cells.

[21] The first antigen-binding molecule according to any one of

[19] to

[20] , wherein the immune-related molecule is CD3.

[22] The first antigen-binding molecule comprises a CD3-binding polypeptide having a combination of amino acid sequences of the heavy chain variable region and the light chain variable region selected from SEQ ID NO: 1 and SEQ ID NO: 122, SEQ ID NO: 114 and SEQ ID NO: 115, SEQ ID NO: 116 and SEQ ID NO: 117, SEQ ID NO: 118 and SEQ ID NO: 119, and SEQ ID NO: 120 and SEQ ID NO: 121, respectively, or a first modified polypeptide modified from the CD3-binding polypeptide, wherein the CD3-binding activity of the first modified polypeptide is lower than that of the CD3-binding polypeptide, as described in

[21] .

[23] The first antigen-binding molecule according to

[18] , wherein the cell metabolite is adenosine or a derivative thereof.

[24] The first antigen-binding molecule comprises an adenosine-binding polypeptide having a combination of amino acid sequences of the heavy chain variable region and the light chain variable region selected from any combination of SEQ ID NO: 106 and SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO: 109, SEQ ID NO: 110 and SEQ ID NO: 111, and SEQ ID NO: 112 and SEQ ID NO: 113, respectively, or a second modified polypeptide modified from the adenosine-binding polypeptide, wherein the adenosine-binding activity of the second modified polypeptide to adenosine is lower or higher than that of the adenosine-binding polypeptide, as described in

[23] .

[25] A first antigen-binding molecule according to any one of

[16] to

[24] , having multiple antigen specificity and further binding to at least a second antigen.

[26] The first antigen-binding molecule according to

[25] , wherein the second antigen is a cancer antigen or an immune-related molecule.

[27] The first antigen-binding molecule according to any one of

[16] to

[26] , wherein the second antigen-binding molecule has multiple antigen specificity and further binds to at least a third antigen.

[28] The first antigen-binding molecule according to

[27] , wherein the third antigen is a cancer antigen or an immune-related molecule.

[29] The first antigen-binding molecule according to any one of

[16] to

[28] , wherein the first antigen-binding molecule has multiple antigen specificity and further binds to at least a second antigen, the second antigen-binding molecule has multiple antigen specificity and further binds to at least a third antigen, and the combination of the first antigen, the second antigen and the third antigen is any of the following combinations (1) to (5). (1) A combination in which the first antigen is an immune-related molecule, the second antigen is the first cancer antigen, and the third antigen is the second cancer antigen. (2) A combination in which the first antigen is a cellular metabolite of a target cell, the second antigen is a cancer antigen, and the third antigen is an immune-related molecule. (3) A combination in which the first antigen is a cellular metabolite of a target cell, the second antigen is an immune-related molecule, and the third antigen is a cancer antigen. (4) A combination in which the first antigen is a first immune-related molecule, the second antigen is a cancer antigen, and the third antigen is a second immune-related molecule. (5) A combination in which the first antigen is a first immune-related molecule, the second antigen is a second immune-related molecule, and the third antigen is a cancer antigen. The combination of the first antigen-binding molecule and the second antigen-binding molecule described in

[30]

[16] .

[31] A combination of the pharmaceutical composition described in

[15] or

[30] .

[32] The combination according to

[31] , wherein the first antigen-binding molecule and the second antigen-binding molecule are administered simultaneously or separately.

[33] A screening method comprising identifying a compound or antibody or fragment thereof as a second antigen-binding molecule when the binding activity of a first antigen-binding molecule to a first antigen using at least one assay selected from SPR, BLI, and ELISA is detectable in the presence of a compound or antibody or fragment thereof, which is arbitrarily selected from a library of compounds or antibodies or fragment thereof, but is undetectable in the absence of the compound or antibody or fragment thereof.

[34] A screening method comprising identifying a compound or antibody or fragment as a second antigen-binding molecule if the binding activity of a first antigen-binding molecule to a first antigen is higher under conditions in which a compound or antibody or fragment arbitrarily selected from a library of compounds or antibodies or fragments is present compared to conditions in which such a compound or antibody or fragment is absent.

[35] (a) A step of immunizing a mammal with an antigen-antigen binding molecule complex consisting of a first antigen and a first antigen-binding molecule, and obtaining a first group of antibody-producing cells that produce monoclonal antibodies bound to the complex, (b) A step of selecting from the first group a second group that produces a monoclonal antibody whose binding activity to either or both of a first antigen and a first antigen-binding molecule that does not form a complex in at least one assay selected from SPR, BLI, and ELISA is undetectable. (c) A screening method comprising the step of selecting from the second group a third group that produces a monoclonal antibody that enhances the binding activity of the first antigen-binding molecule to the first antigen in at least one assay selected from SPR, BLI, and ELISA, and identifying the third group as antibody-producing cells that produce the second antigen-binding molecule.

[36] (a) A step of immunizing a mammal with an antigen-antigen binding molecule complex consisting of a first antigen and a first antigen-binding molecule, and obtaining a first group of antibody-producing cells that produce monoclonal antibodies bound to the complex, (b) A step of selecting from the first group a second group that produces a monoclonal antibody whose binding activity to either or both of a first antigen and a first antigen-binding molecule that does not form a complex in at least one assay selected from SPR, BLI and ELISA is lower than that to binding to the complex. (c) A screening method comprising the step of selecting from the second group a third group that produces a monoclonal antibody that enhances the binding activity of the first antigen-binding molecule to the first antigen in at least one assay selected from SPR, BLI, and ELISA, and identifying the third group as antibody-producing cells that produce the second antigen-binding molecule.

[37] (a) A step of immunizing a mammal with an antigen-antigen-binding domain complex consisting of a first antigen and a first antigen-binding domain, and obtaining a first group of antibody-producing cells that produce monoclonal antibodies bound to the complex, (b) A step of selecting from the first group a second group that produces a monoclonal antibody whose binding activity to either or both of a first antigen and a first antigen-binding region that does not form a complex in at least one assay selected from SPR, BLI, and ELISA is undetectable. (c) A screening method comprising the step of selecting from the second group a third group that produces a monoclonal antibody that enhances the binding activity of the first antigen-binding region to the first antigen in at least one assay selected from SPR, BLI, and ELISA, and identifying the third group as antibody-producing cells that produce a second antigen-binding molecule.

[38] (a) A step of immunizing a mammal with an antigen-antigen-binding domain complex consisting of a first antigen and a first antigen-binding domain, and obtaining a first group of antibody-producing cells that produce monoclonal antibodies bound to the complex, (b) A step of selecting from the first group a second group that produces a monoclonal antibody whose binding activity to either or both of a first antigen and a first antigen-binding region that does not form a complex in at least one assay selected from SPR, BLI, and ELISA is lower than that to binding to the complex. (c) A screening method comprising the step of selecting from the second group a third group that produces a monoclonal antibody that enhances the binding activity of the first antigen-binding region to the first antigen in at least one assay selected from SPR, BLI, and ELISA, and identifying the third group as antibody-producing cells that produce a second antigen-binding molecule.

[39] (d) A step of culturing antibody-producing cells obtained by a screening method including steps (a) to (c) below; (a) A step of immunizing a mammal with an antigen-antigen binding molecule complex consisting of a first antigen and a first antigen-binding molecule, and obtaining a first group of antibody-producing cells that produce monoclonal antibodies bound to the complex, (b) A step of selecting from the first group a second group that produces a monoclonal antibody whose binding activity to either or both of a first antigen and a first antigen-binding molecule that does not form a complex in at least one assay selected from SPR, BLI, and ELISA is undetectable. (c) From the second group, select a third group that produces a monoclonal antibody that enhances the binding activity of the first antigen-binding molecule to the first antigen in at least one assay selected from SPR, BLI, and ELISA, and identify the third group as antibody-producing cells that produce the second antigen-binding molecule; (e) A step of obtaining a culture supernatant or cell disruption product from the antibody-producing cell culture, (f) A method for producing a second antigen-binding molecule, comprising the step of purifying the second antigen-binding molecule from the culture supernatant or cell disrupted product.

[40] (d) A step of culturing antibody-producing cells obtained by a screening method including steps (a) to (c) below; (a) A step of immunizing a mammal with an antigen-antigen binding molecule complex consisting of a first antigen and a first antigen-binding molecule, and obtaining a first group of antibody-producing cells that produce monoclonal antibodies bound to the complex, (b) A step of selecting from the first group a second group that produces a monoclonal antibody whose binding activity to either or both of a first antigen and a first antigen-binding molecule that does not form a complex in at least one assay selected from SPR, BLI and ELISA is lower than that to binding to the complex. (c) From the second group, select a third group that produces a monoclonal antibody that enhances the binding activity of the first antigen-binding molecule to the first antigen in at least one assay selected from SPR, BLI, and ELISA, and identify the third group as antibody-producing cells that produce the second antigen-binding molecule; (e) A step of obtaining a culture supernatant or cell disruption product from the antibody-producing cell culture, (f) A method for producing a second antigen-binding molecule, comprising the step of purifying the second antigen-binding molecule from the culture supernatant or cell disrupted product.

[41] (d) A step of culturing antibody-producing cells obtained by a screening method comprising the following steps (a) to (c); (a) A step of immunizing a mammal with an antigen-antigen-binding domain complex consisting of a first antigen and a first antigen-binding domain, and obtaining a first group of antibody-producing cells that produce monoclonal antibodies bound to the complex, (b) A step of selecting from the first group a second group that produces a monoclonal antibody whose binding activity to either or both of a first antigen and a first antigen-binding region that does not form a complex in at least one assay selected from SPR, BLI, and ELISA is undetectable. (c) From the second group, select a third group that produces a monoclonal antibody that enhances the binding activity of the first antigen-binding region to the first antigen in at least one assay selected from SPR, BLI, and ELISA, and identify the third group as antibody-producing cells that produce the second antigen-binding molecule; (e) A step of obtaining a culture supernatant or cell disruption product from the antibody-producing cell culture, (f) A method for producing a second antigen-binding molecule, comprising the step of purifying the second antigen-binding molecule from the culture supernatant or cell disrupted product.

[42] (d) A step of culturing antibody-producing cells obtained by a screening method including steps (a) to (c) below; (a) A step of immunizing a mammal with an antigen-antigen-binding domain complex consisting of a first antigen and a first antigen-binding domain, and obtaining a first group of antibody-producing cells that produce monoclonal antibodies bound to the complex, (b) A step of selecting from the first group a second group that produces a monoclonal antibody whose binding activity to either or both of a first antigen and a first antigen-binding region that does not form a complex in at least one assay selected from SPR, BLI, and ELISA is lower than that to binding to the complex. (c) From the second group, select a third group that produces a monoclonal antibody that enhances the binding activity of the first antigen-binding region to the first antigen in at least one assay selected from SPR, BLI, and ELISA, and identify the third group as antibody-producing cells that produce the second antigen-binding molecule; (e) A step of obtaining a culture supernatant or cell disruption product from the antibody-producing cell culture, (f) A method for producing a second antigen-binding molecule, comprising the step of purifying the second antigen-binding molecule from the culture supernatant or cell disrupted product.

[43] A first step of modifying a first antigen-binding molecule that binds to a first antigen to obtain a modified version of the first antigen-binding molecule in which binding to the first antigen is reduced or below the detection limit in at least one assay selected from SPR, BLI and ELISA. A second step of obtaining a first phage-display antigen-binding molecule library by removing phages that display antigen-binding molecules that bind to either or both of the first antigen and the modified form from an existing phage-display antigen-binding molecule library, and A method for producing a phage display antigen-binding molecule library, comprising a third step of obtaining a second phage display antigen-binding molecule library from the first phage display antigen-binding molecule library, which is enriched with phages that display antigen-binding molecules that bind to an antigen-antigen-binding molecule complex containing the first antigen and the first antigen-binding molecule.

[44] The method for producing

[43] , wherein the second phage display antigen-binding molecule library is the existing phage display antigen-binding molecule library, and the second and third steps are repeated.

[45] A first step to obtain a first phage display antigen-binding molecule library from an existing phage display antigen-binding molecule library, wherein phages that present antigen-binding molecules (i) can bind to a first antigen but are not bound to the first antigen, and (ii) bind to the first antigen but are not bound to the first antigen-binding molecule are removed, and A method for producing a phage display antigen-binding molecule library, comprising a second step of obtaining a second phage display antigen-binding molecule library obtained by enriching the first phage display antigen-binding molecule library with phages that display antigen-binding molecules that bind to an antigen-antigen-binding molecule complex containing the first antigen and the first antigen-binding molecule.

[46] The method for producing

[45] , wherein the second phage display antigen-binding molecule library is the existing phage display antigen-binding molecule library, and the first and second steps are repeated. [Effects of the Invention]

[0012] According to the present invention, the binding activity of the first antigen-binding molecule to the first antigen can be increased by the second antigen-binding molecule binding to an antigen-antigen-binding molecule complex containing the first antigen and the first antigen-binding molecule. [Brief explanation of the drawing]

[0013] [Figure 1] Figure 1 is a schematic diagram illustrating the binding mechanism of the first antigen-binding molecule and the second antigen-binding molecule when they are used in combination. [Figure 2] Figure 2 is a schematic diagram illustrating the mechanism of action by which one aspect of the first antigen-binding molecule and another aspect of the second antigen-binding molecule crosslink target cells and effector cells. [Figure 3] Figure 3 shows the CD3 signal-inducing ability of the candidate clamping antibodies prepared in Example 3, as observed by a functional assay. [Figure 4] Figure 4 shows the binding activity of the clamping antibody prepared in Example 3. [Figure 5] Figure 5 shows that the CD3-anti-CD3 antibody complex is stabilized by the clamping antibody. [Figure 6] Figure 6 shows the TDCC activity using the same antigen. [Figure 7] Figure 7 shows the TDCC activity against EREG / GPC3 bipositive cells. [Figure 8] Figure 8 shows the binding of adenosine to anti-adenosine antibody and clamping antibody. [Figure 9] Figure 9 shows the affinity of adenosynchronic ramping antibodies. [Figure 10]Figure 10 shows the adenosine concentration-dependent binding of anti-adenosine antibodies and clamping antibodies. [Figure 11] Figure 11 shows the adenosine concentration-dependent cytotoxic activity of a bispecific antibody using an adenosine-linked ramping antibody. [Figure 12] Figure 12 shows the crystal structure of the epitope peptide-fusion anti-CD3 antibody Fab and the clamping antibody. [Figure 13] Figure 13 shows the TDCC activity against GPC3 / CLDN6 bipositive cells. [Figure 14] Figure 14 shows the TDCC activity against GPC3 / Her2 bipositive cells. [Figure 15] Figure 15 shows effector cell-specific activation. [Figure 16] Figure 16 shows the CD8-positive T cell-specific TDCC activity induced by administration of anti-cancer antigen / attenuated CD3 antibody and anti-CD8 clamping antibody. [Figure 17] Figure 17 shows the antitumor effects of administering anti-cancer antigen antibodies / attenuated CD3 antibodies and anti-cancer antigen antibodies / clamping antibodies. [Modes for carrying out the invention]

[0014] A.Definition In this specification, "polypeptide" encompasses all peptides in which multiple amino acids are linked by peptide bonds. In this specification, polypeptides may also be referred to as "peptides" or "proteins." In this specification, "antigen-binding region" means a compound having activity that specifically binds to an antigen. The antigen-binding region may be peptidic or non-peptidic.

[0015] In this specification, "CH1" refers to the single-chain polypeptide of CH1 in an antibody. Specifically, CH1 is the region represented by amino acid residues at positions 118-215 of the heavy chain in the EU numbering system, and this specification includes not only the wild type but also modified versions in which amino acid residues are substituted, added, or deleted from the wild type. In this specification, "CH2" refers to the single-chain polypeptide of the CH2 region of an antibody. Specifically, CH2 is the region represented by amino acid residues at positions 231-340 of the heavy chain in the EU numbering system, and in this specification, this includes not only the wild type but also modified versions in which amino acid residues have been substituted, added, or deleted from the wild type. In this specification, "CH3" refers to the single-chain polypeptide of the CH3 group of an antibody. Specifically, CH3 is the region represented by amino acid residues from position 341 of the heavy chain to the C-terminus in the EU numbering system, and in this specification, it includes not only the wild type but also modified forms in which amino acid residues have been substituted, added, or deleted from the wild type. In this specification, "CL" refers to the single-chain polypeptide of the CL of an antibody. Specifically, CL is the region represented by amino acid residues from position 108 of the light chain to the C-terminus in the EU numbering system, and in this specification, it includes not only the wild type but also modified versions in which amino acid residues are substituted, added, or deleted from the wild type.

[0016] In this specification, "antibody halves" refers to a single molecule obtained by dissociating the bonds between the heavy chains in an antibody. An example of an antibody halve in the case of an IgG antibody is a complex consisting of one heavy chain and one light chain. Antibody halves also include molecules consisting of one heavy chain, obtained by dissociating the bonds between the heavy chains of an antibody consisting of two heavy chains, such as those found in antibodies of camelids, etc., also known as a heavy-chain antibody (VHH (VH originating from heavy-chain antibody) antibody). In one embodiment, antibody halves include those derived from chimeric antibodies or humanized antibodies. In one embodiment, antibody halves include those derived from various isotypes such as IgG, IgM, IgA, IgD, and IgE. Antibody halves are preferably derived from IgG. IgG includes IgG1, IgG2, IgG3, and IgG4. Antibody halves may be derived from any of these subtypes. Antibody halves may be obtained by dissociating the bonds between the heavy chains of naturally occurring antibodies, or they may be genetically modified antibodies in which amino acid residues have been substituted, added, or deleted.

[0017] In this specification, the “hinge region” refers to the region located between CH1 and CH2 in an antibody. Specifically, the hinge region is the region represented by amino acid residues at positions 216-230 in the EU numbering system, and in this specification, it includes not only the wild type but also modified versions in which amino acid residues have been substituted, added, or deleted from the wild type. In this specification, the “hinge region portion in an antibody halve” refers to the hinge region portion in a heavy chain, and consists of a single polypeptide chain.

[0018] In this specification, the term "constant region" refers to the region in an antibody that includes the CH1, CH2, CH3, CL, and hinge regions. In this specification, the term "constant region portion in an antibody halve" refers to the constant region portion in an antibody halve.

[0019] In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes both the native sequence Fc region and mutant Fc regions. In one embodiment, the heavy chain Fc region of human IgG extends from Cys226 or Pro230 to the C-terminus of the heavy chain, provided that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region is present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0020] In this specification, “effector function” encompasses not only the biological activity that varies depending on the antibody isotype, stemming from the antibody’s Fc region, but also the activity that controls the immune cell response by modified antibodies. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); T-cell-dependent cytotoxicity (TDCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0021] An "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR is one that binds to an IgG antibody (gamma receptor) and includes the FcγRI, FcγRII, and FcγRIII subclass receptors, including allelic variants and alternative splicing forms of these receptors. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, for example, Daeron, Annu. Rev. Immunol. 15:203-234 (1997).) FcRs have been reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med 126:330-41 (1995). Other FcRs, including those to be identified in the future, are also included in the term “FcR” as used herein.

[0022] As used herein, "covalent bond" encompasses all commonly known covalent bonds. Examples of covalent bonds include disulfide bonds and carbon-carbon bonds.

[0023] As used herein, the term “cytotoxic agent” means a substance that inhibits or interferes with the function of a cell and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g.,211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 radioisotopes of Pb and Lu); chemotherapeutic agents or chemotherapeutic drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; enzymes such as nuclease and fragments thereof; antibiotics; toxins such as low molecular weight toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including fragments and / or variants thereof); and various antitumor agents or anticancer agents disclosed below.

[0024] As used herein, the term "binding activity" is used to represent the strength of the bond formed between molecules. Covalent bonds are not included in the types of bonds formed between molecules, and bonds formed between molecules such as hydrogen bonds, electrostatic forces, van der Waals forces, and hydrophobic bonds are included. The binding activity between molecules is determined by the sum of these bonds. In this specification, the binding activity is particularly represented by the dissociation constant KD. KD can be determined from the data of known assays for observing the binding between molecules. Examples of assays include surface plasmon resonance (SPR), biolayer interferometry (BLI), enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorter (FACS), etc. Among them, SPR is preferred. For measuring the binding activity by SPR, for example, Biacore (registered trademark) T200 (GE Healthcare) is used. The KD when measured by SPR using Biacore (registered trademark) T200 is about 1×10 -12 to about 1×10 -4 is possible within the range. KD within this range (1×10 -12 to 1×10 -4 ) is such that the larger the KD, the lower the binding activity, and the smaller the KD, the higher the binding activity. In the binding activity of an antigen-binding molecule to an antigen, KD is 1×10-6 In cases exceeding the above, antigen-binding molecules often have difficulty exhibiting their physiological function. For example, if the antigen-binding molecule is an antibody, it often has difficulty exhibiting its effector function. Therefore, in this specification, when measured by SPR, if the KD is 1 × 10⁻⁶, -6 If the value is greater than or equal to 1 × 10, it is considered "low binding activity". -6 A lower value is defined as "high binding activity." The temperature conditions for intermolecular binding assays are typically 25-37°C. For SPR, the temperature conditions are preferably 25°C or 37°C, more preferably 37°C. For BLI, the temperature conditions are preferably 30°C. For ELISA, the temperature conditions are preferably 25°C. The running buffer used in the binding assay may be commercially available or prepared on-site. A commercially available example is HBS-EP+ (GE Healthcare) (0.01M HEPES, 0.15M NaCl, 3mM EDTA, 0.05% (v / v) polyoxyethylene (20) sorbitan monolaurate, pH 7.4). An example of a prepared buffer is ACES buffer (20 mM ACES (Nacalai tesque), 150 mM NaCl, 0.05% (w / v) polyoxyethylene (20) sorbitan monolaurate (pure biochemical), pH 7.4). The test compound is dissolved in the desired buffer. The main component that can affect intermolecular bonding during an assay is NaCl. Although the concentration of NaCl varies depending on the purpose of the experiment, the concentration of NaCl in buffers used in typical experiments that do not involve optimization of salt concentration is 150 mM. In other words, a concentration of NaCl of 150 mM is preferred for commonly used running buffers. pH can also affect intermolecular bonding during an assay, but the buffer used in typical experiments that do not involve optimization of pH is pH 7.4. In other words, a pH of 7.4 is preferred for commonly used running buffers.

[0025] B. First antigen-binding molecule The first antigen-binding molecule of the present invention binds to a first antigen. That is, the first antigen-binding molecule includes a first antigen-binding region that binds to the first antigen. The first antigen-binding molecule binds to the first antigen to form an antigen-antigen-binding molecule complex. In certain embodiments, the first antigen-binding molecule is an antigen-binding molecule that is not expressed in vivo. It is preferable that the first antigen-binding molecule is an antigen-binding molecule that is not expressed in vivo. "An antigen-binding molecule is not expressed in vivo" means that "the antigen-binding molecule is not a protein or fragment thereof that is normally expressed in a living organism without any artificial processing such as drug administration or immunization." In one embodiment, the binding activity of the first antigen-binding molecule or the first antigen-binding region to the first antigen is increased by a second antigen-binding molecule (sometimes referred to herein as a "clamping molecule") described below, which binds to the antigen-antigen-binding molecule complex. The first antigen-binding molecule is not particularly limited as long as its binding activity to the first antigen is increased by the second antigen-binding molecule, and may be a complete antibody consisting of two light chain molecules and two heavy chain molecules, such as a natural antibody, or it may be an antibody halve, diabody (Db), scFv, single-chain antibody, sc(Fv)2, sc(Fab')2, or other antibody fragment. In another embodiment, if the first antigen, as described later, is a receptor, the first antigen-binding molecule or the first antigen-binding region may be a ligand for that receptor. For example, if the first antigen is a T cell receptor complex, a costimulatory molecule, or a coinhibitory molecule, the first antigen-binding molecule or the first antigen-binding region may be their ligands. Specifically, if the first antigen is PD-1, the first antigen-binding molecule or the first antigen-binding region may be PD-L1 or PD-L2.

[0026] Whether the binding activity of the first antigen-binding molecule or the first antigen-binding region to the first antigen is increased by the second antigen-binding molecule that binds to the antigen-antigen-binding molecule complex is determined, for example, in SPR, by the value KD(clamping-) / KD(clamping+), which is obtained by dividing the dissociation constant KD(clamping-) of the first antigen-binding molecule or the first antigen-binding region to the first antigen in the absence of the second antigen-binding molecule (clamping-) by the dissociation constant KD(clamping+) of the first antigen-binding molecule or the first antigen-binding region to the first antigen in the presence of the second antigen-binding molecule (clamping+). The statement above, "The binding activity of the first antigen-binding molecule or the first antigen-binding region to the first antigen is increased by the second antigen-binding molecule that binds to the antigen-antigen-binding molecule complex, as described below," means that KD(clamping-) / KD(clamping+) is greater than 1. A higher KD(clamping-) / KD(clamping+) indicates a greater increase in the binding activity of the first antigen-binding molecule or the first antigen-binding region to the first antigen in the presence of the second antigen-binding molecule compared to the absence of the second antigen-binding molecule. In other words, this means that the on / off switching of the binding of the first antigen-binding molecule or the first antigen-binding region to the first antigen due to the presence of the second antigen-binding molecule is more pronounced.

[0027] In one aspect, the binding activity of the first antigen-binding molecule to the first antigen may be high, low, or so low as to be undetectable by SPR, as measured by SPR. When the first antigen-binding molecule is a bispecific antibody and the first antigen is an immune-related molecule described later, from the viewpoint of reducing side effects, it is preferable that the binding activity of the first antigen-binding molecule to the first antigen is low when measured by SPR, or so low that it is undetectable by SPR. Here, "so low that it is undetectable by SPR" means that the binding activity of the first antigen-binding molecule to the first antigen is undetectable by SPR, but at least a small amount of specific binding of the first antigen-binding molecule to the first antigen occurs. If the binding activity of the first antigen-binding molecule to the first antigen is so low that it is undetectable by SPR, the above-mentioned KD(clamping-) / KD(clamping+) is not used.

[0028] In one embodiment, the binding activity of the second antigen-binding molecule to the first antigen is higher in the presence of the first antigen-binding molecule compared to the absence of the first antigen-binding molecule. This is presumed to be due to one of the following mechanisms: the binding activity of the second antigen-binding molecule to the antigen-antigen-binding molecule complex is higher than that of the free first antigen; the binding activity of the second antigen-binding molecule to the free first antigen is increased by the second antigen-binding molecule binding to the free first antigen-binding molecule; the complex consisting of the first antigen and the second antigen-binding molecule is stabilized by binding to the free first antigen-binding molecule; or a combination of these mechanisms. In this embodiment, as an indicator of the binding activity of the second antigen-binding molecule to the first antigen in the presence of the first antigen-binding molecule compared to the absence of the first antigen-binding molecule, for example, in SPR, the dissociation constant KD(first antigen-binding molecule-) for the second antigen-binding molecule with respect to the free first antigen in the absence of the first antigen-binding molecule is divided by the dissociation constant KD(first antigen-binding molecule+) for the second antigen-binding molecule with respect to the first antigen in the presence of the first antigen-binding molecule, which is KD(first antigen-binding molecule+). The above statement that "the binding activity of the second antigen-binding molecule to the first antigen in the presence of the first antigen-binding molecule is higher than in the absence of the first antigen-binding molecule" means that KD(first antigen-binding molecule-) / KD(first antigen-binding molecule+) is greater than 1.

[0029] In one aspect, the binding activity of the second antigen-binding molecule to the free first antigen may be high, low, or so low as to be undetectable by SPR, as measured by SPR. When the second antigen-binding molecule is a bispecific antibody and the first antigen is an immune-related molecule described later, from the viewpoint of reducing side effects, it is preferable that the binding activity of the second antigen-binding molecule to the free first antigen is low when measured by SPR, or so low that it is undetectable by SPR. Here, "so low that it is undetectable by SPR" means that the binding activity of the second antigen-binding molecule to the free first antigen is undetectable by SPR, but at least a small amount of specific binding of the second antigen-binding molecule to the free first antigen occurs. If the binding activity of the second antigen-binding molecule to the free first antigen is so low that it is undetectable by SPR, the above-mentioned KD (free) / KD (complex) is not used.

[0030] In one embodiment, the KD of the second antigen-binding molecule to the antigen-antigen-binding molecule complex in SPR is used as the binding activity of the second antigen-binding molecule to the antigen-antigen-binding molecule complex. The KD is typically about 1 × 10⁻⁶ -12 From approximately 1 x 10 -4This is shown within a range. A lower KD indicates stronger binding of the second antigen-binding molecule to the antigen-antigen-binding molecule complex. In one embodiment, when measuring the binding activity of the second antigen-binding molecule to the first antigen in the presence of the first antigen-binding molecule, and when measuring the binding activity of the second antigen-binding molecule to the antigen-antigen-binding molecule complex, the first antigen fused with the first antigen-binding molecule may be used. In one embodiment, when comparing the binding activity of the second antigen-binding molecule to the antigen-antigen-binding molecule complex with the binding activity of the second antigen-binding molecule to the first antigen-binding molecule, the binding activity of the second antigen-binding molecule to the first antigen-binding molecule measured in the presence of the first antigen can be compared with the binding activity of the second antigen-binding molecule to the first antigen-binding molecule measured in the absence of the first antigen.

[0031] a. First antigen The first antigen is not particularly limited and includes any antigen. Specific examples of antigens include those listed in International Publication No. 2013 / 180200. The first antigen is preferably an immune-related molecule or cellular metabolite, but is not limited to these. In one embodiment, the first antigen is an extracellular protein. This extracellular protein includes cell membrane proteins. Preferably, the extracellular protein is a cell membrane protein. In another embodiment, the first antigen is a native protein. This native protein is not a protein expressed in a cell through genetic engineering. Preferably, the first antigen is a native cell membrane protein.

[0032] In this specification, immune-related molecules include any molecules produced by immune cells. Immune-related molecules may be, for example, molecules present in the cell membrane or molecules released extracellularly. Specific examples of molecules present in the cell membrane of immune cells include T cell activators, T cell receptor complexes, costimulatory molecules, and coinhibitory molecules. Among these, T cell receptor complexes and costimulatory molecules are preferred. The molecules present in the cell membrane of immune cells are more preferably native proteins rather than genetically engineered proteins. Examples of co-stimulatory molecules include CD2, CD27, CD28, CD40, CD137 (4-1BB), CD40, OX40 (CD134), ICOS (Inducible co-stimulator), DR3, GITR, CD30, TIM1, SLAM, and CD226. CD3 is a component of the T cell receptor complex. CD3 has subtypes: CD3γ, CD3δ, CD3ε, and CD3ζ. Among these, CD3ε is preferred. Examples of co-inhibitory molecules include CTLA4, PD1, TIM3, TIGIT, CD160, LAG3, LAIR1, B7-1, and B7-H1. Molecules released outside the cell include, for example, various cytokines.

[0033] Examples of immune cells mentioned above include granulocytes, macrophages, dendritic cells, T cells, and B cells. Preferably, the immune cell is at least one selected from the group consisting of granulocytes, macrophages, dendritic cells, T cells, and B cells, and more preferably a T cell. Examples of T cell types include CD4-positive, CD8-positive, Th1, Th2, and Th12. Among these, CD8-positive is preferred.

[0034] In this specification, cellular metabolites are cellular metabolites released extracellularly. These cellular metabolites are not particularly limited and encompass any metabolite. Preferably, these cellular metabolites are compounds that are not administered to a living organism but are produced internally from any tissue within the organism. Specific examples of cellular metabolites include cancer tissue-specific metabolites and inflammatory tissue-specific metabolites, as described in International Publication No. 2013 / 180200. Cancer tissue-specific metabolites include primary metabolites of glycolysis or the Krebs cycle such as lactic acid, succinic acid, and citrate; amino acids such as alanine, glutamic acid, and aspartic acid; amino acid metabolites such as kynurenine; arachidonic acid metabolites such as prostaglandin E2; nucleosides having a purine ring structure such as adenosine, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP); uric acid; and 1-methylnicotinamide. Among these, nucleosides having a purine ring structure are preferred, and adenosine is more preferred. Examples of inflammatory tissue-specific metabolites include arachidonic acid metabolites such as prostaglandin E2, nucleosides with a purine ring structure such as adenosine, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP), and uric acid. Among these, nucleosides with a purine ring structure are preferred, and adenosine is more preferred.

[0035] b. Second antigen The first antigen-binding molecule may bind to a single antigen, or it may bind to multiple antigens, thus possessing so-called multi-antigen specificity. If the first antigen-binding molecule has multi-antigen specificity, the first antigen-binding molecule binds to at least the second antigen. That is, the first antigen-binding molecule includes a second antigen-binding region that binds to the second antigen. The second antigen-binding region may be an antibody fragment. The antibody fragment may be any fragment as long as it can bind to the second antigen. Examples of antibody fragments include Fv and Fab.

[0036] The second antigen is not particularly limited and encompasses any antigen. Specific examples of antigens include those listed in International Publication No. 2013 / 180200. Preferably, the second antigen is a cancer antigen or an immune-related molecule. More preferably, the second antigen is a cancer antigen. Specific examples of cancer antigens include cancer-specific antigens exemplified in International Publication No. 2015 / 156268. Immune-related molecules and their examples are the same as those for the first antigen described above. If the first antigen is an immune-related molecule, the second antigen is preferably an antigen other than an immune-related molecule, and more preferably a cancer antigen.

[0037] In one embodiment, the second antigen is an extracellular protein. This extracellular protein also includes cell membrane proteins. Preferably, the extracellular protein is a cell membrane protein.

[0038] c. Other first components In one embodiment, the first antigen-binding molecule may or may not include components other than the antigen-binding region (first other components). These first other components are, for example, antibody fragments, linkers, and cytotoxic agents. From the viewpoint of being able to add various functions to the first antigen-binding molecule, the first antigen-binding molecule preferably includes the first other component. In order to improve the stability of the first antigen-binding molecule in plasma and the manufacturing efficiency, the first other component is preferably an antibody fragment. Examples of antibody fragments include the Fc region of the antibody and the constant region of the antibody. If the first antigen-binding molecule includes the Fc region of an antibody, the Fc region may be a natural Fc region having the same amino acid sequence as the Fc region of a natural antibody, or it may be a modified Fc region derived from the natural Fc region. In this case, the Fc region of the antibody is preferably derived from the Fc region of IgG. The IgG is preferably of human origin. If the first antigen-binding molecule includes the constant region of an antibody, the constant region may be a natural constant region having the same amino acid sequence as the constant region of a natural antibody, or it may be a modified constant region derived from the natural constant region. In this case, it is preferable that the constant region of the antibody is derived from the constant region of IgG. It is preferable that the IgG is of human origin.

[0039] In one embodiment, if the first antigen-binding molecule includes a modified Fc region or a modified constant region as a first other component, the modified Fc region or modified constant region is a modified Fc region or modified constant region in which binding to FcγR is suppressed or does not occur, from the viewpoint of suppressing unwanted immune responses such as cytokine storms. Examples of modified Fc regions or modified steady-state regions in which binding to FcγR is suppressed or absent include the modified Fc regions or modified steady-state regions published in International Publication No. 2012 / 073985.

[0040] In one embodiment, when the first antigen-binding molecule has biantigen specificity and is a heterodimer of a polypeptide containing a first antigen-binding region and a polypeptide containing a second antigen-binding region, from the viewpoint of manufacturing efficiency, the first antigen-binding molecule preferably contains a modified Fc region or a modified constant region.

[0041] In this case, for example, the polypeptide containing the first antigen-binding region and the polypeptide containing the second antigen-binding region each have at least the first CH3 and the second CH3. Specific examples of the modified Fc region or modified constant region in this case include the following modifications (i) to (iii).

[0042] (i) A modified version in which either the first CH3 or the second CH3 has a positively charged region and the other has a negatively charged region, and the positively charged region interacts with the negatively charged region when a heterodimer is formed. (ii) A modified version in which either the first CH3 or the second CH3 has a convex portion and the other has a concave portion, and when a heterodimer is formed, the convex portion fits into the concave portion and interacts. (iii) The first CH3 and the second CH3 are modified IgG CH3s, and a portion of the modified IgG CH3s is replaced by a portion of IgA CH3s, and when a heterodimer is formed, the portion of IgA CH3 replaced by the first CH3 and the portion of IgA CH3 replaced by the second CH3 interact with each other.

[0043] Modifications of the above (i) are published, for example, in International Publication No. 2006 / 106905, International Publication No. 2009 / 089004, International Publication No. 2010 / 129304, and International Publication No. 2014 / 084607. Specifically, this could involve, for example, modifying at least one combination of the EU numbering system amino acids at positions 356 and 439, 357 and 370, and 399 and 409 in the amino acid sequence of one heavy chain constant region to an amino acid with the same charge, or modifying at least one combination of the EU numbering system amino acids at positions 356 and 439, 357 and 370, and 399 and 409 in the EU numbering system of the other heavy chain constant region to an amino acid with the opposite charge to that of the other heavy chain constant region. More specifically, this could involve introducing a mutation that replaces Glu at position 356 in the EU numbering system with Lys in one heavy chain constant region, and introducing a mutation that replaces Lys at position 439 in the EU numbering system with Glu in the other heavy chain constant region.

[0044] Modifications of the above (ii) can be found, for example, in International Publication No. 96 / 027011 and in Margaret Merchant et al., Nature Biotechnology 1998, 16, 677-681. Specifically, combinations include: introducing T366Y to CH3 and Y407A to the other CH3; introducing T366W to one CH3 and Y407A to the other CH3; introducing F405A to one CH3 and T394W to the other CH3; introducing Y407T to one CH3 and T366Y to the other CH3; or introducing T366Y / F405A to one CH3 and T394W / Y407T to the other CH3. Examples of such combinations include introducing T366W / F405W to one CH3 and T394S / Y407A to the other CH3, or introducing F405W / Y407A to one CH3 and T366W / T394S to the other CH3, or introducing F405W to one CH3 and T394S to the other CH3, or introducing T366W to one CH3 and T366S / L368A / Y407V to the other CH3. The modification in (ii) can also be combined with the modification in (i). Examples of such combinations include those published in International Publication No. 2012 / 058768.

[0045] The modification described in (iii) above is a technique that efficiently induces interactions between polypeptides with different sequences through complementary interactions of CH3 by using a strand-exchange engineered domain CH3, in which a portion of the CH3 of one heavy chain is replaced with a sequence derived from IgA corresponding to that portion, and a sequence derived from IgA corresponding to that portion is introduced into the complementary portion of the CH3 of the other heavy chain (Protein Engineering Design & Selection, 23; 195-202, 2010). Even using this known technique, a first antigen-binding molecule with multiple antigen specificity can be efficiently produced. An example of the modification described in (iii) is the modification technique published in International Publication No. 2007 / 110205.

[0046] In addition to the modifications described in (i) to (iii) above, the modifications in CH3 published in International Publication No. 96 / 027011 may be used as a modified Fc region or a modified steady-state region. Furthermore, modifications in the hinge region published in International Publication No. 2011 / 143545 and the FAE technique published in International Publication No. 2014 / 104165 may be used as modified steady-state regions.

[0047] d. Examples of the first antigen-binding molecule Examples of the first antigen-binding molecule or the first antigen-binding region when the first antigen is CD3 are shown below. In this case, the first antigen-binding molecule may be newly constructed or a known molecule, such as those published in International Publication Nos. 2016 / 020444, 2008 / 119565, and 2007 / 042261, as long as it is a molecule that binds to CD3. As a specific example of this example, the first antigen-binding molecule or first antigen-binding region includes a CD3-binding polypeptide in which the combination of amino acid sequences of the heavy chain variable region and the light chain variable region is selected from any combination of SEQ ID NO: 1 and SEQ ID NO: 122, SEQ ID NO: 114 and SEQ ID NO: 115, SEQ ID NO: 116 and SEQ ID NO: 117, SEQ ID NO: 118 and SEQ ID NO: 119, and SEQ ID NO: 120 and SEQ ID NO: 121, or a first modified polypeptide modified from the said CD3-binding polypeptide. The CD3-binding activity of the first modified polypeptide is lower than that of the CD3-binding polypeptide. In this specific example, preferably, the first antigen-binding molecule or the first antigen-binding region includes a first modified polypeptide modified from a CD3-binding polypeptide consisting of any combination selected from SEQ ID NO: 1 and SEQ ID NO: 122, SEQ ID NO: 114 and SEQ ID NO: 115, SEQ ID NO: 116 and SEQ ID NO: 117, SEQ ID NO: 118 and SEQ ID NO: 119, and SEQ ID NO: 120 and SEQ ID NO: 121. The modification includes any modification as long as the CD3-binding activity of the first modified polypeptide is lower than that of the CD3-binding polypeptide. The amino acid sequence homology between the first modified polypeptide and the original CD3-binding polypeptide is preferably 80% or more, and more preferably 90% or more.

[0048] In this specific example, as an indicator of the comparison between the CD3 binding activity of the first modified polypeptide and the CD3 binding activity of the original CD3-binding polypeptide, for example, KD(original) / KD(modified), obtained by dividing the CD3 dissociation constant KD(original) of the original CD3-binding polypeptide in SPR by the KD(modified) of the first modified polypeptide, is used. The statement above that "the CD3 binding activity of the first modified polypeptide is lower than that of the CD3-binding polypeptide" means that KD(original) / KD(modified) is greater than 1.

[0049] The CD3 binding activity of the first modified polypeptide may be high, low, or so low as to be undetectable by SPR, as measured by SPR. Preferably, the CD3 binding activity of the first modified polypeptide is low or so low as to be undetectable by SPR, as measured by SPR. If the CD3 binding activity of the first modified polypeptide is so low that it cannot be detected by SPR, the above-mentioned KD(pre-modification) / KD(post-modification) is not used, and the KD value of the first modified polypeptide to CD3 in the presence of the second antigen-binding molecule, as described later, is used. The CD3 binding activity of the first modified polypeptide in the presence of the second antigen-binding molecule should be, for example, within the range in which the antibody can exert its effector function if the first antigen-binding molecule is an antibody. Preferably, the CD3 binding activity of the first modified polypeptide in the presence of the second antigen-binding molecule is high binding activity. In this specific example, the first antigen-binding molecule is less likely to bind to CD3 in the absence of the second antigen-binding molecule, but becomes more likely to bind to CD3 in the presence of the second antigen-binding molecule. This is useful for the on / off mechanism of the binding of the first antigen-binding molecule to CD3 by the second antigen-binding molecule. For example, when the first and second antigen-binding molecules, both possessing multi-antigen specificity, are combined and used as a pharmaceutical drug, this mechanism allows for more targeted cell-mediated cytotoxic activity by T cells, thus reducing side effects.

[0050] The CD3 subtype used in SPR when determining the KD value for CD3 as described above may be one of CD3γ, CD3δ, CD3ε, and CD3ζ, or a combination thereof. Among these, CD3ε is preferred. Preferably, all CD3 subtypes are of human origin. The CD3ε epitope to which the first antigen-binding molecule binds is not particularly limited, but preferably the CD3ε epitope includes at least the amino acid sequence from the N-terminus to the 27th amino acid of CD3ε, more preferably the CD3ε epitope includes at least the amino acid sequence from the N-terminus to the 8th amino acid of CD3ε, and most preferably the CD3ε epitope includes at least the amino acid sequence from the N-terminus to the 5th amino acid of CD3ε.

[0051] Examples of the first antigen-binding molecule or first antigen-binding region when the first antigen is adenosine are shown below. A specific example of this example is an adenosine-binding polypeptide in which the amino acid sequences of the heavy chain variable region and the light chain variable region are selected from any combination of SEQ ID NOs: 106 and 107, SEQ ID NOs: 108 and 109, SEQ ID NOs: 110 and 111, and SEQ ID NOs: 112 and 113, or a second modified polypeptide modified from the adenosine-binding polypeptide. In this specific example, the adenosine-binding activity of the second modified polypeptide may be lower or higher than that of the adenosine-binding polypeptide. Any modification is included. The amino acid sequence homology between the second modified polypeptide and the adenosine-binding polypeptide from which it was modified is preferably 80% or more, and more preferably 90% or more. In this specific example, if the heavy chain variable region or light chain variable region is derived from an animal other than a human, the second modified polypeptide includes a humanized version.

[0052] In this specific example, as an indicator of the comparison between the adenosine binding activity of the second modified polypeptide and the adenosine binding activity of the original adenosine-binding polypeptide, for example, KD(original) / KD(modified), obtained by dividing the adenosine dissociation constant KD(original) of the original adenosine-binding polypeptide in SPR by the KD(modified) of the second modified polypeptide, is used. The statement above that "the adenosine binding activity of the second modified polypeptide is lower than that of the adenosine-binding polypeptide" means that KD(original) / KD(modified) is greater than 1. Conversely, "the adenosine binding activity of the second modified polypeptide is higher than that of the adenosine-binding polypeptide" means that KD(original) / KD(modified) is less than 1.

[0053] The adenosine binding activity of the second modified polypeptide may be high, low, or so low as to be undetectable by SPR, as measured by SPR. If the adenosine binding activity of the second modified polypeptide is so low that it cannot be detected by SPR, the above-mentioned KD(pre-modification) / KD(post-modification) is not used, and the KD value of the second modified polypeptide to adenosine in the presence of the second antigen-binding molecule, as described later, is used. The adenosine binding activity of the second modified polypeptide in the presence of the second antigen-binding molecule should be, for example, within the range in which the antibody can exert its effector function if the first antigen-binding molecule is an antibody. Preferably, the adenosine binding activity of the second modified polypeptide in the presence of the second antigen-binding molecule is high binding activity.

[0054] In the specific examples above, the first antigen is shown to be CD3 or adenosine. However, it goes without saying that the antibody modification technology of the present invention, which increases the binding activity of the first antigen-binding molecule to the first antigen by a second antigen-binding molecule (described later), can also be applied to first antigens other than CD3 and adenosine.

[0055] e. The third antigen The second antigen-binding molecule, used in combination with the first antigen-binding molecule, may bind to a single antigen or may have so-called multi-antigen specificity, binding to multiple antigens. The third antigen-binding region and the third antigen are the same as those in the second antigen-binding molecule described later.

[0056] C. Second antigen-binding molecule The second antigen-binding molecule of the present invention binds to an antigen-antigen-binding molecule complex. That is, the second antigen-binding molecule includes a complex-binding region that binds to the complex. The complex includes a first antigen and a first antigen-binding molecule that binds to the first antigen. The second antigen-binding molecule is not particularly limited as long as it includes a complex-binding region and increases the binding activity of the first antigen-binding molecule to the first antigen. It may be a complete antibody consisting of two light chain molecules and two heavy chain molecules, such as a natural antibody, or it may be an antibody halve, diabody (Db), scFv, single-chain antibody, sc(Fv)2, sc(Fab')2, or other antibody fragment.

[0057] The mechanism by which the second antigen-binding molecule binds to the complex is not particularly limited as long as it binds to the complex. Preferably, when the second antigen-binding molecule binds to the complex, it binds to both the first antigen and the first antigen-binding molecule. That is, the epitope of the second antigen-binding molecule in the complex is contained in both the first antigen-binding molecule and the first antigen. If the first antigen-binding molecule is an antibody containing a heavy chain variable region and a light chain variable region, the epitope of the second antigen-binding molecule is contained in either or both of the heavy chain variable region or the light chain variable region of the first antigen-binding molecule and the first antigen. In this case, preferably, the epitope to which the second antigen-binding molecule binds is contained in the heavy chain variable region of the first antigen-binding molecule and the first antigen. The second antigen-binding molecule binds to the first antigen and the first antigen-binding molecule before and after the formation of the complex, increasing the binding activity of the first antigen-binding molecule to the first antigen. In other words, the second antigen-binding molecule stabilizes the complex.

[0058] In one embodiment, the second antigen-binding molecule exhibits higher binding activity to the first antigen in the presence of the first antigen-binding molecule compared to the absence of the first antigen-binding molecule. This is presumed to be due to either or both of the following mechanisms: the second antigen-binding molecule has higher binding activity to the complex than the first antigen released from the first antigen-binding molecule, and binding to the first antigen-binding molecule released from the first antigen increases its binding activity to the first antigen released from the first antigen-binding molecule. In this embodiment, the index for comparing the binding activity of the second antigen-binding molecule to the first antigen released from the first antigen-binding molecule with the binding activity to the first antigen in the presence of the first antigen-binding molecule is the same as the index for the first antigen-binding molecule described above. In this embodiment, the binding of the second antigen-binding molecule to the first antigen is enhanced by the presence of the first antigen-binding molecule. This means that the specificity of the second antigen-binding molecule to the first antigen is enhanced by the presence of the first antigen-binding molecule. By utilizing this property, side effects are further reduced, especially when the first and second antigen-binding molecules have dual antigen specificity and are used in combination as a pharmaceutical agent.

[0059] a. Examples of the first antigen-binding molecule Examples of a second antigen-binding molecule or second antigen-binding region when the first antigen is CD3 are shown below. A specific example of this example is a second antigen-binding molecule or second antigen-binding region containing a polypeptide whose heavy chain variable region and light chain variable region are each selected from one of the following combinations of amino acid sequences: SEQ ID NO: 45 and SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50, and SEQ ID NO: 51 and SEQ ID NO: 52.

[0060] Examples of a second antigen-binding molecule or second antigen-binding region when the first antigen is adenosine are shown below. A specific example of this example is a second antigen-binding molecule or second antigen-binding region containing a polypeptide consisting of the following combination of amino acid sequences of the heavy chain variable region and the light chain variable region. A polypeptide in which the amino acid sequences of the heavy chain variable region and the light chain variable region are selected from one of the following combinations: SEQ ID NO: 160 and SEQ ID NO: 161, SEQ ID NO: 162 and SEQ ID NO: 163, and SEQ ID NO: 164 and SEQ ID NO: 165.

[0061] b. The third antigen The second antigen-binding molecule may bind to a single antigen, or it may have so-called multi-antigen specificity, binding to multiple antigens. If the second antigen-binding molecule has multi-antigen specificity, it binds to at least a third antigen. That is, the second antigen-binding molecule includes a third antigen-binding region that binds to the third antigen. The third antigen-binding region can be an antibody fragment. The antibody fragment can be any fragment as long as it can bind to the third antigen. Examples of antibody fragments include Fv and Fab.

[0062] The third antigen is not particularly limited and encompasses any antigen. Specific examples of antigens include those listed in International Publication No. 2013 / 180200. Preferably, the third antigen is a cancer antigen or an immune-related molecule. More preferably, the third antigen is a cancer antigen. If the third antigen is an immune-related molecule, CD8 is an example of such an immune-related molecule. Specific examples of cancer antigens are the same as those for the second antigen described above. However, if both the second and third antigens are cancer antigens, preferably the second and third antigens are heterogeneous cancer antigens. More preferably, the second and third antigens are heterogeneous cancer antigens expressed in the same cancer cells or cancer tissue. The immune-related molecules and their examples are the same as those described above for the first antigen. However, if both the first and third antigens are immune-related molecules, then preferably the first and third antigens are heterogeneous immune-related molecules. Here, "heterogeneous" includes cases where they are different regions on the surface of the primary or higher-order structure of a single protein. In one embodiment, the third antigen is an extracellular protein. This extracellular protein also includes cell membrane proteins. Preferably, the extracellular protein is a cell membrane protein.

[0063] c. Second other configuration In one embodiment, the second antigen-binding molecule may or may not include components other than the antigen-binding region (second other components). The second other components are, for example, an antibody fragment, a linker, and a labeled compound. From the viewpoint of being able to add various functions to the second antigen-binding molecule, the second antigen-binding molecule preferably includes a second other component. In order to improve the stability of the first antigen-binding molecule in plasma and the manufacturing efficiency, the second other component is preferably an antibody fragment. Examples of antibody fragments include the Fc region of the antibody and the constant region of the antibody. If the second antigen-binding molecule includes the Fc region of an antibody, the Fc region may be a natural Fc region having the same amino acid sequence as the Fc region of a natural antibody, or it may be a modified Fc region derived from the natural Fc region. In this case, the Fc region of the antibody is preferably derived from the Fc region of IgG. The IgG is preferably of human origin. If the second antigen-binding molecule includes the constant region of an antibody, the constant region may be a natural constant region having the same amino acid sequence as the constant region of a natural antibody, or it may be a modified constant region modified from the natural constant region. In this case, it is preferable that the constant region of the antibody is derived from the constant region of IgG. It is preferable that the IgG is of human origin.

[0064] In one embodiment, when the second antigen-binding molecule has biantigen specificity and a heterodimer is formed of a polypeptide containing a complex-binding region and a polypeptide containing a third antigen-binding region, from the viewpoint of manufacturing efficiency, the Fc region or constant region is preferably a modified Fc region or a modified constant region, respectively. Specific examples of the modified Fc region or modified constant region in this case include at least one modification from (i) to (iii) of the first other configuration described above, or a modification in the hinge region portion. In this case, modifications in the first other configuration and modifications in the second other configuration can be combined to facilitate heterodimer formation.

[0065] d. The first antigen The first antigen of the first antigen-binding molecule used in combination with the second antigen-binding molecule is the same as that of the first antigen-binding molecule described above.

[0066] e. Second antigen The first antigen-binding molecule used in combination with the second antigen-binding molecule may bind to a single antigen or may have so-called multi-antigen specificity, binding to multiple antigens. The second antigen-binding region and the second antigen are the same as those in the first antigen-binding molecule described above.

[0067] D. Suitable combinations of the first, second, and third antigens. When both the first antigen-binding molecule and the second antigen-binding molecule have multiple antigen specificity, the types of the first antigen, the second antigen, and the third antigen are preferably one of the following combinations (1) to (5). (1) A combination in which the first antigen is an immune-related molecule, the second antigen is the first cancer antigen, and the third antigen is the second cancer antigen. (2) A combination in which the first antigen is a cellular metabolite of the target cell, the second antigen is a cancer antigen, and the third antigen is an immune-related molecule. (3) A combination in which the first antigen is a cellular metabolite of a target cell, the second antigen is an immune-related molecule, and the third antigen is a cancer antigen. (4) A combination in which the first antigen is a first immune-related molecule, the second antigen is a cancer antigen, and the third antigen is a second immune-related molecule. (5) A combination in which the first antigen is a first immune-related molecule, the second antigen is a second immune-related molecule, and the third antigen is a cancer antigen.

[0068] In all of the above (1) to (5), a reduction in side effects can be expected. In particular, in (1) above, from the viewpoint of further reducing side effects, it is preferable that the type of the second cancer antigen is different from that of the first antigen. In this case, more preferably, the first antigen is specifically expressed in the same cancerous or inflammatory tissue as the second antigen. In (4) above, from the viewpoint of further reducing side effects, the type of the second immune-related molecule is preferably different from that of the first immune-related molecule. In this case, the second immune-related molecule is more preferably CD8. Even more preferably the first immune-related molecule is CD3.

[0069] E. Manufacturing method a. First antigen-binding molecule The first antigen-binding molecule can be any compound that contains a first antigen-binding region that binds to the first antigen. The first antigen-binding molecule may be a low-molecular-weight compound, a high-molecular-weight compound, or a fusion molecule thereof. The first antigen-binding region can be, for example, the variable region of an antibody. The cDNA encoding the antigen-binding region can be obtained by common antibody production procedures, such as immunization with purified antigen or DNA immunization, collection of immune cells from immunized animals, hybridoma formation, and cloning of the cDNA encoding the variable region from the hybridoma, as described in International Publication No. 2013 / 180200. The variable region may be humanized. The variable region expressed using the cloned cDNA in a known protein expression system can be used directly as the first antigen-binding molecule.

[0070] If the first antigen-binding molecule further includes a second antigen-binding region or other components of the first antigen-binding molecule, the first antigen-binding molecule may be expressed as a fusion protein with the first antigen-binding region, or as a complex protein formed by intermolecular forces or covalent bonds. For example, if the first antigen-binding molecule is a bispecific antibody, the first antigen-binding molecule is expressed as a complex protein in which the first and second antigen-binding regions are the respective variable regions of the bispecific antibody, and the other components of the first antigen-binding molecule include an Fc region. In this case, the second antigen-binding region is prepared in the same manner as the first antigen-binding region described above. For example, the Fc region described in International Publication No. 2013 / 180200, Bispecific Antibodies and Methods for Their Preparation, can be used.

[0071] In the present invention, the technique for stabilizing an antigen-antigen-binding molecule complex formed by a first antigen and a first antigen-binding molecule with a second antigen-binding molecule may, for example, have high binding activity to the first antigen as measured by SPR, low binding activity, or even so low that it is undetectable by SPR. A first antigen-binding molecule that exhibits low binding activity to the first antigen as measured by SPR, or whose binding activity is so low that it is undetectable by SPR, can be produced by reducing the antigen-binding activity of a molecule that exhibits high binding activity to the first antigen as measured by SPR, using antibody modification techniques such as alanine scan (Biochemistry Vol.32, No.27, 1993, 6828-6835). A first antigen-binding molecule with such low binding affinity that the KD value cannot be calculated from kinetic analysis by SPR is produced by the general antibody production procedure described above. For example, a first antigen-binding molecule with such low binding affinity that it is undetectable by SPR can be produced by first obtaining a group of polypeptide candidates whose binding activity to the first antigen is detected by measuring intermolecular interactions in different modes, such as ELISA, and then using a screening method to identify polypeptides from this group that are undetectable by SPR.

[0072] b. Second antigen-binding molecule The second antigen-binding molecule is identified by screening for molecules that increase the binding activity of the first antigen-binding molecule to the first antigen. The second antigen-binding molecule can be identified, for example, from a library of compounds, antibodies, or fragments thereof by known methods of measuring binding activity. Specifically, these include (Method I) and (Method II) below.

[0073] (Method I) A screening method comprising identifying a compound or antibody or fragment thereof as a second antigen-binding molecule when the binding activity of a first antigen-binding molecule to a first antigen using at least one assay selected from SPR, BLI, and ELISA is detectable in the presence of a compound or antibody or fragment thereof, arbitrarily selected from a library of compounds or antibodies or fragment thereof, but undetectable in the absence of said compound or antibody or fragment thereof.

[0074] (Method II) A screening method comprising identifying a compound, antibody, or fragment as a second antigen-binding molecule if the binding activity of a first antigen-binding molecule to a first antigen is higher under conditions where one compound, antibody, or fragment arbitrarily selected from a library of compounds, antibodies, or fragments is present compared to conditions where such compound, antibody, or fragment is absent.

[0075] A second antigen-binding molecule whose binding activity to a free first antigen is undetectable in at least one assay selected from SPR, BLI, and ELISA, or a second antigen-binding molecule whose binding activity to the first antigen is higher in the presence of the first antigen-binding molecule than in the absence of the first antigen-binding molecule in at least one assay selected from SPR, BLI, and ELISA, can be obtained by the following screening methods (Method III) to (Method VI).

[0076] (Method III) (a) A step of immunizing a mammal with an antigen-antigen binding molecule complex consisting of a first antigen and a first antigen-binding molecule, and obtaining a first group of antibody-producing cells that produce monoclonal antibodies bound to the complex, (b) A step of selecting from the first group a second group that produces a monoclonal antibody whose binding activity to either or both of a first antigen and a first antigen-binding molecule that does not form a complex in at least one assay selected from SPR, BLI, and ELISA is undetectable. (c) A screening method comprising the step of selecting from the second group a third group that produces a monoclonal antibody that enhances the binding activity of the first antigen-binding molecule to the first antigen in at least one assay selected from SPR, BLI, and ELISA, and identifying the third group as antibody-producing cells that produce the second antigen-binding molecule.

[0077] (Method IV) (a) A step of immunizing a mammal with an antigen-antigen binding molecule complex consisting of a first antigen and a first antigen-binding molecule, and obtaining a first group of antibody-producing cells that produce monoclonal antibodies bound to the complex, (b) A step of selecting from the first group a second group that produces a monoclonal antibody whose binding activity to either or both of a first antigen and a first antigen-binding molecule that does not form a complex in at least one assay selected from SPR, BLI and ELISA is lower than that to binding to the complex. (c) A screening method comprising the step of selecting from the second group a third group that produces a monoclonal antibody that enhances the binding activity of the first antigen-binding molecule to the first antigen in at least one assay selected from SPR, BLI, and ELISA, and identifying the third group as antibody-producing cells that produce the second antigen-binding molecule.

[0078] In methods III and IV described above, an antigen-antigen binding molecule complex was immunized in step (a), but it is also possible to immunize an antigen-antigen binding region complex in which the antigen binding molecule is changed to a polypeptide that does not contain any portion other than the antigen binding region.

[0079] (Method V) (a) A step of immunizing a mammal with an antigen-antigen-binding domain complex consisting of a first antigen and a first antigen-binding domain, and obtaining a first group of antibody-producing cells that produce monoclonal antibodies bound to the complex, (b) A step of selecting from the first group a second group that produces a monoclonal antibody whose binding activity to either or both of a first antigen and a first antigen-binding region that does not form a complex in at least one assay selected from SPR, BLI, and ELISA is undetectable. (c) A screening method comprising the step of selecting from the second group a third group that produces a monoclonal antibody that enhances the binding activity of the first antigen-binding region to the first antigen in at least one assay selected from SPR, BLI, and ELISA, and identifying the third group as antibody-producing cells that produce a second antigen-binding molecule.

[0080] (Method VI) (a) A step of immunizing a mammal with an antigen-antigen-binding domain complex consisting of a first antigen and a first antigen-binding domain, and obtaining a first group of antibody-producing cells that produce monoclonal antibodies bound to the complex, (b) A step of selecting from the first group a second group that produces a monoclonal antibody whose binding activity to either or both of a first antigen and a first antigen-binding region that does not form a complex in at least one assay selected from SPR, BLI, and ELISA is lower than that to binding to the complex. (c) A screening method comprising the step of selecting from the second group a third group that produces a monoclonal antibody that enhances the binding activity of the first antigen-binding region to the first antigen in at least one assay selected from SPR, BLI, and ELISA, and identifying the third group as antibody-producing cells that produce a second antigen-binding molecule.

[0081] In methods I through VI described above, instead of "at least one assay selected from SPR, BLI, and ELISA," an assay that can indirectly demonstrate binding activity, such as a pharmacological assay using cells, may be used. Among these (Method I) to (Method VI), Methods III to VI are preferred from the viewpoint of reducing side effects when the first antigen-binding molecule and the second antigen-binding molecule are used as bispecific antibodies in a pharmaceutical product. From the viewpoint of screening efficiency, Methods V and VI are more preferred, and from the viewpoint of further reducing side effects, Methods III and V are more preferred, and from all viewpoints, Method V is the most preferred.

[0082] A method for producing the second antigen-binding molecule includes, for example, culturing antibody-producing cells that produce the second antigen-binding molecule screened in (Method III) to (Method VI) above, and purifying the second antigen-binding molecule from the culture supernatant or cell disrupted product. That is, the second antigen-binding molecule is produced by (Method III') to (Method VI') below.

[0083] (Method III') (d) A step of culturing antibody-producing cells obtained by a screening method including steps (a) to (c) below; (a) A step of immunizing a mammal with an antigen-antigen binding molecule complex consisting of a first antigen and a first antigen-binding molecule, and obtaining a first group of antibody-producing cells that produce monoclonal antibodies bound to the complex, (b) A step of selecting from the first group a second group that produces a monoclonal antibody whose binding activity to either or both of a first antigen and a first antigen-binding molecule that does not form a complex in at least one assay selected from SPR, BLI, and ELISA is undetectable. (c) From the second group, select a third group that produces a monoclonal antibody that enhances the binding activity of the first antigen-binding molecule to the first antigen in at least one assay selected from SPR, BLI, and ELISA, and identify the third group as antibody-producing cells that produce the second antigen-binding molecule; (e) A step of obtaining a culture supernatant or cell disruption product from the antibody-producing cell culture, (f) A method for producing a second antigen-binding molecule, comprising the step of purifying the second antigen-binding molecule from the culture supernatant or cell disrupted product.

[0084] (Method IV') (d) A step of culturing antibody-producing cells obtained by a screening method including steps (a) to (c) below; (a) A step of immunizing a mammal with an antigen-antigen binding molecule complex consisting of a first antigen and a first antigen-binding molecule, and obtaining a first group of antibody-producing cells that produce monoclonal antibodies bound to the complex, (b) A step of selecting from the first group a second group that produces a monoclonal antibody whose binding activity to either or both of a first antigen and a first antigen-binding molecule that does not form a complex in at least one assay selected from SPR, BLI and ELISA is lower than that to binding to the complex. (c) From the second group, select a third group that produces a monoclonal antibody that enhances the binding activity of the first antigen-binding molecule to the first antigen in at least one assay selected from SPR, BLI, and ELISA, and identify the third group as antibody-producing cells that produce the second antigen-binding molecule; (e) A step of obtaining a culture supernatant or cell disruption product from the antibody-producing cell culture, (f) A method for producing a second antigen-binding molecule, comprising the step of purifying the second antigen-binding molecule from the culture supernatant or cell disrupted product.

[0085] (Method V') (d) A step of culturing antibody-producing cells obtained by a screening method including steps (a) to (c) below; (a) A step of immunizing a mammal with an antigen-antigen-binding domain complex consisting of a first antigen and a first antigen-binding domain, and obtaining a first group of antibody-producing cells that produce monoclonal antibodies bound to the complex, (b) A step of selecting from the first group a second group that produces a monoclonal antibody whose binding activity to either or both of a first antigen and a first antigen-binding region that does not form a complex in at least one assay selected from SPR, BLI, and ELISA is undetectable. (c) From the second group, select a third group that produces a monoclonal antibody that enhances the binding activity of the first antigen-binding region to the first antigen in at least one assay selected from SPR, BLI, and ELISA, and identify the third group as antibody-producing cells that produce the second antigen-binding molecule; (e) A step of obtaining a culture supernatant or cell disruption product from the antibody-producing cell culture, (f) A method for producing a second antigen-binding molecule, comprising the step of purifying the second antigen-binding molecule from the culture supernatant or cell disrupted product.

[0086] (Method VI') (d) A step of culturing antibody-producing cells obtained by a screening method including steps (a) to (c) below; (a) A step of immunizing a mammal with an antigen-antigen-binding domain complex consisting of a first antigen and a first antigen-binding domain, and obtaining a first group of antibody-producing cells that produce monoclonal antibodies bound to the complex, (b) A step of selecting from the first group a second group that produces a monoclonal antibody whose binding activity to either or both of a first antigen and a first antigen-binding region that does not form a complex in at least one assay selected from SPR, BLI, and ELISA is lower than that to binding to the complex. (c) From the second group, select a third group that produces a monoclonal antibody that enhances the binding activity of the first antigen-binding region to the first antigen in at least one assay selected from SPR, BLI, and ELISA, and identify the third group as antibody-producing cells that produce the second antigen-binding molecule; (e) A step of obtaining a culture supernatant or cell disruption product from the antibody-producing cell culture, (f) A method for producing a second antigen-binding molecule, comprising the step of purifying the second antigen-binding molecule from the culture supernatant or cell disrupted product.

[0087] The types of cells from which antibody-producing cells are derived in the above-described methods (III) to (VI) and (III') to (VI') include any known types of antibody-producing cells. Examples of cell types from which antibody-producing cells are derived include B cells and hybridomas.

[0088] In another embodiment, the libraries used in Methods I and II described above can be prepared as antigen-binding molecule libraries for phage display by Method VII or Method VIII described below.

[0089] (Method VII) A first step involves modifying a first antigen-binding molecule that binds to a first antigen by modifying its amino acids to obtain a modified version of the first antigen-binding molecule in which binding to the first antigen is reduced or below the detection limit in at least one assay selected from SPR, BLI, and ELISA. A second step of obtaining a first phage-display antigen-binding molecule library by removing phages that display antigen-binding molecules that bind to either or both of the first antigen and the modified form from an existing phage-display antigen-binding molecule library, and A method for producing a phage display antigen-binding molecule library, comprising a third step of obtaining a second phage display antigen-binding molecule library from the first phage display antigen-binding molecule library, which is enriched with phages that display antigen-binding molecules that bind to an antigen-antigen-binding molecule complex containing the first antigen and the first antigen-binding molecule.

[0090] In another embodiment of Method VII, a method for producing a phage display antigen-binding molecule library can be provided, wherein the second phage display antigen-binding molecule library is the existing phage display antigen-binding molecule library, and the second and third steps are repeated. By repeating the second and third steps, a phage display antigen-binding molecule library containing a high density of phages that present the second antigen-binding molecule can be produced.

[0091] (Method VIII) A first step to obtain a first phage display antigen-binding molecule library by removing phages from an existing phage display antigen-binding molecule library that (i) bind to a first antigen-binding molecule that can bind to a first antigen but is not bound to the first antigen, and (ii) bind to the first antigen that is not bound to the first antigen-binding molecule. A method for producing a phage display antigen-binding molecule library, comprising a second step of obtaining a second phage display antigen-binding molecule library obtained by enriching the first phage display antigen-binding molecule library with phages that display antigen-binding molecules that bind to an antigen-antigen-binding molecule complex containing the first antigen and the first antigen-binding molecule.

[0092] In another embodiment of Method VIII, a method for producing a phage display antigen-binding molecule library can be provided, wherein the second phage display antigen-binding molecule library is the existing phage display antigen-binding molecule library, and the first and second steps are repeated. By repeating the first and second steps, a phage display antigen-binding molecule library containing a high density of phages that present the second antigen-binding molecule can be produced.

[0093] F. Combinations The combination of the present invention is a combination of the first antigen-binding molecule described above and the second antigen-binding molecule described above. If the combination of the first antigen, the second antigen, and the third antigen is any of the combinations (1) to (5) in "D. Preferred combinations of the first antigen, the second antigen, and the third antigen" described above, then the combination is preferably a TDCC activity inducer or an ADCC activity inducer, and more preferably a TDCC activity inducer.

[0094] a. Pharmaceutical compositions The combination is preferably a pharmaceutical composition. When the combination is a pharmaceutical composition, the first antigen-binding molecule and the second antigen-binding molecule may be administered simultaneously or separately. Preferably, the first antigen-binding molecule and the second antigen-binding molecule are administered separately.

[0095] The first antigen-binding molecule and the second antigen-binding molecule may be formulated as the same product for simultaneous administration, or they may be formulated separately for separate administration. If the first antigen-binding molecule and the second antigen-binding molecule are formulated separately, they may be combined and packaged as a kit, or the package insert for one formulation may state that it is to be used in combination with the other formulation. An example of the former is to fill the first antigen-binding molecule and the second antigen-binding molecule into separate ampoules and package both ampoules in a single box to form a kit.

[0096] b. Other ingredients The pharmaceutical composition may contain other components in addition to the first antigen-binding molecule and the second antigen-binding molecule. Other components include, for example, pharmaceutically acceptable carriers.

[0097] Pharmaceutical compositions can be formulated using methods known to those skilled in the art. For example, they can be used parenterally in the form of sterile solutions with water or other pharmaceutically acceptable liquids, or as injectable suspensions. For example, they can be formulated by mixing them with pharmacologically acceptable carriers or media, specifically sterile water, saline solution, vegetable oil, emulsifiers, suspensions, surfactants, stabilizers, flavorings, excipients, vehicles, preservatives, binders, etc., in a unit dose form generally accepted for pharmaceutical practice. The amount of active ingredient in these formulations should be set to obtain an appropriate volume within the indicated range.

[0098] Sterile compositions for injection can be formulated using a vehicle such as distilled water for injection, following standard formulation procedures.

[0099] Examples of aqueous solutions for injection include physiological saline, glucose, and isotonic solutions containing other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride). Suitable solubilizers include alcohols (ethanol, etc.), polyalcohols (propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (polysorbate 80). TM (HCO-50, etc.) may be used in combination.

[0100] Examples of oily liquids include sesame oil and soybean oil, and benzyl benzoate and / or benzyl alcohol may be used in combination as solubilizers. It may also be combined with buffers (e.g., phosphate buffer and sodium acetate buffer), analgesics (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants. The prepared injection solution is usually filled into appropriate ampoules.

[0101] c.Dosage form The pharmaceutical composition is preferably administered by parenteral administration. For example, it can be in the form of an injectable, nasal, pulmonary, or transdermal formulation. For example, it can be administered systemically or locally by intravenous, intramuscular, intraperitoneal, or subcutaneous injection.

[0102] d. Target diseases The target diseases of the pharmaceutical composition are not particularly limited. Preferably, the target diseases are those in which, if the first antigen is a cellular metabolite, the cellular metabolite is expressed at a higher level than in normal tissue, and those in which, if the first antigen is an immunometabolite, the second and third antigens are expressed at a higher level than in normal tissue. In other words, the disease is one in which it is desirable for the first antigen-binding molecule and the second antigen-binding molecule to work together to induce effector cells, particularly T cells, in target cells and exert effector function, especially TDCC activity. Specific target diseases include, for example, cell proliferative disorders, hyperimmune disorders, and infectious diseases. Cell proliferative disorders include tumors. Hyperimmune disorders include autoimmune diseases. Infectious diseases include bacterial infections and viral infections.

[0103] e. Other uses The combination of this invention can also be used for applications other than pharmaceutical compositions. In one embodiment, when the first antigen is a low-molecular-weight compound such as a cellular metabolite, the combination of the present invention is useful for a simpler assay for detecting low-molecular-weight compounds. While SPR and HPLC are commonly used for detecting low-molecular-weight compounds, sandwich methods used in ELISA and other methods are often difficult to apply because the antigens are too small. However, with this combination, the first antigen-binding molecule and the second antigen-binding molecule can simultaneously bind to the low-molecular-weight compound by sandwiching it between them, thus providing a simpler detection system for low-molecular-weight compounds.

[0104] In another embodiment, the combination of the present invention is useful for in vivo imaging. As described above, attempts to specifically exert the effector function of conventional antibody drugs on target tissues are still in their early stages of development, and further attempts are expected. Similarly, when performing in vivo imaging specifically on target tissues using antibodies, noise may occur due to the binding of antibodies to tissues other than the target tissue. On the other hand, this noise can be further reduced by using the combination. For example, if the first antigen is a cellular metabolite and the second antigen is a target cell-specific antigen, the combination can be applied to in vivo imaging by including a complex-binding region in the second antigen-binding molecule and a labeling compound (e.g., a radioisotope and a fluorescent dye) as the second other component. In this case, noise can be reduced and detection sensitivity can be improved when imaging target tissues where cellular metabolites are present.

[0105] G. Treatment method When the first antigen-binding molecule and the second antigen-binding molecule described above are used together as a pharmaceutical product, they may be administered simultaneously or separately. The method of administration should be determined based on the pharmacokinetics and mechanism of action of the first and second antigen-binding molecules. The dosage and method of administration will vary depending on the patient's weight, age, symptoms, etc., but a person skilled in the art can determine an appropriate dosage and method of administration by taking these conditions into consideration. [Examples]

[0106] In the following examples, the antibody prepared as one form of the second antigen-binding molecule is referred to as a "clumping antibody" due to its function. Bispecific antibodies are also abbreviated as "BiAb". This embodiment illustrates one aspect of the present invention. The antigen used in the present invention is not necessarily limited to that used in this embodiment.

[0107] [Example 1] The concept of an antibody that recognizes and binds to an antibody that has bound to an antigen. To exert drug efficacy while avoiding side effects, there is a need for drug discovery technologies that do not act systemically in normal tissues or blood, but only act at the lesion site, such as cancer or inflammation. For example, EGFR-BiTE (Non-patent literature: BiTE: Baeuerle PA et. al. Curr. Opin. Mol. Ther. 2009. 11, 22-30) exerts its antitumor effect by recruiting and activating T cells via CD3. If EGFR-BiTE can be given the property of binding to CD3 expressed on T cells near cancer cells but not to CD3 expressed on T cells outside the vicinity of cancer cells, then such a modified EGFR-BiTE can activate T cells only in cancer, thereby exerting a powerful antitumor effect while avoiding side effects.

[0108] It was thought that, not only for antibody drugs against cancer, but also for rheumatoid arthritis, if antibody molecules bind to cytokines only in the synovial fluid of the inflamed joints and inhibit their action without systemic inhibition, it would be possible to exert a high therapeutic effect on inflammatory and autoimmune diseases such as rheumatoid arthritis while avoiding the increased risk of infection due to systemic neutralization of cytokines.

[0109] Antibodies that crosslink antigen-double positive target cells and effector cells, or activate T cells, only in cancer or inflammatory sites, can exert therapeutic effects while avoiding side effects. However, no ideal antibody with such characteristics has been reported to date. Therefore, the inventors hypothesized that by combining a clamping antibody, as shown in Figure 1, which crosslinks via CD3 expressed on T cells near cancer cells or small molecules present in high concentrations in target disease sites such as cancer, with an antibody that recognizes antigens on target disease cells, it would be possible to crosslink cells or induce CD3 signaling in target tissues where small molecules are present in high concentrations, as shown in Figure 2. Therefore, attempts were made to obtain binding antibodies against adenosine, a small molecule known to be present in high concentrations in cancer tissue, and clamping antibodies against anti-CD3 antibodies capable of inducing CD3 signaling.

[0110] [Example 2] Acquisition of attenuated CD3 antibody The anti-CD3 antibody CE115HA000 is a humanized antibody derived from a rat that recognizes CD3ε. By introducing mutations into the antigen-determining region (CDR), variants with different TDCC-inducing abilities have been obtained. Of these, CE115HA146, which has reduced TDCC activity, and CE115HA056, which shows no TDCC activity, were used. The amino acid sequences of the heavy chain variable region are shown in SEQ ID NOs: 1-3.

[0111] [Example 3] Acquisition of clamping antibodies against anti-CD3 antibodies (1) Antigen preparation An antibody (CE115HAPG13-rabCH1hG1m, SEQ ID NO: 6) was constructed by designing the N-terminal heavy chain of CE115HA146 to be linked to the eight amino acid residues of CD3ε (QDGNEEMG, SEQ ID NO: 5) via a GS linker (GGGSGGGS, SEQ ID NO: 4). This gene was incorporated into an expression vector for mammalian cultured cells. The gene encoding the corresponding light chain (GLS3000-rabk, SEQ ID NO: 7) was similarly incorporated into an expression vector for mammalian cultured cells. These expression vectors were introduced into FreeStyle 293F cells (Thermo Fisher Scientific) using the transfection reagent 293fectin Tranfection Reagent (Thermo Fisher Scientific) according to the manufacturer's manual, and the cells were cultured for 5 days before the culture medium was collected. The antibody was purified from the collected culture medium by affinity purification using rProteinA Sepharose Fast Flow resin (GE Healthcare). 10 mg of purified antibody was mixed with 1500 units of protease and FabRICATOR (Genovis) and cleaved at 37°C for 15 hours. Subsequently, the fraction that passed through Eshmuno A resin (Merck Millipore) was subjected to gel filtration to prepare the CD3ε epitope-fused F(ab')2 fragment. The concentration of the purified protein was calculated using the absorbance at 280 nm measured with a spectrophotometer, and the extinction coefficient was determined from the obtained value using the PACE method (Protein Science 1995; 4: 2411-2423).

[0112] (2) Immunization of rabbits, selection of antibody-producing cells, isolation of antibody genes Rabbit immunization was performed by subcutaneous injection of an emulsion prepared by mixing CD3ε epitope fusion F(ab')2 solution with TiterMax Gold (TiterMax USA). After four immunizations, blood and spleen samples were collected from rabbits that showed antibody production. To enrich B cells that present antigen-specific B cell receptors on their surface, peripheral blood mononuclear cells and splenocytes were prepared. These were reacted with CD3ε epitope-fused CE115HA146 (CE115HAPG13rabCH1hG1m / GLS3000-rabk, SEQ ID NOs: 6, 7) and Alexa647 (Invitrogen)-labeled CE115HA146 (CE115HA146-rabCH1hG1m / GLS3000-rabk, SEQ ID NOs: 8, 7). The conjugated antibodies were then stained with DyLite488-labeled anti-human IgG antibody, Goat anti-Human IgG Fc Cross Absorbed DyLight488 conjugate (Thermo Fisher Scientific). Cells stained with DyLite488 alone were isolated using a cell sorter (FACS Aria III, BD), seeded into 96-well plates at a density of 1 cell / well, and cultured in BT medium for 10 days in the presence of 25,000 EL4 cells / well. The EL4 cells used were pre-treated with mytomycin C (Sigma-Aldrich) to suppress cell proliferation. BT medium was prepared by adding Fetal Bovine Serum, ultra-low IgG (Life Technologies), and 1 / 20 volume rabbit T cell culture medium to RPMI 1640 with L-Gln (nacalai tesque). Rabbit T cell culture medium was prepared by culturing rabbit T cells in RPMI-1640 medium supplemented with Phytohemagglutinin-M (Roche), phorbol 12-myristate 13-acetate (Sigma-Aldrich) 2% FBS.

[0113] As a primary screening, the binding affinity of antibodies secreted into B cell culture medium to CD3ε epitope-fused CE115HA146 was evaluated by ELISA. CD3ε epitope-fused CE115HA146 (CE115HAPG13-rabCH1hG1m / GLS3000-rabk, SEQ ID NOs: 6, 7) or CE115HA146 without CD3ε epitope fusion (CE115HA146-rabCH1hG1m / GLS3000-rabk, SEQ ID NOs: 8, 7) was conjugated to a 384-well plate immobilized with the anti-human F(ab')2 antibody, Affipure F(ab')2 Fragment Donkey Anti-Human (Jackson Immuno Research). After adding the culture supernatant of B cells, a peroxidase-labeled anti-rabbit Fc antibody (Biolegend) was reacted, and the absorbance at 405 nm of the antigen-bound rabbit antibody was measured using an ABTS Microwell Peroxidase Substrate (1-Component System) (KPL) plate reader SpectraMax 340PC384 (Molecular device). Secondary screening was performed on clones that showed specific binding to the CD3ε epitope fusion CE115HA146. As part of the secondary screening, the binding affinity to the CD3ε epitope fusion control antibody (hGC33VHGP01-rabCH1hG1m / hGC33VL-rabk, SEQ ID NOs: 9, 10) was similarly evaluated by ELISA to eliminate antibodies that recognize only the peptide sequence, independent of the backbone antibody sequence. 10,560 clones were screened, 352 clones were selected, and RNA was extracted and purified from the selected B cells using the ZR-96 Quick RNA Kit (Zymo Research). RT-PCR was performed using primer sets corresponding to the DNA encoding the heavy chain variable region and light chain variable region of the antibody gene (SEQ ID NOs: 11, 12, and 13, 14) and the PrimeScript II High Fidelity One Step RT-PCR Kit (Takara Bio), and the respective PCR products were obtained.The obtained PCR products of the heavy chain variable region and light chain variable region were cloned into plasmid DNA encoding the human heavy chain constant region and the human light chain constant region, respectively, using the In-fusion HD Cloning Kit (Takara Bio). The nucleotide sequences of the heavy chain constant region and the light chain constant region are shown in SEQ ID NOs. 15 and 16, respectively. Based on these steps, vectors expressing polypeptides fused with the heavy chain variable region and the human heavy chain constant region, and polypeptides fused with the light chain variable region and the human light chain constant region, were constructed.

[0114] (3) Preparation of bispecific antibodies (BiAb) The genes of antibodies selected by B cell cloning and ELISA screening were introduced into FreeStyle 293F cells to induce antibody expression. Transfection was performed in 6-well cell culture plates with 5 mL of culture medium per well, according to the manufacturer's manual. After 4 days of culture, the cell supernatant was prepared, and purified antibodies were obtained by batch purification using rProteinA Sepharose Fast Flow (GE Healthcare) according to a method known to the industry. The antibody concentration was calculated using the same method as for calculating the protein concentration in (1) above. Sufficient expression was achieved in 212 clones.

[0115] The antibody-based antibody (BiAb) was prepared using FAE technology. First, 20 μg each of two antibodies were mixed, and 10 μL of 2-Mercaptoethylamine·HCl (2-MEA, Sigma-Aldrich), prepared to 250 mM using Tris-Buffered saline (TBS, Takara Bio), was added. The total volume was then made up to 100 μL with TBS buffer. After incubating the reaction solution at 37°C for 90 minutes, the 2-MEA was removed and replaced with D-PBS(-) (Wako Pure Chemical Industries) using Zeba 96-well spin plates (Thermo Fisher Scientific). As an example of the first antigen-binding molecule, BiAb1 was prepared using anti-human glypican 3 (GPC3) antibody (GCH065-F760mnN17 / L0011-k0, SEQ ID NOs: 17, 18) and CE115HA146 (CE115HA146-F760mnP17 / GLS3000-k0, SEQ ID NOs: 19, 20), and BiAb2 was prepared using anti-GPC3 antibody (GCH065-F760mnN17 / L0011-k0, SEQ ID NOs: 17, 18) and antibodies derived from rabbit B cells cloned in (2) above.

[0116] (4) CD3 signal reporter assay using Jurkat-luc cells (Jurkat-Luc assay) SK-pca60 cell line, in which human GPC3 is forcibly expressed in SK-HEP-1 cells (ATCC HTB-52), was used as the target cell line, and NFAT-RE-luc2-Jurkat cells (Jurkat-luc cells, Promega), which express Luciferase in response to CD3 signaling, were used as the effector cells. BiAb1 was added at a concentration of 0.09 μg / mL and BiAb2 at a concentration of 0.3 μg / mL to RPMI-1640 (Nacalai teesuque) containing 10% FBS (HyClone), 1×MEM Non-Essential Amino Acids Solution (Gibco), and 1 mM Sodium Pyruvate, in 25 μL of Assay buffer (RPMI-1640 (Nacalai teesuque)). Furthermore, each plate was prepared with separate wells for signal correction and control between plates: one without antibody, one with only BiAb1, and one with only BiAb2. The volume of antibody solution was adjusted to 25 μL, with any shortfall being added using Assay buffer. After adding the antibody solution, target cells were added in 25 μL / well (1 × 10⁶). 4 cells / well), Jurkat-Luc cell solution at 25 μL / well (1×10 4 Cells were seeded (in wells) and cultured at 37°C under 5% CO2 conditions for 6 hours. After allowing the 96-well plate to stand at room temperature for 15 minutes, 75 μL of Bio-Glo Luciferase assay reagent (Promega) was added, mixed, and allowed to react for 10 minutes. Luminescence was measured using a plate reader, EnVision (Perkin Elmer). The average relative luminescence intensity (RLU), used as an indicator of TDCC activity, was corrected using Equation 1 below.

[0117] (Formula 1) TIFF0007862462000001.tif11170

[0118] In Equation 1 above, A represents the average RLU value of wells containing only BiAb1 in multiple different plates, and B represents the average RLU value of wells containing only BiAb1 in each plate. The term obtained by dividing B by A was used as a plate-to-plate correction term for CD3 signal activation by each antibody. The results of the Jurkat-Luc assay are shown in Figure 3. Of 212 clones, 6 clones showed enhancement of more than twice the standard deviation upon addition of BiAb2 in CD3 signal activation by BiAb1. After sequence analysis, four clones, CLA0022, CLA0028, CLA0311, and CLA0344, were obtained as second antigen-binding molecules after removing duplicate sequences. The amino acid sequences of the antigen-determining sites (SEQ ID NOs: 21-44) and variable regions (SEQ ID NOs: 45-52) of the obtained antibodies are shown. In addition, clones that attenuated the CD3 signal were also obtained in this assay.

[0119] [Example 4] Evaluation of binding affinity between anti-CD3 antibody-CD3ε peptide complex and clamping antibody using surface plasmon resonance (SPR). (1) Preparation of analytes and ligands The analytes used were Fab fragments prepared from antibodies of the four clones obtained in Example 3: CLA0022, CLA0028, CLA0311, and CLA0334. Specifically, expression vectors containing genes encoding the respective antibody sequences CLA0022VH-F760mnP17 / CLA0022VL-k0C (SEQ ID NOs: 53, 54), CLA0028VH-F760mnP17 / CLA0028VL-k0C (SEQ ID NOs: 55, 56), CLA0311VH-F760mnP17 / CLA0311VL-k0C (SEQ ID NOs: 57, 58), and CLA0334VH-F760mnP17 / CLA0334VL-k0C (SEQ ID NOs: 59, 60) were introduced into Expi293F using ExpiFectamine293 (Thermo Fisher Scientific). The culture supernatant was collected on day 5 of culture and prepared using HiTrap MabSelect SuRe according to a method known to the industry. Fab fragments were prepared from purified antibodies using the Pierce Fab Preparation Kit (Thermo Fisher Scientific) according to the manufacturer's manual. The concentration of the obtained Fab fragments was calculated using the same method as the protein concentration calculation in Example 3(1).

[0120] The ligands are CD3ε epitope-fused CE115HA146 (CE115HAGP13-rabIgG / GLS3000-rabk, SEQ ID NOs: 61, 7), CD3ε epitope-fused CE115HA056 (CE115HAGP12-rabIgG / GLS3000-rabk, SEQ ID NOs: 62, 7), CD3ε epitope-fused GPC3 antibody (hGC33VHGP01-rabCH1hG1m / hGC33VL-rabk, SEQ ID NOs: 9, 10), and CD3 antibody CE115HA000 (CE115HA000-F760mnP17 / GLS Mammalian expression vectors incorporating genes encoding 3000-k0 (SEQ ID NOs: 63, 20), CE115HA056 (CE115HA056-F760mnP17 / GLS3000-k0, SEQ ID NOs: 64, 20), CE115HA146 (CE115HA146-F760mnP17 / GLS3000-k0, SEQ ID NOs: 19, 20), and the negative control IC17 (IC17Hdk-F760mnP17 / IC17L-k0, SEQ ID NOs: 65, 66) were introduced into Expi293F and prepared in the same manner as antibodies prepared for Fab.

[0121] (2) Evaluation of clamping antibody binding by SPR SuRe protein A (GE Healthcare), prepared at 25 μg / mL with Acetate 4.5 (GE Healthcare), was immobilized on the CM4 sensor chip at a rate of approximately 1000 RU per flow cell using an amine coupling kit (GE Healthcare).

[0122] First, to evaluate the binding specificity of the clamping antibody, the ligand was reacted at 37°C at a flow rate of 10 μL / min for 60 seconds to capture 1000 RU, and then analyte prepared to 100 nM was reacted at a flow rate of 30 μL / min for 180 seconds, and the amount of binding was measured. The amount of binding per RU of ligand was calculated by subtracting the value of the flow cell in which no ligand was captured. As shown in Figure 4, the clamping antibody bound to CD3ε epitope-fused CE115HA146 (CE115HAPG13) and CD3ε epitope-fused CE115HA056 (CE115HAPG12), but showed almost no binding affinity to the CD3 antibody alone (CE115HA000, CE115HA056, CE115HA146), the CD3ε epitope-fused GPC3 antibody, or the negative control IC17.

[0123] Next, to measure affinity, the ligand was reacted at 37°C at a flow rate of 10 μL / min for 60 seconds, capturing 75 RU. Then, Fab fragments derived from clamping antibodies of 0, 25, 50, 100, 200, and 400 nM were reacted as analytes at a flow rate of 30 μL / min for 180 seconds, and dissociation was observed for 300 seconds. The sensor tip was regenerated by flowing 1.5 mM glycine and 25 mM NaOH at a flow rate of 30 μL / min for 15 seconds each. The dissociation constant KD(M) was calculated based on the binding rate constant ka(1 / Ms) and dissociation rate constant kd(1 / s), which are kinetic parameters calculated from the sensorgram obtained from the measurement. Biacore T200 Evaluation Software (GE Healthcare) was used to calculate each parameter. The obtained KD values ​​are shown in Table 1.

[0124] [Table 1] ND not Fre

[0125] The formation of a complex between the CD3ε epitope-fused CD3 antibody and the clamping antibody was also confirmed by crystal structure analysis.

[0126] [Example 5] Evaluation of CD3εδ binding using Bio-layer Interferometry (BLI) (1) Preparation of biotinylated human CD3εδ heterodimer Biotinylated human CD3εδ heterodimer (hereinafter referred to as CD3εδ) was prepared by methods known to the art. Specifically, a gene fragment encoding the extracellular region of human CD3ε was ligated downstream of a gene fragment encoding the constant region of the antibody, a gene fragment encoding a sequence cleaved by TEV protease (ENLYFQG, SEQ ID NO: 67), and a gene fragment encoding the Avi tag (GLNDIFEAQKIEWHE, SEQ ID NO: 68), to which biotin is added by biotin ligase, via a gene fragment encoding a linker composed of glycine and serine. A gene fragment encoding a protein (Fc-fused human CD3ε, SEQ ID NO: 69) in which the extracellular region of human CD3ε, the constant region of the antibody, the TEV protease cleavage sequence, and the Avi tag are linked was incorporated into an animal cell expression vector. Next, a gene fragment encoding the constant region of the antibody and a gene fragment encoding the Flag tag (DYKDDDDK, SEQ ID NO: 70) were ligated downstream of the gene fragment encoding the extracellular region of human CD3δ. A gene fragment encoding a protein (Fc-fused human CD3δ, SEQ ID NO: 71) linked to the extracellular region of human CD3δ, the constant region of an antibody, and a flag tag was incorporated into an animal cell expression vector. The two constructed plasmid vectors were introduced into FreeStyle 293F cells (Invitrogen) using ExpiFectamine 293 (Thermo Fisher Scientific). During gene transfer, a gene expressing biotin ligase (BirA, SEQ ID NO: 72) and biotin were added to biotinylate the Avi tag of CD3ε. The gene-transferred cells were cultured at 37°C and 8% CO2, and the target protein was secreted into the culture supernatant. This cell culture medium was filtered through a 0.22 μm bottle-top filter to obtain the culture supernatant.

[0127] The culture supernatant was added to a column prepared using Eshmuno A resin (Merck Millipore) and the target protein was bound to the column. Elution was then performed using a 20 mM sodium citrate pH 2.7 solution. After neutralizing the eluted fraction, it was added to an Anti-FLAG M2 column prepared using Anti-FLAG M2 agarose resin (Sigma-Aldrich) for adsorption, and the target protein was eluted with FLAG peptide dissolved in D-PBS(-). The eluate was then removed from the aggregate and FLAG peptide by gel filtration chromatography using Superdex 26 / 600 (GE healthcare) to obtain purified CD3εδ. The concentration of the obtained purified protein was calculated using the same method as in Example 3(1).

[0128] (2) Preparation of one-arm antibodies To achieve a 1:1 binding ratio between the antigen CD3εδ and the antibody, the following one-arm antibodies were used: CE115HA000 one arm (CE115HA000-pE22Hh / GLS3000-k0 / / Kn010, SEQ ID NOs: 73, 20, 74), CE115HA056 one arm (CE115HA056-pE22Hh / GLS3000-k0 / / Kn010, SEQ ID NOs: 75, 20, 74), CE115HA146 one arm (CE115HA146-pE22Hh / GLS3000-k0 / / Kn010, SEQ ID NOs: 76, 20, 74), and the negative control anti-KLH antibody IC17 one arm. Arms (IC17Hdk-pE22Hh / IC17L-k0 / / Kn010, SEQ ID NOs: 77, 66, 74) were prepared. The genes encoding the heavy chain and light chain of each antibody, along with the gene encoding the antibody fragment without a variable region, were introduced into Expi293F using ExpiFectamine293 (Thermo Fisher Scientific), and purified antibodies were prepared in the same manner as in Example 4(1).

[0129] (3) Evaluation of ternary complex formation by Octet Using an Octet RED 384 (ForteBio), a streptavidin sensor (ForteBio) was reacted with CD3εδ, prepared in ACES at 0.2 μM, for 300 seconds at 37°C. Next, CE115HA000 one arm, CE115HA056 one arm, CE115HA146 one arm, and a negative control IC17 one arm were reacted for 120 seconds. Dissociation was then monitored for 120 seconds in HBS-EP(+) buffer containing 1 μM clamping antibody Fab fragment (see Example 4(1)). Response values ​​were extracted every 3 seconds, and graphs were created using Microsoft Excel 2013 (Microsoft). As shown in Figure 5, when only running buffer or negative control IC17 Fab was added, rapid dissociation of the CD3 antibody was observed. However, when Fab prepared from clamping antibodies CLA0022, CLA0028, CLA0311, and CLA0334 was added, a further increase in the binding response of the anti-CD3 antibody or a delay in dissociation was observed. Therefore, it was considered that the CD3εδ-anti-CD3 antibody complex was stabilized by the addition of the clamping antibodies.

[0130] [Example 6] Evaluation of TDCC activity using human peripheral blood monocytes (PBMCs) (1) Preparation of human PBMC solution Using a syringe pre-filled with 200 μL of 1000 units / mL heparin solution (Novo Heparin Injection 5000 units, Novo Nordisk), 50 mL of peripheral blood was collected from healthy volunteers belonging to Chugai Pharmaceutical Co., Ltd. The peripheral blood, diluted 2-fold with PBS(-), was divided into four equal parts and added to Leucosep lymphocyte separatory tubes (Greiner bio-one) that had been pre-filled with 15 mL of Ficoll-Paque PLUS and centrifuged. After centrifuging the separatory tubes containing the peripheral blood at 1000 xg for 10 minutes at room temperature, the mononuclear cell fraction was separated. After washing the cells in each layer once with 10 mL of RPMI-1640 Medium containing 10% FBS (hereinafter referred to as 10% FBS / RPMI), the cells were treated with 10% FBS / RPMI, Dulbecco's Modified Eagle's Medium (hereinafter referred to as 10% FBS / D-MEM), or Eagle's Minimal Essential Medium (hereinafter referred to as 10% FBS / E-MEM) depending on the target cells, until the cells reached 5 × 10 5 cells / mL or 1 × 10⁶ 6 The solution was suspended to a cell / mL concentration and used as a human PBMC solution for subsequent experiments.

[0131] (2) Preparation of target cells SK-pca60 (GPC3-positive cells), NCI-H446 (ATCC HTB-171, GPC3-positive cells), SKE-4B2 (human EREG-positive cells) obtained by forcing human EREG into SK-HEP-1 cells, and hEREG / SK-pca60 (human EREG, GPC3-positive cells) obtained by forcing human EREG and human GPC3 into SK-HEP-1 cells were detached from the dish using cell dissociation buffer, and the cell density of SK-pca60 in 10% FBS / D-MEM was 6 × 10⁶. 4 Cells / mL, NCI-H446 has a cell density of 2 × 10 in 10% FBS / RPMI. 5 Cells / mL, SKE-4B2 in 10% FBS / E-MEM, with a cell density of 1 × 10⁶ 5Cells / mL, hEREG / SK-PCA60 in 10% FBS / E-MEM with a cell density of 1 × 10 5 The cells were suspended to a concentration of cells / mL. This cell suspension was used as the target cell for subsequent experiments.

[0132] (3) Preparation of BiAb Each antibody for GCH065-F760mnN17 / L0011-k0 (SEQ ID NOs: 17, 18), EGLVH-F760mnN17 / EGLVL-KT0 (SEQ ID NOs: 78, 79), CE115HA000-F760mnP17 / GLS3000-k0 (SEQ ID NOs: 63, 20), CE115HA056-F760mnP17 / GLS3000-k0 (SEQ ID NOs: 64, 20), CLA0028VH-F760-mnP17 / CLA0028VL-k0C (SEQ ID NOs: 55, 56), and IC17Hdk-F760mnN17 / IC17L-k0 (SEQ ID NOs: 65, 66) incorporates its respective heavy and light chain genes into an expression vector using ExpiFectamine293 (Thermo Fisher). The antibodies were introduced into Expi293F cells (Thermo Fisher Scientific) using a method similar to that used in Example 4(1), and purified antibodies were prepared.

[0133] The obtained antibodies were mixed in equal volumes (200 μg or 500 μg) in the following combinations, and 1 / 10 volume of 2-MEA (Sigma-Aldrich) prepared to 250 mM using TBS (Takara Bio) was added. The reaction was carried out on a 500 μL or 1000 μL scale. After incubating the reaction solution at 37°C for 90 minutes, the 2-MEA was removed and replaced with D-PBS(-) (Wako Pure Chemical Industries) using PD-Minitrap G-25 or PD-Miditrap G25 (GE Healthcare). The prepared BiAb is shown below.

[0134] [Table 2]

[0135] (4) Cytotoxicity assay (TDCC assay) TDCC activity was evaluated by measuring the electrical resistance generated during cell adhesion to electrodes using xCELLigence (ACEA Biosciences). First, 50 μL / well of the culture medium used for target cell preparation was added to RTCA Resistor plate 96 to correct for background values.

[0136] Next, the target cell suspension prepared as in Example 6(2) was added to 50 μL / well (SK-pca60:3×10 3 cells / well, NCI-H446:1×10 4 cells / well, SKE-4B2:5×10 3 cells / well, hEREG / SK-pca60:5×10 3 Cells were seeded (per well), and after placing the plate in xCELLigence, the cells were cultured overnight at 37°C under 5% CO2 conditions. If the target cells were SK-pca60, 25 μL / well of BiAb2, prepared at various concentrations (0, 0.008, 0.08, 0.8, 8, 80 μg / mL), and 25 μL / well of BiAb1, prepared at various concentrations (0, 0.0008, 0.008, 0.08, 0.8, 8 μg / mL), were added the day after seeding. Subsequently, 50 μL / well of a human PBMC suspension containing 10 times the number of target cells was added, and after placing the plate in xCELLigence, the cells were cultured for 36 hours at 37°C under 5% CO2 conditions, during which the electrical resistance (cell index) was measured at 10-minute intervals. On the other hand, when NCI-H446, SKE-4B2, and hEREG / SK-pca60 were used as target cells, 25 μL / well of BiAb2 prepared to each concentration (0, 0.008, 0.08, 0.8, 8 μg / mL) and 25 μL / well of BiAb1 prepared to each concentration (0, 0.008, 0.08, 0.8, 8 μg / mL) were added the day after seeding the cells. Subsequently, 50 μL / well of a human PBMC suspension containing five times the number of target cells was added, and the plate was placed in xCELLigence and cultured for 120 hours at 37°C under 5% CO2 conditions, during which the electrical resistance (cell index) was measured over time at 10-minute intervals. As an indicator of TDCC activity, the cell proliferation inhibition rate (CGI) was calculated using the following formula 2.

[0137] (Formula 2) TIFF0007862462000004.tif11169

[0138] In equation 2 above, all Cell Indices used were calculated using the Delta Cell Index, where the first resistance measurement point after antibody addition is set to 1. X represents the average Delta Cell Index at the final measurement point of the antibody-free well, Y represents the average Delta Cell Index at the final measurement point of the antibody-added well, A represents the average of the average Delta Cell Index values ​​at the final measurement point when only 0.1 μg / mL of the positive control BiAb (SK-pca60, NCI-H446, hEREG / SK-pca60: GCH065 / CE115HA000, SKE-4B2: EGL / CE115HA000) was added to multiple plates of the same target cells, and B represents the average Delta Cell Index value at the final measurement point when only 0.1 μg / mL of the positive control BiAb (SK-pca60, NCI-H446, hEREG / SK-pca60: GCH065 / CE115, SKE-4B2: EGL / CE115) was added to each plate of the same target cells. The term obtained by dividing B by A was used as a plate-to-plate correction term for TDCC activity against the same target cells.

[0139] First, we evaluated antigen-binding-dependent TDCC activity using GCH065 / CE115HA056 as BiAb1 and GCH065 / CL0028 as BiAb2. SK-pca60 cells, which stably express GPC3, were used as target cells, and GCH065 / CE115HA000, which exhibits TDCC activity on its own, was used as a positive control. As shown in Figure 6 (left), GCH065 / CE115HA000 showed significant TDCC activity from an added dose of 0.001 μg / mL, reaching a plateau above 0.01 μg / mL. On the other hand, BiAb1, GCH065 / CE115HA056, did not show TDCC activity on its own, but it was revealed that it showed TDCC activity upon addition of the GPC3-binding clamping antibody BiAb2. Concentration-dependent TDCC activity of BiAb1 was observed in the presence of 0.01 μg / mL of BiAb2. It was revealed that BiAb1 exhibited TDCC activity nearly equivalent to that of the positive control GCH065 / CE115HA000 when BiAb2 concentrations of 0.1–10 μg / mL were added. On the other hand, as shown on the right side of Figure 6, when the clamping antibody IC17 / CLA0028, which does not have antigen-binding ability, was added as BiAb2, no significant TDCC activity was observed.

[0140] Next, we evaluated the TDCC activity specific to double-positive cells using GPC3 and EREG. GCH065 / CE115HA000 and EGL / CE115HA000 were used as positive controls. As shown in Figure 7, GCH065 / CE115HA000 and EGL / CE115HA000 showed TDCC activity against mono-positive cells NCI-H446 (GPC3) and SKE-4B2 (EREG), respectively, and both antibodies showed TDCC activity against the double-positive cell hEREG / SK-pca60 (EREG / GPC3). When BiAb1 GCH065 / CE115HA056 was applied alone, it showed no TDCC activity against any cell line, but in the presence of 1 μg / mL of BiAb2, EGL / CLA0028, it showed TDCC activity only against the double-positive cell hEREG / SK-pca60. In contrast, under conditions where BiAb2 and IC17 / CLA0028, which lack antigen-binding ability, were present at 1 μg / mL, GCH065 / CE115HA056 did not exhibit TDCC activity in any cell line.

[0141] Based on these results, we succeeded in creating an antibody that specifically exhibits TDCC activity in antigen-double positive cells.

[0142] [Example 7] Acquisition of a clamping antibody that recognizes a complex of adenosine-conjugating antibody and adenosine or adenosine derivative from an antibody library. Clamping antibodies that bind to adenosine-conjugating antibodies and adenosine or adenosine derivative complexes were obtained by phage display from the naive human antibody phage display library and synthetic human antibody phage display library described in International Publication No. 2015 / 156268. Referring to the heavy chain variable region and light chain variable region obtained in International Publication No. 2015 / 083764, SMB0002hH-G1m3 / SMB0002hL-k0a (SEQ ID NOs: 80, 81) were used as the adenosine-conjugating antibody. In other words, the adenosine-binding antibody SMB0002hH-G1m3 / SMB0002hL-k0a captured on magnetic beads exhibits binding activity in the presence of adenosine or an adenosine derivative. A variant of SMB0002hH-G1m3 / SMB0002hL-k0a, SMBh068-G1m3 / SMB0002hL-k0a (SEQ ID NO: 82, 8), has a reduced adenosine-binding activity due to a one-amino acid substitution. 1) Phages that did not show binding activity to SMBh508-G1m3 / SMB0002hL-k0a (SEQ ID NO: 83, 81), SMBh606-G1m3 / SMB0002hL-k0a (SEQ ID NO: 84, 81), SMB0002hH-G1m3 / SMBl234-k0a (SEQ ID NO: 80, 85), and SMB0002hH-G1m3 / SMBl255-k0a (SEQ ID NO: 80, 86) were recovered. In this acquisition method, adenosine-binding antibodies and their variants, which were biotin-labeled using EZ-Link Sulfo-NHS-SS-Biotin (Thermo Fisher Scientific) in a manner known to those skilled in the art, were used as panning antigens.

[0143] Escherichia coli (E. coli) containing phage-displaying phagemide vectors from naive or synthetic human antibody libraries constructed by methods known to the art was infected with the helper phage M13KO7TC described in International Publication No. 2015 / 046554, and phages were recovered from the supernatant after overnight incubation at 30°C. An antibody-displaying phage library solution was prepared by adding 1 / 5 volume of 2.5 M NaCl / 10% PEG to the culture medium of the phage-producing E. coli to precipitate the phage population, which was then diluted with TBS. Next, BSA was added to the phage library solution to a final concentration of 4%. Panning was performed using antigens immobilized on magnetic beads. Sera-Mag SpeedBeads NeutrAvidin-coated (Thermo Fisher Scientific), FG beads NeutrAvidin (Tamagawa Seiki), or Dynabeads MyOne Streptavidin T1 (Thermo Fisher Scientific) were used as magnetic beads.

[0144] In the first panning, negative selection was performed using five variants of the adenosine-binding antibody in which adenosine binding was attenuated (SMBh068-G1m3 / SMB0002hL-k0a, SMBh508-G1m3 / SMB0002hL-k0a, SMBh606-G1m3 / SMB0002hL-k0a, SMB0002hH-G1m3 / SMBl234-k0a, and SMB0002hH-G1m3 / SMBl255-k0a) to eliminate phages that bind to adenosine-binding antibodies in the absence of adenosine. Specifically, a solution containing an equimolar mixture of five variants of SMB0002hH-G1m3 / SMB0002hL-k0a, biotin-labeled using the method described above, was added to BSA-blocked Sera-Mag SpeedBeads NeutrAvidin-coated. A total of 2000 pmol of variants was added to the beads and reacted at room temperature for 15 minutes. After washing the beads three times with TBS, 0.5 mL of a BSA-blocked phage library solution was added and allowed to bind at room temperature for 1 hour. By separating the beads using a magnetic stand, antigens and phages that did not bind to the beads were recovered.

[0145] Subsequently, a selection of antibodies that bind to SMB0002hH-G1m3 / SMB0002hL-k0a was performed under the presence of adenosine. The phage library was contacted with the antigen and adenosine for 15 minutes at room temperature by adding 700 pmol of biotin-labeled SMB0002hH-G1m3 / SMB0002hL-k0a and a final concentration of 500 μM adenosine to the phages recovered by the method described above. Then, contact was maintained at 4°C for 45 minutes. Next, magnetic beads FG beads NeutrAvidin or Dynabeads MyOne Streptavidin T1, blocked with BSA, were added to the mixture of the labeled antigen and adenosine and the phage library, and the complex of antigen, adenosine, and phage was conjugated to the magnetic beads for 30 minutes at 4°C. The beads were washed once with 1 mL of ice-cold adenosine / TBST (500 μM adenosine, 0.1% Tween 20, TBS buffer) and once with ice-cold adenosine / TBS (500 μM adenosine, TBS buffer). Then, DTT solution was added to a final concentration of 25 mM, and after stirring at room temperature for 10 minutes, phages were collected from the separated beads using a magnetic stand. Furthermore, trypsin solution was added to the mixture to a final concentration of 1 mg / mL. After stirring the mixture at room temperature for 15 minutes, phages were collected from the separated beads using a magnetic stand. The collected phages were added to 20 mL of E. coli strain ER2738 in the logarithmic growth phase (OD600 0.4-0.7). The E. coli was slowly cultured with stirring at 37°C for 1 hour to infect the E. coli with the phages. Infected E. coli were seeded onto 225 mm x 225 mm plates. Next, the culture medium of the seeded E. coli was infected with M13KO7TC, and the phage was collected from the supernatant after incubation at 30°C overnight to prepare an antibody-presenting phage library.

[0146] Using the prepared antibody-presenting phage library solution, subsequent panning was performed in the same manner until the fifth panning. However, in negative selection, 800 pmol of antigen was used in the second panning, and 400 pmol in the third and subsequent pannings. Furthermore, for SMB0002hH-G1m3 / SMB0002hL-k0a, which was used as the antigen after negative selection, 300 pmol of antigen was used in the second panning, and 150 pmol in the third and subsequent pannings. In the washing procedure of the beads after binding the SMB0002hH-G1m3 / SMB0002hL-k0a complex with adenosine and phage to the magnetic beads, in the second panning, the beads were washed twice with adenosine / TBST followed by one wash with adenosine / TBS. In the third and subsequent pannings, the beads were washed three times with adenosine / TBST followed by two washes with adenosine / TBS. After panning, the recovered phages were used to infect E. coli, and the E. coli were then seeded onto a plate medium to obtain single colonies of E. coli infected with the phages. A similar panning procedure was performed with the addition of an adenosine derivative.

[0147] [Example 8] Evaluation of the binding activity of adenosine-binding antibodies to adenosine complexes using phage ELISA. Phage-containing culture supernatant was recovered from the single E. coli colonies obtained in Example 7, following a conventional method (Methods Mol. Biol. (2002) 178, 133-145). The antibody gene sequence was determined from the single E. coli colonies using a method known to those skilled in the art. The recovered culture supernatant was ultrafiltered using NucleoFast 96 (MACHEREY-NAGEL). Flow-through was removed by centrifugation (6000×g, 4°C, 40 minutes) of NucleoFast 96 to which 200 μL of culture supernatant had been applied to each well. The NucleoFast 96 to which 200 μL of H2O had been added to each well was washed again by centrifugation (6000×g, 4°C, 20 minutes). Finally, 200 μL of TBS was added, and the phages contained in the supernatant of each well of the NucleoFast 96, which was left to stand at room temperature for 5 minutes, were collected as purified phages. The purified phages, or those to which adenosine / TBS was added, were subjected to ELISA using the following procedure: 384-well Streptavidin-coated (Greiner Bio-One) microplates were coated with 10 μL of TBS containing the biotin-labeled SMB0002hH-G1m3 / SMB0002hL-k0a or five variants at a concentration of 25 pmol / mL for at least 1 hour. After removing biotin-labeled antigens not bound to the plate by washing each well of the plate with TBST, the wells were blocked with 80 μL of 2% skim milk-TBS for at least 1 hour. After removing 2% skim milk / TBS, the plate, to which purified phages were added to each well, was allowed to stand at room temperature for 1 hour to allow the antibodies presented by the phages to conjugate to the biotin-labeled antigens present in each well, either in the presence or absence of a final concentration of 500 μM adenosine. Plates to which HRP-conjugated anti-M13 antibody (GE Healthcare), diluted with adenosine / TBST or TBST, was added to each well washed with adenosine / TBST or TBST were incubated for 1 hour.After washing with adenosine / TBST or TBST, the color reaction of the solution in each well to which TMB single solution (ZYMED) was added was stopped by the addition of sulfuric acid, and the color development was measured by absorbance at 450 nm. A similar screening was also performed using adenosine derivatives. As a result, several antibody-presenting phages were identified that bound to SMB0002hH-G1m3 / SMB0002hL-k0a in the presence of adenosine or adenosine derivatives, but did not bind in the absence of adenosine or adenosine derivatives. Furthermore, several phages were identified that did not bind to a plate immobilized with a mixture of five mutants of SMB0002hH-G1m3 / SMB0002hL-k0a in which the adenosine-binding ability was attenuated in the presence of adenosine or adenosine derivatives. These results demonstrate that antibodies exhibiting binding activity to adenosine-binding antibodies only in the presence of adenosine or an adenosine derivative can be obtained from antibody-presenting phage libraries. Of the 768 clones evaluated by phage ELISA, 40 antibodies exhibiting such binding ability and with duplicate sequences removed were obtained as candidates for clamping antibodies that recognize the complex of adenosine-binding antibody with adenosine or an adenosine derivative.

[0148] [Example 9] Preparation of biotin-labeled SMB0002Fab A gene fragment encoding SMB0002hL-k0aTEVBAP (SEQ ID NO: 87), in which a TEV protease cleavage sequence and an AviTag sequence were linked to the C-terminus of the light chain of SMB0002 via a linker, was introduced into an animal expression vector. The animal expression vectors for SMB0002hH-G1m3 and SMB0002hL-k0aTEVBAP were introduced into Expi293 cells (Life Technologies) using 293Fectin (Life Technologies). At the same time, a gene expressing EBNA1 and a gene expressing biotin ligase (BirA) were introduced, and biotin was added to biotin-label the C-terminus of the light chain of SMB0002hH-G1m3 / SMB0002hL-k0aTEVBAP (SEQ ID NO: 80, SEQ ID NO: 87). Cells into which antibody expression vectors were introduced were cultured at 37°C in 8% CO2, and SMB0002hH-G1m3 / SMB0002hL-k0aTEVBAP, in which the C-terminus of the light chain was biotin-labeled, was secreted into the culture supernatant. The cell culture medium was centrifuged, and the supernatant was filtered using a SARTPORE 2 300 (Sartorius) to obtain the culture supernatant. The culture supernatant was added to a 5 mL size Protein A support column HiTrap MabSelect Sure pcc (GE Healthcare) equilibrated with D-PBS(-), and the antibody was eluted by adding 5 mL / min of 50 mM acetate buffer in 4 column volumes. The antibody was then purified by adding 1.5 M Tris-HCl, pH 7.4 to neutralize the solution. The purified antibody fraction was concentrated by replacing the buffer with 100 mM Tris-HCl, pH 8.0 using a Jumbosep 30K disc (Pall), and then diluted to 2 mg / mL with 100 mM Tris-HCl, pH 8.0. To the diluted full-length antibody, 1 / 2000 of a volume of Lys-C (Roche) was added, and the mixture was allowed to stand at 35°C for 2.5 hours. The reaction was then stopped by adding 1 / 10 of a volume of a solution containing 2 tablets of cOmplet EDTA-free Protease Inhibitor Cocktail (Roche) dissolved in 10 mL of MQ.

[0149] Next, this sample was added to 5 mL of HiTrap Mabselect Sure pcc, which had been equilibrated with D-PBS(-), and 5 mL of HiTrap Mabselect Sure, which was connected in tandem. The flow-through was collected. 1 / 6 volume of 1 M Arginine-HCl was added to the collected sample, and it was concentrated using Jambosep 10 K. This was separated and purified using a gel filtration column, Superdex 75 pg 26 / 60 (GE Healthcare), which had been equilibrated with D-PBS(-). This was concentrated using Amicon-Ultra 15 10K (Merck Millipore), and then D-PBS(-) containing 1.6 times the volume of 8M Urea was added. Subsequently, using Slide-A-Lyzer G2 Dialysis Cassettes 20K (Thermo Fisher Scientific), stepwise dialysis was performed in D-PBS(-) containing sufficient volumes of 6M Urea, 4M Urea, and 2M Urea as extradialysis fluid. After that, dialysis was performed twice with D-PBS(-) to refold the prepared Fab fragment. The Fab solution after refolding was concentrated using Amicon-Ultra 4 10K (Merck Millipore) and filtered using a Millex GV filter unit 0.22 um (Merck Millipore) to obtain purified Fab fragment SMB0002hH-G1m3 / SMB0002hL-k0aTEVBAP with biotin-labeled C-terminus of the light chain. This will be designated as biotin-labeled SMB0002Fab.

[0150] [Example 10] Evaluation of the binding of adenosine-binding antibody to adenosine complex using BLI method for acquired antibodies. The variable region sequences of the heavy and light chains of the adenosynchramping antibody obtained in Example 8 were inserted into animal expression plasmids containing the heavy chain antibody constant region, the light chain kappa constant region sequence, or the light chain lambda constant region sequence, respectively, to create antibody expression vectors. The nucleotide sequences of the obtained expression vectors were determined by methods known to the art.

[0151] Antibody expression vectors were transiently introduced into FreeStyle293F cells (Thermo Fisher Scientific) or Expi293 cells (Thermo Fisher Scientific) to induce antibody expression. From the resulting culture supernatant, antibodies were purified using methods known to the art, with rProtein A Sepharose® Fast Flow (GE Healthcare) or Bravo AssayMAP (Agilent) and Protein A (PA-W) Cartridge (Agilent). The concentration of the purified antibody was calculated by measuring the absorbance at 280 nm using a spectrophotometer and using the extinction coefficient calculated by the PACE method from the obtained values ​​(Protein Science 1995; 4: 2411-2423).

[0152] The binding of each prepared purified antibody and adenosine-binding antibody to the adenosine complex was evaluated using OctetHTX (ForteBio). Specifically, Dip and Read TMStreptavidin (SA) Biosensors (ForteBio) were conjugated to biotin-labeled SMB0002Fab prepared by the method described in Example 9 using TBS or adenosine / TBS, or SMB0002hH-G1m3 / SMB0002hL-k0a biotin-labeled with EZ-Link Sulfo-NHS-SS-Biotin. Subsequently, each purified antibody, prepared at 10 μg / mL with TBS or adenosine / TBS, was reacted, and binding at 30°C was evaluated. Figure 8 shows sensorgrams representing the time-dependent binding amount measured with OctetHTX. SC001 (heavy chain / light chain (sequence number: 88, 89)), SC002 (heavy chain / light chain (sequence number: 90, 91)), SC003 (heavy chain / light chain (sequence number: 123, 124)), SC014 (heavy chain / light chain (sequence number: 92, 93)), SC016 (heavy chain / light chain (sequence number: 94, 95)), SC019 (heavy chain / light chain (sequence number: 96, 97)), SC032 (heavy chain / light chain (sequence number: 125, 126)), SC034 (heavy chain / Light chain (SEQ ID NOs: 127, 128), SC044 (heavy chain / light chain (SEQ ID NOs: 129, 130)), SC045 (heavy chain / light chain (SEQ ID NOs: 131, 132)), and SC048 (heavy chain / light chain (SEQ ID NOs: 133, 134)) showed high binding signals to biotin-labeled SMB0002Fab and biotin-labeled SMB0002hH-G1m3 / SMB0002hL-k0 in the presence of adenosine compared to the absence of adenosine. On the other hand, SC009 (heavy chain / light chain (SEQ ID NOs: 98, 99)) showed similar binding signals to biotin-labeled SMB0002Fab and biotin-labeled SMB0002hH-G1m3 / SMB0002hL-k0 in the presence and absence of adenosine.

[0153] [Example 11] Evaluation of the binding of adenosine-binding antibody to adenosine-adenosine complex using the SPR method. The affinity of the clamping antibody that binds to the adenosine-binding antibody and adenosine complex obtained in Example 10 to the adenosine-binding antibody in the presence of adenosine was analyzed using Biacore T200 (GE Healthcare). The target antibody was captured on a Sensor chip CM4 (GE Healthcare) immobilized with an appropriate amount of protein G (Invitrogen) by amine coupling. Two types of running buffers were used: 20 mM ACES, 150 mM NaCl, 0.05% (w / v) Tween20 or 20 mM ACES, 150 mM NaCl, 0.05% (w / v) Tween20, 500 μM adenosine. Biotin-labeled SMB0002Fab prepared in Example 9 was prepared in the respective running buffers at final concentrations of 250 nM, 62.5 nM, and 15.6 nM. Binding of each antibody to SMB0002Fab was measured using the single-cycle kinetic function of Biacore T200 Control Software (GE Healthcare) at a flow rate of 30 μL / min, with a binding time of 3 minutes and a dissociation time of 5 minutes for each ligand concentration. The sensor tip was then regenerated by injecting 10 mM Glycine-HCl (pH 2.5) and 10 mM NaOH at a flow rate of 30 μL / min for 10 seconds each. All measurements were performed at 25°C. The affinity of each antibody to SMB0002Fab in and without 500 μM adenosine (ADO), as measured by the above method, is shown in Figure 9. SC001, SC002, SC003, SC014, SC016, SC019, SC032, SC044, SC045, and SC048 showed lower KD values ​​for binding to the adenosine-binding antibody SMB0002Fab in the presence of adenosine compared to the absence of adenosine, indicating stronger binding to the adenosine-binding antibody in the presence of adenosine. The binding activity of SC001 and SC019 to the adenosine-binding antibody was low in the absence of adenosine, so their KD values ​​could not be determined.

[0154] [Example 12] Evaluation of the ability of adenosine-binding antibody to enhance the binding activity of adenosine using an adenosyncramping antibody with SPR method. The clamping antibody that binds to the adenosine-conjugating antibody and adenosine complex obtained in Example 10 was evaluated for its adenosine concentration-dependent binding to the adenosine-conjugating antibody using Biacore T200 (GE Healthcare). The target antibody was captured on a Sensor chip CM4 (GE Healthcare) immobilized with an appropriate amount of protein G (Invitrogen) by amine coupling. 20 mM ACES, 150 mM NaCl, and 0.05% (w / v) Tween20 were used as the running buffer. The 500 nM biotin-labeled SMB0002Fab prepared in Example 9 was prepared in a running buffer containing final concentrations of 100 μM, 20 μM, 4 μM, 800 nM, 160 nM, 32 nM, 6.4 nM, and 1.28 nM adenosine. The binding of each antibody to SMB0002Fab was measured when injected at a flow rate of 30 μL / min with a binding time of 3 minutes and a dissociation time of 5 minutes. Subsequently, the sensor tip was regenerated by injecting 10 mM Glycine-HCl (pH 2.5) and 10 mM NaOH at a flow rate of 30 μL / min for 10 seconds each. All measurements were performed at 25°C. Figure 10 shows sensorgrams of the binding of each antibody to 500 nM SMB0002Fab in the presence of each concentration of adenosine measured by the above method. Furthermore, based on the results described above, a steady-state analysis was performed using Biacore T200 Evaluation Software, and the KD values ​​of the binding affinity of each antibody to adenosine in the presence of 500 nM SMB0002Fab were calculated. The results are shown in Table 3.

[0155] [Table 3]

[0156] SC001, SC002, SC003, SC014, SC016, SC019, SC032, SC044, SC045, and SC048 were observed to show an increased binding response to SMB0002Fab in an adenosine concentration-dependent manner. The results obtained in Examples 10, 11, and 12 demonstrated that antibodies with adenosine clamping ability can be obtained using the panning method described in Example 7. These results indicate that the acquisition of clamping antibodies is not limited to the CD3 clamping antibody obtained in Example 3, but that clamping antibodies can also be obtained for adenosine and adenosine-binding antibodies.

[0157] [Example 13] Evaluation of adenosine-dependent cytotoxic activity using adenosyncramping antibodies Adenosine-conjugating antibody SMB0002hH-F760mnP17 / SMB0002hL-k0a (heavy chain / light chain (SEQ ID NOs: 100, 81)), CD3 agonist antibody CE115HA000-F760mnN17 / L0011-k0a (heavy chain / light chain (SEQ ID NOs: 135, 105)), adenosine-cramping antibody SC003H-F760mnP17 / SC003L-SCL3 (heavy chain / light chain (SEQ ID NOs: 136, 124)), GPC3-conjugating antibody GCH065-F760mnN17 / L0011-k0a (heavy chain / light chain (SEQ ID NOs: 104, 105)), negative control antibody An animal expression vector containing IC17HdK-F760mnN17 / IC17L-k0a (heavy chain / light chain (SEQ ID NO: 137, 138)) or IC17HdK-F760mnP17 / IC17L-k0a (heavy chain / light chain (SEQ ID NO: 139, 138)) was introduced into Expi293 cells, and the cells were cultured at 37°C under 8% CO2 to induce antibody secretion into the culture supernatant. The antibodies were then purified using a MonoSpin ProA 96-well plate (GL Science) by a method known to those skilled in the art. Bispecific antibodies consisting of adenosine-conjugated antibody and CD3-conjugated antibody were obtained by the method described in Example 3(3). (SMB0002hH-F760mnP17 / SMB0002hL-k0a / / CE115HA000-F760mnN17 / L0011-k0a), and bispecific antibodies of adenosynchramping antibody and GPC3-binding antibody (SC003H-F760mnP17 / SC003L-SCL3 / / GCH065-F760mnN17 / L0011-k0a), as well as bispecific antibodies of adenosynchramping antibody and KLH-binding antibody as a comparative control. (SC003H-F760mnP17 / SC003L-SCL3 / / IC17HdK-F760mnN17 / IC17L-k0a) and bispecific antibodies (IC17HdK-F760mnP17 / IC17L-k0a / / GCH065-F760mnN17 / L0011-k0a) conjugating KLH and GPC3 were prepared.

[0158] The CD3 agonist activity when two types of prepared bispecific antibodies and adenosine were simultaneously added was evaluated using Jurkat-NFAT reporter cells (NFAT luc2_jurkat cells). Jurkat-NFAT reporter cells are a cell line in which an NFAT response element and luciferase (luc2P) are fused to human acute T-cell leukemia-derived cells expressing CD3, and luciferase is expressed when the downstream signal of CD3 is activated. As target cells, the SK-pca60 cell line, which was established by forcibly expressing human GPC3 in the human liver cancer-derived cell line SK-HEP-1, was used. Target cells and reporter cells were added to each well of a white-bottomed, 96-well assay plate (Costar) at concentrations of 1.25E+04 cells / well and 7.50E+04 cells / well, respectively. A mixture of a bispecific antibody consisting of adenosine-conjugated antibody and CD3-conjugated antibody at a final concentration of 50 nM, and a bispecific antibody consisting of adenosine-clamping antibody and GPC3-conjugated antibody at a final concentration of 100 nM, or a bispecific control antibody, was added to each well. Furthermore, adenosine at final concentrations of 1 μM, 10 μM, 100 μM, and 500 μM was added. After incubation at 37°C for 6 hours in the presence of 5% CO2, luciferase enzyme activity was measured using the Bio-Glo luciferase assay system (Promega) according to the provided protocol. A list of the antibodies used is shown in Table 4.

[0159] [Table 4]

[0160] 2104 EnVision was used for detection. As a result, when a mixture of adenosine-binding antibody and CD3-binding antibody, and adenosine-flamping antibody and GPC3-binding antibody, was added, an increase in the luciferase luminescence signal was observed in an adenosine concentration-dependent manner, and the signal was higher than when a mixture of adenosine-binding antibody and CD3-binding antibody and a control bispecific antibody was added (Figure 11). In other words, it was shown that adenosine clamping brings the adenosine-binding antibody and CD3-binding antibody, and the adenosine-flamping antibody and GPC3-binding antibody, closer together, thereby activating CD3.

[0161] [Example 14] Evaluation of binding affinity between anti-CD3 antibody and CD3εδ by SPR in the presence and absence of clamping antibody. (1) Preparation of immobilized antibodies Expression vectors containing genes encoding CE115HA000-BS03a / GLS3000-k0 (SEQ ID NOs: 140, 20), CE115HA056-BS03a / GLS3000-k0 (SEQ ID NOs: 141, 20), CE115HA146-BS03a / GLS3000-k0 (SEQ ID NOs: 142 / 20), IC17Hdk-BS03a / IC17L-k0 (SEQ ID NOs: 143, 66), and CLA0028VH-BS03b / CLA0028VL-k0C (SEQ ID NOs: 144, 56) were introduced into Expi293F using ExpiFectamine293 (Thermo Fisher Scientific). The culture supernatant was collected on day 5 of culture and prepared using HiTrap MabSelect SuRe according to a method known to the industry. The preparation of the BiAb of anti-CD3 antibody and clamping antibody CLA0028 was carried out using the FAE technique shown in Example 3(3). 500 μg each of anti-CD3 antibody or negative control IC17 antibody and clamping antibody CLA0028 were mixed, and 50 μL of 2-Mercaptoethylamine·HCl (2-MEA, Sigma-Aldrich) prepared to 250 mM with D-PBS(-) (Wako Pure Chemical Industries) was added. The total volume was then made up to 500 μL with D-PBS(-) buffer. After incubating the reaction solution at 37°C for 90 minutes, the 2-MEA was removed and replaced with D-PBS(-) (Wako Pure Chemical Industries) using a PD-minitrap G-25 (GE Healthcare). The concentration of the obtained antibody was calculated using the same method as the protein concentration calculation in Example 3(1).

[0162] (2) Preparation of human CD3εδ heterodimer Human CD3εδ heterodimers (hereinafter referred to as CD3εδ) were prepared by methods known to the art. Specifically, a FLAG tag (DYKDDDDK, SEQ ID NO: 70) and a gene fragment encoding a stop codon were ligated to the gene encoding the extracellular region (positions 1-129) of human CD3ε. A His tag (HHHHHH, SEQ ID NO: 166) and a gene fragment encoding a stop codon were ligated to the gene encoding the extracellular region (positions 1-106) of human CD3δ. A gene fragment encoding soluble human CD3ε (SEQ ID NO: 167) with a FLAG tag attached to the C-terminus of the extracellular region of human CD3ε, and a gene fragment encoding soluble human CD3δ (SEQ ID NO: 168) with a His tag attached to the C-terminus were incorporated into animal cell expression vectors. The two constructed plasmid vectors were introduced into FreeStyle 293F cells (Invitrogen) using 293fectin (Thermo Fisher Scientific). Gene-modified cells were cultured at 37°C and 8% CO2 to allow the target protein to be secreted into the culture supernatant. This cell culture medium was filtered through a 0.22 μm bottle-top filter to obtain the culture supernatant.

[0163] The culture supernatant was diluted 3-fold with distilled water and adsorbed onto Q Sepharose HP (GE Healthcare) equilibrated with 20 mM TrisHCl (pH 7.0), and then eluted with a gradient of salt concentration up to 50% using a buffer of 20 mM TrisHCl, 1 M NaCl (pH 7.0). The fraction containing the target protein was adsorbed onto a HisTrap HP column (GE Healthcare) equilibrated with 20 mM NaPhosphate, 500 mM NaCl, 20 mM Imidazol pH 7.5, and eluted with a gradient of imidazole concentration up to 50% using 20 mM NaPhosphate, 500 mM NaCl, 500 mM Imidazol pH 7.5. The fraction containing the target protein was added to and adsorbed onto an Anti-FLAG M2 column prepared using Anti-FLAG M2 agarose resin (Sigma-Aldrich), and the target protein was eluted with FLAG peptide dissolved in D-PBS(-). This eluate was subjected to gel filtration chromatography using Superdex 26 / 600 (GE healthcare) to remove the aggregates and FLAG peptide, and purified CD3εδ was obtained. The concentration of the obtained purified protein was calculated by the same method as the calculation of the protein concentration in Example 3(1).

[0164] (3) Evaluation of the binding property of the clamping antibody by SPR SuRe protein A (GE Healthcare) prepared at 25 μg / mL with Acetate4.5 (GE Healthcare) was immobilized at approximately 1200 RU per flow cell on the sensor chip CM4 using an amine coupling kit (GE healthcare). The running buffer used was HBS-EP+ (GE Healthcare), and the measurement was carried out at 37 °C. Each antibody was reacted for 60 seconds at a flow rate of 10 μL / min to capture 1000 RU, and the analyte CD3εδ prepared at 0 nM, 4.8 nM, 24 nM, 120 nM, 600 nM, 3000 nM, and 15000 nM was allowed to act for 60 seconds at a flow rate of 30 μL / min to monitor the binding phase, and HBS-EP+ was flowed at a flow rate of 30 μL / min for 120 seconds to monitor the dissociation phase. The sensor chip was regenerated by flowing Glycine1.5 and 25 mM NaOH at a flow rate of 30 μL / min for 30 seconds each. The dissociation constant KD (M) was calculated based on the binding rate constant ka (1 / Ms) and the dissociation rate constant kd (1 / s), which are kinetic parameters calculated from the sensorgrams obtained in the measurement. Biacore T200 Evaluation Software (GE Healthcare) was used for the calculation of each parameter. The obtained KD values are shown in Table 5. The affinity enhancement effect was calculated from the values obtained with the BiAb with the IC17 arm excluding the KD values obtained with the BiAb with the CLA0028 arm. As a result, an enhancement of approximately 30-fold for the KD value of the CE115HA000 arm, approximately 200-fold for CE115HA056, and approximately 70-fold for CE115HA146 was observed.

[0165] [Table 5]

[0166] [Example 15] X-ray crystal structure analysis of the clamping antibody (1) Preparation of antibodies The clamping antibody was prepared by introducing an expression vector containing the gene encoding CLA0028VH-F760mnP17 / CLA0028VL-k0C (SEQ ID NOs: 55, 56) into Expi293F using ExpiFectamine293 (Thermo Fisher Scientific). The culture supernatant was collected on day 5 of culture and prepared using HiTrap MabSelect SuRe according to a method known to the industry. CD3 antibodies fused with the CD3ε epitope peptide were prepared by incorporating the genes encoding the CE115HA146 heavy chain (CE115HAPG13-rabCH1hG1m, SEQ ID NO: 6) and the corresponding light chain (GLS3000-rabk, SEQ ID NO: 7) into an expression vector. These were then transfected into FreeStyle 293F cells (Thermo Fisher Scientific) using the transfection reagent 293fectin Tranfection Reagent (Thermo Fisher Scientific) according to the manufacturer's manual, and the culture medium was cultured for 5 days before being prepared by affinity purification using rProteinA Sepharose Fast Flow resin (GE Healthcare).

[0167] (2) Preparation of CLA0028 Fab fragment The CLA0028VH-F760mnP17 / CLA0028VL-k0C samples were fragmented into Fab and Fc using Lys-C (Roche, 11047825001) at 35°C for 2 hours. The Fab samples were then prepared through column purification using HiTrap SP HP 1 ml (GE Healthcare) + HiTrap MabSelect SuRe 1 ml (GE Healthcare) and SEC purification using HiLoad 16 / 600 Superdex 200 pg (GE Healthcare).

[0168] (3) Preparation of the CLA0028 Fab - CE115HAPG13 Fab complex The obtained CLA0028 Fab sample was added to the CE115HAGP13-rabIgG / GLS3000-rabk sample in a molar ratio with a slight excess of CLA0028 Fab. The CLA0028 Fab-CE115HAGP13-rabIgG / GLS3000-rabk complex sample was prepared by SEC purification using Superdex 200 Increase 10 / 300 GL (GE Healthcare) with 20 mM HEPES pH 7.3 and 100 mM NaCl as buffer. The obtained sample was fragmented into Fab and Fc using Lys-C (Roche, 11047825001) at room temperature overnight. Subsequently, the Fc fragments were removed by passing the fragmented sample through HiTrap MabSelect SuRe 1 ml (GE Healthcare). Furthermore, a CLA0028 Fab - CE115HAPG13 Fab complex sample was prepared by SEC purification using a Superdex 200 Increase 10 / 300 GL (GE Healthcare) with 20 mM HEPES pH 7.3 and 100 mM NaCl as buffers, and a complex sample for crystallization was prepared by ultrafiltration concentration of this sample.

[0169] (4) Preparation of CLA0028 Fab - CE115HAPG13 Fab composite crystal Using the obtained crystallization composite sample, crystallization was performed by the sitting drop vapor diffusion method under 21°C conditions, and crystals suitable for X-ray crystal structure analysis were obtained under the reservoir conditions of Morpheus® (Molecular Dimensions) F10.

[0170] (5) X-ray diffraction data measurement and crystal structure determination from CLA0028 Fab - CE115HAPG13 Fab composite crystal The obtained crystals were immersed in a Morpheus® (Molecular Dimensions) F10 reservoir solution, then frozen in liquid nitrogen, and X-ray diffraction data was measured using a Swiss Light Source X10SA. During the measurement, the crystals were kept frozen by constantly placing them under a 100 K nitrogen stream. The obtained diffraction images were processed using autoPROC (Acta Cryst. D67: 293-302 (2011)), and diffraction intensity data up to a resolution of 2.5 Å was acquired. From the obtained X-ray diffraction intensity data, a known Fab crystal structure was used as a search model, and molecular substitution was performed using Phaser (J. Appl. Cryst. (2007) 40, 658-674) to determine the initial structure. Subsequently, model construction and refinement were repeatedly performed using coot (Acta Cryst. D66: 486-501 (2010)), refmac5 (Acta Cryst. D67: 355-367 (2011)), and phenix.refine (Acta Cryst. D68: 352-367 (2012)), and the final refined coordinates were obtained. The crystallographic statistics are shown in Table 6.

[0171] [Table 6]

[0172] (6) Structure of the CLA0028 Fab-CE115HAPG13 Fab complex As shown in Figure 12, the CLA0028 Fab and CE115HAPG13 Fab form a complex with the N-terminal 7-residue peptide of CD3e sandwiched between them, confirming that a clamping antibody was obtained as intended.

[0173] [Example 16] Evaluation of GPC3 and CLDN6-positive cell-specific TDCC activity using human T cells (1) Preparation of effector cells T cells were isolated from PBMC (Stemcell) using a T-cell isolation kit (Stemcell) according to a method known to those skilled in the art, proliferated with CD3 / CD28 beads (Invitrogen), and stored. In subsequent tests, the isolated T cells that had been stored once were thawed, and the cultured suspension was used as effector cells.

[0174] (2) Preparation of target cells NCI-H446 (GPC3-positive cells), AGS (CLDN6-positive cells), and GM5.1 (GPC3 and CLDN6-positive cells) were used as target cells in subsequent experiments.

[0175] (3) Preparation of BiAb BiAbs shown in Table 7 below were prepared according to the method described above.

[0176]

Table 7

[0177] (4) Cytotoxicity assay (TDCC assay) TDCC activity was evaluated by measuring the electrical resistance value generated with cell adhesion to electrodes using xCELLigence (ACEA Biosciences). First, the medium used for preparing target cells was added to a RTCA Resistor plate 96 to correct the background value. Next, the target cell suspension prepared as in Example 6 was seeded, and after installing the plate in xCELLigence, it was cultured overnight at 37°C and 5% CO2. On the day after seeding the cells, BiAb1 was added to each well to a final concentration of (0, 0.4, 2, 10 nM), and BiAb2 was added to a final concentration of (10 nM). Then, an effector cell suspension containing 5 times the number of target cells was added, and after installing the plate in xCELLigence, it was cultured at 37°C and 5% CO2, and the electrical resistance value (Cell index) during that time was measured over time at 10-minute intervals. As an indicator of TDCC activity, the cell proliferation inhibition rate (CGI) was calculated using the following formula 3.

[0178] (Formula 3) TIFF0007862462000010.tif12170

[0179] In equation 3 above, all Cell Indices used were calculated using the Delta Cell Index, where the first resistance measurement point after antibody addition is set to 1. X represents the average value of the Delta Cell Index at the final measurement point of the antibody-free well, and Y represents the average value of the Delta Cell Index at the final measurement point of the antibody-added well.

[0180] Figure 13 shows the TDCC activity mediated by double antigen binding (GPC3 and CLDN6). We evaluated the TDCC activity specific to double-positive cells using GPC3 and CLDN6. GCH065 / CE115HA000 (anti-GPC3 TRAB) and AE3.20 / CE115HA000 (anti-CLDN6 TRAB) were used as positive controls. As shown in Figure 13, GCH065 / CE115HA000 and AE3.20 / CE115HA000 showed TDCC activity against mono-positive cells NCI-H446 (GPC3) and AGS (CLDN6), respectively, and both antibodies showed TDCC activity against double-positive cells GM5.1 (GPC3 / CLDN6). When BiAb1's GCH065 / CE115HA056 and BiAb2's IC17 / CLA0028 were applied, when BiAb1's IC17 / CE115HA056 and BiAb2's AE3.20 / CLA0028 were applied, and when BiAb1's IC17 / CE115HA056 and BiAb2's GCH065 / CLA0028 were applied, none of the cell lines showed TDCC activity. However, in the presence of BiAb2's AE3.20 / CLA0028, BiAb1's GCH065 / CE115HA056 showed TDCC activity only against GM5.1 in double-positive antigen cells. Based on these results, we succeeded in creating an antibody that specifically exhibits TDCC activity in antigen-double positive cells.

[0181] [Example 17] Evaluation of GPC3 and HER2-positive cell-specific TDCC activity using human T cells (1) Preparation of effector cell solution The isolated T cells prepared using the method described above were used as effector cells in subsequent experiments.

[0182] (2) Preparation of target cells NCI-H446 (GPC3-positive cells), NCI-N87 (HER2-positive cells), and GPC3-expressing NCI-N87 (GPC3, HER2-positive cells) were used as target cells in subsequent experiments.

[0183] (3) Preparation of BiAb Following the method described above, the BiAb shown in Table 8 below was prepared.

[0184] [Table 8]

[0185] (4) Cytotoxicity assay (TDCC assay) A cytotoxicity assay using xCELLigence (ACEA Biosciences) as described in Example 16 was performed. On the day after seeding the cells, BiAb1 was added to each well to reach final concentrations (0, 0.08, 0.4, 2 nM), and BiAb2 was added to reach a final concentration (5 nM). Subsequently, an effector cell suspension containing five times the number of target cells was added, and the plate was placed in the xCELLigence. The cells were then cultured at 37°C under 5% CO2 conditions, and the electrical resistance (cell index) was measured over time at 10-minute intervals.

[0186] Figure 14 shows the TDCC activity mediated by double antigen binding (GPC3 and HER2). We evaluated the TDCC activity specific to double-positive cells using GPC3 and HER2. GCH065 / CE115HA000 (anti-GPC3 TRAB) and HER2 / CE115HA000 (anti-Her2 TRAB) were used as positive controls. As shown in Figure 14, GCH065 / CE115HA000 and HER2 / CE115HA000 showed TDCC activity against mono-positive cells NCI-H446 (GPC3) and NCI-N87 (HER2), respectively, and both antibodies showed TDCC activity against double-positive cells GPC3-expressing NCI-N87 (GPC3 / HER2). When BiAb1's GCH065 / CE115HA056 and BiAb2's IC17 / CLA0028 were applied, and when BiAb1's IC17 / CE115HA056 and BiAb2's HER2 / CLA0028 were applied, neither cell line showed TDCC activity. However, in the presence of BiAb2's HER2 / CLA0028, BiAb1's GCH065 / CE115HA056 showed TDCC activity only against double-positive antigen cells expressing GPC3 (NCI-N87). Based on these results, we succeeded in creating an antibody that specifically exhibits TDCC activity in antigen-double positive cells.

[0187] [Example 18] CD8-specific TRAB using clamping antibodies Bispecific antibodies (T cell-redirecting antibodies) (abbreviated as "TRAB") that exert antitumor effects by recruiting and activating T cells via CD3 have potent antitumor effects, but are also known to induce cytokine release syndrome as a side effect (Non-patent literature: Journal for ImmunoTherapy of Cancer 2018. 6, 56). While the antitumor activity induced by TRAB is mainly carried out by CD8-positive T cells, it has been reported that cytokines causing cytokine release syndrome, such as IL-6 induced by TRAB, are mainly released by CD4-positive T cells (Non-patent literature Immunology. 2017 152(3):425-438). If a TRAB can be created that uses only CD8-positive T cells as effector cells, it is expected to be an ideal drug that maintains the potent antitumor activity of TRAB while suppressing the side effects of cytokine release. Thus, by using a technology that utilizes only double-positive cells (in this example, CD3 / CD8-expressing cells), it is possible to develop a superior TRAB. In the following example, the third antigen recognized by the clamping antibody (second antigen-binding molecule) is not a cancer antigen, but the immune-related molecule CD8. It is expected that only when a CD8-positive T cell bound to this clamping antibody approaches a cancer cell to which an antibody (first antigen-binding molecule) that recognizes the first antigen is bound, the clamping antibody recognizes the antigen-antigen-binding molecule complex formed by the antibody that recognizes the first antigen and CD3, thereby inducing TDCC activity. In this case, the second antigen is a cancer antigen.

[0188] Figure 15 is a schematic diagram illustrating the mechanism of action by which one aspect of the first antigen-binding molecule and another aspect of the second antigen-binding molecule crosslink target cells and effector cells. To confirm this concept, we conducted an experiment detailed below.

[0189] (1) Preparation of effector cells CD8-positive T cells and CD4-positive T cells were isolated from human PBMCs using the EasySep Human CD4+ T cell isolation kit (Stemcell) and the EasySep Human CD8+ T cell isolation kit (Stemcell), and were used as effector cells along with the PBMCs in subsequent assays.

[0190] (2) Preparation of target cells SKpca60, a GPC3-positive cell, was used as the target cell for subsequent experiments.

[0191] (3) Preparation of antibodies Following the method described above, the BiAb shown in Table 9 below was prepared.

[0192] [Table 9]

[0193] (4) Cytotoxicity assay (TDCC assay) A cytotoxicity assay using xCELLigence (ACEA Biosciences) as described in Example 16 was performed. Figure 16 shows the CD8-positive T cell-specific TDCC activity mediated by double antigen binding (GPC3 and CD8).

[0194] Since SKpca60, used as the target cell, is GPC3-positive, GCH065 / CE115HA000 (anti-GPC3 TRAB) was used as the positive control. As shown in Figure 16, the positive control GCH065 / CE115HA000 showed TDCC activity regardless of whether PBMC (containing both CD4-positive T cells and CD8-positive T cells), CD4-positive T cells, or CD8-positive T cells were used as effector cells. On the other hand, GCH065 / CE115HA056 + CD8 / CLA0028 showed TDCC activity when CD8-positive T cells were used as effector cells, but did not show TDCC activity when CD4-positive T cells were used. When PBMC was used as the effector cell, it showed about half the TDCC activity compared to when CD8-positive T cells were used as the effector cell, which was thought to be because the number of CD8-positive T cells contained in PBMC was smaller than when CD8-positive T cells were used as the effector cells. In the negative control groups IC17 / CE115HA056 + CD8 / CLA0028 and GCH065 / CE115HA056 + IC17 / CLA0028, no TDCC activity was observed when either cell was used as an effector cell. Based on these results, we successfully created an antibody that specifically exhibits TDCC activity in CD8-positive T cells.

[0195] [Example 19] The in vivo efficacy of some of the antibodies mentioned above was also evaluated using a cancer model. In vivo efficacy evaluations were performed on representative antibodies shown in Table 2 that demonstrated cytotoxic activity in the in vitro assay described in Example 6. The human cancer cell line hEREG / SK-pca60, expressing GPC3 and EREG, was transplanted into NOD scid mice. T cells, proliferated by culturing human PBMCs in vitro, were then transferred to NOD scid mice in which tumor formation was confirmed. These mice were then treated with antibody administration (referred to as a T-cell transfer model). Specifically, in the efficacy study of the antibody using an hEREG / SK-pca60 T cell transfer model, the following tests were conducted: T cells were cultured in large quantities using PBMCs isolated from blood collected from healthy volunteers and Dynabeads Human T-Activator CD3 / CD28 (Thermo Fisher Scientific). Human cancer cell line hEREG / SK-pca60 1×10 7 Cells were mixed with Matrigel basement membrane matrix (BD) and transplanted subcutaneously into the inguinal region of NOD scid mice (CLEA Japan, female, 7 weeks old). The day of transplantation was designated as day 0. On day 21 post-transplantation, the mice were divided into groups according to tumor size and body weight, and then 0.2 mg / mice of anti-asialoGM1 antibody was administered intraperitoneally. The following day, 3 × 10⁶ T cells obtained by the aforementioned expansion culture were cultured. 7 Cells were transplanted into the peritoneal cavity as individual cells. Approximately 4 hours after T cell transplantation, antibodies were administered intravenously at a dose of 1 mg / kg in the tail vein. Antibody administration was performed twice, on Day 0 and Day 7 (Figure 17).

Claims

1. The method includes identifying the first antibody or antigen-binding fragment of a first antibody as a second antibody or antigen-binding fragment of a second antibody when, using at least one assay selected from SPR, BLI, and ELISA, the binding activity of a first antibody or antigen-binding fragment of a first antibody to a first antigen is detectable under conditions where one antibody or antigen-binding fragment of an antibody is arbitrarily selected from a library of antibodies or antigen-binding fragments of antibodies is present, but undetectable under conditions where the said antibody or antigen-binding fragment is absent. A screening method characterized in that the second antibody or the antigen-binding fragment of the second antibody has multiple antigen specificity and further binds to at least a third antigen.

2. The method includes identifying the first antibody or antigen-binding fragment of an antibody as a second antibody or antigen-binding fragment of a second antibody if the binding activity of the first antibody or antigen-binding fragment of the first antibody to a first antigen is higher under conditions where the first antibody or antigen-binding fragment of an antibody is present compared to conditions where the first antibody or antigen-binding fragment of an antibody is absent, using at least one assay selected from SPR, BLI, and ELISA. A screening method characterized in that the second antibody or the antigen-binding fragment of the second antibody has multiple antigen specificity and further binds to at least a third antigen.

3. (a) A step of immunizing a non-human mammal with an antigen-antibody complex or an antigen-antibody complex consisting of a first antigen and a first antibody or an antigen-binding fragment of the first antibody, and obtaining a first group of antibody-producing cells that produce monoclonal antibodies that bind to the complex, (b) A step of selecting from the first group a second group that produces a monoclonal antibody in which the binding activity to a first antigen that does not form a complex in at least one assay selected from SPR, BLI, and ELISA, or the binding activity to a first antibody or an antigen-binding fragment of the first antibody that does not form a complex, or both, is undetectable. (c) Select from the second group a third group that produces a monoclonal antibody that enhances the binding activity of the first antibody or the antigen-binding fragment of the first antibody to the first antigen in at least one assay selected from SPR, BLI and ELISA, and identify the third group as antibody-producing cells that produce the second antibody, A screening method characterized in that the second antibody has multi-antigen specificity and further binds to at least a third antigen.

4. (a) A step of immunizing a non-human mammal with an antigen-antibody complex or an antigen-antibody complex consisting of a first antigen and a first antibody or an antigen-binding fragment of the first antibody, and obtaining a first group of antibody-producing cells that produce monoclonal antibodies that bind to the complex, (b) A step of selecting from the first group a second group that produces a monoclonal antibody in which the binding activity to a first antigen that does not form a complex in at least one assay selected from SPR, BLI, and ELISA, or the binding activity to a first antibody or an antigen-binding fragment of the first antibody that does not form a complex, or both, is lower than the binding activity to the complex. (c) Select from the second group a third group that produces a monoclonal antibody that enhances the binding activity of the first antibody or the antigen-binding fragment of the first antibody to the first antigen in at least one assay selected from SPR, BLI and ELISA, and identify the third group as antibody-producing cells that produce the second antibody, A screening method characterized in that the second antibody has multi-antigen specificity and further binds to at least a third antigen.

5. (d) A step of culturing antibody-producing cells obtained by a screening method including steps (a) through (c) below; (a) A step of immunizing a non-human mammal with an antigen-antibody complex or an antigen-antibody complex consisting of a first antigen and a first antibody or an antigen-binding fragment of the first antibody, and obtaining a first group of antibody-producing cells that produce monoclonal antibodies that bind to the complex, (b) A step of selecting from the first group a second group that produces a monoclonal antibody in which the binding activity to a first antigen that does not form a complex in at least one assay selected from SPR, BLI, and ELISA, or the binding activity to a first antibody or an antigen-binding fragment of the first antibody that does not form a complex, or both, is undetectable. (c) Selecting from the second group a third group that produces a monoclonal antibody that enhances the binding activity of the first antibody or the antigen-binding fragment of the first antibody to the first antigen in at least one assay selected from SPR, BLI, and ELISA, and identifying the third group as antibody-producing cells that produce the second antibody; (e) A step of obtaining a culture supernatant or cell disruption product from the antibody-producing cell culture, (f) A step of purifying a second antibody from the culture supernatant or cell disrupted product, A method for producing a second antibody, characterized in that the second antibody has multiple antigen specificity and further binds to at least a third antigen.

6. (d) A step of culturing antibody-producing cells obtained by a screening method including steps (a) through (c) below; (a) A step of immunizing a non-human mammal with an antigen-antibody complex or an antigen-antibody complex consisting of a first antigen and a first antibody or an antigen-binding fragment of the first antibody, and obtaining a first group of antibody-producing cells that produce monoclonal antibodies that bind to the complex, (b) A step of selecting from the first group a second group that produces a monoclonal antibody in which the binding activity to a first antigen that does not form a complex in at least one assay selected from SPR, BLI, and ELISA, or the binding activity to a first antibody or an antigen-binding fragment of the first antibody that does not form a complex, or both, is lower than the binding activity to the complex. (c) Selecting from the second group a third group that produces a monoclonal antibody that enhances the binding activity of the first antibody or the antigen-binding fragment of the first antibody to the first antigen in at least one assay selected from SPR, BLI, and ELISA, and identifying the third group as antibody-producing cells that produce the second antibody; (e) A step of obtaining a culture supernatant or cell disruption product from the antibody-producing cell culture, (f) A step of purifying a second antibody from the culture supernatant or cell disrupted product, A method for producing a second antibody, characterized in that the second antibody has multiple antigen specificity and further binds to at least a third antigen.

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