Antibody composition

Abstract

The purpose of the present invention is to provide an antibody composition which exerts an effector function more specifically on target cells co-expressing two different antigens and damages the target cells, and which can maintain a sufficiently high affinity for individual target antigens. The present invention pertains to: an antibody composition which is composed of a first IgG halfmer and a second IgG halfmer and against a first antigen and a second antigen that are different from each other; and a first IgG halfmer and a second IgG halfmer that constitute said antibody composition.

Description

[Technical Field]

[0001] The present invention relates to an antibody composition, a method for producing the same, an IgG halfmer, and a kit containing the IgG halfmer. [Background technology]

[0002] Antibody drugs approved to date are known to have various mechanisms of action (Non-Patent Document 1). Representative examples include neutralizing activity, which inhibits the binding of ligands such as growth factors to their receptors; agonistic activity, which activates the bound receptor; and effector functions possessed by IgG class antibody molecules, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Among these, biomarker analysis of clinical trials of rituximab and trastuzumab suggests that ADCC activity is an important mechanism of action for antibody drugs in clinical settings (Non-Patent Documents 2 and 3).

[0003] An antibody is a tetrameric protein of approximately 150 kDa consisting of four polypeptide chain molecules: two immunoglobulin heavy chain (H chain) molecules and two immunoglobulin light chain (L chain) molecules. As shown in Figure 1, an antibody is divided into a variable region (V) containing complementarity-determining regions (CDRs) that are directly involved in antigen binding and whose amino acid sequences differ among antibody clones, and a constant region containing an Fc region (hereinafter also abbreviated as Fc) that controls the above-mentioned effector functions and blood half-life. The Fc region contains CH1 to CH3 domains and a hinge domain.

[0004] Human antibodies are classified into five classes (IgG, IgA, IgM, IgD, and IgE) that differ in function depending on the sequence of the heavy chain constant region. The human IgG class is further divided into four subclasses (IgG1 to IgG4).

[0005] Among these, antibodies of the IgG1 subclass are known to have the highest ADCC activity and CDC activity (Non-Patent Document 4), and many antibody drugs, including rituximab and trastuzumab, are IgG1.

[0006] On the other hand, the IgG4 subclass has weaker effector functions than other subclasses. It also has a unique hinge region amino acid sequence and weaker interaction between the two CH3 domains than other subclasses, resulting in reversible binding and dissociation of the two H chains in the body, a phenomenon known as "Fab arm exchange" (Non-Patent Documents 5 and 6).

[0007] ADCC activity is a mechanism of cell damage caused by natural killer cells (NK cells) and other cells recognizing and activating the Fc of IgG antibodies bound to membrane antigens on the surface of cancer cells via a type of Fc receptor, FcγRIIIA (also referred to as CD16a), resulting in the expression of molecules such as perforin, granzyme, and Fas (Non-Patent Document 1).

[0008] X-ray crystal structure analysis has revealed the binding mode between CD16a and human IgG1 (Non-Patent Documents 7 and 8). Due to the point symmetry of the Fc structure, there are two CD16a-binding regions on the Fc of IgG1, and in reality, Fc and IgG bind in a numerical ratio (stoichiometry) of 1:1. Furthermore, the two CH2 domains that make up the Fc are in contact with each other at different regions (hereinafter referred to as the CD16a-binding regions).

[0009] Specifically, CD16a interacts with L235, G236, G237, P238, S239, D265, V266, S267, H268, E269, E294, Q295, Y296, N297, S298, T299, R301, N325, A327, I332, and the like on one CH2 domain of IgG1. Meanwhile, CD16a also interacts with L235, G236, G237, K326, A327, L328, P329, A330, and the like on the other CH2 domain of IgG1 (numbers indicate amino acid positions on the CH2 domain according to EU numbering) (Non-Patent Documents 7, 8, 9, 10).

[0010] ADCC activity can be enhanced by artificially modifying the CH2 domain of an antibody drug to increase its binding ability to CD16a. In fact, many examples of enhanced ADCC activity through amino acid modifications in the CH2 domain are known (Non-Patent Document 11). An IgG1 antibody in which different amino acid modifications have been made to the CH2 domain of one IgG1 H chain and the CH2 domain of the other H chain, and the two H chains are linked by a disulfide bond, is known to have high ADCC activity (Patent Document 1).

[0011] It is also known that ADCC activity can be enhanced by modifying the sugar chain of the N-linked complex sugar chain that binds to Fc (Non-Patent Document 12). In particular, the technology for enhancing ADCC activity by sugar chain modification has been applied to approved antibody drugs such as mogamulizumab (Non-Patent Document 13) and obinutuzumab (Non-Patent Document 14).

[0012] Bispecific antibodies are artificially engineered antibody molecules that, unlike natural antibodies, are capable of binding to two different antigens, and numerous molecular forms have been reported (Non-Patent Document 15).

[0013] A schematic diagram of the structure of a bispecific antibody is shown in Figure 2A. Applications of bispecific antibodies to medicine include, for example, binding to both cancer cells and CD3 on the surface of T cells (hereafter referred to as CD3 bispecific antibodies), cross-linking the two and thereby damaging cancer cells, and enhancing drug efficacy by neutralizing two types of functional molecules (Non-Patent Document 15).

[0014] In the treatment of cancer and autoimmune diseases, antibody drugs that exert the effector function of IgG antibodies or the T cell recruiting function of CD3 bispecific antibodies are used to eliminate pathogenic cancer cells and autoreactive lymphocytes.

[0015] However, these pathogenic cells are originally derived from normal cells, and it is generally rare to be able to accurately distinguish them from normal cells using a single surface marker molecule. Therefore, removing pathogenic cells using effector functions often attacks normal cells that express the same antigenic molecules, which can lead to side effects.

[0016] For example, CD20, the target antigen of rituximab, which is used to treat lymphoma and various autoimmune diseases, is expressed on normal B cells, while HER2, the target antigen of trastuzumab, which is used to treat breast cancer, is expressed on cardiomyocytes. Therefore, there are concerns about side effects caused by the destruction of normal cells by these antibody drugs.

[0017] On the other hand, Mazor et al. have reported an example in which a bispecific antibody with weakened affinity for each target antigen exerts a relatively strong effector function against both positive cells (Non-Patent Document 16).

[0018] Furthermore, there has been a report on a combination of antigen-binding molecules comprising a first antigen-binding molecule having a first antigen-binding region that binds to a first antigen and a first polypeptide comprising either a first CH2 or a first CH3, and a second antigen-binding molecule having a second antigen-binding region that binds to a second antigen and a second polypeptide comprising either a second CH2 or a second CH3, wherein the first and second antigen-binding molecules are not covalently bound and tend to form heterodimers rather than homodimers when mixed in liquid (Patent Document 2). [Prior art documents] [Patent documents]

[0019] [Patent Document 1] International Publication No. 2013 / 002362 [Patent Document 2] International Publication No. 2018 / 155611 [Non-patent literature]

[0020] [Non-Patent Document 1] Carter P. Nat Rev Cancer 2001; 1: 118-29 [Non-patent document 2] Cartron G, Dacheux L, Salles G, et al. Blood 2002; 99: 754-8 [Non-patent document 3] Weng WK, Levy R. J Clin Oncol 2003; 21: 3940-7 [Non-patent document 4] Birch, JR, Lennox, ES (Eds.), Monoclonal Antibodies: Principles and Applications. Wiley-Liss, Inc., New York, p. 45.(1995) [Non-Patent Document 5] Aalberse RC and Schuurman J, Immunology 2002; 105: 9-19 [Non-Patent Document 6] Labrijn AF, Nat Biotechnol 2009; 27: 767-71 [Non-Patent Document 7] Sondermann P, Nature 2000; 406: 267-73 [Non-Patent Document 8] Radaev S, J Biol Chem 2001; 276: 16469-77 [Non-Patent Document 9] Ferrara C, Proc Natl Acad Sci 2011; 108; 12669-74 [Non-Patent Document 10] Mizushima T, Genes Cells 2011; 16: 1071-80 [Non-Patent Document 11] Strohl WR, Curr Opin Biotechnol 2009; 20: 685-91 [Non-Patent Document 12] Niwa R, J Pharm Sci 2015; 930-41 [Non-Patent Document 13] Beck A, mAbs 2012; 4: 419-25 [Non-Patent Document 14] Goede V, N Engl J Med 2014; 370: 1101-10<00001…(There seems to be an incomplete tag here. Assuming it's a typo and should be )

非特許文献15

非特許文献16

[0021] Please note that there were some potential incomplete or incorrect tags in the original which were adjusted as best as possible in the translation. If the original has more accurate tags, the translation should be adjusted accordingly.One possible solution to the above-mentioned side effects is a technology using bispecific antibodies, which only exert their effector function after recognizing two different antigens, for more selective elimination of pathogenic cells.

[0022] However, as shown in Figure 2B, when target cells are attacked using the effector function of a conventional bispecific antibody, there is a concern that the bispecific antibody may bind to and attack not only target cells that co-express two antigens (hereinafter referred to as "double-positive cells"), but also cells that express only one target antigen (hereinafter referred to as "single-positive cells"). Furthermore, no antibody technology is known that can specifically exert effector functions and damage double-positive cells, regardless of their affinity for the individual target antigens.

[0023] Therefore, the present inventors have an object to provide an antibody composition that more specifically exerts an effector function against and damages target cells that co-express two different antigens. [Means for solving the problem]

[0024] The present inventors conceived the idea that the above-mentioned problems could be solved by an antibody composition having the following elements [1] to [3] in the constant region of the antibody molecule, which are different from those of a normal human IgG1 antibody, as shown in Figure 3. [1] A mixture of antibody halves (first and second IgG halves) that have antigen-binding sites for a first antigen (antigen molecule X) and a second antigen (antigen molecule Y) that are different from each other. In other words, it is a mixture of "HL halves" in which there is no covalent bond between the H chains due to disulfide bonds between the first IgG halves and the second IgG halves. [2] After binding to the surface of target cells expressing both antigen molecules X and Y (X / Y dual-positive cells), the HL molecules react with each other to form an H2L2 complex similar to that of normal IgG, forming the CD16a-binding domain and inducing antibody activity. [3] To prevent antibody activity against cells expressing only a single antigen molecule, even if HL antibodies against antigen molecule X or Y associate with each other on the cell surface to form homo-aggregates, they do not constitute a CD16a-binding domain.

[0025] Based on the above idea, the present inventors discovered the following (1) to (3) and completed the present invention. (1) Regarding [1] above, as shown in Figure 4, the hinge domain of the IgG half-mer is partially or entirely substituted, deleted, or modified to prevent disulfide bonds from forming between the H chains in the hinge domain. (2) As shown in Figure 5, CD16a contacts two CH2 domains in Fc (CH2-A and CH2-B in Figure 5) at different sites (region 1 of CH2-A and region 2 of CH2-B). Therefore, if Region 2 of CH2-A and Region 1 of CH2-B, which are not used for binding, are "destroyed" by amino acid modification or the like to reduce the binding activity, only Region 2 of CH2-A will be absent in a homozygous aggregate between HL forms having such a modified CH2-A, or only Region 1 of CH2-B will be absent in a homozygous aggregate between HL forms having such a modified CH2-B. Therefore, CD16a cannot bind to these homozygous aggregates with sufficient affinity. On the other hand, a combination of HL forms (hetero-associates) each having such modified CH2-A and modified CH2-B as components can provide an antibody composition that satisfies the above conditions [2] and [3]. (3) Hetero-aggregates formed from IgG half-mers in which specific amino acid alterations have been introduced into the CD16a-binding domain or other Fc domains can provide antibody compositions that exhibit enhanced effector function against X / Y dual-positive cells and / or controlled pharmacokinetics.

[0026] That is, the present invention relates to the following. 1. An antibody composition consisting of a first IgG half-mer and a second IgG half-mer, which is directed against a first antigen and a second antigen that are different from each other, and which is one of the following (1A) to (6A): (1A) The first IgG half-mer and the second IgG half-mer each consist of one immunoglobulin light chain (hereinafter abbreviated as L chain) and one immunoglobulin heavy chain (hereinafter abbreviated as H chain), and the H chain comprises an H chain variable region, a hinge domain, a CH1 domain, a CH2 domain, and a CH3 domain, and has a first Fcγ receptor IIIA (hereinafter abbreviated as CD16a)-binding region and a second CD16a-binding region that are different from each other in the CH2 domain. (2A) The first IgG half-mer and the second IgG half-mer contain amino acid residue substitutions of C226A and C229A, respectively, as represented by the EU index. (3A) The first IgG half-mer comprises an antigen-binding domain that binds to the first antigen, and comprises an amino acid residue substitution of D265A as represented by the EU index in the first CD16a-binding region. (4A) The second IgG half-mer comprises an antigen-binding domain that binds to the second antigen, and comprises an amino acid residue substitution of P329Y as represented by the EU index in the second CD16a-binding region. (5A) the first IgG half-mer and the second IgG half-mer each contain (a) amino acid residue substitutions of S239D and K326T, as represented by the EU index; or The first IgG half-mer and the second IgG half-mer each contain amino acid residue substitutions (b) S239D, S298A, E333A, L242C, and K334C, as represented by the EU index. (6A) At least one of the first IgG half-mer and the second IgG half-mer comprises an amino acid residue substitution in the CH3 domain as a modification that weakens the CH3 domain-CH3 domain interaction compared to that of the IgG1 subclass. 2. The antibody composition according to 1, which specifically exerts an effector function on target cells that co-express the first antigen and the second antigen, thereby damaging them, compared to the effector function on target cells that express only the first antigen and target cells that express only the second antigen. 3. The antibody composition according to 1 or 2 above, wherein the first IgG half-mer and the second IgG half-mer each contain at least one amino acid residue substitution selected from Y349A, L351A, T366A, L368A, D399A, F405A, Y407A, K409A, and K409R, as represented by the EU index, which is a modification that weakens CH3 domain-to-domain interaction compared to that of the IgG1 subclass. 4. The antibody composition according to 3, wherein the first IgG half-mer and the second IgG half-mer each contain the amino acid residue substitution K409R, as represented by the EU index, which is a modification that weakens the interaction between CH3 domains compared to the interaction between CH3 domains of the IgG1 subclass. 5. The antibody composition according to any one of 1 to 4 above, wherein the first IgG half-mer and the second IgG half-mer each contain the amino acid residue substitutions (a) S239D and K326T. 6. The H-chain constant region (hereinafter abbreviated as CH) of the first IgG halfmer comprises the amino acid sequence shown in SEQ ID NO: 248; 6. The antibody composition according to any one of 1 to 5 above, wherein the CH of the second IgG halfmer comprises the amino acid sequence shown in SEQ ID NO:252. 7. The antibody composition according to any one of 1 to 6 above, wherein the proportion of sugar chains in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end is 20% or more of all N-glycoside-linked sugar chains bound to the Fc region in the first IgG half-mer and the second IgG half-mer. 8. The antibody composition according to any one of 1 to 7 above, wherein the immunoglobulin subclass of the first IgG half-mer and the second IgG half-mer is IgG1. 9. A first IgG halfer that associates with a second IgG halfer, wherein the first IgG halfmer and the second IgG halfmer are any of the following (1B) to (7B): (1B) The first IgG half-mer and the second IgG half-mer form an antibody composition against a first antigen and a second antigen that are different from each other. (2B) The first IgG half-mer and the second IgG half-mer each consist of one L chain and one H chain, and the H chain includes an H chain variable region, a hinge domain, a CH1 domain, a CH2 domain, and a CH3 domain, and has a first CD16a-binding region and a second CD16a-binding region that are different from each other in the CH2 domain. (3B) The first IgG half-mer and the second IgG half-mer contain amino acid residue substitutions of C226A and C229A, respectively, as represented by the EU index. (4B) The first IgG half-mer comprises an antigen-binding domain that binds to the first antigen, and comprises an amino acid residue substitution of D265A as represented by the EU index in the first CD16a-binding region. (5B) The second IgG half-mer comprises an antigen-binding domain that binds to the second antigen, and comprises an amino acid residue substitution of P329Y as represented by the EU index in the second CD16a-binding region. (6B) the first IgG half-mer and the second IgG half-mer each contain (a) amino acid residue substitutions S239D and K326T as represented by the EU index, or The first IgG half-mer and the second IgG half-mer each contain amino acid residue substitutions (b) S239D, S298A, E333A, L242C, and K334C, as represented by the EU index. (7B) At least one of the first IgG half-mer and the second IgG half-mer comprises an amino acid residue substitution in the CH3 domain as a modification that weakens the CH3 domain-CH3 domain interaction compared to that of the IgG1 subclass. 10. A second IgG halfer that associates with a first IgG halfer, wherein the first IgG halfer and the second IgG halfmer are any of (1C) to (7C) below. (1C) The first IgG half-mer and the second IgG half-mer form an antibody composition against a first antigen and a second antigen that are different from each other. (2C) The first IgG half-mer and the second IgG half-mer each consist of one L chain and one H chain, and the H chain includes an H chain variable region, a hinge domain, a CH1 domain, a CH2 domain, and a CH3 domain, and has a first CD16a-binding region and a second CD16a-binding region in the CH2 domain that are different from each other. (3C) The first IgG half-mer and the second IgG half-mer contain amino acid residue substitutions of C226A and C229A, respectively, as represented by the EU index. (4C) The first IgG half-mer comprises an antigen-binding domain that binds to the first antigen, and comprises an amino acid residue substitution of D265A as represented by the EU index in the first CD16a-binding region. (5C) The second IgG half-mer comprises an antigen-binding domain that binds to the second antigen, and comprises an amino acid residue substitution of P329Y as represented by the EU index in the second CD16a-binding region. (6C) the first IgG half-mer and the second IgG half-mer each contain (a) amino acid residue substitutions S239D and K326T as represented by the EU index, or The first IgG half-mer and the second IgG half-mer each contain amino acid residue substitutions (b) S239D, S298A, E333A, L242C, and K334C, as represented by the EU index. (7C) At least one of the first IgG half-mer and the second IgG half-mer comprises an amino acid residue substitution in the CH3 domain as a modification that weakens the CH3 domain-to-CH3 domain interaction compared to that of the IgG1 subclass. 11. DNA encoding the amino acid sequence of a) or b) below. a) The amino acid sequence of the first IgG half-mer described in 9 above b) The amino acid sequence of the second IgG half-mer according to 10 above. 12. A recombinant vector comprising at least one of a DNA encoding the amino acid sequence of a) described in 11 above and a DNA encoding the amino acid sequence of b). 13. A transformant into which the recombinant vector according to 12 above has been introduced. 14. A kit comprising the first IgG half-mer described in 9 above and the second IgG half-mer described in 10 above. 15. A method for inducing effector function specifically in target cells co-expressing the first antigen and the second antigen, using the antibody composition described in any one of 1 to 8 above, by comparing the effector function in target cells expressing only the first antigen and target cells expressing only the second antigen. 16. A pharmaceutical composition comprising the antibody composition described in any one of 1 to 8 above. 17. The pharmaceutical composition according to 16 above, for use in treating cancer, autoimmune diseases or allergic diseases. 18. Use of the antibody composition according to any one of 1 to 8 above for the manufacture of a pharmaceutical composition for treating cancer, an autoimmune disease, or an allergic disease. 19. Use of the antibody composition according to any one of 1 to 8 above for the treatment of cancer, an autoimmune disease, or an allergic disease. 20. The antibody composition according to any one of 1 to 8 above, for use in treating cancer, an autoimmune disease, or an allergic disease. 21. A method for treating cancer, an autoimmune disease, or an allergic disease, which comprises administering to a subject an effective amount of the antibody composition described in any one of 1 to 8 above. 22. A first IgG half-mer to be used in combination with a second IgG half-mer, wherein the first IgG half-mer and the second IgG half-mer are any of the following (1B) to (7B): (1B) The first IgG half-mer and the second IgG half-mer form an antibody composition against a first antigen and a second antigen that are different from each other. (2B) The first IgG half-mer and the second IgG half-mer each consist of one L chain and one H chain, and the H chain includes an H chain variable region, a hinge domain, a CH1 domain, a CH2 domain, and a CH3 domain, and has a first CD16a-binding region and a second CD16a-binding region that are different from each other in the CH2 domain. (3B) The first IgG half-mer and the second IgG half-mer contain amino acid residue substitutions of C226A and C229A, respectively, as represented by the EU index. (4B) The first IgG half-mer comprises an antigen-binding domain that binds to the first antigen, and comprises an amino acid residue substitution of D265A as represented by the EU index in the first CD16a-binding region. (5B) The second IgG half-mer comprises an antigen-binding domain that binds to the second antigen, and comprises an amino acid residue substitution of P329Y as represented by the EU index in the second CD16a-binding region. (6B) the first IgG half-mer and the second IgG half-mer each contain (a) amino acid residue substitutions S239D and K326T as represented by the EU index, or The first IgG half-mer and the second IgG half-mer each contain amino acid residue substitutions (b) S239D, S298A, E333A, L242C, and K334C, as represented by the EU index. (7B) At least one of the first IgG half-mer and the second IgG half-mer comprises an amino acid residue substitution in the CH3 domain as a modification that weakens the CH3 domain-CH3 domain interaction compared to that of the IgG1 subclass. 23. A second IgG half-mer to be used in combination with a first IgG half-mer, wherein the first IgG half-mer and the second IgG half-mer are any of (1C) to (7C) below. (1C) The first IgG half-mer and the second IgG half-mer form an antibody composition against a first antigen and a second antigen that are different from each other. (2C) The first IgG half-mer and the second IgG half-mer each consist of one L chain and one H chain, and the H chain includes an H chain variable region, a hinge domain, a CH1 domain, a CH2 domain, and a CH3 domain, and has a first CD16a-binding region and a second CD16a-binding region in the CH2 domain that are different from each other. (3C) The first IgG half-mer and the second IgG half-mer contain amino acid residue substitutions of C226A and C229A, respectively, as represented by the EU index. (4C) The first IgG half-mer comprises an antigen-binding domain that binds to the first antigen, and comprises an amino acid residue substitution of D265A as represented by the EU index in the first CD16a-binding region. (5C) The second IgG half-mer comprises an antigen-binding domain that binds to the second antigen, and comprises an amino acid residue substitution of P329Y as represented by the EU index in the second CD16a-binding region. (6C) the first IgG half-mer and the second IgG half-mer each contain (a) amino acid residue substitutions S239D and K326T as represented by the EU index, or The first IgG half-mer and the second IgG half-mer each contain amino acid residue substitutions (b) S239D, S298A, E333A, L242C, and K334C, as represented by the EU index. (7C) At least one of the first IgG half-mer and the second IgG half-mer comprises an amino acid residue substitution in the CH3 domain as a modification that weakens the CH3 domain-to-CH3 domain interaction compared to that of the IgG1 subclass. 24. A first or second IgG halfer contained in the antibody composition according to any one of 1 to 8 above. 25. A method for specifically inducing effector function in positive cells expressing both the first and second antigens, using the IgG halfmer according to 9, 10 or 24 above.

[0027] 26. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CADM1 and the second antigen is CCR4. 27. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is EGFR and the second antigen is HER2. 28. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD52 and the second antigen is CD70. 29. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD2 and the second antigen is CD70. 30. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD19 and the second antigen is CD70. 31. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD2 and the second antigen is CD40 ligand. 32. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is PD-L1 and the second antigen is one selected from CD19, CD30, CCR4, CD20, CD22, and CD79b. 33. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD8 and the second antigen is CCR4. 34. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CTLA-4 and the second antigen is one selected from CD4, CCR4 and GITR. 35. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is TIGIT and the second antigen is one selected from CD4, CCR4 and GITR. 36. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is PD-1 and the second antigen is one selected from CD4, CCR4, and GITR. 37. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is OX40 and the second antigen is one selected from CD127, CD26, CD70, and CD15s. 38. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is 4-1BB and the second antigen is one selected from CD127, CD26, CD70, and CD15s. 39. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is GITR and the second antigen is one selected from CD127, CD26, CD70, and CD15s. 40. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD40 ligand and the second antigen is one selected from CD127, CD26, CD70 and CD15s. 41. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD4 and the second antigen is CD69.

[0028] 42. A pharmaceutical composition for use in treating ATL, comprising the antibody composition according to 26 above. 43. A pharmaceutical composition for use in treating at least one of gastric cancer and breast cancer, comprising the antibody composition according to 27 above. 44. A pharmaceutical composition for use in treating Sjogren's syndrome, comprising the antibody composition according to 31 above. 45. A pharmaceutical composition for use in treating at least one of lymphoma and leukemia, comprising the antibody composition according to 32 above. 46. ​​A pharmaceutical composition for use in treating cancer, comprising the antibody composition according to any one of 33 to 36 above. 47. A pharmaceutical composition for use in treating an autoimmune disease, comprising the antibody composition according to any one of 37 to 40 above. 48. A pharmaceutical composition for use in treating ANCA-associated glomerulonephritis, comprising the antibody composition according to 41 above.

[0029] 49. Use of the antibody composition according to 26 above for the manufacture of a pharmaceutical composition for the treatment of ATL. 50. Use of the antibody composition according to 27 above for the manufacture of a pharmaceutical composition for treating at least one of gastric cancer and breast cancer. 51. Use of the antibody composition according to 31 above for the manufacture of a pharmaceutical composition for treating Sjogren's syndrome. 52. Use of the antibody composition according to 32 above for the manufacture of a pharmaceutical composition for treating at least one of lymphoma and leukemia. 53. Use of the antibody composition according to any one of 33 to 36 above for the manufacture of a pharmaceutical composition for treating cancer. 54. Use of the antibody composition according to any one of 37 to 40 above for the manufacture of a pharmaceutical composition for treating an autoimmune disease. 55. Use of the antibody composition according to 41 above for the manufacture of a pharmaceutical composition for treating ANCA-associated glomerulonephritis.

[0030] 56. Use of the antibody composition according to 26 for the treatment of ATL. 57. Use of the antibody composition according to 27 above for the treatment of at least one of gastric cancer and breast cancer. 58. Use of the antibody composition according to 31 above for the treatment of Sjogren's syndrome. 59. Use of the antibody composition according to 32 for the treatment of at least one of lymphoma and leukemia. 60. Use of the antibody composition according to any one of 33 to 36 for the treatment of cancer. 61. Use of the antibody composition according to any one of 37 to 40 for the treatment of an autoimmune disease. 62. Use of the antibody composition according to 41 above for the treatment of ANCA-associated glomerulonephritis.

[0031] 63. The antibody composition according to 26 above for use in treating ATL. 64. The antibody composition according to 27 above, for use in treating at least one of gastric cancer and breast cancer. 65. The antibody composition according to 31 above for use in treating Sjogren's syndrome. 66. The antibody composition according to 32 above for use in treating at least one of lymphoma and leukemia. 67. The antibody composition according to any one of 33 to 36, for use in treating cancer. 68. The antibody composition according to any one of 37 to 40, for use in treating an autoimmune disease. 69. The antibody composition according to 41 above for use in treating ANCA-associated glomerulonephritis.

[0032] 70. A method for treating ATL, comprising administering to a subject an effective amount of the antibody composition according to 26 above. 71. A method for treating at least one of gastric cancer and breast cancer, which comprises administering to a subject an effective amount of the antibody composition according to 27 above. 72. A method for treating Sjogren's syndrome, comprising administering an effective amount of the antibody composition according to 31 above to a subject. 73. A method for treating at least one of lymphoma and leukemia, which comprises administering to a subject an effective amount of the antibody composition according to 32 above. 74. A method for treating cancer, comprising administering an effective amount of the antibody composition according to any one of 33 to 36 above to a subject. 75. A method for treating an autoimmune disease, comprising administering an effective amount of the antibody composition according to any one of 37 to 40 above to a subject. 76. A method for treating ANCA-associated glomerulonephritis, comprising administering to a subject an effective amount of the antibody composition according to 41 above.

[0033] 77. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is PD-1 and the second antigen is CD3. 78. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is PD-1 and the second antigen is CD4. 79. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is integrin α4β7, and the second antigen is one selected from CCR6, CXCR3, CD161, and CD127. 80. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is integrin α4β7 and the second antigen is CD40 ligand. 81. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is integrin α4β1 and the second antigen is CD40 ligand. 82. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CXCR5 and the second antigen is CD127. 83. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is a TPO receptor (c-mpl) and the second antigen is CD34 or CD123. 84. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CXCR3 and the second antigen is CD3. 85. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CXCR3 and the second antigen is CD127 or CD40 ligand. 86. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CCR4 and the second antigen is CD127 or CD40 ligand. 87. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CCR6 and the second antigen is CD127 or CD40 ligand. 88. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CRTH2 and the second antigen is CD127 or CD40 ligand. 89. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CRTH2 and the second antigen is CCR4. 90. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CRTH2 and the second antigen is ST2. 91. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CRTH2 and the second antigen is CCR6 or CCR3. 92. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD207 and the second antigen is CD11b or CD1a. 93. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD123 and the second antigen is HLA-DR or ASCT2. 94. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CSF1R and the second antigen is CD14. 95. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CSF1R and the second antigen is CD33. 96. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD19 and the second antigen is CD38. 97. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD3 and the second antigen is IL-23R. 98. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD3 and the second antigen is CX3CR1. 99. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CXCR4 and the second antigen is Type 1 collagen. 100. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CXCR4 and the second antigen is CD14. 101. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CXCR4 and the second antigen is CD16. 102. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CLEC10A and the second antigen is CD14 or CD16. 103. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD70 and the second antigen is CD38. 104. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD70 and the second antigen is one selected from CD4, CD127 and TIM-1. 105. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD4 and the second antigen is TIM-1. 106. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD70 and the second antigen is CD52. 107. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CB1 and the second antigen is AT1R. 108. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD4 or PD-1, and the second antigen is CD153. 109. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is FcεRI and the second antigen is CD34 or C-KIT. 110. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD34 and the second antigen is CD203 or MRGPRX2. 111. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD52 and the second antigen is CD127. 112. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD69 and the second antigen is CD21. 113. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD106 and the second antigen is one selected from CD11c, CD19, CD21 and CD72. 114. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is 4-1BB and the second antigen is one selected from CD11c, CD19, CD21 and CD72. 115. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is BLT1 and the second antigen is CD49b. 116. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD226 and the second antigen is CD8. 117. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CXCR3 and the second antigen is one selected from CD8, CD49a, IL-15R and NKG2D. 118. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD49a and the second antigen is one selected from CD8, IL-15R and NKG2D. 119. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is IL-15R and the second antigen is CD8 or NKG2D. 120. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is NKG2D and the second antigen is CD8. 121. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD177 and the second antigen is PR3. 122. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD4 and the second antigen is CD127. 123. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD40 ligand and the second antigen is IL-6. 124. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is IL-17R and the second antigen is membrane-type TNF. 125. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is IL-23R and the second antigen is CCR6.

[0034] 126. Use of the antibody composition according to 77 above for the manufacture of a pharmaceutical composition for treating rheumatoid arthritis. 127. Use of the antibody composition according to 78 for the manufacture of a pharmaceutical composition for the treatment of celiac disease. 128. Use of the antibody composition according to 79 above for the manufacture of a pharmaceutical composition for treating inflammatory bowel disease. 129. Use of the antibody composition according to item 80 for the manufacture of a pharmaceutical composition for treating at least one of multiple sclerosis and inflammatory bowel disease. 130. Use of the antibody composition according to 81 for the manufacture of a pharmaceutical composition for treating at least one of multiple sclerosis and inflammatory bowel disease. 131. Use of the antibody composition according to 82 for the manufacture of a pharmaceutical composition for the treatment of allergic diseases. 132. Use of the antibody composition according to 83 above for the manufacture of a pharmaceutical composition for the treatment of primary myelofibrosis. 133. Use of the antibody composition according to 84 above for the manufacture of a pharmaceutical composition for treating at least one disease selected from autoimmune diseases, arteriosclerosis and ischemic heart disease. 134. Use of the antibody composition according to 85 above for the manufacture of a pharmaceutical composition for the treatment of systemic lupus erythematosus. 135. Use of the antibody composition according to 86 above for the manufacture of a pharmaceutical composition for the treatment of allergic diseases. 136. Use of the antibody composition according to 87 above for the manufacture of a pharmaceutical composition for the treatment of psoriasis. 137. Use of the antibody composition according to 88 for the manufacture of a pharmaceutical composition for the treatment of allergic diseases. 138. Use of the antibody composition according to 89 above for the manufacture of a pharmaceutical composition for treating at least one disease selected from asthma, eosinophilic sinusitis, and atopic dermatitis. 139. Use of the antibody composition according to 90 above for the manufacture of a pharmaceutical composition for treating at least one of asthma and eosinophilic sinusitis. 140. Use of the antibody composition according to 91 above for the manufacture of a pharmaceutical composition for the treatment of allergic diseases. 141. Use of the antibody composition according to 92 above for the manufacture of a pharmaceutical composition for the treatment of Langerhans Cell Histiocytosis (LCH). 142. Use of the antibody composition according to 93 above for the manufacture of a pharmaceutical composition for the treatment of systemic lupus erythematosus. 143. Use of the antibody composition according to 94 above for the manufacture of a pharmaceutical composition for the treatment of idiopathic pulmonary fibrosis. 144. Use of the antibody composition according to 95 above for the manufacture of a pharmaceutical composition for the treatment of cancer. 145. Use of the antibody composition according to 96 or 97 above for the manufacture of a pharmaceutical composition for the treatment of systemic lupus erythematosus. 146. Use of the antibody composition according to 98 above for the manufacture of a pharmaceutical composition for treating at least one disease selected from autoimmune diseases, arteriosclerosis and ischemic heart disease. 147. Use of the antibody composition according to 99 above for the manufacture of a pharmaceutical composition for treating at least one fibrotic disease selected from various fibrotic diseases such as idiopathic pulmonary fibrosis, systemic sclerosis, and liver cirrhosis. 148. Use of the antibody composition according to 100 for the manufacture of a pharmaceutical composition for the treatment of idiopathic pulmonary fibrosis. 149. Use of the antibody composition according to 101 for the manufacture of a pharmaceutical composition for treating at least one disease selected from neutrophilic asthma, chronic obstructive pulmonary disease, and Alzheimer's disease. 150. Use of the antibody composition according to 102 for the manufacture of a pharmaceutical composition for treating at least one disease selected from Crohn's disease, rheumatoid arthritis, and asthma. 151. Use of the antibody composition according to 103 for the manufacture of a pharmaceutical composition for treating at least one of multiple sclerosis and neuromyelitis optica. 152. Use of the antibody composition according to 104 or 105 for the manufacture of a pharmaceutical composition for treating systemic lupus erythematosus. 153. Use of the antibody composition according to 106 for the manufacture of a pharmaceutical composition for the treatment of multiple sclerosis. 154. Use of the antibody composition according to 107 for the manufacture of a pharmaceutical composition for treating liver cirrhosis. 155. Use of the antibody composition according to 108 for the manufacture of a pharmaceutical composition for the treatment of systemic lupus erythematosus. 156. Use of the antibody composition according to 109 for the manufacture of a pharmaceutical composition for treating mast cell activation syndromes (MCAS), including at least one selected from urticaria, food allergies, and mastocytosis. 157. Use of the antibody composition according to 110 for the manufacture of a pharmaceutical composition for treating mast cell activation syndrome (MCAS), including at least one selected from urticaria, food allergy, and mastocytosis. 158. Use of the antibody composition according to 111 for the manufacture of a pharmaceutical composition for the treatment of multiple sclerosis. 159. Use of the antibody composition according to 112 above for the manufacture of a pharmaceutical composition for treating at least one disease selected from multiple sclerosis, type 1 diabetes, and rheumatoid arthritis. 160. Use of the antibody composition according to 113 for the manufacture of a pharmaceutical composition for treating at least one disease selected from secondary progressive multiple sclerosis (SPMS), rheumatoid arthritis in which anti-TNF treatment is ineffective, transplantation, and systemic lupus erythematosus. 161. Use of the antibody composition according to 114 for the manufacture of a pharmaceutical composition for treating at least one disease selected from secondary progressive multiple sclerosis (SPMS), rheumatoid arthritis ineffective against anti-TNF treatment, transplantation, and systemic lupus erythematosus. 162. Use of the antibody composition according to 115 above for the manufacture of a pharmaceutical composition for the treatment of asthma. 163. Use of the antibody composition according to 116 for the manufacture of a pharmaceutical composition for the treatment of systemic sclerosis. 164. Use of the antibody composition according to 117 above for the manufacture of a pharmaceutical composition for treating at least one of vitiligo vulgaris and psoriasis. 165. Use of the antibody composition according to 118 for the manufacture of a pharmaceutical composition for treating at least one of vitiligo vulgaris and psoriasis. 166. Use of the antibody composition according to 119 above for the manufacture of a pharmaceutical composition for treating at least one of vitiligo vulgaris and psoriasis. 167. Use of the antibody composition according to 120 for the manufacture of a pharmaceutical composition for treating at least one of vitiligo vulgaris and psoriasis. 168. Use of the antibody composition according to 121 above for the manufacture of a pharmaceutical composition for treating at least one of ANCA (Antineutrophil Cytoplasmic Antibody)-associated vasculitis and systemic lupus erythematosus. 169. Use of the antibody composition according to 122 for the manufacture of a pharmaceutical composition for the treatment of a T cell-dependent immune-related disease. 170. Use of the antibody composition according to 123 for the manufacture of a pharmaceutical composition for treating an immune-related disease. 171. Use of the antibody composition according to 124 for the manufacture of a pharmaceutical composition for the treatment of psoriatic arthritis. 172. Use of the antibody composition according to 125 above for the manufacture of a pharmaceutical composition for treating autoimmune diseases including at least one of Sjogren's syndrome and psoriasis.

[0035] 173. Use of the antibody composition according to 77 above for the treatment of rheumatoid arthritis. 174. Use of the antibody composition according to 78 for the treatment of celiac disease. 175. Use of the antibody composition according to 79 for the treatment of inflammatory bowel disease. 176. Use of the antibody composition according to item 80 for the treatment of at least one of multiple sclerosis and inflammatory bowel disease. 177. Use of the antibody composition according to 81 for the treatment of at least one of multiple sclerosis and inflammatory bowel disease. 178. Use of the antibody composition according to 82 for the treatment of allergic diseases. 179. Use of the antibody composition according to 83 for the treatment of primary myelofibrosis. 180. Use of the antibody composition according to 84 above for the treatment of at least one disease selected from autoimmune diseases, arteriosclerosis and ischemic heart disease. 181. Use of the antibody composition according to 85 above for the treatment of systemic lupus erythematosus. 182. Use of the antibody composition according to 86 for the treatment of allergic diseases. 183. Use of the antibody composition according to 87 for the treatment of psoriasis. 184. Use of the antibody composition according to 88 for the treatment of allergic diseases. 185. Use of the antibody composition according to 89 above for the treatment of at least one selected from asthma, eosinophilic sinusitis, and atopic dermatitis. 186. Use of the antibody composition according to 90 for the treatment of at least one of asthma and eosinophilic sinusitis. 187. Use of the antibody composition according to 91 for the treatment of allergic diseases. 188. Use of the antibody composition according to 92 for the treatment of Langerhans cell histiocytosis (LCH). 189. Use of the antibody composition according to 93 for the treatment of systemic lupus erythematosus. 190. Use of the antibody composition according to 94 for the treatment of idiopathic pulmonary fibrosis. 191. Use of the antibody composition according to 95 above for the treatment of cancer. 192. Use of the antibody composition according to 96 or 97 for the treatment of systemic lupus erythematosus. 193. Use of the antibody composition according to 98 for the treatment of at least one disease selected from autoimmune diseases, arteriosclerosis and ischemic heart disease. 194. Use of the antibody composition according to 99 above for the treatment of fibrotic diseases including at least one selected from idiopathic pulmonary fibrosis, systemic sclerosis, and liver cirrhosis. 195. Use of the antibody composition according to 100 for the treatment of idiopathic pulmonary fibrosis. 196. Use of the antibody composition according to 101 for the treatment of at least one selected from neutrophilic asthma, chronic obstructive pulmonary disease and Alzheimer's disease. 197. Use of the antibody composition according to 102 for the treatment of at least one selected from Crohn's disease, rheumatoid arthritis and asthma. 198. Use of the antibody composition according to 103 for the treatment of at least one of multiple sclerosis and neuromyelitis optica. 199. Use of the antibody composition according to 104 or 105 for the treatment of systemic lupus erythematosus. 200. Use of the antibody composition according to 106 for the treatment of multiple sclerosis. 201. Use of the antibody composition according to 107 for the treatment of liver cirrhosis. 202. Use of the antibody composition according to 108 for the treatment of systemic lupus erythematosus. 203. Use of the antibody composition according to 109 for the treatment of mast cell activation syndrome (MCAS), including at least one selected from urticaria, food allergy, and mastocytosis. 204. Use of the antibody composition according to 110 for the treatment of mast cell activation syndrome (MCAS), including at least one selected from urticaria, food allergy, and mastocytosis. 205. Use of the antibody composition according to 111 for the treatment of multiple sclerosis. 206. Use of the antibody composition according to 112 for the treatment of at least one selected from multiple sclerosis, type 1 diabetes and rheumatoid arthritis. 207. Use of the antibody composition according to 113 for the treatment of at least one selected from secondary progressive multiple sclerosis (SPMS), rheumatoid arthritis ineffective against anti-TNF treatment, transplantation, and systemic lupus erythematosus. 208. Use of the antibody composition according to 114 for the treatment of at least one selected from secondary progressive multiple sclerosis (SPMS), rheumatoid arthritis in which anti-TNF treatment is ineffective, transplantation, and systemic lupus erythematosus. 209. Use of the antibody composition according to 115 for the treatment of asthma. 210. Use of the antibody composition according to 116 for the treatment of systemic sclerosis. 211. Use of the antibody composition according to 117 for the treatment of at least one of vitiligo vulgaris and psoriasis. 212. Use of the antibody composition according to 118 for the treatment of at least one of vitiligo vulgaris and psoriasis. 213. Use of the antibody composition according to 119 for the treatment of at least one of vitiligo vulgaris and psoriasis. 214. Use of the antibody composition according to 120 for the treatment of at least one of vitiligo vulgaris and psoriasis. 215. Use of the antibody composition according to 121 for the treatment of at least one of ANCA-associated vasculitis and systemic lupus erythematosus. 216. Use of the antibody composition according to 122 for the treatment of a T cell-dependent immune-related disease. 217. Use of the antibody composition according to 123 for the treatment of immune-related diseases. 218. Use of the antibody composition according to 124 for the treatment of psoriatic arthritis. 219. Use of the antibody composition according to 125 for the treatment of autoimmune diseases including at least one of Sjogren's syndrome and psoriasis.

[0036] 220. The antibody composition according to 77 above for use in treating rheumatoid arthritis. 221. The antibody composition according to 78 for use in treating celiac disease. 222. The antibody composition according to 79 above for use in treating inflammatory bowel disease. 223. The antibody composition according to 80 above for use in treating at least one of multiple sclerosis and inflammatory bowel disease. 224. The antibody composition according to 81 above for use in treating at least one of multiple sclerosis and inflammatory bowel disease. 225. The antibody composition according to 82 for use in treating an allergic disease. 226. The antibody composition according to 83 for use in treating primary myelofibrosis. 227. The antibody composition according to 84 above, for use in treating at least one disease selected from autoimmune diseases, arteriosclerosis, and ischemic heart disease. 228. The antibody composition according to 85 above for use in treating systemic lupus erythematosus. 229. The antibody composition according to claim 86 for use in treating an allergic disease. 230. The antibody composition according to claim 87 for use in treating psoriasis. 231. The antibody composition according to claim 88 for use in treating an allergic disease. 232. The antibody composition according to 89 above, for use in treating at least one selected from asthma, eosinophilic sinusitis, and atopic dermatitis. 233. The antibody composition according to 90 above for use in treating at least one of asthma and eosinophilic sinusitis. 234. The antibody composition according to 91 above for use in treating an allergic disease. 235. The antibody composition according to 92 for use in treating Langerhans cell histiocytosis (LCH). 236. The antibody composition according to 93 for use in treating systemic lupus erythematosus. 237. The antibody composition according to 94 above for use in treating idiopathic pulmonary fibrosis. 238. The antibody composition according to claim 95 for use in treating cancer. 239. The antibody composition according to claim 96 or 97, for use in treating systemic lupus erythematosus. 240. The antibody composition according to 98 above, for use in treating at least one disease selected from autoimmune diseases, arteriosclerosis, and ischemic heart disease. 241. The antibody composition according to 99 above, for use in treating a fibrotic disease including at least one selected from idiopathic pulmonary fibrosis, systemic sclerosis, and liver cirrhosis. 242. The antibody composition according to 100 above for use in treating idiopathic pulmonary fibrosis. 243. The antibody composition according to 101 for use in treating at least one selected from neutrophilic asthma, chronic obstructive pulmonary disease, and Alzheimer's disease. 244. The antibody composition according to 102 for use in treating at least one selected from Crohn's disease, rheumatoid arthritis and asthma. 245. The antibody composition according to 103 for use in treating at least one of multiple sclerosis and neuromyelitis optica. 246. The antibody composition according to 104 or 105 for use in treating systemic lupus erythematosus. 247. The antibody composition according to 106 for use in treating multiple sclerosis. 248. The antibody composition according to 107 for use in treating liver cirrhosis. 249. The antibody composition according to 108 for use in treating systemic lupus erythematosus. 250. The antibody composition according to 109 for use in treating mast cell activation syndrome (MCAS), including at least one selected from urticaria, food allergy, and mastocytosis. 251. The antibody composition according to 110 for use in treating mast cell activation syndrome (MCAS), including at least one selected from urticaria, food allergy, and mastocytosis. 252. The antibody composition according to 111 for use in treating multiple sclerosis. 253. The antibody composition according to 112 above for use in treating at least one selected from multiple sclerosis, type 1 diabetes, and rheumatoid arthritis. 254. The antibody composition according to 113, for use in the treatment of at least one selected from secondary progressive multiple sclerosis (SPMS), rheumatoid arthritis refractory to anti-TNF treatment, transplantation, and systemic lupus erythematosus. 255. The antibody composition according to 114, for use in the treatment of at least one selected from secondary progressive multiple sclerosis (SPMS), rheumatoid arthritis refractory to anti-TNF treatment, transplantation, and systemic lupus erythematosus. 256. The antibody composition according to 115 above for use in treating asthma. 257. The antibody composition according to 116 above for use in treating systemic sclerosis. 258. The antibody composition according to 117 above for use in treating at least one of vitiligo vulgaris and psoriasis. 259. The antibody composition according to 118 for use in treating at least one of vitiligo vulgaris and psoriasis. 260. The antibody composition according to 119 above for use in treating at least one of vitiligo vulgaris and psoriasis. 261. The antibody composition according to 120 for use in treating at least one of vitiligo vulgaris and psoriasis. 262. The antibody composition according to 121 for use in treating at least one of ANCA-associated vasculitis and systemic lupus erythematosus. 263. The antibody composition according to 122 for use in treating a T cell-dependent immune-related disease. 264. The antibody composition according to 123 for use in treating an immune-related disease. 265. The antibody composition according to 124 for use in treating psoriatic arthritis. 266. The antibody composition according to 125 for use in treating autoimmune diseases including at least one of Sjogren's syndrome and psoriasis.

[0037] 267. A method for treating rheumatoid arthritis, comprising administering to a subject an effective amount of the antibody composition according to 77 above. 268. A method for treating celiac disease, comprising administering to a subject an effective amount of the antibody composition according to 78. 269. A method for treating inflammatory bowel disease, comprising administering to a subject an effective amount of the antibody composition according to 79 above. 270. A method for treating at least one of multiple sclerosis and inflammatory bowel disease, comprising administering to a subject an effective amount of the antibody composition according to 80 above. 271. A method for treating at least one of multiple sclerosis and inflammatory bowel disease, which comprises administering to a subject an effective amount of the antibody composition according to 81 above. 272. A method for treating allergic diseases, comprising administering an effective amount of the antibody composition according to 82 to a subject. 273. A method for treating primary myelofibrosis, comprising administering to a subject an effective amount of the antibody composition according to 83. 274. A method for treating at least one disease selected from autoimmune diseases, arteriosclerosis and ischemic heart disease, which comprises administering an effective amount of the antibody composition according to 84 to a subject. 275. A method for treating systemic lupus erythematosus, comprising administering to a subject an effective amount of the antibody composition according to 85 above. 276. A method for treating an allergic disease, comprising administering to a subject an effective amount of the antibody composition according to 86 above. 277. A method for treating psoriasis, comprising administering to a subject an effective amount of the antibody composition according to 87 above. 278. A method for treating an allergic disease, comprising administering to a subject an effective amount of the antibody composition according to 88. 279. A method for treating at least one of asthma, eosinophilic sinusitis, and atopic dermatitis, which comprises administering an effective amount of the antibody composition according to 89 to a subject. 280. A method for treating at least one of asthma and eosinophilic sinusitis, comprising administering to a subject an effective amount of the antibody composition according to 90 above. 281. For the production of a pharmaceutical composition for treating allergic diseases, which comprises administering to a subject an effective amount of the antibody composition described in 91. 282. A method for treating Langerhans cell histiocytosis (LCH), comprising administering to a subject an effective amount of the antibody composition according to 92. 283. A method for treating systemic lupus erythematosus, comprising administering to a subject an effective amount of the antibody composition according to 93 above. 284. A method for treating idiopathic pulmonary fibrosis, comprising administering an effective amount of the antibody composition according to 94 to a subject. 285. A method for treating cancer, comprising administering to a subject an effective amount of the antibody composition according to 95 above. 286. A method for treating systemic lupus erythematosus, comprising administering an effective amount of the antibody composition according to 96 or 97 to a subject. 287. A method for treating at least one disease selected from autoimmune diseases, arteriosclerosis and ischemic heart disease, which comprises administering an effective amount of the antibody composition according to 98 to a subject. 288. A method for treating fibrotic diseases including at least one selected from idiopathic pulmonary fibrosis, systemic sclerosis, and liver cirrhosis, which comprises administering an effective amount of the antibody composition according to 99 above to a subject. 289. A method for treating idiopathic pulmonary fibrosis, comprising administering an effective amount of the antibody composition according to 100 to a subject. 290. A method for treating at least one disease selected from neutrophilic asthma, chronic obstructive pulmonary disease, and Alzheimer's disease, which comprises administering an effective amount of the antibody composition according to 101 to a subject. 291. A method for treating at least one disease selected from Crohn's disease, rheumatoid arthritis, and asthma, which comprises administering to a subject an effective amount of the antibody composition according to 102 above. 292. A method for treating at least one of multiple sclerosis and neuromyelitis optica using the antibody composition described in 103. 293. A method for treating systemic lupus erythematosus, comprising administering an effective amount of the antibody composition according to 104 or 105 to a subject. 294. A method for treating multiple sclerosis, comprising administering to a subject an effective amount of the antibody composition according to 106 above. 295. A method for treating cirrhosis, comprising administering to a subject an effective amount of the antibody composition according to 107 above. 296. A method for treating systemic lupus erythematosus, comprising administering to a subject an effective amount of the antibody composition according to 108 above. 297. A method for treating mast cell activation syndrome (MCAS), including at least one selected from urticaria, food allergy, and mastocytosis, comprising administering to a subject an effective amount of the antibody composition according to 109. 298. A method for treating mast cell activation syndrome (MCAS), including at least one selected from urticaria, food allergy, and mastocytosis, comprising administering to a subject an effective amount of the antibody composition described in 110. 299. A method for treating multiple sclerosis, comprising administering to a subject an effective amount of the antibody composition according to 111 above. 300. A method for treating at least one disease selected from multiple sclerosis, type 1 diabetes, and rheumatoid arthritis, which comprises administering to a subject an effective amount of the antibody composition according to 112. 301. A method for treating at least one condition selected from secondary progressive multiple sclerosis (SPMS), rheumatoid arthritis refractory to anti-TNF treatment, transplantation, and systemic lupus erythematosus, comprising administering to a subject an effective amount of the antibody composition described in 113. 302. A method for treating at least one condition selected from secondary progressive multiple sclerosis (SPMS), rheumatoid arthritis refractory to anti-TNF treatment, transplantation, and systemic lupus erythematosus, comprising administering to a subject an effective amount of the antibody composition described in 114. 303. A method for treating asthma, comprising administering to a subject an effective amount of the antibody composition according to 115 above. 304. A method for treating systemic sclerosis, comprising administering to a subject an effective amount of the antibody composition according to 116 above. 305. A method for treating at least one of vitiligo vulgaris and psoriasis, comprising administering an effective amount of the antibody composition according to 117 above to a subject. 306. A method for treating at least one of vitiligo vulgaris and psoriasis, comprising administering an effective amount of the antibody composition according to 118 to a subject. 307. A method for treating at least one of vitiligo vulgaris and psoriasis, comprising administering an effective amount of the antibody composition according to 119 above to a subject. 308. A method for treating at least one of vitiligo vulgaris and psoriasis, comprising administering an effective amount of the antibody composition according to 120 to a subject. 309. A method for treating at least one of ANCA-associated vasculitis and systemic lupus erythematosus, which comprises administering to a subject an effective amount of the antibody composition according to 121 above. 310. A method for treating a T cell-dependent immune-related disease, comprising administering to a subject an effective amount of the antibody composition according to 122 above. 311. A method for treating an immune-related disease, comprising administering to a subject an effective amount of the antibody composition according to 123 above. 312. A method for treating psoriatic arthritis, comprising administering to a subject an effective amount of the antibody composition according to 124 above. 313. A method for treating autoimmune diseases including at least one of Sjogren's syndrome and psoriasis, which comprises administering to a subject an effective amount of the antibody composition according to 125 above.

[0038] 314. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD64 and the second antigen is one selected from CD206, CD163 and CD68. 315. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD163 and the second antigen is CD206 or CD68. 316. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD206 and the second antigen is CD68. 317. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD14 and the second antigen is one selected from CD48, CD84, CD97 and CD305. 318. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD15 and the second antigen is one selected from CD48, CD84, CD97 and CD305. 319. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD33 and the second antigen is one selected from CD48, CD84, CD97 and CD305. 320. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD11b and the second antigen is one selected from CD48, CD84, CD97 and CD305. 321. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CCR4 and the second antigen is one selected from CADM1, CD30 and CD70. 322. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD38 and the second antigen is CD138 or BCMA. 323. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is BCMA and the second antigen is CD56 or CS1. 324. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD40 ligand and the second antigen is one selected from CD36, CD62P and CD63. 325. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is TIM-3 and the second antigen is one selected from CD123, CD33, CD47, CD70 and CLEC12A. 326. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD123 and the second antigen is one selected from CD33, CD47, CD70 and CLEC12A. 327. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD5 and the second antigen is CD23. 328. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD10 or CD5, and the second antigen is CD20. 329. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CD40 and the second antigen is selected from CD80, CD86, ICOS ligand, 4-1BB ligand, OX40 ligand, CD70, GITR, PD-L1, PD-L2, B7-DC, B7H3, B7H4, B7H5, B7H6 and B7H7. 330. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is PTPRS and the second antigen is one selected from IL-21R, CD38 and CD32a. 331. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is OX40 and the second antigen is CD127 or CD40 ligand. 332. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is OX40 and the second antigen is CD8 or NKG2D. 333. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is OX40 and the second antigen is CD226. 334. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is OX40 and the second antigen is CRTH2. 335. The antibody composition according to any one of 1 to 8 above, wherein the first antigen is CRTH2 and the second antigen is any one selected from CD2, CD7 and CD45.

[0039] 336. A pharmaceutical composition for use in treating cancer, comprising the antibody composition according to any one of 314 to 320. 337. A pharmaceutical composition for use in treating leukemia, comprising the antibody composition according to any one of 321 to 328. 338. A pharmaceutical composition for use in treating lymphoma, comprising the antibody composition according to any one of 321 to 328. 339. A pharmaceutical composition for use in treating an inflammatory disease, comprising the antibody composition according to 329 above. 340. A pharmaceutical composition for use in treating an allergic disease, comprising the antibody composition according to any one of 331, 334 and 335. 341. A pharmaceutical composition for use in treating at least one of vitiligo vulgaris and psoriasis, comprising the antibody composition according to 332 above. 342. A pharmaceutical composition for use in treating scleroderma, comprising the antibody composition according to 333 above.

[0040] 343. Use of the antibody composition according to any one of 314 to 320 for the manufacture of a pharmaceutical composition for treating cancer. 344. Use of the antibody composition according to any one of the above 321 to 328 for the manufacture of a pharmaceutical composition for treating leukemia. 345. Use of the antibody composition according to any one of the above 321 to 328 for the manufacture of a pharmaceutical composition for treating lymphoma. 346. Use of the antibody composition according to 329 for the manufacture of a pharmaceutical composition for the treatment of an inflammatory disease. 347. Use of the antibody composition according to any one of 331, 334 and 335 for the manufacture of a pharmaceutical composition for the treatment of allergic diseases. 348. Use of the antibody composition according to 332 for the manufacture of a pharmaceutical composition for treating at least one of vitiligo vulgaris and psoriasis. 349. Use of the antibody composition according to 333 for the manufacture of a pharmaceutical composition for the treatment of scleroderma. 350. Use of the antibody composition according to any one of 314 to 320 for the treatment of cancer. 351. Use of the antibody composition according to any one of 321 to 328 for the treatment of leukemia. 352. Use of the antibody composition according to any one of 321 to 328 for the treatment of lymphoma. 353. Use of the antibody composition according to 329 for the treatment of an inflammatory disease. 354. Use of the antibody composition according to any one of 331, 334 and 335 for the treatment of an allergic disease. 355. Use of the antibody composition according to 332 for the treatment of at least one of vitiligo vulgaris and psoriasis. 356. Use of the antibody composition according to 333 for the treatment of scleroderma.

[0041] 357. The antibody composition according to any one of 314 to 320, for use in treating cancer. 358. The antibody composition according to any one of 321 to 328, for use in treating leukemia. 359. The antibody composition according to any one of 321 to 328, for use in treating lymphoma. 360. The antibody composition according to 329 for use in treating an inflammatory disease. 361. The antibody composition according to any one of 331, 334 and 335 for use in treating an allergic disease. 362. The antibody composition according to 332 for use in treating at least one of vitiligo vulgaris and psoriasis. 363. The antibody composition according to 333 for use in treating scleroderma.

[0042] 364. A method for treating cancer, comprising administering to a subject an effective amount of the antibody composition according to any one of 314 to 320. 365. A method for treating leukemia, comprising administering to a subject an effective amount of the antibody composition according to any one of 321 to 328. 366. A method for treating lymphoma, comprising administering to a subject an effective amount of the antibody composition according to any one of 321 to 328. 367. A method for treating an inflammatory disease, comprising administering to a subject an effective amount of the antibody composition according to 329 above. 368. A method for treating an allergic disease, comprising administering to a subject an effective amount of the antibody composition according to any one of 331, 334 and 335. 369. A method for treating at least one of vitiligo vulgaris and psoriasis, comprising administering to a subject an effective amount of the antibody composition according to 332 above. 370. A method for treating scleroderma, comprising administering to a subject an effective amount of the antibody composition according to 333 above.

[0043] 371. A pharmaceutical composition for use in treating rheumatoid arthritis, comprising the antibody composition according to 77 above. 372. A pharmaceutical composition for use in treating celiac disease, comprising the antibody composition according to 78. 373. A pharmaceutical composition for use in treating inflammatory bowel disease, comprising the antibody composition according to 79 above. 374. A pharmaceutical composition for use in treating at least one of multiple sclerosis and inflammatory bowel disease, comprising the antibody composition according to item 80. 375. A pharmaceutical composition for use in treating at least one of multiple sclerosis and inflammatory bowel disease, comprising the antibody composition according to 81 above. 376. A pharmaceutical composition for use in treating allergic diseases, comprising the antibody composition according to item 82. 377. A pharmaceutical composition for use in treating primary myelofibrosis, comprising the antibody composition according to 83 above. 378. A pharmaceutical composition for use in treating at least one disease selected from autoimmune diseases, arteriosclerosis and ischemic heart disease, comprising the antibody composition according to 84 above. 379. A pharmaceutical composition for use in treating systemic lupus erythematosus, comprising the antibody composition according to 85 above. 380. A pharmaceutical composition for use in treating allergic diseases, comprising the antibody composition according to item 86. 381. A pharmaceutical composition for use in treating psoriasis, comprising the antibody composition according to item 87. 382. A pharmaceutical composition for use in treating allergic diseases, comprising the antibody composition according to item 88. 383. A pharmaceutical composition for use in treating at least one disease selected from asthma, eosinophilic sinusitis, and atopic dermatitis, comprising the antibody composition according to 89 above. 384. A pharmaceutical composition for use in treating at least one of asthma and eosinophilic sinusitis, comprising the antibody composition according to 90 above. 385. A pharmaceutical composition for use in treating allergic diseases, comprising the antibody composition according to 91 above. 386. A pharmaceutical composition for use in treating Langerhans cell histiocytosis (LCH), comprising the antibody composition according to 92 above. 387. A pharmaceutical composition for use in treating systemic lupus erythematosus, comprising the antibody composition according to 93 above. 388. A pharmaceutical composition for use in treating idiopathic pulmonary fibrosis, comprising the antibody composition according to 94 above. 389. A pharmaceutical composition for use in treating cancer, comprising the antibody composition according to 95 above. 390. A pharmaceutical composition for use in treating systemic lupus erythematosus, comprising the antibody composition according to 96 or 97 above. 391. A pharmaceutical composition for use in treating at least one disease selected from autoimmune diseases, arteriosclerosis, and ischemic heart disease, comprising the antibody composition according to 98 above. 392. A pharmaceutical composition for use in treating fibrotic diseases including at least one selected from idiopathic pulmonary fibrosis, systemic sclerosis, and liver cirrhosis, comprising the antibody composition according to 99 above. 393. A pharmaceutical composition for use in treating idiopathic pulmonary fibrosis, comprising the antibody composition according to 100 above. 394. A pharmaceutical composition for use in treating at least one disease selected from neutrophilic asthma, chronic obstructive pulmonary disease, and Alzheimer's disease, comprising the antibody composition according to 101 above. 395. A pharmaceutical composition for use in treating at least one disease selected from Crohn's disease, rheumatoid arthritis, and asthma, comprising the antibody composition according to 102 above. 396. A pharmaceutical composition for use in treating at least one of multiple sclerosis and neuromyelitis optica, comprising the antibody composition according to 103 above. 397. A pharmaceutical composition for use in treating systemic lupus erythematosus, comprising the antibody composition according to 104 or 105 above. 398. A pharmaceutical composition for use in treating multiple sclerosis, comprising the antibody composition according to 106 above. 399. A pharmaceutical composition for use in treating liver cirrhosis, comprising the antibody composition according to 107 above. 400. A pharmaceutical composition for use in treating systemic lupus erythematosus, comprising the antibody composition according to 108. 401. A pharmaceutical composition for use in treating mast cell activation syndrome (MCAS), including at least one selected from urticaria, food allergy, and mastocytosis, comprising the antibody composition according to 109. 402. A pharmaceutical composition for use in treating mast cell activation syndrome (MCAS), including at least one selected from urticaria, food allergy, and mastocytosis, comprising the antibody composition according to 110. 403. A pharmaceutical composition for use in treating multiple sclerosis, comprising the antibody composition according to 111 above. 404. A pharmaceutical composition for use in treating at least one disease selected from multiple sclerosis, type 1 diabetes, and rheumatoid arthritis, comprising the antibody composition according to 112 above. 405. A pharmaceutical composition for use in treating at least one disease selected from secondary progressive multiple sclerosis (SPMS), rheumatoid arthritis in which anti-TNF treatment is ineffective, transplantation, and systemic lupus erythematosus, comprising the antibody composition according to 113. 406. A pharmaceutical composition for use in treating at least one selected from secondary progressive multiple sclerosis (SPMS), rheumatoid arthritis in which anti-TNF treatment is ineffective, transplantation, and systemic lupus erythematosus, comprising the antibody composition according to 114. 407. A pharmaceutical composition for use in treating asthma, comprising the antibody composition according to 115 above. 408. A pharmaceutical composition for use in treating systemic sclerosis, comprising the antibody composition according to 116 above. 409. A pharmaceutical composition for use in treating at least one of vitiligo vulgaris and psoriasis, comprising the antibody composition according to 117 above. 410. A pharmaceutical composition for use in treating at least one of vitiligo vulgaris and psoriasis, comprising the antibody composition according to 118 above. 411. A pharmaceutical composition for use in treating at least one of vitiligo vulgaris and psoriasis, comprising the antibody composition according to 119 above. 412. A pharmaceutical composition for use in treating at least one of vitiligo vulgaris and psoriasis, comprising the antibody composition according to 120 above. 413. A pharmaceutical composition for use in treating at least one of ANCA-associated vasculitis and systemic lupus erythematosus, comprising the antibody composition according to 121 above. 414. A pharmaceutical composition for use in treating a T cell-dependent immune-related disease, comprising the antibody composition according to 122 above. 415. A pharmaceutical composition for use in treating immune-related diseases, comprising the antibody composition according to 123 above. 416. A pharmaceutical composition for use in treating psoriatic arthritis, comprising the antibody composition according to 124 above. 417. A pharmaceutical composition for use in treating autoimmune diseases including at least one of Sjogren's syndrome and psoriasis, comprising the antibody composition according to 125 above. [Effects of the Invention]

[0044] The antibody compositions of the present invention consist of two types of IgG halves, first and second IgG halves, which have antigen-binding domains for two different antigens and have attenuated CD16a-binding activity at their different CD16a-binding regions. Therefore, even if a homomeric antibody structure is formed between the first IgG halves or the second IgG halves, it cannot bind to CD16a and therefore cannot exert its antibody activity. On the other hand, when a heteromeric antibody structure is formed between the first and second IgG halves, it can bind to CD16a via the second CD16a-binding region of the first IgG halfmer and the first CD16a-binding region of the second IgG halfmer.

[0045] Furthermore, the hinge domains of the first and second IgG half-mers are modified so that disulfide bonds are not formed. As a result, disulfide bonds are not formed between the H chains of the first and second IgG half-mers. Therefore, when the first and second IgG half-mers are mixed, the first and second IgG half-mers can exist in an equilibrium state as an aggregate or half-mer. This enables the antibody composition of the present invention to more specifically exert effector function on and damage dual-positive cells compared to single-positive cells.

[0046] Furthermore, in the antibody compositions of the present invention, specific amino acid modifications have been introduced into the CD16a-binding region and other Fc regions of the first and second IgG half-mers, so that hetero-aggregates formed by the first and second IgG half-mers exhibit enhanced effector function against CD16a-positive cells and / or controlled in vivo kinetics. [Brief explanation of the drawings]

[0047] [Figure 1] FIG. 1 is a schematic diagram showing the structures of an antibody, VHH-Fc, and scFv-Fc. [Figure 2A] FIG. 2A shows a schematic diagram of the structure of a typical bispecific antibody. [Figure 2B] Figure 2B shows a schematic diagram of the binding mode of a typical bispecific antibody. [Figure 3] FIG. 3 shows a schematic diagram of one embodiment of the binding mode of the antibody composition of the present invention. [Figure 4] FIG. 4 shows a schematic diagram of one embodiment of the structure of the antibody composition of the present invention. [Figure 5] FIG. 5 shows a schematic diagram of the binding mode between normal human IgG and CD16a. [Figure 6] FIG. 6 shows a schematic diagram of the binding mode between the antibody composition of the present invention and CD16a. [Figure 7] FIG. 7 is a schematic diagram showing candidate sites for amino acid modification in the format of a normal human IgG1. [Figure 8] FIG. 8 shows the ADCC activity of human IgG1 anti-CCR6 antibodies in which the CD16a binding region has been "destroyed." [Figure 9] FIG. 9 is a schematic diagram of a monovalent antibody for evaluating ADCC activity in which the above-mentioned modifications are asymmetrically introduced into only CH2-A and CH2-B. [Figure 10] FIG. 10 shows the ADCC activity of CD16a-binding asymmetric modified monovalent antibodies. [Figure 11] FIG. 11 is a schematic diagram of the IgG1114_AA_AAA_D265A( / P329Y) type IgG halfmer used in Example 3. [Figure 12] FIG. 12 shows the results of evaluating the degree of purification by SDS-PAGE of the anti-CD4 antibody half-mer and the anti-CD70 antibody half-mer. [Figure 13] FIG. 13 shows the results of measuring the expression of CD4 and CD70 antigens in CD4 single positive cells, CD70 single positive cells, and CD4 / CD70 dual positive cells. [Figure 14] FIG. 14 shows the ADCC activity of anti-CD4 IgG1 and anti-CD70 IgG1 and half-antibody against CD4 single-positive cells, CD70 single-positive cells, and CD4 / CD70 dual-positive cells. [Figure 15A] FIG. 15A shows the ADCC activity of each half-mer added to CD4 / CD70 dual-positive cells (TL-Om1), CD70 single-positive cells (MT-1), and CD4 single-positive cells (CD4 / EL-4). [Figure 15B] FIG. 15B shows the ADCC activity upon addition of each half-mer to CD4 / CD70 dual-positive cells (TL-Om1), CD70 single-positive cells (MT-1), and CD4 single-positive cells (CD4 / EL-4). [Figure 15C] FIG. 15C shows the ADCC activity of each half-mer added to CD4 / CD70 dual-positive cells (TL-Om1), CD70 single-positive cells (MT-1), and CD4 single-positive cells (CD4 / EL-4). [Figure 15D]FIG. 15D shows the ADCC activity of each half-mer added to CD4 / CD70 dual-positive cells (TL-Om1), CD70 single-positive cells (MT-1), and CD4 single-positive cells (CD4 / EL-4). [Figure 15E] FIG. 15E shows the ADCC activity upon addition of each half-mer to CD4 / CD70 dual-positive cells (TL-Om1), CD70 single-positive cells (MT-1), and CD4 single-positive cells (CD4 / EL-4). [Figure 15F] FIG. 15F shows the ADCC activity upon addition of each half-mer to CD4 / CD70 dual-positive cells (TL-Om1), CD70 single-positive cells (MT-1), and CD4 single-positive cells (CD4 / EL-4). [Figure 15G] FIG. 15G shows the ADCC activity upon addition of each half-mer to CD4 / CD70 dual-positive cells (TL-Om1), CD70 single-positive cells (MT-1), and CD4 single-positive cells (CD4 / EL-4). [Figure 15H] Figure 15H shows the ADCC activity of each half-mer, anti-CD4 IgG1, or anti-CD70 IgG1 on CD4 / CD70 dual-positive cells (TL-Om1), CD70 single-positive cells (MT-1), and CD4 single-positive cells (CD4 / EL-4). [Figure 16A] 16A shows the ADCC activity of each half-mer against CD4 / CD70 dual-positive cells (TL-Om1), CD70 mono-positive cells (MT-1), and CD4 mono-positive cells (CD4 / EL-4). The amino acid alterations introduced into the CH3 domain of each half-mer are shown above each graph. [Figure 16B] Figure 16B shows the ADCC activity of each half-antibody, anti-CD4 IgG1, or anti-CD70 IgG1 on CD4 / CD70 dual-positive cells (TL-Om1), CD70 mono-positive cells (MT-1), and CD4 mono-positive cells (CD4 / EL-4). The abbreviations for the antibodies added (for half-antibodies, the names of the amino acid modifications introduced into the CH3 domain) are shown above each graph. [Figure 17] FIG. 17 is a graph showing the time course of serum antibody concentration when 1 mg / kg of an anti-DNP antibody (wild-type) or a mixture of each half-mer was administered to mice. [Figure 18A] 18A shows the ADCC activity in an immunoglobulin-added system when each half-mer was added to CD4 / CD70 dual-positive cells (TL-Om1), CD70 mono-positive cells (MT-1), and CD4 mono-positive cells (CD4 / EL-4). The abbreviations for the amino acid modifications that enhance CD16a binding for each added half-mer are shown above each graph. [Figure 18B] Figure 18B shows the ADCC activity in an immunoglobulin-added system when each half-mer was added to CD4 / CD70 dual-positive cells (TL-Om1), CD70 mono-positive cells (MT-1), and CD4 mono-positive cells (CD4 / EL-4). The abbreviation for the amino acid modification that enhances CD16a binding for each added half-mer is shown above each graph. [Figure 18C] 18C shows the ADCC activity of CD4 / CD70 dual-positive cells (TL-Om1), CD70 mono-positive cells (MT-1), and CD4 mono-positive cells (CD4 / EL-4) in an immunoglobulin-added system when half-antibodies, anti-CD4 IgG1, or anti-CD70 IgG1 were added. The abbreviations of the antibodies added (for half-antibodies, the name of amino acid modifications that enhance CD16a binding) are shown above each graph. [Figure 19A] Figure 19A shows the ADCC activity (cell viability) of each half-mer in a human blood reconstitution system. The horizontal axis shows the concentration of the added antibody (the half-mer is the total concentration of the mixture), and the vertical axis shows the proportion (%) of each target cell (listed at the top of the graph) among all CD3-positive T cells or all lymphocytes when no antibody was added. [Figure 19B] Figure 19B shows the ADCC activity (cell viability) of each half-mer in a human blood reconstitution system. The horizontal axis shows the concentration of the added antibody (the half-mer is the total concentration of the mixture), and the vertical axis shows the proportion (%) of each target cell (listed at the top of the graph) among all CD3-positive T cells or all lymphocytes when no antibody was added. [Figure 20] FIG. 20 shows the ADCC activity of CD4 / CCR4 dual-positive cells (TL-Om1), CCR4 single-positive cells (MT-1), and CD4 single-positive cells (CD4 / EL-4) when each half-mer, anti-CD4 IgG1, or anti-CCR4 IgG1 was added. [Figure 21] FIG. 21 shows the ADCC activity of CD4 / CCR4 dual-positive cells (HH), CCR4 single-positive cells (L428), and CD4 single-positive cells (TALL1) when each half-mer, anti-CD4 IgG1, or anti-CCR4 IgG1 was added. [Figure 22] FIG. 22 shows the ADCC activity of CCR4 / CD70 dual-positive cells (L428), CCR4 single-positive cells (PEER), and CD70 single-positive cells (SUP-M2) when each half-mer, anti-CCR4 IgG1, or anti-CD70 IgG1 was added. [Figure 23] FIG. 23 shows the binding activity of each half-mer and anti-DNP antibody to CD16a. DETAILED DESCRIPTION OF THE INVENTION

[0048] 1. Structure of the antibody composition In the present invention, antibody molecules are also referred to as immunoglobulins (hereinafter referred to as Ig), and human antibodies are classified into isotypes such as IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM depending on differences in molecular structure. IgG1, IgG2, IgG3, and IgG4, which have relatively high amino acid sequence homology, are collectively referred to as IgG.

[0049] An antibody molecule is composed of polypeptides called heavy chains (hereinafter referred to as H chains) and light chains (hereinafter referred to as L chains).An antibody is a tetrameric protein consisting of two H chains and two L chains.

[0050] Furthermore, the H chain is composed of an H chain variable region (also referred to as VH) and an H chain constant region (also referred to as CH) from the N-terminus, while the L chain is composed of an L chain variable region (also referred to as VL) and an L chain constant region (also referred to as CL) from the N-terminus. The CH subclasses are known to include α, δ, ε, γ, and μ chains, respectively. The CL subclasses are known to include λ and κ chains.

[0051] A domain refers to a functional structural unit that constitutes each polypeptide of an antibody molecule. In addition, the Fc region (Fc) in the present invention refers to a partial sequence and partial structure of the heavy chain constant region consisting of a hinge domain, a CH2 domain, and a CH3 domain.

[0052] The CH is composed of, from the N-terminus, a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain. In the present invention, the CH1 domain, hinge domain, CH2 domain, CH3 domain, and Fc can be identified by the amino acid residue number from the N-terminus according to the EU index [Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services (1991)].

[0053] Specifically, CH1 is identified as the amino acid sequence of positions 118 to 215 in the EU index, the hinge as the amino acid sequence of positions 216 to 230 in the EU index, CH2 as the amino acid sequence of positions 231 to 340 in the EU index, and CH3 as the amino acid sequence of positions 341 to 447 in the EU index.

[0054] The IgG of the present invention also includes artificial subspecies modified molecules that have at least an antigen-binding domain and Fc and have functions similar to those of IgG. Specifically, these include modified molecules in which IgG amino acid residues have been substituted, deleted, added, or modified, as well as adducts of polypeptides or domains. Furthermore, these include molecules in which part or all of the antigen-binding site consisting of the VH, VL, or Fab of IgG has been replaced with another antigen-binding domain. Specifically, these include single-chain Fv (scFv)-Fc and variable domain of heavy chain of heavy chain antibody (VHH)-Fc shown in Figure 1 (Brinkmann U et al., MABS 2017, 9: 182-212; Fernandes CFC et al., Frontiers in Immunology 2017, 8: 653).

[0055] In the present invention, the term "antibody" includes not only monoclonal antibodies obtained from hybridomas but also recombinant antibodies produced by genetic engineering. Recombinant antibodies include chimeric antibodies in which a human antibody constant region is linked to a non-human antibody variable region, humanized antibodies, and human antibodies produced using human antibody-producing animals.

[0056] Chimeric antibodies can be produced by obtaining cDNA encoding VH and VL from a hybridoma derived from non-human animal cells that produces monoclonal antibodies, inserting them into an expression vector for animal cells containing DNA encoding CH and CL of a human antibody to construct a human chimeric antibody expression vector, and introducing the vector into animal cells to express the antibody.

[0057] A humanized antibody is an antibody produced by inserting the complementarity determining regions (hereinafter abbreviated as CDRs) of the heavy and light chain variable regions of a non-human antibody into the framework regions (hereinafter abbreviated as FRs) of a human antibody variable region.

[0058] Humanized antibodies (or CDR-grafted antibodies) can be produced by the following method: A cDNA encoding a VH amino acid sequence consisting of the amino acid sequence of the CDR of the VH of a non-human animal antibody and the amino acid sequence of the FR of the VH of any human antibody, and a cDNA encoding a VL amino acid sequence consisting of the amino acid sequence of the CDR of the VL of a non-human animal antibody and the amino acid sequence of the FR of the VL of any human antibody are constructed. These cDNAs are inserted into an expression vector for animal cells carrying DNA encoding the CH and CL of a human antibody to construct a humanized antibody expression vector, and the humanized antibody can be produced by expressing the vector by introducing it into animal cells.

[0059] Human antibodies originally refer to antibodies that can exist naturally in the human body or antibodies consisting of an amino acid sequence encoded by a human gene, but also include human antibody phage libraries produced through recent advances in genetic engineering, cell engineering, and developmental engineering technologies, antibodies obtained from cloning of immortalized human peripheral blood lymphocytes, or antibodies obtained from transgenic animals producing human antibodies.

[0060] Human antibodies can be obtained by immunizing mice carrying human immunoglobulin genes with a desired antigen (Tomizuka K. et al., Proc Natl Acad Sci USA. 97, 722-7, 2000). Alternatively, human antibodies can be obtained without immunization by selecting human antibodies with the desired binding activity using a phage display library in which antibody genes are amplified from human-derived B cells (Winter G. et al., Annu Rev Immunol. 12: 433-55, 1994).

[0061] Furthermore, by immortalizing human B cells using EB virus, cells that produce human antibodies with the desired binding activity can be created, and human antibodies can be obtained (Rosen A. et al., Nature 267, 52-54. 1977).

[0062] Antibodies present in the human body can be produced, for example, by immortalizing lymphocytes isolated from human peripheral blood by infecting them with EB virus or the like, followed by cloning, to culture the lymphocytes that produce the antibodies, and then purifying the antibodies from the culture.

[0063] A human antibody phage library is a library of phages in which antibody genes prepared from human B cells are inserted into the phage genome to express antibody fragments such as Fab or scFv on their surface. Phages expressing antibody fragments with the desired antigen-binding activity can be recovered from the library using their binding activity toward an antigen-immobilized substrate as an indicator. These antibody fragments can also be converted into human antibody molecules consisting of two complete heavy chains and two complete light chains by genetic engineering techniques.

[0064] A human antibody-producing transgenic animal is an animal in which a human antibody gene has been integrated into the chromosome of a host animal. Specifically, a human antibody-producing transgenic animal can be produced by introducing a human antibody gene into mouse ES cells, transplanting the ES cells into the early embryo of another mouse, and then allowing the ES cells to develop.

[0065] A method for producing human antibodies from human antibody-producing transgenic animals involves obtaining human antibody-producing hybridomas by hybridoma production methods typically used in non-human mammals, and culturing them to produce and accumulate human antibodies in the culture.

[0066] The amino acid sequences of VH and VL may be any of the amino acid sequences of VH and VL of a human antibody, the amino acid sequences of VH and VL of a non-human animal antibody, the amino acid sequence of a humanized antibody in which the CDR of a non-human animal antibody is grafted onto the framework of a human antibody, and the amino acid sequences of VH and VL derived from a human antibody.

[0067] Specific examples include the amino acid sequences of VH and VL of non-human animal antibodies, humanized antibodies, and human antibodies produced by hybridomas or antibody-producing cells.

[0068] The amino acid sequence of CL may be either the amino acid sequence of a human antibody or the amino acid sequence of a non-human animal antibody, but is preferably the amino acid sequence of Cκ or Cλ of a human antibody.

[0069] Any CH may be used as long as it belongs to the immunoglobulin class, but preferably any of the subclasses belonging to the human IgG class, γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), and γ4 (IgG4), can be used.

[0070] The "antigen-binding domain" may be a recombinant binding protein that utilizes the binding domain of a known binding molecule such as an antibody, a ligand, or a receptor, and specific examples include recombinant proteins comprising the CDR of an antibody that binds to each antigen, an antibody variable region comprising the CDR, and a recombinant protein comprising an antibody variable region and a binding domain of a ligand that binds to each antigen. Of these, in the present invention, the antigen-binding domain is preferably an antibody variable region.

[0071] The antibody composition of the present invention is an antibody composition consisting of a first IgG halfmer and a second IgG halfmer, which are directed against a first antigen and a second antigen that are different from each other, and has the following properties 1) to 6): 1) The first IgG half-mer and the second IgG half-mer each consist of one L chain and one H chain, and the H chain includes an H chain variable region, a hinge domain, and CH1, CH2, and CH3 domains, with first and second CD16a-binding regions in the CH2 domain that differ from each other. 2) The hinge domains of the first IgG half-mer and the second IgG half-mer each contain partial or full substitutions or deletions, or modified alterations that prevent disulfide bond formation between the H chains of the first IgG half-mer and the second IgG half-mer. 3) The first IgG half-mer comprises an antigen-binding domain that binds to a first antigen and comprises a modification in the first CD16a-binding region that attenuates CD16a-binding activity. 4) The second IgG half-mer comprises an antigen-binding domain that binds to a second antigen and comprises a modification in the second CD16a-binding region that attenuates CD16a-binding activity. 5) The first IgG half-mer and the second IgG half-mer each contain a modification that enhances CD16a binding activity. 6) At least one of the first IgG half-mer and the second IgG half-mer contains a modification that weakens the CH3 domain-to-domain interaction compared to that of the IgG1 subclass.

[0072] The term "antibody composition against a first antigen and a second antigen that are different from each other" refers to a composition containing an IgG half-mer against the first antigen and an IgG half-mer against the second antigen.

[0073] Possible embodiments of the IgG half-mers in the antibody compositions of the present invention include, for example, monomer half-mers and aggregates of the half-mers. Examples of the half-mers include a first IgG half-mer and a second IgG half-mer. Examples of the aggregates include an aggregate of the first IgG half-mer, an aggregate of the second IgG half-mer, and an aggregate of the first IgG half-mer and the second IgG half-mer. Specifically, the first and second halves contained in the antibody composition may be in equilibrium with an aggregate of the first half-mer and the second half-mer, and an aggregate of the first IgG half-mer and the second IgG half-mer.

[0074] The "antibody composition against a first antigen and a second antigen that are different from each other" of the present invention also includes an antibody composition containing an IgG half-mer against a first antigenic determinant (epitope) and an IgG half-mer against a second antigenic determinant (epitope) in the same antigen.

[0075] An "IgG half-mer" is a dimeric protein consisting of one L chain and one H chain, and the H chain contains an H chain variable region, CH1 to CH3 domains, and a hinge domain. The CH2 domain of the H chain of an IgG half-mer contains two distinct CD16a-binding regions (first and second CD16a-binding regions).

[0076] The term "IgG halfmer" also includes halfmers of artificial subspecies modified molecules that have an antigen-binding domain and Fc and are functionally similar to IgG. Specifically, it includes halfmers of modified molecules in which IgG amino acids have been substituted, deleted, or added, or modified, as well as halfmers of adducts of polypeptides or domains. It also includes halfmers in which part or all of the antigen-binding site consisting of IgG VH, VL, or Fab has been replaced with another antigen-binding domain, specifically halfmers such as single-chain Fv (scFv)-Fc and VHH-Fc shown in Figure 1.

[0077] The "CD16a-binding region" refers to the region present in the Fc of IgG that binds to CD16a. Due to the point symmetry of the Fc structure, there are two CD16a-binding regions on the Fc of IgG1, and each region contacts CD16a at a different region in the CH2 domain composed of the two polypeptide chains that make up the Fc (see Figure 5).

[0078] The first CD16a-binding region may include a region containing at least one amino acid residue selected from the amino acid residues at positions 235, 236, 237, 238, 239, 265, 266, 267, 268, 269, 294, 295, 296, 297, 298, 299, 301, 325, 327, and 332 according to the EU index.

[0079] When the immunoglobulin subclass of the CH2 domain is IgG1, examples of the first CD16a-binding region include a region containing at least one amino acid residue selected from Leu at position 235, Gly at position 236, Gly at position 237, Pro at position 238, Ser at position 239, Asp at position 265, Val at position 266, Ser at position 267, His at position 268, Glu at position 269, Glu at position 294, Gln at position 295, Tyr at position 296, Asn at position 297, Ser at position 298, Thr at position 299, Arg at position 301, Asn at position 325, Ala at position 327, and Ile at position 332, as defined by the EU index.

[0080] The second CD16a-binding region includes a region containing at least one amino acid residue selected from the amino acid residues at positions 235, 236, 237, 326, 327, 328, 329, and 330 according to the EU index.

[0081] When the immunoglobulin subclass of the CH2 domain is IgG1, the second CD16a-binding region can be a region containing at least one amino acid residue selected from the following amino acid residues according to the EU index: Leu at position 235, Gly at position 236, Gly at position 237, Lys at position 326, Ala at position 327, Leu at position 328, Pro at position 329, and Ala at position 330.

[0082] "CD16a binding activity" refers to the activity of IgG Fc binding to CD16a. The CD16a binding activity of an IgG halfmer can be confirmed by combining two IgG halfmer molecules to form an IgG, reacting it with a recombinant CD16a protein, and measuring the binding activity (U.S. Patent Application Publication No. 2004 / 0259150).

[0083] In one embodiment of the method for measuring the CD16a-binding activity of IgG halfmers, for example, binding activity to CD16a expressed on the cell membrane can be measured by fluorescent antibody assay (Cancer Immunol. Immunother., 36, 373, 1993). Furthermore, binding activity to purified CD16a protein can be measured according to immunological quantification methods such as Western staining, radioimmunoassay (RIA), viroimmunoassay (VIA), enzyme immunoassay (EIA), fluoroimmunoassay (FIA), and metalloimmunoassay (MIA) described in the literature (Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., 1995; Enzyme Immunoassay, 3rd Edition, Igaku-Shoin (1987); Revised Edition, Enzyme Immunoassay, Gakusai Kikaku (1985)).

[0084] Specifically, for example, the binding activity to purified CD16a protein can be quantified by EIA as follows: FcγRIIIa is immobilized on a plastic plate for EIA and reacted with a sample containing an antibody composition. Next, the amount of bound antibody composition is measured using an appropriate secondary antibody.

[0085] The binding activity to purified CD16a protein can also be measured using a biosensor [e.g., BIAcore (BIACORE)] [J. Immunol. Methods, 200, 121 (1997)] or the isothermal titration calorimetry method [Proc. Natl. Acad. Sci. USA, 97, 9026 (2000)].

[0086] Alternatively, the CD16a-binding activity of an IgG halfmer can be confirmed by providing the Fc of the IgG halfmer with an effector function (such as ADCC activity) described below, combining two molecules of the IgG halfmer to form an IgG, and then measuring the effector function of the IgG.

[0087] "Effector function" refers to an antibody-dependent function mediated by the Fc of an antibody. Examples of effector functions include ADCC activity, CDC activity, and antibody-dependent cellular phagocytosis (ADCP) activity by phagocytes such as macrophages or dendritic cells. Effector functions can be measured, for example, by the method described in Cancer Immunol. Immunother., 36, 373 (1993).

[0088] One embodiment of the method for measuring ADCC activity or CDC activity is, for example, the following method. Specific examples include the method described below in the Examples. 1) Prepare effector cells (human peripheral blood mononuclear cells (PBMCs) or a cell line stably expressing human CD16a) and target cells in medium (e.g., RPMI medium). For CDC activity measurements, prepare a solution of human complement proteins diluted to an appropriate concentration by mixing with the target cells. 2) Dispense the antibody solution, target cells, and effector cells into a cell culture vessel (e.g., a 96-well plate). Maintain a constant ratio of effector cells to target cells (E / T ratio). 3) Place the plate in a CO2 incubator and let it stand for 2-6 hours. At the same time, add a solubilizing solution (e.g., an aqueous solution containing acid, alkali, surfactant, etc.) to the 100% reaction well to completely dissolve the target cells. After centrifuging the reaction vessel, collect the supernatant and dispense it into an ELISA plate. After applying the color-developing solution and allowing it to react, add a stop solution and measure the absorbance (A450) using a plate reader. 4) Calculate ADCC activity (%) or CDC activity (%) using the following formula.

[0089] ADCC activity (%) or CDC activity (%) = 100 × (SET) / (Max-T) S = absorbance of sample reaction well - absorbance of medium well E = Effector well absorbance - Medium well absorbance T = target well absorbance - medium well absorbance Max = 100% reaction well - 100% reaction control well

[0090] As the ADCC measurement kit, a known kit can be used, for example, CytoTox96® Non-Radioactive Cytotoxicity Assay (Promega).

[0091] ADCC activity refers to the activity of an antibody bound to an antigen on a target cell, activating immune cells (such as natural killer cells) by binding to the Fc receptor of the immune cell via the antibody's Fc, thereby damaging the target cell.

[0092] Fc receptors (hereinafter sometimes referred to as FcRs) are receptors that bind to the Fc of antibodies, and induce various effector functions upon antibody binding.

[0093] FcRs correspond to antibody subclasses, with IgG, IgE, IgA, and IgM specifically binding to FcγR, FcεR, FcαR, and FcμR, respectively. Furthermore, FcγR subtypes include FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), which exist as isoforms: FcγRIA, FcγRIB, FcγRIC, FcγRIIA, FcγRIIB, FcγRIIC, FcγRIIIA (CD16a), and FcγRIIIB, respectively. These different FcγRs are present on different cell types [Annu. Rev. Immunol. 9: 457-492 (1991)].

[0094] In humans, FcγRIIIB is specifically expressed on neutrophils, whereas FcγRIIIA is expressed on monocytes, natural killer cells (NK cells), and a subset of T cells. Antibody binding via FcγRIIIA induces NK cell-dependent ADCC.

[0095] CDC activity refers to the ability of antibodies bound to antigens on target cells to activate a series of cascades (complement activation pathway) consisting of complement-related proteins in the blood, resulting in target cell damage. In addition, protein fragments generated by complement activation can induce the migration and activation of immune cells.

[0096] The CDC activity cascade begins when C1q, which has an antibody Fc-binding domain, binds to Fc and then binds to two serine proteases, C1r and C1s, to form the C1 complex.

[0097] The phrase "the CD16a-binding activity of the CD16a-binding region is attenuated" means that the CD16a-binding activity and / or effector function (such as ADCC activity) of an IgG formed by combining two IgG half-mers that have been modified in the Fc to attenuate the CD16a-binding activity of the first and / or second CD16a-binding region is attenuated compared to the CD16a-binding activity and / or effector function of an IgG formed by combining two IgG half-mer molecules before the modification.

[0098] In one embodiment in which the CD16a-binding activity of the CD16a-binding region is attenuated, for example, the CD16a-binding activity of an IgG formed by combining two molecules of the IgG half-mers having the above-described modifications is preferably 60% or less, more preferably 50% or less, and even more preferably 40% or less, 30% or less, 20% or less, or 10% or less, in that order, when the CD16a-binding activity of an IgG formed by combining two molecules of the IgG half-mers before the above-described modifications is taken as 100%.

[0099] In one embodiment in which the CD16a-binding activity of the CD16a-binding region is attenuated, for example, the ADCC activity of an IgG formed by combining two molecules of the IgG half-mers into which the above-described modifications have been added is preferably 80% or less, more preferably 70% or less, and even more preferably 60% or less, 50% or less, 40% or less, or 30% or less, in that order, when the ADCC activity of an IgG formed by combining two molecules of the IgG half-mers before the modification is taken as 100%.

[0100] The CD16a-binding activity of the CD16a-binding region is attenuated by modification of the CD16a-binding region. As used herein, "modification" refers to a modification of an amino acid residue from the wild-type amino acid sequence, and means, for example, substitution, deletion, addition, or modification of an amino acid residue. As used herein, amino acid residue substitution is also abbreviated as amino acid residue substitution. As used herein, modifications that attenuate the CD16a-binding activity of the CD16a-binding region are not particularly limited by the type of antigen to which the antibody of the present invention binds; antibodies having any antigen-binding domain may be combined with any modification as long as the effects of the present invention are achieved.

[0101] Examples of modifications that attenuate the CD16a-binding activity of the first CD16a-binding region include substitution of at least one amino acid residue selected from the amino acid residues at positions 235, 236, 237, 238, 239, 265, 266, 267, 268, 269, 294, 295, 296, 297, 298, 299, 301, 325, 327, and 332 according to the EU index, and modifications that attenuate the CD16a-binding activity of the first CD16a-binding region include substitution of at least one amino acid residue selected from the amino acid residues at positions 235, 238, 239, 265, 266, 267, 268, 269, 294, 295, 296, 297, 298, 299, 301, 325, 327, and 332 according to the EU index. Substitution of at least one amino acid residue selected from amino acid residues at positions 235, 238, 239, 265, 267, 268, 269, 294, 295, 296, 297, 298, 299, 301, 325, 327, and 332 is preferred, and substitution of at least one amino acid residue selected from amino acid residues at positions 235, 238, 239, 265, 267, 269, 296, 298, 299, and 327 is more preferred. Examples of amino acid residue substitution combinations include the combination of positions 238 and 265 (hereinafter, such combinations will be referred to as 238 / 265, etc.), substitution of amino acid residues at positions 238 / 267, 265 / 267, and 238 / 265 / 267.

[0102] When the immunoglobulin subclass of the CH2 domain is IgG1, examples of modifications that attenuate the CD16a-binding activity of the first CD16a-binding region include substitution of at least one amino acid residue selected from Leu at position 235, Gly at position 236, Gly at position 237, Pro at position 238, Ser at position 239, Asp at position 265, Val at position 266, Ser at position 267, His at position 268, Glu at position 269, Glu at position 294, Gln at position 295, Tyr at position 296, Asn at position 297, Ser at position 298, Thr at position 299, Arg at position 301, Asn at position 325, Ala at position 327, and Ile at position 332, as defined by the EU index. Substitution of at least one amino acid residue selected from Pro at position 238, Ser at position 239, Asp at position 265, Val at position 266, Ser at position 267, His at position 268, Glu at position 269, Glu at position 294, Gln at position 295, Tyr at position 296, Asn at position 297, Ser at position 298, Thr at position 299, Arg at position 301, Asn at position 325, Ala at position 327, and Ile at position 332 is preferred, and substitution of at least one amino acid residue selected from Leu at position 235, Pro at position 238, Ser at position 239, Asp at position 265, Ser at position 267, Glu at position 269, Tyr at position 296, Ser at position 298, Thr at position 299, and Ala at position 327 is more preferred. Specific examples include substitutions of at least one amino acid residue selected from L235R, P238A, S239R, D265A, D265N, D265E, S267L, S267K, E269P, Y296P, S298E, T299A, and A327I.

[0103] Modifications that attenuate the CD16a-binding activity of the second CD16a-binding domain include substitutions of at least one amino acid residue selected from amino acid residues at positions 235, 236, 237, 326, 327, 328, 329, and 330 according to the EU index, with substitutions of at least one amino acid residue selected from amino acid residues at positions 326, 328, 329, and 330 being preferred, and substitutions of at least one amino acid residue selected from amino acid residues at positions 326, 328, and 329 being more preferred. Examples of combinations of amino acid residue substitutions include substitutions of amino acid residues at positions 326 / 328, 326 / 329, 328 / 329, and 326 / 328 / 329.

[0104] When the immunoglobulin subclass of the CH2 domain is IgG1, modifications that attenuate the CD16a-binding activity of the second CD16a-binding region include substitution of at least one amino acid residue selected from Leu at position 235, Gly at position 236, Gly at position 237, Lys at position 326, Ala at position 327, Leu at position 328, Pro at position 329, and Ala at position 330 according to the EU index; substitution of at least one amino acid residue selected from Lys at position 326, Leu at position 328, Pro at position 329, and Ala at position 330 is preferred, and substitution of at least one amino acid residue selected from Lys at position 326, Leu at position 328, and Pro at position 329 is more preferred. Specifically, at least one amino acid residue substitution selected from K326W, K326G, L328V, L328R, P329Y, P329K, P329W, and A330P is included.

[0105] The combination of an alteration that attenuates the CD16a-binding activity in the CD16a-binding region of the first IgG half-mer and an alteration that attenuates the CD16a-binding activity in the CD16a-binding region of the second IgG half-mer is not particularly limited as long as the alterations are asymmetric, and the above alterations can be combined as appropriate. Specific examples include combinations of substitutions of the following amino acid residues: Substitution of amino acid residues at position 265 in the first IgG half-mer and at position 329 in the second IgG half-mer Substitution of amino acid residues at position 329 in the first IgG half-mer and at position 265 in the second IgG half-mer Amino acid residue substitutions at positions 238 / 267 in the first IgG half-mer and at position 329 in the second IgG half-mer Amino acid residue substitutions at position 329 in the first IgG half-mer and positions 238 / 267 in the second IgG half-mer

[0106] The combination of a modification (first modification) that attenuates the CD16a-binding activity of the CD16a-binding region of the first IgG half-mer and a modification (second modification) that attenuates the CD16a-binding activity of the CD16a-binding region of the second IgG half-mer is preferably one shown in Table 1 below, and more preferably one in which the first modification and the second modification are S267K and P329Y, Y296P and P329Y, S298E and P329Y, D265A and P329Y, or S239R and P329Y.

[0107] [Table 1]

[0108] The amino acid residues after the above substitution may be amino acids that can be substituted for each other. Examples of amino acids that can be substituted for each other are shown below. Amino acids that belong to the same group can be substituted for each other. Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid Group C: asparagine, glutamine D group: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid Group E: proline, 3-hydroxyproline, 4-hydroxyproline Group F: serine, threonine, homoserine Group G: phenylalanine, tyrosine

[0109] The amino acids to be substituted may be natural or non-natural. Natural amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and L-cysteine. Non-natural amino acids include various amino acids having an amino group and a carboxyl group, with derivatives of various natural amino acids being preferred. Many non-natural amino acids are available from various reagent companies (Sigma-Aldrich, TCI, etc.). Many unnatural amino acids have been disclosed in the literature (Chem. Today 2003, 65; Curr Opin Chem Biol. 2000, 6, 645).

[0110] In the first IgG half-mer, the CD16a-binding activity of the first CD16a-binding region is attenuated by modification, and in the second IgG half-mer, the CD16a-binding activity of the second CD16a-binding region is attenuated by modification. Therefore, even if a homo-aggregate antibody structure is formed by two first IgG halves or two second IgG halves, it is unable to bind to CD16a and therefore is unable to exert its activity.

[0111] On the other hand, when a hetero-aggregate antibody structure is formed by the first and second IgG half-mers, it becomes capable of binding to CD16a via the second CD16a-binding region in the first IgG half-mer and the first CD16a-binding region in the second IgG half-mer (see Figure 5).

[0112] The first and second IgG half-mers have different antigen-binding domains. Therefore, by forming a hetero-aggregate antibody structure consisting of the first and second IgG half-mers, an antibody composition can bind to CD16a and specifically exert effector functions such as ADCC activity only when bound to a target cell expressing two different antigens on the same cell surface.

[0113] The hinge domains of each of the first and second IgG halves are altered by partial or complete substitution, deletion, or modification so that disulfide bonds cannot be formed, thereby preventing the formation of disulfide bonds between the H chains of the first and second IgG halves.

[0114] Therefore, the coexistence of the first and second IgG half-mers allows the first and second IgG half-mers to exist in an equilibrium state as an aggregate or half-mer, and high specificity can be exerted against both positive cells.

[0115] As a result, the antibody composition of the present invention can specifically exert effector function on target cells that co-express the first antigen and the second antigen, thereby damaging them, compared to the effector function on target cells that express only the first antigen and target cells that express only the second antigen.

[0116] In the present invention, the phrase "to exhibit an effector function specifically on target cells co-expressing the first and second antigens, compared to the effector function on target cells expressing only the first antigen and target cells expressing only the second antigen" means that the effector function on target cells co-expressing the first and second antigens (double-positive cells) is stronger than the effector function on target cells expressing only the first antigen and target cells expressing only the second antigen (single-positive cells).

[0117] The presence of specific effector function for dual-positive cells compared with single-positive cells can be evaluated by the methods described in the sections [1] High specificity for dual-positive cells and [2] Enhanced effector function.

[0118] Examples of partial substitutions in the hinge domain to prevent the formation of disulfide bonds between the H chains of the first IgG half-mer and the second IgG half-mer include substitutions of amino acid residues at positions 226 and 229 of the EU index. Alternatively, examples include deletion of part or all of the hinge region including the cysteine ​​site. Specific examples of partial substitutions in the hinge domain to prevent the formation of disulfide bonds between the H chains of the first IgG half-mer and the second IgG half-mer include substitutions of amino acid residues C226A and C229A of the EU index.

[0119] To maintain the above-mentioned equilibrium, it is effective to weaken the non-covalent interaction between the H chains of the first and second IgG half-mers, and it is particularly preferable that the interaction between the CH3 domains is weakened.

[0120] Specifically, for example, the interaction between the H chain CH3 domains in the first and second IgG half-mers is preferably weaker than the interaction between the CH3 domains of the IgG1 subclass.

[0121] A preferred method for weakening the CH3 domain interaction compared to that of the IgG1 subclass is to introduce amino acid alterations (such as substitution, deletion, addition, or modification of amino acid residues) into the CH3 domain, and introduction of amino acid residue substitutions is particularly preferred. Examples of CH3 domains into which amino acid alterations can be introduced to weaken the CH3 domain interaction compared to that of the IgG1 subclass include the IgG1 CH3 domain and the IgG4 CH3 domain.

[0122] It has been reported that substituting Arg at position 409 (EU index) in the CH3 domain of human IgG4 with Lys derived from human IgG1 enhances the interaction between CH3 domains (Structure 2011 19: 9: 1274-1282).Furthermore, it has been reported that substituting a specific amino acid residue in the CH3 domain of human IgG1 with Ala identified an amino acid site important for the interaction between CH3 domains of human IgG1 based on the change in free energy (Biochemistry 1998: 37: 9266-9273).

[0123] The amino acid residue substitution that attenuates the interaction between CH3 domains may be any that attenuates the interaction between CH3 domains based on the amino acid sequence of the CH3 domain used, and may be introduced into at least one of the first IgG half-mer and the second IgG half-mer, but is preferably introduced into both. Amino acid residue substitutions that attenuate the interaction between CH3 domains can be identified by evaluating molecules containing the desired amino acid residue substitutions using binding assays such as binding ELISA and SPR, or molecular weight analyses such as SDS-PAGE and native mass spectrometry.

[0124] Specific examples of amino acid residue substitutions that attenuate the interaction between CH3 domains include substituting at least one amino acid residue selected from positions 349, 351, 366, 368, 399, 405, 407, and 409 in the EU index in the CH3 domain with another amino acid residue.

[0125] More specifically, Y349A, L351A, T366A, L368A, D399A, F405A, Y407A, K409A, and K409R are preferred, L368A, Y407A, and K409R are more preferred, and K409R is most preferred. Any one of these amino acid residue substitutions may be introduced, or two or more may be introduced in combination.

[0126] 2. Regulation of effector functions of antibody compositions The antibody composition of the present invention can also confer Fc-dependent effector functions to the first and second IgG half-mers. The effector functions of the antibody composition can be controlled by various methods.

[0127] For example, the first and second IgG half-mers preferably further enhance their CD16a-binding activity by including at least one amino acid residue substitution in the CH2 domain that further enhances their CD16a-binding activity, thereby enhancing the effector function of the antibody composition.

[0128] The amino acid residue substitution that enhances CD16a-binding activity may be introduced into each of the first and second IgG halves, or into only one of them. When the amino acid residue substitution that enhances CD16a-binding activity is introduced into both the first and second IgG halves, the amino acid residue substitution in the first IgG half-mer and the amino acid residue substitution in the second IgG half-mer may be the same or different, but is preferably the same.

[0129] When amino acid residue substitutions for enhancing the CD16a-binding activity of the antibody composition are introduced into both the first and second IgG half-mers, the amino acid residue substitutions will be in regions different from the regions modified to attenuate the CD16a-binding activity of the first and second IgG half-mers.

[0130] Methods for regulating the effector function of the antibody composition of the present invention include the following methods.

[0131] For example, the effector function of an antibody composition can be controlled by using the amino acid sequence of the Fc of the IgG1 subclass in the first and second IgG half-mers to control the amount of fucose (also called core fucose) that is α-1,6-linked to N-acetylglucosamine (GlcNAc) present at the reducing end of the N-linked complex glycan (hereinafter sometimes simply referred to as complex glycan) that is linked to Asn at position 297 in the EU index (WO 2005 / 035586, WO 2002 / 31140, WO 00 / 61739), or by substituting amino acid residues in the Fc of the antibody.

[0132] 1) Regulation of effector function by glycosylation The effector function of the antibody composition can be enhanced or reduced by controlling the content of fucose added to the N-acetylglucosamine at the reducing end of the complex sugar chains bound to the Fc of the first and second IgG half-mers.

[0133] To reduce the amount of fucose added to the N-linked complex glycans attached to the Fc of an IgG halfmer, fucose-free IgG halfmers can be obtained by expressing the IgG halfmers using CHO cells lacking the α1,6-fucosyltransferase gene (FUT8). An antibody composition consisting of fucose-free IgG halfmers has high ADCC activity.

[0134] On the other hand, one method for increasing the content of fucose added to the N-linked complex sugar chains bound to the Fc of an IgG halfmer is to express the IgG halfmer using a host cell transfected with an α1,6-fucosyltransferase gene, thereby obtaining a fucose-bound IgG halfmer. An antibody composition consisting of an IgG halfmer bound to fucose has lower ADCC activity than an antibody composition consisting of an IgG halfmer not bound to fucose.

[0135] In the Fc of an IgG half-mer, an N-glycosylated glycan is bound to the Asn residue at position 297 in the EU index, but no glycans are known to be bound to other Asn residues in the Fc. Therefore, two N-glycosylated glycans are usually bound to one antibody molecule.

[0136] Known N-linked glycans include high mannose, complex, and hybrid types, and any N-linked glycan that does not have fucose bound thereto can exhibit higher ADCC activity than glycans that do have fucose bound thereto.

[0137] Complex sugar chains that bind to the Fc of an IgG half-mer include sugar chains in which one or more N-acetylglucosamines (GlcNAc) or galactose-N-acetylglucosamines (hereinafter referred to as Gal-GlcNAc) are linked via α1-2 or α1-4 bonds to the mannose (Man) at the non-reducing end of the core structure (trimannosyl core structure).

[0138] Further examples include complex-type sugar chains having sialic acid, bisecting N-acetylglucosamine (hereinafter referred to as bisecting GlcNAc) or the like at the non-reducing end of Gal-GlcNAc.

[0139] In the present invention, core fucose or α1,6-fucose refers to a sugar chain structure in which the 6-position of N-acetylglucosamine (hereinafter sometimes referred to as GlcNAc) at the reducing end of an N-glycosidically linked complex sugar chain is α-linked to the 1-position of fucose (hereinafter sometimes referred to as Fuc). Furthermore, an N-glycosidically linked complex sugar chain in which fucose is not bound to N-acetylglucosamine at the reducing end is simply referred to as a sugar chain lacking fucose or lacking core fucose.

[0140] In the present invention, the core structure or trimannosyl core structure refers to a Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc structure.

[0141] The sugar chain bound to the IgG half-mer, a biantennary N-glycoside-linked complex sugar chain (also called a biantennary complex sugar chain), is represented by the following chemical formula:

[0142] [ka]

[0143] The first and second IgG half-mers preferably have an Fc in which a complex sugar chain is bound to Asn at position 297 on the EU index. As long as they have the above-mentioned sugar chain structure, they may have a single sugar chain or multiple different sugar chain structures. Specific examples include antibody compositions in which, of all N-glycoside-linked sugar chains bound to Fc in the first and second IgG half-mers, the proportion of sugar chains in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end (sugar chains lacking core fucose) is 20% or more.

[0144] The proportion of glycans lacking core fucose may be any proportion that increases the ADCC activity of the antibody composition, but is preferably 20% or more, more preferably 51% to 100%, even more preferably 80% to 100%, particularly preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and most preferably 100%.

[0145] A 50% proportion of glycans lacking core fucose includes, for example, an antibody composition containing 100% molecules in which one of the N-glycoside-linked glycans bound to the first and second IgG half-mers does not have fucose bound to it, or an antibody composition containing 50% molecules in which both of the N-glycoside-linked glycans bound to the first and second IgG half-mers do not have fucose bound to it, and 50% molecules in which both of the N-glycoside-linked glycans bound to the first and second IgG half-mers have fucose bound to them.

[0146] In the present invention, the sugar chain lacking fucose may have any structure on the non-reducing end as long as fucose is not bound to N-acetylglucosamine on the reducing end in the chemical formula shown above.

[0147] In the present invention, "no fucose bound to N-acetylglucosamine at the reducing end of the sugar chain (no core fucose)" means that no fucose is substantially bound. Specifically, an IgG half-mer with substantially no fucose bound thereto refers to an IgG half-mer in which fucose is substantially undetectable in the sugar chain analysis described below. "Substantially undetectable" means below the detection limit of measurement. An antibody composition consisting of first and second IgG half-mers in which all sugar chains lack core fucose has the highest ADCC activity.

[0148] The proportion of IgG half-mers having sugar chains lacking fucose in IgG half-mers having Fc to which N-glycoside-linked complex sugar chains are bound can be determined by releasing the sugar chains from the IgG half-mers using a known method such as hydrazinolysis or enzymatic digestion [Biochemical Experimental Methods 23 - Glycoprotein Sugar Chain Research Methods (Academic Press Center), edited by Takahashi Reiko (1989)], fluorescently or isotope-labeling the released sugar chains, and separating the labeled sugar chains by chromatography.

[0149] Furthermore, the proportion of IgG half-mers with fucose-free glycans bound to them in IgG half-mers containing Fc with complex glycans bound to them can be determined by analyzing the released glycans using the HPAED-PAD method (J. Liq. Chromatogr., 6, 1577, 1983).

[0150] 2) Regulation of effector function by amino acid residue substitution The antibody composition of the present invention can enhance or attenuate effector functions such as ADCC activity, ADCP activity, and CDC activity by converting the antibody subclass of Fc or substituting amino acid residues in Fc of the first IgG half-mer and the second IgG half-mer.

[0151] Antibodies of the IgG1 subclass are known to have the highest ADCC activity and CDC activity among IgG subclasses, and the immunoglobulin subclass of the CH2 domain is preferably IgG1.

[0152] For example, the CDC activity of an antibody can be increased by substituting amino acid residues in Fc using the Fc amino acid sequence described in U.S. Patent Application Publication No. 2007 / 0148165. Furthermore, the ADCC or CDC activity of an antibody composition can be enhanced or attenuated by substituting amino acid residues described in U.S. Patent Nos. 6,737,056, 7,297,775, and 7,317,091.

[0153] Specific amino acid residue substitutions that enhance ADCC activity include P247I, A339D, F243L, R292P, Y300L, P396L, T393A, H433P, S239D, S298A, A330L, I332E, E333A, and K334A, while specific amino acid residue substitutions that decrease ADCC activity include L235E, P238A, N297A, K322A, and P331S.

[0154] ADCC activity can be enhanced by combining two or more of the above amino acid residue substitutions, and the number of substituted amino acid residues can be increased depending on the purpose. Examples of amino acid residue substitution combinations that can enhance ADCC activity include S298A / E333A / K334A / P247L, S298A / E333A / K334A / H268E, S298A / E333A / K334A / P247L / N421K, S298A / E333A / K334A / E294W, S298A / E333A / K334A / K326T, and S298A / E333A / R292L / K 334E, S298A / E333A / K334A / S239D, S298A / E333A / K334A / K248M, S239D / I332E, S239D / A330F, S239D / K326T, S239D / K326E, S239D / K326I, S239D / I332D, S239E / I332Y, S239E / I332E, S239E / K326T, and S239E / K326I are preferred.

[0155] Because amino acid residue substitutions that enhance ADCC activity can affect the pharmacokinetics of an antibody, it is preferable that an antibody with amino acid residue substitutions has pharmacokinetics equivalent to that of a wild-type IgG antibody without amino acid residue substitutions. Specific amino acid substitutions include, but are not limited to, S239D, S239E, S239D / K326T, S239D / A330F, S239D / K326E, S239E / I332E, S298A / E333A / K334A, S298A / E333A / K334A / H268E, and S239D / S298A / E333A / L242C / K334C, with S239D / K326T and S239D / S298A / E333A / L242C / K334C being more preferred. Equivalent pharmacokinetics means that the maximum blood concentration (Cmax), blood half-life (t1 / 2), or area under the blood concentration-time curve (AUC) is between 50% and 150% of that of a wild-type IgG antibody.

[0156] Specific amino acid residue substitutions that increase CDC activity include at least one amino acid residue substitution selected from K326A, S267E, H268F, S324T, K274Q, N276K, Y296F, Y300F, K326W, K326Y, E333A, E333S, A339T, D356E, L358M, N384S, K392N, T394F, T394Y, V397M, and V422I.

[0157] CDC activity can also be increased by combining two or more of the above amino acid residue substitutions, and the number of substituted amino acid residues can be increased depending on the purpose. Preferred amino acid residue substitutions that increase CDC activity include at least one amino acid residue substitution selected from N276K, A339T, T394F, and T394Y, N276K and A339T, and K274Q, N276K, Y296F, Y300F, A339T, D356E, L358M, N384S, V397M, and V422I. Specific amino acid residue substitutions that decrease CDC activity include L235E, N297A, K322A, P329A, and P331S.

[0158] Furthermore, the blood half-life can be extended by introducing amino acid residue substitutions such as T250Q, M428L, M252Y, S254T, or T256E into Fc of the human IgG1 subclass. Furthermore, cytotoxic activities such as ADCC activity, ADCP activity, and CDC activity can be reduced or eliminated by using Fc from which N-linked glycosylation has been removed by introducing an amino acid residue substitution at position N297, Fc of the human IgG2 or IgG4 subclass, or a chimeric Fc of IgG2 and IgG4.

[0159] 3. Method for producing antibody composition The antibody composition of the present invention can be produced by a method comprising the following steps 1 to 3. Step 1: A step of introducing a recombinant vector containing DNA encoding the amino acid sequence of an IgG half-mer (hereinafter also referred to as a recombinant vector for expressing an IgG half-mer) into cells to obtain a transformant. Step 2: A step of culturing the transformant obtained in step 1, allowing IgG half-mers to accumulate in the culture, and collecting the IgG half-mers from the culture. Step 3: A step of obtaining an antibody composition consisting of the IgG half-mers collected in Step 2. Each step will be described below.

[0160] [Process 1] Step 1 is a step of introducing a recombinant vector containing DNA encoding the amino acid sequence of at least one of the first and second IgG half-mers into cells to obtain a transformant. Specifically, step (1) includes the following steps (1-1) to (1-3). (1-1) introducing a modification that attenuates the CD16a-binding activity into the first CD16a-binding region of the first IgG half-mer. (1-2) introducing a modification that attenuates the CD16a-binding activity into the second CD16a-binding region of the second IgG half-mer; (1-3) A step of substituting or deleting part or all of the hinge domain, or introducing an alteration that modifies the hinge domain of the first and second IgG half-mers so that disulfide bonds are not formed between the H chains.

[0161] An example of step (1-1) is a step in which, in the preparation of a recombinant vector for expressing a first IgG halfmer, at least one amino acid residue selected from the amino acid residues at positions 235, 236, 237, 238, 239, 265, 266, 267, 268, 269, 294, 295, 296, 297, 298, 299, 301, 325, 327, and 332 according to the EU index is substituted, as appropriate for the subclass of the CH2 domain.

[0162] An example of step (1-2) is substituting at least one amino acid residue selected from the amino acid residues at positions 235, 236, 237, 326, 327, 328, 329, and 330 of the EU index in the construction of a recombinant vector for expressing a second IgG halfmer, which is performed as appropriate depending on the subclass of the CH2 domain.

[0163] An example of step (1-3) is the step of substituting the amino acid residues at positions 226 and 229 of the EU index in the preparation of a recombinant vector for expressing an IgG half-mer, which is carried out appropriately depending on the subclass of the hinge domain.

[0164] IgG half-mers can be obtained using methods described in, for example, Molecular Cloning, 2nd Edition; Current Protocols in Molecular Biology; Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; Monoclonal Antibodies: principles and practice, Third Edition, Acad. Press, 1993; Antibody Engineering, A Practical Approach, IRL Press at Oxford University Press, 1996, for example, by expression in a transformant as follows.

[0165] (1) Construction of recombinant vector for expression of IgG half-mers The recombinant vector for expressing an IgG halfmer is an expression vector for animal cells into which a gene encoding the amino acid sequence of the IgG halfmer that constitutes the antibody composition of the present invention has been incorporated.

[0166] The recombinant vector can be constructed by cloning DNA encoding the amino acid sequence of an IgG half-mer into an expression vector for animal cells.

[0167] The DNA can be synthesized either entirely or by polymerase chain reaction (PCR) (Molecular Cloning, 2nd ed.). Furthermore, by combining these methods, genes encoding IgG half-mers can be generated.

[0168] When animal cells are used as hosts, any expression vector can be used as long as it can function in animal cells. For example, pcDNAI, pCDM8 (Funakoshi), pAGE107 [JP Patent Publication No. 3-22979; Cytotechnology, 3, 133 (1990)], pAS3-3 (JP Patent Publication No. 2-227075), pCDM8 [Nature, 329, 840 (1987)], pcDNAI / Amp (Invitrogen), pcDNA3.1 (Invitrogen), pREP4 (Invitrogen), pAGE103 [J. Biochemistry, 101, 1307], etc. (1987)], pAGE210, pME18SFL3, pKANTEX93 (WO 97 / 10354), N5KG1val (U.S. Pat. No. 6,001,358), and Tol2 transposon vector (WO 2010 / 143698).

[0169] Any promoter can be used as long as it is functional in animal cells. Examples include the promoter of the immediate early (IE) gene of cytomegalovirus (CMV), the SV40 early promoter, retrovirus promoters, metallothionein promoters, heat shock promoters, SRα promoters, and promoters or enhancers of Moloney murine leukemia virus. The enhancer of the IE gene of human CMV may also be used in combination with the promoter.

[0170] The expression vectors that can be used may be either a type in which the antibody H chain and L chain are present on separate vectors or a type in which they are present on the same vector (hereinafter referred to as tandem type).

[0171] (2) Obtaining cDNA encoding the variable region cDNA encoding the VH and VL of any antibody can be obtained as follows: cDNA is synthesized using mRNA extracted from hybridoma cells producing the antibody as a template, and the synthesized cDNA is inserted into a vector such as a phage or plasmid to prepare a cDNA library.

[0172] From the library, recombinant phages or plasmids carrying cDNA encoding VH and recombinant phages or plasmids carrying cDNA encoding the L-chain variable region are isolated using DNA encoding the constant or variable regions of existing antibodies as probes. The full nucleotide sequences of the VH and VL of the antibody of interest on the recombinant phages or recombinant plasmids are determined, and the full amino acid sequences of the VH and VL are deduced from the nucleotide sequences.

[0173] Hybridoma cells producing any non-human animal antibody can be obtained by immunizing a non-human animal with an antigen to which the antibody binds, and then generating hybridomas by combining antibody-producing cells from the immunized animal with myeloma cells according to well-known methods [Molecular Cloning, 2nd Edition; Current Protocols in Molecular Biology; Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; Monoclonal Antibodies: Principles and Practice, Third Edition, Acad. Press, 1993; Antibody Engineering, A Practical Approach, IRL Press at Oxford University Press, 1996]. Single-cell cloned hybridomas are then selected, cultured, and purified from the culture supernatant.

[0174] Any non-human animal can be used, such as a mouse, rat, hamster, or rabbit, as long as it is possible to produce hybridoma cells.

[0175] Methods for preparing total RNA from hybridoma cells include, for example, the guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymol., 154, 3 (1987)] and the RNeasy kit (QIAGEN). Methods for preparing mRNA from total RNA include the oligo(dT)-immobilized cellulose column method [Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab. Press, New York, 1989].

[0176] Furthermore, examples of kits for preparing mRNA from hybridoma cells include Fast Track mRNA Isolation Kit (manufactured by Invitrogen) and Quick Prep mRNA Purification Kit (manufactured by Pharmacia).

[0177] Methods for synthesizing cDNA and preparing a cDNA library include conventional methods (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab. Press, New York, 1989; Current Protocols in Molecular Biology, Supplement 1-34) and methods using commercially available kits, such as Super Script (registered trademark) Plasmid System for cDNA Synthesis and Plasmid Cloning (GIBCO BRL) or ZAP-cDNA Synthesis Kit (Stratagene).

[0178] When preparing a cDNA library, any vector can be used to incorporate the cDNA synthesized using mRNA extracted from hybridoma cells as a template, as long as it is capable of incorporating the cDNA.

[0179] For example, ZAP Express (Strategies, 5, 58, 1992), pBluescript II SK(+) (Nucleic Acids Research, 17, 9494, 1989), λZAP II (Stratagene), λgt10, λgt11 (DNA Cloning: A Practical Approach, I, 49, 1985), Lambda BlueMid (Clontech), λExCell, pT7T3 18U (Pharmacia), pcD2 (Mol. Cell. Biol., 3, 280, 1983), and pUC18 (Gene, 33, 103, 1985) can be used.

[0180] Any E. coli can be used to introduce a cDNA library constructed using a phage or plasmid vector, as long as it allows the introduction, expression, and maintenance of the cDNA library.

[0181] For example, XL1-Blue MRF (Strategies, 5, 81, 1992), C600 (Genetics, 39, 440, 1954), Y1088, Y1090 (Science, 222, 778, 1983), NM522 Journal of Molecular Biology (J. Mol. Biol., 166, 1, 1983), K802 (J. Mol. Biol., 16, 118, 1966), and JM105 (Gene, 38, 275, 1985) are used.

[0182] cDNA clones encoding the VH and VL of non-human animal antibodies can be selected from a cDNA library by colony hybridization or plaque hybridization using probes labeled with isotopes or fluorescent labels (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab. Press, New York, 1989).

[0183] Alternatively, cDNAs encoding VH and VL can be prepared by preparing primers and using cDNA or a cDNA library as a template by PCR (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab. Press, New York, 1989; Current Protocols in Molecular Biology, Supplements 1-34).

[0184] The cDNA selected by the above method is cleaved with an appropriate restriction enzyme or the like, and then cloned into a plasmid such as pBluescript II SK(-) (Stratagene). The cDNA is then subjected to a commonly used base sequence analysis method, such as the dideoxy method of Sanger et al. (Proc. Natl. Acad. Sci., USA, 74, 5463, 1977), and analyzed using an automatic base sequence analyzer, such as the ABI PRISM377 DNA sequencer (Applied Biosystems), to determine the base sequence of the cDNA.

[0185] The entire amino acid sequences of VH and VL are deduced from the determined nucleotide sequence and compared with the entire amino acid sequences of VH and VL of known antibodies (Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services, 1991), thereby confirming whether the obtained cDNA encodes an amino acid sequence that completely contains the VH and VL of an antibody, including the secretory signal sequence.

[0186] Furthermore, when the amino acid sequence of the antibody variable region or the nucleotide sequence of the DNA encoding the variable region is already known, it can be produced using the following method.

[0187] When the amino acid sequence is known, the base sequence of the DNA encoding the variable region can be designed taking into consideration codon usage (Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services, 1991), and several synthetic DNAs of approximately 100 to 150 bases in length can be synthesized based on the base sequence of the designed DNA. These can then be used to perform PCR or to synthesize full-length DNA, thereby obtaining DNA. When the base sequence is known, DNA can be obtained based on that information in the same manner as described above.

[0188] (3) Analysis of the amino acid sequence of the antibody variable region The complete amino acid sequences of antibody VH and VL, including the secretory signal sequence, can be compared with the amino acid sequences of VH and VL of known antibodies (Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services, 1991) to estimate the length of the secretory signal sequence and the N-terminal amino acid sequence, and further to determine the subgroup to which the antibody belongs. The amino acid sequences of each CDR of VH and VL can also be determined in a similar manner.

[0189] (4) Construction of cDNA encoding the variable region of the humanized antibody cDNA encoding the VH and VL of a humanized antibody can be constructed as follows. First, the amino acid sequences of the framework regions (hereinafter referred to as FR) of the VH and VL of a human antibody into which the CDRs of the VH and VL of a non-human animal antibody of interest are grafted are selected. Any amino acid sequences of the FRs of the VH and VL of a human antibody can be used as long as they are those of a human antibody.

[0190] Examples include the amino acid sequences of the VH and VL FRs of human antibodies registered in databases such as the Protein Data Bank, and the consensus amino acid sequences of each subgroup of the VH and VL FRs of human antibodies (Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services, 1991).

[0191] Among these, in order to produce a humanized antibody with sufficient activity, it is preferable to select an amino acid sequence that has as high a homology as possible (at least 60% or more) with the amino acid sequences of the FRs of VH and VL of the target non-human animal antibody.

[0192] Next, the amino acid sequences of the VH and VL CDRs of the target non-human animal antibody are grafted onto the amino acid sequences of the VH and VL FRs of the selected human antibody to design the VH and VL amino acid sequences of the humanized antibody. The designed amino acid sequences are converted into DNA nucleotide sequences taking into account the codon usage frequency found in the nucleotide sequence of the antibody gene (Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 1991), and the DNA nucleotide sequences encoding the VH and VL amino acid sequences of the humanized antibody are designed. The designed DNA nucleotide sequences are then fully synthesized.

[0193] Furthermore, by introducing appropriate restriction enzyme recognition sequences into the 5'-ends of the synthetic DNAs at both ends, they can be easily cloned into the recombinant IgG half-mer expression vector constructed in 3(1) above. After PCR, the amplified product is cloned into a plasmid such as pBluescript II SK(-) (Stratagene), and the nucleotide sequence is determined by the method described in 3(2) above to obtain a plasmid containing the nucleotide sequence of DNA encoding the amino acid sequences of VH and VL of the desired humanized antibody.

[0194] (5) Modification of the amino acid sequence of the variable region of the humanized antibody It is known that humanized antibodies, in which only the CDRs of the VH and VL of a non-human animal antibody are grafted onto the FRs of the VH and VL of a human antibody, have reduced antigen-binding activity compared to the original non-human animal antibody (BIO / TECHNOLOGY, 9, 266, 1991).

[0195] This is thought to be because, in the VH and VL of the original non-human animal antibody, not only the CDRs but also some amino acid residues in the FRs are directly or indirectly involved in antigen-binding activity, and these amino acid residues are changed to different amino acid residues in the FRs of the VH and VL of the human antibody upon CDR transplantation.

[0196] To solve this problem, in humanized antibodies, amino acid residues in the amino acid sequences of the VH and VL FRs of a human antibody that are directly involved in antigen binding or that interact with amino acid residues in the CDRs to maintain the three-dimensional structure of the antibody are identified and then modified with amino acid residues derived from the original non-human animal antibody to increase the reduced antigen-binding activity (BIO / TECHNOLOGY, 9, 266, 1991).

[0197] In the production of humanized antibodies, the most important point is how efficiently to identify the amino acid residues in the FRs that are involved in the antigen-binding activity. For this purpose, the construction and analysis of the three-dimensional structure of antibodies has been carried out by X-ray crystallography (J. Mol. Biol., 112, 535, 1977) or computer modeling (Protein Engineering, 7, 1501, 1994), etc.

[0198] This information on antibody three-dimensional structures has provided much useful information for the production of humanized antibodies. However, a method for producing humanized antibodies that is applicable to all antibodies has not yet been established. Currently, various trial and error processes are required, such as producing several variants of each antibody and examining the correlation with each variant's antigen-binding activity.

[0199] Amino acid residues in the FRs of VH and VL of a human antibody can be modified by PCR using synthetic DNA for modification as described in 3(4). The nucleotide sequence of the PCR amplified product is determined by the method described in 3(2) to confirm that the desired modification has been achieved.

[0200] (6) Expression of IgG half-mers By introducing the recombinant vector for expressing the IgG half-mer described in 3(1) above into suitable animal cells, it is possible to obtain a transformant that transiently or stably produces at least one of the first and second IgG half-mers.

[0201] (6-a) Transient expression of antibody composition The recombinant vectors for expressing IgG subunits obtained in (3) and (6), or modified recombinant vectors thereof, can be used to transiently express antibody compositions, and the antigen-binding activity of the various antibody compositions produced can be efficiently evaluated.

[0202] Any host cells capable of expressing at least one of the first and second IgG halfmers can be used as the host cells into which the recombinant IgG halfmer-expressing vector is introduced, such as COS-7 cells [American Type Culture Collection (ATCC) No.: CRL1651] (Methods in Nucleic Acids Res., CRC Press, 283, 1991).

[0203] The recombinant vector for expressing IgG half-mers can be introduced into COS-7 cells using the DEAE-dextran method (Methods in Nucleic Acids Res., CRC press, 1991) or the lipofection method (Proc. Natl. Acad. Sci. USA, 84, 7413, 1987).

[0204] After introduction of the recombinant vector for expressing IgG halfmers, the expression level and antigen-binding activity of IgG halfmers in the culture supernatant are measured using enzyme-linked immunosorbent assays [Monoclonal Antibodies—Principles and practice, Third Edition, Academic Press (1996); Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory (1988); Monoclonal Antibody Experimental Manual, Kodansha Scientific (1987)], or the like.

[0205] (6-b) Stable expression of IgG half-mers By introducing the recombinant vector for expressing IgG half-mer obtained in (1) into an appropriate host cell, a transformant that stably expresses IgG half-mer can be obtained.

[0206] Any method for introducing DNA into host cells can be used to introduce a recombinant vector into host cells, including, for example, electroporation (Cytotechnology, 3, 133, 1990), calcium phosphate method (JP Patent Publication No. 2-227075), lipofection (Proc. Natl. Acad. Sci. USA, 84, 7413, 1987), injection method [Manipulating the Mouse Embryo: A Laboratory Manual], particle gun (gene gun) method (JP Patent Nos. 2606856 and 2517813), DEAE-dextran method [Biomanual Series 4: Gene Transfer and Expression / Analysis Methods (Yodosha), edited by Yokota Takashi and Arai Kenichi (1994)], and viral vector method (Manipulating the Mouse Embryo, 2nd ed.).

[0207] Any host cells can be used to introduce the recombinant vector, as long as they are capable of expressing at least one of the first and second IgG half-mers, including human leukemia Namalwa cells, monkey COS cells, Chinese hamster CHO cells, HBT5637 (Japanese Patent Publication No. 63-299), rat myeloma cells, mouse myeloma cells, Syrian hamster kidney-derived cells, embryonic stem cells, and fertilized egg cells.

[0208] Specifically, for example, PER.C6, CHO-K1 (ATCC CCL-61), DUKXB11 (ATCC CCL-9096), Pro-5 (ATCC CCL-1781), CHO-S (Life Technologies, Cat #11619), Lec13 cells, rat myeloma cells YB2 / 3HL.P2.G11.16Ag.20 (ATCC No.: CRL1662, also referred to as YB2 / 0), mouse myeloma cells NS0, mouse myeloma cells SP2 / 0-Ag14 (ATCC No.: CRL1581), mouse P3X63-Ag8.653 cells (ATCC No.: CRL1580), and CHO cells (CHO / DG44) lacking the dihydrofolate reductase gene (hereinafter referred to as dhfr) [Proc. Natl. Acad. Sci. Sci. USA, 77, 4216 (1980)], Syrian hamster cells BHK, HBT563 cells, sublines of the above cell lines, cells of the above cell lines acclimatized under serum-free culture, and cells acclimatized under non-adherent culture conditions.

[0209] Cells used for producing IgG halfmers can also be cells in which the amount of core fucose in the glycan linked to Asn in Fc at EU index 297 has been reduced or deleted. Specifically, cells in which the amount of enzyme involved in the synthesis of GDP-L-fucose or transport to the Golgi apparatus or the enzyme involved in the binding of core fucose has been reduced or deleted can be selected, or cells obtained by various artificial techniques can be used as host cells.

[0210] Specifically, cells with controlled core fucose can be prepared by methods such as reducing or deleting enzyme activity associated with core fucose glycosylation, or increasing core fucose-cleaving enzyme activity.

[0211] Examples of enzymes involved in core fucose sugar chain modification include enzymes involved in the synthesis or transport of GDP-L-fucose, and enzymes involved in the binding of core fucose to complex N-glycoside-linked sugar chains.

[0212] Specific examples of enzymes involved in the synthesis of GDP-L-fucose or its transport to the Golgi apparatus include GDP-mannose 4,6-dehydratase (hereinafter referred to as GMD), GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase (hereinafter referred to as Fx), GDP-beta-L-fucose-pyrophosphorylase (GFPP), fucokinase, and GDP-L-fucose transporter.

[0213] An example of an enzyme involved in binding of core fucose is α1,6-fucosyltransferase (hereinafter referred to as FUT8).

[0214] In cells that produce IgG halfmers, one of the above enzyme activities may be reduced or deleted, or a combination of multiple enzyme activities may be reduced or deleted.

[0215] Methods for reducing or eliminating the above-mentioned enzyme activity include, for example, (a) gene disruption techniques targeting the enzyme gene; (b) introducing a dominant-negative form of the enzyme gene; (c) introducing a mutation into the enzyme; (d) suppressing the transcription or translation of the enzyme gene; and (e) selecting strains that are resistant to lectins that recognize glycan structures in which the 6th position of N-acetylglucosamine at the reducing end of the N-glycoside-linked glycan is α-linked to the 1st position of fucose.

[0216] Examples of lectins include lectins that bind to α1,6 fucose, such as lentil lectin LCA (lentil agglutinin derived from Lens Culinaris), pea lectin PSA (pea lectin derived from Pisum sativum), broad bean lectin VFA (aggregate agglutinin derived from Vicia faba), and aleuria aurantia lectin AAL (lectin derived from Aleuria aurantia).

[0217] Specific examples of cells include CHO cells deficient in the FUT8 gene (WO 2005 / 035586, WO 2002 / 31140, WO 2000 / 061739), lectin-resistant Lec13 (Somatic Cell and Molecular Genetics, 12, 55, 1986), cells deficient in the GDP-fucose transporter gene (WO 2003 / 085102), cells deficient in the GDP-mannose 4,6-dehydratase (GMD) gene (WO 2002 / 31140), WGA lectin-resistant cells, and LCA lectin-resistant cells (WO 2002 / 31140).

[0218] In addition to the above methods, it is also possible to express IgG half-mers with high mannose-type N-linked glycans and reduced core fucose content by inhibiting enzymes involved in the synthesis of N-linked glycans, such as mannosidase I and mannosidase II.

[0219] Furthermore, by using host cells overexpressing N-acetylglucosamine transferase III (GnTIII), it is possible to produce IgG half-mers with complex and hybrid glycans linked to bisecting GlcNAc and reduced core fucose content.

[0220] After introduction of the recombinant vector, transformants that stably express IgG half-mers are selected by culturing them in an animal cell culture medium containing drugs such as G418 sulfate (hereinafter abbreviated as G418), cycloheximide (hereinafter abbreviated as CHX), and methotrexate (hereinafter abbreviated as MTX) (Japanese Patent Laid-Open Publication No. 2-257891).

[0221] Examples of media for animal cell culture include RPMI1640 medium (manufactured by Invitrogen), GIT medium (manufactured by Nippon Pharmaceutical Co., Ltd.), EX-CELL301 medium, EX-CELL302, EX-CELL325 medium (manufactured by JRH), IMDM medium (manufactured by Invitrogen), Hybridoma-SFM medium (manufactured by Invitrogen), and media obtained by adding various additives such as fetal bovine serum (hereinafter abbreviated as FBS) to these media.

[0222] The resulting transformant is cultured in a medium to express and accumulate IgG half-mers in the culture supernatant. The expression level and antigen-binding activity of the IgG half-mers in the culture supernatant can be measured by ELISA or other methods. Furthermore, the expression level of the IgG half-mers produced by the transformant can be improved by using a dhfr gene amplification system (Japanese Patent Laid-Open Publication No. 2-257891).

[0223] The above describes a method for expressing IgG half-mers using animal cells as a host. However, IgG half-mers can also be expressed in yeast, insect cells, plant cells, or individual animals or plants using methods similar to those used in animal cells, based on known techniques.

[0224] When yeast is used as the host cell, examples of suitable microorganisms include those belonging to the genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, and Schwanniomyces, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, and Schwanniomyces alluvius.

[0225] Any method for introducing a recombinant vector can be used as long as it is a method for introducing DNA into yeast, and examples thereof include the electroporation method (Methods. Enzymol., 194, 182, 1990), the spheroplast method (Proc. Natl. Acad. Sci. USA, 84, 1929, 1978), and the lithium acetate method (J. Bacteriology, 153, 163, 1983; Proc. Natl. Acad. Sci. USA, 75, 1929, 1978).

[0226] When insect cells are used as hosts, IgG half-mers can be expressed by the method described in, for example, Current Protocols in Molecular Biology (Baculovirus Expression Vectors, A Laboratory Manual, W.H. Freeman and Company, New York, 1992), Bio / Technology, 6, 47, 1988, etc.

[0227] [Process 2] Step 2 is a step of culturing the transformant obtained in step 1 to produce and accumulate IgG half-mers in the culture, and collecting and purifying the IgG half-mers from the culture.

[0228] The first and second IgG half-mers may be collected from the same transformant, or from transformants expressing each of them independently. Typically, the first and second IgG half-mers are collected from transformants expressing each of them independently, purified, and then mixed to prepare an antibody composition.

[0229] When the host cells prepared in step 1 have the ability to express an IgG halfmer, the IgG halfmer can be introduced into the host cells described below, and then the cells can be cultured, and the desired IgG halfmer can be collected from the culture.

[0230] Furthermore, cells of a gene-introduced animal or plant may be redifferentiated to create an animal individual (transgenic non-human animal) or a plant individual (transgenic plant) into which the gene has been introduced, and IgG half-mers may be collected from these individuals.

[0231] When the transformant is an animal or plant, it can be bred or cultivated according to a conventional method to produce and accumulate IgG half-mers, and the IgG half-mers can be collected from the animal or plant.

[0232] Examples of methods for producing IgG half-mers using animal individuals include methods in which a gene is introduced into an animal constructed in accordance with known methods (American Journal of Clinical Nutrition, 63, 639S, 1996; American Journal of Clinical Nutrition, 63, 627S, 1996; Bio / Technology, 9, 830, 1991), and the desired IgG half-mer is produced in the animal.

[0233] In the case of individual animals, for example, transgenic non-human animals into which DNA encoding an IgG half-mer has been introduced are bred, and the IgG half-mer is produced and accumulated in the animal, from which the IgG half-mer can be collected.

[0234] Examples of the site of production and accumulation in an animal include the animal's milk (Japanese Patent Application Laid-Open No. 63-309192) or eggs. Any promoter that can be expressed in an animal can be used in this case. For example, mammary gland cell-specific promoters such as the α-casein promoter, β-casein promoter, β-lactoglobulin promoter, and whey acidic protein promoter are preferably used.

[0235] Examples of methods for producing IgG half-mers using plant individuals include cultivating transgenic plants into which DNA encoding IgG half-mers has been introduced in accordance with known methods [Tissue Culture, 20 (1994); Tissue Culture, 21 (1995); Trends in Biotechnology, 15, 45 (1997)], allowing the IgG half-mers to be produced and accumulated in the plants, and then collecting the IgG half-mers from the plants.

[0236] The IgG half-mer can be purified as follows: When the IgG half-mer is produced by a transformant into which a gene encoding the IgG half-mer has been introduced, for example, and the IgG half-mer is expressed intracellularly as a soluble protein, the cells are collected by centrifugation after the end of the culture, suspended in an aqueous buffer solution, and then disrupted using an ultrasonic disrupter, French press, Manton-Gaulin homogenizer, Dynomill, or the like to obtain a cell-free extract.

[0237] The IgG half-mer can be purified from the supernatant obtained by centrifuging the cell-free extract using conventional enzyme isolation and purification methods, i.e., solvent extraction, salting out with ammonium sulfate or the like, desalting, precipitation with an organic solvent, anion exchange chromatography using a resin such as diethylaminoethyl (DEAE)-Sepharose or DIAION HPA-75 (Mitsubishi Chemical Corporation), cation exchange chromatography using a resin such as S-Sepharose FF (Pharmacia), hydrophobic chromatography using a resin such as butyl Sepharose or phenyl Sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, electrophoresis such as isoelectric focusing, or the like, either alone or in combination.

[0238] In the present invention, affinity chromatography using a CH binder or an Fc binder is used (Monoclonal Antibodies—Principles and practice, Third Edition, Academic Press, 1996; Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).

[0239] Alternatively, if the IgG half-mer is expressed as an insoluble body within the cells, the cells are similarly recovered, disrupted, and centrifuged to recover the insoluble IgG half-mer as a precipitate fraction. The recovered insoluble IgG half-mer is solubilized with a protein denaturant. The solubilized solution is diluted or dialyzed to restore the IgG half-mer to its normal conformation, after which the IgG half-mer can be purified by the same isolation and purification method as above.

[0240] When IgG half-mers are secreted outside the cells, the IgG half-mers or derivatives thereof can be recovered in the culture supernatant. That is, the culture is treated by the same method as above, such as centrifugation, to obtain a culture supernatant, and the IgG half-mers can be purified from the culture supernatant using the same isolation and purification method as above.

[0241] Specifically, the CH binder or Fc binder may be any protein, resin, or the like that binds to CH or Fc, and examples thereof include an Fc-binding protein and an antibody that binds to an antibody heavy chain constant region (CH).

[0242] Examples of Fc-binding proteins include Protein A derived from Staphylococcus Aureus, Protein G derived from hemolytic Streptococcus, Fc receptors and their subclasses (FcγRI, IIA, IIB, IIIA, IIIB), and binding partial fragments thereof.

[0243] Examples of antibodies that bind to the CH include antibodies that bind to the CH1 domain, hinge domain, CH2 domain, or CH3 domain.

[0244] In the present invention, more preferred examples of CH binders include Protein A, Protein G, anti-CH1 antibodies, and binding partial fragments thereof.

[0245] As a method for purifying IgG half-mers, for example, the culture supernatant obtained by culturing the transformant described above in [Step 1] (6) is loaded onto a Protein A column or a Protein G column, and the column is then washed with phosphate buffer saline (hereinafter abbreviated as PBS).

[0246] The IgG half-mer is then eluted from the column with a low pH (pH 2.0 to 6.0) citrate buffer or the like, and the eluate is neutralized with an alkaline Tris buffer or the like. The neutralized eluate is dialyzed against a sufficient amount of PBS or the like to obtain purified IgG half-mer.

[0247] The molecular weight of the purified IgG half-mer can be measured using polyacrylamide gel electrophoresis [Nature, 227, 680 (1970)] or Western blotting [Monoclonal Antibodies—Principles and practice, Third Edition, Academic Press (1996); Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory (1988)], or the like.

[0248] [Process 3] Step 3 is a step of obtaining an antibody composition by mixing the first and second IgG half-mers collected and purified in step 2. The mixing ratio of the first and second IgG half-mers is preferably set appropriately depending on factors such as their antigen-binding activity, their binding activity to an antigen-positive cultured cell line, the strength of the CD16a-binding activity of their respective CD16a-binding domains, and the interaction between the CH3s of the first and second IgG half-mers.

[0249] The antibody composition of the present invention can also be produced as an antibody composition containing a mixture of the first and second IgG halves by simultaneously expressing the above-mentioned recombinant vectors for expressing the first and second IgG halves in host cells and purifying the resulting antibody composition. In this case, the desired mixture ratio of the first and second IgG halves can be obtained by controlling the expression levels of the genes encoding each half. The genes encoding the first and second IgG halves may be expressed using the same recombinant expression vector, or may be expressed using separate recombinant expression vectors.

[0250] 4. Evaluation of antibody composition activity Methods for measuring the protein amount of purified IgG halfmers, and the FcR-binding activity, C1q-binding activity, antigen-binding activity, or cytotoxic activity such as ADCC activity or CDC activity of antibody compositions composed of IgG halfmers include known methods described in, for example, Molecular Cloning 2nd Edition, Current Protocols in Molecular BioloGy; Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; Monoclonal Antibodies: principles and practice, Third Edition, Acad. Press, 1993; Antibody Engineering, A Practical Approach, IRL Press at Oxford University Press, 1996, and the like.

[0251] Specific examples of the antibody composition's binding activity to an antigen and its binding activity to an antigen-positive cultured cell line can be measured by ELISA, fluorescent antibody testing (Cancer Immunol. Immunother, 36, 373, 1993), etc. Cytotoxic activity against an antigen-positive cultured cell line can be evaluated by measuring CDC activity, ADCC activity, etc. (Cancer Immunol. Immunother, 36, 373, 1993; U.S. Patent Application Publication No. 2004 / 0259150).

[0252] Whether an antibody composition has binding activity to CD16a can be confirmed by producing a recombinant CD16a protein and measuring the binding activity (US Patent Application Publication No. 2004 / 0259150).

[0253] Methods for measuring ADCC activity include, for example, a method in which target cells labeled with a radioisotope, a fluorescent substance, a dye, or the like, an antibody composition, and an effector cell are contacted, and then the activity of the labeled substance released from the damaged target cells or the physiological activity of the released enzyme is measured.

[0254] Methods for measuring CDC activity include, for example, contacting target cells labeled with a radioisotope, fluorescent substance, dye, or the like with a biological sample such as an antibody composition and serum containing a complement component, and then measuring the activity of the labeled substance released from the damaged target cells or the physiological activity of the released enzyme.

[0255] 5. Analysis of Glycan Structure The sugar chain structure of the IgG half-mer expressed in various cells can be analyzed in accordance with the analysis of the sugar chain structure of a normal glycoprotein.

[0256] For example, the glycans bound to IgG half-mers are composed of neutral sugars such as galactose (Gal), mannose (Man), or fucose (Fuc), amino sugars such as N-acetylglucosamine (GlcNAc), or acidic sugars such as sialic acid (Sial), and can be analyzed using techniques such as glycan composition analysis and glycan structure analysis using two-dimensional glycan mapping.

[0257] (1) Neutral and amino sugar composition analysis The composition of the sugar chains of the IgG half-mer can be analyzed by acid hydrolysis of the sugar chains with trifluoroacetic acid or the like to liberate neutral sugars or amino sugars, and then analyzing their composition ratios.

[0258] A specific example of such a method is to use a sugar composition analyzer manufactured by Dionex BioLC, which analyzes sugar composition using HPAEC-PAD (high performance anion-exchange chromatography-pulsed amperometric detection) (J. Liq. Chromatogr., 6, 1577, 1983).

[0259] Alternatively, the composition ratio can be analyzed by fluorescent labeling with 2-aminopyridine. Specifically, a sample hydrolyzed with acid according to a known method [Agric. Biol. Chem., 55(1), 283-284, 1991] is fluorescently labeled with 2-aminopyridyl and analyzed by HPLC to calculate the composition ratio.

[0260] (2) Glycan structure analysis Structural analysis of glycans in IgG half-mers can be performed by the two-dimensional glycan mapping method (Anal. Biochem., 171, 73, 1988; Biochemical Experimental Methods 23 - Glycoprotein Glycan Research Methods, Academic Press, edited by Takahashi Reiko, 1989). The two-dimensional glycan mapping method is a method in which, for example, the retention time or elution position of a glycan obtained by reversed-phase chromatography is plotted on the X-axis and the retention time or elution position of a glycan obtained by normal-phase chromatography on the Y-axis, and the glycan structure is estimated by comparing the results with those of known glycans.

[0261] Specifically, IgG half-mers are hydrolyzed with hydrazinolysis to release the glycans from the IgG half-mers. The glycans are then fluorescently labeled with 2-aminopyridine (hereafter abbreviated as PA) (J. Biochem., 95, 197, 1984). The glycans are then separated from excess PA reagent by gel filtration and subjected to reversed-phase chromatography. Each peak of the separated glycans is then subjected to normal-phase chromatography. Based on these results, the glycan structures can be estimated by plotting the glycan structure on a two-dimensional glycan map and comparing the spots with those of glycan standards (TaKaRa) and literature (Anal. Biochem., 171, 73, 1988).

[0262] Furthermore, mass spectrometry such as MALDI-TOF-MS can be performed on each glycan to confirm the structure predicted by the two-dimensional glycan mapping method.

[0263] The part of the Fc of the IgG half-mer to which the glycan is bound can be confirmed by treating the reductively alkylated IgG half-mer with endoproteases such as trypsin, pepsin, Lys-C, and Asp-N, separating the IgG half-mer by reverse-phase chromatography (LC), and then analyzing it with a mass spectrometer (MS) or other device.

[0264] That is, based on the amino acid sequence of the Fc of the target IgG half-mer, whether or not a glycan is actually bound can be confirmed by checking whether or not the molecular weights of the peptides that can be generated by protease treatment and the molecular weights of the peptides bound to glycans match the MS analysis values.

[0265] 6. Methods for identifying glycan structures The proportion of glycans lacking core fucose among all complex-type N-glycoside-linked glycans bound to Fc in an IgG half-mer can be determined by the glycan structure analysis method described in Section 5 above. It can also be determined by immunological quantification using lectins.

[0266] Identification of the sugar chain structure in IgG half-mers using immunological quantification methods using lectins can be carried out, for example, as follows, in accordance with immunological quantification methods such as Western staining, RIA (Radioimmunoassay), VIA (Viroimmunoassay), EIA (Enzymeimmunoassay), FIA (Fluoroimmunoassay), and MIA (Metalloimmunoassay) described in the literature [Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., (1995); Enzyme Immunoassay, 3rd Edition, Igaku-Shoin (1987); Revised Edition, Enzyme Immunoassay, Gakusai Kikaku (1985)].

[0267] A lectin that recognizes the sugar chain structure of an IgG half-mer is labeled, and the labeled lectin is reacted with a sample antibody composition. The amount of the complex between the labeled lectin and the antibody composition is then measured.

[0268] Examples of lectins that can be used to identify the sugar chain structure of IgG half-mers include WGA (wheat-germ agglutinin derived from T. vulgaris), ConA (concanavalin A derived from C. ensiformis), RIC (toxin derived from R. communis), L-PHA (leukoagglutinin derived from P. vulgaris), LCA (lentil agglutinin derived from L. culinaris), and PSA (P.sativum extract Pea Lectin, AAL (Aleuria aurantia Lectin), ACL (Amaranthus caudatus Lectin), BPL (Bauhinia purpurea Lectin), DSL (Datura stramonium Lectin), DBA (Dolichos biflorus Agglutinin), EBL (Elderberry). Balk Lectin, ECL (Erythrina cristagalli Lectin), EEL (Euonymus europaeus Lectin), LNG (Galanthus nivalis Lectin), GSL (Griffonia simplicifolia Lectin), HPA (Helix pomatia Agglutinin), HHL (Hippeastrum Hybrid). Lectin)、Jacalin、LTL(Lotus tetragonolobus Lectin)、LEL(Lycopersicon esculentum Lectin)、MAL(Maackia amurensis Lectin)、MPL(Maclura pomifera Lectin)、NPL(Narcissus pseudonarcissus Lectin)、PNA(Peanut Agglutinin、E-PHA(Phaseolus vulgaris Erythroagglutinin)、PTL(Psophocarpus tetragonolobus Lectin)、RCA(Ricinus communis Agglutinin)、STL(Solanum tuberosum Lectin)、SJA(Sophora japonica Agglutinin)、SBA(Soybean Agglutinin, UEA (Ulex europaeus Agglutinin), VVL (Vicia villosa Lectin), WFA (Wisteria floribunda Agglutinin).

[0269] It is preferable to use a lectin that specifically recognizes core fucose. Specific examples include lentil lectin LCA (lentil agglutinin derived from Lens Culinaris), pea lectin PSA (pea lectin derived from Pisum sativum), broad bean lectin VFA (aggregate agglutinin derived from Vicia faba), and aleuria aurantia lectin AAL (lectin derived from Aleuria aurantia).

[0270] 7. Uses of the antibody compositions of the present invention The antibody composition of the present invention is composed of first and second IgG half-mers having antigen-binding domains for different antigens, and is therefore capable of recognizing two different antigens. Therefore, pharmaceutical compositions containing the antibody composition of the present invention can adopt molecular forms suited to the two different target antigens, and can therefore exhibit high specificity for positive cells expressing both of the two antigens.

[0271] When the antibody composition of the present invention is used in a pharmaceutical composition, it is preferable that the composition has the following properties [1] to [3]. The following properties [1] to [3] can serve as indicators for selecting an excellent antibody composition of the present invention. [1] High specificity for both positive and negative cells [2] Enhanced effector function [3] Serum half-life equivalent to that of wild-type IgG antibodies Each property will be explained below.

[0272] [1] High specificity for both positive and negative cells The antibody composition of the present invention having higher specificity for dual-positive cells than for single-positive cells can be selected, for example, by a method comprising the following steps (1a) to (1d).

[0273] (1a) A first IgG half-mer is prepared by introducing amino acid modifications that eliminate disulfide bonds between H chains in the hinge region, amino acid modifications that reduce the CD16a-binding activity in the first CD16a-binding region, and amino acid modifications that reduce the interaction between CH3 domains.

[0274] (1b) In the same manner as in (1a), a second IgG half-mer is prepared by introducing amino acid modifications that eliminate disulfide bonds between the heavy chains in the hinge region, amino acid modifications that reduce the CD16a-binding activity in the second CD16a-binding region, and amino acid modifications that reduce the interaction between the CH3 domains.

[0275] (1c) Dual-positive cells expressing the first and second antigens, single-positive cells expressing the first antigen, and single-positive cells expressing the second antigen are prepared. To use cells in which the expression level of each antigen is adjusted, transfectants for each antigen may be prepared. The effector function of an antibody composition comprising a mixture of the first IgG half-mer and the second IgG half-mer is evaluated for each cell type.

[0276] (1d) Based on the results of the evaluation in (1c), an antibody composition having a stronger effector function on dual-positive cells than on each single-positive cell is selected, thereby obtaining an antibody composition having higher specificity for dual-positive cells than for single-positive cells.

[0277] Specific methods include, for example, the method described below in Example 3. Specifically, for example, when the effector function is ADCC activity, an antibody composition having higher specificity for dual-positive cells than for single-positive cells can be obtained by selecting an antibody composition having stronger effector function for dual-positive cells compared to single-positive cells, as described below. 1-i) As target cells, single-positive cells expressing a first antigen, single-positive cells expressing a second antigen, and double-positive cells expressing both the first and second antigens are used. Using a flow cytometer, the expression levels of the first and second antigens in each cell are measured to confirm that the target antigens are expressed in the cells. 1-ii) Human peripheral blood mononuclear cells (PBMCs) or a human CD16a-expressing cell line stably expressing human CD16a by introducing a gene encoding human CD16a are used as effector cells, and target cells are prepared using culture medium (e.g., RPMI medium). 1-iii) Place the well in a CO2 incubator and allow to stand for 2-6 hours. At the same time, add a solubilizing solution (e.g., an aqueous solution containing acid, alkali, surfactant, etc.) to the 100% reaction well to completely dissolve the target cells. After centrifuging the reaction vessel, collect the supernatant and dispense it into an ELISA plate. After applying the color-developing solution and allowing it to react, add a stop solution and measure the absorbance (A450) using a plate reader. 1-iv) Calculate ADCC activity (%) using the following formula: ADCC activity (%)=100×(SET) / (Max-T) S = absorbance of sample reaction well - absorbance of medium well E = Effector well absorbance - Medium well absorbance T = target well absorbance - medium well absorbance Max = 100% reaction well - 100% reaction control well 1-vi) As positive control antibodies, an antibody against the first antigen, usually an IgG1 type, and an antibody against the second antigen are used. It is confirmed that the antibody against the first antigen, usually an IgG1 type, exerts ADCC activity against single-positive cells expressing the first antigen, and that the antibody against the second antigen, usually an IgG1 type, exerts ADCC activity against single-positive cells expressing the second antigen.

[0278] On the other hand, if an antibody solution comprising a mixture of antibody halves against a first antigen and antibody halves against a second antigen does not exhibit ADCC activity against single-positive cells expressing the first antigen or single-positive cells expressing the second antigen, but exhibits ADCC activity specifically only against dual-positive cells expressing the first antigen and the second antigen, the antibody composition is evaluated as having a stronger effector function against dual-positive cells than against each single-positive cell.

[0279] An example of "an antibody solution comprising a mixture of antibody halves against a first antigen and antibody halves against a second antigen does not exhibit ADCC activity against single-positive cells expressing the first antigen or single-positive cells expressing the second antigen, but specifically exhibits ADCC activity only against positive cells expressing both the first and second antigens" is when the ADCC activity against single-positive cells expressing the first antigen and single-positive cells expressing the second antigen is comparable to that in a negative control, and when the ADCC activity against positive cells expressing both the first and second antigens is comparable to that in a positive control. Another example is when the ADCC activity against single-positive cells expressing the first antigen and single-positive cells expressing the second antigen is weaker than that in a positive control, and when the ADCC activity against positive cells expressing both the first and second antigens is comparable to that in a positive control.

[0280] Here, "similar ADCC activity" means that the ADCC activity is preferably within a range of ±0 to 50%, more preferably ±0 to 30%, of the target ADCC activity, and "attenuated ADCC activity" means that the ADCC activity is preferably within a range of 0 to 60%, more preferably 0 to 30%, of the target ADCC activity.

[0281] In this evaluation, it is preferable to use an antibody against the first antigen and an antibody against the second antigen as negative controls. The antibody against the first antigen exhibits effector function against both positive cells and single positive cells expressing the first antigen, and the antibody against the second antigen exhibits effector function against both positive cells and single positive cells expressing the second antigen.

[0282] Alternatively, instead of cells expressing each antigen, beads immobilized with each antigen can be used for evaluation. For example, double-positive beads immobilized with both the first and second antigens, single-positive beads immobilized with only the first antigen, and single-positive beads immobilized with only the second antigen are prepared. After adding the antibody composition of the present invention to each bead, recombinant CD16a protein is then added. The binding activity of the recombinant CD16a protein to the antibody compositions bound to the double-positive beads and the two single-positive beads can then be examined to select antibody compositions with high selectivity for both antigens of interest. Methods for measuring the interaction with the recombinant CD16a protein include binding ELISA and surface plasmon resonance.

[0283] [2] Enhanced effector function The antibody composition of the present invention having an enhanced effector function can be selected, for example, by a method comprising the following steps (2a) to (2c).

[0284] (2a) The effector function of an antibody composition selected by the method described in [1] above, which has higher specificity for double-positive cells than for single-positive cells, is enhanced. Means for enhancing the effector function of the antibody composition of the present invention, which has high specificity for double-positive cells selected by the method described in [1] above, include, for example, amino acid modifications that enhance CD16a-binding activity and / or reducing or deleting the content of core fucose bound to Fc.

[0285] When amino acid residue substitutions are introduced to enhance CD16a-binding activity, the amino acid residues may be any amino acid residues contained in the CD16a-binding region formed in the hetero-association of IgG halfmers.

[0286] Furthermore, the amino acid residue substitutions may be introduced into both the first and second IgG half-mers or into either one of them, but in order to achieve the effects of the present invention, it is preferable that the position of the amino acid residue substitution that enhances CD16a-binding activity is different from the position of the amino acid residue substitution that attenuates CD16a-binding activity within the same IgG half-mer.

[0287] In other words, as long as the IgG half-mers are different from each other, the position of the amino acid residue substitution for enhancing CD16a-binding activity may be the same as the position of the amino acid residue substitution for attenuating CD16a-binding activity.

[0288] The position at which an amino acid residue substitution to enhance CD16a-binding activity should be introduced can be determined by preparing hetero-associates consisting of various IgG half-mers in combination with the position of an amino acid residue substitution that attenuates CD16a-binding activity, measuring ADCC activity, etc., and evaluating whether the activity is specific to both CD16a-positive and CD16b-positive cells.

[0289] In addition to substituting amino acid residues to enhance CD16a-binding activity, effector function can be enhanced by reducing or deleting the content of core fucose bound to Fc.

[0290] (2b) The antibody composition whose effector function has been enhanced in (2a) is evaluated for effector function (e.g., ADCC activity) against single-positive cells expressing the first antigen, single-positive cells expressing the second antigen, and positive cells expressing both the first and second antigens.

[0291] (2c) Based on the results of the evaluation in (2b), an antibody composition having a stronger effector function on double-positive cells than on each single-positive cell is selected, thereby obtaining an antibody composition having an enhanced specific effector function on double-positive cells.

[0292] By the above-mentioned method, an antibody composition that has enhanced effector function and exhibits high specificity for both positive and negative cells can be selected.

[0293] The effector function of an antibody composition with enhanced effector function specific to both positive and negative cells can be evaluated for clinical efficacy using an evaluation system that mimics human blood. Examples of evaluation systems that mimic human blood include an ADCC evaluation system supplemented with blood protein components such as human albumin, immunoglobulins, human plasma, and human serum, as well as human NK cells, human peripheral blood mononuclear cells (PBMCs), or human granulocytes, and a cytotoxicity evaluation system using whole human blood.

[0294] Specific examples of evaluation methods that mimic human blood include a method using an ADCC evaluation system described below in [Example 8] and a method using a cytotoxic activity evaluation system described below in [Example 9].

[0295] Specific examples of methods using an ADCC evaluation system include the following: In the above 1-ii), the ADCC activity is measured and evaluated in the same manner as in 1-i) to 1-vi), except that an evaluation system simulating the human body is constructed by adding immunoglobulin to a final concentration of preferably 0.01 to 10% by mass, more preferably 0.1 to 10% by mass, and even more preferably 1 to 4% by mass.

[0296] Specific examples of methods using a cytotoxic activity evaluation system based on a human blood reconstitution system include the following. 2-i) As target cells, single-positive cells expressing the first antigen, single-positive cells expressing the second antigen, and double-positive cells expressing both the first and second antigens are used. Using a flow cytometer, the expression levels of the first and second antigens in each cell are measured to confirm that the target antigens are expressed in the cells. 2-ii) Human blood is centrifuged and the supernatant is collected to obtain plasma. Furthermore, peripheral blood mononuclear cells and granulocyte fractions are obtained from the human blood using an erythrocyte removal agent (e.g., Hetasep: Stemcell Technology, #07806). 2-iii) The plasma collected in 2-ii) and the peripheral blood mononuclear cell and granulocyte fractions are mixed and seeded on a cell culture plate. An antibody composition containing a mixture of antibody halves against the first antigen and antibody halves against the second antigen is then added, and the plate is cultured at 37°C for approximately 12 to 48 hours. 2-iv) The proportion of cells expressing both the first and second antigens is measured by flow cytometry. A wild-type IgG antibody is used as a negative control.

[0297] As a result, when an antibody composition comprising a mixture of antibody halves against a first antigen and antibody halves against a second antigen eliminates both positive cells expressing the first antigen and the second antigen in an antibody concentration-dependent manner, but does not eliminate single positive cells expressing the first antigen or single positive cells expressing the second antigen, it is evaluated that the effector function specific to both positive cells is enhanced.

[0298] "Removal of double-positive cells" means that the proportion of double-positive cells expressing the first antigen and the second antigen when the antibody composition of the present invention is added is lower than the proportion in a negative control. "A low proportion of double-positive cells" means that the proportion is preferably within the range of 0 to 70%, more preferably 0 to 50%, of the proportion of double-positive cells in the negative control.

[0299] "Not removing single-positive cells" means that the proportion of single-positive cells expressing the first antigen and the second antigen when the antibody composition of the present invention is added is approximately the same as that in the negative control. "Similar proportion of single-positive cells" means that the proportion of single-positive cells is preferably within ±0 to 40%, more preferably ±0 to 20%, of the proportion of single-positive cells in the negative control.

[0300] This evaluation makes it possible to select antibody compositions that exhibit enhanced effector functions even in human blood.

[0301] (3a) Antibody compositions with high specificity for both positive cells and enhanced effector functions, selected using the methods described above in [1] and [2], will be subjected to PK (pharmacokinetic) testing using animals such as mice and monkeys. A wild-type IgG antibody is used as a negative control.

[0302] (3b) Based on the results of the PK test in (3a), antibody compositions that exhibit kinetics similar to those of wild-type IgG antibodies are selected as antibody compositions with a half-life in blood equivalent to that of the wild-type IgG antibody. Specific methods include, for example, the method described below in [Example 7].

[0303] Specific examples of PK testing methods include the following. 3-i) An antibody composition (preferably 0.1-10 mg / kg) that combines high specificity for both positive and negative cells and enhanced effector function is administered into the tail vein of a mouse (e.g., a BALB / c mouse), and blood is collected preferably 1 hour, 4 hours, 24 hours, and 72 hours later. Under isoflurane anesthesia, the cheek is pricked with a lancet to collect blood, and serum is collected by centrifugation in a micro-collection tube (e.g., BD Microtina®) at room temperature, preferably at 4,000-16,000 rpm, for 5-20 minutes. A wild-type IgG antibody is used as a positive control antibody. 3-ii) Measuring the antibody concentration present in serum. The antibody concentration in serum can be measured by known methods, for example, using the AlphaLISA Human Kappa light chain immunoassay kit (AL3023, PerkinElmer), and a calibration curve can be prepared using the kappa light chain included in the kit. 3-iii) An antibody composition whose blood half-life (t1 / 2), which can be calculated from the antibody concentration measured in 3-ii), is preferably within the range of 50% to 150% compared to the positive control wild-type IgG, is evaluated as having a blood half-life equivalent to that of a wild-type IgG antibody.

[0304] Instead of PK testing using animals, antibody compositions with long blood half-lives can also be selected by measuring the denaturation midpoint (Tm) value using differential scanning calorimetry (DSC) as a physicochemical characteristic of the antibody.

[0305] Among the antibody compositions of the present invention, those having a Tm value (midpoint temperature of denaturation) equivalent to that of a wild-type IgG antibody exhibit good PK. Specific methods for measuring the Tm value include the method described below in Example 7. Specifically, the Tm value can be measured by differential scanning fluorimetry (DSF), and an example of a measuring device is NanoTemper's Prometheus NT.48. The Tm value of an antibody can be measured by starting from preferably 10°C to 30°C and raising the temperature to 80°C to 100°C, preferably at a heating rate of 0.5°C to 2°C / min.

[0306] It is also known that the higher the binding activity of an antibody to FcRn (fetal Fc receptor) at pH 6.0, the longer its half-life in blood. Therefore, antibody compositions with long blood half-lives can be selected by measuring their binding activity to FcRn. FcRn binding activity can be measured, for example, by the method disclosed in J. Immunol. 2002;169:5171-80.

[0307] Antibody compositions having properties favorable for use in pharmaceuticals can be selected by evaluating the above-mentioned [1] to [3]. Preferred combinations of amino acid residue substitutions possessed by antibody compositions of the present invention having these properties are preferably those shown in Table 2, more preferably those shown in Tables 3A and 3B, and most preferably those shown in Table 4.

[0308] [Table 2]

[0309] [Table 3A]

[0310] [Table 3B]

[0311] That is, the combinations of amino acid residue substitutions shown in Tables 3A and 3B are as follows:

[0312] <First IgG half-mer> 1)C226A, C229A, D265A, S239D, K326T, L368A 2)C226A, C229A, D265A, S239D, K326T, Y407A 3)C226A, C229A, D265A, S239D, K326T, K409R 4)C226A, C229A, D265A, S239D, S298A, E333A, L242C, K334C, L368A 5)C226A, C229A, D265A, S239D, S298A, E333A, L242C, K334C, Y407A 6)C226A, C229A, D265A, S239D, S298A, E333A, L242C, K334C, K409R 7)C226A, C229A, S298E, S239D, K326T, L368A 8)C226A, C229A, S298E, S239D, K326T, Y407A 9)C226A, C229A, S298E, S239D, K326T, K409R 10)C226A, C229A, S298E, S239D, E333A, L242C, K334C, L368A 11)C226A, C229A, S298E, S239D, E333A, L242C, K334C, Y407A 12)C226A, C229A, S298E, S239D, E333A, L242C, K334C, K409R

[0313] <Second IgG half-mer> 1)C226A, C229A, P329Y, S239D, K326T, L368A 2)C226A, C229A, P329Y, S239D, K326T, Y407A 3)C226A, C229A, P329Y, S239D, K326T, K409R 4)C226A, C229A, P329Y, S239D, S298A, E333A, L242C, K334C, L368A 5)C226A, C229A, P329Y, S239D, S298A, E333A, L242C, K334C, Y407A 6)C226A, C229A, P329Y, S239D, S298A, E333A, L242C, K334C, K409R

[0314] The antibody composition of the present invention preferably contains the second IgG halfmers [1) to 6)] of the same number as each of the first IgG halfmers [1) to 6)]. Furthermore, the antibody composition of the present invention preferably contains the second IgG halfmers in the following combinations with respect to each of the first IgG halfmers [7) to 12)]. First IgG half-mer 7) and second IgG half-mer 1) First IgG half-mer 8) and second IgG half-mer 2) First IgG half-mer 9) and second IgG half-mer 3) First IgG half-mer 10) and second IgG half-mer 4) First IgG half-mer 11) and second IgG half-mer 5) First IgG half-mer 12) and second IgG half-mer 6)

[0315] More specifically, preferred embodiments of the amino acid residue substitution combinations in the antibody composition of the present invention include, for example, the following: <1> ~ <12> Examples include:

[0316] <1> (2A) The first IgG half-mer and the second IgG half-mer contain amino acid residue substitutions of C226A and C229A, respectively, as represented by the EU index. (3A) The first IgG half-mer comprises an antigen-binding domain that binds to a first antigen, and comprises an amino acid residue substitution of D265A as represented by the EU index in the first CD16a-binding region. (4A) The second IgG half-mer comprises an antigen-binding domain that binds to a second antigen and comprises an amino acid residue substitution of P329Y as represented by the EU index in the second CD16a-binding region. (5A) The first IgG half-mer and the second IgG half-mer each contain amino acid residue substitutions of (a) S239D and K326T, as represented by the EU index. (6A) The first IgG half-mer and the second IgG half-mer each contain L368A, which is represented by the EU index.

[0317] <2> (2A) The first IgG half-mer and the second IgG half-mer contain amino acid residue substitutions of C226A and C229A, respectively, as represented by the EU index. (3A) Each of the first IgG half-mers comprises an antigen-binding domain that binds to a first antigen and comprises an amino acid residue substitution of D265A, as represented by the EU index, in the first CD16a-binding region. (4A) The second IgG half-mer comprises an antigen-binding domain that binds to a second antigen and comprises an amino acid residue substitution of P329Y as represented by the EU index in the second CD16a-binding region. (5A) The first IgG half-mer and the second IgG half-mer each contain amino acid residue substitutions of (a) S239D and K326T, as represented by the EU index. (6A) The first IgG half-mer and the second IgG half-mer each contain Y407A, which is represented by the EU index.

[0318] <3> (2A) The first IgG half-mer and the second IgG half-mer contain amino acid residue substitutions of C226A and C229A, respectively, as represented by the EU index. (3A) Each of the first IgG half-mers comprises an antigen-binding domain that binds to a first antigen and comprises an amino acid residue substitution of D265A, as represented by the EU index, in the first CD16a-binding region. (4A) The second IgG half-mer comprises an antigen-binding domain that binds to a second antigen and comprises an amino acid residue substitution of P329Y as represented by the EU index in the second CD16a-binding region. (5A) The first IgG half-mer and the second IgG half-mer each contain amino acid residue substitutions of (a) S239D and K326T, as represented by the EU index. (6A) The first IgG half-mer and the second IgG half-mer each contain K409R, as represented by the EU index.

[0319] <4> (2A) The first IgG half-mer and the second IgG half-mer contain amino acid residue substitutions of C226A and C229A, respectively, as represented by the EU index. (3A) Each of the first IgG half-mers comprises an antigen-binding domain that binds to a first antigen and comprises an amino acid residue substitution of D265A, as represented by the EU index, in the first CD16a-binding region. (4A) The second IgG half-mer comprises an antigen-binding domain that binds to a second antigen and comprises an amino acid residue substitution of P329Y as represented by the EU index in the second CD16a-binding region. (5A) The first IgG half-mer and the second IgG half-mer each contain amino acid residue substitutions of (b) S239D, S298A, E333A, L242C, and K334C, as represented by the EU index. (6A) The first IgG half-mer and the second IgG half-mer each contain L368A, which is represented by the EU index.

[0320] <5> (2A) The first IgG half-mer and the second IgG half-mer contain amino acid residue substitutions of C226A and C229A, respectively, as represented by the EU index. (3A) The first IgG half-mer comprises an antigen-binding domain that binds to a first antigen, and comprises an amino acid residue substitution of D265A as represented by the EU index in the first CD16a-binding region. (4A) The second IgG half-mer comprises an antigen-binding domain that binds to a second antigen and comprises an amino acid residue substitution of P329Y as represented by the EU index in the second CD16a-binding region. (5A) The first IgG half-mer and the second IgG half-mer each contain amino acid residue substitutions of (b) S239D, S298A, E333A, L242C, and K334C, as represented by the EU index. (6A) The first IgG half-mer and the second IgG half-mer each contain Y407A, which is represented by the EU index.

[0321] <6> (2A) The first IgG half-mer and the second IgG half-mer contain amino acid residue substitutions of C226A and C229A, respectively, as represented by the EU index. (3A) The first IgG half-mer comprises an antigen-binding domain that binds to a first antigen, and comprises an amino acid residue substitution of D265A as represented by the EU index in the first CD16a-binding region. (4A) The second IgG half-mer comprises an antigen-binding domain that binds to a second antigen and comprises an amino acid residue substitution of P329Y as represented by the EU index in the second CD16a-binding region. (5A) The first IgG half-mer and the second IgG half-mer each contain amino acid residue substitutions of (b) S239D, S298A, E333A, L242C, and K334C, as represented by the EU index. (6A) The first IgG half-mer and the second IgG half-mer each contain K409R, as represented by the EU index.

[0322] <7> (2A) The first IgG half-mer and the second IgG half-mer contain amino acid residue substitutions of C226A and C229A, respectively, as represented by the EU index. (3A) The first IgG half-mer comprises an antigen-binding domain that binds to a first antigen and comprises an amino acid residue substitution of S298E, as represented by the EU index, in the first CD16a-binding region. (4A) The second IgG half-mer comprises an antigen-binding domain that binds to a second antigen and comprises an amino acid residue substitution of P329Y as represented by the EU index in the second CD16a-binding region. (5A) The first IgG half-mer and the second IgG half-mer each contain amino acid residue substitutions of (a) S239D and K326T, as represented by the EU index. (6A) The first IgG half-mer and the second IgG half-mer each contain K409R, as represented by the EU index.

[0323] <8> (2A) The first IgG half-mer and the second IgG half-mer contain amino acid residue substitutions of C226A and C229A, respectively, as represented by the EU index. (3A) The first IgG half-mer comprises an antigen-binding domain that binds to a first antigen and comprises an amino acid residue substitution of S298E, as represented by the EU index, in the first CD16a-binding region. (4A) The second IgG half-mer comprises an antigen-binding domain that binds to a second antigen and comprises an amino acid residue substitution of P329Y as represented by the EU index in the second CD16a-binding region. (5A) The first IgG half-mer and the second IgG half-mer each contain amino acid residue substitutions of (a) S239D and K326T, as represented by the EU index. (6A) The first IgG half-mer and the second IgG half-mer each contain L368A, which is represented by the EU index.

[0324] <9> (2A) The first IgG half-mer and the second IgG half-mer contain amino acid residue substitutions of C226A and C229A, respectively, as represented by the EU index. (3A) Each of the first IgG half-mers comprises an antigen-binding domain that binds to a first antigen and contains an amino acid residue substitution of S298E, as represented by the EU index, in the first CD16a-binding region. (4A) The second IgG half-mer comprises an antigen-binding domain that binds to a second antigen and comprises an amino acid residue substitution of P329Y as represented by the EU index in the second CD16a-binding region. (5A) The first IgG half-mer and the second IgG half-mer each contain amino acid residue substitutions of (a) S239D and K326T, as represented by the EU index. (6A) The first IgG half-mer and the second IgG half-mer each contain Y407A, which is represented by the EU index.

[0325] <10> (2A) The first IgG half-mer and the second IgG half-mer contain amino acid residue substitutions of C226A and C229A, respectively, as represented by the EU index. (3A) Each of the first IgG half-mers comprises an antigen-binding domain that binds to a first antigen and contains an amino acid residue substitution of S298E, as represented by the EU index, in the first CD16a-binding region. (4A) The second IgG half-mer comprises an antigen-binding domain that binds to a second antigen and comprises an amino acid residue substitution of P329Y as represented by the EU index in the second CD16a-binding region. (5A) The first IgG half-mer contains the amino acid residue substitutions S239D, E333A, L242C, and K334C as indicated by the EU index, and the second IgG half-mer contains the amino acid residue substitutions S239D, S298A, E333A, L242C, and K334C as indicated by the EU index. (6A) The first IgG half-mer and the second IgG half-mer each contain L368A, which is represented by the EU index.

[0326] <11> (2A) The first IgG half-mer and the second IgG half-mer contain amino acid residue substitutions of C226A and C229A, respectively, as represented by the EU index. (3A) The first IgG half-mer comprises an antigen-binding domain that binds to a first antigen and comprises an amino acid residue substitution of S298E, as represented by the EU index, in the first CD16a-binding region. (4A) The second IgG half-mer comprises an antigen-binding domain that binds to a second antigen and comprises an amino acid residue substitution of P329Y as represented by the EU index in the second CD16a-binding region. (5A) The first IgG half-mer contains the amino acid residue substitutions S239D, E333A, L242C, and K334C as indicated by the EU index, and the second IgG half-mer contains the amino acid residue substitutions S239D, S298A, E333A, L242C, and K334C as indicated by the EU index. (6A) The first IgG half-mer and the second IgG half-mer each contain Y407A, which is represented by the EU index.

[0327] <12> (2A) The first IgG half-mer and the second IgG half-mer contain amino acid residue substitutions of C226A and C229A, respectively, as represented by the EU index. (3A) The first IgG half-mer comprises an antigen-binding domain that binds to a first antigen and comprises an amino acid residue substitution of S298E, as represented by the EU index, in the first CD16a-binding region. (4A) The second IgG half-mer comprises an antigen-binding domain that binds to a second antigen and comprises an amino acid residue substitution of P329Y as represented by the EU index in the second CD16a-binding region. (5A) The first IgG half-mer contains the amino acid residue substitutions S239D, E333A, L242C, and K334C as indicated by the EU index, and the second IgG half-mer contains the amino acid residue substitutions S239D, S298A, E333A, L242C, and K334C as indicated by the EU index. (6A) The first IgG half-mer and the second IgG half-mer each contain K409R, as represented by the EU index.

[0328] [Table 4]

[0329] Furthermore, the antibody composition of the present invention is preferably an antibody composition comprising a first IgG halfmer and a second IgG halfmer shown in Table 5. In Table 5, each SEQ ID NO: indicates the SEQ ID NO of the amino acid sequence of the CH domain.

[0330] [Table 5]

[0331] One embodiment of the present invention is the first IgG halfmer or the second IgG halfmer that constitutes the above-mentioned antibody composition.

[0332] The first IgG halfmer of the present invention can associate with a second IgG halfmer in positive cells expressing both the desired first and second antigens, thereby exerting effector function specifically in both positive cells. The first IgG halfmer of the present invention can be used in combination with the second IgG halfmer, in the production of an antibody composition consisting of the first IgG halfmer and the second IgG halfmer, and in the production of a pharmaceutical composition comprising an antibody composition consisting of the first IgG halfmer and the second IgG halfmer.

[0333] Furthermore, the second IgG halfer of the present invention includes a second IgG halfer characterized by associating with the first IgG halfmer, which can associate with the first IgG halfmer in positive cells expressing both the desired first and second antigens and thereby exert effector function specifically in both positive cells. The second IgG halfmer of the present invention can be used in combination with the first IgG halfmer, for producing an antibody composition consisting of the first IgG halfmer and the second IgG halfmer, and for producing a pharmaceutical composition containing an antibody composition consisting of the first IgG halfmer and the second IgG halfmer.

[0334] The first IgG halfmer or second IgG halfmer contained in the above-exemplified antibody compositions is an IgG halfmer characterized by associating with the corresponding IgG halfmer, and is therefore included in the first or second IgG halfmer of the present invention.

[0335] Furthermore, the first or second IgG half-mer of the present invention may be an IgG half-mer that specifically binds to any antigen, as long as the antigen to which the first IgG half-mer binds is different from the antigen to which the second IgG half-mer binds.

[0336] The antigen to which the antibody composition of the present invention binds may be any antigen, preferably an antigen molecule associated with cancer, immune disease, allergic disease, cardiovascular disease, etc. Examples include cytokines, chemokines, growth factors and their receptors, and CD antigens.

[0337] Examples of cytokine or growth factor receptors include receptors for interferon (hereinafter referred to as IFN)-α, IFN-β, IFN-γ, interleukin (hereinafter referred to as IL)-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, IL-27, granulocyte colony-stimulating factor (G-CSF), granulocyte / macrophage colony-stimulating factor (GM-CSF), or macrophage colony-stimulating factor (M-CSF).

[0338] Examples of chemokine receptors include receptors for SLC, ELC, I-309, TARC, MDC, MIP-3α, and CTACK.

[0339] Examples of growth factor receptors include receptors for epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), angiopoietin, fibroblast growth factor (FGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), erythropoietin (EPO), thrombopoietin (TPO), TGFβ, ephrin, angiopoietin, Frizzled ligand, and SDF-1.

[0340] As Cluster of Differentiation (hereinafter referred to as CD) antigens, there are CD1a, CD1c (BDCA1), CD1d, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26 (DPP-4), CD27, CD28, CD30, CD32, CD32a, CD33, CD34, CD37, CD38, CD39, CD40, CD43, CD44, CD45, CD47, CD48, CD49, CD49a, CD49b, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD59, CD62E, CD62L, CD62P, CD63, CD64, CD66a (CEACAM1), CD66b (NCA-95), CD66c (NCA-50 / 90), CD66d (CGM1), CD66e (CEA), CD66f (PSG), CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD76, CD77, CD78, CD79a, CD79b, CD80 (B7.1), CD81, CD82, CD83, CD84 (SLAMF5), CD85a (ILT-5), CD85b (ILT8), CD85c (LIR8), CD85d (ILT4), CD85f (ILT11), CD85g (ILT7), CD85h (ILT1), CD85i (LIR6a), CD85j (ILT2), CD85k (ILT3), CD85m (ILT10), CD86 (B7.2), CD87, CD89, CD94 (NKG2), CD95 (Fas), CD97, CD98, CD103, CD106, CD107a (LAMP1), CD114 (G-CSFR), CD115 (M-CSFR, CSF1R), CD116 (GM-CSFR), CD117 (SCF-R,C-KIT)、CD119(IFNGR1)、CD121a(IL-1R1)、CD122(IL-2Rb)、CD123(IL-3Ra)、CD124(IL-4Ra)、CD125(IL-5Ra)、CD126(IL-6Ra)、CD127(IL-7Ra)、CD134(OX40)、CD135(FLT3)、CD137(4-1BB)、CD138(Syndecan-1)、CD140(PDGFR)、CD146(MUC18)、CD147(EMMRRIN)、CD152(CTLA-4)、CD153(CD30リガンド)、CD158a(KIR2DL1)、CD158b1(KIR2DL2)、CD158b2(KIR2DL3)、CD158c(KIR2DS6)、CD158d(KIR2DL4)、CD158e1(KIR3DL1)、CD158e2(KIR3DS1)、CD158f(KIR2DL5)、CD158g(KIR2DS5)、CD158h(KIR2DS1)、CD158i(KIR2DS4)、CD158j(KIR2DS2)、CD158k(KIR3DL2)、CD159a(NKG2A)、CD159c(NKG2C)、CD161(NKRP1A)、CD162(PSGL-1)、CD163、CD169(SIGLEC1)、CD177、CD178(FasL)、CD183(CXCR3)、CD184(CXCR4)、CD185(CXCR5)、CD191(CCR1)、CD193(CCR3)、CD194(CCR4)、CD195(CCR5)、CD196(CCR6)、CD197(CCR7)、CD198(CCR8)、CD199(CCR9)、CD200(OX2)、CD206(MMR)、CD207(Langerin),CD209(DC-SIGN), CD212(IL-12Rβ1), CD213a1(IL-13Ra1), CD213a2(IL-13Ra2), CD215(IL- 15RA, CD217(IL-17R), CD218a(IL-18Ra), CD218b(IL-18Rβ), CD223(LAG3), CD226(DNAM-1). )、CD229(SLAMF3), CD252(OX40L), CD269(BCMA), CD272(BTLA), CD274(PD-L1), CD275(ICOS). CD276(B7H3), CD278(ICOS), CD279(PD-1), CD281(TLR1), CD282(TLR2), CD283(TLR3) CD284(TLR4), CD286(TLR6), CD288(TLR8), CD289(TLR9), CD294(CRTH2), CD301(CLEC10A) MGL), CD302(DCL1), CD303(BDCA2), CD304(BDCA4), CD305, CD314(NKG2D), CD317(BST2), CD 319 (CS1), CD324 (E-cadherin), CD326 (EpCAM), CD357 (GITR), CD358 (DR6), CD360 (IL-21R) . CD365(TIM-1), CD366(TIM-3), CD369(DECTIN-1), CD370(CLEC9A), CD371(CLEC12A), human leukocyte antigen(HLA)-Class II(HLA-DR) and HLA-I.

[0341] Further, examples of antigens involved in the pathogenesis of tumors or antigens of antibodies that regulate immune function include gangliosides GM1, GM2, GD2, GD3, Lewis X (CD15s), Lewis Y, CD3, CD4, CD40, CD40 ligand, B7 family molecules (e.g., CD80, CD86, CD274, B7-DC, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7), ligands of B7 family molecules (e.g., CD28, CTLA-4, ICOS, PD-1, or BTLA), OX-40, OX-40 ligand, CD137, tumor necrosis factor (TNF) receptor family molecules (e.g., DR3, DR4, DR5, BAFFR, LIGHT, TNFR1, or TNFR2), and TNF-related apoptosis-inducing ligand. receptor (TRAIL) family molecules, receptor family of TRAIL family molecules (e.g., TRAIL-R1, TRAIL-R2, TRAIL-R3, or TRAIL-R4), receptor activator of nuclear factor kappa B ligand (RANK), RANK ligand, CD25, folate receptor, mesothelin, SIGLEC8, cytokine and chemokine receptors [e.g., IL-1RII, IL-12Rβ2, IL-17RB, IL-23R, IL-27Rα, IL-31R, IL-33Rα, IL-36R, transforming growth factor (TGF)βRII, CCR2, CCR10, CXCR1, CXCR2, leukotriene B4 receptor (BLT1)], NK cell receptors (e.g., NKG2D, E4BP4, NKp30, NKp44, NKp46, AhR), T cell receptors (e.g., TCRα / β, TCRVβ11, TCRγ / δ, TSLPR, SLAM, SLAMF6, LAP, GARP, SR-A1, CD200R, DCR3, TIGIT), B cell receptors (e.g., BLYS, APRIL, TSLPR), dendritic cell receptors (e.g., FCER1A, TLR7, CADM1, XCR1, BTLA, SIRPA, DCIR, TROP2, AXL, SIGLEC6, SIGLEC15, CX3CR1, S100A8, S100A9, ASGR1), amino acid transporters (e.g., ASCT2), serine proteases (e.g., Proteinase-3, PR3), and the like.

[0342] Examples of receptor tyrosine kinases include EGF receptors, insulin receptors, IGF-1 receptors, NGF receptors, PDGF receptors, M-CSF receptors, FGF receptors, VEGF receptors, and Eph receptors. Examples of tyrosine kinase-associated receptors include cytokine receptors and Fc receptors. Examples of cell adhesion molecules include cadherins and integrins. Examples of G protein-coupled receptors include adenosine receptors, cannabinoid receptors, and glucagon receptors.

[0343] Epidermal Growth Factor Receptor (EGFR) V-ERB-Avian Leukemia Viral Oncogene Homolog2(HER2)、V-ERB-B2 Avian Erythroblastic Leukemia Viral Oncogene Homolog3(HER3)、V-ERB-B2 Avian Erythroblastic Leukemia Viral Oncogene Homolog4(HER4)、Insulin Receptor-Insulin Growth Factor-I Receptor(IGF1R)、Nerve Growth Factor Receptor(NGFR)、Platelet-derived Growth Factor Receptor, Alpha(PDGFRA)、Platelet-derived Growth Factor Receptor,Beta(PDGFRB)、Colony-stimulating Factor Receptor(CSF1R)-2 Receptor,Alpha(CSF2RA)、Colony-stimulating Factor3 Receptor,Granulocyte(CSF3R)、Fibroblast Growth Factor Receptor1(FGFR1)、Fibroblast Growth Factor Receptor2(FGFR2)、Fibroblast Growth Factor Receptor2(FGFR2)、Fibroblast Growth Factor Receptor(FGFR3) Factor Receptor4(FGFR4)、Kinase Insert Domain Receptor(KDR)、Ephrin Receptor EphA1(EPHA1)、Ephrin Receptor EphA2(EPHA2)、Ephrin Receptor EphA3(EPHA3)、

[0344] Also, Interleukin-1 Receptor 1 (IL-1R1) Interleukin-1 Receptor Accessory Protein(IL-1RAP)、Interleukin-1 Receptor Like 1(IL-1RL1、ST2)、Hepatocyte Growth Factor Receptor(c-Met)、Macrophage Stimulating 1 Receptor(RON)、Platelet-Derived Growth Factor Receptor(PDGFR)、Junctional Adhesion Molecule-Like(JAML)、Nectin-like Protein 5(Necl-5)、Tumor Necrosis Factor Receptor 1(TNF-R1)、Tumor Necrosis Factor Receptor 2(TNF-R2)、TNF-Related Apoptosis-Inducing Ligand Receptor 1(TRAIL-R1)、TNF-Related Apoptosis-Inducing Ligand Receptor 2(TRAIL-R2)、Death Receptor 3(DR3)、Death Receptor 6(DR6)、Receptor Activator of NF-kB(RANK)、Nerve Growth Factor Receptor(NGFR)、Lymphotoxin-beta Receptor (LTβR), OX40 (TNFRSF4), Fas (TNFRSF6), 4-1BBL (TNFRSF9), Fn14 (TNFRSF12A), TACI (TNFRSF13B). BAFF-R(TNFRSF13C), HVEM(TNFRSF14), BCMA(TNFRSF17), GITR(TNFRSF18), TROY(TNFRSF19), Ectodysplasin A1 Receptor(EDAR), Ectodysplasin A2 Receptor(XEDAR), Receptor Expressed in Lymphoid Tissues (RELT), CD3, CD27, CD30, CD40, FcαRI, FcγRIII, FcεRI, FcExamples include Fragment of IgG, Receptor Transporter, Alpha (FCGRT), etc.

[0345] In addition, cell adhesion molecules include integrin Alpha 9 (ITGA9), P-selectin Glycoprotein Ligand-1 (PSGL-1), Cadherin 11 (CDH11), Mucosal Vascular Addressing Cell Adhesion Molecule 1 (MADCAM1), integrin Alpha 4 (ITGA4), integrin Beta 4 (ITGB4), integrin Alpha 4 Beta 7, integrin Alpha 4 Beta 1, and collagen (Type I Collagen). G protein-coupled receptors include adenosine A2A receptor (ADORA2A), adenosine A2B receptor (ADORA2B), repulsive guidance molecule (RGMA), glucagon receptor (GCGR), prolactin receptor (PRLR), glucagon-like peptide 1 receptor (GLP1R), and cannabinoid These include Receptor1 (CB1) and Mas-related G protein coupled receptor-X2 (MRGPRX2).

[0346] A pharmaceutical composition comprising the antibody composition of the present invention is preferably used to treat a disease involving cells expressing the antigen molecule on their surface, and more preferably used to treat cancer, autoimmune diseases, and allergic diseases.

[0347] Examples of cancer include leukemia, lymphoma, multiple myeloma, brain tumor, breast cancer, uterine cancer, cervical cancer, ovarian cancer, esophageal cancer, stomach cancer, colon cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, adrenal cancer, gastrointestinal stromal tumor, mesothelioma, head and neck cancer, kidney cancer, lung cancer, sarcoma, prostate cancer, testicular tumor, bladder cancer, skin cancer, and childhood cancer. Also included are various cancer treatments that rely on the removal of suppressive immune cells.

[0348] Examples of autoimmune diseases or allergies include Guillain-Barré syndrome, myasthenia gravis, multiple sclerosis, chronic gastritis, chronic atrophic gastritis, autoimmune hepatitis, primary biliary cholangitis, ulcerative colitis, Crohn's disease, autoimmune pancreatitis, Takayasu's arteritis, Goodpasture's syndrome, rapidly progressive glomerulonephritis, IgA nephropathy, megaloblastic anemia, autoimmune hemolytic anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Graves' disease, Hashimoto's disease, primary hypothyroidism, idiopathic Addison's disease, type I diabetes, chronic discoid lupus erythematosus, localized scleroderma, pemphigus, pustular psoriasis, psoriatic arthritis, plaque psoriasis, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, and acquired These include epidermolysis bullosa, alopecia areata, vitiligo, Harada's disease, autoimmune optic neuropathy, autoimmune inner ear disorder, idiopathic azoospermia, habitual abortion, rheumatoid arthritis, systemic lupus erythematosus, antiphospholipid syndrome, polymyositis, dermatomyositis, scleroderma, Sjögren's syndrome, IgG4-related disease, vasculitis syndrome, mixed connective tissue disease, autoimmune hemolytic anemia, incompatible blood transfusion, idiopathic thrombocytopenic purpura, atopic dermatitis, polyarteritis nodosa, allergic rhinitis (hay fever), allergic conjunctivitis, allergic gastroenteritis, bronchial asthma, childhood asthma, food allergies, drug allergies, urticaria, anaphylactic shock, transplant rejection, contact dermatitis, and Behcet's disease.

[0349] Specific combinations of first and second antigens bound by the antibody compositions of the present invention, target cells, and diseases to be treated with pharmaceutical compositions containing the antibody compositions are shown below, with corresponding relationships shown in Tables 6A to 6E.

[0350] c1) An antibody composition, wherein the first antigen is CADM1, the second antigen is CCR4, and the target cells are ATL (Adult T-cell Leukemia-lymphoma) cells. The disease to be treated with the pharmaceutical composition containing the antibody composition includes ATL. c2) An antibody composition, wherein the first antigen is EGFR, the second antigen is HER2, and the target cell is a cancer cell. Pharmaceutical compositions containing the antibody composition include those for solid cancers such as stomach cancer and breast cancer. c3) An antibody composition, wherein the first antigen is CD52, the second antigen is CD70, and the target cell is an activated T cell, an activated B cell, or an activated APC. c4) An antibody composition in which the first antigen is CD2, the second antigen is CD70, and the target cell is an activated T cell. c5) An antibody composition, wherein the first antigen is CD19, the second antigen is CD70, and the target cell is an activated B cell. c6) An antibody composition, wherein the first antigen is CD2, the second antigen is CD40 ligand, and the target cell is an activated effector T cell (activated Teff). The disease to be treated with the pharmaceutical composition containing the antibody composition includes Sjogren's syndrome. c7) An antibody composition, wherein the first antigen is PD-L1, the second antigen is one selected from CD19, CD30, CCR4, CD20, CD22 and CD79b, and the target cell is a lymphoma. Diseases that can be treated with pharmaceutical compositions containing the antibody composition include lymphoma. c8) An antibody composition, wherein the first antigen is PD-L1, the second antigen is one selected from CD19, CD30, CCR4, CD20, CD22 and CD79b, and the target cell is a leukemia. The disease to be treated with the pharmaceutical composition containing the antibody composition includes leukemia. c9) An antibody composition, wherein the first antigen is CD8, the second antigen is CCR4, and the target cell is a regulatory T cell (Treg). The pharmaceutical composition containing the antibody composition can be used to treat diseases such as cancer, and the pharmaceutical composition is preferably used for cancer immunotherapy. c10) An antibody composition, wherein the first antigen is CTLA-4, the second antigen is one selected from CD4, CCR4 and GITR, and the target cell is a regulatory T cell (Treg). The pharmaceutical composition containing the antibody composition can be used to treat diseases such as cancer, and the pharmaceutical composition is preferably used for cancer immunotherapy. c11) An antibody composition, wherein the first antigen is TIGIT, the second antigen is one selected from CD4, CCR4 and GITR, and the target cell is a regulatory T cell (Treg). The pharmaceutical composition containing the antibody composition can be used to treat diseases such as cancer, and the pharmaceutical composition is preferably used for cancer immunotherapy. c12) An antibody composition, wherein the first antigen is PD-1, the second antigen is one selected from CD4, CCR4 and GITR, and the target cell is a regulatory T cell (Treg). The pharmaceutical composition containing the antibody composition can be used to treat diseases such as cancer, and the pharmaceutical composition is preferably used for cancer immunotherapy. c13) An antibody composition, wherein the first antigen is OX40, the second antigen is one selected from CD127, CD26, CD70 and CD15s, and the target cell is an effector T cell (Teff). Diseases that can be treated with a pharmaceutical composition containing the antibody composition include autoimmune diseases. c14) An antibody composition, wherein the first antigen is 4-1BB, the second antigen is one selected from CD127, CD26, CD70 and CD15s, and the target cell is an effector T cell (Teff). Diseases that can be treated with a pharmaceutical composition containing the antibody composition include autoimmune diseases. c15) An antibody composition, wherein the first antigen is GITR, the second antigen is one selected from CD127, CD26, CD70 and CD15s, and the target cell is an effector T cell (Teff). Diseases that can be treated with a pharmaceutical composition containing the antibody composition include autoimmune diseases. c16) An antibody composition, wherein the first antigen is CD40 ligand, the second antigen is one selected from CD127, CD26, CD70 and CD15s, and the target cell is an effector T cell (Teff). Diseases that can be treated with a pharmaceutical composition containing the antibody composition include autoimmune diseases. c17) An antibody composition, wherein the first antigen is CD4, the second antigen is CD69, and the target cell is a tissue-resident memory T (TRM) cell. The disease to be treated with a pharmaceutical composition containing the antibody composition includes ANCA (Anti-Neutrophil Cytoplasmic Antibody)-associated glomerulonephritis. c18) An antibody composition, wherein the first antigen is PD-1, the second antigen is CD3, and the target cells are at least one of peripheral helper T (TPH) cells and PD-1highCD8-positive T cells. The disease to be treated with a pharmaceutical composition containing the antibody composition includes rheumatoid arthritis. c19) An antibody composition, wherein the first antigen is PD-1, the second antigen is CD4, and the target cell is a PD-1-positive T cell. The disease to be treated with the pharmaceutical composition containing the antibody composition includes celiac disease. c20) An antibody composition wherein the first antigen is integrin α4β7, the second antigen is one selected from CCR6, CXCR3 and CD161, and the target cell is at least one selected from effector T cells (Teff) and innate lymphoid cells (ILCs). The disease to be treated with a pharmaceutical composition containing the antibody composition includes inflammatory bowel disease. c21) An antibody composition wherein the first antigen is integrin α4β7, the second antigen is CD127, and the target cells are all innate lymphoid cells (ILCs) and intestinal infiltrating T cells other than Tregs. The disease to be treated with a pharmaceutical composition containing the antibody composition includes inflammatory bowel disease. c22) An antibody composition, wherein the first antigen is integrin α4β7, the second antigen is CD40 ligand, and the target cells are locally migrating activated T cells. The target disease for treatment with a pharmaceutical composition containing the antibody composition includes at least one of multiple sclerosis and inflammatory bowel disease. c23) An antibody composition, wherein the first antigen is integrin α4β1, the second antigen is CD40 ligand, and the target cells are locally migrating activated T cells. The target disease for treatment with a pharmaceutical composition containing the antibody composition includes at least one of multiple sclerosis and inflammatory bowel disease. c24) An antibody composition, wherein the first antigen is CXCR5, the second antigen is CD127, and the target cell is an innate lymphoid cell (ILC). Diseases that can be treated with a pharmaceutical composition containing the antibody composition include allergic diseases. c25) An antibody composition, wherein the first antigen is the TPO receptor (c-mpl), the second antigen is CD34, and the target cell is an abnormally proliferative hematopoietic stem cell. The disease to be treated with a pharmaceutical composition containing the antibody composition includes primary myelofibrosis. c26) An antibody composition, wherein the first antigen is a TPO receptor (c-mpl), the second antigen is CD123, and the target cell is a CD123-positive leukemia stem cell. The disease to be treated with a pharmaceutical composition containing the antibody composition includes primary myelofibrosis. c27) An antibody composition in which the first antigen is CXCR3, the second antigen is CD3, and the target cell is a CX3CR1-positive cell (such as a cytotoxic effector T cell, excluding an NK cell). The disease to be treated with a pharmaceutical composition containing the antibody composition includes at least one selected from autoimmune diseases (for example, Crohn's disease, etc.), arteriosclerosis, and ischemic heart disease. c28) An antibody composition, wherein the first antigen is CXCR3, the second antigen is CD127 or CD40 ligand, and the target cell is a Th1 cell. The disease to be treated with a pharmaceutical composition containing the antibody composition includes systemic lupus erythematosus. c29) An antibody composition, wherein the first antigen is CCR4, the second antigen is CD127 or CD40 ligand, and the target cells are CCR4-positive T cells other than Tregs. Diseases that can be treated with a pharmaceutical composition containing the antibody composition include allergic diseases. c30) An antibody composition, wherein the first antigen is CCR6, the second antigen is CD127 or CD40 ligand, and the target cell is a Th17 cell. The disease to be treated with the pharmaceutical composition containing the antibody composition includes psoriasis. c31) An antibody composition, wherein the first antigen is CRTH2, the second antigen is CD127 or CD40 ligand, and the target cell is a Th2 cell. Diseases that can be treated with a pharmaceutical composition containing the antibody composition include allergic diseases. c32) An antibody composition, wherein the first antigen is CRTH2, the second antigen is CCR4, and the target cell is a Th2 cell. The disease to be treated by the pharmaceutical composition containing the antibody composition is at least one selected from asthma, eosinophilic sinusitis, and atopic dermatitis. c33) An antibody composition, wherein the first antigen is CRTH2, the second antigen is ST2, and the target cell is a Tpath2 cell. The disease to be treated with the pharmaceutical composition containing the antibody composition includes at least one of asthma and eosinophilic sinusitis. c34) An antibody composition, wherein the first antigen is CRTH2, the second antigen is CCR6, and the target cells are Th2 / Th17 cells (heterozygous cells that appear only in severe asthma). Diseases that can be treated with a pharmaceutical composition containing the antibody composition include allergic diseases. c35) An antibody composition, wherein the first antigen is CRTH2, the second antigen is CCR3, and the target cells are eosinophils, basophils, or Th2 cells. Diseases that can be treated with a pharmaceutical composition containing the antibody composition include allergic diseases. c36) An antibody composition, wherein the first antigen is CD207, the second antigen is CD11b or CD1a, and the target cell is a Langerhans cell precursor. The disease to be treated with a pharmaceutical composition containing the antibody composition includes Langerhans cell histiocytosis (LCH). c37) An antibody composition, wherein the first antigen is CD123, the second antigen is HLA-DR, and the target cell is a plasmacytoid dendritic cell. The disease to be treated with a pharmaceutical composition containing the antibody composition includes systemic lupus erythematosus. c38) An antibody composition, wherein the first antigen is CD123, the second antigen is ASCT2, and the target cell is at least one selected from plasmacytoid dendritic cells, Th1 cells, and Th17 cells. The disease to be treated with a pharmaceutical composition containing the antibody composition includes systemic lupus erythematosus. c39) An antibody composition, wherein the first antigen is CSF1R, the second antigen is CD14, and the target cell is a monocytic myeloid-derived suppressor cell. The disease to be treated with a pharmaceutical composition containing the antibody composition includes idiopathic pulmonary fibrosis. c40) An antibody composition, wherein the first antigen is CSF1R, the second antigen is CD33, and the target cell is a myeloid-derived suppressor cell. Diseases that can be treated with pharmaceutical compositions containing the antibody composition include cancer. c41) An antibody composition, wherein the first antigen is CD19, the second antigen is CD38, and the target cells are plasma cells. The disease to be treated with a pharmaceutical composition containing the antibody composition includes systemic lupus erythematosus. c42) An antibody composition, wherein the first antigen is CD3, the second antigen is IL-23R, and the target cell is at least one selected from Th1 cells, Th17 cells, and Tfh cells. The disease to be treated with a pharmaceutical composition containing the antibody composition includes systemic lupus erythematosus. c43) An antibody composition in which the first antigen is CD3, the second antigen is CX3CR1, and the target cell is a CX3CR1-positive cell (e.g., a cytotoxic effector T cell, etc., excluding NK cells). The disease to be treated with a pharmaceutical composition containing the antibody composition includes at least one selected from autoimmune diseases (for example, Crane's disease), arteriosclerosis, and ischemic heart disease. c44) An antibody composition, wherein the first antigen is CXCR4, the second antigen is Type 1 collagen, and the target cells are fibrocytes. The disease to be treated by the pharmaceutical composition containing the antibody composition includes at least one selected from various fibrotic diseases such as idiopathic pulmonary fibrosis, systemic sclerosis, and liver cirrhosis. c45) An antibody composition, wherein the first antigen is CXCR4, the second antigen is CD14, and the target cell is at least one of a monocyte and a fibrocyte. The disease to be treated with a pharmaceutical composition containing the antibody composition includes idiopathic pulmonary fibrosis. c46) An antibody composition, wherein the first antigen is CXCR4, the second antigen is CD16, and the target cell is an activated neutrophil. The disease to be treated by the pharmaceutical composition containing the antibody composition includes at least one selected from neutrophilic asthma, chronic obstructive pulmonary disease, and Alzheimer's disease. c47) An antibody composition, wherein the first antigen is CLEC10A, the second antigen is CD14 or CD16, and the target cell is an intermediate monocyte. The disease to be treated by the pharmaceutical composition containing the antibody composition includes at least one selected from Crohn's disease, rheumatoid arthritis, and asthma. c48) An antibody composition, wherein the first antigen is CD70, the second antigen is CD38, and the target cells are at least one of activated T cells and activated B cells. The disease to be treated by the pharmaceutical composition containing the antibody composition includes at least one of multiple sclerosis and neuromyelitis optica. c49) An antibody composition, wherein the first antigen is CD70, the second antigen is one selected from CD4, CD127 and TIM-1, and the target cell is at least one selected from Th1 cells, Th17 cells and plasmacytoid dendritic cells. The disease to be treated with a pharmaceutical composition containing the antibody composition includes systemic lupus erythematosus. c50) An antibody composition, wherein the first antigen is CD4, the second antigen is TIM-1, and the target cell is at least one selected from Th1 cells, Th17 cells, and plasmacytoid dendritic cells. The disease to be treated with a pharmaceutical composition containing the antibody composition includes systemic lupus erythematosus. c51) An antibody composition, wherein the first antigen is CD70, the second antigen is CD52, and the target cell is at least one of an activated T cell and an activated B cell. The target disease for treatment with a pharmaceutical composition containing the antibody composition includes multiple sclerosis. c52) An antibody composition wherein the first antigen is CB1, the second antigen is AT1R, and the target cells are GPCR heteromer-expressing rogue stellate cells. An example of a disease to be treated with a pharmaceutical composition containing the antibody composition is cirrhosis of the liver. c53) An antibody composition, wherein the first antigen is CD4 or PD-1, the second antigen is CD153, and the target cell is a senescent T cell. The disease to be treated with a pharmaceutical composition containing the antibody composition includes systemic lupus erythematosus. c54) An antibody composition, wherein the first antigen is FcεRI, the second antigen is CD34 or C-KIT, and the target cells are mast cells and / or mast cell precursor cells. Diseases that can be treated with a pharmaceutical composition containing the antibody composition include mast cell activation syndromes (MCAS), which include at least one selected from urticaria, food allergies, and mastocytosis. c55) An antibody composition, wherein the first antigen is CD34, the second antigen is CD203 or MRGPRX2, and the target cell is a mast cell precursor cell. Diseases to be treated with a pharmaceutical composition containing the antibody composition include mast cell activation syndrome (MCAS), which includes at least one selected from urticaria, food allergy, and mastocytosis. c56) An antibody composition, wherein the first antigen is CD52, the second antigen is CD127, and the target cells are at least one of T cells other than Tregs and B cells. The target disease for treatment with a pharmaceutical composition containing the antibody composition includes multiple sclerosis. c57) An antibody composition, wherein the first antigen is CD69, the second antigen is CD21, and the target cells are activated mature naive B cells. The disease to be treated by the pharmaceutical composition containing the antibody composition includes at least one selected from multiple sclerosis, type 1 diabetes, and rheumatoid arthritis. c58) An antibody composition, wherein the first antigen is CD106, the second antigen is one selected from CD11c, CD19, CD21 and CD72, and the target cells are follicular dendritic cells. The target disease for treatment with a pharmaceutical composition containing the antibody composition includes at least one selected from secondary progressive multiple sclerosis (SPMS), rheumatoid arthritis for which anti-TNF treatment is ineffective, transplantation, and systemic lupus erythematosus. c59) An antibody composition, wherein the first antigen is 4-1BB, the second antigen is one selected from CD11c, CD19, CD21 and CD72, and the target cells are follicular dendritic cells. The disease to be treated by a pharmaceutical composition containing the antibody composition includes at least one selected from secondary progressive multiple sclerosis (SPMS), rheumatoid arthritis for which anti-TNF treatment is ineffective, transplantation, and systemic lupus erythematosus. c60) An antibody composition wherein the first antigen is BLT1, the second antigen is CD49b, and the target cells are BLT1-positive CD8-positive cells. The disease to be treated with the pharmaceutical composition containing the antibody composition includes asthma. c61) An antibody composition in which the first antigen is CD226, the second antigen is CD8, and the target cells are CD226-positive CD8-positive effector memory cells. The disease to be treated with a pharmaceutical composition containing the antibody composition includes systemic sclerosis. c62) An antibody composition, wherein the first antigen is CXCR3, the second antigen is one selected from CD8, CD49a, IL-15R and NKG2D, and the target cells are effector memory CD8-positive cells. The disease to be treated with the pharmaceutical composition containing the antibody composition includes at least one of vitiligo vulgaris and psoriasis. c63) An antibody composition, wherein the first antigen is CD49a, the second antigen is one selected from CD8, IL-15R and NKG2D, and the target cells are effector memory CD8-positive cells. The disease to be treated with the pharmaceutical composition containing the antibody composition includes at least one of vitiligo vulgaris and psoriasis. c64) An antibody composition, wherein the first antigen is IL-15R, the second antigen is CD8 or NKG2D, and the target cells are effector memory CD8-positive cells. The disease to be treated with the pharmaceutical composition containing the antibody composition includes at least one of vitiligo vulgaris and psoriasis. c65) An antibody composition, wherein the first antigen is NKG2D, the second antigen is CD8, and the target cells are effector memory CD8-positive cells. The disease to be treated with the pharmaceutical composition containing the antibody composition includes at least one of vitiligo vulgaris and psoriasis. c66) An antibody composition wherein the first antigen is CD177, the second antigen is PR3, and the target cells are at least one of extravascularly infiltrating neutrophils and PR3-presenting neutrophils. The disease to be treated by a pharmaceutical composition containing the antibody composition includes at least one of ANCA (Antineutrophil Cytoplasmic Antibody)-associated vasculitis and systemic lupus erythematosus. c67) An antibody composition in which the first antigen is CD4, the second antigen is CD127, and the target cells are CD4-positive T cells other than Tregs. Diseases that can be treated with a pharmaceutical composition containing the antibody composition include T cell-dependent immune-related diseases. c68) An antibody composition, wherein the first antigen is CD40 ligand, the second antigen is IL-6, and the target cell is an activated T cell. Diseases that can be treated with a pharmaceutical composition containing the antibody composition include immune-related diseases. c69) An antibody composition, wherein the first antigen is IL-17R, the second antigen is membrane-type TNF, and the target cells are cells expressing IL17R and membrane-type TNF. The disease to be treated with the pharmaceutical composition containing the antibody composition includes psoriatic arthritis. c70) An antibody composition, wherein the first antigen is IL-23R, the second antigen is CCR6, and the target cell is a Th17 cell. Diseases that can be treated with a pharmaceutical composition containing the antibody composition include autoimmune diseases, including at least one of Sjogren's syndrome and psoriasis. c71) An antibody composition, wherein the first antigen is CD64, the second antigen is one selected from CD206, CD163 and CD68, and the target cell is an M2 macrophage. Diseases that can be treated with pharmaceutical compositions containing the antibody composition include cancer. c72) An antibody composition, wherein the first antigen is CD163, the second antigen is CD206 or CD68, and the target cell is an M2 macrophage. Diseases that can be treated with pharmaceutical compositions containing the antibody composition include cancer. c73) An antibody composition, wherein the first antigen is CD206, the second antigen is CD68, and the target cell is an M2 macrophage. Diseases that can be treated with pharmaceutical compositions containing the antibody composition include cancer. c74) An antibody composition, wherein the first antigen is CD14, the second antigen is one selected from CD48, CD84, CD97 and CD305, and the target cell is a myeloid-derived suppressor cell. Diseases that can be treated with pharmaceutical compositions containing the antibody composition include cancer. c75) An antibody composition, wherein the first antigen is CD15, the second antigen is one selected from CD48, CD84, CD97 and CD305, and the target cell is a myeloid-derived suppressor cell. Diseases that can be treated with pharmaceutical compositions containing the antibody composition include cancer. c76) An antibody composition, wherein the first antigen is CD33, the second antigen is one selected from CD48, CD84, CD97 and CD305, and the target cell is a myeloid-derived suppressor cell. Diseases that can be treated with pharmaceutical compositions containing the antibody composition include cancer. c77) An antibody composition, wherein the first antigen is CD11b, the second antigen is one selected from CD48, CD84, CD97 and CD305, and the target cell is a myeloid-derived suppressor cell. Diseases that can be treated with pharmaceutical compositions containing the antibody composition include cancer. c78) An antibody composition, wherein the first antigen is CCR4, the second antigen is one selected from CADM1, CD30 and CD70, and the target cell is a tumor cell. The disease to be treated with the pharmaceutical composition containing the antibody composition includes at least one of leukemia and lymphoma. c79) An antibody composition, wherein the first antigen is CD38, the second antigen is CD138 or BCMA, and the target cell is a tumor cell. The disease to be treated with the pharmaceutical composition containing the antibody composition includes at least one of leukemia and lymphoma. c80) An antibody composition, wherein the first antigen is BCMA, the second antigen is CD56 or CS1, and the target cell is a tumor cell. The disease to be treated with the pharmaceutical composition containing the antibody composition includes at least one of leukemia and lymphoma. c81) An antibody composition, wherein the first antigen is CD40 ligand, the second antigen is one selected from CD36, CD62P and CD63, and the target cell is a megakaryocyte. The disease to be treated with the pharmaceutical composition containing the antibody composition includes at least one of leukemia and lymphoma. c82) An antibody composition, wherein the first antigen is TIM-3, the second antigen is one selected from CD123, CD33, CD47, CD70 and CLEC12A, and the target cell is a tumor cell. The disease to be treated with the pharmaceutical composition containing the antibody composition includes at least one of leukemia and lymphoma. c83) An antibody composition, wherein the first antigen is CD123, the second antigen is one selected from CD33, CD47, CD70 and CLEC12A, and the target cell is a tumor cell. The disease to be treated with the pharmaceutical composition containing the antibody composition includes at least one of leukemia and lymphoma. c84) An antibody composition, wherein the first antigen is CD5, the second antigen is CD23, and the target cell is a tumor cell. The disease to be treated with the pharmaceutical composition containing the antibody composition includes at least one of leukemia and lymphoma. c85) An antibody composition, wherein the first antigen is CD10 or CD5, the second antigen is CD20, and the target cell is a tumor cell. The disease to be treated with the pharmaceutical composition containing the antibody composition includes at least one of leukemia and lymphoma. c86) An antibody composition wherein the first antigen is CD40, the second antigen is selected from CD80, CD86, ICOS ligand, 4-1BB ligand, OX40 ligand, CD70, GITR, PD-L1, PD-L2, B7-DC, B7H3, B7H4, B7H5, B7H6 and B7H7, and the target cell is an activated antigen-presenting cell. Diseases that can be treated with a pharmaceutical composition containing the antibody composition include inflammatory diseases. c87) An antibody composition, wherein the first antigen is PTPRS, the second antigen is IL-21R, and the target cell is at least one selected from plasmacytoid dendritic cells (pDCs), T cells, and B cells. Diseases that can be treated with a pharmaceutical composition containing the antibody composition include autoimmune diseases. c88) An antibody composition, wherein the first antigen is PTPRS, the second antigen is CD38, and the target cell is at least one selected from plasmacytoid dendritic cells (pDCs), activated T cells, B cells, and plasma cells. Diseases that can be treated with a pharmaceutical composition containing the antibody composition include autoimmune diseases. c89) An antibody composition, wherein the first antigen is PTPRS, the second antigen is CD32a, and the target cell is at least one of a plasmacytoid dendritic cell (pDC) and a myeloid cell. Diseases that can be treated with a pharmaceutical composition containing the antibody composition include autoimmune diseases. c90) An antibody composition in which the first antigen is OX40, the second antigen is CD127 or CD40 ligand, and the target cell is a helper T cell other than a Treg. Diseases that can be treated with a pharmaceutical composition containing the antibody composition include allergic diseases. c91) An antibody composition, wherein the first antigen is OX40, the second antigen is CD8 or NKG2D, and the target cell is a CD8 memory T cell. The disease to be treated with the pharmaceutical composition containing the antibody composition includes at least one of vitiligo vulgaris and psoriasis. c92) An antibody composition wherein the first antigen is OX40, the second antigen is CD226, and the target cells are CD8 memory T cells. The disease to be treated with the pharmaceutical composition containing the antibody composition includes scleroderma. c93) An antibody composition, wherein the first antigen is OX40, the second antigen is CRTH2, and the target cell is an activated Th2 cell. Diseases that can be treated with a pharmaceutical composition containing the antibody composition include allergic diseases. c94) An antibody composition wherein the first antigen is CRTH2, the second antigen is CD2, and the target cells are at least one of Th2 cells and innate lymphoid cells (ILC2 cells). Diseases that can be treated with a pharmaceutical composition containing the antibody composition include allergic diseases. c95) An antibody composition, wherein the first antigen is CRTH2, the second antigen is CD7, and the target cells are at least one of Th2 cells and innate lymphoid cells (ILC2 cells). Diseases that can be treated with a pharmaceutical composition containing the antibody composition include allergic diseases. c96) An antibody composition, wherein the first antigen is CRTH2, the second antigen is CD45, and the target cells are CRTH2-positive blood cells. Diseases that can be treated with a pharmaceutical composition containing the antibody composition include allergic diseases.

[0351] [Table 6A]

[0352] [Table 6B]

[0353] [Table 6C]

[0354] [Table 6D]

[0355] [Table 6E]

[0356] Although a pharmaceutical composition containing the antibody composition of the present invention can be administered alone as a therapeutic agent, it is usually preferable to mix it with one or more pharmacologically acceptable carriers and provide it as a pharmaceutical formulation prepared by any method well known in the technical field of pharmaceuticals.

[0357] The route of administration is preferably the most effective for treatment, and examples thereof include oral administration, and parenteral administration such as oral, tracheal, rectal, subcutaneous, intramuscular, and intravenous administration, and in the case of antibody composition formulations, intravenous administration is preferred.

[0358] Examples of dosage forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes, and the like.

[0359] Examples of formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, and granules.

[0360] Liquid preparations such as emulsions and syrups can be produced using additives such as water, sugars such as sucrose, sorbitol, fructose, etc., glycols such as polyethylene glycol or propylene glycol, oils such as sesame oil, olive oil, soybean oil, etc., preservatives such as p-hydroxybenzoic acid esters, or flavors such as strawberry flavor or peppermint.

[0361] Capsules, tablets, powders, granules, etc. can be produced using additives such as excipients such as lactose, glucose, sucrose, or mannitol; disintegrating agents such as starch or sodium alginate; lubricants such as magnesium stearate or talc; binders such as polyvinyl alcohol, hydroxypropyl cellulose, or gelatin; surfactants such as fatty acid esters; or plasticizers such as glycerin.

[0362] Examples of formulations suitable for parenteral administration include injections, suppositories, sprays, and the like.

[0363] Injectable preparations are prepared using a carrier such as a salt solution, a glucose solution, or a mixture of both. Alternatively, a powdered injectable preparation can be prepared by lyophilizing the antibody composition in accordance with a conventional method and adding sodium chloride thereto.

[0364] Suppositories are prepared using carriers such as cocoa butter, hydrogenated fats or carboxylic acids.

[0365] A spray is prepared using the antibody composition itself or a carrier that does not irritate the recipient's oral and respiratory mucosa and disperses the antibody composition as fine particles to facilitate absorption.

[0366] Specific examples of carriers include lactose and glycerin. Depending on the properties of the antibody composition and the carrier used, formulations such as aerosols and dry powders are possible. Furthermore, the components exemplified as additives for oral preparations can also be added to these parenteral preparations.

[0367] The dosage or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but the amount of active ingredient is usually 10 μg / kg to 20 mg / kg per day for adults.

[0368] Furthermore, methods for examining the antitumor effect of antibody compositions against various tumor cells include, as in vitro experiments, CDC activity measurement methods, ADCC activity measurement methods, etc., and as in vivo experiments, antitumor experiments using tumor systems in experimental animals such as mice, etc.

[0369] One embodiment of the present invention is a kit comprising first and second IgG half-mers. The kit may optionally include other materials, including suitable containers (e.g., bottles, vials, test tubes), labels with instructions, filters, needles, syringes, and instructions for use.

[0370] In addition to the IgG half-mer as an active ingredient, the above kit may contain, as needed, for example, sterilized water, physiological saline, vegetable oil, surfactant, lipid, solubilizing agent, buffer, protein stabilizer (e.g., BSA or gelatin), preservative, blocking solution, reaction solution, reaction stop solution, reagent for treating the sample, and the like.

[0371] Examples of usage modes of the above kit include (1) a method in which the first IgG half-mer and the second IgG half-mer are mixed in advance and then administered, and (2) a method in which the first IgG half-mer and the second IgG half-mer are administered separately. The (2) method in which the first IgG half-mer and the second IgG half-mer are administered separately includes, for example, a method in which the first IgG half-mer and the second IgG half-mer are administered simultaneously or sequentially. The amounts and ratios of the first IgG half-mer and the second IgG half-mer can be adjusted as appropriate.

[0372] Another aspect of the present invention is a first IgG halfmer, which is composed of a first IgG halfmer and a second IgG halfmer and is used for producing an antibody composition against a first antigen and a second antigen that are different from each other.

[0373] Another embodiment of the present invention relates to a second IgG halfmer, which is composed of a first IgG halfmer and a second IgG halfmer and is used for producing an antibody composition against a first antigen and a second antigen that are different from each other.

[0374] One embodiment of the present invention includes a first IgG halfmer used in combination with a second IgG halfmer. Another embodiment of the present invention includes a second IgG halfmer used in combination with a first IgG halfmer. Here, "combined use" of a first IgG halfmer and a second IgG halfmer refers to administering the first IgG halfmer and the second IgG halfmer simultaneously or sequentially to form an antibody composition comprising the first IgG halfmer and the second IgG halfmer, and containing halves that bind to a first antigen and a second antigen that are different from each other. Use of the antibody composition of the present invention includes all administrations, including simultaneous or sequential administration of the first halfmer and the second halfmer, mixing them in an injection vial, mixing them in an infusion bag, or administering them directly to a patient.

[0375] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples. [Example]

[0376] The present inventors conceived that the constant region of the antibody molecule of the present invention requires the following elements that differ from those of a typical human IgG1 antibody. 1) As shown in Figure 4, it is a mixture of antibody halves against different antigen molecules X and Y, i.e., it is an "HL body" in which there is no covalent bond between the H chains due to disulfide bonds in the hinge region. 2) As shown in Figure 3, the HL molecules for antigen molecules X and Y bind to the surface of target cells expressing both X and Y, and then associate to form an H2L2 complex similar to that of normal IgG, which constitutes the CD16a-binding domain and triggers the antibody effector function (e.g., ADCC activity). 3) As shown in Figure 3, even if HL antibodies against X or Y associate with each other on the cell surface, they do not constitute a CD16a-binding region, so that the antibody does not activate cells expressing only a single antigen molecule.

[0377] In this example, the above 1) was attempted by substituting cysteine ​​in the hinge domain with alanine.

[0378] X-ray crystal structure analysis has shown that the Fc of human IgG1 and CD16a bind asymmetrically (Nature 2000; 406: 267-73, J Biol Chem 2001; 276: 16469-77, Mizushima T, Genes Cells 2011; 16: 1071-80). Therefore, in order to achieve the above-mentioned 2) and 3), the present inventors conceived of introducing the amino acid alterations described below, which take advantage of this asymmetric binding mode, into the CH2 domain of the above-mentioned HL form.

[0379] As shown in Figure 5, CD16a contacts the two CH2 domains in Fc at separate sites. The two CH2 domains are tentatively named CH2-A and CH2-B, and the site on CH2-A that interacts with CD16a is designated Region 1, and the site on CH2-B that interacts with CD16a is designated Region 2.

[0380] Region 1 includes L235, G236, G237, P238, S239, D265, V266, S267, H268, E269, E294, Q295, Y296, N297, S298, T299, R301, N325, A327, and I332, while region 2 includes L235, G236, G237, K326, A327, L328, P329, and A330.

[0381] As shown in Figure 5, due to the symmetry of the Fc structure, there is another binding region, consisting of region 2 of CH2-A and region 1 of CH2-B, on the opposite side of the binding region where the actual Fc binds to CD16a, which is not actually used for binding to CD16a.

[0382] As shown in Figure 6, Region 2 of CH2-A and Region 1 of CH2-B, which are not used for binding to CD16a, are "destroyed" by introducing amino acid modifications. In this case, when HL bodies (X) having such modified CH2-A or HL bodies (Y) having such modified CH2-B homozygously associate to form an H2L2 complex (XX, YY), only Region 1 or only Region 2 is present. Therefore, these homozygous complexes may not be able to bind CD16a with sufficient affinity. In contrast, an H2L2 complex (XY), which is a heterozygous complex of HL bodies having such modified CH2-A and modified CH2-B as components, may satisfy the above conditions 2) and 3).

[0383] [Example 1] Generation of a human IgG1 anti-CCR6 antibody that "disrupts" CD16a binding To identify sites on CH2 that can attenuate CD16a binding, we generated antibodies in which the above-mentioned region 1 or region 2 was "destroyed" by amino acid modification. That is, as shown in Figure 7, amino acid residues at candidate sites were modified in the format of a normal human IgG1.

[0384] In this case, to express human IgG1 as a recombinant antibody, the two H chains are encoded by the same gene on the expression vector, and there is no distinction between CH2-A and CH2-B, and amino acid residues are modified in both simultaneously. As the CD16a binding sites on both the front and back are simultaneously destroyed, we must search for modification sites that weaken ADCC activity.

[0385] Unless otherwise specified, all antibody molecules produced in this example lack α1,6-fucose in the N-linked glycan attached to asparagine at position 297 of the H chain, thereby enhancing ADCC activity. For this purpose, fucosyltransferase (FUT8) knockout CHO cells (WO 2005 / 035586, WO 02 / 31140) were used as host cells for antibody expression.

[0386] (1) Construction of a human IgG1 anti-CCR6 antibody expression vector with CD16a binding disrupted Figures 5 and 6 show schematic diagrams of "Region 1" and "Region 2" on the CH2 domain. As shown in Figure 6, to "disrupt" binding to CD16a, an expression vector for a human IgG1 anti-human CCR6 antibody (WO 2013 / 005649) was constructed in which amino acid modifications were introduced into amino acid sites P238, D265, and S267, which bind to CD16a in "Region 1" on the CH2 domain, and amino acid sites K326, L328, and P329, which bind to CD16a in "Region 2" on the CH2 domain. The gene sequences and amino acid sequences of the antibodies used in the construction are shown in Table 7.

[0387] [Table 7]

[0388] Using the human IgG1 anti-human CCR6 antibody expression vector pCI-IgG1_KG1684, consisting of the nucleotide sequences shown in SEQ ID NOs: 1, 3, 5, and 7, as a template, PCR was performed using PrimeSTAR Max DNA Polymerase (Takara Bio) and primers (Sigma Oligo) containing modified sites, according to the instructions attached to the PrimeSTAR Max DNA Polymerase.

[0389] PCR was performed using a GeneAmp PCR System 9700 (Applied Biosystems). After heat denaturation at 98°C for 1 minute, 30 cycles of 98°C for 10 seconds, 58°C for 5 seconds, and 72°C for 5 seconds were performed. The reaction mixture was subjected to electrophoresis using a 0.8% agarose gel, and the amplified fragment was recovered using a QIAquick Gel Extraction Kit (Qiagen). A ligation reaction with the plasmid pCI vector (Promega) was performed using an In-Fusion HD Cloning Kit (Clontech), and the reaction mixture was used to transform Escherichia coli DH5α competent cells (Takara Bio).

[0390] Plasmid DNA was prepared from the resulting clones of the transformed strains and reacted using the Big Dye Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems) according to the attached instructions. The base sequence of the DNA inserted into the plasmid was then analyzed using the company's DNA sequencer, ABI PRISM 3700 DNA Analyzer.

[0391] (2) Expression of a human IgG1 anti-CCR6 antibody that "destroyed" CD16a binding The expression vector prepared in (1) was introduced into host cells using the following method. FUT8 knockout CHO cells (WO 2005 / 035586, WO 02 / 31140) were used as host cells. The plasmid was introduced according to the attached instructions.

[0392] The culture volume was 200 mL, and the cells were cultured for 5 days under the set conditions of 37°C, 5% CO2, and 125 rpm. After culture, the cell suspension was centrifuged, and the culture supernatant containing the modified antibody was collected through a 0.2 μm filter (ThermoScientific).

[0393] The names of the purified antibody samples for each antibody are shown in Table 8.

[0394] [Table 8]

[0395] (3) Purification of a human IgG1 anti-CCR6 antibody that "destroys" CD16a binding The modified antibodies were purified by affinity purification using MabSelect SuRe (GE Healthcare) as follows. After equilibrating the resin with PBS, the culture supernatant obtained in (2) was loaded onto the resin and washed twice with PBS. After washing, the antibodies were eluted using elution buffer (100 mM citric acid, pH 3.5) and neutralized with 1 / 10 volume of neutralization buffer (2 M Tris-HCl, pH 8.0).

[0396] Subsequently, the solution was concentrated by ultrafiltration using Amicon Ultra-4 Central Filter Units (Millipore) and buffer (10 mM citric acid, 150 mM NaCl, pH 6.0) was replaced. The absorbance at 280 nM (A280) was measured using a Nanodrop8000 (ThermoScientific) to determine the concentration of the antibody solution and prepare it.

[0397] (4) Purification of human IgG1 anti-CCR6 antibodies that "disrupt" CD16a binding by SDS-PAGE To evaluate the purity of various purified human IgG1 anti-CCR6 antibody samples lacking CD16 binding, approximately 1 μg of the antibody purified sample was subjected to SDS-denaturing polyacrylamide gel electrophoresis (hereinafter referred to as SDS-PAGE) according to a known method [Nature, 227, 680 (1970)].

[0398] As a result, under reducing conditions, the human IgG1 anti-CCR6 antibodies lacking various CD16 binding functions showed bands at approximately 50 kilodaltons (hereinafter referred to as kDa) for the H chain and approximately 25 kDa for the L chain, similar to normal IgG1 types. Furthermore, under non-reducing conditions, bands were observed at approximately 150 kDa, confirming that the anti-CCR6 domain-exchanged antibodies produced were composed of the desired H chain and L chain.

[0399] The above results confirmed that the purified samples of various CD16-binding-deficient human IgG1 anti-CCR6 antibodies obtained in Section 3 of this Example contained sufficient proportions of the target IgG molecules, each composed of an H chain and an L chain.

[0400] (5) ADCC activity of a human IgG1 anti-CCR6 antibody with a disrupted CD16a binding domain The effector cells were NK-92 / CD16 transfectants, which were NK cell lines (ATCC) transfected with DNA encoding human CD16a (Val type) and allowed to stably express the gene, and the target cells were human CCR6 / CHO transfectants. The cultured cells were harvested and counted, and 8 × 10 5 cells / mL, 2 × 10 5 The cells were adjusted to 100 cells / mL using RPMI medium (phenol red-free RPMI1640 medium supplemented with 5% FBS / 1% PS).

[0401] After dispensing 50 μL of antibody solution into a 96-well plate using a repeat dispenser, 50 μL of target cells were dispensed into each well. 50 μL of effector cells were dispensed into each well and centrifuged at 1800 rpm for 2 minutes to confirm that the cells were evenly distributed.

[0402] After incubation in a CO2 incubator at 37°C for 3 hours and 15 minutes, one-tenth the volume of solubilization solution was dispensed into the total control and incubated at 37°C for 45 minutes. After centrifugation at 1800 rpm for 2 minutes, 50 μL of the supernatant was dispensed into an ELISA plate. The substrate (powder) was dissolved in 12 mL of suspension buffer to prepare a color development solution, which was applied at 50 μL / well for the reaction. 50 μL / well of stop solution was added, and the absorbance (A450) was measured using a plate reader.

[0403] The ADCC measurement kit used was CytoTox96® Non-Radioactive Cytotoxicity Assay (Promega). ADCC activity (%) was calculated using the following formula.

[0404] ADCC activity (%)=100x(SET) / (Max-T) S = absorbance of sample reaction well - absorbance of medium well E = Effector well absorbance - Medium well absorbance T = target well absorbance - medium well absorbance Max = 100% reaction well - 100% reaction control well

[0405] The results are shown in Figure 8. As shown in Figure 8, it was confirmed that in region 1 of CH2, the modified forms consisting of IgG1_D265A, IgG1_P238A / S267L, and IgG1_D265A / S267L had significantly reduced ADCC activity against target cells.

[0406] Furthermore, in region 2 of CH2, the variants consisting of IgG1_P329Y, IgG1_K326W / P329Y, IgG1_L328V / P329Y, and IgG1_K326W / L328V / P329Y were confirmed to have significantly reduced ADCC activity, indicating that all of the variants contain the P329Y modification.

[0407] From the above results, it was revealed that the CD16a binding site can be destroyed by introducing the amino acid alteration D265A or P238A / S267L in "region 1" on the CH2 domain, and by introducing at least the amino acid alteration P329Y in "region 2" on the CH2 domain.

[0408] [Example 2] Development of a human IgG1 anti-CCR6 monovalent antibody with asymmetric CD16a binding modifications In Example 1, as examples of amino acid sequences capable of disrupting the asymmetric binding site between CD16a and Fc, D265A or P238A / S267L derived from "region 1" was selected for CH2-A, and P329Y derived from "region 2" was selected for CH2-B.

[0409] In this section, to evaluate ADCC activity by asymmetrically introducing the above-mentioned modifications into only CH2-A and CH2-B, we used a "monovalent antibody" (WO 2011 / 108502) as a basic framework, which allows the expression of separate genes encoding two H chain molecules and the resulting hetero-association. Figure 9 shows a schematic diagram of one embodiment of such a monovalent antibody. In Figure 9, the hetero-association is designated "asymmetric variant #1."

[0410] (1) Construction of an expression vector for a CD16a-binding asymmetrically engineered monovalent antibody We constructed expression vectors for human IgG1 anti-CCR6 monovalent antibodies (WO 2011 / 108502) into which CH2-A (D265A or P238A / S267L) and CH2-B (P329Y) were introduced asymmetrically or symmetrically.

[0411] Using the human IgG1 anti-CCR6 monovalent antibody expression vector pCI-mvG1_KG1684 as a template, PCR was performed using PrimeSTAR Max DNA Polymerase (Takara Bio) and primers (Sigma Oligo) containing modified sites, according to the instructions attached to the PrimeSTAR Max DNA Polymerase.

[0412] The PCR reaction was carried out using a GeneAmp PCR System 9700 (Applied Biosystems) by heat denaturing at 98°C for 1 minute, followed by 30 cycles of reaction at 98°C for 10 seconds, 58°C for 5 seconds, and 72°C for 5 seconds.

[0413] The reaction mixture was subjected to electrophoresis on a 0.8% agarose gel, and the amplified fragment was recovered using a QIAquick Gel Extraction Kit (Qiagen). The resulting fragment was ligated with the plasmid pCI vector (Promega) using an In-Fusion HD Cloning Kit (Clontech), and the resulting reaction mixture was used to transform Escherichia coli DH5α competent cells (Takara Bio).

[0414] Plasmid DNA was prepared from the resulting clones of the transformed strains and reacted using the Big Dye Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems) according to the attached instructions. The base sequence of the DNA inserted into the plasmid was then analyzed using the company's DNA sequencer, ABI PRISM 3700 DNA Analyzer.

[0415] (2) Expression of CD16a-binding asymmetrically engineered monovalent antibodies The antibodies were expressed and the culture supernatant containing the antibodies was collected in the same manner as in Section 2 of Example 1. Each purified antibody sample and its modified site are shown in Table 9. The amino acid residue substitution at the modified site was performed in the same manner as in Example 1.

[0416] [Table 9]

[0417] (3) Purification of CD16a-binding asymmetrically engineered monovalent antibodies The antibody was purified using the same method as in Section 3 of Example 1. The elution buffer used was 100 mM citric acid, pH 3.9. A monomer fraction was then isolated from the antibody solution using an AKTA FPLC (GE Healthcare) and a Superdex High-performance Column (GE Healthcare). Purified antibody was obtained by filtration sterilization using an AKTA system 0.22 μm pore size membrane filter (Millex-GV, Millipore). Absorbance at 280 nm (A280) was measured using a Nanodrop8000 (ThermoScientific).

[0418] (4) Purification of human IgG1 anti-CCR6 monovalent antibodies with CD16a binding asymmetric modifications by SDS-PAGE To evaluate the purity of purified samples of human IgG1 anti-CCR6 monovalent antibodies into which various CD16a-binding asymmetric modifications had been introduced, SDS-PAGE was performed using approximately 1 μg of the purified antibody samples.

[0419] As a result, under reducing conditions, all variants showed bands at approximately 50 kDa for the H chain and Fc-fused L chain, similar to the wild-type monovalent antibody. Furthermore, under non-reducing conditions, bands were observed at approximately 100 kDa, confirming that the asymmetric and symmetric variants prepared were composed of the desired H chain and L chain.

[0420] The above results confirmed that the purified samples of human IgG1 anti-CCR6 monovalent antibodies into which various CD16a-binding asymmetric alterations were introduced, obtained in Section 3 of this Example, contained sufficient proportions of the target monovalent antibody molecules each composed of an H chain and an Fc-fused L chain.

[0421] (5) ADCC activity of CD16a-binding asymmetrically engineered monovalent antibodies ADCC activity was measured using the same method as in Section 5 of Example 1. The results are shown in Figure 10. As shown in Figure 10, it was confirmed that the monovalent antibodies into which asymmetric alterations in CD16a binding had been introduced (asymmetric variants #1 to #4) cytotoxicized target cells in an antibody concentration-dependent manner. On the other hand, it was confirmed that the monovalent antibodies into which symmetric alterations in CD16a binding had been introduced (symmetric variants #1 to #5) did not exhibit ADCC activity against target cells.

[0422] These results demonstrate that the monovalent antibody into which asymmetric alterations in CD16a binding have been introduced maintains its binding activity to human CD16a and can exert ADCC activity against target cells.

[0423] On the other hand, when the same modification is introduced into both CH2 domains, ADCC activity is lost, confirming that ADCC activity is not induced when antibodies with the same modified CH2 form homophilic associations on the cell surface. Thus, a modified CH2 domain was found to construct an HL antibody that exhibits ADCC activity only when it hetero-associates with the two antigens on the cell surface, as aimed at by the present invention.

[0424] [Example 3] In this example, the modified CH2 obtained in Examples 1 and 2, which can only induce ADCC activity through hetero-association, was incorporated into halves (HL bodies) of two types of antibodies against different antigens, and it was verified whether ADCC activity can be induced specifically in target cells co-expressing the two types of antigens.

[0425] The interaction between CH3 domains has been reported in J Immunol 2011; 187: 3238-3246. The interaction between CH3 domains of human IgG1 (KD value) is 3.0 x 10 -9 M, K of the CH3 domain of human IgG4 D The value is 4.8 x 10 -8 At M, the interaction of the CH3 domain of human IgG4 is approximately 6-7 times weaker than that of IgG1.

[0426] Taking advantage of this property and the amino acid alteration (C226A / C229A:AA) that breaks the disulfide bond between the heavy chains in the hinge region, we attempted to create a half-antibody. The heavy chain constant region of the half-antibody was based on human IgG1, and only the CH3 domain was exchanged with the human IgG4 sequence. Furthermore, an amino acid alteration (AA) was added to the hinge region to create a domain-exchanged antibody (hereafter referred to as IgG1114_AA type).

[0427] In addition, to enhance ADCC activity, known ADCC-enhancing amino acid modifications were made to the CH2 domain, and the α1,6-fucose N-linked glycan attached to Asn297 was removed. Finally, a CD16a-binding asymmetric amino acid modification was introduced. Figure 11 shows a schematic diagram of the IgG1114_AA_AAA_D265A( / P329Y) IgG halfmer. CD4 and CD70 were used as model antigens.

[0428] Anti-CD4 antibodies (J. Immunol. 1992; 149:1779-1787) and anti-CD70 antibodies (WO 2007 / 03637) incorporating the above-described alterations were produced according to the following procedure. Anti-CD4 and CD70 antibodies with heavy chain constant regions composed of amino acid sequences derived from IgG1114_AA-type antibodies incorporating known ADCC activity-enhancing amino acid alterations (S298A / E333A / K334A:AAA) and CD16a-binding asymmetric amino acid alterations (D265A or P329Y) are referred to as IgG1114_AA_AAA_D265A( / P329Y)-type anti-CD4 antibody halfmers and IgG1114_AA_AAA_D265A( / P329Y)-type anti-CD70 antibody halfmers, respectively.

[0429] The subclasses from which each domain of the various designed anti-CD4 antibody halfmers and anti-CD70 antibody halfmers is derived, and the corresponding amino acid sequences of the H-chain constant regions, are shown in Table 10. The nucleotide and amino acid sequences of the antibodies against CD4 and CD70 used are shown in Table 11.

[0430] [Table 10]

[0431] [Table 11]

[0432] (1) Construction of expression vectors for half-anti-CD4 antibody and half-anti-CD70 antibody A DNA fragment of approximately 9 kbp was excised from the human IgG1 anti-CD4 antibody expression vector pCI-IgG1_CD4(ibalizumab) (SEQ ID NOs: 5, 7, 41, 43) or the human IgG1 anti-CD70 antibody expression vector pCI-IgG1_CD70(2H5) (SEQ ID NOs: 5, 7, 45, 47) using the restriction enzymes NheI and NotI, and purified.

[0433] The purified DNA fragment was ligated with an artificially synthesized gene consisting of the CH1 domain, hinge domain (with C226A / C229A modifications), CH2 domain (with S298A / K333A / E334A modifications, D265A, or P329Y modifications) of a human IgG1 antibody, and the CH3 domain of a human IgG4 antibody using the In-Fusion HD Cloning Kit (Clontech). The reaction mixture was then used to transform Escherichia coli DH5α competent cells (Takara Bio). Plasmid DNA was prepared from the resulting clones of the transformants, and the DNA sequences were analyzed using FASMAC.

[0434] (2) Expression of various anti-CD4 IgG1 antibodies and anti-CD70 IgG1 antibodies and their half-antibody forms The antibody was expressed in the same manner as in item 2 of Example 1, and the culture supernatant containing the antibody was collected.

[0435] The names of the purified antibody samples are shown in Table 12.

[0436] [Table 12]

[0437] (3) Purification of anti-CD4 antibody half-mers and anti-CD70 antibody half-mers The antibody was purified, and the concentration of the antibody solution was measured and prepared in the same manner as in item 3 of Example 1.

[0438] (4) Purification of anti-CD4 antibody half-mers and anti-CD70 antibody half-mers by SDS-PAGE To evaluate the purity of each antibody sample, approximately 1 μg of the purified antibody sample was subjected to SDS-PAGE. The results are shown in Figure 12.

[0439] As shown in Figure 12, for all purified antibodies, bands were observed at approximately 50 kDa for the H chain and approximately 25 kDa for the L chain under reducing conditions. Furthermore, under non-reducing conditions, bands were observed at approximately 150 kDa for the anti-CD4 IgG1 antibody and anti-CD70 IgG1 antibody, and bands at approximately 75 kDa for the anti-CD4 halfmer and anti-CD70 halfmer. This confirmed that the prepared anti-CD4 IgG1 antibody and anti-CD70 IgG1 antibody have antibody structures composed of the desired H chains and L chains, and that the anti-CD4 antibody halfmer and anti-CD70 antibody halfmer are composed of the desired H chains and L chains and have antibody structures that facilitate halfmer formation.

[0440] The above results confirmed that the purified anti-CD4 antibody half-mer and anti-CD70 antibody half-mer samples obtained in Section 3 of this Example contained sufficient amounts of the target antibody molecules.

[0441] (5) ADCC activity of anti-CD4 IgG1 antibody, anti-CD70 IgG1 antibody, and their half-antibody complexes against CD4 / CD70 dual-positive cells and CD4 and CD70 single-positive cells ADCC activity was measured in the same manner as in Section 5 of Example 1. Target cells used were CD4 / EL-4 transfectants as CD4 mono-positive cells, MT-1 as CD70 mono-positive cells, and TL-Om1 as CD4 / CD70 dual-positive cells. The expression levels of CD4 and CD70 in these cells were measured using a flow cytometer, and the results are shown in Figure 13.

[0442] As shown in Figure 13, it was confirmed that the target antigen was expressed on the cells. The results of the ADCC activity evaluation are shown in Figure 14. Anti-CD4 IgG1 antibody and anti-CD70 IgG1 antibody were used as positive control antibodies.

[0443] As expected, as shown in Figure 14, anti-CD4 antibody exerted ADCC activity against CD4 / EL-4 transfectants, anti-CD70 antibody against MT-1, and both anti-CD4 and anti-CD70 antibodies against TL-Om1.

[0444] On the other hand, the antibody solution containing a mixture of anti-CD4 antibody half-mer 1 and anti-CD70 antibody half-mer 2, or the antibody solution containing a mixture of anti-CD4 antibody half-mer 2 and anti-CD70 antibody half-mer 1, did not exhibit ADCC activity against CD4 and CD70 single-positive cells, but exhibited ADCC activity specifically against CD4 / CD70 dual-positive cells.

[0445] [Example 4] The modified CH2 disrupted CD16a binding by altering the amino acid residues at positions 235, 239, 265, 267, 269, 296, 298, 299, and 327 (EU index) in the first CD16a-binding domain, and at positions 326, 328, 329, and 330 (EU index) in the second CD16a-binding domain. These modified CH2s were then loaded onto two antibody halves (HL bodies) against different antigens to examine whether they could induce ADCC activity specifically in target cells co-expressing the two antigens.

[0446] The various designed anti-CD4 antibody halves and anti-CD70 antibody halves were prepared in the same manner as in Example 3, and their AD...

Claims

1. An antibody composition consisting of a first IgG half-mer and a second IgG half-mer, which are directed against a first antigen and a second antigen that are different from each other, and which satisfies the following (1A) to (6A): (1A) The first IgG half-mer and the second IgG half-mer each comprise one immunoglobulin light chain (hereinafter abbreviated as L chain) and one immunoglobulin heavy chain (hereinafter abbreviated as H chain), the H chain comprising an H chain variable region, a hinge domain, a CH1 domain, a CH2 domain, and a CH3 domain, and the CH2 domain has a first Fcγ receptor IIIA (hereinafter abbreviated as CD16a)-binding region and a second CD16a-binding region that are different from each other; (2A) the first IgG half-mer and the second IgG half-mer contain amino acid residue substitutions of C226A and C229A, respectively, as represented by the EU index; (3A) the first IgG halfmer comprises an antigen-binding domain that binds to the first antigen, and comprises an amino acid residue substitution of D265A as represented by the EU index in the first CD16a-binding region; (4A) the second IgG halfmer comprises an antigen-binding domain that binds to the second antigen, and comprises an amino acid residue substitution of P329Y as represented by the EU index in the second CD16a-binding region; (5A) the first IgG half-mer and the second IgG half-mer each contain (a) amino acid residue substitutions of S239D and K326T as represented by the EU index, or the first IgG half-mer and the second IgG half-mer each contain (b) amino acid residue substitutions of S239D, S298A, E333A, L242C, and K334C, as represented by the EU index; and (6A) At least one of the first IgG half-mer and the second IgG half-mer comprises an amino acid residue substitution in the CH3 domain as a modification that weakens the CH3 domain-to-CH3 domain interaction compared to the CH3 domain-to-CH3 domain interaction of the IgG1 subclass.

2. The antibody composition according to claim 1, which specifically exerts an effector function on target cells that co-express the first antigen and the second antigen, thereby damaging them, compared to the effector function on target cells that express only the first antigen and target cells that express only the second antigen.

3. The antibody composition of claim 1 or 2, wherein the first IgG half-mer and the second IgG half-mer each comprise at least one amino acid residue substitution selected from Y349A, L351A, T366A, L368A, D399A, F405A, Y407A, K409A, and K409R, as represented by the EU index, which is a modification that weakens CH3 domain-to-CH3 domain interaction compared to that of the IgG1 subclass.

4. The antibody composition of claim 3, wherein the first IgG half-mer and the second IgG half-mer each contain an amino acid residue substitution of K409R, as represented by the EU index, as a modification that weakens CH3 domain-to-CH3 domain interaction compared to IgG1 subclass CH3 domain interaction.

5. The antibody composition according to any one of claims 1 to 4, wherein the first IgG half-mer and the second IgG half-mer each contain the amino acid residue substitutions (a) S239D and K326T.

6. The antibody composition according to any one of claims 1 to 5, wherein the first IgG half-mer and the second IgG half-mer contain amino acid residue substitutions of S239D, S298A, E333A, L242C, and K334C, respectively, as represented by the EU index.

7. The antibody composition according to any one of claims 1 to 6, wherein the proportion of glycans in which fucose is not bound to N-acetylglucosamine at the glycan reducing end is 20% or more of all N-glycoside-linked glycans bound to the Fc region in the first IgG half-mer and the second IgG half-mer.

8. The antibody composition according to any one of claims 1 to 7, wherein the immunoglobulin subclass of the first IgG half-mer and the second IgG half-mer is IgG1.

9. The following DNA combinations a) and b): a) DNA encoding the amino acid sequence of the first IgG halfmer according to any one of claims 1 to 8. b) DNA encoding the amino acid sequence of the second IgG halfmer according to any one of claims 1 to 8.

10. A set of recombinant vectors comprising the set of DNAs according to claim 9.

11. A set of transformants into which the set of recombinant vectors according to claim 10 has been introduced.

12. 1. A kit comprising a first IgG half-mer and a second IgG half-mer, (1B) the first IgG half-mer and the second IgG half-mer form an antibody composition against a first antigen and a second antigen that are different from each other; (2B) the first IgG half-mer and the second IgG half-mer each comprise one L chain and one H chain, the H chain comprising an H chain variable region, a hinge domain, a CH1 domain, a CH2 domain, and a CH3 domain, and the CH2 domain comprises a first CD16a-binding region and a second CD16a-binding region that are different from each other; (3B) the first IgG half-mer and the second IgG half-mer contain amino acid residue substitutions of C226A and C229A, respectively, as represented by the EU index; (4B) the first IgG halfmer comprises an antigen-binding domain that binds to the first antigen, and comprises an amino acid residue substitution of D265A as represented by the EU index in the first CD16a-binding region; (5B) the second IgG half-mer comprises an antigen-binding domain that binds to the second antigen, and comprises an amino acid residue substitution of P329Y as represented by the EU index in the second CD16a-binding region; (6B) the first IgG half-mer and the second IgG half-mer each contain amino acid residue substitutions (a) S239D and K326T as represented by the EU index, or the first IgG half-mer and the second IgG half-mer each contain amino acid residue substitutions (b) S239D, S298A, E333A, L242C, and K334C as represented by the EU index; (7B) A kit, wherein at least one of the first IgG half-mer and the second IgG half-mer includes an amino acid residue substitution in the CH3 domain as a modification that weakens CH3 domain-to-domain interaction compared to CH3 domain-to-domain interaction of the IgG1 subclass.

13. A method for inducing effector function specifically against target cells that co-express the first antigen and the second antigen in vitro, using the antibody composition described in any one of claims 1 to 8, by comparing the effector function against target cells that express only the first antigen and target cells that express only the second antigen.

14. A pharmaceutical composition comprising the antibody composition according to any one of claims 1 to 8.

15. 15. The pharmaceutical composition according to claim 14 for use in the treatment of cancer, autoimmune diseases or allergic diseases.

16. A method for producing the antibody composition according to any one of claims 1 to 8, comprising the steps of culturing a set of transformants according to claim 11 in a medium, allowing the first IgG half-mer according to any one of claims 1 to 8 and the second IgG half-mer according to any one of claims 1 to 8 to accumulate in the culture, and collecting the first IgG half-mer and the second IgG half-mer from the culture.

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