ANTIBODY COMPOSITION

TECHNICAL FIELD

The present invention relates to an antibody composition and a method for producing the same, an IgG half-molecule, and a kit including the IgG half-molecule.

BACKGROUND ART

It is known that antibody preparations approved so far have various action mechanisms (Non-Patent Literature 1). Representative examples thereof include effector functions possessed by an IgG class antibody molecule such as a neutralization activity of inhibiting binding of a ligand for a growth factor or the like and a receptor, an agonistic activity of activating a bound receptor, an antibody-dependent cellular cytotoxicity (hereinafter ADCC) activity, and a complement-dependent cytotoxicity (hereinafter CDC) activity. Among these, from biomarker analysis of a clinical study of rituximab or trastuzumab, the ADCC activity is suggested to be a clinically important action mechanism of an antibody preparation (Non-Patent Literatures 2 and 3).

An antibody is a tetrameric protein of about 150 kDa composed of a total of four molecules of polypeptide chains: two molecules of immunoglobulin heavy chain (H chain) and two molecules of immunoglobulin light chain (L chain). A shown in FIG. 1, an antibody is divided into a variable region (V) including a complementarity-determining region (CSR) that is directly involved in antigen binding and has a different amino acid sequence for each antibody clone, and a constant region including an Fc region (hereinafter also abbreviated as Fc) that controls the above-mentioned effector function or a blood half-life. The Fc includes CH1 to CH3 domains and a hinge domain.

A human antibody is classified into 5 classes: IgG, IgA, IgM, IgD, and IgE having different functions according to the sequence of the H chain constant region. Further, a human IgG class is divided into 4 subclasses from IgG1 IgG4.

Among these, antibody of the IgG1 subclass is known to have the highest ADCC activity and CDC activity (Non-Patent Literature 4), and many antibody preparations including rituximab and trastuzumab are IgG1.

On the other hand, the IgG4 subclass is known to have characteristics such that the effector function is weak as compared with other subclasses, and further it has an amino acid sequence of an intrinsic hinge domain, and reversible association and dissociation of two H chains in the body called “Fab arm exchange” occurs due to a weak interaction between two CH3 domains as compared with other subclasses (Non-Patent Literatures 5 and 6).

The ADCC activity, is a mechanism of cytotoxicity caused through the expression of a molecule such as perform, granzyme, or Fas as a result of activation of a natural killer cell (hereinafter NK cell) or the like by recognizinig Fc of an IgG-type antibody bound to a membrane antigen on a cancer cell surface via a type of an Fc receptor, FcγRIIIa (hereinafter also abbreviated as CD16a) (Non-Patent Literature 1).

By X-ray crystallography, the binding mode between CD16a and human IgG1 has been revealed (Non-Patent Literatures 7 and 8), The CD16a-binding domain on Fc of IgG1 is present at two sites due to the point symmetry of the structure of Fc, and in fact, Fc and IgG bind at a number ratio (stoichiometry) of 1.1 Then, in the two CH2 domains constituting Fc, contact is made in regions (hereinafter CD16a-binding domains) that are different from each other.

Specifically, CD16a interacts with L235, G236, G237, P238, S239, D265, V266, S267, H268, E269, E294, Q295, Y296, N297S298, T299, R301, N325, A327, I332, or the like on one of the CH2 domains of IgG1. On the other hand, CD16a simultaneously interacts with L235, G236, G237,K326, A327, L328, P329, A330, or the like on the other CH2 domain of IgG1 (the number represent the position of an amino acid on the CH2 domain according to EU numbering) (Non -Patent Literatures 7, 8, 9, and 10).

It is possible to enhance an ADCC activity by artificially altering the CH2 domain of an antibody preparation to increase the binding ability to CD16a. In fact, there are many known examples of enhancing n ADCC activity by altering an amino acid of the CH2 domain (Non-Patent Literature 11). It is known that an IgG1 antibody in which different amino acid alterations are added to the CH2 domain of one H chain of IgG1 and the CH2 domain of the other H chain, and the two H chains are bound through a disulfide bond has a high ADCC activity (Patent Literature 1).

Further, it is also known that an ADCC activity can be enhanced by altering a sugar chain of an N-linked complex sugar chain that is bound to Fc (Non Patent Literature 12). Particularly, the technique for enhancing an ADCC activity by a sugar chain alteration is applied to approved antibody preparations such as mobgamulizumab (Non-Patent Literature 13) and obinutuzumab (Non-Patent Literature 14).

A bispecific antibody is an artificially altered antibody molecule configured to be able to bind to two different types of antigens unlike natural antibodies, and many molecular forms have been reported (Non-Patent Literature 15).

A schematic diagram of a structure at a bispecific antibody is shown FIG. 2A. As an application of the bispecific antibody to a medicine, for example, damage to a cancer cell by binding to the cancer cell and CD3 of a T cell surface (hereinafter, a CD3 bispecific antibody) so as to crosslink both, and enhancement of a drug efficacy by neutralizing two types of functional molecules are exemplified (Non-Patent Literature 15).

In a treatment of a cancer or an autoimmune disease, in order to remove a cancer coil that is an etiologic cell or a self-reactive lymphocyte, an antibody preparation exhibiting an effector function of an IgG-type antibody or a T cell recruiting function of a CD3 bispecific antibody is used.

However, such an etiologic cell is originally derived from a normal cell, and it is generally rare that an etiologic cell can be accurately distinguished from a normal cell by a single surface marker molecule. Therefore, the removal of an etiologic cell utilizing an effector function like also attacks a normal cell expressing the same antigen molecule, and may result in adverse effects in many cases.

For example, CD20 that is target antigen of rituximab to be used in a treatment of lymphomas at various autoimmune diseases is expressed in normal B cell, and HER2 that is a target antigen of trastuzumab be used in a treatment of breast cancer is expressed in a cardiomyocyte. Therefore, there are concerns about adverse effects by disrupting a normal cell with such an antibody preparation.

On the other hand, Maxor et al. have reported a case where a bispecific antibody having reduced affinity for individual target antigens exhibits a relatively strong effector function for a double-positive (Non-Patent Literature 16).

Further, there has been a report on a combination of antigen-binding molecules including a first antigen-binding molecule having a first antigen-binding domain that binds to a first antigen and a first polypeptide including either one or both of first CH2 and first CH3, and a second antigen-binding molecule having a second antigen-binding domain that binds to a second antigen and a second polypeptide including either one or both of second CH2 and second CH3, wherein the first antigen binding molecule and the second antigen-binding molecule are not bound through a covalent bond, and are more likely to form a heterodimer than a homodimer when mixed in a liquid (Patent Literature 2).

CITATION LIST
Patent Literature

Patent Literature WO2013/002362

Patent Literature 2: WO2018/155611

Non-Patent Literature

Non-Patent Literature 1: Carter P. Nat Rev Cancer 2001; 1: 118-29

Non-Patent Literature 2: Cartron G, Dacheux Salles G, et al. Blood 2002: 99: 754-8

Non-Patent Literature 3: Weng W. K. Levy R. J Clin Oncol 2003; 21: 3940-7

Non-Patent Literature 4: Birch, J. R., Lennox, E. S. (Eds.), Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., New York, p. 45 (1995)

Non-Patent Literature 5: Aalberse R C and Schuurman J, Immunology 2002; 105:9-

Non-Patent Literature 6: Labrijn A F, Nat Biotechnol 2009; 27: 767-71

Non-Patent Literature 7: Sondermann P. Nature 2000; 406: 267-73

Non-Patent Literature 8: Radaev S, J Biol. Chem 2001; 276: 16469-77

Non-Patent Literature 9: Ferrara C, Proc Natl Acad Sci 2011; 108; 12669-74

Non-Patent Literature 10: Mizushima T, Genes Cells 2011; 16: 1071-80

Non-Patent Literature 11: Strohl W R, Curr Opin Biotechnol 2009; 20: 685-91

Non-Patent Literature 12: Niwa R, J Pharm Sci 2015; 930-41

Non-Patent Literature 13: Beck A, mAbs 2012; 4: 419-25

Non-Patent Literature 14: Goede V, N Engl J Med 2014; 370: 1101-10

Non-Patent Literature 15: Byrne Trends Biotechnol 2011; 31: 621-32

Non-Patent Literature 16: Mazor Y, MAbs 2015; 7: 377-89

SUMMARY OF INVENTION
Technical Problem

As one method for solving the above-mentioned adverse effects, a technique using a bispecific antibody that exhibits an effector function only when it recognizes two different types of antigens for removing an etiologic cell with higher selectivity is conceivable.

However, as shown in FIG. 2B, when a target cell is attacked using an effector function by as normal bispecific antibody, there is a concern that the bispecific antibody may bind to and attack not only a target cell coexpressing two types of antigens (hereinafter also abbreviated as double-positive cell), but also a expressing only one type of target antigen (hereinafter also abbreviated as single-positive cell). Further, a technique for an antibody that exhibits an effector function specifically for a double-positive cell and damages the cell regardless of affinity for individual target antigens has not been known.

Therefore, present inventors aim to provide an antibody composition which exhibits an effector function more specifically fora target cell coexpressing two types of antigens that are different from each other and damages the target cell.

Solution to Problem

The present inventors conceived that the above problem can be solved by an antibody composition having the following elements [1] to [3] that are different from a normal human IgG1 antibody in a constant region of an antibody molecule as shown in FIG. 3.

[1] It is a mixture of antibody half-molecules (first and second IgG half-molecules) having antigen binding sites for a first antigen (antigen molecule) and a second antigen (antigen molecule Y) that are different front each other. That is, it is a mixture of “HL molecules” in which a covalent bond formed by an inter-H chain disulfide bond is not present between the first IgG half-molecule and the second IgG half-molecule.

The HL molecules against each of the antigen molecules X and Y bind to the surface of a target cell (X/Y double positive cell) expressing both the antigen molecules X and Y and thereafter are associated with each other to form an H2L2 molecule in the same manner as normal IgG and form a CD16a-binding domain, and thus cause an antibody activity.

[3] A CD16a-binding domain is not formed even if the HL molecules against the antigen molecule X or Y are associated with each other en a cell surface to form a homo assembly so that an antibody activity is not caused against a cell expressing only a single antigen molecule.

Based on the above conception, the present inventors found the following (1) to (3) and thus completed the present invention.

(1) With respect to the above [1], as shown in FIG. 4, a part or the whole of a hinge domain of an IgG half-molecule is altered so as not to form a disulfide bond between H chains in the hinge domain by substitution or deletion or modification.

(2) As shown in FIG. 5, CD16a comes into contact with two CH2 domains (in FIG. 5, CH2-A and CH2-B) in Fc at different sites (a region 1 of CH2-A and a region 2 of CH2-B), respectively,

Therefore, when each of the region 2 of CH2-A and the region 1 of CH2-B that are not used far binding are “disrupted” by alteration or the like of an amino acid to attenuate the binding activity, in a homo assembly of HL molecules having such altered CH2-A, only the region 2 of CH2-A or in a homo assembly of HL molecules having such altered CH2-B, only the region 1 of CH2-B is not present. Therefore, to such a homo assembly, CD16a cannot bind with sufficient affinity.

On the other hand, according to a combination of HL molecules having such altered CH2-A and altered CH2-B as constituent elements (hetero assembly), an antibody composition satisfying the above conditions [2] and [3] is obtained.

(3) According to the hetero assembly formed from the IgG half-molecules in which a specific amino acid alteration is introduced into a CD16a-binding domain or another Fc region, an antibody composition having an enhanced effector function for the X/Y double-positive cell and/or exhibiting controlled biokinetics is obtained.

That is, the present invention relates to the following.

1. An antibody composition, which is an antibody composition against a first antigen and a second antigen that are different from each other, including a first IgG half-molecule and a second IgG half-molecule, wherein the following (1A) to (6A) are satisfied:

(1A) each of the first IgG half-molecule and the second IgG half-molecule is composed of one immunoglobulin light chain (hereinafter abbreviated as chain) and one immunoglobulin heavy chain (hereinafter abbreviated as 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 Fcγ receptor IIIA (hereinafter CD16a)-binding domain and a second CD16a-binding domain that are different from each other in the CH2 domain,

(2A) each of the first IgG half-molecule and the second IgG half-molecule includes amino acid residue substitutions of C226A and C229A numbered according to the EU index,

(3A) the first IgG half-molecule includes an antigen-binding domain that binds to the first antigen, and includes an amino acid residue substitution or D265A numbered according to toe EU index in the first CD16a-binding domain,

(4A) the second IgG half-molecule includes an antigen-binding domain that binds to the second antigen, and includes an amino acid residue substitution of P329Y numbered according to the EU index in the second CD16a-binding domain,

(5A) each of the first IgG half-molecule and the second IgG half-molecule includes amino acid residue substitutions of (a) S239D and K326T numbered according to the EU index, or

each of the first IgG half-molecule and the second IgG half-molecule includes amino acid residue substitutions of (b) S239D, S298A E333A, L242C, and K334C numbered according to the EU index, and

(6A) at least one of the first IgG half-molecule and the second IgG half-molecule includes an amino acid residue substitution in the CH3 domain as an alteration for attenuating an inter-CH3 domain interaction as compared with an inter-CH3 domain interaction of the IgG1 subclass.

2. The antibody composition according to the above 1, wherein the antibody composition exhibits an effector function specifically for a target cell coexpressing the first antigen and the second antigen and damages the target cell as compared with an effector function for a target cell ex pressing only the first antigen and a target cell expressing only the second antigen.

3. The antibody composition according to the above 1 or 2, wherein each of the first IgG half-molecule and the second IgG half-molecule includes at least one amino acid residue substitution selected from Y349A, L351A, T366A, L368A, D399A, F405A, Y407A, K409A, and K409R numbered according to the EU index as the alteration for attenuating the inter-CH3 domain interaction as compared with the inter-CH3 domain interaction of the IgG1 subclass.

4. The antibody composition according to the above 3, wherein each of the first IgG half-molecule and the second IgG half-molecule includes an amino acid residue substitution of K409R numbered according to the EU index as the alteration for attenuating the inter-CH3 domain interaction as compared with the inter-CH3 domain interaction of the IgG1 subclass.

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

6. The antibody composition according to any one of the above 1 to 5, wherein

an H chain constant region (also abbreviated as CH) of the fast IgG half-molecule contains an amino acid sequence represented by SEQ ID NO: 248, and

a CH of the second IgG half-molecule contains an amino acid sequence represented by SEQ ID NO: 252.

7. The antibody composition according to any one of the above 1 to 6, wherein a ratio of sugar chains in which fucose is not bound to N-acetylglucosamine at a reducing end of the sugar chain among the total N-glycoside-linked type sugar chains bound to an Fc region in the first IgG half-molecule and the second IgG half-molecule is 20% or more.

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

9. A first IgG half-molecule, which is a first IgG half-molecule characterized by being associated with a second IgG half-molecule, wherein the first IgG half-molecule and the second IgG half-molecule satisfy the following (1B) to (7B):

(1B) the first IgG half-molecule and the second IgG half-molecule form an antibody composition against a first antigen and a second antigen that are different from each other,

(2B) each of the first IgG half-molecule and the second IgG half-molecule composed 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 domain and a second CD16a-binding domain that are different from each other in the CH2 domain,

(3B) each of the first IgG half-molecule and the second IgG half-molecule includes amino acid residue substitutions of C226A and C229A numbered according to the EU index,

(4B) the first IgG half-molecule includes an antigen-binding domain that binds to the first antigen, and includes an amino acid residue substitution of D265A numbered according to the EU index in the first CD16a-binding domain,

(5B) the second IgG half-molecule includes an antigen-binding domain that binds to the second antigen, and includes an amino acid residue substitution of P329Y numbered according to the EU index in the second CD16a-binding domain,

(6B) each of the first IgG half-molecule and the second half-molecule includes amino acid residue substitutions of (a) S239D and K326T numbered according to the EU index, or

each of the first IgG half -molecule and the second IgG half -molecule includes amino acid residue substitutions of (b) S239D, S298A, E,333A, L242C, and K334C numbered according to the EU index, and

(7B) at least one of the first IgG half-molecule and the second IgG half-molecule includes an amino acid residue substitution in the CH3 domain as an alteration the attenuating an inter-CH3 domain interaction as compared with an inter-CH3 domain interaction of the IgG1 subclass.

10. A second IgG half-molecule, which is a second IgG half-molecule characterized by being associated with a first IgG half-molecule, wherein the first IgG half-molecule and the second IgG half-molecule satisfy the following (1C) to (7C):

(1C) the first IgG half-molecule and the second IgG half-molecule form an antibody composition against a first antigen and a second antigen that are different from each other,

(2C) each of the first IgG half-molecule and the second IgG half-molecule is composed of one L chain and one H chain, and the H chain in an H chain variable region, a hinge domain, a CH1 domain, a CH2 domain, and a CH3 domain, and has a first CD16a-binding domain and a second CD16a-binding domain that are different from each other in the CH2 domain,

(3C) each of the first IgG half-molecule and the second IgG half-molecule includes amino acid residue substitutions of C226A and C229A numbered according to the EU index,

(4C) the first IgG half-molecule includes an antigen-binding domain that binds to the first antigen, and includes an amino acid residue substitution of D265A numbered according in the EU index in the first CD16a-binding domain,

(5C) the second IgG half-molecule includes an antigen-binding domain that binds to the second antigen, and includes an ammo acid residue substitution of P329Y numbered according to the EU index in the second CD16a-binding domain,

(6C) each of the first IgG half-molecule and the second IgG half-molecule includes amino acid residue substitutions of (a) S239D and K326T numbered according to the EU index, or

each of the first IgG half-molecule and the second IgG half-molecule includes amino acid residue substitutions of (b) S239D, S298A, E333A, L242C, and K334C numbered according to the EU index, and

(7C) at least one of the first IgG half-molecule and the second IgG half-molecule includes an amino acid residue substitution in the CH3 domain as an alteration for attenuating inter-CH3 domain interaction as compared with an inter-CH3 domain interaction of the IgG1 subclass.

11. A DNA encoding the following amino acid sequence a) or b):

a) an amino acid sequence of the first IgG half-molecule according to the above 9 or

b) an amino acid sequence of the second IgG half-molecule according to the above 10.

12. A recombinant vector, containing at least one of the DNA encoding the amino acid sequence a) and the DNA encoding the amino acid sequence b) according to the above 11.

13. A transformant, in which the recombinant vector according to the above 12 is introduced.

14. A kit, including the first IgG half-molecule according to the above 9 and the second IgG half-molecule according to the above 10.

15. A method for inducing an effector function specifically for a target cell coexpressing the first antigen and the second antigen as compared with an effector function for a target cell expressing only the first antigen and a target cell expressing only the second antigen using the antibody composition according to any one of the above 1 to 8.

16. A pharmaceutical composition, containing the antibody compositions according to any one of the above 1 to 8.

17. The pharmaceutical composition according to the above 16, for use in a treats; em of a cancer, an autoimmune disease, or an allergic disease.

18. Use of the antibody composition according to any one of the above 1 to 8 for producing is pharmaceutical composition fora treatment of a cancer, an autoimmune disease, or an allergic disease.

19. Use of the antibody composition according to an one of the above1 to 8 for treating a cancer, an autoimmune disease, or allergic disease.

20. The antibody composition according to any one of the above 1 to 8 for use in a treatment of a cancer, an autoimmune disease, or an allergic disease.

21. A method for treating a cancer, an autoimmune disease, or an allergic disease, including administering an effective amount of the antibody composition according to any one of the above 1 to 8 to a subject.

22. A first IgG half-molecule, which is a first IgG hue-molecule for use in combination with a second IgG half-molecule, wherein the first IgG half-molecule and the second IgG half-molecule satisfy the following (1B) to (7B):

(1B) the first IgG half-molecule and the second IgG half-molecule form an antibody composition against a first antigen and a second antigen that are different from each other,

(2B) each of the first IgG half-molecule and the second IgG half-molecule is composed 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 domain and a second CD16a-binding domain that are different from each other in the CH2 domain,

(3B) each of the first IgG half-molecule and the second IgG half-molecule includes amino acid residue substitutions of C226A and C229A numbered according to the EU index,

(6B) each of the first IgG half-molecule and the second IgG half-molecule includes amino acid residue substitutions of (a) S239D and K326T numbered according to the EU index, or

each of the first IgG half-molecule and the second IgG half-molecule includes amino acid residue substitutions of (b) S239D, S298A, E333A, L242C, and K334C numbered according to the EU index, and

(7B) at least one of the first IgG half-molecule and the second IgG half-molecule includes an amino acid residue substitution in the CH3 domain as an alteration for attenuating an inter-CH3 domain interaction as compared with an inter-CH3 domain interaction of the IgG1 subclass.

23. second IgG half-molecule, which is a second IgG half-molecule for use in combination with a first IgG half-molecule, wherein the first IgG half-molecule and the second IgG half-molecule satisfy the following (1C) to (7C):

(1C) the first IgG half-molecule and the second IgG half-molecule form an antibody composition against a first antigen and a second antigen that are different from each other,

(2C) each of the first IgG half-molecule and the second IgG half-molecule is composed of one L chain and one chain, and the H chain includes an H chain variable region, a hinge domain, a CH1 domain, a CH2 domain, an a CH3 domain, and has a first CD16a-binding domain and a second CD16a-binding domain that are different from each other in the CH2 domain,

(3C) each of the first IgG half-molecule and the second IgG half-molecule includes amino acid residue substitutions of C226A and C229A numbered according to the EU index,

(5C) the second IgG half-molecule includes an antigen-binding domain that binds to the second antigen, and includes an amino acid residue substitution of P329Y numbered according to the EU index in the second CD16a-binding domain,

(6C) each of the first IgG half-molecule and the second IgG half-molecule includes amino acid residue substitutions of (a) S239D and K326 T numbered according to the EU index, or

each of the first IgG half-molecule and the second IgG half-molecule includes amino acid residue substitutions of (b) S239D, S298A, E333A, L242C, and K334C numbered according to the EU index, and

(7C) at least one of the first IgG half-molecule and the second IgG half-molecule includes an amino acid residue substitution in the CH3 domain as an alteration for attenuating an inter-CH3 domain interaction as compared with an inter-CH3 domain interaction of the IgG1 subclass.

24. The first or second IgG half-molecule contained in the antibody composition according to any one of the above 1 to 8.

25. A method for inducing an effector function specifically for a double-positive cell expressing the first and second antigens using the IgG half-molecule according to the above 9, 10, or 24.

26. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CADM1 and the second antigen is CCR4.

27. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is EGFR and the second antigen is HER2.

28. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CD52 and the second antigen CD70.

29. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CD2 and the second antigen is CD70.

30. he antibody composition according to any one of the above 1 to 8, wherein the first antigen is CD19 and the second antigen is CD70.

31. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CD2 and the second antigen is CD40 ligand.

32. The antibody composition according to any one of the above 1 to 8, 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 the above 1 to 8, wherein the first antigen is CD8 and the second antigen is CCR4.

34. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CTLA 4 and the second antigen is one selected from CD4, CCR4, and GITR,

35. The antibody compost non according to any one of the above 1 to 8, 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 the above 1 to 8, 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 the above 1 to 8, 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 the above 1 to 8, wherein the first antigen is 4-1BB and die second antigen is one selected from CD127, CD26, CD70, and CD15s.

39. The antibody composition according to any one of the above 1 to 8, 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 the above 1 to 8, 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 the above 1 to 8, wherein the first antigen is CD4 arid the second. antigen is CD69.

42. A pharmaceutical composition for use in a treatment of ATL, containing the antibody composition according to the above 26.

43. A pharmaceutical composition for use in a treatment of at least one of gastric cancer and breast cancer, containing the antibody composition according to the above 27.

44. A pharmaceutical composition for use in a treatment of Sjogren's syndrome, containing the antibody composition according to the above 31.

45. A pharmaceutical composition for use in a treatment of at least one of lymphoma and leukemia, containing the antibody composition according to the above 32.

46. A pharmaceutical composition for use in a treatment of a cancer, containing the antibody composition according to any one of the above 33 to 36.

47. A pharmaceutical composition or use in a treatment of an autoimmune disease, containing the antibody composition according to any one of the above 37 to 40.

48. A pharmaceutical composition for use in a treatment of ANCA-associated glomerulonephritis, containing the antibody composition according to the above 41.

49. Use of the antibody composition according to the above 26 for producing a pharmaceutical composition for treating ATL.

50. Use of the antibody composition according to the above 27 for producing a pharmaceutical composition for treating at least one of gastric cancer and breast cancer.

51. Use of the antibody composition according to the above 31 for producing a pharmaceutical composition for treating Sjogren's syndrome,

52. Use of the antibody composition according to the above 32 for producing a pharmaceutical composition for treating at least one of lymphoma and leukemia.

53. Use of the antibody composition according to any one of the above 33 to 36 for producing a pharmaceutical composition for treating a cancer.

54. Use of the antibody composition according to any one of the above 37 to 40 for producing a pharmaceutical composition for treating an autoimmune disease.

55. Use of the antibody composition according to the above 41 for producing a pharmaceutical composition for treating ANCA-associated glomerulonephritis.

56. Use of the antibody composition according to the above 26 for treating ATL.

57. Use of the antibody composition according to the above 27 for treating at least one of gastric cancer and breast cancer.

58. Use of the antibody composition according to the above 31 for treating Sjogren's syndrome.

59. Use of the antibody composition according to the above 32 for treating at least one of lymphoma and leukemia.

60. Use of the antibody composition according to any one of the above 33 to 36 for treating a cancer.

61. Use of the antibody composition according to any one of the above 37 to 40 for treating an autoimmune disease.

62. Use of the antibody composition according to the above 41 for treating ANCA-associated glomerulonephritis.

63. The antibody composition according to the above 26 for use in a treatment of ATL.

64. The antibody composition according to the above 27 for use in a treatment of at least one of gastric cancer and breast cancer.

65. The antibody composition according to the above 31 for use in a treatment of Sjogren's syndrome.

66. The antibody composition according to the above 32 for use in a treatment of at least one of lymphoma and leukemia.

67. The antibody composition according to any one of the above 33 to 36 for use in a treatment of a cancer.

68. The antibody composition according to any one of the above 37 to 40 for use in a treatment of an autoimmune disease.

69. The antibody composition according to the above 41 for use in a treatment of ANCA-associated glomerulonephritis.

70. A method for treating ATL, including administering an effective amount of the antibody composition according to the above 26 to a subject.

71. A method for treating at least one of gastric cancer and breast cancer, including administering an effective amount of the antibody composition according to the above 27 to a subject.

72. A method for treating Sjogren's syndrome, including administering an effective amount of the antibody composition according to the above 31 to a subject.

73. A method for treating at least one of lymphoma and leukemia, including administering an effective amount of the antibody composition according to the above 32 to a subject.

74. A method for treating a cancer, including administering an effective amount of the antibody composition according to any one of the above 33 to 36 to a subject.

75. A method for treating an autoimmune disease, including administering an effective amount of the antibody composition according to any one of the above 37 to 40 to a subject.

76. A method for treating ANCA-associated glomerulonephritis, including administering an effective amount of the antibody composition according to the above 41 to a subject.

77. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is PD-1 and the second antigen is CD3.

78. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is PD-1 and the second antigen is CD4.

79. The antibody composition according to any one of the above 1 to 8, 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 the above 1 to 8, wherein the first antigen is integrin α4β7 and the second antigen is CD40 ligand.

81. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is integrin α4β1 and the second antigen is CD40 ligand.

82. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CXCR5 and the second antigen is CD127.

83. The antibody composition according to any one of the above 1 to 8, 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 the above 1 to 8, wherein the first antigen is CXCR3 and the second antigen is CD3.

85. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CXCR3 and the second antigen is CD127 or CD40 ligand.

86. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CCR4 and the second antigen is CD127 or CD40 ligand.

87. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CCR6 and the second antigen is CD127 or CD40 ligand.

88. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CRTH2 and the second antigen is CD127 or CD40 ligand.

89. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CRTH2 and the second antigen is CCR4.

90. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CRTH2 and the second antigen is ST2.

91. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CRTH2 and the second antigen CCR6 or CCR3.

92. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CD207 and the second antigen is CD11b or CD1a.

93. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CD123 and the second antigen is HLA-DR or ASCT2.

94. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CSF1R and the second antigen is CD14.

95. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CSF1R and the second antigen CD33.

96. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CD19 and the second antigen is CD38.

97. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CD3 and the second antigen is IL-23R.

98. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CD3 and the second antigen is CX3CR1.

99. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CXCR4 and the second antigen is type 1 collagen.

100. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CXCR4 and the second antigen is CD14.

101. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CXCR4 and the second antigen is CD16.

102. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CLEC10A and the second antigen is CD14 or CD16.

103. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CD70 and the second antigen is CD38.

104. The antibody composition according to any one of the above 1 to 8, 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 the above 1 to 8, wherein the first antigen is CD4 and the second antigen is TIM-1.

106. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CD70 and the second antigen is CD52.

107. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CB1 and the second antigen is AT1R.

108. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CD4 or PD-1 and the second antigen is CD153.

109. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is FcεRI and the second antigen is CD34 or C-KIT.

110. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CD34 and the second antigen is CD203 MRGPRX2.

111. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CD52 and the second antigen is CD127.

112. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CD69 and the second antigen is CD21.

113. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CD106 and the second antigen is one selected from CD11c, CD19, CD 21, and CD 72.

114. The antibody composition according to any one of the above 1 to 8, wherein the tint antigen is 4-1BS and the second antigen is one selected from CD11c, CD19, CD21, and CD72.

115. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is BLT1 and the second antigen is CD49b.

116. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CD226 and the second antigen is CD8.

117. The antibody composition according to any one of the above 1 to 8, 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 the above 1 to 8, wherein the first antigen is CD49a and the. second antigen is one selected from CD8, IL-15R, and NKG2 D.

119. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is IL-15R and the second antigen is CD8, or NKG2D.

120. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is NIKG2D and the second antigen is CD8.

121. The antibody composition according to any one of the above 1 to 8, wherein the first antigen s CD177 and the, second antigen is PR3.

122. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CD4 and the second antigen is CD127.

123. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CD40 ligand mind the second antigen is IL-6.

124. The antibody composition according to any one of the above 1 to 8, wherein the first antigen, is IL-17R and the second antigen is membrane-associated TNF.

125. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is IL-23R and the second antigen is membrane-associated CCR6.

126. Use of the antibody composition according to the above 77 for producing a pharmaceutical composition for treating rheumatoid arthritis.

127. Use of the antibody composition according to the above 78 for producing a pharmaceutical composition for treating celiac disease.

128. Use of the antibody composition according to the above 79 for producing a pharmaceutical composition for treating an inflammatory bowel disease.

129. Use of the antibody composition according to the above 80 for producing a pharmaceutical composition for treating at least one of multiple sclerosis and an inflammatory bowel disease.

130. Use of the antibody composition according to the above 81 for producing a pharmaceutical composition for treating at least one of multiple sclerosis and an inflammatory bowel disease.

131. Use of the antibody composition according to the above 82 for producing a pharmaceutical composition for treating an allergic disease.

132. Use of the antibody composition according to the above 83 for producing a pharmaceutical composition for treating primary myelofibrosis.

133. Use of the antibody composition according to the above 84 for producing a pharmaceutical composition for treating at least one selected from an autoimmune disease, arteriosclerosis, and an ischemic heart disease.

134. Use of the antibody composition according to the above 85 for producing a pharmaceutical composition for treating systemic lupus erythematosus.

135. Use of the antibody composition according to the above 86 for producing a pharmaceutical composition for treating an allergic disease.

136. Use of the antibody composition according to the above 87 for producing a pharmaceutical composition for treating psoriasis.

137. Use of the antibody composition according to the above 88 for producing a pharmaceutical composition for treating an allergic disease.

138. Use of the antibody composition according to the above 89 for producing a pharmaceutical composition for treating at least one selected from asthma, eosinophilic sinusitis, and atopic dermatitis.

139. Use of the antibody composition according to the above 90 for producing a pharmaceutical composition for treating at least one of asthma and eosinophilic sinusitis.

140. Use of the antibody composition according to the above 91 for producing a pharmaceutical composition for treating an allergic disease.

141. Use of the antibody composition according to the above 92 for producing a pharmaceutical composition for treating langerhans cell histiocytosis (LCH).

142. Use of the antibody composition according to the above 93 for producing a pharmaceutical composition for treating systemic lupus erythematosus.

143. Use of the antibody composition according to the above 94 for producing a pharmaceutical composition for treating idiopathic pulmonary fibrosis.

144. Use of the antibody composition according to the above 95 for producing a pharmaceutical composition for treating a cancer.

145. Use of the antibody composition according to the above 96 or 97 for producing a pharmaceutical composition for treating systemic lupus erythematosus.

146. Use of the antibody composition according to the above 98 for producing a pharmaceutical composition for treating at least one selected from an autoimmune disease, arteriosclerosis, and an ischemic heart disease.

147. Use of the antibody composition according to the above 99 for producing a pharmaceutical composition for treating at least one selected from various fibrotic diseases such as idiopathic pulmonary fibrosis, systemic sclerosis, and hepatic cirrhosis.

148. Use of the antibody composition according to the above 100 for producing a pharmaceutical composition for treating idiopathic pulmonary fibrosis.

149. Use of the antibody composition according to the above 101 for producing a pharmaceutical composition for treating at least one selected from neutrophilic asthma, chromic obstructive pulmonary disease, and Alzheimer's disease.

150. Use of the antibody composition according to the above 102 for producing a pharmaceutical composition for treating at least one selected from Crohn's disease, rheumatoid arthritis, and asthma.

151. Use of the antibody composition according to the above 103 for producing pharmaceutical composition for treating at least one of multiple sclerosis and neuromyelitis optica.

152. Use of the antibody composition according to the above 104 or 105 for producing a pharmaceutical composition for treating systemic lupus erythematosus.

153. Use of the antibody composition according to the above 106 or producing a pharmaceutical composition for treating multiple sclerosis.

154. Use of the antibody composition according to the above 107 for producing a pharmaceutical composition for treating hepatic cirrhosis.

155. Use of the antibody composition according to the above 108 for producing a pharmaceutical composition for treating systemic lupus erythematosus.

156. Use of the antibody composition according to the above 109 for producing a pharmaceutical composition for treating a mast cell activation syndrome (MCAS) including at least one selected from urticaria, a food allergy, and mastocytosis.

157. Use of the antibody composition according to the above 110 for producing a pharmaceutical composition for treating a mast cell activation syndrome (MCAS) including at least one selected from urticaria, a food allergy, and mastocytosis.

158. Use of the antibody composition according to the above 111 for producing a pharmaceutical composition tor treating multiple sclerosis.

159. Use of the antibody composition according to the above 112 for producing a pharmaceutical composition for treating at least one selected from multiple sclerosis, type 1 diabetes mellitus, and rheumatoid arthritis.

160. Use of the antibody composition according to the above 113 for producing a pharmaceutical composition for treating at least one selected from secondary progressive multiple sclerosis (SPMS), rheumatoid arthritis for which anti-TNF therapy is not effective, transplantation, and systemic lupus erythematosus.

161. Use of the antibody composition according to the above 114 for producing a pharmaceutical composition for treating at least one selected from secondary progressive multiple sclerosis (SPMS), rheumatoid arthritis for which anti-TNF therapy is not effective, transplantation, and systemic lupus erythematosus.

162. Use of the antibody composition according to the above 115 for producing a pharmaceutical composition for treating asthma.

163. Use of the antibody composition according to the above 116 for producing a pharmaceutical composition for treating systemic scleroderma.

164. Use of the antibody composition according to the above 117 for producing a pharmaceutical composition for treating at least one of vitiligo and psoriasis.

165. Use of the antibody composition according to the above for pharmaceutical composition for treating at least one of vitiligo and psoriasis.

166. Use of the antibody composition according to the above 119 for producing a pharmaceutical composition for treating at least one of vitiligo and psoriasis.

167. Use of the antibody composition according to the above 120 for producing a pharmaceutical composition for treating at least one of vitiligo and psoriasis.

168. Use of the antibody composition according to the above 121 for producing 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 the above 122 for producing a pharmaceutical composition for treating a cell-dependent immune-related disease.

170. Use of the antibody composition according to the above 123 for producing a pharmaceutical composition for treating an immune-related disease.

171. Use of the antibody composition according to the above 124 for producing a pharmaceutical composition tor treating psoriatic arthritis.

172. Use of the antibody composition according to the above 125 for producing a pharmaceutical composition for treating a autoimmune disease including at least one of Sjogren's syndrome and psoriasis.

173. Use of the antibody composition according to the above 77 for treating rheumatoid arthritis.

174. Use of the antibody composition is according to the above 78 for treating celiac disease.

175. Use of the antibody composition according to the above 79 for treating an inflammatory bowel disease.

176. Use of the antibody composition according to the above 80 for treating at least one of multiple sclerosis and an inflammatory bowel disease.

177. Use of the antibody composition according to the above 81 tor treating at least one of multiple sclerosis, and an inflammatory bowel disease.

178. Use of the antibody composition according to the above 82 for treating an allergic disease.

179. Use of the antibody composition according to the above 83 the treating primary myelofibrosis.

180. Use of the antibody composition according to the above 84 for treating at least one selected from an autoimmune disease, arteriosclerosis, and an ischemic heart disease.

181. Use of the antibody composition according to the above 85 for treating systemic lupus erythematosus.

182. Use of the antibody composition according to the above 86 for treating an allergic disease.

183. Use of the antibody composition according to the above 87 for treating psoriasis.

184. Use of the antibody composition according to the above 88 for treating an allergic disease.

185. Use of the antibody composition according to the above 89 for treating at least one ticketed from asthma, eosinophilic sinusitis, and atopic dermatitis.

186. Use of the antibody composition according to the above 90 for treating at least one of asthma and eosinophilic sinusitis.

187. Use of the antibody composition according to the above 91 for treating an allergic disease.

188. Use of the antibody composition according to the above 92 for treating langerhans cell histiocytosis (LCH).

189. Use of the antibody composition according to the above 93 for treating systemic lupus erythematosus.

190. Use of the antibody composition according to the above 94 for treating idiopathic pulmonary fibrosis.

191. Use of the antibody composition according to the above 95 for treating a cancer.

192. Use of the antibody composition according to the above 96 or 97 for treating systemic lupus erythematosus.

193. Use of the antibody composition according to the above 98 for treating at least one selected from an autoimmune disease, arteriosclerosis, and an ischemic heart disease.

194. Use of the antibody composition according to the above 99 for treating a fibrotic disease including at least one selected from idiopathic pulmonary fibrosis, systemic sclerosis, and hepatic cirrhosis.

195. Use of the antibody composition according to the above 100 for treating idiopathic pulmonary fibrosis.

196. Use of the antibody composition according to the above 101 for treating at least one selected from neutrophilic asthma, chrome obstructive pulmonary disease, and Alzheimer's disease.

197. Use of the antibody composition according to the above 102 for treating at least one selected from Crohn's disease, rheumatoid arthritis, and asthma.

198. Use of the antibody composition according to the above 103 for treating at least one of multiple sclerosis and neuromyelitis optica.

199. Use of the antibody composition according to the above 104 or 105 for treating systemic lupus erythematosus.

200. Use of the antibody composition according to the above 106 for treating multiple sclerosis.

201. Use of the antibody composition according to the above 107 for treating hepatic cirrhosis.

202. Use of the antibody composition according to the above 108 for treating systemic lupus erythematosus.

203. Use of the antibody composition according to the above 109 for treating a mast cell activation syndrome (MCAS) including at least one selected from urticaria, a food allergy, and mastocytosis.

204. Use of the antibody composition according to the above 110 for treating a mast cell activation syndrome (MCAS) including at least one selected from urticaria, a food allergy, and mastocytosis.

205. Use of the antibody composition according to the above 111 for treating multiple sclerosis.

206. Use of the antibody composition according to the above 112 tor treating at least one selected from multiple sclerosis, type 1 diabetes mellitus, and rheumatoid arthritis.

207. Use of the antibody composition according to the above 113 for treating at least one selected from secondary progressive multiple sclerosis (SPMS), rheumatoid arthritis for which anti-TNF therapy is not effective, transplantation, and systemic lupus erythematosus.

208. Use of the antibody composition according to the above 114 for treating at least one selected from secondary progressive multiple sclerosis (SPMS) rheumatoid arthritis for which anti-TNF therapy is not effective, transplantation, and systemic lupus erythematosus.

209. Use of the antibody composition according the above 115 for treating asthma.

210. Use of the antibody composition according to the above 116 for treating systemic scleroderma.

211. Use of the antibody composition according to the above 117 tor treating at least one of vitiligo and psoriasis.

212. Use of the antibody composition according to the above 118 for treating at least one of vitiligo and psoriasis.

213. Use of the antibody composition according to the above 119 for treating at least one of vitiligo and psoriasis.

214. Use of the antibody composition according to the above 120 for treating at least one of vitiligo and psoriasis.

215. Use of the antibody composition according to the above 121 for treating at least one of ANCA-associated vasculitis and systemic lupus erythematosus.

216. Use of the antibody composition according to the above 122 for treating a T cell-dependent immune-related disease.

217. Use of the antibody composition according to the above 123 for treating an immune-related disease.

218. Use of the antibody composition according to the above 124 for treating psoriatic arthritis.

219. Use of the antibody composition according to the above 125 for treating an autoimmune disease including at least one of Sjogren's syndrome and psoriasis.

220. The antibody composition according to the above 77 for use in a treatment of rheumatoid arthritis.

221. The antibody composition according to the above 78 for use in a treatment of celiac disease.

222. The antibody composition according to the above 79 for use in a treatment of an inflammatory bowel disease.

223. The. antibody composition according to the above 80 for use in a treatment of at least one of multiple sclerosis and an inflammatory bowel disease.

224. The antibody composition according to the above 81 for use in a treatment of at least one of multiple sclerosis and an inflammatory bowel disease.

225. The antibody composition according to the above 82 for use in a treatment of an allergic disease.

226. The antibody composition according to the above 83 for use in a treatment of primary myelofibrosis.

227. The antibody composition according to the above 84 for use in a treatment of at least one selected from an autoimmune disease, arteriosclerosis, and an ischemic heart disease.

228. The antibody composition according to the above 85 for use in a treatment of systemic lupus erythematosus.

229. The antibody composition according to the above 86 for use in a treatment of an allergic disease.

230. The antibody composition according to the above 87 for use in a treatment of psoriasis.

231. The antibody composition according to the above 88 for use in a treatment of an allergic disease.

232. The antibody composition according to the above 89 for use in a treatment of at least one selected from asthma, eosinophilic sinusitis, and atopic dermatitis.

233. The antibody composition according to the above 90 for use in a treatment of at least one of asthma and eosinophilic sinusitis.

234. The antibody composition according to the above 91 for use in a treatment of an allergic disease.

235. The antibody composition a:cording to the above 92 for use in a treatment of langerhans cell histiocytosis (LCH).

236. The antibody composition according to the above 93 for use in a treatment of systemic lupus erythematosus.

237. The antibody composition according to the above 94 for use in a treatment of idiopathic pulmonary fibrosis.

238. me antibody composition according to the above 95 for use in a treatment of a cancer.

239. The antibody composition according to the above 96 or 97 for use in a treatment of systemic lupus erythematosus.

240. The antibody composition according to the above 98 for use in a treatment of at least one selected front an autoimmune disease, arteriosclerosis, and an ischemic heart disease.

241. The antibody composition according to the above 99 for use in a treatment of a fibrotic disease including at least one selected from idiopathic pulmonary fibrosis, systemic sclerosis, and hepatic cirrhosis.

242. The antibody composition according o the above 100 for use in a treatment of idiopathic pulmonary fibrosis.

243. The antibody composition according to the above 101 for use in a treatment of at least one selected from neutrophilic asthma, chronic obstructive pulmonary disease, and Alzheimer's disease.

244. The antibody composition according to the above 102 for use in a treatment of at least one selected from Crohn's disease, rheumatoid arthritis, and asthma.

245. The antibody composition according to the above 103 for use in a treatment of at least one of multiple sclerosis and neuromyelitis optics.

246. The antibody composition according to the above 104 or 105 for use in a treatment of systemic lupus erythematosus.

247. The antibody composition according to the above 106 for use in a treatment of multiple sclerosis.

248. The antibody composition according to the above 107 for use in a treatment of hepatic cirrhosis.

249. The antibody composition according to the above 108 for use in a treatment of systemic lupus erythematosus.

250. The antibody composition according to the above 109 for use in a treatment of a mast cell activation syndrome (MCAS) including at least one selected from urticaria, a food allergy, and mastocytosis.

251. The antibody composition according to the above 110 for use in a treatment of a mast cell activation syndrome (MCAS) including at least one selected from urticaria, a food allergy, and mastocytosis.

252. The antibody composition according to the above 111 for use in a treatment of multiple sclerosis.

253. The antibody composition according to the above 112 for use in a treatment of at least one selected from multiple sclerosis, type 1 diabetes mellitus, and rheumatoid arthritis.

254. The antibody composition according to the above 113 for use in a treatment of at least one selected from secondary progressive multiple sclerosis (SPMS), rheumatoid arthritis for which anti-TNF therapy is not effective, transplantation, and systemic lupus erythematosus.

255. The antibody composition according to the above 114 for use in a treatment of at least one selected from secondary progressive multiple sclerosis (SPMS), rheumatoid arthritis for winch anti-TNF therapy is not effective, transplantation, and systemic lupus erythematosus.

256. The antibody composition according to the above 115 for use in a treatment of asthma.

257. The antibody composition according to the above 116 for use in a treatment of systemic scleroderma.

258. The antibody composition according to the above 117 for use in a treatment of at least one of vitiligo and psoriasis.

259. The antibody composition according to the above 118 for use in a treatment of at least one of vitiligo and psoriasis.

260. The antibody composition according to the above 119 tin use in a treatment of at least one of vitiligo and psoriasis.

261. The antibody composition according to the above 120 for use in a treatment of at least one of vitiligo and psoriasis.

262. The antibody composition according to the above 121 for use in a treatment of at least one of ANCA-associated vasculitis and systemic lupus erythematosus.

263. The antibody composition according to the above 122 for use in a treatment of a T cell-dependent immune-related disease.

264. The antibody composition according to the above 123 for use in a treatment of an immune-related disease.

265. The antibody composition according to the above 124 for use in a treatment of psoriatic arthritis.

266. The antibody composition according to the above 125 for use in a treatment of an autoimmune disease including at least one of Sjogren's syndrome and psoriasis.

267. A method for treating rheumatoid arthritis, including administering an effective amount of the antibody composition according to the above 77 to a subject.

268. A method fir treating celiac disease, including administering an effective amount of the antibody composition according to the above 78 to a subject.

269. A method for treating an inflammatory bowel disease, including administering an effective amount of the antibody composition according to the above 79 to a subject.

270. A method for treating at least one of multiple sclerosis and an inflammatory bowel disease, including administering an effective amount of the antibody composition according to the above 80 to a subject.

271. A method for treating at least one of multiple sclerosis and an inflammatory bowel disease, including administering an effective amount of the antibody composition according to the above 81 to a subject.

272. A method for treating an allergic disease, including administering an effective amount of the antibody composition according to the above 82 to a subject.

273. A method for treating primary myelofibrosis, including administering an effective amount of the antibody composition according to the above 83 to a subject.

274. A method for treating at least one selected from an autoimmune disease, arteriosclerosis, and an ischemic heart disease, including administering an effective amount of the antibody composition according to the above 84 to a subject.

275. A method for treating systemic lupus erythematosus, including administering an effective amount of the antibody composition according to the above 85 to a subject.

276. A method for treating an allergic disease, including administering an effective amount of the antibody composition according to the above 86 to a subject.

277. A method for treating psoriasis, including administering an effective amount of the antibody composition according to the above 87 to a subject.

278. A method for treating an allergic disease, including administering an effective amount of the antibody composition according to the above 88 to a subject.

279. A method for treating at least one selected from asthma, eosinophilic sinusitis, and atopic dermatitis, including administering an effective amount of the antibody composition according to the above 89 to a subject.

280. A method for treating at least one of asthma and eosinophilic sinusitis, including administering an effective amount of the antibody composition according to the above 90 to a subject.

281. toe production of a pharmaceutical composition for treating an allergic disease, including administering an effective amount of the antibody composition according to the above 91 to a subject.

282. A method the treating langerhans cell histiocytosis (LCH), including administering an effective amount of the antibody composition according to the above 92 to a subject.

283. A method for treating systemic lupus erythematosus, including administering an effective amount of the antibody composition according to the above 93 to a subject.

284. A method for treating idiopathic pulmonary fibrosis, including administering an effective amount of the antibody composition according to the above 94 to a subject.

285. A method for treating a cancer, including administering an effective amount of the antibody composition according to the above 95 to a subject.

286. A method for treating systemic lupus erythematosus, including administering an effective amount of the antibody composition according to the above 96 or 97 to a subject.

287. A method for treating at least one selected from an autoimmune disease, arteriosclerosis, and an ischemic heart disease, including administering an of amount of the antibody composition according to the above 98 to a subject.

288. A method for treating a fibrotic disease including at least one selected from idiopathic pulmonary fibrosis, systemic sclerosis, and hepatic cirrhosis, including administering an effective amount of the antibody composition according to the above 99 to a subject.

289. A method for treating idiopathic pulmonary fibrosis, including administering an effective amount of the antibody composition according to the above 100 to a subject.

290. A method for treating at least one selected from neutrophilic asthma, chronic obstructive pulmonary disease, and Alzheimer's disease, including administering an effective amount of the antibody composition according to the above 101 to a subject.

291. A method for treating at least one selected from Crohn's disease, rheumatoid arthritis, and asthma, including administering an effective amount of the antibody composition according to the above 102 to a subject.

292. A method for treating at least one of multiple sclerosis and neuromyelitis optics, using the antibody composition according to the above 103.

293. A method for treating systemic lupus erythematosus, including administering an effective amount of the antibody composition according to the above 104 or 105 to a subject.

294. A method for treating multiple sclerosis, including administering an effective amt um of the antibody composition according to the above 106 to a subject.

295. A method for treating hepatic cirrhosis, including administering an effective amount of the antibody composition according to the above 107 to a subject.

296. A method or treating systemic lupus erythematosus, including administering an effective amount of the antibody composition according to the above 108 to a subject.

297. A method for treating a mast cell activation syndrome (MCAS) including at least one selected from urticaria, a food allergy and mastocytosis, including administering an effective amount of the antibody composition according to the above 109 to a subject.

298. A method for treating a mast cell activation syndrome (MCAS) including at least one selected from urticaria, a food allergy, and mastocytosis, including administering an effective amount of the antibody composition according to the above 110 to a subject.

299. A method for treating multiple sclerosis, including administering an effective amount of the antibody composition according to the above 111 to a subject.

300. A method for treating at least one selected from multiple sclerosis, type 1 diabetes mellitus, and rheumatoid arthritis including administering an effective amount of the antibody composition according to the above 112 to a subject.

301. A method for treating at least one selected secondary progressive multiple sclerosis (SPMS), rheumatoid arthritis for which anti-TNF therapy is not effective, transplantation, and systemic lupus erythematosus, including administering an effective amount of the antibody composition according to the above 113 to a subject.

302. A method for treating at least one selected from secondary progressive multiple sclerosis (SPMS), rheumatoid arthritis for which anti-TNF therapy is not effective, transplantation, and systemic lupus erythematosus, including administering an effective amount of the antibody composition according to the above 114 to a subject.

303. A method for treating asthma, including administering an effective amount of the antibody composition according to the above 115 to a subject.

304. A method for treating systemic scleroderma, including administering an effective amount of the antibody composition according to the above 116 to a subject.

305, A method for treating at least one of vitiligo and psoriasis, including administering an effective amount of the antibody composition according to the above 117 to a subject.

306. A method for treating at least one of vitiligo and psoriasis, including administering an effective amount of the antibody composition according to the above 118 to a subject.

307. A method for treating at least one of vitiligo and psoriasis, including administering an effective amount of the antibody composition according to the above 119 to a subject.

308. A method for treating at least one of vitiligo and psoriasis, including administering an effective amount of the antibody composition according to the above 120 to a subject.

309. A method for treating et least one of ANCA-associated vasculitis and systemic lupus erythematosus, including administering an effective amount of the antibody composition according to the above 121 to a subject.

310. A method for treating a T cell-dependent immune-related disease, including administering an effective amount of the antibody composition according to the above 122 to a subject.

311. A method for treating an immune-related disease, including administering an effective amount of the antibody composition according to the above 123 to a subject.

312. A method for treating psortatic arthritis, including administering an effective amount of the antibody composition according to the above 124 to a subject.

313. A method for treating an autoimmune disease including at least one of Sjogren's syndrome and psoriasis, including administering an effective amount of the antibody composition according to the above 125 to a subject.

314. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CD64 and the second antigen is one selected born CD206, CD163, and CD68.

315. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CD163 and the second antigen is CD206 or CD68.

316. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CD206 and the second antigen is CD68.

317. The antibody composition according to any one of the above 1 to 8, 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 the above 1 to 8, wherein the first antigen is CD15 and the second antigen is one selected from CD48, CD84, CD97, and CD305.

119. The antibody composition according to any one of the above 1 to 8, 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 my one of the above 1 to 8, wherein the first antigen is CD11b and the second antigen is one selected front CD48, CD84, CD97, and CD305.

321. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CCR4 and the second antigen is one selected from CADM1, CD 30, and CD70.

322. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CD38 and the second antigen is CD138 or BCMA.

323. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is BCMA and the second antigen is CD56 or CS1.

324. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CD40 ligand and the second antigen is one selected from CD36, CD162P, and CD63.

325. The antibody composition according to any one of the above 1 to 8, 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 the above 1 to 8, 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 the above 1 to 8, wherein the first antigen is CD5 and the second antigen is CD23.

328. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CD10 or CD5 and the second antigen is CD20.

329. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CD40 and the second antigen is one 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 the above 1 to 8, 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 the above 1 to 8, wherein the first antigen is OX40 and the second antigen is CD127 or CD40 ligand.

332. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is OX40 and the second antigen is CD8 or NKG2D,

333. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is OX40 and the second antigen is CD 226.

334. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is OX40 and the second antigen is CRTH2.

335. The antibody composition according to any one of the above 1 to 8, wherein the first antigen is CRTH2 and the second antigen is any one selected from CD2, CD7, and CD45.

336. A pharmaceutical composition for use in a treatment of a cancer, containing the antibody composition according to any one of the above 314 to 320.

337. A pharmaceutical composition for use in a treatment of leukemia, containing the antibody composition according to any one of the above 321 to 328.

338. A pharmaceutical composition for use in a treatment of lymphoma, containing the antibody composition according to any one of the above 321 to 328.

339. A pharmaceutical composition tor use in a treatment of an inflammatory disease, containing the antibody composition according to the above 329.

340. A pharmaceutical composition for use in a treatment of an allergic disease, containing the antibody composition according to any one of the above 331, 334, and 335.

341. A pharmaceutical composition for use in a treatment of at least one of vitiligo and psoriasis, containing the antibody composition according to the above 332.

342. A pharmaceutical composition for use in a treatment of scleroderma, containing the antibody composition according to the above 333.

343. Use of the antibody composition according to any one of the above 314 to 320 for producing a pharmaceutical composition for treating a cancer.

344. Use of the antibody composition according to any one of the above 321 to 328 for producing a pharmaceutical composition for treating leukemia.

345. Use of the antibody composition according to any one of the above 321 to 328 for producing a pharmaceutical composition for treating lymphoma.

346. Use of the antibody composition according to the above 329 for producing a pharmaceutical composition for treating an inflammatory disease.

347. Use of the antibody composition according to any one of the above 331, 334, and 335 for producing a pharmaceutical composition for treating an allergic disease.

348. Use of the antibody composition according to the above 332 for producing a pharmaceutical composition for treating at least one of vitiligo and psoriasis.

349. Use of the antibody composition according to the above 333 for producing a pharmaceutical composition for treating scleroderma.

350. Use of the antibody composition according to any one of the above 314 to 320 for treating a cancer.

351. Use of the antibody composition according to any one of the above 321 to 328 for treating leukemia.

352. Use of the antibody composition according to any one of the above 321 to 328 for treating lymphoma.

353. Use of the antibody composition according to the above 329 for treating an inflammatory disease.

354. Use of the antibody composition according to any one of the above 331, 334, and 335 for treating an allergic disease.

355. Use of the antibody composition according to the above 332 for treating at least one of vitiligo and psoriasis.

356. Use of the antibody composition according to the above 333 for treating scleroderma.

357. The antibody composition according to any one of the above 314 to 320 for use in a treatment of a cancer.

358. The antibody composition according to any one of the above 321 to 328 for use in a treatment of leukemia.

359. The antibody composition according to any one of the above 321 to 328 for use in a treatment of lymphoma.

360. The antibody composition according to the above 329 for use in a treatment of an inflammatory disease.

361. The antibody composition according to any one of the above 331, 334, and 335 for use in a treatment of an allergic disease.

362. The antibody composition according to the above 332 for use in a treatment of at least one of vitiligo and psoriasis.

363. The antibody composition according to the above 333 for use in a treatment of scleroderma.

364. A method for treating a cancer, including administering an effective amount of the antibody composition according to any one of the above 314 to 320 to a subject.

365. A method for treating leukemia, including administering an effective amount of the antibody composition according to any one of the above 321 to 328 to a subject.

366. A method for treating lymphoma, including administering an effective amount of the antibody composition according to any one of the above 321 to 328 to a subject.

367. A method for treating an inflammatory disease, including administering an effective amount of the antibody composition according to the above 329 to a subject.

368. A method fin treating an allergic disease, including administering an effective amount of the antibody composition according to any one of the above 331, 334, and 335 to a subject.

369. A method for treating at least one of vitiligo and psoriasis, including administering an effective amount of the antibody composition according to the above 332 to a subject.

370. A method for treating scleroderma, including administering an effective amount of the antibody composition according to the above 333 to a subject.

371. A pharmaceutical composition for use in a treatment of rheumatoid arthritis, containing the antibody composition according to the above 77.

372. A pharmaceutical composition for use in a treatment of celiac disease, containing the antibody composition according to the above 78.

373. A pharmaceutical composition for use in a treatment of an inflammatory bowel disease, containing the antibody composition according to the above 79.

374. A pharmaceutical composition for use in a treatment of at least one of multiple sclerosis and an inflammatory bowel disease, containing the antibody composition according to the above 80.

375. A pharmaceutical composition for use in a treatment of at least one of multiple sclerosis and an inflammatory bowel disease, containing the antibody composition according to the above 81.

376. A pharmaceutical composition for use in a treatment of an allergic disease, containing the antibody composition according to the above 82.

377. A pharmaceutical composition for use in a treatment of primary myelofibrosis, containing the antibody composition according to the above 83.

378. A pharmaceutical composition for use in a treatment of at least one selected from an autoimmune disease, arteriosclerosis, and an ischemic heart disease, containing the antibody composition according to the above 84.

379. A pharmaceutical composition for use in a treatment of systemic lupus erythematosus, containing the antibody composition according to the above 85.

380. A pharmaceutical composition for use in a treatment of an allergic disease, containing the antibody composition according to the above 86.

381. A pharmaceutical composition for use in a treatment of psoriasis, containing the antibody composition according to the above 87.

382. A pharmaceutical composition fin use in a treatment of an allergic disease, containing the antibody composition according to the above 88.

383. A pharmaceutical composition for use in a treatment of at least one selected from asthma, eosinophilic sinusitis, and atopic dermatitis, containing the antibody composition according to the above 89.

384. A pharmaceutical composition for use in a treatment of at least one of asthma and eosinophilic sinusitis, containing the antibody composition according to the above 90.

385. A. pharmaceutical composition for use in a treatment of an allergic disease, containing the antibody composition according to the above 91.

386. A pharmaceutical composition for use in a treatment of langerhans cell histiocytosis (LCH), containing the antibody composition according to the above 92.

387. A pharmaceutical composition for use in a treatment of systemic lupus erythematosus, containing the antibody composition according to the above 93.

388. A pharmaceutical composition for use in a treatment of idiopathic pulmonary fibrosis, containing the antibody composition according to the above 94.

389. A pharmaceutical composition for use in a treatment of a cancer, containing the antibody composition according to the above 95.

390. A pharmaceutical composition far use in a treatment of systemic lupus erythematosus, containing the antibody composition according to the above 96 or 97.

391. A pharmaceutical composition for use in a treatment of at least one selected from an autoimmune disease, arteriosclerosis, and an ischemic heart disease, containing the antibody composition according to the above 98.

392. A pharmaceutical composition for use in a treatment of a fibrotic disease including at least one selected from idiopathic pulmonary fibrosis, systemic sclerosis, and hepatic, cirrhosis, containing the antibody composition according to the above 99.

393. A pharmaceutical composition for use in a treatment of idiopathic pulmonary fibrosis, containing the antibody composition according to the above 100.

394. A pharmaceutical composition for use in a treatment of at least one selected from neutrophilic asthma, chronic obstructive pulmonary disease, and Alzheimer's disease, containing the antibody composition according to the above 101.

395. A pharmaceutical composition for use in a treatment of at least one selected from Crohn's disease, rheumatoid arthritis, and asthma, containing the antibody composition according to the above 102.

396. A pharmaceutical composition for use in a treatment of at least one of multiple sclerosis and neuromyelitis optica, containing the antibody composition according to the above 103.

397. A pharmaceutical composition for use in a treatment of systemic lupus erythematosus, containing the antibody imposition according to the above 104 or 105.

398. A pharmaceutical composition for use in a treatment of multiple sclerosis, containing the antibody composition according to the above 106.

399. A pharmaceutical composition for use in a treatment of hepatic cirrhosis, containing the antibody composition according to the above 107.

400. A pharmaceutical composition for use in a treatment of systemic lupus erythematosus, containing the antibody composition according to the above 108.

401. A pharmaceutical composition for use in a treatment of a mast cell activation syndrome (MCAS) including at least one selected from urticaria, a food allergy, and mastocytosis, containing the antibody composition according to the above 109.

402. A pharmaceutical composition for use in a treatment of a mast cell activation syndrome (MCAS) including at least one selected from urticaria, a food allergy, and mastocytosis, containing the antibody composition according to the above 110.

403. A pharmaceutical composition for use in a treatment of multiple sclerosis, containing the antibody composition according to the above 111.

404. A pharmaceutical composition for use in a treatment of at least one selected from multiple sclerosis, type 1 diabetes mellitus, and rheumatoid arthritis, containing the antibody composition according to the above 112.

405. A pharmaceutical composition or use in a treatment of at least one selected from secondary progressive multiple sclerosis (SPMS), rheumatoid arthritis for which anti-TNF therapy is no effective, transplantation, and systemic lupus erythematosus, containing the antibody composition according to the above 113.

406. A pharmaceutical composition for use in a treatment of at least one selected from secondary progressive multiple sclerosis (SPMS), rheumatoid arthritis for which and TNF therapy is not effective, transplantation, and systemic lupus erythematosus, containing the antibody composition according to the above 114.

407. A pharmaceutical composition for use in a treatment of asthma, containing the antibody composition according to the above 115.

408. A pharmaceutical composition for use in a treatment of systemic scleroderma, containing the antibody composition according to the above 116.

409. A pharmaceutical composition for use in a treatment of at least one of vitiligo and psoriasis, containing the antibody composition according to the above 117.

410. A pharmaceutical composition for use in a treatment of at least one of vitiligo and psoriasis, containing the antibody composition according to the above 118.

411. A pharmaceutical composition for use in a treatment of at least one of vitiligo and psoriasis, containing the antibody composition according to the above 119.

412. A pharmaceutical composition for use in a treatment of at least one of vitiligo and psoriasis, containing the antibody composition according to the above 120.

413. A pharmaceutical composition for use in a treatment of at least one of ANCA-associated vasculitis and systemic lupus erythematosus, containing the antibody composition according to the above 121.

414. A pharmaceutical composition for use in a treatment of a T cell-dependent immune-related disease, containing the antibody composition according to the above 122.

415. A pharmaceutical composition for use in a treatment of art immune-related disease, containing the antibody composition according to the above 123.

416. A pharmaceutical composition for use hi a treatment of psoriatic arthritis, containing the antibody composition according to the above 124.

417. A pharmaceutical composition for use in a treatment of an autoimmune disease including at least one of Sjogen's syndrome and psoriasis, containing the antibody composition according to the above 125.

Advantageous Effects of Invention

The antibody composition of the present invention includes first and second IgG half-molecules that are two types of IgG half-molecules, which have antigen-binding domains for two types a antigens that are different from each other, and in which a CD16a-binding activity in CD16a-binding domains that are different from each other is attenuated. Accordingly, even if an antibody structure of a homo assembly is constituted the first IgG half-molecules or the second IgG half-molecules, it cannot bind to CD16a, and therefore cannot exhibit an activity of an antibody. On the other hand, when an antibody structure of a hetero assembly is constituted by the first and second IgG half-molecules, it can bind to CD16a through the second CD16a-binding domain in the first IgG half-molecule and the first CD16a -binding domain in the second IgG half-molecule.

Further, a hinge domain in the first and second IgG half-molecules is altered so as not to form a disulfide bond. Accordingly, an inter-H chain disulfide bond is not formed between the first IgG half-molecule and the second IgG half-molecule. Therefore, when the first and second IgG half-molecules are mixed, it becomes possible to make the first and second IgG half-molecules exist in an equilibrium state of an assembly or half-molecules. Accordingly, the antibody composition of the present invention can exhibit an effector function more specifically for a double-positive cell and damage the cell as compared with a single-positive cell.

In addition, in the antibody composition of the present invention, a specific amino acid alteration is introduced into a CD16a-binding domain or another Fc region in the first and second IgG half-molecules. Due to this, the hetero assembly formed by the first and second IgG half-molecules exhibits an enhanced effector function for a double-positive cell and/or controlled biokinetics,

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram showing the structures of antibody, VHH-Fc, and scFv-Fc.

FIG. 2A shows a schematic diagram of the structure of a general bispecific antibody.

FIG. 2B shows a schematic diagram of a binding mode by a general bispecific antibody.

FIG. 3 shows a schematic diagram of an embodiment of a binding mode by an antibody composition of the present invention.

FIG. 4 shows a schematic diagram of an embodiment of the structure of the antibody composition of the present invention.

FIG. 5 shows a schematic diagram of a binding mode between normal human IgG1 and CD16a.

FIG. 6 shows a schematic diagram of a binding mode between the antibody composition of the present invention and CD16a.

FIG. 7 is a schematic diagram showing a candidate site of an amino acid alteration on a format of normal human IgG1.

FIG. 8 is a view showing the ADCC activity of human IgG1 anti-CCR6 antibodies in which a CD16a-binding domain was “disrupted”.

FIG. 9 is a schematic diagram of a monovalent antibody for evaluating an ADCC activity by asymmetrically introducing the alteration into only each of CH2-A and CH2-B.

FIG. 10 is a view showing the ADCC activity of CD16a-binding asymmetrically altered monovalent antibodies.

FIG. 11 is a schematic diagram of an IgG1114_AA_AAA_D265A (/P329Y)-type IgG half-molecule used in Example 3.

FIG. 12 shows the results of evaluating the purification degree by SDS-PAGE of anti-CD4 antibody half-molecules and anti-CD70 antibody half-molecules.

FIG. 13 shows the results of measuring the expression of CD4 antigen and CD70 antigen in CD4 single-positive cells, CD70 single-positive and CD4/CD70 double-positive cells.

FIG. 14 is a view showing the ADCC activity of anti-CD4 IgG1 and anti-CD70 IgG1 and half-molecules against CD4 single-positive cells, CD 70 single-positive cells, and CD4/CD70 double-positive cells.

FIG. 15A is a view showing the ADCC activity when adding each half-molecule against CD4/CD70 double-positive cells (TL-Om1), CD70 single-positive cells (MT-1), and CD4 single-positive cells (CD4. EL-4).

FIG. 15B is a view showing the ADCC activity when adding each half-molecule against CD4/CD70 double-positive can (TL-Om1), CD70 single-positive cells (MT-1), and CD4 single-positive cells (CD4/EL-4).

FIG. 15C is a view showing the ADCC activity when adding each half-molecule against CD4/CD70 double-positive cells (TL-Om1), CD70 single-positive cells (MT-1), CD4 single-positive cells (CD4/EL-4).

FIG. 15D is a view showing the ADCC activity when adding each half-molecule against CD4/CD70 double-positive cells (TL-Om1), CD70 single-positive cells (MT-1), and CD4 single-positive cells (CD4/EL-4).

FIG. 15E is a view showing the ADCC activity when adding each half-molecule against CD4/CD70 double-positive cells (TL-Om1), CD70 single-positive cells (MT-1), and CD4 single-positive cells (CD4/EL-4).

FIG. 15F is a view showing the ADCC activity when adding each half-molecule against CD4/CD70 double-positive cells (TL-Om1), CD70 single-positive cells (MT-1), and CD4 single-positive cells (CD4/EL-4).

FIG. 15G is a view showing the ADCC activity when adding each half-molecule against CD4/CD70 double-positive cells (TL-Om1), CD70 single-positive cells (MT-1), and CD4 single-positive cells (CD4/EL-4).

FIG. 15H is a view showing the ADCC activity when adding each half-molecule, anti -CD4 IgG1,or anti-CD70 IgG1 against CD4/C170 double-positive cells (TL-Om1) CD70 single-positive cells (MT-1), and CD4 single-positive cells (CD4/EL-4).

FIG. 16A is a view showing the ADCC activity when adding each half-molecule against CD4/CD70 double-positive cells (TL-Om1), CD70 single-positive cells (MT-1), and CD4 single-positive cells (CD4/EL-4). The name of the amino acid alteration introduced into the CH3 domain of the added half-molecule is shown above each graph.

FIG. 16B is a view showing the ADCC activity when adding each half-molecule, anti-CD4 IgG1, or anti-CD70 IgG1 against CD4/CD70 double-positive cells (TL-Om1), CD70 single-positive cells (MT-1), and CD4 single-positive cells (CD4/EL-4). The abbreviation of the added antibody (in the case or the half-molecule, the name of the amino acid alteration introduced into the CH3 domain) is shown above each graph.

FIG. 17 is a view showing the changes in serum antibody concentration when 1 mg/kg of an anti-DNP antibody (wild type) or each mixture of half-molecules was administered to mice.

FIG. 18A is a view showing the ADCC activity in an immunoglobulin-added system when adding each half-molecule against CD4/CD70 double-positive cells (TL-Om1), CD70 single-positive cells (MT-1), and CD4 single-positive cells (CD4/EL-4). As the abbreviation of the added half-molecule, the name of the CD16a-binding enhancing amino acid alteration is shown above each graph.

FIG. 18B is a view showing the ADCC activity in an immunoglobulin-added system when adding each half-molecule against CD4/CD*70 double-positive cells (TL-Om1) CD 70 single-positive cells (MT-1), and CD4 single-positive cells (CD4/EL-4). As the abbreviation of the added half-molecule, the name of the CD16a-binding enhancing amino acid alteration is shown above each graph.

FIG. 18C is a view showing the ADCC activity in an immunoglobulin-added system when adding a half-molecule, anti-CD4 IgG1, anti-CD70 IgG1 against CD4/CD70 double-positive cells (TL-Om1), CD70 single-positive cells (MT-1), and CD4 single-positive cells (CD4/EL-4). The abbreviation of the added antibody (in the case of the half-molecule, the name of the CD16a-binding enhancing amino acid alteration) is shown above each graph.

FIG 19A is a view showing the ADCC activity (cell viability) of each half-molecule in a human blood reconstitution system. The horizontal axis represents the concentration of the added antibody (in the case of the half-molecule, the total concentration of the mixture), and the vertical axis represents the existence ratio (%) of each target cell (shown above the graph) in the total CD3-positive T cells or the total lymphocytes when no antibody was added.

FIG. 19B is a view showing the ADCC activity (cell viability) of each half-molecule in a human blood reconstitution system. The horizontal axis represents the concentration of the antibody (in the case of the half-molecule, the total concentration of the mixture), and the vertical axis represents the existence ratio (%) of each target cell (shown above the graph) in the total CD3-positive T cells or the total lymphocytes when no antibody was added.

FIG. 20 is a view showing the ADCC activity when adding each half-molecule, anti-CCR4 IgG1, anti-CCR4 IgG1 against CD4/CCR4 double-positive cells (TL-Om1), CCR4 single-positive cells (MT-1), and CD4 single-positive cells (CD4/EL-4).

FIG. 21 is a view showing the ADCC activity when adding each half-molecule, anti-CD4 IgG1, or anti-CCR4 IgG1 against CD4/CCR4 double-positive cells (HH), CCR4 single-positive cells (L428), and CD4 single-positive cells (TALL1).

FIG. 22 is a view showing the ADCC activity when adding each half-molecule, anti-CCR4 IgG1, anti-CD70 IgG1 against CCR4/CD70 double-positive cells (LA28), CCR,4 single-positive cells (PEER), and CD70 single-positive cells (SUP-M2).

FIG. 23 is a view showing the binding activity to CD16a of each half-molecule and an anti-DNA antibody.

DESCRIPTION OF EMBODIMENTS

1. Structure of Antibody Composition

In the present invention, an antibody molecule, is also referred to as an immunoglobulin (hereinafter referred to as Ig), and a human antibody is classified into isotypes of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM according to a difference in molecular structure. IgG1, IgG2, IgG3, and IgG4 haying relatively high homology of an amino acid sequence are also collectively referred to as IgG.

An antibody molecule is constituted by polypeptides called heavy chain (hereinafter referred to as H chain) and light chain (hereinafter referred to L chain). An antibody tetrameric protein composed of two H chains and two L chains.

Further, the H chain is constituted by each region of an H chain variable region also referred to VH) and an H chain constant region (also referred CH) from the N terminal side and the L chain is constituted by each region 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 terminal side. In the CH, α, δ, ε, γ and μ chains are known for each subclass. In the CL, λ and κ are known.

A domain is a functional structural unit that constitutes each polypeptide of an antibody molecule. Further, an Fc region (Fc) in the present invention refers to a partial sequence and a partial structure of an H chain constant region composed of a hinge domain, a CH2 domain, and a CH3 domain.

The CH is constituted by each domain of a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain from the N terminal side. The CH1 domain, the hinge domain, the CH2 domain, the CH3 domain, and the Fc in the present invention can be specified by the amino acid residue number from the N terminus according to the EU index [Kabat et al., Sequences of Proteins of immunological interest, US Dept. Health and Human Services (1991)].

Specifically, CH1 is specified as an amino acid sequence at positions 118 to 215 according to the EU index, the hinge is specified as an amino acid sequence at positions 216 to 230 according to the EU index, CH2 is specified as an amino acid sequence at positions 231 to 340 according to the EU index, and CH3 is specified as an amino acid sequence at positions 341 to 447 according to the EU index.

As the IgG in the present invention, an artificial subspecies of an altered molecule that includes at least an antigen-binding domain and Fc and has a similar function to IgG is also included. Specifically, an altered molecule obtained by substitution, deletion or addition, or modification of an amino acid residue of IgG, and further an adduct of a polypeptide or a domain, and the like are included. Further, those obtained by substitution of a part or the whole of an antigen-binding site composed of VH, VL, or Fab of IgG with another antigen-binding domain are also included. Specifically, single-chain (scFv)-Fc, a variable domain of heavy chain of heavy chain antibody (VHH)-Fc shown in FIG. 1, and the like are also included (Brinkmann U et al., MABS 2017, 9: 182-212; Fernandes C F C et al., Frontiers in Immunology 2017, 8: 653).

As the antibody in the present invention, other than a monoclonal antibody obtained from a hybridoma, a recombinant antibody produced by a genetic recombination technique is also included. As the recombinant antibody, a chimeric, antibody obtained by binding a human antibody constant region to a non-human antibody variable region, a humanized antibody, and a human antibody produced using a human antibody-producing animal, or the like are included.

The chimeric antibody can be produced by obtaining cDNAs encoding VH and VL from a hybridoma that produces a monoclonal antibody and is derived from a non-human animal cell, inserting each of the cDNAs into an expression vector the an animal cell having DNAs encoding CH and CL of a human antibody, thereby constructing a human chimeric antibody expression vector, and introducing the vector into an animal cell and expressing the antibody.

The humanized antibody refers to an antibody produced by inserting complementarity determining regions (hereinafter abbreviated as CDRs) of the H chain and the L chain of a non-human antibody variable region into a framework region (hereinafter abbreviated as FR) of a human antibody variable region.

The humanized antibody (or a CDR-grafted antibody) can be produced by the following method. A cDNA encoding the amino acid sequence of a CDR of VH of a non-human animal antibody and the amino acid sequence of VH composed of the amino acid sequence of FR of VH of an arbitrary human antibody, and a cDNA encoding the amino acid sequence of a CDR of VL of a non-human animal antibody and the amino acid sequence of VL composed of the amino acid sequence of FR of VL of an arbitrary human antibody are constructed. Each of the cDNAs is inserted into an expression vector for an animal cell having DNAs encoding CH and CL of a human antibody to construct a humanized antibody expression vector, and introducing the vector into an animal cell and expressing the antibody, whereby the humanized antibody can be produced.

The human antibody originally refers to an antibody that can be naturally present in the human body or an antibody composed of an amino acid sequence encoded by a human gene, but also includes antibodies that are obtained from human antibody phage libraries, cloning of immortalized human peripheral blood lymphocytes, or human antibody-producing transgenic animals, which are produced by recent advancement of genetic engineering, cellular engineering, and developmental engineering technologies, and the like.

The human antibody can be obtained by immunizing a mouse having a human immunoglobulin gene (Tomizuka et al., Proc Natl Acad Sci USA, 97, 722-7, 2000) with a desired antigen. Further, the human antibody can be obtained without immunization by selecting a human antibody having a desired binding activity using a phage display library obtained by amplifying antibody genes from human-derived B cells (Winter G. et al., Annu Rev Immunol. 12: 433-55, 1994).

Further, the human antibody can be obtained by producing a cell that produces a human antibody having a desired binding activity by immortalizing a human B cell using EB virus (Rosen A. et al., Nature 267, 52-54, 1977).

As for the antibody that is present in the human body, for example, a lymphocyte isolated from human peripheral blood is infected with EB virus or the like so as to immortalize it, followed by cloning, whereby a lymphocyte that produces the antibody can be cultured, and the antibody can be purified from the culture.

The human antibody phage library is a library of phages in which an antibody fragment such as Fab or scFv is expressed on the surface thereof by inserting an antibody gene prepared from a human B cell into a phage gene. It is possible to collect phages that express an antibody fragment having a desired antigen-binding activity from the library using a binding activity to a substrate onto which an antigen is immobilized as an index. The antibody fragment further can also be converted into a human antibody molecule composed of two complete H chains and two complete chains using a genetic engineering technique.

The human antibody-producing transgenic animal refers to an animal in which a human antibody gene is incorporated into the chromosome of a host animal. Specifically, the human antibody-producing transgenic animal can be produced by introducing a human antibody gene into a mouse ES cell, implanting the ES cell to an early embryo of another mouse, and then allowing the embryo to develop.

As for a method for producing a human antibody from a human antibody-producing transgenic animal, a human antibody-producing hybridoma is obtained by a general method for producing a hybridoma performed for mammals other than humans, and cultured, whereby human antibody can be produced and accumulated in the culture.

The amino acid sequences of VH and VL may be any of the amino acid sequences of VH and VL a human antibody, the amino acid sequences of VH and VL of a non-human animal antibody, the amino acid sequences of a humanized antibody in which a CDR of a non-human animal antibody is implanted to a framework of a human antibody, and the amino acid sequences of VH and VL derived from a human antibody.

Specifically, the ammo acid sequences of VH and VL of a non-human animal antibody, a humanized antibody, and a human antibody that a hybridoma or an antibody-producing cell produces, and the like are exemplified.

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.

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

The “antigen-binding domain” may be a binding protein recombined by utilizing a binding domain of a known binding molecule such as an antibody, a ligand, a receptor, or the like, and specific examples include a recombinant protein including a CDR of an antibody that binds to each antigen, an antibody variable region including a CDR, a recombinant protein including an antibody variable region and a binding domain of a ligand that binds to each antigen, and the like. Among these, the antigen-binding domain is preferably an antibody variable region in the present invention.

The antibody composition of the present invention is an antibody composition against a first antigen and a second antigen that are different from each other, including a first IgG half-molecule and a second half-molecule, and has the following properties 1) to 6).

1) Each of the first IgG half-molecule and the second IgG half-molecule is composed 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, and has first and second CD16a-binding domains that are different from each other in the CH2 domain.

2) The hinge domain of each of the first IgG half-molecule and the second IgG half-molecule includes an alteration of a substitution or a deletion of a part or the whole or a modification so as not to form an inter-H chain disulfide bond between the first IgG half-molecule and the second IgG half-molecule.

3) The first IgG half-molecule includes an antigen-binding domain that binds to the first antigen, and includes an alteration for attenuating a CD16a-binding activity in the first CD16a-binding domain.

4) The second IgG half-molecule includes an antigen-binding domain that binds to the second antigen, and includes an alteration for attenuating a CD16a-binding activity in the second CD16a-binding domain.

5) Each of the first IgG half-molecule and the second IgG half-molecule includes an alteration for enhancing a CD16a-binding activity.

6) At least one of the first IgG half-molecule and the second IgG half-molecule includes an alteration for attenuating an inter-CH3 domain interaction as compared with an inter-CH3 domain interaction of the IgG1 subclass.

The “antibody composition against a first antigen and a second antigen that are different from each other” denotes a composition containing an IgG half-molecule against a first antigen and an IgG half-molecule against a second antigen.

As a possible state of the IgG half-molecule in the antibody composition of the present invention, for example, a monomeric half-molecule and an assembly composed of half-molecules are exemplified. Examples of the half-molecule include the first IgG half-molecule and the second IgG half-molecule. Examples of the assembly include an assembly of the first IgG half-molecules, an assembly of the second IgG half-molecules, and an assembly of the first IgG half-molecule and the second IgG half-molecule. Specifically, the first and second half-molecules contained in the antibody composition may be in a state of equilibrium between an assembly composed of the first IgG half-molecules and an assembly composed of the second IgG half-molecules, and an assembly composed of the first IgG half-molecule and the second IgG half-molecule.

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-molecule against first antigenic determinant (epitope) and an IgG half-molecule against a second antigenic determinant (epitope) in the same antigen.

The “IgG half-molecule” is a dimeric protein composed of one L chain and one H chain, and the H chain includes an H chain variable region, CH1 to CH3 domains, and a hinge domain. In the CH2 domain of the H chain of the IgG half-molecule, two CD16a-binding domains (first and second CD 16a-binding domains) that are different from each other are present.

Further, the “IgG half-molecule” also includes a half-molecule of an artificial subspecies of altered molecule that includes an antigen-binding domain and Fc and has a similar function to IgG. Specifically, a half-molecule of an altered molecule obtained by substitution, deletion or addition, or modification of an amino acid of IgG, and further a half-molecule of an adduct of a polypeptide or a domain, and the like are included. Further, half-molecules of those obtained by substitution of a part or the whole of an antigen-binding site composed of VH, VL, or Fab of IgG with another antigen-binding domain, speeilically, half-molecules of single-chain Fv (scFv)-Fc and VHH-Fc shown in FIG. 1, and the like are also included.

The “CD16a-hinding domain” refers to a domain that is present in Fc of IgG and binds to CD16a. The CD16a-binding domain on Fc of IgG1 is present at two sites due to the point symmetry of the structure of Fc, and in the CH2 domain constituting Fc and composed of two polypeptide chains, contact with CD16a is made in regions that are different from each other (see FIG. 5).

Examples of the first CD16a-binding domain include a domain including at least one selected from amino acid residues at position 235, position 236, position 237, position 238, position 239, position 265, position 266, position 267, position 268, position 269, position 294, position 295, position 296, position 297, position 298, position 299, position 301, position 325, position 327, and position 332 numbered according to the EU index.

When the immunoglobulin subclass of the CH2 domain is IgG1, examples of the first CD16a-binding domain include a domain including at least one selected from amino acid residues of Leu at position 235, Gly at position 236, Gly at position 237, Pro at position 238, Ser at position 219, 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 numbered according to the EU index.

Examples of the second CD16a-binding domain include a domain including at least one selected from amino acid residues at position 235, position 226, position 237, position 326, position 327, position 328, position 329, and position 330 numbered according to the EU index.

When the immunoglobulin subclass of the CH2 domain is IgG1, examples of the second CD16a-binding domain include a domain including at least one selected from amino acid residues of Leu at position 235, Gly position 236, Gly at position 237, Lys at position 324, Ala at position 327, Leu at position 328, Pro at position 329, and Ala at position 330 numbered according to the EU index.

The “CD16a-binding activity” refers to an activity of binding of Fc of IgG CD16a. The CD16a-binding activity of the IgG half-molecule can be confirmed by combining two IgG half-molecules to form IgG, and thereafter allowing the IgG to react with a recombinant CD16a protein, and measuring the binding activity (US Patent Application Publication No. 2004/0259150).

As one aspect of the method for measuring the CD16a-binding activity of the IgG half-molecule, for example, the binding activity to CD16a expressed on a cell membrane can be measured by a fluorescent antibody method (Cancer Immunol. Immunother, 36, 373, 1993), or the like. Further, for example, the binding activity to a purified CD16a protein can be measured according to an immunological quantitative method such as Western staining, RIA (radioimmunoassay), VIA (viroimmunoassay), EIA (enzymoimmunoassay), FIA (fluoroimmunoassay), or MIA (metalloimmunoassay) described in literature [Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., 1995; Enzyme Immunoassay, 3rd ed., IGAKU-SHION Ltd. (1987); Enzyme Antibody Techinque, Revised Edition, Gakusai Kikaku (1985)], or the like.

Specifically, for example, when the binding activity to the purified CD16a protein is quantified by EIA, the quantification can be carried out as follows. FcγRIIIa is immobilized on a plastic plate for EIA, and is allowed to react with a sample containing the antibody composition. Subsequently, the amount of the antibody composition bound using an appropriate secondary antibody is measured.

The binding activity to the purified CD16a protein can also be measured by measurement using a biosensor [for example, BIAcore (manufactured by Biacore, Inc.)] [J. Immunol. Methods, 200, 121 (1997)] or by an isothermal titration calorimetry method [Proc. Natl. Acad. Sci. U.S.A., 97, 9026 (2000)], or the like.

Further, the CD16a-binding activity of the IgG half-molecule can also be confirmed by allowing the ce of the IgG half-molecule to have the below-mentioned effector function (ADCC activity or the like), constituting IgG in which two molecules of the IgG half-molecule are combined, and measuring the effector function of the IgG.

The “effector function” refers to an antibody-dependent function caused via Fc of an antibody. As the effector function, for example, an ADCC activity; a CDC activity, or antibody-dependent phagocytosis (antibody-dependent cellular phagocytosis activity: ADCP activity) by a phagocyte such as a macrophage or a dendritic cell is exemplified. As a method for measuring the effector function, for example, an ADCC activity and a CDC activity can be measured using a method described in Cancer Immunother, 36, 373 (1993).

As one aspect of the method for measuring an ADCC activity or a CDC activity, for example, the following method is exemplified. Specifically, for example, a method described later in Examples is exemplified.

1) As an effector cell, a human peripheral blood mononuclear cell (PBMC) or a cell line made to stably express human CD16a by gene transfer of human CD16a, and a target cell are prepared using a culture medium (for example, RPlMI medium). In the case of measurement of a CDC activity a solution obtained by diluting a human complement protein to an appropriate concentration is mixed with a target cell and adjusted.

2) Into a cell culture container (for example, 96-well plate), an antibody solution, the target cells, and the effector cells are dispensed. The number ratio of the effector cells and the target cells (E/T ratio) is made constant.

3) The resultant is left to stand for about 2 to 6 hours in a incubator. At the same time, a solubilizing solution (for example, an aqueous solution containing an acid, an alkali, a surfactant, or the like) is added to a 100% reaction well, and the target cells are completely lysed. After the reaction container is centrifuged, the supernatant is collected and dispensed into an ELISA plate. A coloring solution is applied thereto to cause a reaction, and thereafter, a stopping solution is added thereto, and an absorbance (A450) is measured using a plate reader.

4) The ADCC activity (%) and the CDC activity (%) are calculated using the following formula.

ADCC activity(%)or CDC activity(%)=100×(S−E−T)(Max−T)

S=absorbance of sample reaction well—absorbance of culture medium well

E=absorbance of effector well—absorbance of culture medium well

T=absorbance of target well—absorbance of culture medium well

Max100% reaction well—100% reaction control well

As a kit for ADCC measurement, a known kit can be used, and for example, CytoTox 96(R) Non-Radioactive Cytotoxicity Assay (Promega) is exemplified.

The ADCC activity refers to an activity in which an antibody bound to an antigen on a target cell binds to an Fc receptor of an immune cell via Fc of the antibody so as to activate the immune cell (a natural killer cell or the like) and damage the target cell.

The Fc receptor (hereinafter sometimes referred to as FcR) is a receptor that binds to Fc of an antibody, and the binding of the antibody induces various effector functions.

The FcR corresponds to the subclass of an antibody, and IgG, IgE, IgA, and IgM bind specifically to FcγR, FcεR, and FcαR, respectively. Further, in the FcγR, there exist FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16) subtypes, and there exist FcγRIA, FcγRIB, FcγRIC, FcγRIIA, FcγRIIB, FcγRIIC, FcγRIIIA (CD16a), and FcγRIIIB isoforms, respectively. The different types of FcγRs are present on different cells [Anna. Rev. Immunol. 9: 457-492 (1991)].

In humans, FcγRIIIB is expressed specifically in neutrophils, and FcγRIIIA is expressed in monocytes, natural killer cells (NK cells), and some T cells. Binding of the antibody via FcγRIIIA induces NK cell-dependent ADCC.

The CDC activity refers to an activity in which an antibody bound to an antigen on a target cell activates a series of cascades (complement activation pathways) composed of complement-related protein groups in the blood, and damages the target cell. In addition, a protein fragment generated by the activation of the complement can induce the migration and activation of an immune cell.

The cascade of CDC activity starts by forming a C1 complex through binding of C1q having a binding domain for Fc of an antibody to Fc, and its binding to C1r and C1s that are two serine proteases.

The state where “the CD16a-binding activity in the CD16a-binding domain is attenuated” refers to a state where the CD16a-binding activity and/or the effector function (ADCC activity or the like) of IgG obtained by combining two molecules of the IgG half-molecule to which an alteration for attenuating the CD16a-binding activity in the first and/or second CD16a-binding domain in Fc is added is attenuated as compared with the CD16a-binding activity and/or the effector function of IgG obtained by combining two molecules a the IgG half-molecule before the alteration.

As one aspect of the state where the CD16a-binding activity in the CD16a-binding domain is attenuated, for example, a state where the CD16a-binding activity of IgG obtained by combining two molecules of the IgG half-molecule to which the alteration is added is preferably 60% or less, more preferably 50% or less, further more preferably, in the following order, 40% or less, 30% or less, 20% or less, and 10% or leas when the CD 16a-binding activity of IgG obtained by combining two molecules of the IgG half-molecule before the alteration is taken as 100% is exemplified.

As one aspect of the state where the CD16a-binding activity in the CD 16a-binding domain is attenuated, for example, a state where the ADCC activity of IgG obtained by combining, two molecules of the IgG half-molecule to which the alteration is added is preferably 80% or less, more preferably 70% or less, further more preferably, in the following order, 60% or less, 50% or less, 40% or less, and 30% or less when the ADCC activity of IgG obtained by combining two molecules of the IgG half-molecule before the alteration is taken as 100% is exemplified.

The CD16a-binding activity in the CD16a-binding domain is attenuated by alteration in the CD16a-binding domain. The “alteration” in the present description refers to alteration of an amino acid residue from the wild-type amino acid sequence, and means, for example, substitution, deletion, or addition, or modification of an amino acid residue. In the present description substitution of an amino acid residue is also abbreviated as amino acid residue substitution. The alteration for attenuating the CD16a-binding activity in the CD16a-binding domain in the present description is not particularly limited by the type of antigen to which the antibody of the present invention binds, and an antibody having any antigen-binding domain may be combined with any alteration as long as the effect of the present invention is exhibited.

Examples of the alteration for attenuating the CD16a-binding activity in the first CD16a-binding domain include substitution of at least one amino acid residue selected from amino acid residues at position 235, position 236, position 237, position 238, position 239, position 265, position 266, position 267, position 268, position 269, position 294, position 295, position 296, position 297, position 298, position 299, position 301, position 325, position 327, and position 332 numbered according to the EU index, and preferred is substitution of at least one amino acid residue selected from amino acid residues at position 235, position 238, position 239, position 265, position 266, position 267, position 268, position 269, position 294, position 295, position 296, position 297, position 298, position 299, position 301, position 325, position 327, and position 332, and more preferred is substitution of at least one amino acid residue selected from amino acid residues at position 235, position 238, position 239, position 265, position 267, position 269, position 296, position 298, position 299, and position 327. As a combination of amino acid residue substitutions, for example, amino acid residue substitutions at position 238 and position 265 in combination (hereinafter such a combination is represented by position 238/position 265, or the like), at position 238/position 267, at position 265/position 267, or at position 238/position 265/position 267 is exemplified.

When the immunoglobulin subclass of the CH2 domain is IgG1, examples of the alteration for attenuating the CD16a-binding activity in the first CD16a-binding domain 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 numbered according to the EU index, and preferred is 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, Val at position 266, Ser at position 267, His at position 268, Glu at position 269, Gln 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 and preferred is 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, Gln at position 269, Tyr at position 296, Ser at position 298, Thr at position 299, and Ala at position 327. Specifically, for example, at least one amino acid residue substitution selected from L235R, P238A, S239R, D265A, D265N, D265E, S267L, S267K, E269P, Y296P, S298E, T299A, and A327I is exemplified.

Examples of the alteration for attenuating the CD16a-binding activity in the second CD16a-binding domain include substitution of at least one amino acid residue selected from amino acid residues at position 235, position 236, position 237, position 326, position 327, position 328, position 329, and position 330 numbered according to the EU index, and preferred is substitution of at least one amino acid residue selected from amino acid residues at position 326, position 328, position 329, and position 330, and more preferred is substitution of at least one amino acid residue selected from amino acid residues at position 326, position 328, and position 329. As a combination of amino acid residue substitutions, for example, amino acid residue substitutions at position 326/position 328, at position 326/position 329, at position 328/position 329, and at position 126/position 328(position 129 are exemplified.

When the immunoglobulin subclass of the CH2 domain is examples of the alteration for attenuating the CD16a-binding activity in the second CD16a-binding domain 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 numbered according to the EU index, and preferred is substitution of in least on amino acid residue selected from Lys at position 326, Leu at position 328, Pro at position 329, and Ala at position 130, and more preferred is substitution of at least one amino acid residue selected from Lys at position 326, Leu at position 328, and Pro at position 329. Specifically, at least one amino acid residue substitution selected from K326W, K326G, L328V, L328R, P329Y, P329K, P329W, and A330P is exemplified.

A combination of the alteration for attenuating the CD16a-binding activity to CD16a-binding domain of the first half-molecule and the alteration for attenuating the CD16a-binding activity in the CD16a-binding domain of the second IgG half-molecule is not particularly limited as long as asymmetric alterations are brought about, and the above-mentioned alterations can be appropriately combined. Specific examples include the following combinations of amino acid residue substitutions.

- amino acid residue substitutions at position 265 in the first IgG half-molecule and at position 329 in the second IgG half-molecule
- amino acid residue substitutions at position 329 in the first IgG half-molecule and at position 265 in the second IgG half-molecule
- amino acid residue substitutions at position 238/position 267 in the first IgG half-molecule and at position 329 the second IgG half-molecule
- amino acid residue substitutions at position 329 in the first IgG half-molecule and at position 238/Position 267 in the second IgG half-molecule

The combination of the alteration to attenuating the CD16a- binding activity in the CD16a -binding domain of the first IgG half-molecule (first alteration) and the alteration for attenuating the CD16a-binding activity in the CD16a-binding domain of the second IgG half-molecule (second alteration) is preferably combinations shown in the following Table 1, and more preferably a combination of the first alternation and the second alteration as follows: S267K and P329Y, Y296P and P329 Y, S298E and P329Y, D265A and P329Y, or S239R and P329Y.

TABLE 1

First
Second
First
Second

alteration
alteration
alteration
alteration

D265A
P329Y
Y296P
P329K

P238A/S267L
P329Y
S298E
P329K

S239R
K326G
T299A
P329K

S239R
L328R
S239R
P329W

D265A
L328R
D265A
P329W

D265E
L328R
D265N
P329W

S267K
L328R
D265E
P329W

E269P
L328R
S267K
P329W

Y296P
L328R
E269P
P329W

S298E
L328R
Y296P
P329W

T299A
L328R
S298E
P329W

S239R
P329Y
T299A
P329W

D265E
P329Y
L235R
P329W

S267K
P329Y
A327I
P329W

Y296P
P329Y
S239R
A330P

S298E
P329Y
D265A
A330P

T299A
P329Y
D265E
A330P

L235R
P329Y
S267K
A330P

A327I
P329Y
E269P
A330P

S239R
P329K
Y296P
A330P

D265A
P329K
S298E
A330P

D265E
P329K
T299A
A330P

S267K
P329K
L235R
A330P

A327I
A330P

An amino acid residue after substitution described above may be a mutually substitutable amino acid. Hereinafter, examples of the mutually substitutable amino acid are shown. Amino acids included in the same group can be mutually substituted.

group A: leucine, isoleneine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butyl glycine, t-buyl alanine, and cyclohexylalanine

group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, and 2-aminosuberic acid

group C: asparagine and glutamine

group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, and 2,3-diaminopropionic acid

group E: proline, 3-hydroxyproline, and 4-hydroxyproline

group F: serine, threonine and homoserine

group G: phenylalarnine and tyrosine

The above-mentioned amino acids to be substituted may be either a natural type or an unnatural type. Examples of the natural amino acid include L-alanine, 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, L-cysteine, and the like. Examples of the unnatural amino acid include various amino acids having an amino group and as carboxyl group, however, preferably, derivatives of various types of natural amino acids are desired. Many unnatural amino acids are available from respective reagent companies (Sigma-Aldrich Co. LLC, TCI Co., Ltd., and the like). With respect to the unnatural amino acids, there are many disclosures in literature (Chem. Today 2003, 65; Curr Opin Chem Biol. 2000, 6, 645).

In the first IgG half-molecule, the CD16a-binding activity in the first CD16a-binding domain is attenuated by alteration, and also in the second IgG half-molecule, the CD16a-binding activity in the second CD16a-binding domain is attenuated by alteration. Accordingly, even if an antibody structure of a homo assembly is constituted by the first IgG half-molecules or the second IgG half-molecules, it cannot bind to CD16a, and therefore cannot exhibit the activity of an antibody.

On the other hand, when an antibody structure of a hetero assembly is constituted by the first and second IgG half-molecules, it can bind to CD16a through the second CD16a-binding domain in the first IgG half-molecule and the first CD16a-binding domain in the second IgG half-molecule (see FIG. 5).

The first and second IgG half-molecules have antigen-binding domains that are different front each other. Therefore, by constituting an antibody structure of a hetero assembly composed of the first and second IgG half-molecules, the assembly becomes an antibody composition which CD16a binds to and thus can specifically exhibit an effector function such as an ADCC activity only when it binds to a target cell expressing two types of antigens that are different from each other on the same cell.

A hinge domain in each of the first and second IgG half-molecules is altered so as not to form a disulfide bond by substitution or deletion of a part or the whole thereof or modification. Accordingly, inter-H chain disulfide bond is not formed between the first IgG half-molecule and the second IgG half-molecule.

Therefore, by the coexistence of the first and second IgG half-molecules, it becomes possible to make the first and second IgG half-molecules exist in an equilibrium state of an assembly or half-molecules, so that high specificity for a double-positive cell can be exhibited.

Accordingly, the antibody composition of the present invention can exhibit an effector function specifically for a target cell coexpressing a first antigen and a second antigen and damage the cell as compared with an effector function for a target cell expressing only the first antigen and a target cell expressing only the second antigen.

In the present invention, the phrase “exhibit an effector function specifically for a target cell coexpressing a first antigen and a second antigen as compared with an effector function for a target cell expressing only the first antigen and a target cell expressing only the second antigen” means that the effector function for a target cell coexpressing a first antigen and a second antigen (double-positive cell) is strong as compared with the effector function for a target cell expressing only the first antigen and a target cell expressing only the second antigen (single-positive cell).

Whether an effector function specific for a double-positive cell is exhibited as compared with a singles-positive cell can be evaluated by a method described in the sections [1] High Specificity for Double-Positive Cell and [2] Enhanced Effector Function.

As the substitution of a part of a hinge domain so as not to them an inter-H chain disulfide bond between the fast IgG half-molecule and the second IgG half-molecule, for example, amino acid residue substitutions at position 226 and position 229 numbered according to the EU index are exemplified. Alternatively, deletion of a part or the whole of a hinge domain containing the cysteine site is exemplified. As the substitution of a part of a binge domain so as not to form an inter-H chain disulfide bond between the first IgG half-molecule and the second IgG half-molecule, specifically for example, amino acid residue substitutions of C226A and C229A numbered according to the EU index are preferred.

In order to make the first and second IgG half-molecules exist in the above-mentioned equilibrium state, it is effective that the non-covalent bonding interaction between the H chains in the first and second IgG half-molecules is attenuated, and in particular, it is preferred that the inter-CH3 domain interaction is attenuated.

Specifically, for example, it is preferred that the inter-CH3 domain interaction of the H chains in first and second IgG half-molecules is weaker than the inter-CH3 domain interaction of the IgG1 subclass.

As a method for attenuating the inter-CH3 domain interaction as compared with the inter-CH3 domain interaction of the IgG1 subclass, it is preferred to introduce an amino acid alteration (an amino acid residue substitution, deletion, or addition, or modification or the like) in the CH3 domain, and it is particularly preferred to introduce an amino acid residue substitution. Examples of the CH3 domain into which an amino acid alteration is introduced to attenuate the inter-CH3 domain interaction as compared with the inter-CH3 domain interaction of the IgG1 subclass include a CH3 domain of IgG1 and a CH3 domain of IgG4.

It has been reported that the interaction between CH3 domains is enhanced by substituting Arg at position 409 numbered according to the EU index in the CH3 domain of human IgG4 with Lys derived from human (Structure 2011 19: 9; 1274-1282). Further, there has also been a report that an amino acid site important for the interaction between the CH3 domains of human IgG1 was identified based on changes in free energy by substituting a specific amino acid residue in the CH3 domain of human IgG1 with Ala (Biochemistry 1998: 37: 9266-9273).

The amino acid residue substitution for attenuating the inter-CH3 domain interaction may be any as long as it attenuates the inter-CH3 domain interaction based on the amino acid sequence of the CH3 domain to he used, and it may be introduced into at least one of the first IgG half-molecule and the second IgG half-molecule, but is preferably introduced into each of them. The amino acid residue substitution for attenuating the inter-CH3 domain interaction can be identified by evaluating a molecule including a desired amino acid residue substitution using a binding assay such as binding ELISA or an SPR method, a molecular weight analysis such as SDS-PAGE or native mass spectrometry, or the like.

As the ammo acid residue substitution for attenuating the inter-CH3 domain interaction specifically, substitution of at least one amino acid residue at position selected from position 349, position 351, position 366, position 368, position 399, position 405, position 407, and position 409 numbered according to the EU index in CH3 domain with another amino acid residue is exemplified.

More specifically, Y349A, L351A, T366 A, L368A, D399A, F405A, Y1407A, K409A, and K409R are preferred, L368A, Y407A, and K409R are more preferred, and K409R is most preferred. Among these amino acid residue substitutions, any one substitution may be introduced, or two or more substitutions may be introduced in combination.

2. Control of Effector Function of Antibody Composition

The antibody composition of the present invention can also be imparted with an effector function dependent on Fc in the first and second IgG half-molecules. The effector function of the antibody composition can be controlled by various methods.

For example, it is preferred to further enhance the CD16a-binding activity by further including at least one amino acid residue substitution fur enhancing the CD16a-binding activity in the CH2 domain in the first and second IgG half-molecules. Accordingly, the effector function of the antibody composition can be enhanced.

The amino acid residue substitution for enhancing the CD16a-binding activity may be introduced into each of the first and second IgG half-molecules or may be introduced into only either one of them. When the amino acid residue substitution for enhancing the CD16a-binding activity is introduced into both the first and second IgG half-molecules, the amino acid residue substitution in the first IgG half-molecule and the amino acid residue substitution in the second IgG half-molecule may be the same or different, but are preferably the same.

When the amino acid residue substitution for enhancing the CD16a -binding activity of the antibody composition is introduced into both the first and second IgG half-molecules, the amino acid residue substitutions at in regions different from the regions altered for attenuating the CD16a-binding activity in the first and second IgG half-molecules.

Examples of a method for controlling the effector function of the antibody composition of the present invention include a method as described below.

For example, the effector function of the antibody composition can be controlled by a method for controlling the amount of fucose (also referred to as core fucose) that is α1,6-linked to N-acetylglucosamine (GlcNAc) present at the reducing end side of an N-linked complex sugar chain (hereinafter sometimes simply abbreviated as complex sugar chain) that is bound to Asn at position 297 according to the EU index using the amino acid sequence of Fe of the IgG1 subclass in the first and second IgG half-molecules (WO 2005/035586, WO 2002/31140, or WO 00/61739), or b substituting an amino acid residue of Fc of the antibody.

1) Control of Effector Function by Sugar Chain Alteration

The effector function of the antibody composition can be enhanced or decreased by controlling the content of fucose to he added to N-acetylglucosamine at the reducing end of the complex sugar chain bound to Fc of the first and second IgG half-molecules.

As for a method for decreasing the content of fucose to be added to the N-linked complex sugar chain bound to Fc of the IgG half-molecule the IgG half-molecule in which fucose is not bound can be obtained by expressing the IgG half-molecule using an α1,6-fucosyltransferase (FUT8) gene-deficient CHO cell. The antibody composition composed of the IgG half-molecule in which fucose is not bound has a high ADCC activity.

On the other hand, as for a method for increasing the content fucose to be added to the N-linked complex sugar chain bound to Fc of the IgG half-molecule, the IgG half-molecule in which fucose is bound can be obtained by expressing the IgG half-molecule using a host cell into which an α1,6-fucosyltransferase gene has been introduced. The antibody composition composed of the IgG half-molecule in which fucose is bound has a lower ADCC activity than the antibody composition composed of the IgG half-molecule in which fucose is not bound.

In Fc of the IgG half-molecule, the N-linked sugar chain is bound to the Asn residue at position 297 according to the EU index, however, it is not known that the sugar chain is bound to other Asn residues of Fc. Therefore, generally, two N-glycoside-linked sugar chains are bound per molecule of the antibody.

As the N-linked sugar chain, a high mannose type, a complex type, and a hybrid type are known, and a higher ADCC activity as compared with a sugar chain to which fucose is bound can be obtained by any N-linked sugar chain as long as it is a sugar chain to which fucose is not bound.

As the complex sugar chain to be bound to Fc of the IgG half-molecule, a sugar chain in which one or more N-acetylglucosamine (GlcNAc) residues or galactose-N-acetylglucosamine (hereinafter referred to as Gal-GlcNAc) residues are α1,2-linked or α1,4-linked to mannose (Man) at the non-reducing end side of a core structure (trimannosyl core structure) is exemplified.

Further, a complex type sugar chain having sialic acid, bisecting N-acetylglucosamine (hereinafter referred to as bisecting GlcNAc), or the like at the non-reducing end side of Gal-GlcNAc can be exemplified.

In the present invention, the core fucose or the 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 the N-glycoside-linked complex sugar chain and the 1-position of fucose (hereinafter sometimes referred to as Fuc) are α-linked. Further, the sugar chain in which fucose is not bound to N-acetylglucosamine at the reducing end of the N-glycoside-linked complex sugar chain is simply referred to as a sugar chain having no fucose or no core fucose.

Further, in the present invention, the core structure or the trimatmosyl core structure refers to a Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc structure.

As the sugar chain bound to the IgG half-molecule, a double-stranded N-glycoside-linked complex sugar chain (also referred to as a biantennary complex sugar chain) is represented by the following chemical formula.

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The first and second IgG half-molecules preferably have Fc in which the complex sugar chain is bound to Asn at position 297 according to the EU index. They may have a single sugar chain structure or a plurality of different sugar chain structures as long as they have the above-mentioned sugar chain structure. Specifically, an antibody composition in which the ratio of the sugar chain in which fucose is not bound to N-acetylglucosamine at the reducing end or the sugar chain (sugar chain having no core fucose) among all the N-glycoside-linked sugar chains bound to Fc in the first and second IgG half-molecules is 20% or more is exemplified.

The ratio of the sugar chain having no core fucose includes any ratio as long as the ADCC activity of antibody composition is increased, but a ratio of preferably 20% or more, more preferably 51% to 100%, further more preferably 80% to 100%, particularly preferably 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and most preferably, 100% can be exemplified.

The ratio of the sugar chain having no core fucose being 50% includes, for example, both an antibody composition containing a molecule in which fucose is not bound to one sugar chain of the N-glycoside-linked sugar chains bound to the first and second IgG half-molecules at 100%, and an antibody composition containing a molecule in which fucose is not bound to both sugar chains of the N-glycoside-linked sugar chains bound to the first and second IgG half-molecules at 50% and also containing a molecule in which fucose is bound to both sugar chains of the N-glycoside-linked sugar chains bound to the first and second IgG half-molecules at 50%.

In the present invention, as the sugar chain having no fucose, the structure of the sugar chain at the non-reducing end side may be any as long as fucose is not bound to N-acetylglucosamine at the reducing end side in the chemical formula shown above.

In the present invention, the state where fucose is not bound to N-acetylglucosamine at the reducing end of the sugar chain (having no core fucose) refers to a state where fucose is not substantially bound thereto. The IgG half-molecule in which fucose is not substantially bound specifically refers to a case where it is an IgG half-molecule to such an extent that fucose cannot be substantially detected in the below-mentioned sugar chain analysis. The extent that fucose cannot be substantially detected refers to that it is the detection limit or less in the measurement. The antibody composition including the first and second IgG half-molecules having no core fucose in all the sugar chains has the highest ADCC activity.

The ratio of the IgG half-molecule including a sugar chain having no fucose in the IgG half-molecule having Fc to which the N-glycoside-linked complex sugar chain is bound can be determined by releasing the sugar chains using a known method such as hydrazinolysis or enzyme digestion [Biochemical Experimentation Methods 23-Method for Studying Glycoprotein Sugar Chain (Japan Scientific Societies Press) edited by Reiko Takahashi (1989)] from the IgG half-molecule, subjecting the released sugar chains to fluorescence labeling or radioisotope labeling, and separating the labeled sugar chains by chromatography.

In addition, the ratio of the IgG half-molecule in which a sugar chain having no fucose is bound included in the IgG half-molecule containing Fc in which the complex sugar chain is bound can be determined by analyzing the released sugar chains using an HPAED-PAD method (J. Chromatogr., 6, 1577, 1983).

2) Control of Effector Function by Amino Acid Residue Substitution

The antibody composition of the present invention can enhance or attenuate an effector function such as an ADCC activity, an ADCP activity and a CDC activity by changing the antibody subclass of Fc or by amino acid residue substitution in Fc in the first IgG half-molecule and the second IgG half-molecule.

The IgG1 subclass antibody is known to have the highest ADCC activity and CDC activity in the IgG subclasses, and the immunoglobulin subclass of the CH2 domain is preferably IgG1.

As the amino acid residue substitution in Fc, for example, by using the amino acid to sequence of Fc described in US Patent Application Publication No. 2007/0148165, the CDC activity of the antibody can be increased. Further, by performing an amino acid residue substitution described in U.S. Pat. No. 6,737,056, 7,297,775 and 7,317,091, the ADCC activity or the CDC activity of the antibody composition can be enhanced or attenuated.

Specific examples of the amino acid residue substitution for enhancing the ADCC activity include P247I, A339D, F243L, R292P, Y300L, P396L, T393A, H433P, S239D, S298A, A330L, I1332E, E333A, K334A, and the like. On the other hand, specific examples of the amino acid residue substitution for decreasing the ADCC activity include L235E, P238A, N297A, K322A, P331S, and the like.

The ADCC activity can be enhanced by combining any two or more of the above-mentioned amino acid residue substitutions, and the number of amino acid residues to be substituted can be increased according to the purpose. As a combination of amino acid residue substitutions capable of enhancing the ADCC activity, S298A/E333A/K334A/P247L, S298A/E333A/K334A/H268E, S298A/E333A/K334A/P247L/N421K, S298A/E333A/K334A/E294W, S298A/E333A/K334A/K326T, S298A/E333A/R292L/K334E, S298A/E333A/K334A/S239D, S298A/E333A/K334A/K248M, S239D/I332E, S239D/A330P, S239D/K326T, S239D/K326E, S239D/K326I, S219D/I332D, S239E/I332Y, S239E/I332E, S239E/K326T, and S239E/K326I are preferred.

The amino acid residue substitution for enhancing ADCC activity may affect the pharmacokinetics of the antibody, and therefore, it is preferred that the antibody in which the amino acid residue substitution is introduced has pharmacokinetics comparable to that of the wild-type IgG antibody in which the amino acid residue substitution is not introduced. As the specific amino acid substitution, S239D, S239E, S239D/K326T, S239D/A330F, S239D/K326E, S239E/I332E, S298A/E333A/K334A, S298A/E333A/K334A/H268E, and S239D/S298A/E333A/L242C/K334C are preferred, and S239D/K326T and S239D/S298A/E333A/L242C/K334C are more preferred. The comparable pharmacokinetics means 50% or more and 150% or less as compared with the wild-type IgG antibody in any index of maximum blood concentration (Cmax), blood half-life (t1/2), or area under the blood concentration-time curve (AUC).

As a specific amino acid residue substitution for increasing the CDC activity, at least one amino acid residue substitution selected from K326A, S267E, H268F, S324T, K274Q, N276K, Y296F, Y300F, K326W, K226Y, E333A, E333S, A339T, D356E, L358M, N384S, K392N, T394F, T394Y, V397M, and V422I is exemplified.

The CDC activity can also be increased by combining any two or more of the above-mentioned amino acid residue substitutions, and the number of amino acid residues to be substituted can be increased according to the purpose. As the amino acid residue substitution tor increasing the CDC activity, preferably at least one amino acid residue substitution selected from N276K, A339T, T394F, and T394Y, amino acid residue substitutions of N276K and A339T, amino acid residue substitutions of K274Q, N 276K, Y296F, Y300F, A339T, D356E, L358M, N384S, V397M, and V422I, and the like are exemplified. On the other hand, as a specific amino acid residue substitution for decreasing the CDC activity, L235E, N297A, K322A, P329 A, P331S, and the like are exemplified.

In addition, the blood half-life can be extended by introducing an amino acid residue substitution such as T250Q, M428L, M252Y, S254T, or T256E into Fc of the human IgG1 subclass. Moreover, cellular cytotoxicity such as an ADCC activity, an ADCP activity, or a CDC activity can be attenuated or deleted using Fc from which an N-linked sugar chain has been removed, Fc of human IgG2 or IgG4 subclass, chimeric Fc of IgG2 and IgG4, or the like by introducing an amino acid residue substitution at position N297.

3. Method for Producing Antibody Composition

As the method for producing an antibody composition of the present invention, a production method including, the following steps 1 to 3 is exemplified.

Step 1: a step of introducing a recombinant vector containing a DNA encoding the amino acid sequence of the IgG half-molecule (hereinafter also abbreviated as a recombinant vector for expression of the IgG half-molecule) into a cell, thereby obtaining a transformant

Step 2: a step of culturing the transformant obtained in the step to accumulate the IgG half-molecule in a culture, and collecting the IgG half-molecule from the culture

Step 3: a step of obtaining an antibody composition composed of the IgG half-molecule collected in the step 2

Hereinafter, the respective steps will be described.

[Step 1]

The step 1 is a step of introducing a recombinant vector containing a DNA encoding the amino acid sequence of at least one of the first and second IgG half-molecules into a cell, thereby obtaining a transformant.

The step (1) specifically includes the following steps (1-1) to (1-3).

(1-1) a step of introducing an alteration for attenuating a CD16a-binding activity in the first CD16a-binding domain in the first IgG half-molecule

(1-2) a step of introducing an alteration for attenuating a CD16a-binding activity in the second CD16a-binding domain in the second IgG half-molecule

(1-3) a step of introducing an alteration for performing substitution or deletion of a part or the whole or modification of a hinge domain so as not to form an inter-H chain disulfide bond in the hinge domains of the first and second IgG half-molecules.

As for the step (1-1), for example, in the production of a recombinant vector for expression of the first IgG half-molecule, performing a step of substituting at least one amino acid residue selected from amino acid residues at position 235, position 236, position 237, position 238, position 239, position 265, position 266, position 267, position 268, position 269 position 294, position 295, position 296, position 297, position 298, position 299, position 301, position 325 position 327, and position 332 numbered according to the EU index as appropriate according to the subclass of the CH2 domain is exemplified.

As for the step (1-2), for example, in the production of a recombinant vector for expression of the second IgG half-molecule, performing a step of substituting at least one amino acid residue selected from amino acid residues at position 235, position 236, position 237, position 326, position 327, position 328, position 329, and position 330 numbered according to the EU index as appropriate according to tire subclass of the CH2 domain is exemplified.

As for the step (1-3), for example, in the production of a recombinant vector for expression of the IgG half-molecule, performing a step of adding substitutions of amino acid residues at position 226 and position 229 numbered according to the EU index as appropriate according to the subclass of the hinge domain is exemplified.

The IgG half-molecule can be obtained, for example, by expressing it in a transformant in the following manner using a method described in Molecular Cloning, Second 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, or the like.

(1) Construction of Recombinant Vector for Expression of IgG Half-Molecule

The recombinant vector for expression of the IgG half-molecule is an expression vector for animal cells into which a gene encoding the amino acid sequence of the IgG half-molecule constituting the antibody composition of the present invention is incorporated.

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

As for the DNA, the total DNA may be synthesized or synthesis by a polymerase chain reaction (PCR method) is also possible (Molecular Cloning, Second Edition). Further, it is also possible to produce the gene encoding the IgG half-molecule by combining a plurality of such methods.

When an animal cell is used as a host, as the expression vector, any vector can be used as long as it can exhibit its function in the animal cell. Examples thereof include pcDNAI, pCDM8 (manufactured by Funakoshi Co., Ltd.), pAGE107 [JP-A-H3-22979; and Cytotechnology, 3, 133 (1990)], pAS3-3 (JP-A-H2-227075), pCDM8 [Nature, 329, 840 (1987)], pcDNAI/Amp (manufactured by Invitrogen, Inc.), pcDNA3.1 (manufactured by Invitrogen, Inc.), pREP4 (manufactured by Invitrogen, Inc.), pcGE103 [J Biochemistry, 101, 1307 (1987)], pAGE210, pME18SFL3, pKANTEX93 (WO 97/10354), N5KG1 val (U.S. Pat. No. 6,001,358), a Tol2 transposon vector (WO 2010/143698), and the like.

As a promoter, any promoter can be used as long as it can exhibit its functions in an animal cell. Examples thereof include a cytomegalovirus (CMV) immediate early (IE) gene promoter, an SV40 early promoter, a retrovirus promoter, a metallothionein promoter, a heat-shock promoter, an SRα promoter, and a Moloney murine leukemia virus promoter or enhancer. In addition, a human CMV IE gene enhancer may be used together with the promoter.

As the expression vector, either of a type in which the H chain and the L chain of the antibody exist on separate vectors or a type in which both the H chain and the L chain of the antibody exist on the same vector (hereinafter referred to as tandem type) can be used.

(2) Acquisition of cDNA Encoding Variable Region

cDNAs encoding VH and VL of an arbitrary antibody can be obtained in the following manner. The cDNAs are synthesized using mRNA extracted from a hybridoma cell that produces the arbitrary antibody as a template. The synthesized cDNAs are inserted into a vector such as a phage or a plasmid, thereby producing a cDNA library.

A recombinant phage or a recombinant plasmid having a cDNA encoding VH and a recombinant phage or a recombinant plasmid having a cDNA encoding an L chain variable region are each isolated from the library using a DNA encoding a constant region or a variable region of an existing antibody as a probe. The entire base sequences of VH and VL of the target antibody on the recombinant phage or the recombinant plasmid are determined, and then, the entire amino acid sequences of VH or VL are deduced from the base sequences.

The hybridoma cell that produces an arbitrary non-human animal antibody can be obtained as follows. A non-human animal is immunized with an antigen to which the antibody binds, and a hybridoma is produced from an antibody-producing cell of the immunized animal and as myeloma cell according to a well-known method [Molecular Cloning, Second 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]. Subsequently, a single-cell cloned hybridoma is selected and cultured, followed by purification from the culture supernatant.

As the non-human animal, any animal such as a mouse, a rat, a hamster, or a rabbit can be used as long as it can produce a hybridoma cell.

Examples of the method for preparing total RNA from the hybridoma cell include a guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymol. 154, 3 (1987)], and an RNeasy Kit (Manufactured by QIAGEN, Inc.), and also examples of the method for preparing mRNA from total RNA include an oligo (dT) immobilized cellulose column method [Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab. Press New York, 1989] and the like.

Further, examples of a kit for preparing mRNA from the hybridoma cell include Fast Track mRNA isolation Kit (manufactured by Invitrogen, Inc.), Quick Prep mRNA Purification Kit (manufactured by Pharmacia Company), and the like.

Examples of the methods for synthesizing a cDNA and preparing a cDNA library include conventional methods (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab, Press New York, 1989 and Current Protocols in Molecular Biology, Supplement 1-34), and a method using a commercially available kit. Examples of the commercially available kit include Super Script (registered trademark) Plasmid System for cDNA Synthesis and Plasmid Cloning (manufactured by GIBCO BBL, Inc.) and ZAP-cDNA Synthesis Kit (manufactured by Stratagene, Inc.).

When the cDNA library is produced, as the vector into which a cDNA synthesized using mRNA extracted from a hybridoma cell as a template is incorporated, any vector can be used as long as it can incorporate the cDNA.

For example, ZAP Express (Strategies, 5, 58, 1992) pBluescript II SK(+) (Nucleic Acids Research 17, 9494, 1989), λZAP II (manufactured by Stratagene, Inc.) λgt 10 and λgt 11 (DNA Cloning: A Practical Approach, I, 49, 1985), Lambda BlueMid (manufactured by Clontech Laboratories, Inc.), λExCell, pT7T3 18U (manufactured by Pharmacia, Inc.), pcD2 (Mol. Cell. Biol., 3, 280, 1983), pUC18 (Gene, 33, 103, 1985) or the like can be used.

As Escherichia coli into which a cDNA library constructed by a phage or plasmid vector is introduced, any Escherichia coli can be used as long as it can introduce, express, and maintain the cDNA library.

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), JM105 (Gene, 38. 275, 1985), or the like is used.

As for the method for selecting cDNA clones encoding VH and VL of the non-human antibody from the cDNA library, the selection can be carried out by a colony hybridization method or a plaque hybridization method using a probe labeled with an isotope, fluoresce, oil the like (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab. Press New York, 1989).

Further, it is also possible to prepare cDNAs encoding VH and VL by preparing primers and performing PCR (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab. Press New York 1989; and Current Protocols in Molecular Biology, Supplement 1-34) using the cDNAs or the cDNA library as a template.

The cDNA selected by the above-mentioned method is cleaved with an appropriate restriction enzyme or the like, and then cloned into a plasmid such as pBluescript II SK(−) (manufactured by Stratagene, Inc.), and the base sequence of the cDNA can be determined by a commonly used base sequence analysis method, for example, by performing a reaction of a dideoxy method by Sanger et al. (Proc. Natl. Acad. Sci. U.S.A, 74, 5463, 1977) or tile like, and then performing an analysis using an automatic base sequence analyzer, for example, a base sequence analyzer such as an ABI PRISM 377 DNA sequencer (manufactured by Applied Biosystems, Inc.).

By deducing the entire amino acid sequence of each of VH and VL from the determined base sequence and comparing it with the entire amino acid sequence of each of VH and VL of a known antibody (Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 1991), it can be confirmed whether the obtained cDNA encodes the amino acid sequence completely including each of VH and VL of the antibody containing a secretion signal sequence.

Further, when the amino acid sequence of an antibody variable region or the base sequence of a DNA encoding the variable region is already known, it can be produced using the following method.

When the amino acid sequence is known, the DNA can be obtained by designing the base sequence of a DNA encoding the variable region in consideration of the codon usage frequency (Sequences of Proteins of Immunological interest, US Dept. Health and Human Services, 1991), synthesizing several synthetic DNAs composed of approximately 100 to 150 bases based on the base sequence of the designed DNA, and carrying out the PCR method using them or synthesizing a full-length DNA. When the base sequence is known, the DNA can be obtained in the same manner as described above based on the information.

(3) Analysis of Amino Acid Sequence of Variable Region of Antibody

With respect to the complete amino acid sequences of VH and VL of the antibody containing a secretion signal sequence, by comparing them with the amino acid sequences of VH and VL of a known antibody (Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 1991), the length of the secretion signal sequence and the N-terminal amino acid sequence can be deduced, and further the subgroup to which the antibody belongs can be known. In addition, also the amino acid sequences of the respective CDRs of VH and VL can be found out by a similar method.

(4) Construction of cDNA Encoding Variable Region of Humanized Antibody

The cDNAs encoding VH and VL of a humanized antibody can be constructed in the following manner. First, the amino acid sequences of framework regions (hereinafter referred to as FRs) of VH and VL of a human antibody for grafting CDRs of VH and VL of a target non-human animal antibody are selected. As the amino acid sequences of FRs of VH and VL of the human antibody, any can be used as long as they are derived from a human antibody.

Examples thereof include amino, acid sequences of FRs of VH and VL of human antibodies registered in a database such as Protein Data Bank, common amino acid sequences in each subgroup of FRs of VH and VL of human antibodies (Sequences Proteins of Immunological Interest, US Dept. Health and Human Services, 1991), and the like.

Among them, in order to produce the humanized antibody having a sufficient activity, it is preferred to select amino acid sequences having a homology as high as possible (at least 60% or more) with the amino acid sequences of FRs of VH and VL of the target non-human animal antibody.

Subsequently, the amino acid sequences of CDRs of VH and VL of the target non-human animal antibody are grafted to the selected amino acid sequences of FRs of VH and VL of the human antibody, and the amino acid sequences of VH and VL of the humanized antibody are designed. The designed amino acid sequences are converted into base sequences of DNAs in consideration of the usage frequency of codons found in the base sequences of the antibody genes (Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 1991), and the base sequences of DNAs encoding the ammo acid sequences of VH and VL of the humanized antibody are designed. The designed base sequences of the DNAs are fully synthesized.

Further, cloning into the recombinant vector for expression of the IgG half-molecule constructed in the above 3(1) can be easily carried out by introducing an appropriate restriction enzyme recognition sequence at the 5′ ends of the synthetic DNA located at both ends. After PCR, each of the amplification products is cloned into a plasmid such as pBluescript II SK(−) manufactured by Stratagene, Inc.) and the base sequences are determined by the method described in the above 3(2), thereby obtaining a plasmid having the base sequences of the DNAs encoding the amino acid sequences of VH and VL of the desired humanized antibody.

(5) Alteration of Amino Acid Sequence of Variable Region of Humanized Antibody

It is known that the antigen-binding activity of a humanized antibody prepared merely by grafting only CDRs of VH and VL of a non-human animal antibody to FRs of VH and VL of a human antibody is decreased as compared with that of the original non-human animal antibody (BIO/TECHNOLOGY, 9, 266 1991).

The reason for this is probably because in VH and VL of the original non-human animal antibody not only CDRs but also some of the amino acid residues of FRs are directly or indirectly involved in the antigen-binding activity, and such amino acid residues are changed to different amino acid residues of FRs of VH and VL of the human antibody by grafting the CDRs.

In order to solve this problem, an attempt has been made for a humanized antibody to raise the lowered antigen-binding activity by identifying the amino acid residues directly involved in the binding to an antigen or the amino acid residues indirectly involved in the binding to an antigen through an interaction with the amino acid residues of CDRs to maintain the conformation of the antibody in the amino acid sequences of FRs of VH and VL of the human antibody, and altering them to amino acid residues derived from the original non-human animal antibody (BIO/TECHNOLOGY, 9, 266, 1991).

In the production of the humanized antibody, it is most important how efficiently the amino acid residues of FRs involved in the antigen-binding activity are identified, and therefore, the construction and analysis of the conformation of the antibody have been carried out by X-ray crystallography (J. Mol. Biol., 112, 535, 1977), computer modeling (Protein Engineering, 7, 1501, 1994), or the like.

Such information of the conformation of the antibody has provided much useful information for the production of humanized antibodies. However, there has not yet been established any method for producing a humanized antibody adaptable to all types of antibodies. At present, it is still necessary to make various trial-and-error approaches such as production of several types of variants for each antibody and examination of the correlation with the antigen-binding activity among the variants.

Alteration of the amino acid residues of FRs of VH and VL of a human antibody can be achieved by performing the PCR method described in the above 3(4) using synthetic DNAs for alteration. With respect to the amplification product after the PCR, the base sequence is determined by the method described in 3(2) to confirm that the desired alteration has been carried out.

(6) Expression of IgG Half-Molecule

A transformant that transiently or stably produces at least one of the first and second half-molecules can be obtained by introducing the recombinant vector for expression of the IgG half-molecule of the above 3(1) into an appropriate animal cell.

(6-a) Transient Expression of Antibody Composition

By performing transient expression of antibody compositions using the recombinant vector for expression of the IgG half-molecule obtained in (3) and (6) or a recombinant vector obtained by alteration thereof the antigen-binding activity of many types of antibody compositions produced can be efficiently evaluated.

As a host cell into which the recombinant vector for expression of the IgG half-molecule is introduced, any cell can be used as long as it is a host cell capable of expressing at least one of the first and second IgG half-molecules. For example, COS-7 cells [American Type Culture Collection (ATCC) number: CRL1651] are exemplified (Methods in Nucleic Acids Res, CRC press, 283, 1991).

In the introduction of the recombinant vector for expression of the IgG half-molecule into COS-7 cells, a DEAE-dextran method (Methods in Nucleic Acids Res., CRC press, 1991), a lipofection method (Proc. Natl. Acad, Sci. USA, 84, 7413, 1987), or the like is used.

After the introduction of the recombinant vector for expression of the IgG half-molecule, the expression level and the antigen-binding activity of the IgG half-molecule in a culture supernatant are measured using an enzyme immunoassay method [Monoclonal Antibodies-Principles and practice, Third edition, Academic Press (1996), Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory (1988) and A manual for monoclonal antibody experiments, Kodansha scientific books (1987)] or the like.

(6-b) Stable Expression of IgG Half-Molecule

A transformant that stably expresses the IgG half-molecule can be obtained by introducing the recombinant vector for expression of the IgG half-molecule obtained in (1) into an appropriate host cell.

In the introduction of the recombinant vector into a host cell, any method can be used as long as it is a method for introducing a DNA into the host cell, and examples thereof include an electroporation method (Cytotechnology, 3, 133, 1990), a calcium phosphate method (JP-A-H2-227075), a lipofection method (Proc. Natl. Acad. Sci. U.S.A., 84, 7413, 1987), an injection method [Manipulating the Mouse Embryo A Laboratory Manual], a method using a particle gun (gene gun) (Japanese Patent No. 2606856 and Japanese Patent No. 2517813), a DEAE-dextran method [Biomanual Series 4-Methods of Gene Transfer, Expression and Analysis (Yodosha), edited by Takashi Yokota and Kenichi Arai (1994)], a virus vector method (Manipulating Mouse Embryo, Second Edition), and the like.

As the host cell into which the recombinant vector is introduced, any cell can be used as long as it is a host cell capable of expressing at least one the first and second IgG half-molecules. Examples thereof include human leukemia cell Namalwa cells, monkey COS cells, CHO cells that are Chinese hamster cells, HBT5637 (JP-A-S63-299), rat myeloma cells, mouse myeloma cells, cells derived from Syrian hamster kidney, embryonic stem cells, fertilized egg cells, and the like.

Specific examples thereof include PER.C6, CHO-K1 (AFCC CCL-61), DUKXB11 (ATCC CCL-9096), Pro-5 (ATCC CCL-1781), CHO-S (Life Technologies, Cat #11619), Lec13 cells, rat myelorna cells YB2/3HL.P2.G11.16Ag20 (ATCC NO: CRL1662, or also called YB2/0), mouse myeloma cells NS0, mouse myeloma cells SP2/0-Ag14 (ATCC NO: CRL1581), mouse P3X63-Ag8,653 cells (ATCC NO: CRL1580), dihydrofolate reductase gene (dihydroforate reductase, hereinafter referred to as dhfr)-deficient CHO cells (CHO/DG44) [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)], Syrian Hamster cells BHK, HBT563 cells, of substrains of the above-mentioned cell lines and cells prepared by acclimating the above-mentioned cell tines under serum-free culture, cells prepared by acclimating the above-mentioned cell lines under non-adhesion culture conditions, and the like.

As a cell used for the production of the IgG half-molecule, a cell for decreasing or deleting the amount of core fucose of the sugar chain bound to Asn at position 297 according to the EU index in Fc can also be used. Specifically, a cell in which an enzyme involved in the synthesis of GDP-L-fucose or transport thereof to the Golgi body, or an enzyme involved in the binding of core fucose has been decreased or deleted is selected, or a cell obtained by any of various artificial methods can also be used as the host cell.

Specifically, a cell in which core fucose is controlled can be produced by a method for decreasing or deleting an enzyme activity involved in the sugar chain modification of core fucose, a method for increasing an activity of a core fucose cleavage enzyme, or the like.

Examples of the enzyme involved in the sugar chain modification of core fucose include an enzyme involved in the synthesis or transport of GDP-L-fucose, and an enzyme involved in the binding of core fucose to an N-glycoside-linked complex sugar chain.

Specific examples of the enzyme involved in the synthesis of GDP-L-fucose or the transport thereof to the Golgi body 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, GDP-L-fucose transporter, and the like.

Examples of the enzyme involved in the binding of core fucose include α1,6-fucosyltransferase (hereinafter referred to as FUT8), and the like.

In the cell that produces the IgG half-molecule, one of the above-mentioned enzyme activities may be decreased or deleted or a plurality of enzyme activities may be combined and decreased or deleted.

Examples of the method for decreasing or deleting the above-mentioned enzyme activities include (a) a method for gene disruption targeting the gene of the enzyme; (b) a method for introducing a dominant-negative mutant of the gene of the enzyme; (c) a method for introducing a mutation into the enzyme; (d) a method for suppressing transcription or translation of the gene of the enzyme; (e) a method for selecting a cell line that is resistant to a lectin and recognizes a sugar chain structure in which the 6-position of N-acetylglucosamine at the reducing end of the N-glycoside-linked sugar chain and the 1-position of fucose are α-linked; and the like.

Examples of the lectin include a lectin that is bound 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 (agglutinin derived from Vicia faba), and Aleuria aurantia lectin AAL (lectin derived from Aleuria aurantia).

Specific examples of the cell include FUT8 gene-deficient CHO cells (WO 2005/035556, WO 2002/31140, and WO 2000/061739), Lec13 that has acquired lectin resistance (Somatic Cell and Molecular genetics, 12, 55, 1986), GDP-fucose transporter gene-deficient cells (WO 2003/055102), GDP-mannose 4,6-dehydratase (GMD) gene-deficient cells (WO 2002/31140), WGA lectin resistant cells, LCA lectin resistant cells (WO 2002/31140), and the like.

In addition to the above-mentioned methods, the IgG half -molecule in which high mannose-two N-linked sugar chain is bound and the amount of core fucose is decreased can also be expressed by inhibiting an enzyme such as mannosidase I or mannosidase II that is an enzyme involved in an N-linked sugar chain synthesis system.

Further, by using a host cell made to overexpress N-acetylglueosamine transferase III (GnTIII), half-molecule in which a bisecting GlcNAc-bound complex and a hybrid sugar chain are bound, and the amount of core fucose is decreased can also be produced.

After introduction of the recombinant vector, a transformant that stably expresses the IgG half-molecule is selected by culturing it in a culture medium for animal cell culture containing an agent such as G418 sulfate (hereinafter referred to as G418), cycloheximide (hereinafter abbreviated as CHX), methetrexate (hereinafter abbreviated as MTX) (JP-A-H2-257891).

Examples of the culture medium for animal cell culture include RPMI 1640 medium (manufactured by Invitrogen, Inc.), GIT medium (manufactured by Nihon Pharmaceutical Co., Ltd.), EX-CELL301 medium, EX-CELL302, EX-CELL325 medium (manufactured by JRH), IMDM medium (manufactured by Invitrogen, Inc.), Hybridoma-SFM medium (manufactured by Invitrogen, Inc.), culture media obtained by adding various. additives such as fetal bovine serum (hereinafter abbreviated as FBS) to these culture media, and the like.

The IgG half-molecule is expressed and accumulated in a culture supernatant by culturing the obtained transformant in a culture medium. The expression level and the antigen-binding activity of the IgG half-molecule in the culture supernatant can be measured by an ELISA method or the like. In addition, the expression level of the IgG half-molecule produced by the transformant can be improved using a dhfr gene amplification system (JP-A-H2-257891) or the like.

The method for expressing the IgG half-molecule using an animal cell as the host has been described above, however, the IgG half-molecule can be expressed also in yeast, an insect cell, a plant cell, or an animal individual or a plant individual in the same manner as in the animal cell based on a known technique.

When yeast is used as the host cell, a microorganism belonging to the genus Saccharomyces, the genus Schizosaccharomyces, the genus Kluyveromyces, the genus Trichosporon, the genus Schwanniomyces, or the like, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pollulans, Schwanniomyces alluviuns, or the like can be examplified.

As a method for introducing the recombinant vector, any method can be used as long as it is a method for introducing a DNA into yeast, and for example, methods described in an electroporation method (Methods Enzymol., 194, 182, 1990), a spheroplast method (Proc. Natl. Acad. Sci, U.S.A., 84, 1929, 1978), a lithium acetate method a (J. Bacteriology, 153, 163, 1983, Proc. Natl. Acad. Sci. U.S.A., 75, 1929, 1978), and the like are exemplified.

When an insect cell is used as the host, the IgG half-molecule can be expressed by, for example, a method described in current Protocols in Molecular Biology (Baculovirus Expression Vectors, A Laboratory Manual, W. H. Freeman and Company, New York, 1992), Bio/Technology, 47, 1988, or the like.

[Step 2]

The step 2 is a step of culturing the transformant obtained in the step 1 to produce and accumulate the IgG half-molecule in a culture, and collecting the IgG half-molecule from the culture and purifying the IgG half-molecule.

The first and second IgG half-molecules may be collected from the same transformant, or may be collected from transformants individually expressing each of the first and second IgG half-molecules. Generally, the first and second IgG half-molecules are collected from transformants individually expressing each of the first and second IgG half-molecules, and mixed after purification, whereby the antibody composition is prepared.

When the host cell prepared in the step 1 has an ability to express the IgG half-molecule, after the IgG half-molecule is introduced into the host cell described below, the cell is cultured, and the target IgG half-molecule can be collected from the culture.

Further, an animal individual into which a gene is introduced (transgenic non-human animal) or a plant individual into which a gene is introduced (transgenic plant) is prepared by redifferentiation of an animal or plant cell into which a gene is introduced, and the IgG half-molecule may be collected using such an individual.

When the transformant is an animal individual or a plant individual, the individual is raised or cultivated according to a conventional method to produce and accumulate the IgG half-molecule, and the IgG half-molecule can be collected from the animal individual or the plant individual.

Examples of the method for producing the IgG half-molecule using an animal individual include a method for producing the target IgG half-molecule in an animal prepared by introducing a gene according to a known method (American Journal of Clinical Nutrition, 63, 639S, 1996; American Journal Nutrition, 63, 627S, 1996; Bio/Technology, 9, 830, 1991).

In the case of an animal individual, for example, a transgenic non-human animal into which a DNA encoding the IgG half-molecule is introduced is raised to produce and accumulate the IgG half-molecule in the animal, and the IgG half-molecule can be collected from the inside of the animal.

As a place of production and accumulation in the animal, for example, milk (JP-A-S63-309192), egg, or the like of the animal can be exemplified. As the promoter used at that time, any promoter can be used as long as it enables expression in an animal. For example, an α-casein promoter, a β-casein promoter, a β-lactoglobulin promoter, and a whey acidic protein promoter that are mammary gland cell-specific promoters, and the like are preferably used.

Examples of the method for producing the half-molecule using a plant individual include a method for cultivating a transgenic plant into which a DNA encoding the IgG half-molecule is introduced according to a known method [Tissue Culture, 20 (1994); Tissue Culture, 21 (1995); and Trends in Biotechnology 15, 45 (1997)] to produce and accumulate the IgG half-molecule in the plant, and collecting the IgG half-molecule from the inside of the plant.

The IgG half-molecule can be purified in the following manner. When the IgG half-molecule produced by the transformant into which the gene encoding IgG half-molecule is introduced is, for example, expressed as a soluble protein in the cells, after culture is completed, the cells are collected by centrifugation and suspended in an aqueous buffer solution, and thereafter, the cells are homogenized using an ultrasonic homogenizer, a French press, a Manton Gaulin homogenizer, a dynomill, or the like, whereby a cell-free extract is obtained.

From the supernatant obtained by centrifugation of the edl-free extract, the IgG half-molecule can be purified by a general enzyme isolation and purification method, that is, by using methods such as a solvent extraction method, a salting-out method using ammonium sulfate or the like, a desalting method, a precipitation method using an organic solvent, anion exchange chromatography using a resin such as diethylaminoethyl (DEAE)-sepharose or DIAION HPA-75 (manufactured by Mitsubishi Chemical Corporation), cation exchange chromatography using a resin such as S-Sepharose FF (Pharmacia, Inc.), hydrophobic chromatography using a resin such as butyl sepharose or phenyl sepharose, a gel filtration method using a molecular sieve, affinity chromatography, a chromatofocusing method, and electrophoresis such as isoelectric focusing electrophoresis alone or in combination.

In the present invention, as the affinity chromatography, affinity chromatography using a CH-binding body or an Fc-binding body is used (Monoclonal Antibodies-Principles and practice, Third edition, Academic Press, 1996, Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).

Further, when the IgG half-molecule is expressed within the cells forming insoluble body, the cells are collected and homogenized in the same manner, followed by centrifugation, whereby the insoluble body of the IgG half-molecule is collected as a precipitated fraction. The collected insoluble body of the IgG half-molecule is solubilized with a protein denaturing agent. The IgG half-molecule is returned to a normal conformation by diluting or dialyzing the solubilized solution, and thereafter, the IgG half-molecule can be purified by the same isolation and purification method as described above.

When the IgG half -molecule is secreted extracellularly, the IgG half-molecule or a derivative thereof can be recovered in the culture supernatant. That is, the culture supernatant is obtained by treating the culture by a method such as centrifugation in the same manner as described above, and the IgG half-molecule can be purified from the culture supernatant using the same isolation and purification method as described above.

Specifically, the CH-binding body or the Fc-binding body may be any material such as a protein or a resin as long as it binds to CH or Fc, and examples thereof include an Fc-binding protein, an antibody that binds to an H chain constant region (CH) of an antibody, and the like.

Example of the Fc-binding protein include Protein A derived from Staphylococcus Aureus Protein G derived from hemolytic Streptococcus, an Fc receptor and the subclasses thereof (FcγRI, IIA, IIB, IIIA, and IIIB) and binding portion fragments thereof, and the like.

Examples of the antibody that binds to CH include antibodies that bind to a CH1 domain, a hinge domain, a CH2 domain, or a CH3 domain.

In the present invention, more preferred examples of the CH-binding body include Protein A, Protein G, an anti-CH1 antibody, and binding portion fragments thereof.

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

Thereafter, the IgG half-molecule is eluted from the column using a citric acid buffer at low pH (pH 2.0 to 6.0) or the like, and the eluate is neutralized with an alkaline Tris buffer or the like. The neutralized eluate is subjected to dialysis using a sufficient amount of PBS or the like, whereby the purified IgG half-molecule can be obtained.

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

[Step 3]

The step 3 is a step of mixing the first and second IgG half-molecules collected and purified in the step 2, thereby obtaining the antibody composition. It is preferred that the mixing ratio of the first and second IgG half-molecules is appropriately set according to a binding activity to an antigen, a binding activity to an antigen-positive cultured cell line, the strength of the CD16a-binding activity in each CD16a-binding domain, an interaction between the CH3 of the first IgG half-molecule and the CH3 of the second IgG half-molecule, or the like.

Further, the antibody composition of the present invention can also be produced as an antibody composition in which the first and second IgG half-molecules are mixed by simultaneously expressing the recombinant vectors for expression of the first and second IgG half-molecules in the host cell and performing purification. In this case, as for the mixing ratio of the first and second IgG half-molecules, an antibody composition having a desired mixing ratio can be produced by controlling the expression level of the gene encoding each half-molecule. In addition, the genes encoding the first second IgG half-molecules may be expressed by the same recombinant expression vector or may be expressed by separate recombinant expression vectors.

4. Evaluation or Activity of Antibody Composition

As a method for measuring the protein amount of the purified IgG half-molecule, an FcR-binding activity, a C1q-binding activity, an antigen-binding activity, or cellular cytotoxicity such as an ADCC activity or a CDC activity of the antibody composition constituted by the IgG half-molecule, 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 are exemplified.

As a specific example, the binding activity of the antibody composition to an antigen or the binding activity thereof to an antigen-positive cultured cell line can be measured by an ELISA method, a fluorescent antibody method (Cancer Immunol. Immunother, 36, 373, 1993), or the like. The cellular cytotoxicity against an antigen-positive cultured cell line be evaluated by measuring a CDC activity, an ADCC activity, or the like (Cancer Immunol. Immunother, 36, 373, 1993; US Patent Application Publication No. 2004/0259150).

Whether or not the antibody composition has a binding activity to CD16a can be confirmed by producing a recombinant CD16a protein and then measuring binding activity (US Patent Application Publication No. 2004/0259150).

Example of the method for measuring an ADCC activity include a method in which a target cell labeled with a radioisotope, a fluorescent substance, a dye, or the like, the antibody composition, and an effector cell are brought into contact with one another, and then, the activity of the labeling substance released from the damaged target cell or the biological activity or the like of an enzyme released therefrom is measured, and the like.

Example of the method for measuring a CDC activity include a method in which a target cell labeled with a radioisotope, a fluorescent substance, a dye, or the like, the antibody composition, and a biological sample such as serum containing a complement component are brought into contact with one another, and then, the activity of the labeling substance released from the damaged target cell or the biological activity of an enzyme released therefrom is measured, and the like.

5. Analysis of Sugar Chain Structure

The sugar chain structure of the IgG half-molecule expressed in various types of cells can be analyzed according to a general analysis of a sugar chain structure of a glycoprotein.

For example, a sugar chain bound to the IgG half-molecule is constituted by a neutral sugar such as galactose (Gal), mannose (Man), or fucose (Fuc), an amino sugar such as N-acetylglucosamine (GlcNAc), or an acidic sugar such as sialic acid (Sial), and can be analyzed using a technique such as a sugar chain structure analysis using a sugar composition analysis and a two-dimensional sugar chain mapping method.

(1) Neutral Sugar and Amino Sugar Composition Analysis

A composition analysis of a sugar chain of the IgG half-molecule can be carried out by performing acid hydrolysis of a sugar chain with trifluoroacetic acid or the like to release a neutral sugar or an amino sugar and analyzing the composition ratio thereof.

Specific examples of the method include a method using a sugar composition analyzer manufactured by Dionex Corporation, BioLC is a device for analyzing a sugar composition by an HPAEC-PAD (high performance anion-exchange chromatography-pulsed amperometric detection) method (J. Liq. Chromatogr. 6, 1577, 1983).

Further, a composition ratio can also be analyzed by a fluorescence labeling method using 2-aminopyridine. Specifically, a sample that is acid-hydrolyzed according to a known method [Agric. Biol. Chem., 55(1), 283-284, 1991] is fluorescently labeled by 2-aminopyridylation, and a composition ratio can be calculated by performing an HPLC analysis.

(2) Sugar Chain Structure Analysis

The structure analysis of a sugar chain in the IgG half-molecule can be carried out by a two-dimensional sugar chain mapping method (Anal. Biochem., 171, 73, 1988; Biochemical Experimentation Methods 23-Methods of Studies on Glycoprotein Sugar Chains, Japan Scientific Societies Press, edited by Reiko Takahashi, 1989). The two-dimensional sugar chain mapping method is, for example, a method for deducing a sugar chain structure by plotting the retention time or elution position of a sugar chain by reverse-phase chromatography on the X axis and the retention time or elution position of the sugar chain by normal-phase chromatography on the Y axis, respectively, and comparing them with results of those of known sugar chains.

Specifically, a sugar chain is released from the IgG half-molecule by hydrazinolysis of the IgG half-molecule and fluorescence labeling of the sugar chain with 2-aminopyridine (hereinafter abbreviated as PA) (J. Biochem., 95, 197, 1984) is performed, and thereafter, the sugar chain is separated from an excess amount of a PA-treating reagent by gel filtration, followed by reverse-phase chromatography, Then, each peak of the fractionated sugar chain is subjected to normal-phase chromatography. The sugar chain structure can be deduced by plotting on a two-dimensional sugar chain map based on these results and comparing them with the spots of a sugar chain standard (manufactured by TaKaRa, Inc.) or those in literature (Anal. Biochem., 171, 73, 1988).

Further, mass spectrometry such as MALDI-TOF-MS of each sugar chain is performed, and the structure deduced by the two-dimensional sugar chain mapping method can be continued.

Which portion of Fc of the IgG half-molecule a sugar chain is bound to can be confirmed by treating the IgG half-molecule subjected to reductive alkylation with an endoprotease such as trypsin, pepsin, Lys-C, Asp-N, and separating the resultant by reverse-phase chromatography (LC), followed by an analysis using a mass spectrometer (MS) or the like.

In other words whether or not a sugar chain is actually bound can be confirmed by whether or not the molecular weight of a peptide that can be produced by a protease treatment and the molecular weight of a peptide to which the sugar chain is bound match the MS analytical values based on the amino acid sequence of the Fc of the target IgG half-molecule.

6. Method for Identifying Sugar Chain Structure

The ratio of sugar chains having no core fucose among all the N-glycoside-linked complex sugar chains bound to Fc in the IgG half-molecule can be identified using the method for analyzing a sugar chain structure described in the above 5. In addition, it can also be identified by an immunological quantitative method using a lectin.

The identification of a sugar chain structure in the IgG half-molecule by an immunological quantitative method using a lectin can be carried out according to an immunological quantitative method such as Western staining, RIA (radioimmunoassay), VIA (viroimmunoassay), EIA (enzymoimmunoassay), FIA (fluoroimmunoassay), or MIA (metalloimmunoassay) described in literature [Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc. (1995); Enzyme Immunoassay; 3rd Ed., IGAKU-SHOIN Ltd. (1987); Enzyme Antibody Technique, Revised Edition, Gakusai Kikaku (1985)], or the like, for example, in the following manner.

A lectin that recognizes the sugar chain structure of the IgG half-molecule is labeled, and the labeled lectin and the antibody composition that is a sample are allowed to react with each other. Subsequently, the amount of a complex of the labeled lectin with the antibody composition is measured.

Examples of the lectin used for identification of the sugar chain structure of the IgG half-molecule include WGA (wheat-germ agglutinin derived from T. vulgaris), ConA (concanavalin A derived from C. ensiformis), RIC (a toxin derived from R. communis), L-PHA (leucoagglutinin derived from P. vulgaris), LCA (lentil agglutinin derived from L. culinaris), PSA (pea lectin derived from P. sativum), AAL (Aleuria aurantia Lectin), ACL (Amaranthus caudatus Lectin), BPL (Bauhinia pupurea Lectin), DSL (Datura stramonium Lectin), DBA (Dolichos biflorus Agglutinin), EBL (Elderberry Balk Lectin), ECL (Erythrina cristagalli Lectin), EEL (Euonymus europaeus Lectin), (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 (Solamum tuberosum Lectin), SJA (Sophora japonica Agglutinin), SBA (Soybean Agglutinin), UEA (Ulex europaeus Agglutinin), VVL (Vicia villosa Lectin), and WFA (Wisteria floribunda Agglutinin).

A lectin that specifically recognizes core fucose is preferably used, and specific examples thereof include lentil lectin LCA (lentil agglutinin derived from Lens culinaris), pea lectin PSA (pea lectin derived from Pisum sativum), broad bean lectin VFA (agglutinin derived from Vicia faba), and Aleuria aurantia lectin AAL (lectin derived from Aleuria aurautia).

7. Use of Antibody Composition of the Present Invention

The antibody composition of the present invention can recognize two types of antigens that are different from each other by including the first and second IgG half-molecules having antigen-binding domains for antigens that are different from each other. Therefore, a pharmaceutical composition containing the antibody composition of the present invention can have a molecular form in accordance with two different types of target antigens, and thus can exhibit high specificity for a double-positive cell that expresses the two types of antigens.

When the antibody composition of the present invention is used for a pharmaceutical composition, it is preferred that the antibody composition has the following properties [1] to [3]. The following properties [1] to [3] can serve as indices for selecting excellent antibody composition in the present invention.

[1] High Specificity for Double-Positive Cell

[2] Enhanced Effector Function

[3] Blood Half-Life Comparable to that of Wild-Type IgG Antibody

Hereinafter, the respective properties wiil be described.

[1] High Specificity for Double-Positive Cell

The antibody composition of the present invention having high specificity for a double-positive cell as compared with a single-positive cell can be selected, for example, by a method including the following steps (1a) to (1d).

(1a) The first IgG half-molecule in which each of an amino acid alteration for deleting an inter-H chain disulfide bond in a hinge domain, an amino acid alteration for attenuating a CD16a-binding activity in a first CD16a-binding domain, and an amino acid alteration for attenuating inter-CH3 domain interaction is introduced is produced.

(1b) The second IgG half-molecule in which each of an amino acid alteration for deleting an inter-H chain disulfide bond in a hinge domain, an amino acid alteration for attenuating is CD16a -binding activity in a second CD16a-binding domain, and an amino acid alteration the attenuating an inter-CH3 domain interaction is introduced is produced in the same manner as in (1a).

(1c) A double-positive cell that expresses first and second antigens, a single-positive cell that expresses the first antigen, and a single-positive cell that expresses the second antigen are prepared.

In order to use cells in which the expression level of the antigen in each cell is adjusted, each antigen transfectant may be prepared. The effector function of the antibody composition in which the first IgG half-molecule and the second IgG half-molecule are mixed is evaluated for each cell.

(1d) An antibody composition having high specificity for the double-positive cell as compared with the single-positive cells is obtained by selecting an antibody composition having a strong effector function for the double-positive cell as compared with the effector function for each of the single-positive cells based on the results of evaluation in (1c).

As a specific method, for example, a method described below in [Example 3] is exemplified. Specifically, for example, when the effector function is an ADCC activity, an antibody composition having high specificity for the double-positive cell as compared with the singe positive cells can be obtained by selecting an antibody composition having a strong effector function for the double-positive cell as compared with the effector function for each of the single-positive cells as follows.

1-i) As the target cell, the single-positive cell that expresses the first antigen, the single-positive cell that expresses the second antigen, and the double-positive cell that expresses the first antigen and the second antigen are used. The expression levels of the first antigen and the second antigen in each cell are measured using a flow cytometer, and it is confirmed that the target antigen is expressed in the cell.

1-ii) As the effector cell, a human peripheral blood mononuclear (PBMC) or a human CD16a-expressing which is transfected with a gene encoding human CD16a to stably express the gene, and the target cell are prepared using a culture medium (for example, RPMI medium).

1-iii) The cells are left to stand for about 2 to 6 hours in a CO₂incubator. At the same time, a solubilizing solution (for example, an aqueous solution containing an acid, an alkali, a surfactant, or the like) is added to a 100% reaction well, and the target cells are completely lysed. After the reaction container is centrifuged, the supernatant is collected and dispensed into an ELISA plate. A coloring solution is applied thereto to cause a reaction, and thereafter, a stopping solution is added thereto, and an absorbance (A450) is measured using a plate reader.

1-iv) The ADCC activity (%) is calculated using the following formula.

ADCC activity(%)=100×(S−E−T)/(Max−T)

S=absorbance of sample reaction—absorbance of culture medium well

E=absorbance of effector—absorbance of culture medium well

T=absorbance of target well—absorbance of culture medium well

Max=100% reaction well—100% reaction control well

1-vi the positive control antibody, a normal IgG1-type antibody against the first antigen and a normal IgG1-type antibody against the second antigen are used. It is confirmed that the normal IgG1-type antibody against the first antigen exhibits ADCC activity against the single-positive cell that expresses the first antigen, and the normal IgG1-type antibody against the second antigen exhibits an ADCC activity against the single-positive cell that expresses the second antigen.

On the Other band, when an antibody solution obtained by mixing a half-molecule of the antibody against the first antigen and a half-molecule of the antibody against the second antigen does not exhibit an ADCC activity against the single-positive cell that expresses the first antigen and the single-positive cell that expresses the second antigen, and specifically exhibits an ADCC activity only against the double-positive cell that expresses the first antigen and the second antigen, it in evaluated to he an antibody composition having a strong effector function fur the double-positive cell as compared with the effector function for each of the single-positive cells.

As one aspect of the case where “an antibody solution obtained by mixing a half-molecule of the antibody against the first antigen and a half-molecule of the antibody against the second antigen does not exhibit an ADCC activity against the single-positive cell that expresses the first antigen and the single-positive cell that expresses the second antigen, and specifically exhibits ADCC activity only against the double-positive cell that expresses the first antigen and the second antigen”, for example, a ease where the ADCC activity against the single-positive cell that expresses the first antigen and the single-positive cell that expresses the second antigen is comparable to the ADCC activity of the negative control, and also the ADCC activity against the double-positive cell that expresses the first antigen and the second antigen is comparable to the ADCC activity of the positive control is exemplified. As another aspect, for example, a case where the ADCC activity against the single-positive cell that expresses the first antigen and the single-positive cell that expresses the second antigen is attenuated as compared with the ADCC activity of the positive control, and also the ADCC activity against the double-positive cell that expresses the first antigen and the second antigen is comparable to the ADCC activity of the positive control is exemplified.

Here, the description “the ADCC activity is comparable” means that the ADCC activity is within a range of preferably ±0 to 50%, and more preferably ±0 to 30% with respect to the ADCC activity of the control, and the description “the ADCC activity is attenuated” means that the ADCC activity is within a range of preferably 0 to 60%, and more preferably 0 to 30% with respect to the ADCC activity of the control.

As the negative control at this evaluation, it is preferred to use the antibody against the first antigen and the antibody against the second antigen. The antibody against the first antigen exhibits an effector function for the double-positive cell and the single-positive cell that expresses the first antigen, and the antibody against the second antigen exhibits an effector function for the double-positive cell and the single-positive cell that expresses the second antigen.

Further, in place of each antigen-expressing cell, a bead or the like with each antigen fixed thereto can be used. For example, a double-positive bead with the first antigen and the second antigen fixed thereto, a single-positive bead with only the first antigen fixed thereto, and a single-positive bead with only the second antigen fixed thereto are prepared. To each bead, the antibody composition of the present invention is added, and thereafter, further a recombinant CD16a protein is added thereto. Thereafter, by examining the binding activity of the recombinant CD16a protein to the antibody composition bound onto the double-positive bead and two single-positive beads, an antibody composition having high selectivity for both target antigens can be selected. As a method for measuring an interaction with the recombinant CD16a protein, a binding ELISA method, a surface plasmon resonance method, or the like can be used.

[2] Enhanced Effector Function

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

(2a) The effector function of the antibody composition having high specificity for the double-positive cell as compared with the single-positive cells selected by the method described in the above [1] is enhanced.

As a means for enhancing the effector function of the antibody composition of the present invention having high specificity for the double-positive cell selected by the method described in the above [1], for example, performing an amino acid alteration for enhancing the CD16a-binding activity, and/or decreasing the content of the core fucose bound to Fc or deleting the core fucose is exemplified.

When an amino acid residue substitution for enhancing the CD16a-binding activity is introduced, the amino acid residue may be any as long as it is an amino acid residue contained in the CD16a domain formed in the hetero assembly of the IgG half-molecules.

Further, the amino acid residue substitution may be introduced into both the first and second IgG half-molecules or may be introduced into either one of them. However, in order to achieve the effect of the present invention in, the same IgG half-molecule, it is preferred that the position of the amino acid residue substitution for enhancing the CD16a-binding activity and the position of the amino acid residue substitution for attenuating the CD16a-binding activity different.

In other words, if the IgG half-molecules are mutually different, the position of the amino acid residue substitution for enhancing the CD16a-binding activity and the position of the amino acid residue substitution for attenuating the CD16a-binding activity may be the same.

Which position the amino acid residue substitution for enhancing me CD16a-binding activity is introduced into can be determined by producing hetero assemblies composed of various IgG half-molecules in combination with the position or the amino acid residue substitution for attenuating the CD16a-binding activity measuring the ADCC activity or the like, and evaluating that the activity is an activity specific for the double-positive cells

The effector function can be enhanced by decreasing the content of the core fucose bound to Fc or deleting the core fucose in addition to the amino acid residue substitution for enhancing the CD16a-binding activity.

(2 b) With respect to the antibody composition with a effector function enhanced in (2a), the effector function (for example, the ADCC activity or the like) for the single-positive cell that expresses the first antigen, the single-positive cell that expresses the second antigen and the double-positive cell that expresses the first and second antigens is evaluated.

(2c) An antibody composition having an enhanced effector function specific for the double-positive cell is obtained by selecting an antibody composition having a strong effector function for the double-positive cell as compared with the effector function for each of the single-positive cells based on the results of evaluation in (2b).

By the above-mentioned method, an antibody composition having an enhanced effector function and exhibiting high specificity for the double-positive cell can be selected.

The antibody composition having an enhanced effector function specific for the double-positive cell can be evaluated for an effector function closer to a clinical effect by an evaluation system mimicking human blood. As the evaluation system mimicking human blood, for example, an ADCC evaluation system in which a protein component of blood such as human albumin, an immunoglobulin, human plasma, or human serum and human NK cells, human peripheral blood mononuclear cells (PBMC), human granulocytes, or the like are added, a cellular cytotoxicity evaluation system using human whole blood, and the like are exemplified.

Examples of a specific method for the evaluation system mimicking human blood include a method using an ADCC evaluation system described below in [Example 8] and a method using a cellular cytotoxicity evaluation system described below in [Example 9].

As the method using an ADCC evaluation system, specifically, for example, the following method is exemplified. The ADCC activity is measured and evaluated in the same manner as in 1-i) to 1-vi) except that in the above-mentioned 1-ii), an evaluation system mimicking the internal human body is constructed by adding immunoglubulin at a final concentration of preferably 0.01 to 10 mass %, more preferably 0.1 to 10 mass %, and further more preferably 1 to 4 mass %.

As the method using a cellular cytotoxicity evaluation system by a human blood reconstitution system, specifically, for example, the following method is exemplified.

2-i) As the target cell, the single-positive cell that expresses the first antigen, the single-positive cell that expresses the second antigen, and the double-positive cell that expresses the first antigen and the second antigen are used. The expression levels of the first antigen and the second antigen in each cell are measured using a flow cytometer, and it is confirmed that the target antigen is expressed in the cell.

2-ii) Plasma is obtained by centrifuging human blood and recovering the supernatant. Further, a fraction of peripheral blood mononuclear cells and granulocytes is obtained from the human blood using a red blood cell depletion reagent (for example, Hetasep: Stemcell technology, #07806).

2-iii) The plasma and the fraction of peripheral blood mononuclear cells and granulocytes recovered in 2-ii) are mixed, and the mixture is seeded in a cell culture plate, and thereafter, an antibody composition obtained by mixing a half-molecule of the antibody against the first antigen and a half-molecule of the antibody against the second antigen is added thereto, and the cells are cultured at 37°0 C. for about 12 to 48 hours.

2-iv) The ratio of the double-positive cell that expresses the first antigen and the second antigen is measured by flow cytometry. As the negative control, a wild-type IgG antibody is used.

As a result, when the antibody composition obtained by mixing a half-molecule the antibody against the first antigen and a half-molecule of the antibody against the second antigen removes the double-positive cell that expresses the first antigen and the second antigen in an antibody concentration-dependent manner, but does not remove the single-positive cell that expresses the first antigen and the single-positive cell that expresses the second antigen, it is evaluated that the effector function specific for the double-positive cell is enhanced.

The description “removes the double-positive cell” means that the ratio of the double-positive cell that expresses the first antigen and the second antigen when the antibody composition of the present invention is added is lower than the ratio in the case of the negative control. The description “the ratio or the double-positive cell is lower” means that it is within a range of preferably 0 to 70%, and more preferably 0 to 50% with respect to the ratio of the double-positive cell in the case of the negative control.

The description “does not remove the single-positive cell” means that the ratio of the single-positive cell that expresses the first antigen and the single-positive cell that expresses the second antigen when the antibody composition of the present invention is added is comparable to the ratio in the of the negative control. The description “the ratio of the single-positive cell is comparable” means that it is within a range of preferably ±0 to 40%, and more preferably ±0 to 20% with respect to the ratio of the single-positive cell in the case of the negative control.

By this evaluation, an antibody composition that exhibits an enhanced effector function even in human blood can he selected.

(3) The antibody composition having both high specificity for the double-positive cell and an enhanced effector function selected using the above-mentioned method in [1] and [2] is subjected to PK studies (pharmacokineties and pharmacodynamics studies) using an animal such as a mouse or a monkey.

As the negative control, a wild-type IgG antibody is used.

(3b). As a result of the PK studies in (3a), an antibody composition that exhibits kinetics close to that of the wild-type IgG antibody is selected as an antibody composition haying a blood half-life comparable to that of the wild-type IgG antibody. As a specific method, for example, a method described below in [Example 7] is exemplified.

As the method for the PK studies, specifically, for example, the following method is exemplified.

3-i) After the antibody composition (preferably 0.1 to 10 mg/kg) having both high specificity for the double-positive coil and an enhanced effector function is administered to a mouse (for example, BALB/c mouse) through the tail vein, the blood is collected preferably after 1 hour, 4 hours, 24 hours, and 72 hours. After piercing the cheek with a lancet under isoflurane anesthesia and collecting the blood, serum is recovered by centrifuging the blood at preferably 4,000 to 16,000 rpm at room temperature preferably for 5 to 20 minutes with a micro blood collection tube [for example, Microtainer (registered trademark) manufactured by BD Company]. As the positive control antibody, a wild-type IgG antibody is used.

3-ii) The concentration of the antibody present in the serum is measured. The concentration of the antibody in the serum can be measured by a known method, and for example, the measurement can be carried out using AlphaLISA Human Kappa light chain immunoassay kit (AL3023, PerkinElmer), and a calibration curve can be created with Kappa light chain in the kit.

3-iii) An antibody composition in which the blood half-life (t1/2) that can be calculated from the concentration of the antibody measured in 3-ii) is within a range of preferably 50% to 150% as compared with that of the wild-type IgG antibody as the positive control is evaluated to be an antibody composition having a blood half-life comparable to that of the wild-type IgG antibody.

The antibody composition having a long blood half-life can be selected also by measuring the denaturation midpoint (Tm) value by differential scanning calorimetry (DSC) as a physicochemical characteristic of an antibody in place of the PK studies using an animal.

Among the antibody compositions of the present invention, those having a Tm value (denaturation midpoint temperature) comparable to that of the wild-type IgG antibody exhibit favorable PK. As specific method for measuring the Tm value, for example, a method described below in [Example 7] is exemplified. Specifically, the measurement can be carried out by differential scanning fluorimetry (DSF), and as a measuring apparatus, for example, Prometheus NT48 of NanoTemper Technologies GmbH is exemplified. The measurement of the Tm value of the antibody can be carried out preferably by elevating the temperature to 80° C. to 100° C. starting from 10° C. to 30° C. at a temperature elevation rate of preferably 0.5° C. to 2° C./min.

Further, it is known that the higher the binding activity at pH 6.0 of the antibody to FcRn (neonatal Fc receptor) is, the longer the blood half-life is. Therefore, by measuring the binding activity to FcRn, an antibody composition having a long blood half-life can be selected. The binding activity to FcRn can be measured, for example, by a method disclosed in J. Immunol. 2002; 169: 5171-80.

By the evaluation in [1] to [3] described above, an antibody composition having preferred properties when it is used in a pharmaceutical product can be selected. A combination of amino acid residue substitutions included in the preferred antibody composition of the present invention having such properties is preferably a combination shown in Table 2, more preferably a combination shown in Table 3A or 3B, and most. preferably a combination shown in Table 4.

TABLE 2

Domain
Purpose of amino acid
Combination of amino acid residue substitutions

name
residue substitution
First IgG half-molecule
Second IgG half-molecule

Hinge
To form hetero assembly
C226A/C229A

CH2
To bind to CD16a only when
D265A, S267K, Y296P, S298E, or S239R
P329Y

hetero assembly is formed

CH2
To enhance CD16a binding
S239D, S239E, S239D/K326T, S239D/A330F, S239D/K326E, S239E/I332E,

activity
S208A/E33A/K334A/H268E, or S239D/S298A/E333A/L242C/K334C

CH3
To form hetero assembly
Y349A, L351A, T366A, L368A, D399A, F405A, Y407A, K409A, or K409R

TABLE 3A

Domain
Purpose of amino acid
Combination of amino acid residue substitutions

name
residue substitution
First IgG half-molecule
Second IgG half-molecule

Hinge
To form hetero assembly
C226A/C229A

CH2
To bind to CD16a only when
D265A
P329Y

hetero assembly is formed

CH2
To enhance CD16a binding
S293D/K326T or S239D/S296A/E333A/L242C/K334C

activity

CH3
To form hetero assembly
L368A, Y407A, or K490R

TABLE 3B

Domain
Purpose of amino acid
Combination of amino acid residue substitutions

name
residue substitution
First IgG half-molecule
Second IgG half-molecule

Hinge
To form hetero assembly
C226A/C229A

CH2
To bind to CD16a only when
S296E
P329Y

hetero assembly is formed

CH2
To enhance CD16a binding
S293D/K326T or S239D/S298A/E333A/L242C/K334C

activity

CH3
To form hetero assembly
L368A, Y407A, or K409R

That is, combinations of amino residue substitutions shown in Tables 3A and 3B are as follows.

First IgG Half-Molecule>

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, S219D, 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

Second IgG Half-Molecule>

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) C226 A, C229A , P329Y, S239D, S295A, E333A, L242C, K334C, Y407A

6) C226A, C229A, P329Y, S239D, S298A, E333A, L242C, K334C, K409R

The antibody composition of the present invention preferably contains the second IgG half-molecule [1) to 6)] with the same number with respect to the first IgG half-molecule [1) to 6)]. Further, the antibody composition of the present invention preferably contains the second IgG half-molecule so that the combination thereof with the first IgG half-molecule [7) to 12)] becomes as follows.

- the first IgG half-molecule 7) and the second IgG half-molecule 1)
- the first IgG half-molecule 8) and the second IgG half-molecule 2)
- the first IgG half-molecule 9) and the second IgG half-molecule 3)
- the first IgG half-molecule 10) and the second IgG half-molecule 4)
- the first IgG half-molecule 11) and the second IgG half-molecule 5)
- the first IgG half-molecule 12) and the second IgG half-molecule 6)

More specifically, as preferred embodiments of the combination of amino acid residue substitutions included in the antibody composition of the present invention, for example, the following <1> to <12> are exemplified.

<1>