Heterodimerized polypeptide

[0001] This application is the National Stage of International Application Serial No. PCT/JP2012/066665, filed on Jun. 29, 2012, which claims the benefit of Japanese Patent Application Serial No. 2011-146778, filed on Jun. 30, 2011, and Japanese Patent Application Serial No. 2011-288240, filed on Dec. 28, 2011.

[0002] The present invention provides antibody constant regions whose amino acid sequence is modified from a naturally-occurring antibody constant region, antibodies comprising such constant regions, pharmaceutical compositions comprising such antibodies, and methods for producing them.

[0003] Antibodies are drawing attention as pharmaceuticals since they are highly stable in blood and have few side effects (Non-patent Documents 1 and 2). Almost all antibody pharmaceuticals currently on the market are antibodies of the human IgG1 subclass. So far, many studies have been carried out on antibody-dependent cellular cytotoxicity (hereinafter, referred to as ADCC) and complement-dependent cytotoxicity (hereinafter, referred to as CDC) which are effector functions of the IgG class antibodies; and in human IgG class, antibodies of the IgG1 subclass have been reported to have the highest ADCC activity and CDC activity (Non-Patent Document 3). Furthermore, antibody-dependent cell-mediated phagocytosis (ADCP), which is phagocytosis of target cells mediated by IgG class antibodies, is also shown as one of the antibody effector functions (Non-Patent Documents 4 and 5).

[0004] For an IgG antibody to exhibit ADCC, CDC, or ADCP, the antibody Fc region must bind to an antibody receptor which is present on the surface of effector cells such as killer cells, natural killer cells, and activated macrophages (hereinafter denoted as FcγR) and various complement components. In humans, the FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb isoforms have been reported as the FcγR protein family, and the respective allotypes have also been reported (Non-patent Document 6).

[0005] Enhancement of cytotoxic effector functions such as ADCC, ADCP, and CDC is drawing attention as a promising means for enhancing the anti-tumor effects of antibodies. The importance of FcγR-mediated effector functions of antibodies for their antitumor effects has been reported using mouse models (Non-patent Documents 7 and 8). Furthermore, correlation was observed between clinical effects in humans and the high-affinity polymorphic allotype (V158) and the low-affinity polymorphic allotype (F158) of FcγRIIIa (Non-patent Document 9). These reports showed that antibodies having an Fc region that has been optimized for specific FcγR-binding mediate a stronger effector function, as a result demonstrate more effective antitumor effects.

[0006] The balance between the binding activities of an antibody towards an activating receptor consisted of FcγRIa, FcγRIIa, FcγRIIIa, and FcγRIIIb, and an inhibitory receptor consisted of FcγRIIb is an important element when optimizing antibody effector function. Use of an Fc region that enhances binding activity to activating receptors and decreases binding activity to inhibitory receptors may be able to confer antibodies with optimum effector functions (Non-patent Document 10). Conversely, use of an Fc region that has sustained or decreased binding activity to activating receptors and enhanced binding activity to inhibitory receptors may be able to confer immunosuppressive effect to antibodies (Non-patent Document 11). For the binding between an Fc region and FcγR, several amino acid residues in the antibody hinge region and CH2 domain, and the sugar chain added to Asn at position 297 (EU numbering) which is bound to the CH2 domain have been shown to be important for the binding between the Fc region and FcγR (Non-patent Documents 12, 13, and 14). There has been research on Fc region variants that have various FcγR-binding properties mainly at this binding site, and Fc region variants that have higher binding activities to activating FcγR have been obtained (Patent Documents 1 and 2). For example, Lazar et al. have successfully increased human FcγRIIIa (V158) binding approximately 370-fold by substituting Ser at position 239, Ala at position 330, and Ile at position 332 (EU numbering) of human IgG1 with Asp, Leu, and Glu, respectively (Non-patent Document 15 and Patent Document 2). The ratio of FcγRIIIa-binding to FcγIIb-binding (A/I ratio) of this variant is increased approximately 9-fold as compared to that of the wild type. Furthermore, Lazar et al. have successfully enhanced the binding to FcγIIb approximately 430-fold (Non-patent Document 16). Shinkawa et al. have successfully enhanced the FcγRIIIa-binding approximately 100-fold by removing fucose from the sugar chain added to Asn at position 297 (EU numbering) (Non-patent Document 17).

[0007] However, the functional modifications of antibody Fc regions reported so far have limitations, and there is a demand for better functional modifications of Fc regions.

• [0008] [Patent Document 1] WO 2000/042072
• [0009] [Patent Document 2] WO 2006/019447
• [0010] [Patent Document 3] WO 2009/041062
• [0011] [Patent Document 4] WO 2006/106905

• [0012] [Non-patent Document 1] Nature Biotechnology, 23, 1073-1078 (2005)
• [0013] [Non-patent Document 2] Eur. J. Pharm. Biopharm, 59(3), 389-96 (2005)
• [0014] [Non-patent Document 3] Chemical Immunology, 65, 88 (1997)
• [0015] [Non-patent Document 4] Cancer Res., 68, 8049-8057 (2008)
• [0016] [Non-patent Document 5] Blood, 113, 3735-3743 (2009)
• [0017] [Non-patent Document 6] Immunol. Lett. 82, 57-65 (2002)
• [0018] [Non-patent Document 7] Pro. Nat. Acad. Sci. 95: 652-656 (1998)
• [0019] [Non-patent Document 8] Nature Medicine, 6: 443-446 (2000)
• [0020] [Non-patent Document 9] Blood 99:754-758 (2002)
• [0021] [Non-patent Document 10] Science, 310, 1510-1512 (2005)
• [0022] [Non-patent Document 11] Science, 291, 484-486 (2001)
• [0023] [Non-patent Document 12] Chemical Immunology, 65, 88 (1997)
• [0024] [Non-patent Document 13] Eur. J. Immunol. 23, 1098 (1993)
• [0025] [Non-patent Document 14] Immunology, 86, 319 (1995)
• [0026] [Non-patent Document 15] Pro. Nat. Acad. Sci., 103, 4005-4010 (2006)
• [0027] [Non-patent Document 16] Mol. Immun. 45, 3926-3933 (2008)
• [0028] [Non-patent Document 17] J. Biol. Chem., 278, 3466-3473 (2003)

[0029] The present invention was achieved in view of the above circumstances. An objective of the present invention is to provide polypeptides with improved Fc region function over conventional homodimerized polypeptides having an Fc region; pharmaceutical compositions comprising the polypeptides; therapeutic agents or preventive agents for immunoinflammatory diseases comprising the pharmaceutical compositions; therapeutic agents or preventive agents for various types of cancers; and methods for producing them. Furthermore, an objective of the present invention is to provide methods of improving the Fc region function over conventional homodimerized polypeptides having an Fc region.

[0030] The present inventors performed dedicated research to solve the above-mentioned problems. As a result, the present inventors produced heterodimerized polypeptides having an Fc region consisting of two polypeptides with different amino acid sequences (a first polypeptide and a second polypeptide), and thus successfully produced a heterodimerized polypeptide comprising an Fc region with improved Fc region function over conventional homodimers in which the Fc region is composed of only the first polypeptide or only the second polypeptide. More specifically, the present invention provides [1] to [78] below:

[0031] [1] a polypeptide comprising an Fc region, wherein the polypeptide is characterized in that the Fc region is composed of a heterodimer comprising a first polypeptide and a second polypeptide, and wherein the polypeptide is characterized in that a function of the Fc region is altered compared to that of a polypeptide characterized in that the Fc region is composed of a homodimer comprising only the first polypeptide and compared to that of a polypeptide characterized in that the Fc region is composed of a homodimer comprising only the second polypeptide;
[2] the polypeptide of [1], wherein at least one or more amino acid mutation is introduced into the Fc region;
[3] the polypeptide of [1] or [2], wherein the amino acid mutation includes at least one amino acid mutation that improves the Fc region function when the mutation is introduced into only one of the Fc regions compared to when the mutation is introduced into the Fc regions of both the first polypeptide and the second polypeptide and when the mutation is not introduced;
[4] the polypeptide of [2] or [3], wherein at least one or more amino acid mutation is introduced into a CH2 domain of the Fc region;
[5] the polypeptide of [4], wherein at least one or more amino acid mutations are introduced into an amino acid site selected from the group consisting of
Ala at position 231 (EU numbering);
Pro at position 232 (EU numbering);
Glu at position 233 (EU numbering);
Leu at position 234 (EU numbering);
Leu at position 235 (EU numbering);
Gly at position 236 (EU numbering);
Gly at position 237 (EU numbering);
Pro at position 238 (EU numbering);
Ser at position 239 (EU numbering);
Val at position 240 (EU numbering);
Asp at position 265 (EU numbering);
Val at position 266 (EU numbering);
Ser at position 267 (EU numbering);
His at position 268 (EU numbering);
Glu at position 269 (EU numbering);
Asp at position 270 (EU numbering);
Pro at position 271 (EU numbering);
Gln at position 295 (EU numbering);
Tyr at position 296 (EU numbering);
Ser at position 298 (EU numbering);
Tyr at position 300 (EU numbering);
Ser at position 324 (EU numbering);
Asn at position 325 (EU numbering);
Lys at position 326 (EU numbering);
Ala at position 327 (EU numbering);
Leu at position 328 (EU numbering);
Pro at position 329 (EU numbering);
Ala at position 330 (EU numbering);
Pro at position 331 (EU numbering);
Ile at position 332 (EU numbering);
Glu at position 333 (EU numbering);
Lys at position 334 (EU numbering);
Thr at position 335 (EU numbering);
Ile at position 336 (EU numbering); and
Ser at position 337 (EU numbering) in the CH2 domain of the Fc region;
[6] the polypeptide of any one of [1] to [5], wherein the alteration of Fc region function is at least one or more alterations selected from the group consisting of enhancement of binding activity and improvement of selectivity of binding to an Fcγ receptor;
[7] the polypeptide of [6], wherein the alteration of Fc region function is enhancement of binding activity to an Fcγ receptor;
[8] the polypeptide of [7], wherein the Fcγ receptor is at least one or more receptors selected from the group consisting of FcγRIa, FcγRIIa R, FcγRIIa H, FcγRIIb, and FcγRIIIa;
[9] the polypeptide of [8], wherein the Fcγ receptor is FcγRIa;
[10] the polypeptide of [9], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region i of Tables 2-1 and 2-2 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[11] the polypeptide of [9], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region ii of Tables 2-1, 2-2, and 2-3 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[12] the polypeptide of [8], wherein the Fcγ receptor is FcγRIIa R;
[13] the polypeptide of [12], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region i of Tables 3-1 and 3-2 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[14] the polypeptide of [12], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region ii of Tables 3-1 and 3-2 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[15] the polypeptide of [8], wherein the Fcγ receptor is FcγRIIa H;
[16] the polypeptide of [15], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region i of Table 4 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[17] the polypeptide of [15], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region ii of Table 4 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[18] the polypeptide of [8], wherein the Fcγ receptor is FcγRIIb;
[19] the polypeptide of [18], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region i of Table 5 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[20] the polypeptide of [18], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region ii of Table 5 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[21] the polypeptide of [8], wherein the Fcγ receptor is FcγRIIIa;
[22] the polypeptide of [21], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region i of Table 6 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[23] the polypeptide of [21], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region ii of Table 6 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[24] the polypeptide of [6], wherein the alteration of Fc region function is improvement of selectivity of binding activity to an Fcγ receptor;
[25] the polypeptide of [24], wherein the improvement of selectivity of binding activity to an Fcγ receptor refers to selectivity between an activating Fcγ receptor and an inhibitory Fcγ receptor;
[26] the polypeptide of [25], wherein among the Fey receptors, the activating Fcγ receptor is at least one or more receptors selected from the group consisting of FcγRIa, FcγRIIa R, FcγRIIa H, and FcγRIIIa, and the inhibitory Fcγ receptor is FcγRIIb;
[27] the polypeptide of [26], wherein the activating Fcγ receptor is FcγRIa, and the inhibitory Fcγ receptor is FcγRIIb, which is characterized in that the FcγRIa-binding activity is selectively enhanced compared to the FcγRIIb-binding activity;
[28] the polypeptide of [27], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region a of Tables 19-1, 19-2, 19-3, and 19-4 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[29] the polypeptide of [27], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region b of Tables 19-1, 19-2, 19-3, 19-4, and 19-5 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[30] the polypeptide of [26], wherein the activating Fcγ receptor is FcγRIa, and the inhibitory Fcγ receptor is FcγRIIb, which is characterized in that the binding activity to FcγRIa is selectively decreased compared to the FcγRIIb-binding activity;
[31] the polypeptide of [30], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region c of Tables 23-1 and 23-2 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[32] the polypeptide of [30], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region d of Tables 23-1 and 23-2 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[33] the polypeptide of [26], wherein the activating Fcγ receptor is FcγRIIa R, and the inhibitory Fcγ receptor is FcγRIIb, which is characterized in that the binding activity to FcγRIIa R is selectively enhanced compared to the FcγRIIb-binding activity;
[34] the polypeptide of [33], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region a of Table 20-1 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[35] the polypeptide of [33], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region b of Tables 20-1, 20-2, and 20-3 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[36] the polypeptide of [26], wherein the activating Fcγ receptor is FcγRIIa R, and the inhibitory Fcγ receptor is FcγRIIb, which is characterized in that the FcγRIIa R-binding activity is selectively decreased compared to the FcγRIIb-binding activity;
[37] the polypeptide of [36], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region c of Table 24-1 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[38] the polypeptide of [36], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region d of Tables 24-1 and 24-2 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[39] the polypeptide of [26], wherein the activating Fcγ receptor is FcγRIIa H, and the inhibitory Fcγ receptor is FcγRIIb, which is characterized in that the FcγRIIa H-binding activity is selectively enhanced compared to the FcγRIIb-binding activity;
[40] the polypeptide of [39], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region a of Table 21-1 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[41] the polypeptide of [39], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region b of Tables 21-1, 21-2, and 21-3 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[42] the polypeptide of [26], wherein the activating Fcγ receptor is FcγRIIa H, and the inhibitory Fcγ receptor is FcγRIIb, which is characterized in that the FcγRIIa H-binding activity is selectively decreased compared to the FcγRIIb-binding activity;
[43] the polypeptide of [42], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region c of Tables 25-1 and 25-2 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[44] the polypeptide of [42], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region d of Tables 25-1, 25-2, and 25-3 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[45] the polypeptide of [26], wherein the activating Fcγ receptor is FcγRIIIa, and the inhibitory Fcγ receptor is FcγRIIb, which is characterized in that the FcγRIIIa-binding activity is selectively enhanced compared to the FcγRIIb-binding activity;
[46] the polypeptide of [45], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region a of Table 22-1 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[47] the polypeptide of [45], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region b of Tables 22-1, 22-2, and 22-3 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[48] the polypeptide of [26], wherein the activating Fcγ receptor is FcγRIIIa, and the inhibitory Fcγ receptor is FcγRIIb, which is characterized in that the FcγRIIIa-binding activity is selectively decreased compared to the FcγRIIb-binding activity;
[49] the polypeptide of [48], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region c of Tables 26-1 and 26-2 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[50] the polypeptide of [48], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region d of Tables 26-1, 26-2, 26-3, and 26-4 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[51] the polypeptide of [1], [21], or [45], wherein at least one or more amino acid mutations selected from the group consisting of
substitution of amino acid L at position 234 (EU numbering) with Y;
substitution of amino acid L at position 235 (EU numbering) with Y or Q;
substitution of amino acid G at position 236 (EU numbering) with W;
substitution of amino acid S at position 239 (EU numbering) with M;
substitution of amino acid H at position 268 (EU numbering) with D;
substitution of amino acid D at position 270 (EU numbering) with E;
substitution of amino acid S at position 298 (EU numbering) with A;
substitution of amino acid K at position 326 (EU numbering) with D;
substitution of amino acid A at position 327 (EU numbering) with D;
substitution of amino acid L at position 328 (EU numbering) with W;
substitution of amino acid A at position 330 (EU numbering) with M or K; and
substitution of amino acid K at position 334 (EU numbering) with E or L are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[52] the polypeptide of [1], [21], or [45], wherein a mutation is introduced into at least one amino acid selected from Leu at position 234, Leu at position 235, Gly at position 236, Ser at position 239, His at position 268, Asp at position 270, Ser at position 298, Lys at position 326, Ala at position 327, Leu at position 328, Ala at position 330, and Lys at position 334 (EU numbering) in the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
[53] the polypeptide of [1], [21], or [45], wherein a mutation is introduced into at least one amino acid selected from Leu at position 234, Leu at position 235, Gly at position 236, Ser at position 239, His at position 268, Asp at position 270, Ser at position 298, Ala at position 327, Leu at position 328, and Lys at position 334 (EU numbering) in the amino acid sequence of either polypeptide of the first polypeptide and the second polypeptide constituting the Fc region; and a mutation is introduced into at least one amino acid selected from Asp at position 270, Lys at position 326, Ala at position 330, and Lys at position 334 (EU numbering) in the amino acid sequence of the other polypeptide;
[54] the polypeptide of [1], [21], or [45], wherein at least one amino acid mutation selected from the group consisting of
substitution of amino acid L at position 234 (EU numbering) with Y;
substitution of amino acid L at position 235 (EU numbering) with Y or Q;
substitution of amino acid G at position 236 (EU numbering) with W;
substitution of amino acid S at position 239 (EU numbering) with M;
substitution of amino acid H at position 268 (EU numbering) with D;
substitution of amino acid D at position 270 (EU numbering) with E;
substitution of amino acid S at position 298 (EU numbering) with A;
substitution of amino acid A at position 327 (EU numbering) with D;
substitution of amino acid L at position 328 (EU numbering) with W; and
substitution of amino acid K at position 334 (EU numbering) with L
is introduced into the amino acid sequence of either polypeptide of the first polypeptide and the second polypeptide constituting the Fc region; and at least one amino acid mutation selected from the group consisting of
substitution of amino acid D at position 270 (EU numbering) with E;
substitution of amino acid K at position 326 (EU numbering) with D;
substitution of amino acid A at position 330 (EU numbering) with M or K; and
substitution of amino acid K at position 334 (EU numbering) with E
is introduced into the amino acid sequence of the other polypeptide;
[55] the polypeptide of [1], [21], or [45], wherein any one set of mutations of (i) to (vi) is introduced into the amino acid sequence of either polypeptide of the first polypeptide and the second polypeptide constituting the Fc region, and any one set of mutations of (vii) to (ix) is introduced into the amino acid sequence of the other polypeptide:

• • [0032] (i) substitution of amino acid L at position 234 (EU numbering) with Y;
    • [0033] substitution of amino acid L at position 235 (EU numbering) with Y;
    • [0034] substitution of amino acid G at position 236 (EU numbering) with W;
    • [0035] substitution of amino acid H at position 268 (EU numbering) with D; and
    • [0036] substitution of amino acid S at position 298 (EU numbering) with A;
  • [0037] (ii) substitution of amino acid L at position 234 (EU numbering) with Y;
    • [0038] substitution of amino acid L at position 235 (EU numbering) with Y;
    • [0039] substitution of amino acid G at position 236 (EU numbering) with W;
    • [0040] substitution of amino acid H at position 268 (EU numbering) with D;
    • [0041] substitution of amino acid D at position 270 (EU numbering) with E; and
    • [0042] substitution of amino acid S at position 298 (EU numbering) with A;
  • [0043] (iii) substitution of amino acid L at position 234 (EU numbering) with Y;
    • [0044] substitution of amino acid L at position 235 (EU numbering) with Q;
    • [0045] substitution of amino acid G at position 236 (EU numbering) with W;
    • [0046] substitution of amino acid S at position 239 (EU numbering) with M;
    • [0047] substitution of amino acid H at position 268 (EU numbering) with D;
    • [0048] substitution of amino acid D at position 270 (EU numbering) with E; and
    • [0049] substitution of amino acid S at position 298 (EU numbering) with A;
  • [0050] (iv) substitution of amino acid L at position 234 (EU numbering) with Y;
    • [0051] substitution of amino acid L at position 235 (EU numbering) with Y;
    • [0052] substitution of amino acid G at position 236 (EU numbering) with W;
    • [0053] substitution of amino acid H at position 268 (EU numbering) with D;
    • [0054] substitution of amino acid S at position 298 (EU numbering) with A; and
    • [0055] substitution of amino acid A at position 327 (EU numbering) with D;
  • [0056] (v) substitution of amino acid L at position 234 (EU numbering) with Y;
    • [0057] substitution of amino acid L at position 235 (EU numbering) with Y;
    • [0058] substitution of amino acid G at position 236 (EU numbering) with W;
    • [0059] substitution of amino acid S at position 239 (EU numbering) with M;
    • [0060] substitution of amino acid H at position 268 (EU numbering) with D;
    • [0061] substitution of amino acid S at position 298 (EU numbering) with A; and
    • [0062] substitution of amino acid A at position 327 (EU numbering) with D;
  • [0063] (vi) substitution of amino acid L at position 234 (EU numbering) with Y;
    • [0064] substitution of amino acid L at position 235 (EU numbering) with Y;
    • [0065] substitution of amino acid G at position 236 (EU numbering) with W;
    • [0066] substitution of amino acid S at position 239 (EU numbering) with M;
    • [0067] substitution of amino acid H at position 268 (EU numbering) with D;
    • [0068] substitution of amino acid S at position 298 (EU numbering) with A;
    • [0069] substitution of amino acid A at position 327 (EU numbering) with D;
    • [0070] substitution of amino acid L at position 328 (EU numbering) with W; and
    • [0071] substitution of amino acid K at position 334 (EU numbering) with L;
  • [0072] (vii) substitution of amino acid K at position 326 (EU numbering) with D;
    • [0073] substitution of amino acid A at position 330 (EU numbering) with M; and
    • [0074] substitution of amino acid K at position 334 (EU numbering) with E;
  • [0075] (viii) substitution of amino acid D at position 270 (EU numbering) with E;
    • [0076] substitution of amino acid K at position 326 (EU numbering) with D;
    • [0077] substitution of amino acid A at position 330 (EU numbering) with M; and
    • [0078] substitution of amino acid K at position 334 (EU numbering) with E;
  • [0079] (ix) substitution of amino acid D at position 270 (EU numbering) with E;
    • [0080] substitution of amino acid K at position 326 (EU numbering) with D;
    • [0081] substitution of amino acid A at position 330 (EU numbering) with K; and
    • [0082] substitution of amino acid K at position 334 (EU numbering) with E;
      [56] a polypeptide comprising an Fc region, wherein the polypeptide is characterized in that the Fc region is composed of a heterodimer comprising a first polypeptide and a second polypeptide, and wherein the polypeptide is characterized in that a function of the Fc region is altered compared to that of a polypeptide characterized in that the Fc region is composed of a homodimer comprising only the first polypeptide or compared to that of a polypeptide characterized in that the Fc region is composed of a homodimer comprising only the second polypeptide;
      [57] the polypeptide of [56], wherein the alteration of Fc region function is at least one or more alterations selected from the group consisting of enhancement of binding activity, reduction of binding, and improvement of binding selectivity of the polypeptide to an Fcγ receptor;
      [58] the polypeptide of [57], wherein the alteration of Fc region function is additionally an alteration that improves physicochemical stability;
      [59] the polypeptide of [58], wherein the alteration that improves physicochemical stability means that a polypeptide characterized in that the Fc region is composed of a heterodimer comprising a first polypeptide and a second polypeptide has a higher Tm than that of a polypeptide characterized in that the Fc region is composed of a homodimer comprising only the first polypeptide or that of a polypeptide characterized in that the Fc region is composed of a homodimer comprising only the second polypeptide;
      [60] the polypeptide of any one of [57] to [59], wherein the alteration of Fc region function is enhancement of binding activity to an Fcγ receptor, and the Fcγ receptor is at least one or more receptors selected from the group consisting of FcγRIa, FcγRIIa R, FcγRIIa H, FcγRIIb, and FcγRIIIa;
      [61] the polypeptide of [60], wherein the Fcγ receptor is FcγRIa;
      [62] the polypeptide of [61], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Tables 31-1, 31-2, and 31-3 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
      [63] the polypeptide of [60], wherein the Fcγ receptor is FcγRIIa R;
      [64] the polypeptide of [63], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Tables 32-1 and 32-2 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
      [65] the polypeptide of [60], wherein the Fcγ receptor is FcγRIIa H;
      [66] the polypeptide of [65], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Tables 33-1 and 33-2 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
      [67] the polypeptide of [60], wherein the Fcγ receptor is FcγRIIb;
      [68] the polypeptide of [67], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Tables 34-1 and 34-2 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
      [69] the polypeptide of [60], wherein the Fcγ receptor is FcγRIIIa;
      [70] the polypeptide of [69], wherein at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Tables 35-1 and 35-2 of this specification are introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region;
      [71] the polypeptide of any one of [1] to [70], wherein an amino acid alteration is additionally introduced to impart difference in isoelectric points between the first polypeptide and the second polypeptide;
      [72] the polypeptide of [71], wherein the amino acid alteration to confer difference in isoelectric points is characterized by the introduction of at least one amino acid mutation at an amino acid site selected from the group consisting of Gly at position 137, Gly at position 138, Thr at position 139, Lys at position 147, Ser at position 192, Leu at position 193, Gln at position 196, Tyr at position 198, Ile at position 199, Asn at position 203, Lys at position 214, Val at position 263, Glu at position 272, Lys at position 274, Tyr at position 278, Lys at position 288, Lys at position 290, Gly at position 316, Lys at position 317, Lys at position 320, Lys at position 324, Thr at position 335, Ser at position 337, Lys at position 340, Leu at position 358, Lys at position 360, Gln at position 362, Ser at position 364, Ser at position 383, Asn at position 384, Gly at position 385, Gln at position 386, Pro at position 387, Asn at position 390, Val at position 397, and Val at position 422 (EU numbering) in the amino acid sequence of the first polypeptide and/or the second polypeptide;
      [73] the polypeptide of [71], wherein the amino acid alteration to confer difference in isoelectric points is characterized by the introduction of a mutation to at least one amino acid selected from the group consisting of Gln at position 196, Ile at position 199, Val at position 263, Glu at position 272, Gly at position 316, Leu at position 358, Ser at position 364, Ser at position 383, Pro at position 387, and Val at position 397 (EU numbering) in the amino acid sequence of either polypeptide of the first polypeptide and the second polypeptide; and introduction of a mutation to at least one amino acid selected from the group consisting of Gly at position 137, Gly at position 138, Thr at position 139, Lys at position 147, Ser at position 192, Leu at position 193, Tyr at position 198, Ile at position 199, Asn at position 203, Lys at position 214, Lys at position 274, Tyr at position 278, Lys at position 288, Lys at position 290, Gly at position 316, Lys at position 317, Lys at position 320, Lys at position 324, Thr at position 335, Ser at position 337, Lys at position 340, Leu at position 358, Lys at position 360, Gln at position 362, Ser at position 383, Asn at position 384, Gly at position 385, Gln at position 386, Asn at position 390, and Val at position 422 (EU numbering) in the amino acid sequence of the other polypeptide;
      [74] the polypeptide of any one of [1] to [73], wherein the polypeptide is an antigen-binding molecule;
      [75] the polypeptide of [74], wherein the antigen-binding molecule is an antibody, a bispecific antibody, or an Fc fusion molecule such as a peptide Fc fusion protein or scaffold Fc fusion protein;
      [76] a pharmaceutical composition comprising the polypeptide of [74] or [75] and a medically acceptable carrier;
      [77] a method for altering the function of a polypeptide comprising an Fc region, which comprises the steps of heterodimerizing the Fc region by introducing an amino acid mutation into the first polypeptide and/or the second polypeptide constituting the Fc region, and introducing an amino acid mutation to alter the Fc region function compared to when the Fc region forms a homodimer; and
      [78] a method for producing a polypeptide comprising an Fc region, which comprises the steps of heterodimerizing the Fc region by introducing an amino acid mutation into the first polypeptide and/or the second polypeptide constituting the Fc region, and introducing an amino acid mutation to alter the Fc region function compared to when the Fc region forms a homodimer.

[0083] The present invention provides polypeptides that are suitable as pharmaceuticals, wherein their binding activities and physical properties (for example, stability and homogeneity) have been improved by altering the amino acid sequence of the antibody constant region.

[0138] The following definitions are provided to facilitate understanding of the present invention described herein.

[0139] The present invention provides polypeptides comprising an Fc region, wherein the polypeptide is characterized in that the Fc region is composed of a heterodimer comprising a first polypeptide and a second polypeptide, wherein the polypeptide is characterized in that a function of the Fc region is altered when compared to that of a polypeptide characterized in that the Fc region is composed of a homodimer comprising only the first polypeptide, and when compared to that of a polypeptide characterized in that the Fc region is composed of a homodimer comprising only the second polypeptide. Furthermore, the present invention provides methods for producing the polypeptide, and methods for altering functions of an Fc region-containing polypeptide.

[0140] In the present invention, the “polypeptide comprising an Fc region, wherein the polypeptide is characterized in that the Fc region is composed of a heterodimer comprising a first polypeptide and a second polypeptide” may be a polypeptide complex composed of a first polypeptide and a second polypeptide, as well as other multiple polypeptides.

[0141] Herein, “first polypeptide” and “second polypeptide” mean polypeptides constituting an antibody Fc region. The terms “first polypeptide” and “second polypeptide” means that their sequences are different from each other, and preferably at least the CH2 domain sequences are different. The polypeptides may be, for example, polypeptides that constitute the Fc region of a naturally-occurring IgG, or polypeptides produced by altering the polypeptides constituting the Fc region of a naturally-occurring IgG.

[0142] “Naturally-occurring IgGs” refers to polypeptides that belong to a class of antibodies practically encoded by immunoglobulin gamma genes and comprise an amino acid sequence identical to those of IgGs found in nature. For example, a naturally-occurring human IgG refers to a naturally-occurring human IgG1, naturally-occurring human IgG2, naturally-occurring human IgG3, naturally-occurring human IgG4, or such. Naturally-occurring IgGs also include mutants spontaneously produced from them.

[0143] “Polypeptides” of the present invention generally refers to peptides or proteins approximately ten amino acids or more in length. Furthermore, they are generally polypeptides derived from organisms, but are not particularly limited, and for example, they may be polypeptides comprising an artificially designed sequence. Furthermore, they may be any of naturally-occurring polypeptides, synthetic polypeptides, recombinant polypeptides, or such. Protein molecules of the present invention refer to molecules comprising the polypeptide.

[0144] Preferred examples of the polypeptides of the present invention include antibodies. More preferred examples include naturally-occurring IgGs, particularly naturally-occurring human IgGs. “Naturally-occurring IgGs” refers to polypeptides belonging to a class of antibodies practically encoded by immunoglobulin gamma genes and comprising an amino acid sequence identical to those of IgGs found in nature. For example, a naturally-occurring human IgG means a naturally-occurring human IgG1, naturally-occurring human IgG2, naturally-occurring human IgG3, naturally-occurring human IgG4, or such. Naturally-occurring IgGs also include mutants spontaneously produced from them.

[0145] While an IgK (Kappa, κ chain), IgL1, IgL2, IgL3, IgL6, and IgL7 (Lambda, λ chain)-type constant region is present in the antibody light chain constant region, it may be any light chain constant region. For the human IgK (Kappa) constant region and human IgL7 (Lambda) constant region, a plurality of allotype sequences due to genetic polymorphism are described in “Sequences of proteins of immunological interest”, NIH Publication No. 91-3242, and any of them may be used in the present invention. Furthermore, in the present invention, a light chain constant region may be a light chain constant region that has been altered with amino acid substitutions, additions, deletions, insertions, and/or modifications or such. For the antibody Fc region, for example, Fc regions of the IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM types exist. For example, a human IgG antibody Fc region can be used as the antibody Fc region of the present invention, and human IgG1 antibody Fc regions are preferred. Fc regions that can be used as an Fc region of the present invention are, for example, those derived from naturally-occurring IgG constant regions, or specifically, a constant region derived from naturally-occurring human IgG1 (SEQ ID NO: 76), a constant region derived from naturally-occurring human IgG2 (SEQ ID NO: 77), a constant region derived from naturally-occurring human IgG3 (SEQ ID NO: 78), and a constant region derived from naturally-occurring human IgG4 (SEQ ID NO: 79). FIG. 32 shows the constant region sequences of the naturally-occurring IgG1, IgG2, IgG3, and IgG4. Constant regions of naturally-occurring IgGs also include mutants spontaneously produced from them. For the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, a plurality of allotype sequences due to genetic polymorphism are described in “Sequences of proteins of immunological interest”, NIH Publication No. 91-3242, and any of them may be used in the present invention. In particular, for the human IgG1 sequence, the amino acid sequence at positions 356 to 358 (EU numbering) may be either DEL or EEM.

[0146] Furthermore, the strength of interaction between an antibody Fc region and FcγR has been reported to depend on Zn²⁺ ion concentration (Immunology Letters 143 (2012) 60-69). The antibody shows a stronger interaction between the Fc region and FcgR when the Zn²⁺ ion concentration of the Fc region is higher. Chelation of Zn²⁺ by His310 and His435 present in CH3 of the antibody Fc region opens up each CH2 domain of the Fc region at a distal position. This facilitates interaction between the CH2 domain and FcgR, and the interaction between the Fc region and FcgR is enhanced. A non-limiting embodiment of the Fc region of the present invention includes an Fc region in which His at position 310, His at position 435, His at position 433, and/or Asn at position 434 (EU numbering) is chelated with Zn²⁺.

[0147] In the present invention, “Fc region” refers to a region consisted of a hinge region or a part thereof, a CH2 domain, and a CH3 domain in an antibody molecule. According to EU numbering (herein, also called the EU INDEX), an IgG-class Fc region refers to, for example, a region from cysteine at position 226 to the C terminus, or from proline at position 230 to the C terminus, but is not limited thereto.

[0148] The Fc region may be obtained preferably by re-eluting fractions adsorbed onto a protein A column or protein G column after partially digesting IgG1, IgG2, IgG3, IgG4 monoclonal antibodies or such using a protease such as pepsin. The protease is not particularly limited as long as it can digest a full-length antibody so as to produce Fab and F(ab′)2 in a restrictive manner by appropriately setting the enzyme reaction conditions such as pH; and examples include pepsin and papain.

[0149] Herein, “heterodimerization” means constituting a single polypeptide from two polypeptides with different amino acid sequences, and “heterodimer” means a polypeptide composed of two polypeptides with different amino acid sequences. Furthermore, “homodimerization” means constituting a single polypeptide from two polypeptides having the same amino acid sequences, and “homodimer” means a polypeptide composed of two polypeptides having the same amino acid sequences; or a polypeptide composed of polypeptides having the same amino acid sequences excluding alterations made for the purpose of efficient heterodimerization or alterations made for the purpose of efficient purification of heterodimers; or a polypeptide composed of polypeptides comprising the same amino acids excluding alterations that have not been made for the purpose of improving Fc function. In the present invention, “heterodimer” or “homodimer” preferably means “heterodimerization” or “homodimerization” for the Fc region, or preferably means “heterodimerization” or “homodimerization” for CH2 in the Fc region. Furthermore, “parent polypeptide” means a polypeptide before introduction of alterations such as amino acid mutations.

[0150] The amino acid mutation of the present invention may be used alone or in combination with multiple amino acid mutations.

[0151] When multiple amino acid mutations are used in combination, the number of mutations combined is not particularly limited, and can be set appropriately within a range that can achieve the objectives of the invention, and examples include two or more to 30 or less, and preferably two or more to 15 or less.

[0152] When multiple amino acid mutations are combined, the amino acid mutations may be introduced into only one of the two polypeptides constituting the Fc region, or they may be appropriately distributed to both of the two polypeptides.

[0153] Furthermore, in the present invention, to achieve a higher function-modifying effect in the Fc region, it is preferable to introduce at least one amino acid mutation that improves the Fc region function when the mutation is introduced into only one of the polypeptides, compared to when no mutation is introduced and when the mutation is introduced into both Fc regions of the two polypeptides.

[0154] The site to be altered is not particularly limited as long as it is in an Fc region, and it can be appropriately set within a range that can achieve the objectives of the present invention; and examples include the hinge region, CH2 region, and CH3 region.

[0155] More preferably, the site to be altered is a CH2 domain. CH2 domain refers to positions 231 to 340 (EU numbering), and CH3 domain refers to positions 341 to 447 (EU numbering). For example, when introducing mutations into the amino acid sequence of a constant region derived from human IgG1, amino acid residues at one or more positions selected from among 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, and 447 (EU numbering) may be altered.

[0156] More specifically, when introducing alterations into the amino acid sequence of a human IgG1 constant region, amino acid residues at one or more positions selected from among 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, and 447 (EU numbering) may be altered.

[0157] More specifically, when introducing alterations into the amino acid sequence of a human IgG1 constant region, amino acid residues at one or more positions selected from among 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, and 340 (EU numbering) may be altered.

[0158] More specifically, when introducing alterations into the amino acid sequence of a human IgG1 constant region, amino acid residues at one or more positions selected from among 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 265, 266, 267, 268, 269, 270, 271, 295, 296, 298, 300, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, and 337 (EU numbering) may be altered.

[0159] In the present invention, amino acid alteration means any of substitution, deletion, addition, insertion, and modification, or a combination thereof. In the present invention, amino acid alteration may be rephrased as amino acid mutation, and they are used synonymously.

Substitution

[0160] When substituting amino acid residues, substitution to a different amino acid residue is carried out with the objective of altering aspects such as (a)-(c) described below:

(a) polypeptide backbone structure in the sheet-structure or helical-structure region;

(b) electric charge or hydrophobicity at the target site; or

(c) size of the side chain.

[0161] Amino acid residues are classified into the following groups based on their general side chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, and ile;

(2) neutral hydrophilic: cys, ser, thr, asn, and gln;

(3) acidic: asp and glu;

(4) basic: his, lys, and arg;

(5) residues that affect the chain orientation: gly and pro; and

(6) aromatic: tip, tyr, and phe.

[0162] Substitution between amino acid residues within each of these amino acid groups is referred to as conservative substitution, and amino acid residue substitution between different groups is referred to as non-conservative substitution.

[0163] Substitutions in the present invention may be conservative substitutions or non-conservative substitutions, or a combination of conservative substitutions and non-conservative substitutions.

[0164] In addition to the amino acid mutations introduced based on the present invention, polypeptides of the present invention may further comprise additional alterations. For example, additional alterations may be selected from any of amino acid substitutions, deletions, and modifications, or combinations thereof.

[0165] For example, polypeptides of the present invention can be arbitrarily altered within a range that does not practically change the intended function of the polypeptide. When the polypeptide of the present invention is an antibody, its heavy chain and light chain can be altered. For example, such mutations can be performed through conservative substitution of amino acid residues. Furthermore, even if an alteration changes the intended function of a polypeptide of the present invention, the alteration may also be carried out as long as the change in the function is in a range within the objectives of the present invention.

[0166] Amino acid sequence alteration of the present invention includes post-translational modification. A specific post-translational modification may be addition or deletion of a sugar chain. For example, in the IgG1 constant region, the amino acid residue at position 297 (EU numbering) may be sugar chain-modified. The sugar-chain structure for the modification is not limited. Generally, antibodies expressed in eukaryotic cells comprise glycosylation in the constant region. Therefore, antibodies expressed in cells such as those below are normally modified by some type of sugar chain:

• • [0167] antibody-producing cells of mammals
  • [0168] eukaryotic cells transformed with an expression vector comprising a DNA encoding an antibody

[0169] Eukaryotic cells shown here include yeast and animal cells. For example, CHO cells and HEK293H cells are representative animal cells used in transformation with an expression vector comprising an antibody-encoding DNA. On the other hand, those without glycosylation at this site are also included in the antibody of the present invention. Antibodies whose constant region is not glycosylated can be obtained by expressing an antibody-encoding gene in prokaryotic cells such as Escherichia coli.

[0170] In the present invention, additional alterations specifically include, for example, addition of sialic acid to the sugar chain of an Fc region (MAbs. 2010 September-October; 2(5): 519-27).

[0171] When the polypeptide of the present invention is an antibody, for example, amino acid substitutions that improve FcRn-binding activity (J. Immunol. 2006 Jan. 1; 176(1): 346-56; J Biol Chem. 2006 Aug. 18; 281(33): 23514-24; Int. Immunol. 2006 December; 18(12): 1759-69; Nat Biotechnol. 2010 February; 28(2): 157-9; WO/2006/019447; WO/2006/053301; and WO/2009/086320), and amino acid substitutions for improving antibody heterogeneity or stability (WO/2009/041613) may be introduced into an antibody constant region portion.

[0172] To produce a heterodimerized polypeptide of the present invention, it is required that polypeptides having amino acids that differ from each other are associated, or a heterodimerized polypeptide of interest is separated from other homodimerized polypeptides.

[0173] For association of polypeptides having different amino acids from each another and comprising an Fc region, a technique of suppressing unintended association between H chains by introducing electrostatic repulsion into the interface of the second constant region of the antibody H chain (CH2) or the third constant region of the H chain (CH3) (WO 2006/106905) can be applied.

[0174] In the technology of suppressing unintended association between H chains by introducing electrostatic repulsion into the interface of CH2 or CH3, examples of amino acid residues in contact at the interface of other constant regions of the H chain include the residue at position 356 (EU numbering), the residue at position 439 (EU numbering), the region facing the residue at position 357 (EU numbering), the residue at position 370 (EU numbering), the residue at position 399 (EU numbering), and the residue at position 409 (EU numbering) in the CH3 domain.

[0175] More specifically, for example, in an antibody containing two types of H chain CH3 domains, the antibody in which one to three pairs of amino acid residues selected from the amino acid residues shown below in (1) to (3) in the first H chain CH3 domain have the same type of charge can be produced:

• [0176] (1) amino acid residues at positions 356 and 439 (EU numbering) which are amino acid residues contained in the H chain CH3 domain;
• [0177] (2) amino acid residues at positions 357 and 370 (EU numbering) which are amino acid residues contained in the H chain CH3 domain; and
• [0178] (3) amino acid residues at positions 399 and 409 (EU numbering) which are amino acid residues contained in the H chain CH3 domain.

[0179] Furthermore, an antibody can be produced in which one to three pairs of amino acid residues corresponding to the amino acid residue pairs indicated above in (1) to (3) having the same type of charge in the first H chain CH3 domain have charges opposite to the corresponding amino acid residues in the aforementioned first H chain CH3 domain, wherein the amino acid residue pairs are selected from the amino acid residue pairs indicated above in (1) to (3) in the second H chain CH3 domain which differs from the first H chain CH3 domain.

[0180] The respective amino acid residues of (1) to (3) mentioned above are positioned close to each other when associated. Those skilled in the art can find sites that correspond to the above-mentioned amino acid residues of (1) to (3) by homology modeling and such using commercially available software for the desired H chain CH3 domain or H chain constant region, and amino acid residues of these sites can be altered when appropriate.

[0181] In the above-mentioned antibodies, for example, “charged amino acid residues” are preferably selected from amino acid residues included in either of groups (X) or (Y) below:

(X) glutamic acid (E) and aspartic acid (D); and

(Y) lysine (K), arginine (R), and histidine (H).

[0182] In the above-mentioned antibodies, the phrase “having the same type of charge” means that, for example, all of the two or more amino acid residues are amino acid residues included in either of the above-mentioned groups (X) and (Y). The phrase “having the opposite charge” means that, for example, when at least one of the two or more amino acid residues is an amino acid residue included in either one of the above-mentioned groups (X) and (Y), the remaining amino acid residues are amino acid residues included in the other group.

[0183] In a preferred embodiment of the above-mentioned antibody, the first H chain CH3 domain and the second H chain CH3 domain may be cross-linked by disulfide bonds.

[0184] In the present invention, the amino acid residues to be altered are not limited to amino acid residues of the antibody constant region or antibody variable region described above. Those skilled in the art can find amino acid residues that form the interface in polypeptide mutants or heteromultimers through homology modeling and such using commercially available software, and can alter the amino acid residues at those sites to regulate association.

[0185] Other known technologies can also be used for the association of polypeptides of the present invention having different amino acids and comprising an Fc region. Polypeptides having different amino acids and comprising an Fc region can be efficiently associated with each other by substituting an amino acid side chain present in one of the H chain variable regions of the antibody with a larger side chain (knob), and substituting an amino acid side chain present in the opposing variable region of the other H chain with a smaller side chain (hole), to allow placement of the knob within the hole (WO 1996/027011; and Ridgway J B et al., Protein Engineering (1996) 9, 617-621; Merchant A M et al. Nature Biotechnology (1998) 16, 677-681).

[0186] In addition, other known technologies can also be used for the association of polypeptides having different amino acids and comprising an Fc region. Association of polypeptides having different sequences can be induced efficiently by complementary association of CH3, by using a strand-exchange engineered domain CH3 produced by changing part of the CH3 in one of the H chains of an antibody into an IgA-derived sequence corresponding to that portion, and introducing a corresponding IgA-derived sequence into the complementary portion of the CH3 on the other H chain (Protein Engineering Design & Selection, 23; 195-202, 2010). This known technology can also be used to efficiently induce association of polypeptides having different amino acids and comprising an Fc region.

[0187] In addition, one can also use technologies for heterodimerized antibody production using association of antibody CH1 and CL, and association of VH and VL, which are described in WO 2011/028952.

[0188] Furthermore, even in cases where heterodimerized polypeptides cannot be formed efficiently, heterodimerized polypeptides can be obtained by separating and purifying them from homodimerized polypeptides. When producing a heterodimerized polypeptide consisting of a first polypeptide and a second polypeptide which have different sequences from each other, homodimerized polypeptides consisting of only two first polypeptides, and homodimerized polypeptide consisting of only two second polypeptide are mixed in as impurities. Known technologies can be used as a method for efficiently removing these two types of homodimerized polypeptides. A method has been reported to be able to purify two types of homodimers and the heterodimerized antibody of interest by ion exchange chromatography, by creating a difference in isoelectric points by introducing amino acid substitutions into the variable regions of the two types of H chains (WO 2007114325). To date, as a method for purifying heterodimerized antibodies, a method using Protein A has been reported to purify a heterodimerized antibody comprising a mouse IgG2a H chain that binds to Protein A and a rat IgG2b H chain that does not bind to Protein A (WO 98050431 and WO 95033844).

[0189] Furthermore, a heterodimerized antibody alone can be efficiently purified by using H chains in which amino acid residues at the IgG-Protein A binding site, positions 435 and 436 (EU numbering), are substituted with amino acids yielding different Protein A affinities such as Tyr or His to change interaction of each of the H chains with Protein A, and using a Protein A column.

[0190] A plurality of these substitutions and technologies, for example, two or more of them can be used in combination. Furthermore, these alterations can be made separately to the first polypeptide and the second polypeptide when necessary. Polypeptides of the present invention may also be those produced based on the products of the above-mentioned alterations.

[0191] Amino acid sequence can be altered by various methods known to those skilled in the art. Such methods include the site-directed mutagenesis method (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis. Gene 152: 271-275; Zoller, M J, and Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 100: 468-500; Kramer, W, Drutsa, V, Jansen, H W, Kramer, B, Pflugfelder, M, and Fritz, H J (1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12: 9441-9456; Kramer W, and Fritz H J (1987) Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154, 350-367; and Kunkel, T A (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA. 82: 488-492), the PCR mutation method, and the cassette mutation method, but are not limited thereto.

[0192] In the present invention, the phrase “Fc region function” refers to, for example, Fcγ receptor-binding activity of an Fc region (enhancement of binding activity or reduction of binding activity), selectivity of an Fc region for Fcγ receptor isoform (improvement of binding selectivity), physicochemical stability of an Fc region, ADCC activity, and ADCP activity. Here, Fcγ receptor isoform selectivity of an Fc region means selective binding to specific isoforms of Fcγ receptor. Physicochemical stability of an Fc region means, for example, an Fc region's thermal stability, stability to protease, stability to chemical treatment, stability to freeze thawing, storage stability, stability under acidic conditions, photostability, stability to shaking- or concentration-associated stress, or maintenance of solubility in a wide range of solution conditions. Furthermore, an Fc region function may refer to a function combining two or more functions from Fcγ receptor-binding activity of an Fc region, Fcγ receptor isoform selectivity of an Fc region, and physicochemical stability of an Fc region; and this means, for example, a function combining Fcγ receptor-binding activity of an Fc region and Fcγ receptor isoform selectivity of an Fc region, a function combining Fcγ receptor-binding activity of an Fc region and physicochemical stability of an Fc region, a function combining Fcγ receptor isoform selectivity of an Fc region and physicochemical stability of the Fc region, and a function combining Fcγ receptor-binding activity of an Fc region, Fcγ receptor isoform selectivity of an Fc region, and physicochemical stability of an Fc region.

[0193] In the present invention, “alteration of Fc region function” means, for example, enhancement, reduction, or such of Fcγ receptor-binding activity of an Fc region when the Fc region function refers to the Fcγ receptor-binding activity of the Fc region. Improvement of selectivity means, for example, enhancing binding activity to an Fcγ receptor and at the same time maintaining or lowering binding activities to other Fcγ receptors. Alternatively, improvement of selectivity means, for example, lowering binding activity to certain Fcγ receptors while maintaining or enhancing binding activities to other Fcγ receptors. Furthermore, for example, when the Fc region function refers to Fcγ receptor subtype selectivity of an Fc region, “alteration of Fc region function” means improving or lowering the Fcγ receptor subtype selectivity of the Fc region. Alternatively, for example, when the Fc region function refers to physicochemical stability of an Fc region, “alteration of Fc region function” means improving or lowering the physicochemical stability of the Fc region, suppressing a decrease in stability, or such; and more specifically it means, for example, improving or lowering the Tm value of the CH2 domain, suppressing a decrease in the Tm value, or such.

[0194] For example, improvement of combined functions of Fcγ receptor-binding activity of an Fc region, Fcγ receptor isoform selectivity of an Fc region, and physicochemical stability of an Fc region does not necessarily have to be improvement in every one of the Fcγ receptor-binding activity of an Fc region, the Fcγ receptor isoform selectivity of an Fc region, and the physicochemical stability of an Fc region when compared to a control, and the improvement is acceptable as long as the Fc region function is improved as a whole. Conversely, for example, lowering of combined functions of the Fcγ receptor-binding activity of an Fc region, the Fcγ receptor isoform selectivity of an Fc region, and the physicochemical stability of an Fc region does not necessarily have to be decrease in every one of the Fcγ receptor-binding activity of an Fc region, the Fcγ receptor isoform selectivity of an Fc region, and the physicochemical stability of an Fc region when compared to a control, and the decrease is acceptable as long as the Fc region function is lowered as a whole.

[0195] In the present invention, “Fcγ receptors” (herein, referred to as Fcγ receptors, FcγR, or FcgR) refers to receptors that may bind to the Fc region of IgG1, IgG2, IgG3, and IgG4, and practically means any member of the family of proteins encoded by the Fcγ receptor genes. In humans, this family includes FcγRI (CD64) including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32) including isoforms FcγRIIa (including allotypes H131 (type H) and R131 (type R)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16) including isoforms FcγRIIIa (including allotypes V158 and F158), and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), and any human FcγRs, FcγR isoforms or allotypes yet to be discovered, but is not limited thereto. The FcγRs include human, mouse, rat, rabbit, and monkey-derived FcγRs but is not limited thereto, and may be derived from any organism. Mouse FcγRs include FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2 or FcγRIV), and any mouse FcγRs, or FcγR isoforms or allotypes yet to be discovered, but are not limited thereto. Favorable examples of such Fcγ receptors include human FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16), and/or FcγRIIIb (CD16).

[0196] For FcγRs, there are activating receptors carrying the immunoreceptor tyrosine-based activation motif (ITAM) and inhibitory receptor carrying the immunoreceptor tyrosine-based inhibitory motif (ITIM). FcγRs are classified into activating FcγRs: FcγRI, FcγRIIa R, FcγRII H, FcγRIIIa, and FcγRIIIb, and inhibitory FcγR: FcγRIIb.

[0197] The polynucleotide sequence and amino acid sequence of FcγRI are set forth in NM_000566.3 and NP_000557.1, respectively;

the polynucleotide sequence and amino acid sequence of FcγRIIa are set forth in BC020823.1 and AAH20823.1, respectively;

the polynucleotide sequence and amino acid sequence of FcγRIIb are set forth in BC146678.1 and AAI46679.1, respectively;

the polynucleotide sequence and amino acid sequence of FcγRIIIa are set forth in BC033678.1 and AAH33678.1, respectively; and

the polynucleotide sequence and amino acid sequence of FcγRIIIb are set forth in BC128562.1 and AAI28563.1, respectively (the RefSeq Registration number).

[0198] In FcγRIIa, there are two allotypes, one where the amino acid at position 131 of FcγRIIa is histidine (type H) and the other where this amino acid is substituted with arginine (type R) (J. Exp. Med, 172: 19-25, 1990). In FcγRIIb, there are two allotypes, one where the amino acid at position 232 of FcγRIIb is isoleucine (type I) and the other where this amino acid is substituted with threonine (type T) (Arthritis. Rheum. 46: 1242-1254 (2002)). In FcγRIIIa, there are two allotypes, one where the amino acid at position 158 of FcγRIIIa is valine (type V) and the other where this amino acid is substituted with phenylalanine (type F) (J. Clin. Invest. 100(5): 1059-1070 (1997)). Furthermore, in FcγRIIIb, there are two allotypes, the NA1 type and the NA2 type (J. Clin. Invest. 85: 1287-1295 (1990)).

[0199] One of the substances (the ligand) in observation of an interaction is immobilized onto a gold thin film on a sensor chip, and by shining light from the reverse side of the sensor chip so that total reflection takes place at the interface between the gold thin film and glass, a portion of reduced reflection intensity is formed in part of the reflected light (SPR signal). When the other one of the substances (the analyte) in observation of an interaction is made to flow on the sensor chip surface and the ligand binds to the analyte, the mass of the immobilized ligand molecule increases and the refractive index of the solvent on the sensor chip surface changes. The position of the SPR signal shifts as a result of this change in refractive index (on the other hand, the signal position returns when this binding dissociates). The Biacore system indicates the amount of shift mentioned above, or more specifically the time variable of mass by plotting the change in mass on the sensor chip surface on the ordinate as the measurement data (sensorgram). The amount of analyte bound to the ligand captured on the sensor chip surface is determined from the sensorgram (amount of change in the response on the sensorgram before and after interaction with the analyte). However, since the amount of binding also depends on the amount of ligand, it is necessary to make the comparison under conditions where the amount of ligand is considered to be practically the same. Kinetic parameters such as association rate constants (ka) and dissociation rate constants (kd) are determined from the curve of the sensorgram, and the affinities (KD) are determined from the ratio of these constants. In the BIACORE method, a method for measuring inhibition is preferably used. An example of the method for measuring inhibition is described in Proc. Natl. Acad. Sci USA (2006) 103 (11), 4005-4010.

[0200] In the present invention, whether or not the binding activity towards each type of Fcγ receptor is enhanced or decreased in a polypeptide or in an Fc region of the present invention can be determined, for example, by using Biacore (GE Healthcare) which is an interaction analyzer that utilizes the surface plasmon resonance (SPR) phenomena, as shown in the Examples. Biacore includes any models such as Biacore T100, T200, X100, A100, 4000, 3000, 2000, 1000, and C. For the sensor chip, any sensorchips for Biacore such as CM7, CM5, CM4, CM3, C1, SA, NTA, L1, HPA, and Au chip can be used. For the running buffer, besides HBE-EP+, a buffer produced by using HEPES, phosphoric acid, ACES, Tris, citric acid, and such to adjust the pH to a near neutral pH such as 7.4 can be used. The measurements can be performed in the range of 4° C. to 37° C. A protein for capturing antibodies such as Protein A, Protein G, or Protein L, which captures an antibody, anti-human IgG antibody, anti-human IgG-Fab, anti-human L chain antibody, anti-human Fc antibody, antigen protein, or antigen peptide is immobilized onto a sensor chip by coupling methods such as amine coupling, disulfide coupling, or aldehyde coupling. Various types of Fcγ receptors such as Fcγ receptor I, IIa R type, Ha H type, IIb, IIIc F type, V type, and IIIb are applied as analytes, and interactions were measured to obtain sensorgrams. At this point, measurements can be carried out by adjusting the Fcγ receptor concentration to a level in the range of several uM to several pM according to the strength of interaction such as KD of the sample to be measured. The dissociation constant (KD) is obtained based on the measurement, and by observing whether the KD value is decreased or increased, one can determine whether the binding activity of an Fc region or a polypeptide of the present invention to various Fcγ receptors is increased or decreased. When capturing is carried out by the antibody-capturing protein immobilized onto the sensor chip, the level of change in the sensorgram values before and after applying various Fcγ receptors as analytes for the antibodies on the sensor chip is used as an indicator; and according to the degree of the value increase, one can determine whether the binding activity of the polypeptide or Fc region of the present invention to various Fcγ receptors is increased or decreased. Alternatively, it is also possible to immobilize various Fcγ receptors, instead of antibodies, onto the sensor chips, and make them interact with an antibody sample to be evaluated. Whether the binding activity of the Fc region or the polypeptide of the present invention to the various Fcγ receptors is enhanced or decreased can be determined from the decrease or increase in KD values calculated from the interaction sensorgrams, or from the degree of increase in the sensorgrams before and after allowing the antibody sample to react.

[0201] Specifically, the binding activity of an Fc region towards an Fcγ receptor can be measured by the Amplified Luminescent Proximity Homogeneous Assay (ALPHA) screening, the BIACORE method which utilizes the surface plasmon resonance (SPR) phenomena, or such, in addition to ELISA or fluorescence activated cell sorting (FACS) (Proc. Natl. Acad. Sci. USA (2006) 103 (11): 4005-4010).

[0202] ALPHA screening is performed by ALPHA technology which uses two beads, a donor and an acceptor, based on the following principles. Luminescent signals are detected only when molecules bound to donor beads physically interact with molecules bound to the acceptor beads, and the two beads are in close proximity to each other. Laser-excited photosensitizer in the donor beads converts ambient oxygen to excited-state singlet oxygen. Singlet oxygen is dispersed around the donor beads, and when it reaches the adjacent acceptor beads, chemiluminescent reaction is induced in the beads, and light is ultimately emitted. When the molecules bound to the donor beads do not interact with the molecules bound to the acceptor beads, the chemiluminescent reaction does not take place because singlet oxygen produced by the donor beads does not reach the acceptor beads.

[0203] For example, a biotinylated test polypeptide is bound to streptavidin on the donor beads, and Fcγ receptor tagged with glutathione S transferase (GST) is linked to the acceptor beads. In the absence of a competing polypeptide, the test polypeptide interacts with the Fcγ receptor and produces 520-620 nm signals. An untagged polypeptide competes with the test polypeptide for interaction with the Fcγ receptor. Relative binding activities can be determined by quantifying the decrease in fluorescence observed as a result of the competition. Biotinylation of polypeptide using Sulfo-NHS-biotin and such is well known. The method of expressing the Fcγ receptor and GST in a cell carrying a fusion gene produced by fusing a polynucleotide encoding the Fcγ receptor in frame with a polynucleotide encoding GST in an expressible vector, and performing purification using a glutathione column is appropriately adopted as a method for tagging an Fcγ receptor with GST. The obtained signals are preferably analyzed, for example, by fitting them to a one-site competition model which uses a non-linear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).

[0204] Tagging is not limited to the use of GST, and any tag such as histidine tag, MBP, CBP, Flag tag, HA tag, V5 tag, and c-myc tag, may be used. Furthermore, the binding of a test polypeptide to donor beads is not limited to binding using the biotin-streptavidin reaction. In particular, when a test polypeptide is an antibody or peptide carrying Fc such as an Fc fusion polypeptide, a method of binding a test polypeptide through an Fc-recognizing protein such as Protein A or Protein G on the donor beads can be considered.

[0205] Reduction of binding to FcγR or FcγR-binding activity refers to binding to FcγR with a substantially weaker binding activity than that of the parent polypeptide when assays are performed using practically the same amount of polypeptides to be compared.

[0206] Heterodimerized polypeptides with attenuated, decreased, or lowered binding to FcγR or FcγR-binding activity refers to those that bind to FcγR with a substantially weaker binding activity than that of homodimerized polypeptides when assays are performed using practically the same amount of polypeptides to be compared.

[0207] For example, in the KD values measured by the above-mentioned measurement methods, the KD value ratio (KD value of the parent polypeptide/KD value of the mutated polypeptide) is preferably 0.99 or less, 0.95 or less, 0.9 or less, 0.8 or less, 0.7 or less, 0.5 or less, 0.3 or less, and 0.1 or less, and more preferably, 0.08 or less, 0.05 or less, 0.02 or less, 0.01 or less, and 0.001 or less. Herein, the KD value ratio is also called the KD ratio.

[0208] The KD values measured by the above-mentioned measurement methods are preferably increased by 1 μM or more, and more preferably increased by 10 μM, 100 μM, 1 nM or more, 2 nM or more, 3 nM or more, 5 nM or more, 10 nM or more, 20 nM or more, 50 nM or more, 100 nM or more, or 1 μM or more. The KD values measured by the above-mentioned measurement methods are preferably 1 μM or more, and preferably 10 μM or more, 100 μM or more, 1 nM or more, 10 nM or more, 100 nM or more, 500 nM or more, 1 μM or more, 3 μM or more, or 5 μM or more.

[0209] Enhancement, increase, or improvement of binding to FcγR or FcγR-binding activity refers to binding to FcγR with a substantially stronger binding activity than that of the parent polypeptide when assays are performed using practically the same amount of polypeptides to be compared.

[0210] Heterodimerized polypeptides with enhanced, increased, or improved binding to FcγR or FcγR-binding activity refers to those that bind to FcγR with a substantially stronger binding activity than that of homodimerized polypeptides when assays are performed using practically the same amount of polypeptides to be compared.

[0211] For example, in the KD values measured by the above-mentioned measurement methods, the KD value ratio (KD value of the parent polypeptide/KD value of the mutated polypeptide) is preferably 1.1 or more, 1.2 or more, 1.3 or more, 1.5 or more, 1.8 or more, 2 or more, or 3 or more, and more preferably, 5 or more, 10 or more, 100 or more, 250 or more, or 1000 or more. Herein, the KD value ratio is also called KD ratio.

[0212] The KD values measured by the above-mentioned measurement methods are preferably decreased by 1 μM or more, and more preferably decreased by 10 μM, 100 μM, 1 nM or more, 2 nM or more, 3 nM or more, 5 nM or more, 10 nM or more, 20 nM or more, 50 nM or more, 100 nM or more, or 1 μM or more.

[0213] The KD values measured by the above-mentioned measurement methods are preferably 5 μM or less, and more preferably, 3 μM or less, 1 μM or less, 0.5 μM or less, 0.1 μM or less, 0.01 μM or less, 1 nM or less, 0.1 nM or less, 0.001 nM or less, or 1 μM or less.

[0214] In the present invention, when alteration of Fc region function of the polypeptide is enhancement of binding activity to an Fcγ receptor, amino acid mutations may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the aforementioned Fc region. The type and range of the amino acid mutation to be introduced are not particularly limited.

[0215] The strength of interaction between an antibody Fc region and FcγR has been reported to depend on the Zn²⁺ ion concentration (Immunology Letters 143 (2012) 60-69). The antibody shows a stronger interaction between the Fc region and FcgR when the Zn²⁺ ion concentration of the Fc region is higher. Chelation of Zn²⁺ by His310 and His435 present in CH3 of the antibody Fc region opens up each CH2 domain of the Fc region at a distal position. This facilitates interaction between the CH2 domain and FcgR, and the interaction between the Fc region and FcgR is enhanced. A non-limiting embodiment of the antibody of the present invention includes an Fc region in which His at position 310, His at position 435, His at position 433, and/or Asn at position 434 (EU numbering) is chelated with Zn²⁺.

[0216] When the Fcγ receptor is FcγRIa, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region i of Tables 2-1 and 2-2 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region. Furthermore, when the Fcγ receptor is FcγRIa, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region ii of Tables 2-1, 2-2, and 2-3 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region.

[0217] When the Fcγ receptor is FcγRIIa R, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region i of Tables 3-1 and 3-2 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region. Furthermore, when the Fcγ receptor is FcγRIIa R, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region ii of Tables 3-1 and 3-2 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region.

[0218] When the Fcγ receptor is FcγRIIa H, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region i of Table 4 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region. Furthermore, when the Fcγ receptor is FcγRIIa H, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region ii of Table 4 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region.

[0219] When the Fcγ receptor is FcγRIIb, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region i of Table 5 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region. Furthermore, when the Fcγ receptor is FcγRIIb, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region ii of Table 5 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region.

[0220] When the Fcγ receptor is FcγRIIIa, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region i of Table 6 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region. Furthermore, when the Fcγ receptor is FcγRIIIa, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region ii of Table 6 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region. Furthermore, when the Fcγ receptor is FcγRIIIa, more specifically, at least one or more (for example, two or three) amino acid mutations selected from the group consisting of substitution of amino acid L at position 234 (EU numbering) with Y; substitution of amino acid L at position 235 (EU numbering) with Y or Q; substitution of amino acid G at position 236 (EU numbering) with W; substitution of amino acid S at position 239 (EU numbering) with M; substitution of amino acid H at position 268 (EU numbering) with D; substitution of amino acid D at position 270 (EU numbering) with E; substitution of amino acid S at position 298 (EU numbering) with A; substitution of amino acid K at position 326 (EU numbering) with D; substitution of amino acid A at position 327 (EU numbering) with D; substitution of amino acid L at position 328 (EU numbering) with W; substitution of amino acid A at position 330 (EU numbering) with M or K; and substitution of amino acid K at position 334 (EU numbering) with E or L may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region. Even more specifically, when the Fcγ receptor is FcγRIIIa, at least one or more (for example, two or three) amino acid mutations selected from the group consisting of substitution of amino acid S at position 239 (EU numbering) with D; substitution of amino acid A at position 330 (EU numbering) with L; and substitution of amino acid I at position 332 (EU numbering) with E may be introduced into the amino acid sequence of either one of the polypeptides constituting the Fc region, the first polypeptide or the second polypeptide, and at least one or more (for example, two or three) amino acid mutations selected from the group consisting of substitution of amino acid L at position 234 (EU numbering) with Y; substitution of amino acid G at position 236 (EU numbering) with W; and substitution of amino acid S at position 298 (EU numbering) with A may be introduced into the amino acid sequence of the other polypeptide.

[0221] Even more specifically, when the Fcγ receptor is FcγRIIIa, a mutation may be introduced into at least one or more (for example, two or three) amino acids selected from among Leu at position 234, Leu at position 235, Gly at position 236, Ser at position 239, His at position 268, Asp at position 270, Ser at position 298, Ala at position 327, Leu at position 328, and Lys at position 334 (EU numbering) in the amino acid sequence of either one of the polypeptides constituting the Fc region, the first polypeptide or the second polypeptide; and a mutation may be introduced into at least one or more (for example, two or three) amino acids selected from among Asp at position 270, Lys at position 326, Ala at position 330, and Lys at position 334 (EU numbering) in the amino acid sequence of the other polypeptide.

[0222] The amino acid to be altered may be selected appropriately, and preferably at least one or more (for example, two or three) amino acid mutations selected from the group consisting of:

substitution of amino acid L at position 234 (EU numbering) with Y;

substitution of amino acid L at position 235 (EU numbering) with Y or Q;

substitution of amino acid G at position 236 (EU numbering) with W;

substitution of amino acid S at position 239 (EU numbering) with M;

substitution of amino acid H at position 268 (EU numbering) with D;

substitution of amino acid D at position 270 (EU numbering) with E;

substitution of amino acid S at position 298 (EU numbering) with A;

substitution of amino acid A at position 327 (EU numbering) with D;

substitution of amino acid L at position 328 (EU numbering) with W; and

[0223] substitution of amino acid K at position 334 (EU numbering) with L may be introduced into the amino acid sequence of either one of the polypeptides constituting the Fc region, the first polypeptide or the second polypeptide; and at least one or more (for example, two or three) amino acid mutations selected from the group consisting of:
substitution of amino acid D at position 270 (EU numbering) with E;
substitution of amino acid K at position 326 (EU numbering) with D;
substitution of amino acid A at position 330 (EU numbering) with M or K; and
substitution of amino acid K at position 334 (EU numbering) with E may be introduced into the amino acid sequence of the other polypeptide.

[0224] More preferably, any one set of mutations of (i) to (vi) may be introduced into the amino acid sequence of either one of the polypeptides constituting the Fc region, the first polypeptide or the second polypeptide; and any one set of mutations of (vii) to (ix) may be introduced into the amino acid sequence of the other polypeptide:

(i) substitution of amino acid L at position 234 (EU numbering) with Y;

• • [0225] substitution of amino acid L at position 235 (EU numbering) with Y;
  • [0226] substitution of amino acid G at position 236 (EU numbering) with W;
  • [0227] substitution of amino acid H at position 268 (EU numbering) with D; and
  • [0228] substitution of amino acid S at position 298 (EU numbering) with A;
    (ii) substitution of amino acid L at position 234 (EU numbering) with Y;
  • [0229] substitution of amino acid L at position 235 (EU numbering) with Y;
  • [0230] substitution of amino acid G at position 236 (EU numbering) with W;
  • [0231] substitution of amino acid H at position 268 (EU numbering) with D;
  • [0232] substitution of amino acid D at position 270 (EU numbering) with E; and
  • [0233] substitution of amino acid S at position 298 (EU numbering) with A;
    (iii) substitution of amino acid L at position 234 (EU numbering) with Y;
  • [0234] substitution of amino acid L at position 235 (EU numbering) with Q;
  • [0235] substitution of amino acid G at position 236 (EU numbering) with W;
  • [0236] substitution of amino acid S at position 239 (EU numbering) with M;
  • [0237] substitution of amino acid H at position 268 (EU numbering) with D;
  • [0238] substitution of amino acid D at position 270 (EU numbering) with E; and
  • [0239] substitution of amino acid S at position 298 (EU numbering) with A;
    (iv) substitution of amino acid L at position 234 (EU numbering) with Y;
  • [0240] substitution of amino acid L at position 235 (EU numbering) with Y;
  • [0241] substitution of amino acid G at position 236 (EU numbering) with W;
  • [0242] substitution of amino acid H at position 268 (EU numbering) with D;
  • [0243] substitution of amino acid S at position 298 (EU numbering) with A; and
  • [0244] substitution of amino acid A at position 327 (EU numbering) with D;
    (v) substitution of amino acid L at position 234 (EU numbering) with Y;
  • [0245] substitution of amino acid L at position 235 (EU numbering) with Y;
  • [0246] substitution of amino acid G at position 236 (EU numbering) with W;
  • [0247] substitution of amino acid S at position 239 (EU numbering) with M;
  • [0248] substitution of amino acid H at position 268 (EU numbering) with D;
  • [0249] substitution of amino acid S at position 298 (EU numbering) with A; and
  • [0250] substitution of amino acid A at position 327 (EU numbering) with D;
    (vi) substitution of amino acid L at position 234 (EU numbering) with Y;
  • [0251] substitution of amino acid L at position 235 (EU numbering) with Y;
  • [0252] substitution of amino acid G at position 236 (EU numbering) with W;
  • [0253] substitution of amino acid S at position 239 (EU numbering) with M;
  • [0254] substitution of amino acid H at position 268 (EU numbering) with D;
  • [0255] substitution of amino acid S at position 298 (EU numbering) with A;
  • [0256] substitution of amino acid A at position 327 (EU numbering) with D;
  • [0257] substitution of amino acid L at position 328 (EU numbering) with W; and
  • [0258] substitution of amino acid K at position 334 (EU numbering) with L;
    (vii) substitution of amino acid K at position 326 (EU numbering) with D;
  • [0259] substitution of amino acid A at position 330 (EU numbering) with M; and
  • [0260] substitution of amino acid K at position 334 (EU numbering) with E;
    (viii) substitution of amino acid D at position 270 (EU numbering) with E;
  • [0261] substitution of amino acid K at position 326 (EU numbering) with D;
  • [0262] substitution of amino acid A at position 330 (EU numbering) with M; and
  • [0263] substitution of amino acid K at position 334 (EU numbering) with E;
    (ix) substitution of amino acid D at position 270 (EU numbering) with E;
  • [0264] substitution of amino acid K at position 326 (EU numbering) with D;
  • [0265] substitution of amino acid A at position 330 (EU numbering) with K; and
  • [0266] substitution of amino acid K at position 334 (EU numbering) with E.

[0267] Selectivity of binding activity can be determined by measuring the binding activities of the polypeptide towards the respective Fcγ receptor isoforms, and then determining their ratios. For example, the amount of binding and KD value towards an FcγR can be used as an indicator of binding activity.

[0268] Herein, “improvement of selectivity of binding activity” means that, for example, the ratio of binding activities of a test polypeptide to Fcγ receptor isoforms (binding activity of the test polypeptide to a first Fcγ receptor isoform/binding activity of the test polypeptide to the second Fcγ receptor isoform) is increased by 0.1 or more, or preferably 0.2 or more, 0.5 or more, 1 or more, 2 or more, 3 or more, 5 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 30 or more, 50 or more, 70 or more, 100 or more, 150 or more, 200 or more, 500 or more, or 1000 or more when compared to the ratio of binding activities of a parent polypeptide of the test polypeptide to the Fcγ receptor isoforms (binding activity of the parent polypeptide of the test polypeptide to the first Fcγ receptor isoform/binding activity of the parent polypeptide of the test polypeptide to the second Fcγ receptor isoform) determined based on the above-mentioned measurement method. Furthermore, decreased Fcγ receptor isoform selectivity means that, for example, the ratio of binding activities of a test polypeptide to Fcγ receptor isoforms is reduced by 0.1 or more, or preferably 0.2 or more, 0.5 or more, 1 or more, 2 or more, 3 or more, 5 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 30 or more, 50 or more, 70 or more, 100 or more, 150 or more, 200 or more, 500 or more, or 1000 or more when compared to the ratio of binding activities of the parent polypeptide of the test polypeptide to the Fcγ receptor isoforms determined based on the above-mentioned measurement method.

[0269] Herein, as an indicator of selectivity, for example, the A/I ratio which shows the ratio of binding activities towards activating FcγR and inhibitory FcγR can also be used. The values obtained by dividing the KD of the test polypeptide for FcγRIIb by the KD of the test polypeptide for FcγRIIa H type or R type were used as the respective A/I ratios. The A/I ratio is preferably 1.1 or more, 1.5 or more, 2 or more, 3 or more, or 5 or more, and more preferably 6 or more, 8 or more, or 9 or more.

[0270] Herein, as an indicator of selectivity, for example, the FcγRIIIa F/FcγRIIb ratio which is a value obtained by dividing the KD for FcγRIIb by the KD for FcγRIIIa F can be used. The values obtained by dividing the KD of the test polypeptide for FcγRIIb by the KD of the test polypeptide for FcγRIIIa were defined as the respective FcγRIIIa F/FcγRIIb ratios. The FcγRIIIa F/FcγRIIb ratio is preferably 1.1 or more, 1.5 or more, 2 or more, 3 or more, or 5 or more, and more preferably 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 200 or more, 210 or more, 220 or more, 230 or more, or 240 or more.

[0271] In the present invention, when the alteration of Fc region function of the polypeptide is the improvement of selectivity of binding activity to an Fey receptor, an amino acid mutation may be introduced into the amino acid sequence of the first polypeptide and/or the second polypeptide constituting the Fc region. The type and range of the amino acid mutation to be introduced is not particularly limited.

[0272] In a case where the activating Fey receptor is FcγRIa, the inhibitory Fey receptor is FcγRIIb, and the improvement of selectivity is selective enhancement of binding activity to FcγRIa than to FcγRIIb, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region a of Tables 19-1, 19-2, 19-3, and 19-4 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region. Furthermore, when the improvement of selectivity is selective enhancement of binding activity to FcγRIa than to FcγRIIb, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region b of Tables 19-1, 19-2, 19-3, 19-4, and 19-5 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region.

[0273] In a case where the activating Fey receptor is FcγRIa, the inhibitory Fey receptor is FcγRIIb, and the improvement of selectivity is selective reduction of binding activity to FcγRIa than to FcγRIIb, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region c of Tables 23-1 and 23-2 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region. Furthermore, when the improvement of selectivity is selective reduction of binding activity to FcγRIa than to FcγRIIb, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region d of Tables 23-1 and 23-2 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region.

[0274] In a case where the activating Fey receptor is FcγRIIa R, the inhibitory Fey receptor is FcγRIIb, and the improvement of selectivity is selective enhancement of binding activity to FcγRIIa R than to FcγRIIb, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region a of Table 20-1 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region. Furthermore, when the improvement of selectivity is selective enhancement of binding activity to FcγRIIa R than to FcγRIIb, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region b of Tables 20-1, 20-2, and 20-3 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region.

[0275] In a case where the activating Fcγ receptor is FcγRIIa R, the inhibitory Fcγ receptor is FcγRIIb, and the improvement of selectivity is selective reduction of binding activity to FcγRIIa R than to FcγRIIb, at least one amino acid mutations selected from the group consisting of the amino acid mutations described in Region c of Table 24-1 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region. Furthermore, when the improvement of selectivity is selective reduction of binding activity to FcγRIIa R than to FcγRIIb, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region d of Tables 24-1 and 24-2 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region.

[0276] In a case where the activating Fcγ receptor is FcγRIIa H, the inhibitory Fcγ receptor is FcγRIIb, and the improvement of selectivity is selective enhancement of binding activity to FcγRIIa H than to FcγRIIb, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region a of Table 21-1 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region. Furthermore, when the improvement of selectivity is selective enhancement of binding activity to FcγRIIa H than to FcγRIIb, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region b of Tables 21-1, 21-2, and 21-3 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region.

[0277] In a case where the activating Fcγ receptor is FcγRIIa H, the inhibitory Fcγ receptor is FcγRIIb, and the improvement of selectivity is selective reduction of binding activity to FcγRIIa H than to FcγRIIb, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region c of Tables 25-1 and 25-2 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region. Furthermore, when the improvement of selectivity is selective reduction of binding activity to FcγRIIa H than to FcγRIIb, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region d of Tables 25-1, 25-2, and 25-3 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region.

[0278] In a case where the activating Fcγ receptor is FcγRIIIa, the inhibitory Fcγ receptor is FcγRIIb, and the improvement of selectivity is selective enhancement of binding activity to FcγRIIIa than to FcγRIIb, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region a of Table 22-1 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region. Furthermore, when the improvement of selectivity is selective enhancement of binding activity to FcγRIIIa than to FcγRIIb, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region b of Tables 22-1, 22-2, and 22-3 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region.

[0279] In a case where the activating Fcγ receptor is FcγRIIIa, the inhibitory Fcγ receptor is FcγRIIb, and the improvement of selectivity is selective reduction of binding activity to FcγRIIIa than to FcγRIIb, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region c of Tables 26-1 and 26-2 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region. Furthermore, when the improvement of selectivity is selective reduction of binding activity to FcγRIIIa than to FcγRIIb, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Region d of Tables 26-1, 26-2, 26-3, and 26-4 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region.

[0280] Herein, the selective enhancement of binding activity to a desired Fcγ receptor means any of the following cases:

• [0281] (i) binding activity to a desired Fcγ receptor is enhanced, and binding activities to receptors other than the desired Fcγ receptor are unchanged or decreased;
• [0282] (ii) binding activity to a desired Fcγ receptor is enhanced, and binding activities to receptors other than the desired Fcγ receptor are also enhanced, but the degree of enhancement of binding activity to receptors other than the desired Fcγ receptor is less than the degree of enhancement of binding activity to the desired Fcγ receptor; or
• [0283] (iii) binding activity to a desired Fcγ receptor is decreased, but the degree of reduction of binding activity is less than the degree of reduction of binding activities to Fcγ receptors other than the desired Fcγ receptor.

[0284] Furthermore, selective reduction of binding activity to a desired Fcγ receptor means any of the following cases:

• [0285] (i) binding activity to a desired Fcγ receptor is decreased, and binding activities to receptors other than the desired Fcγ receptor are unchanged or enhanced;
• [0286] (ii) binding activity to a desired Fcγ receptor is decreased, and binding activities to receptors other than the desired Fcγ receptor are also decreased, but the degree of reduction of binding activity to receptors other than the desired Fcγ receptor is less than the degree of reduction of binding activity to the desired Fcγ receptor; or
• [0287] (iii) binding activity to a desired Fcγ receptor is enhanced, but the degree of enhancement of binding activity is less than the degree of enhancement of binding activities to Fcγ receptors other than the desired Fcγ receptor.

[0288] Herein, physicochemical stability of a polypeptide means, for example, thermodynamic stability of a polypeptide, which can be determined using, for example, the Tm value of the CH2 domain as an indicator. Tm values can be measured by circular dichroism (CD), differential scanning calorimeter (DSC), or differential scanning fluorimetry (DSF).

[0289] The change in mean residue molar ellipticity (θ) that accompanies a rise in temperature is measured by CD to calculate the Tm value. The measuring instrument includes, for example, a circular dichroism dispersion meter (JASCO Corporation). When CD spectra are measured at a suitable wavelength (for example, 208 nm or 222 nm) while increasing the temperature, θ increases at a certain temperature, and becomes a constant value at higher temperatures. The temperature at which a midpoint value between low-temperature θ and high-temperature θ is taken as Tm. For measurement, it is possible to use, for example, a protein solution prepared using citric acid, Tris, phosphate solution, or such, at a concentration of several hundred ug/mL.

[0290] DSC measures the change in calorie that accompanies a rise in temperature to calculate the Tm value. The measuring instrument includes MicroCal VP-DSC and Micro Cal Capillary DSC (both from DKSH Japan). A protein solution and a buffer are filled in measurement cells, and when temperature differences among the cells are measured while raising the temperature, a change to endothermic reaction is observed starting at a certain temperature. This temperature is taken to be Tm. For measurement, it is possible to use, for example, a protein solution prepared using citrate buffer, TBS, PBS, histidine buffer, or such at a concentration of several ten ug/mL to several hundred ug/mL.

[0291] DSF detects exposure of hydrophobic residues that accompanies a rise in temperature by using a fluorescent reagent (for example, SYPRO Orange) that specifically binds to hydrophobic residues to calculate the Tm value. A protein solution and a fluorescence reagent are mixed at appropriate ratios, and when fluorescence intensities are measured while raising the temperature using an RT-PCR instrument, increase in fluorescence intensity is observed at a certain temperature. This temperature is taken to be Tm. Examples of the measuring instrument include Rotor-Gene Q (QIAGEN), and CFX96 real-time PCR analysis system (Bio-Rad). For measurement, it is possible to use, for example, a protein solution prepared using PBS, histidine buffer, or such at a concentration of several ten ug/mL to several hundred ug/mL.

[0292] Herein, improved physicochemical stability of a polypeptide means that, for example, the Tm value of the CH2 domain in the Fc region of a test polypeptide determined based on the above-mentioned measurement method is increased by 0.1 degrees or more, preferably 0.2 degrees or more, 0.3 degrees or more, 0.4 degrees or more, 0.5 degrees or more, 1 degree or more, 2 degrees or more, 3 degrees or more, 4 degrees or more, 5 degrees or more, or 10 degrees or more compared to the Tm value of the CH2 domain in the Fc region of a control polypeptide. Furthermore, improved physical stability of a polypeptide refers to suppressed reduction of physical stability of a polypeptide; and for example, reduction of the Tm value of the CH2 domain in the Fc region of a test polypeptide is suppressed relative to the Tm value of the CH2 domain in the Fc region of a control polypeptide by 0.1 degrees or more, preferably 0.2 degrees or more, 0.3 degrees or more, 0.4 degrees or more, 0.5 degrees or more, 1 degree or more, 2 degrees or more, 3 degrees or more, 4 degrees or more, 5 degrees or more, or 10 degrees or more, as determined based on the above-mentioned measurement method.

[0293] Herein, “reduction of physical stability of a polypeptide” means that the Tm value of the CH2 domain in the Fc region of a test polypeptide determined based on the above-mentioned measurement method is decreased by 0.1 degrees or more, preferably 0.2 degrees or more, 0.3 degrees or more, 0.4 degrees or more, 0.5 degrees or more, 1 degree or more, 2 degrees or more, 3 degrees or more, 4 degrees or more, 5 degrees or more, or 10 degrees or more compared to the Tm value of CH2 domain in the Fc region of a control peptide.

[0294] The present invention also comprises a polypeptide comprising an Fc region, wherein the polypeptide is characterized in that the Fc region is composed of a heterodimer comprising a first polypeptide and a second polypeptide, and wherein a function of the Fc region is altered compared to that of a polypeptide characterized in that the Fc region is composed of a homodimer comprising only the first polypeptide or when compared to that of a polypeptide characterized in that the Fc region is composed of a homodimer comprising only the second polypeptide.

[0295] In the polypeptide, the alteration of Fc region function may be an alteration that further improves physicochemical stability, in addition to at least one or more alterations selected from the group consisting of enhancement of binding activity, reduction of binding, and improvement of selectivity of binding activity of the polypeptide to an Fcγ receptor; and as long as any of these functions is altered, it can be said that the Fc region function of the present invention is altered.

[0296] In the present invention, the phrase “when amino acid mutations are introduced into the Fc region in both the first polypeptide and the second polypeptide, the Fc region function is not altered” means that when the same amino acid mutations are introduced into both the first polypeptide and the second polypeptide, the desired function is not improved. For example, it means that when one intends to enhance the binding activity of a polypeptide to an Fcγ receptor, the binding activity does not change or is decreased; when one intends to reduce the binding activity, the binding activity does not change or is enhanced; when one intends to improve selectivity of the binding activity, the selectivity is not improved; and when one intends to improve physicochemical stability of the polypeptide, the stability does not change or is decreased. Regarding the amino acid mutation, the phrase “when it is introduced into only one of the Fc regions, the Fc region function is altered” means that the desired function is improved when the amino acid mutation is introduced only into either the first polypeptide or the second polypeptide. For example, it means that when one intends to enhance the binding activity of a polypeptide to an Fcγ receptor, the binding activity is enhanced; when one intends to reduce the binding activity, the binding activity is decreased; when one intends to improve selectivity of binding activity, the selectivity is improved; and when one intends to improve physicochemical stability of a polypeptide, the stability is improved.

[0297] The present invention also includes a polypeptide which is characterized in that the Fc region is composed of a heterodimer comprising a first polypeptide and a second polypeptide, and which is characterized in having a higher Tm than that of a polypeptide characterized in that the Fc region is composed of a homodimer comprising only the first polypeptide or that of a polypeptide characterized in that the Fc region is composed of a homodimer comprising only the second polypeptide. Along with an alteration that improves physicochemical stability, i.e., having a high Tm, the polypeptide may also have additional alterations to the Fc region function.

[0298] In a case where the additional alteration of Fc region function is enhancement of FcγRIa-binding activity, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Tables 31-1, 31-2, and 31-3 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region.

[0299] In a case where the additional alteration of Fc region function is enhancement of FcγRIIa R-binding activity, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Tables 32-1 and 32-2 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region.

[0300] In a case where the additional alteration of Fc region function is enhancement of FcγRIIa H-binding activity, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Tables 33-1 and 33-2 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region.

[0301] In a case where the additional alteration of Fc region function is enhancement of FcγRIIb-binding activity, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Tables 34-1 and 34-2 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region.

[0302] In a case where the additional alteration of Fc region function is enhancement of FcγRIIIa-binding activity, at least one or more amino acid mutations selected from the group consisting of the amino acid mutations described in Tables 35-1 and 35-2 herein may be introduced into the amino acid sequences of the first polypeptide and/or the second polypeptide constituting the Fc region.

[0303] In the present invention, the combination of the first polypeptide and the second polypeptide into which amino acid mutations are introduced is not particularly limited, and examples include combinations of different types/or the same type of polypeptides selected from the polypeptides described in SEQ ID NOs: 2 to 4 and 6 to 60. In addition, the preferred examples include a combination of polypeptides comprising the first polypeptide and the second polypeptide described in the Examples herein (a combination of the H chains of two antibodies and the L chain of a single antibody).

[0304] The polypeptide of the present invention may be an antigen-binding molecule. In the present invention, while the antibody-binding molecule is not particularly limited in type, preferred examples include an antibody, a bispecific antibody, or an Fc fusion molecule such as a peptide Fc fusion protein or a scaffold Fc fusion protein.

<Antibody>

[0305] Furthermore, an antibody is provided as a polypeptide of the present invention.

[0306] The term “antibody/antibodies” in the present invention is used in the broadest sense, and as long as the desired biological activity is shown, it comprises any antibody such as monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody variants, antibody fragments, polyspecific antibodies (for example, bispecific antibodies), chimeric antibodies, and humanized antibodies.

[0307] Regarding the antibodies of the present invention, the antigen type and antibody origin are not limited, and they may be any type of antibodies. The origin of the antibodies is not particularly limited, but examples include human antibodies, mouse antibodies, rat antibodies, and rabbit antibodies.

[0308] Methods for producing the antibodies are well known to those skilled in the art, and for example, monoclonal antibodies may be produced by the hybridoma method (Kohler and Milstein, Nature 256: 495 (1975)), or the recombination method (U.S. Pat. No. 4,816,567). Alternatively, they may be isolated from a phage antibody library (Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1991)). Alternatively, they may be isolated from a single B cell clone (N. Biotechnol. 28(5): 253-457 (2011)).

[0309] A humanized antibody is also called a reshaped human antibody. Specifically, humanized antibodies prepared by grafting the CDRs of a non-human animal antibody such as a mouse antibody to a human antibody and such are known. Common genetic engineering technologies for obtaining humanized antibodies are also known. Specifically, for example, overlap extension PCR is known as a method for grafting mouse antibody CDRs to human FRs.

[0310] A vector for expressing a humanized antibody can be produced by inserting a DNA encoding an antibody variable region in which three CDRs and four FRs are ligated and a DNA encoding a human antibody constant region into an expression vector so that these DNAs are fused in frame. After this integration vector is transfected into a host to establish recombinant cells, these cells are cultured, and the DNA encoding the humanized antibody is expressed to produce the humanized antibody in the culture of the cells (see, European Patent Publication No. EP 239,400, and International Patent Publication No. WO 1996/002576).

[0311] As necessary, an amino acid residue in an FR may be substituted so that the CDRs of a reshaped human antibody form an appropriate antigen-binding site. For example, a mutation can be introduced into the amino acid sequence of an FR by applying the PCR method used for grafting mouse CDRs to human FRs.

[0312] A desired human antibody can be obtained by DNA immunization using a transgenic animal having the complete repertoire of human antibody genes (see International Publication Nos. WO 1993/012227, WO 1992/003918, WO 1994/002602, WO 1994/025585, WO 1996/034096, and WO 1996/033735) as an animal for immunization.

[0313] Furthermore, technologies for obtaining a human antibody by panning using a human antibody library are known. For example, a human antibody V region is expressed on the surface of a phage as a single-chain antibody (scFv) by the phage display method. The scFv-expressing phage that binds to the antigen can be selected. The DNA sequence that encodes the V region of the antigen-bound human antibody can be determined by analyzing the genes of the selected phage. After determining the DNA sequence of the scFv that binds to the antigen, an expression vector can be prepared by fusing the V-region sequence in-frame with the sequence of a desired human antibody C region, and then inserting this into a suitable expression vector. The expression vector is introduced into suitable expression cells such as those described above, and the human antibody can be obtained by expressing the human antibody-encoding gene. These methods are already known (see, International Publication Nos. WO 1992/001047, WO 1992/020791, WO 1993/006213, WO 1993/011236, WO 1993/019172, WO 1995/001438, and WO 1995/15388).

[0314] Variable regions constituting the antibodies of the present invention can be variable regions that recognize any antigen.

[0315] Herein, there is no particular limitation on the antigen, and it may be any antigens. Examples of the antigen include 17-IA, 4-1 BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 Adenosine Receptor, A33, ACE, ACE-2, Activin, Activin A, Activin AB, Activin B, Activin C, Activin RIA, Activin RIA ALK-2, Activin RIB ALK-4, Activin RITA, Activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAMS, ADAMS, ADAMTS, ADAMTS4, ADAMTS5, Addressins, adiponectin, ADP ribosyl cyclase-1, aFGF, AGE, ALCAM, ALK, ALK-1, ALK-7, allergen, alpha1-antichemotrypsin, alpha1-antitrypsin, alpha-synuclein, alpha-V/beta-1 antagonist, aminin, amylin, amyloid beta, amyloid immunoglobulin heavy chain variable region. amyloid immunoglobulin light chain variable region, Androgen, ANG, angiotensinogen, Angiopoietin ligand-2, antithrombinIII, Anthrax, APAF-1, APE, APJ, apo A1, apo serum amyloid A, Apo-SAA, APP, APRIL, AR, ARC, ART, Artemin, ASPARTIC, Atrial natriuretic factor, Atrial natriuretic peptide, atrial natriuretic peptides A, atrial natriuretic peptides B, atrial natriuretic peptides C, av/b3 integrin, Ax1, B7-1, B7-2, B7-H, BACE, BACE-1, Bacillus anthracis protective antigen, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, BcI, BCMA, BDNF, b-ECGF, beta-2-microglobulin, betalactamase, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, B-lymphocyte Stimulator (BIyS), BMP, BMP-2 (BMP-2a), BMP-3 (Osteogenin), BMP-4 (BMP-2b), BMP-5, BMP-6 (Vgr-1), BMP-7 (OP-1), BMP-8 (BMP-8a), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BMPR-II (BRK-3), BMPs, BOK, Bombesin, Bone-derived neurotrophic factor, bovine growth hormone, BPDE, BPDE-DNA, BRK-2, BTC, B-lymphocyte cell adhesion molecule, C10, C1-inhibitor, C1q, C3, C3a, C4, C5, C5a (complement 5a), CA125, CAD-8, Cadherin-3, Calcitonin, cAMP, Carbonic anhydrase-IX, carcinoembryonic antigen (CEA), carcinoma-associated antigen, Cardiotrophin-1, Cathepsin A, Cathepsin B, Cathepsin C/DPPI, Cathepsin D, Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin O, Cathepsin S, Cathepsin V, Cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1/1-309, CCL11/Eotaxin, CCL12/MCP-5, CCL13/MCP-4, CCL14/HCC-1, CCL15/HCC-2, CCL16/HCC-4, CCL17/TARC, CCL18/PARC, CCL19/ELC, CCL2/MCP-1, CCL20/MIP-3-alpha, CCL21/SLC, CCL22/MDC, CCL23/MPIF-1, CCL24/Eotaxin-2, CCL25/TECK, CCL26/Eotaxin-3, CCL27/CTACK, CCL28/MEC, CCL3/M1P-1-alpha, CCL3L1/LD-78-beta, CCL4/MIP-1-beta, CCL5/RANTES, CCL6/C10, CCL7/MCP-3, CCL8/MCP-2, CCL9/10/MTP-1-gamma, CCR, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD10, CD105, CD11a, CD11b, CD11c, CD123, CD13, CD137, CD138, CD14, CD140a, CD146, CD147, CD148, CD15, CD152, CD16, CD164, CD18, CD19, CD2, CD20, CD21, CD22, CD23, CD25, CD26, CD27L, CD28, CD29, CD3, CD30, CD30L, CD32, CD33 (p67 proteins), CD34, CD37, CD38, CD3E, CD4, CD40, CD40L, CD44, CD45, CD46, CD49a, CD49b, CD5, CD51, CD52, CD54, CD55, CD56, CD6, CD61, CD64, CD66e, CD7, CD70, CD74, CD8, CD80 (B7-1), CD89, CD95, CD105, CD158a, CEA, CEACAM5, CFTR, cGMP, CGRP receptor, CINC, CKb8-1, Claudin18, CLC, Clostridium botulinum toxin, Clostridium difficile toxin, Clostridium perfringens toxin, c-Met, CMV, CMV UL, CNTF, CNTN-1, complement factor 3 (C3), complement factor D, corticosteroid-binding globulin, Colony stimulating factor-1 receptor, COX, C-Ret, CRG-2, CRTH2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1/Fractalkine, CX3CR1, CXCL, CXCL1/Gro-alpha, CXCL10, CXCL11/1-TAC, CXCL12/SDF-1-alpha/beta, CXCL13/BCA-1, CXCL14/BRAK, CXCL15/Lungkine. CXCL16, CXCL16, CXCL2/Gro-beta CXCL3/Gro-gamma, CXCL3, CXCL4/PF4, CXCL5/ENA-78, CXCL6/GCP-2, CXCL7/NAP-2, CXCL8/IL-8, CXCL9/Mig, CXCL10/IP-10, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cystatin C, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Decay accelerating factor, Delta-like protein ligand 4, des(1-3)-IGF-1 (brain IGF-1), Dhh, DHICA oxidase, Dickkopf-1, digoxin, Dipeptidyl peptidase IV, DK1, DNAM-1, Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EGF like domain containing protein 7, Elastase, elastin, EMA, EMMPRIN, ENA, ENA-78, Endosialin, endothelin receptor, endotoxin, Enkephalinase, eNOS, Eot, Eotaxin, Eotaxin-2, eotaxini, EpCAM, Ephrin B2/EphB4, Epha2 tyrosine kinase receptor, epidermal growth factor receptor (EGFR), ErbB2 receptor, ErbB3 tyrosine kinase receptor, ERCC, EREG, erythropoietin (EPO), Erythropoietin receptor, E-selectin, ET-1, Exodus-2, F protein of RSV, F10, F11, F12, F13, F5, F9, Factor Ia, Factor IX, Factor Xa, Factor VII, factor VIII, Factor VIIIc, Fas, FcalphaR, FcepsilonRI, FcgammaIIb, FcgammaRI, FcgammaRIIa, FcgammaRIIIa, FcgammaRIIIb, FcRn, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF-2 receptor, FGF-3, FGF-8, FGF-acidic, FGF-basic, FGFR, FGFR-3, Fibrin, fibroblast activation protein (FAP), fibroblast growth factor, fibroblast growth factor-10, fibronectin, FL, FLIP, Flt-3, FLT3 ligand, Folate receptor, follicle stimulating hormone (FSH), Fractalkine (CX3C), free heavy chain, free light chain, FZD1, FZD10, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, G250, Gas 6, GCP-2, GCSF, G-CSF, G-CSF receptor, GD2, GD3, GDF, GDF-1, GDF-15 (MIC-1), GDF-3 (Vgr-2), GDF-5 (BMP-14/CDMP-1), GDF-6 (BMP-13/CDMP-2), GDF-7 (BMP-12/CDMP-3), GDF-8 (Myostatin), GDF-9, GDNF, Gelsolin, GFAP, GF-CSF, GFR-alpha1, GFR-alpha2, GFR-alpha3, GF-β1, gH envelope glycoprotein, GITR, Glucagon, Glucagon receptor, Glucagon-like peptide 1 receptor, Glut 4, Glutamate carboxypeptidase II, glycoprotein hormone receptors, glycoprotein IIb/IIIa (GP IIb/IIIa), Glypican-3, GM-CSF, GM-CSF receptor, gp130, gp140, gp72, granulocyte-CSF (G-CSF), GRO/MGSA, Growth hormone releasing factor, GRO-β, GRO-γ, H. pylori, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCC 1, HCMV gB envelope glycoprotein, HCMV UL, Hemopoietic growth factor (HGF), Hep B gp120, heparanase, heparin cofactor II, hepatic growth factor, Bacillus anthracis protective antigen, Hepatitis C virus E2 glycoprotein, Hepatitis E, Hepcidin, Her1, Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HGF, HGFA, High molecular weight melanoma-associated antigen (HMW-MAA), HIV envelope proteins such as GP120, HIV MIB gp120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HMGB-1, HRG, Hrk, HSP47, Hsp90, HSV gD glycoprotein, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (hGH), human serum albumin, human tissue-type plasminogen activator (t-PA), Huntingtin, HVEM, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFN-alpha, IFN-beta, IFN-gamma, IgA, IgA receptor, IgE, IGF, IGF binding proteins, IGF-1, IGF-1 R, IGF-2, IGFBP, IGFR, IL, IL-1, IL-10, IL-10 receptors, IL-11, IL-11 receptors, IL-12, IL-12 receptors, IL-13, IL-13 receptors, IL-15, IL-15 receptors, IL-16, IL-16 receptors, IL-17, IL-17 receptors, IL-18 (IGIF), IL-18 receptors, IL-1alpha, IL-1beta, IL-1 receptors, IL-2, IL-2 receptors, IL-20, IL-20 receptors, IL-21, IL-21 receptors, IL-23, IL-23 receptors, IL-2 receptors, IL-3, IL-3 receptors, IL-31, IL-31 receptors, IL-3 receptors, IL-4, IL-4 receptors IL-5, IL-5 receptors, IL-6, IL-6 receptors, IL-7, IL-7 receptors, IL-8, IL-8 receptors, IL-9, IL-9 receptors, immunoglobulin immune complex, immunoglobulins, INF-alpha, INF-alpha receptors, INF-beta, INF-beta receptors, INF-gamma, INF-gamma receptors, IFN type-I, IFN type-I receptor, influenza, inhibin, Inhibin α, Inhibin β, iNOS, insulin, Insulin A-chain, Insulin B-chain, Insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor binding proteins, integrin, integrin alpha2, integrin alpha3, integrin alpha4, integrin alpha4/beta1, integrin alpha-V/beta-3, integrin alpha-V/beta-6, integrin alpha4/beta7, integrin alpha5/beta1, integrin alpha5/beta3, integrin alpha5/beta6, integrin alphaσ (alphaV), integrin alphaθ, integrin beta1, integrin beta2, integrin beta3 (GPIIb-IIIa), IP-10, I-TAC, JE, kalliklein, Kallikrein 11, Kallikrein 12, Kallikrein 14, Kallikrein 15, Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein L1, Kallikrein L2, Kallikrein L3, Kallikrein L4, kallistatin, KC, KDR, Keratinocyte Growth Factor (KGF), Keratinocyte Growth Factor-2 (KGF-2), KGF, killer immunoglobulin-like receptor, kit ligand (KL), Kit tyrosine kinase, laminin 5, LAMP, LAPP (Amylin, islet-amyloid polypeptide), LAP (TGF-1), latency associated peptide, Latent TGF-1, Latent TGF-1 bpl, LBP, LDGF, LDL, LDL receptor, LECT2, Lefty, Leptin, leutinizing hormone (LH), Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, LFA-3 receptors, Lfo, LIF, LIGHT, lipoproteins, LIX, LKN, Lptn, L-Selectin, LT-a, LT-b, LTB4, LTBP-1, Lung surfactant, Luteinizing hormone, Lymphotactin, Lymphotoxin Beta Receptor, Lysosphingolipid receptor, Mac-1, macrophage-CSF (M-CSF), MAdCAM, MAG, MAP2, MARC, maspin, MCAM, MCK-2, MCP, MCP-1, MCP-2, MCP-3, MCP-4, MCP-I (MCAF), M-CSF, MDC, MDC (67 a.a.), MDC (69 a.a.), megsin, Mer, MET tyrosine kinase receptor family, METALLOPROTEASES, Membrane glycoprotein OX2, Mesothelin, MGDF receptor, MGMT, MHC(HLA-DR), microbial protein, MIF, MIG, MIP, MIP-1α, MIP-1β, MIP-3α, MIP-3β, MIP-4, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, monocyte attractant protein, monocyte colony inhibitory factor, mouse gonadotropin-associated peptide, MPIF, Mpo, MSK, MSP, MUC-16, MUC18, mucin (Mud), Muellerian-inhibiting substance, Mug, MuSK, Myelin associated glycoprotein, myeloid progenitor inhibitor factor-1 (MPIF-I), NAIP, Nanobody, NAP, NAP-2, NCA 90, NCAD, N-Cadherin, NCAM, Neprilysin, Neural cell adhesion molecule, neroserpin, Neuronal growth factor (NGF), Neurotrophin-3, Neurotrophin-4, Neurotrophin-6, Neuropilin 1, Neurturin, NGF-beta, NGFR, NKG20, N-methionyl human growth hormone, nNOS, NO, Nogo-A, Nogo receptor, non-structural protein type 3 (NS3) from the hepatitis C virus, NOS, Npn, NRG-3, NT, NT-3, NT-4, NTN, OB, OGG1, Oncostatin M, OP-2, OPG, OPN, OSM, OSM receptors, osteoinductive factors, osteopontin, OX40L, OX40R, oxidized LDL, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-Cadherin, PCNA, PCSK9, PDGF, PDGF receptor, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-D, PDK-1, PECAM, PEDF, PEM, PF-4, PGE, PGF, PGI2, PGD2, P1GF, PIN, PLA2, Placenta growth factor, placental alkaline phosphatase (PLAP), placental lactogen, plasminogen activator inhibitor-1, platelet-growth factor, plgR, PLP, poly glycol chains of different size (e.g. PEG-20, PEG-30, PEG40), PP14, prekallikrein, prion protein, procalcitonin, Programmed cell death protein 1, proinsulin, prolactin, Proprotein convertase PC9, prorelaxin, prostate specific membrane antigen (PSMA), Protein A, Protein C, Protein D, Protein S, Protein Z, PS, PSA, PSCA, PsmAr, PTEN, PTHrp, Ptk, PTN, P-selectin glycoprotein ligand-1, R51, RAGE, RANK, RANKL, RANTES, relaxin, Relaxin A-chain, Relaxin B-chain, renin, respiratory syncytial virus (RSV) F, Ret, reticulon 4, Rheumatoid factors, RLI P76, RPA2, RPK-1, RSK, RSV Fgp, S100, RON-8, SCF/KL, SCGF, Sclerostin, SDF-1, SDF1α, SDF1β, SERINE, Serum Amyloid P, Serum albumin, sFRP-3, Shh, Shiga like toxin II, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, sphingosine 1-phosphate receptor 1, Staphylococcal lipoteichoic acid, Stat, STEAP, STEAP-II, stem cell factor (SCF), streptokinase, superoxide dismutase, syndecan-1, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TB, TCA-3, T-cell receptor alpha/beta, TdT, TECK, TEM1, TEMS, TEM7, TEM8, Tenascin, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific, TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta R1 (ALK-5), TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, TGF-I, Thrombin, thrombopoietin (TPO), Thymic stromal lymphoprotein receptor, Thymus Ck-1, thyroid stimulating hormone (TSH), thyroxine, thyroxine-binding globulin, Tie, TIMP, TIQ, Tissue Factor, tissue factor protease inhibitor, tissue factor protein, TMEFF2, Tmpo, TMPRSS2, TNF receptor I, TNF receptor II, TNF-alpha, TNF-beta, TNF-beta2, TNFc, TNF-RT, TNF-RII, TNFRSF 10A (TRAIL R1 Apo-2/DR4), TNFRSF10B (TRAIL R2 DR5/KILLER/TRICK-2A/TRICK-B), TNFRSF10C (TRAIL R3 DcRl/LIT/TRID), TNFRSF10D (TRAIL R4 DcR2/TRUNDD), TNFRSF11A (RANK ODF R/TRANCE R), TNFRSF11B (OPG OCIF/TR1), TNFRSF12 (TWEAK R FN14), TNFRSF12A, TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR/HveA/LIGHT R/TR2), TNFRSF 16 (NGFR p75NTR), TNFRSF 17 (BCMA), TNFRSF 18 (GITR ATTR), TNFRSF19 (TROY TAJ/TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF R1 CD120a/p55-60), TNFRSF1B (TNF RII CD120b/p75-80), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRSF25 (DR3 Apo-3/LARD/TR-3/TRAMP/WSL-1), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII/TNFC R), TNFRSF4 (OX40 ACT35/TXGP1R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1/APT1/CD95), TNFRSF6B (DcR3 M68/TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1 BB CD137/ILA), TNFRST23 (DcTRAIL R1 TNFRH1), TNFSF10 (TRAIL Apo-2 Ligand/TL2), TNFSF11 (TRANCE/RANK Ligand ODF/OPG Ligand), TNFSF12 (TWEAK Apo-3 Ligand/DR3 Ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS/TALL1/THANK/TNFSF20), TNFSF14 (LIGHT HVEM Ligand/LTg), TNFSF15 (TL1A/VEGI), TNFSF18 (GITR Ligand AITR Ligand/TL6), TNFSF1A (TNF-a Conectin/DIF/TNFSF2), TNFSF1B (TNF-b LTa/TNFSF1), TNFSF3 (LTb TNFC/p33), TNFSF4 (OX40 Ligand gp34/TXGP1), TNFSF5 (CD40 Ligand CD154/gp39/HIGM1/IMD3/TRAP), TNFSF6 (Fas Ligand Apo-1 Ligand/APT1 Ligand), TNFSF7 (CD27 Ligand CD70), TNFSF8 (CD30 Ligand CD153), TNFSF9 (4-1 BB Ligand CD137 Ligand), TNF-α, TNF-β, TNIL-1, toxic metabolite, TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, transforming growth factors (TGF) such as TGF-alpha and TGF-beta, Transmembrane glycoprotein NMB, Transthyretin, TRF, Trk, TROP-2, Trophoblast glycoprotein, TSG, TSLP, Tumor Necrosis Factor (TNF), tumor-associated antigen CA 125, tumor-associated antigen expressing Lewis Y related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VAP-1, vascular endothelial growth factor (VEGF), vaspin, VCAM, VCAM-1, VECAD, VE-Cadherin, VE-Cadherin-2, VEFGR-1 (flt-1), VEFGR-2, VEGF receptor (VEGFR), VEGFR-3 (flt-4), VEGI, VIM, Viral antigens, VitB12 receptor, Vitronectin receptor, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand Factor (vWF), WIF-1, WNT1, WNT10A, WNT10B, WNT11, WNT16, WNT2, WNT2B/13, WNT3, WNT3A, WNT4, WNTSA, WNTSB, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, XCL1, XCL2/SCM-1-beta, XCL1/Lymphotactin, XCR1, XEDAR, XIAP, XPD.

[0316] One or more amino acid residue alterations are allowed in the amino acid sequences constituting the variable regions as long as their antigen-binding activities are maintained. When altering a variable region amino acid sequence, there is no particularly limitation on the site of alteration and number of amino acids altered. For example, amino acids present in CDR and/or FR can be altered appropriately. When altering amino acids in a variable region, the binding activity is preferably maintained without particular limitation; and for example, as compared to before alteration, the binding activity is 50% or more, preferably 80% or more, and more preferably 100% or more. Furthermore, the binding activity may be increased by amino acid alterations. For example, the binding activity may be 2-, 5-, 10-times higher or such than that before alteration. In the antibodies of the present invention, alteration of amino acid sequence may be at least one of amino acid residue substitution, addition, deletion, insertion, and modification.

[0317] For example, the modification of the N-terminal glutamine of a variable region into pyroglutamic acid by pyroglutamylation is a modification well known to those skilled in the art. Thus, when the heavy-chain N terminus is glutamine, the antibodies of the present invention comprise the variable regions in which the glutamine is modified to pyroglutamic acid.

[0318] Antibody variable regions of the present invention may have any sequences, and they may be antibody variable regions of any origin, such as mouse antibodies, rat antibodies, rabbit antibodies, goat antibodies, camel antibodies, humanized antibodies produced by humanizing these non-human antibodies, and human antibodies. “Humanized antibodies”, also referred to as “reshaped human antibodies”, are antibodies in which the complementarity determining regions (CDRs) of an antibody derived from a non-human mammal, for example, a mouse antibody, are transplanted into the CDRs of a human antibody. Methods for identifying CDRs are known (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al., Nature (1989) 342: 877). Their common genetic recombination technologies are also known (see, European Patent Application Publication No. EP 125023 and WO 96/02576). Furthermore, these antibodies may have various amino acid substitutions introduced into their variable regions to improve their antigen binding, pharmacokinetics, stability, and antigenicity. Variable regions of the antibodies of the present invention may be able to bind antigens repeatedly due to their pH dependability in antigen binding (WO/2009/125825).

[0319] κ chain and λ chain-type constant regions are present in antibody light-chain constant regions, but either one of the light chain constant regions is acceptable. Furthermore, light-chain constant regions of the present invention may be light-chain constant regions with amino acid alterations such as substitutions, additions, deletions, insertions, and/or modifications.

[0320] For example, for the heavy chain constant regions of an antibody of the present invention, heavy chain constant regions of human IgG antibodies may be used and heavy chain constant regions of human IgG1 antibodies are preferred.

[0321] The variable regions constituting an antibody of the present invention can be variable regions that recognize any antigen. One or several amino acid residues in the amino acid sequence constituting a heavy chain variable region can be altered as long as the antigen-binding activity is maintained.

[0322] Furthermore, alteration of variable regions is carried out with the objective of increasing binding activity, improving specificity, lowering pI, conferring a pH-dependent property to antigen binding, improving thermal stability of binding, improving solubility, providing stability to chemical modification, improving sugar-chain-derived heterogeneity, avoiding T cell epitope that reduces immunogenicity identified by in silico prediction, or by an in vitro assay using T cells, introducing T cell epitope that activates regulatory T cells, or such (mAbs 3: 243-247, 2011).

[0323] Furthermore, a polypeptide of the present invention may be an Fc fusion protein molecule produced by linking an Fc region with another protein, a biologically active peptide, or such (peptide Fc fusion protein), or an Fc fusion protein molecule produced by linking an Fc region with an extracellular matrix composed of polymers such as collagen or polylactic acid (scaffold Fc fusion protein).

[0324] Examples of another protein or biologically active peptide include receptors, adhesion molecules, ligands, and enzymes, but are not limited thereto.

[0325] Preferred examples of Fc fusion protein molecules of the present invention include proteins with Fc domain fused to a receptor protein that binds to a target, and such examples include TNFR-Fc fusion protein, IL1R-Fc fusion protein, VEGFR-Fc fusion protein, and CTLA4-Fc fusion protein (Nat. Med. 2003 January; 9(1): 47-52; BioDrugs. 2006; 20(3): 151-60). Furthermore, a protein to be fused to a polypeptide of the present invention may be any molecule as long as it binds to a target molecule, and examples include scFv molecules (WO 2005/037989), single-domain antibody molecules (WO 2004/058821; WO 2003/002609), antibody-like molecules (Current Opinion in Biotechnology 2006, 17: 653-658; Current Opinion in Biotechnology 2007, 18: 1-10; Current Opinion in Structural Biology 1997, 7: 463-469; and Protein Science 2006, 15: 14-27) such as DARPins (WO 2002/020565), Affibody (WO 1995/001937), Avimer (WO 2004/044011; WO 2005/040229), and Adnectin (WO 2002/032925). Furthermore, antibodies and Fc fusion protein molecules may be multispecific antibodies that bind to multiple types of target molecules or epitopes such as bispecific antibodies.

[0326] Furthermore, the antibodies of the present invention include antibody modification products. Such antibody modification products include, for example, antibodies linked with various molecules such as polyethylene glycol (PEG) and cytotoxic substances. Such antibody modification products can be obtained by chemically modifying antibodies of the present invention. Methods for modifying antibodies are already established in this field.

[0327] The antibodies of the present invention may also be bispecific antibodies. “Bispecific antibody” refers to an antibody that has in a single antibody molecule variable regions that recognize different epitopes. The epitopes may be present in a single molecule or in different molecules.

[0328] The polypeptides of the present invention can be prepared by the methods known to those skilled in the art. For example, the antibodies can be prepared by the methods described below, but the methods are not limited thereto.

[0329] There are various known host cell/expression vector combinations for antibody preparation by introducing isolated genes encoding the polypeptide into appropriate hosts. All of these expression systems are applicable to isolation of the antigen-binding molecules of the present invention. Appropriate eukaryotic cells used as host cells include animal cells, plant cells, and fungal cells. Specifically, the animal cells include, for example, the following cells.

[0330] (1) mammalian cells: CHO (Chinese hamster ovary cell line), COS (Monkey kidney cell line), myeloma (Sp2/O, NS0 and such), BHK (baby hamster kidney cell line), HEK293 (human embryonic kidney cell line with sheared adenovirus (Ad)5 DNA), PER.C6 cell (human embryonic retinal cell line transformed with the Adenovirus Type 5 (Ad5) E1A and E1B genes), Hela, Vero, or such (Current Protocols in Protein Science (May, 2001, Unit 5.9, Table 5.9.1));
(2) amphibian cells: Xenopus oocytes, or such; and
(3) insect cells: sf9, sf21, Tn5, or such.

[0331] A DNA encoding an antibody heavy chain in which one or more amino acid residues in the Fc region have been substituted with other amino acids of interest and DNA encoding an antibody light chain, are expressed. A DNA encoding a heavy chain in which one or more amino acid residues in the Fc region are substituted with other amino acids of interest can be prepared, for example, by obtaining a DNA encoding the Fc region of a natural heavy chain, and introducing an appropriate substitution so that a codon encoding a particular amino acid in the Fc region encodes another amino acid of interest.

[0332] Alternatively, a DNA encoding a heavy chain in which one or more amino acid residues in the Fc region are substituted with other amino acids of interest can also be prepared by designing and then chemically synthesizing a DNA encoding a protein in which one or more amino acid residues in the Fc region of the natural heavy chain are substituted with other amino acids of interest. The position and type of amino acid substitution are not particularly limited. Furthermore, alteration is not limited to substitution, and alteration may be any of deletion, addition, or insertion, or combination thereof.

[0333] Alternatively, a DNA encoding a heavy chain in which one or more amino acid residues in the Fc region are substituted with other amino acids of interest can be prepared as a combination of partial DNAs. Such combinations of partial DNAs include, for example, the combination of a DNA encoding a variable region and a DNA encoding a constant region, and the combination of a DNA encoding an Fab region and a DNA encoding an Fc region, but are not limited thereto. Furthermore, a DNA encoding a light chain can similarly be prepared as a combination of partial DNAs.

[0334] Methods for expressing the above-described DNAs include the methods described below. For example, a heavy chain expression vector is constructed by inserting a DNA encoding a heavy chain variable region into an expression vector along with a DNA encoding a heavy chain constant region. Likewise, a light chain expression vector is constructed by inserting a DNA encoding a light chain variable region into an expression vector along with a DNA encoding a light chain constant region. Alternatively, these heavy and light chain genes may be inserted into a single vector.

[0335] When inserting a DNA encoding the antibody of interest into an expression vector, the DNA is inserted so that the antibody is expressed under the control of an expression-regulating region such as an enhancer or promoter. Next, host cells are transformed with this expression vector to express the antibody. In such cases, an appropriate combination of host and expression vector may be used.

[0336] Examples of the vectors include M13 vectors, pUC vectors, pBR322, pBluescript, and pCR-Script. Alternatively, when aiming to subclone and excise cDNA, in addition to the vectors described above, pGEM-T, pDIRECT, pT7, and such can be used.

[0337] Expression vectors are particularly useful when using vectors for producing the antibodies of the present invention. For example, when a host cell is E. coli such as JM109, DH5α, HB101, and XL1-Blue, the expression vectors must carry a promoter that allows efficient expression in E. coli, for example, lacZ promoter (Ward et al., Nature (1989) 341: 544-546; FASEB J. (1992) δ: 2422-2427; its entirety are incorporated herein by reference), araB promoter (Better et al., Science (1988) 240: 1041-1043; its entirety are incorporated herein by reference), T7 promoter, or such. Such vectors include pGEX-5X-1 (Pharmacia), “QIAexpress system” (QIAGEN), pEGFP, or pET (in this case, the host is preferably BL21 that expresses T7 RNA polymerase) in addition to the vectors described above.

[0338] The vectors may contain signal sequences for polypeptide secretion. As a signal sequence for polypeptide secretion, a pelB signal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169: 4379; its entirety are incorporated herein by reference) may be used when a polypeptide is secreted into the E. coli periplasm. The vector can be introduced into host cells by lipofectin method, calcium phosphate method, and DEAE-Dextran method, for example.

[0339] In addition to E. coli expression vectors, the vectors for producing the polypeptides of the present invention include mammalian expression vectors (for example, pcDNA3 (Invitrogen), pEGF-BOS (Nucleic Acids. Res. 1990, 18(17): p5322; its entirety are incorporated herein by reference), pEF, and pCDM8), insect cell-derived expression vectors (for example, the “Bac-to-BAC baculovirus expression system” (GIBCO BRL) and pBacPAK8), plant-derived expression vectors (for example, pMH1 and pMH2), animal virus-derived expression vectors (for example, pHSV, pMV, and pAdexLcw), retroviral expression vectors (for example, pZIPneo), yeast expression vectors (for example, “Pichia Expression Kit” (Invitrogen), pNV11, and SP-Q01), and Bacillus subtilis expression vectors (for example, pPL608 and pKTH50), for example.

[0340] When aiming for expression in animal cells such as CHO, COS, NIH3T3, and HEK293 cells, the vectors must have a promoter essential for expression in cells, for example, SV40 promoter (Mulligan et al., Nature (1979) 277: 108; its entirety are incorporated herein by reference), MMTV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18: 5322; its entirety are incorporated herein by reference), CAG promoter (Gene. (1990) 18: 5322; its entirety are incorporated herein by reference), and CMV promoter, and more preferably they have a gene for selecting transformed cells (for example, a drug resistance gene that allows evaluation using an agent (neomycin, G418, or such)). Vectors with such characteristics include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13, for example. Also, in some cases, the EBNA1 protein may be further co-expressed for the purpose of increasing the gene copy number, and in this case, a vector having a replication initiation point OriP is used. (Biotechnol. Bioeng. 2001 Oct. 20; 75(2): 197-203; and Biotechnol. Bioeng. 2005 Sep. 20; 91(6): 670-7.)

[0341] In addition, the following method can be used for stable gene expression and gene copy number amplification in cells: CHO cells deficient in a nucleic acid synthesis pathway are introduced with a vector that carries a DHFR gene which compensates for the deficiency (for example, pCHOI), and the vector is amplified using methotrexate (MTX). Alternatively, the following method can be used for transient gene expression: COS cells with a gene expressing SV40 T antigen on their chromosome are transformed with a vector with an SV40 replication origin (pcD and such). Replication origins derived from polyoma virus, adenovirus, bovine papilloma virus (BPV), and such can also be used. To amplify gene copy number in host cells, the expression vectors may further carry selection markers such as aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, and dihydrofolate reductase (dhfr) gene.

[0342] Antibodies can be collected, for example, by culturing transformed cells, and then separating the antibodies from the inside of the transformed cells or from the culture media. Antibodies can be separated and purified using an appropriate combination of methods such as centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, 1q, FcRn, protein A, protein G column, affinity chromatography, ion exchange chromatography, and gel filtration chromatography.

[0343] As an efficient method for producing bispecific antibodies, the Knobs-into-holes technology may be used. Specifically, to produce a heterodimerized polypeptide of the present invention, it is necessary to have association between polypeptides having amino acids that differ from each other, or to separate the heterodimerized polypeptide of interest from the other homodimerized polypeptides.

[0344] For association of polypeptides having different amino acids from each another and comprising an Fc region, a technology of suppressing unintended association between H chains by introducing electrostatic repulsion into the interface of the second constant region of the antibody H chain (CH2) or the third constant region of the H chain (CH3) (WO 2006/106905) can be applied.

[0345] In the technology of suppressing unintended association between H chains by introducing electrostatic repulsion into the interface of CH2 or CH3, examples of amino acid residues in contact at the interface of other constant regions of the H chain include the residue at position 356 (EU numbering), the residue at position 439 (EU numbering), the region facing the residue at position 357 (EU numbering), the residue at position 370 (EU numbering), the residue at position 399 (EU numbering), and the residue at position 409 (EU numbering) in the CH3 domain.

[0346] More specifically, for example, in an antibody containing two types of H chain CH3 domains, the antibody in which one to three pairs of amino acid residues selected from the amino acid residues shown below in (1) to (3) in the first H chain CH3 domain have the same type of charge can be produced:

• [0347] (1) amino acid residues at positions 356 and 439 (EU numbering) which are amino acid residues contained in the H chain CH3 domain;
• [0348] (2) amino acid residues at positions 357 and 370 (EU numbering) which are amino acid residues contained in the H chain CH3 domain; and
• [0349] (3) amino acid residues at positions 399 and 409 (EU numbering) which are amino acid residues contained in the H chain CH3 domain.

[0350] Furthermore, an antibody can be produced in which one to three pairs of amino acid residues corresponding to the amino acid residue pairs indicated above in (1) to (3) having the same type of charge in the first H chain CH3 domain have charges opposite to the corresponding amino acid residues in the aforementioned first H chain CH3 domain, wherein the amino acid residue pairs are selected from the amino acid residue pairs indicated above in (1) to (3) in the second H chain CH3 domain which differs from the first H chain CH3 domain.

[0351] The respective amino acid residues of (1) to (3) mentioned above are positioned close to each other when associated. Those skilled in the art can find sites that correspond to the above-mentioned amino acid residues of (1) to (3) by homology modeling and such using commercially available software for the desired H chain CH3 domain or H chain constant region, and amino acid residues of these sites can be altered when appropriate.

[0352] In the above-mentioned antibodies, for example, “charged amino acid residues” are preferably selected from amino acid residues included in either of groups (X) or (Y) below:

(X) glutamic acid (E) and aspartic acid (D); and

(Y) lysine (K), arginine (R), and histidine (H).

[0353] In the above-mentioned antibodies, the phrase “having the same type of charge” means that, for example, all of the two or more amino acid residues are amino acid residues included in either of the above-mentioned groups (X) and (Y). The phrase “having the opposite charge” means that, for example, when at least one of the two or more amino acid residues is an amino acid residue included in either one of the above-mentioned groups (X) and (Y), the remaining amino acid residues are amino acid residues included in the other group.

[0354] In a preferred embodiment of the above-mentioned antibody, the first H chain CH3 domain and the second H chain CH3 domain may be cross-linked by disulfide bonds.

[0355] In the present invention, the amino acid residues to be altered are not limited to amino acid residues of the antibody constant region or antibody variable region described above. Those skilled in the art can find amino acid residues that form the interface in polypeptide mutants or heteromultimers through homology modeling and such using commercially available software, and can alter the amino acid residues at those sites to regulate association.

[0356] Other known technologies can also be used for the association of polypeptides of the present invention having different amino acids and comprising an Fc region. Polypeptides having different amino acids and comprising an Fc region can be efficiently associated with each other by substituting an amino acid side chain present in one of the H chain variable regions of the antibody with a larger side chain (knob), and substituting an amino acid side chain present in the opposing variable region of the other H chain with a smaller side chain (hole), to allow placement of the knob within the hole (WO 1996/027011; and Ridgway J B et al., Protein Engineering (1996) 9, 617-621; Merchant A M et al. Nature Biotechnology (1998) 16, 677-681).

[0357] In addition, other known technologies can also be used for the association of polypeptides having different amino acids and comprising an Fc region. Association of polypeptides having different sequences can be induced efficiently by complementary association of CH3, by using a strand-exchange engineered domain CH3 produced by changing part of the CH3 in one of the H chains of an antibody into an IgA-derived sequence corresponding to that portion, and introducing a corresponding IgA-derived sequence into the complementary portion of the CH3 on the other H chain (Protein Engineering Design & Selection, 23; 195-202, 2010). This known technology can also be used to efficiently induce association of polypeptides having different amino acids and comprising an Fc region.

[0358] In addition, one can also use technologies for heterodimerized antibody production using association of antibody CH1 and CL, and association of VH and VL, which are described in WO 2011/028952.

[0359] Furthermore, even in cases where heterodimerized polypeptides cannot be formed efficiently, heterodimerized polypeptides can be obtained by separating and purifying them from homodimerized polypeptides. When producing a heterodimerized polypeptide consisting of a first polypeptide and a second polypeptide which have different sequences from each other, homodimerized polypeptides consisting of only two first polypeptides, and homodimerized polypeptide consisting of only two second polypeptide are mixed in as impurities. Known technologies can be used as a method for efficiently removing these two types of homodimerized polypeptides. A method has been reported to be able to purify two types of homodimers and the heterodimerized antibody of interest by ion exchange chromatography, by creating a difference in isoelectric points by introducing amino acid substitutions into the variable regions of the two types of H chains (WO 2007114325).

[0360] Regarding amino acid alteration for conferring difference in isoelectric points, the amino acid alteration to be introduced is not particularly limited as long as a difference is produced between the isoelectric points of the two associating polypeptides, and it may also include amino acid alterations made for other purposes such as lowering immunogenicity. The altered amino acids are preferably amino acids at positions with little influence on the binding activity towards an Fcγ receptor. Furthermore, it may be an amino acid alteration that increases binding activity to a desired Fcγ receptor. For such alterations, it is preferable to introduce at least one amino acid mutation into an amino acid position selected from specifically the group consisting of Gly at position 137, Gly at position 138, Thr at position 139, Lys at position 147, Ser at position 192, Leu at position 193, Gln at position 196, Tyr at position 198, Ile at position 199, Asn at position 203, Lys at position 214, Val at position 263, Glu at position 272, Lys at position 274, Tyr at position 278, Lys at position 288, Lys at position 290, Gly at position 316, Lys at position 317, Lys at position 320, Lys at position 324, Thr at position 335, Ser at position 337, Lys at position 340, Leu at position 358, Lys at position 360, Gln at position 362, Ser at position 364, Ser at position 383, Asn at position 384, Gly at position 385, Gln at position 386, Pro at position 387, Asn at position 390, Val at position 397, and Val at position 422 (EU numbering) in the amino acid sequence of the first polypeptide and/or the second polypeptide. Furthermore, it is preferable to introduce a mutation into at least one amino acid selected from the group consisting of Gly at position 137, Gly at position 138, Thr at position 139, Lys at position 147, Ser at position 192, Leu at position 193, Gln at position 196, Ile at position 199, Asn at position 203, Lys at position 214, Glu at position 272, Lys at position 274, Lys at position 288, Lys at position 290, Leu at position 358, Lys at position 360, Gln at position 362, Ser at position 383, Asn at position 384, Gly at position 385, Gln at position 386, Asn at position 390, Val at position 397, and Val at position 422 (EU numbering). Further, it is more preferable to introduce a mutation into at least one amino acid selected from the group consisting of Gly at position 137, Gly at position 138, Lys at position 147, Ser at position 192, Leu at position 193, Gln at position 196, Ile at position 199, Asn at position 203, Lys at position 214, Lys at position 274, Lys at position 288, Leu at position 358, Asn at position 384, and Val at position 397 (EU numbering).

[0361] More specifically, it is preferable that a mutation is introduced into at least one amino acid selected from the group consisting of Gln at position 196, Ile at position 199, Val at position 263, Glu at position 272, Gly at position 316, Leu at position 358, Ser at position 364, Ser at position 383, Pro at position 387, and Val at position 397 (EU numbering) in the amino acid sequence of either polypeptide of the first polypeptide and the second polypeptide; and preferably a mutation is introduced into at least one amino acid selected from the group consisting of Gly at position 137, Gly at position 138, Thr at position 139, Lys at position 147, Ser at position 192, Leu at position 193, Tyr at position 198, Ile at position 199, Asn at position 203, Lys at position 214, Lys at position 274, Tyr at position 278, Lys at position 288, Lys at position 290, Gly at position 316, Lys at position 317, Lys at position 320, Lys at position 324, Thr at position 335, Ser at position 337, Lys at position 340, Leu at position 358, Lys at position 360, Gln at position 362, Ser at position 383, Asn at position 384, Gly at position 385, Gln at position 386, Asn at position 390, and Val at position 422 (EU numbering) in the amino acid sequence of the other polypeptide. Furthermore, it is preferable that a mutation is introduced into at least one amino acid selected from the group consisting of Gln at position 196, Ile at position 199, Glu at position 272, Leu at position 358, Ser at position 383, and Val at position 397 (EU numbering) in the amino acid sequence of one of the polypeptides; and preferably a mutation is introduced into at least one amino acid selected from the group consisting of Gly at position 137, Gly at position 138, Thr at position 139, Lys at position 147, Ser at position 192, Leu at position 193, Ile at position 199, Asn at position 203, Lys at position 214, Lys at position 274, Lys at position 288, Lys at position 290, Leu at position 358, Lys at position 360, Gln at position 362, Ser at position 383, Asn at position 384, Gly at position 385, Gln at position 386, Asn at position 390, and Val at position 422 (EU numbering) in the amino acid sequence of the other polypeptide. Furthermore, it is more preferable that a mutation is introduced into at least one amino acid selected from the group consisting of Gln at position 196, Ile at position 199, Leu at position 358, and Val at position 397 (EU numbering) in the amino acid sequence of one of the polypeptides; and that a mutation is introduced into at least one amino acid selected from the group consisting of Gly at position 137, Gly at position 138, Lys at position 147, Ser at position 192, Leu at position 193, Ile at position 199, Asn at position 203, Lys at position 214, Lys at position 274, Lys at position 288, and Asn at position 384 (EU numbering) in the amino acid sequence of the other polypeptide.

[0362] The amino acid alteration is not particularly limited as long as the alteration is made to produce a difference in isoelectric points between the two associating polypeptides after the alteration.

[0363] Examples of a preferred alteration for increasing the isoelectric point include substitution of the amino acid at position 196 with Lys, substitution of the amino acid at position 263 with Lys, substitution of the amino acid at position 272 with Lys, substitution of the amino acid at position 316 with Lys, substitution of the amino acid at position 364 with Lys, substitution of the amino acid at position 358 with Lys, substitution of the amino acid at position 383 with Lys, substitution of the amino acid at position 387 with Lys, and substitution of the amino acid at position 397 with Lys (EU numbering). Examples of a preferred alteration for decreasing the isoelectric point include substitution of the amino acid at position 137 with Glu, substitution of the amino acid at position 138 with Glu, substitution of the amino acid at position 139 with Glu, substitution of the amino acid at position 147 with Glu, substitution of the amino acid at position 198 with Glu, substitution of the amino acid at position 203 with Asp, substitution of the amino acid at position 214 with Thr, substitution of the amino acid at position 274 with Gln, substitution of the amino acid at position 278 with Glu, substitution of the amino acid at position 288 with Glu, substitution of the amino acid at position 290 with Glu, substitution of the amino acid at position 316 with Glu, substitution of the amino acid at position 317 with Glu, substitution of the amino acid at position 320 with Glu, substitution of the amino acid at position 324 with Glu, substitution of the amino acid at position 335 with Glu, substitution of the amino acid at position 337 with Asp, substitution of the amino acid at position 340 with Glu, substitution of the amino acid at position 358 with Glu, substitution of the amino acid at position 360 with Glu, substitution of the amino acid at position 362 with Glu, substitution of the amino acid at position 383 with Glu, substitution of the amino acid at position 384 with Glu, substitution of the amino acid at position 385 with Glu, substitution of the amino acid at position 386 with Glu, substitution of the amino acid at position 390 with Glu, and substitution of the amino acid at position 422 with Glu (EU numbering).

[0364] When combining amino acid alterations that are made for a purpose other than producing a difference in isoelectric points, for example, to lower antigenicity, substitution of the amino acid at position 138 with Ser, substitution of the amino acid at position 192 with Asn, substitution of the amino acid at position 193 with Phe, and substitution of the amino acid at position 199 with Thr (EU numbering) may be combined.

[0365] So far, as a method for purifying a heterodimerized antibody, a method that uses protein A for purifying a heterodimerized antibody comprising a mouse IgG2a H chain that binds to protein A and a rat IgG2b H chain that does not bind to protein A has been reported (WO 98050431 and WO 95033844).

[0366] Furthermore, a heterodimerized antibody alone can be efficiently purified by using H chains in which the amino acid residues at positions 435 and 436 (EU numbering), which are the binding site between IgG and Protein A, are substituted with amino acids such as Tyr and His which have different affinity to Protein A to change the interaction between each of the H chains and Protein A, and by using a Protein A column. A plurality, for example, two or more, of these substitutions and technologies can be used in combination. Furthermore, when appropriate, these alterations can be applied separately to the first polypeptide and the second polypeptide. Polypeptides of the present invention may be those produced based on the polypeptides to which the above-mentioned alterations have been applied.

[0367] The present invention also provides a method for producing a polypeptide comprising an Fc region, which comprises the steps of heterodimerizing the Fc region by introducing an amino acid mutation into the first polypeptide and/or the second polypeptide constituting the Fc region, and introducing an amino acid mutation to alter the Fc region function compared to when the Fc region forms a homodimer.

[0368] Examples include a production method comprising the following steps of:

• [0369] (a) in a polypeptide comprising an Fc region, introducing an amino acid mutation into the first polypeptide and/or the second polypeptide constituting the Fc region;
• [0370] (b) determining the Fc region function of the heterodimer consisting of the first polypeptide and second polypeptide into which a mutation is introduced in step (a); and
• [0371] (c) selecting a polypeptide with altered Fc region function compared to the parent polypeptide or compared to when the Fc region is homodimerized by introduction of the amino acid mutation.

[0372] In this production method, the following step may be performed after step (a):

• [0373] (d) displaying the Fc region-containing heterodimerized polypeptide consisting of the first polypeptide and the second polypeptide on the presented ribosomes, phages, or yeast.

[0374] A preferred embodiment is a method for producing a polypeptide comprising an Fc region which comprises the steps of:

• [0375] (a) altering a nucleic acid encoding the polypeptide so that the Fc region function is altered compared to the parent polypeptide or compared to when the Fc region forms a homodimer by introduction of amino acid mutation;
• [0376] (b) introducing the nucleic acid into a host cell and culturing the cell to express the polypeptide; and
• [0377] (c) collecting the polypeptide from a host cell culture.

[0378] Antibodies and Fc fusion protein molecules produced by the production method are also included in the present invention.

[0379] The type and range of amino acid mutations introduced by the present method are not particularly limited, but examples include amino acid mutations involved in alteration of each Fc region function described herein (more specifically, amino acid mutations disclosed specifically in the Tables in the Examples).

[0380] The present invention also provides a method for altering the function of a polypeptide comprising an Fc region, which comprises the steps of heterodimerizing the Fc region by introducing an amino acid mutation into the first polypeptide and/or the second polypeptide constituting the Fc region to alter the Fc region function compared to when the Fc region forms a homodimer by introduction of the amino acid mutation.

[0381] Examples include alteration methods comprising the following steps of:

• [0382] (a) in a polypeptide comprising an Fc region, introducing an amino acid mutation into the first polypeptide and/or the second polypeptide constituting the Fc region;
• [0383] (b) determining the Fc region function of the heterodimer consisting of the first polypeptide and second polypeptide into which a mutation is introduced in step (a); and
• [0384] (c) selecting a polypeptide with altered Fc region function compared to the parent polypeptide or compared to when the Fc region is homodimerized by introduction of the amino acid mutation.

[0385] In this alteration method, the following step may be performed after step (a):

• [0386] (d) displaying the Fc region-containing heterodimerized polypeptide consisting of the first polypeptide and the second polypeptide presented on ribosomes, phages, or yeast.

[0387] A preferred embodiment is a method for altering a polypeptide comprising an Fc region which comprises the steps of:

• [0388] (a) altering a nucleic acid encoding the polypeptide so that the Fc region function is altered compared to the parent polypeptide or compared to when the Fc region forms a homodimer by introduction of the amino acid mutation;
• [0389] (b) introducing the nucleic acid into a host cell and culturing the cell to express the polypeptide; and
• [0390] (c) collecting the polypeptide from a host cell culture.

[0391] Antibodies and Fc fusion protein molecules altered by the alteration method are also included in the present invention.

[0392] The type and range of amino acid mutations introduced by the present method are not particularly limited, but examples include amino acid mutations involved in alteration of each Fc region function described herein (more specifically, amino acid mutations disclosed specifically in the Tables in the Examples).

[0393] Furthermore, the present invention provides a nucleic acid encoding a polypeptide comprising an Fc region, wherein the polypeptide is characterized in that the Fc region is composed of a heterodimer comprising a first polypeptide and a second polypeptide, and wherein the polypeptide is characterized in that an Fc region function is altered compared to that of a polypeptide characterized in that the Fc region is composed of a homodimer comprising only the first polypeptide and/or compared to that of a polypeptide characterized in that the Fc region is composed of a homodimer comprising only the second polypeptide. The nucleic acid of the present invention may be in any form such as DNA or RNA.

[0394] The present invention also provides vectors carrying the above-described nucleic acids of the present invention. The type of vector can be appropriately selected by those skilled in the art depending on the host cells to be introduced with the vector. The vectors include, for example, those described above.

[0395] Furthermore, the present invention relates to host cells transformed with the above-described vectors of the present invention. Appropriate host cells can be selected by those skilled in the art. The host cells include, for example, those described above.

<Pharmaceutical Compositions>

[0396] The present invention provides pharmaceutical compositions comprising the polypeptide of the present invention.

[0397] The pharmaceutical compositions of the present invention can be formulated, in addition to the antibodies or Fc-fusion protein molecules, which are the polypeptides of the present invention, with pharmaceutically acceptable carriers by known methods. For example, the compositions can be used parenterally, when the antibodies or Fc-fusion protein molecules are formulated in a sterile solution or suspension for injection using water or any other pharmaceutically acceptable liquid. For example, the compositions can be formulated by appropriately combining the antibodies or Fc-fusion protein molecules with pharmacologically acceptable carriers or media, specifically, sterile water or physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binding agents, and such, by mixing them at a unit dose and form required by generally accepted pharmaceutical implementations. Specific examples of the carriers include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain triglyceride, polyoxyethylene hardened castor oil 60, saccharose, carboxymethyl cellulose, corn starch, inorganic salt, and such. The content of the active ingredient in such a formulation is adjusted so that an appropriate dose within the required range can be obtained.

[0398] Sterile compositions for injection can be formulated using vehicles such as distilled water for injection, according to standard protocols.

[0399] Aqueous solutions used for injection include, for example, physiological saline and isotonic solutions containing glucose or other adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride. These can be used in conjunction with suitable solubilizers such as alcohol, specifically ethanol, polyalcohols such as propylene glycol and polyethylene glycol, and non-ionic surfactants such as Polysorbate 80™ and HCO-50.

[0400] Oils include sesame oils and soybean oils, and can be combined with solubilizers such as benzyl benzoate or benzyl alcohol. These may also be formulated with buffers, for example, phosphate buffers or sodium acetate buffers; analgesics, for example, procaine hydrochloride; stabilizers, for example, benzyl alcohol or phenol; or antioxidants. The prepared injections are typically aliquoted into appropriate ampules.

[0401] The administration is preferably carried out parenterally, and specifically includes injection, intranasal administration, intrapulmonary administration, and percutaneous administration. For example, injections can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection.

[0402] Furthermore, the method of administration can be appropriately selected according to the age and symptoms of the patient. A single dosage of the pharmaceutical composition containing a polypeptide or a polynucleotide encoding a polypeptide can be selected, for example, from the range of 0.0001 to 1,000 mg per kg of body weight. Alternatively, the dosage may be, for example, in the range of 0.001 to 100,000 mg/patient. However, the dosage is not limited to these values. The dosage and method of administration vary depending on the patient's body weight, age, and symptoms, and can be appropriately selected by those skilled in the art.

[0403] In the present invention, the pharmaceutical compositions comprising the polypeptides of the present invention described above are useful as active ingredients for therapeutic agents or preventive agents for cancer, immune inflammatory diseases, and such.

[0404] As used herein, the three-letter and single-letter codes for respective amino acids are as follows:

Alanine: Ala (A)

Arginine: Arg (R)

Asparagine: Asn (N)

Aspartic acid: Asp (D)

Cysteine: Cys (C)

Glutamine: Gln (O)

Glutamic acid: Glu (E)

Glycine: Gly (G)

Histidine: His (H)

Isoleucine: Ile (I)

Leucine: Leu (L)

Lysine: Lys (K)

Methionine: Met (M)

Phenylalanine: Phe (F)

Proline: Pro (P)

Serine: Ser (S)

Threonine: Thr (T)

Tryptophan: Trp (W)

Tyrosine: Tyr (Y)

Valine: Val (V)

[0405] All prior art documents cited herein are incorporated by reference in their entirety.

[0406] Herein below, the present invention will be specifically described further with reference to the Examples, but it is not to be construed as being limited thereto.

[0407] An antibody interacts via its Fc region with various molecules such as FcRn, FcγR, and complements. A single molecule of FcRn, which is a ligand of Fc, binds to each one of the heavy chains (H chains) of an antibody. Thus, two molecules of FcRn bind to a single antibody molecule (FIG. 1). In vivo, FcRn is expressed on the cell membrane. Thus, an antibody recognizes two molecules of FcRn in a symmetrical manner via the identical sites in its respective H chains in vivo (Nature, 372: 379-383, 1994). Furthermore, in a manner similar to the relationship between IgG and FcRn, a single molecule of IgA, which belongs to the same immunoglobulin family as IgG, recognizes in a symmetrical manner two molecules of FcαR, which is an IgA receptor (FIG. 2) (Nature, 423: 614-620, 2003).

[0408] However, unlike FcRn and others, only one molecule of FcγR binds to one molecule of an antibody (FIG. 3) (JBC, 276: 16469-16477, 2001). IgG recognizes FcγR via the CH2 domains of the two H chains; however, the FcγR-interaction sites are different between the two H chains. For example, when the H chain shown at the left side of FIG. 3 is defined as H_A chain, and the one at the right side as H_B chain, Ala at position 327 (EU numbering) in each of the H_A and H_B chains interacts with FcγR. However, there is a difference between the properties of the partner residues with which the respective H chains interact (FIG. 4). The H_A chain interacts with FcγRIII in a hydrophobic manner at Trp of positions 87 and 110 (EU numbering), while the H_B chain interacts with FcγRIII at His of position 131 (EU numbering). Hence, when Ala at position 327 (EU numbering) is substituted with a highly-hydrophobic amino acid such as Trp, it can reduce the FcγR-binding activity of H_B chain even if it has the effect of improving the FcγR-binding activity of H_A chain. Thus, the asymmetric effect of the two H chains on FcγR needs to be considered to optimize the interaction of the Fc region of IgG with FcγR by amino acid alteration. Nevertheless, in the prior art, the same alteration has been introduced into the two H chains to optimize the interaction of the Fc region of IgG with FcγR (WO 2006/019447 and WO 2000/042072). However, when considering the asymmetric interaction the Fc region of IgG with FcγR, the interaction between IgG and FcγR can be optimized more finely by introducing different alterations into the H chains. That is, the interaction with FcγR can be more finely optimized by using a heterodimerized antibody resulting from the introduction of different alterations into the two H chains to optimize the interaction of the Fc region with FcγR as compared to a homodimerized antibody resulting from the introduction of the same alteration into the H chains, which has been performed in the prior art.

[0409] It was assessed whether the FcγR-binding activity of an antibody can be optimized more finely by using a heterodimerized antibody introduced with different alterations into the two H chains as compared to a homodimerized antibody of the prior art.

[0410] Conventionally, alterations that enhance the FcγR binding have been sought by using a homodimerized antibody resulting from the introduction of the same alteration into both H chains of an antibody. However, as described in Example 1, an antibody interacts with FcγR in an asymmetric manner, and when the same alteration is introduced into the two H chains, the alteration in one H chain could enhance the FcγR-binding activity while the alteration in the other H chain could rather inhibit the binding. The FcγR-binding activity is not necessarily increased in a homodimerized antibody resulting from the introduction of such an alteration into both H chains. However, a heterodimerized antibody resulting from the introduction of the alteration into only one of the two H chains can have increased FcγR-binding activity.

[0411] In order to test this hypothesis, with respect to FcγR binding, a heterodimerized antibody comprising a first polypeptide in which only one H chain has been introduced with an alteration that is thought to alter the FcγR-binding activity and a second polypeptide without the above alteration was compared with a homodimerized antibody comprising the first polypeptide in which only one H chain has been introduced with the alteration that is thought to alter the FcγR-binding activity. Based on the previous concept, when the FcγR-binding activity is increased by the alteration, the homodimerized antibody is always superior to the heterodimerized antibody. However, if an antibody Fc recognizes FcγR in an asymmetric manner, the heterodimerized antibody is expected to show greater FcγR-binding activity than the homodimerized antibody depending on the type of alteration.

[0412] The H chain variable region of an antibody used was the variable region of an anti-glypican-3 antibody which contains CDR of pH7 of the anti-glypican-3 antibody with improved kinetics in plasma disclosed in WO 2009/041062. The variable region is named GpH7 (SEQ ID NO: 1). The constant regions of antibody H chains described below were used in combination with GpH7. When the H chain constant region of an antibody is named H1, the sequence of the antibody H chain having the variable region GpH7 is referred to as GpH7-H1 Amino acid alterations are indicated in a manner such as D356K. The first alphabetical letter (for example, “D” of D356K) is a one-letter code representing the amino acid residue before alteration, and the following numeral (for example, “356” of D356K) indicates the position of alteration (EU numbering). The last alphabetical letter (for example, “K” of D356K) is a one-letter code representing the amino acid residue after alteration. GpH7-G1d (SEQ ID NO: 2) resulting from the removal of the C-terminal Gly and Lys from an IgG1 having the variable region GpH7; GpH7-A5 (SEQ ID NO: 3) resulting from the introduction of mutations D356K and H435R into GpH7-G1d; and GpH7-B3 (SEQ ID NO: 4) resulting from the introduction of K439E into GpH7-G1d were prepared according to the method described in Reference Example 1. The mutations D356K and K439E were introduced into each H chain to allow effective formation of heterodimer of the respective H chains when producing a heterodimerized antibody comprising two types of H chains (WO 2006/106905). H435R, which is an alteration that inhibits Protein A binding, was introduced to allow efficient separation of the heterodimerized and homodimerized forms (see Reference Examples 3, 4, and 5). Meanwhile, the antibody L chain used was GpL16-k0 (SEQ ID NO: 5), which is the L chain of the glypican-3 antibody with improved kinetics in plasma disclosed in WO 2009/041062.

[0413] Mutations for proving the concept of the heterodimerized antibody were introduced into parental polypeptides GpH7-A5 and GpH7-B3 to construct modified variants, and the variants were assessed. The constructed expression vectors were used to transfect FreeStyle293 cells (Invitrogen) according to the method described in Reference Example 1. The expressed antibodies were purified according to the method described in Reference Example 1. When expressing a homodimerized antibody, an expression vector inserted with the antibody L chain GpL16-k0 was used together with an expression vector inserted with one type of antibody H chain sequence. When a heterodimerized antibody was expressed, an expression vector inserted with the antibody L chain GpL16-k0, as used for the above homodimerized antibody, and an expression vector inserted with a sequence resulting from the introduction of an additional alteration into GpH7-A5 having the alteration D356K as one antibody H chain, and an expression vector inserted with a sequence resulting from the introduction of an additional alteration into GpH7-B3 having the alteration K439E as the other antibody H chain, were used to achieve efficient expression of the heterodimerized antibody. The antibody expressed and purified is referred to, for example, as GpH7-H1/GpH7-H2/GpL16-k0, when the expression vector used to express one antibody H chain of the heterodimerized antibody is GpH7-H1, the expression vector for the other antibody H chain is GpH7-H2, and the expression vector for the antibody L chain is GpL16-k0. In this system, a sequence introduced with the alterations D356K and H435R corresponds to H1, and a sequence introduced with the alteration K439E corresponds to H2. For example, when the expression vectors used to express the antibody H chain and L chain of a homodimerized antibody are respectively GpH7-H1 and GpL16-k0, the homodimerized antibody is referred to as GpH7-H1/GpL16-k0. Prepared antibodies were used to measure the FcγR-binding activity by the method described in Reference Example 2.

[0414] First, it was assessed whether the alterations D356K and H435R introduced into GpH7-A5, and the alteration K439E introduced into GpH7-B3 to form and purify heterodimers had an effect on the FcγR-binding activity as compared to native IgG. As a control, GpH7-G1d/GpL16-k0 (SEQ ID NOs: 2 and 5, respectively) was expressed using plasmids inserted with GpH7-G1d and GpL16-k0 as the antibody H chain and L chain, respectively, and purified according to the method of Reference Example 1. Likewise, the homodimerized antibody GpH7-A5/GpL16-k0 (SEQ ID NOs: 3 and 5, respectively) whose two H chains were introduced with D356K and H435R, the homodimerized antibody GpH7-B3/GpL16-k0 (SEQ ID NOs: 4 and 5, respectively) whose two H chains were introduced with K439E, and the heterodimerized antibody GpH7-A5/GpH7-B3/GpL16-k0 (SEQ ID NOs: 3, 4, and 5, respectively) in which one of the H chains was introduced with D356K and H435R and the other was introduced with K439E, were prepared. These antibodies and their binding activity to each FcγR were compared according to the method described in Reference Example 2, and the result is summarized in FIG. 5.

[0415] The measurement result showed that there was no significant difference in the binding activity to each FcγR between GpH7-G1d/GpL16-k0 and GpH7-A5/GpH7-B3/GpL16-k0. Furthermore, with respect to each FcγR, GpH7-A5/GpL16-k0 and GpH7-B3/GpL16-k0 retained at least about 80% of the binding activity of GpH7-G1d/GpL16-k0. Based on the above result, it was determined that the FcγR binding of GpH7-A5/GpH7-B3/GpL16-k0, GpH7-A5/GpL16-k0, and GpH7-B3/GpL16-k0 was not significantly reduced as compared to GpH7-G1d/GpL16-k0, and thus, variants resulting from the introduction of mutations into each H chain of these antibodies can be compared for the binding activity to each FcγR.

[0416] Then, GpH7-A26 (SEQ ID NO: 6) resulting from the introduction of mutation G237A into GpH7-A5 was constructed according to the method described in Reference Example 1. Using GpL16-k0 as the L chain, and GpH7-A26 and GpH7-B3 as the H chain, the heterodimerized antibody GpH7-A26/GpH7-B3/GpL16-k0 (SEQ ID NOs: 6, 4, and 5, respectively) in which only one of the H chains has been introduced with G237A was expressed according to the method described in Reference Example 1. Likewise, using GpH7-A26 as the H chain and GpL16-k0 as the L chain, the homodimerized antibody GpH7-A26/GpL16-k0 (SEQ ID NOs: 6 and 5, respectively) whose two H chains were introduced with G237A was expressed according to the method described in Reference Example 1. These antibodies were assessed for the binding activity to each FcγR according to the method described in Reference Example 2 (FIG. 6). The result showed that the heterodimerized antibody GpH7-A26/GpH7-B3/GpL16-k0 had increased binding activities to FcγRIIa R and FcγRIIb as compared to GpH7-A5/GpH7-B3/GpL16-k0. Meanwhile, the homodimerized antibody GpH7-A26/GpL16-k0 in which the same alteration was introduced into both H chains had reduced binding activities to FcγRIIa R and FcγRIIb as compared to GpH7-A5/GpH7-B3/GpL16-k0. These results demonstrate that, G237A is an alteration that increases the binding activities to FcγRIIa R and FcγRIIb when introduced into only one H chain, while reduces the binding activities to FcγRIIa R and FcγRIIb when introduced into both H chains.

[0417] Then, GpH7-A29 (SEQ ID NO: 7) resulting from the introduction of the mutation G237L into GpH7-A5 was constructed according to the method described in Reference Example 1. Using GpL16-k0 as the L chain, and GpH7-A29 and GpH7-B3 as the H chain, the heterodimerized antibody GpH7-A29/GpH7-B3/GpL16-k0 (SEQ ID NOs: 7, 4, and 5, respectively) in which only one of the H chains has been introduced with G237L was expressed according to the method described in Reference Example 1. Likewise, using GpH7-A29 as the H chain and GpL16-k0 as the L chain, the homodimerized antibody GpH7-A29/GpL16-k0 (SEQ ID NOs: 7 and 5, respectively) whose two H chains were introduced with G237L was expressed according to the method described in Reference Example 1. These antibodies and their binding activities to each FcγR were assessed according to the method described in Reference Example 2 (FIG. 7). The heterodimerized antibody GpH7-A29/GpH7-B3/GpL16-k0 had increased FcγRIIa R-binding and FcγRIIb-binding activities as compared to GpH7-A5/GpH7-B3/GpL16-k0. Meanwhile, the homodimerized antibody GpH7-A29/GpL16-k0 whose two H chains had the same alteration had reduced binding activity to FcγRIIa R and FcγRIIb as compared to GpH7-A5/GpH7-B3/GpL16-k0. These results demonstrate that, G237L is an alteration that has an effect of increasing the binding activities to FcγRIIa R and FcγRIIb when introduced into only one H chain, while reducing the binding activities to FcγRIIa R and FcγRIIb when introduced into both H chains.

[0418] Then, GpH7-A42 (SEQ ID NO: 8) resulting from the introduction of the mutation L328E into GpH7-A5 was constructed according to the method described in Reference Example 1. Using GpL16-k0 as the L chain, and GpH7-A42 and GpH7-B3 as the H chain, the heterodimerized antibody GpH7-A42/GpH7-B3/GpL16-k0 (SEQ ID NOs: 8, 4, and 5, respectively) in which only one of the H chains has been introduced with L328E was expressed according to the method described in Reference Example 1. Likewise, using GpH7-A42 as the H chain and GpL16-k0 as the L chain, the homodimerized antibody GpH7-A42/GpL16-k0 (SEQ ID NO: 8 and 5, respectively) whose two H chains were introduced with L328E was expressed according to the method described in Reference Example 1. These antibodies were assessed for the binding activity to each FcγR according to the method described in Reference Example 2 (FIG. 8). The heterodimerized antibody GpH7-A42/GpH7-B3/GpL16-k0 had increased binding activity to FcγRIIa R and FcγRIIb as compared to GpH7-A5/GpH7-B3/GpL16-k0. Meanwhile, the homodimerized antibody GpH7-A42/GpL16-k0 whose two H chains were introduced with the same alteration had reduced FcγRIIa R-binding activity and increased FcγRIIb-binding activity as compared to GpH7-A5/GpH7-B3/GpL16-k0; however, the degree of increase was greater in GpH7-A42/GpH7-B3/GpL16-k0 in which only one of the H chains was introduced with L328E. These results demonstrate that L328E is an alteration that is more effective to increase the binding activities to FcγRIIa R and FcγRIIb when introduced into only one H chain than when introduced into both H chains.

[0419] Then, GpH7-A43 (SEQ ID NO: 9) resulting from the introduction of the mutation L328D into GpH7-A5 was constructed according to the method described in Reference Example 1. Using GpL16-k0 as the L chain, and GpH7-A43 and GpH7-B3 as the H chain, the heterodimerized antibody GpH7-A43/GpH7-B3/GpL16-k0 (SEQ ID NOs: 9, 4, and 5, respectively) in which only one of the H chains has been introduced with L328D was expressed according to the method described in Reference Example 1. Likewise, using GpH7-A43 as the H chain and GpL16-k0 as the L chain, the homodimerized antibody GpH7-A43/GpL16-k0 (SEQ ID NOs: 9 and 5, respectively) whose two H chains were introduced with L328D was expressed according to the method described in Reference Example 1. These antibodies were assessed for the binding activity to each FcγR according to the method described in Reference Example 2 (FIG. 9). The heterodimerized antibody GpH7-A43/GpH7-B3/GpL16-k0 had increased binding activity to FcγRIIa R and FcγRIIb as compared to GpH7-A5/GpH7-B3/GpL16-k0. Meanwhile, the homodimerized antibody GpH7-A43/GpL16-k0 whose two H chains were introduced with the same alteration had reduced FcγRIIa R-binding activity and increased FcγRIIb-binding activity as compared to GpH7-A5/GpH7-B3/GpL16-k0; however, the degree of increase was greater in GpH7-A43/GpH7-B3/GpL16-k0 in which only one of the H chains was introduced with L328D. These results demonstrate that L328D is an alteration that is more effective to increase the binding activities to FcγRIIa R and FcγRIIb when introduced into only one H chain than when introduced into both H chains.

[0420] Then, GpH7-B16 (SEQ ID NO: 10) resulting from the introduction of the mutation L234E into GpH7-B3 was constructed according to the method described in Reference Example 1. Using GpL16-k0 as the L chain, and GpH7-A5 and GpH7-B16 as the H chain, the heterodimerized antibody GpH7-A5/GpH7-B16/GpL16-k0 (SEQ ID NOs: 3, 10, and 5, respectively) in which only one of the H chains has been introduced with L234E was expressed according to the method described in Reference Example 1. Likewise, using GpH7-B16 as the H chain and GpL16-k0 as the L chain, the homodimerized antibody GpH7-B16/GpL16-k0 (SEQ ID NOs: 10 and 5, respectively) whose two H chains were introduced with L234E was expressed according to the method described in Reference Example 1. These antibodies were assessed for the binding activity to each FcγR according to the method described in Reference Example 2 (FIG. 10). The heterodimerized antibody GpH7-A5/GpH7-B16/GpL16-k0 had increased binding activities to FcγRIIIa F and FcγRIIb as compared to GpH7-A5/GpH7-B3/GpL16-k0. Meanwhile, the homodimerized antibody GpH7-B16/GpL16-k0 whose two H chains were introduced with the same alteration had reduced binding activities to FcγRIIIa F and FcγRIIb as compared to GpH7-A5/GpH7-B3/GpL16-k0. These results demonstrate that L234E is an alteration that is effective to increase the binding activities to FcγRIIIa F and FcγRIIb when introduced into only one H chain, while it reduces the binding activities to FcγRIIIa F and FcγRIIb when introduced into both H chains.

[0421] Then, GpH7-B17 (SEQ ID NO: 11) resulting from the introduction of the mutation L234D into GpH7-B3 was constructed according to the method described in Reference Example 1. Using GGpL16-k0 as the L chain, and GpH7-A5 and GpH7-B17 as the H chain, the heterodimerized antibody GpH7-A5/GpH7-B17/GpL16-k0 (SEQ ID NOs: 3, 11, and 5, respectively) whose two H chains were introduced with L234D was expressed according to the method described in Reference Example 1. Likewise, using GpH7-B17 as the H chain and GpL16-k0 as the L chain, the homodimerized antibody GpH7-B17/GpL16-k0 (SEQ ID NOs: 11 and 5, respectively) whose two H chains were introduced with L234D was expressed according to the method described in Reference Example 1. These antibodies were assessed for the binding activity to each FcγR according to the method described in Reference Example 2 (FIG. 11). The heterodimerized antibody GpH7-A5/GpH7-B17/GpL16-k0 had increased binding activity to FcγRIIa R and FcγRIIb as compared to GpH7-A5/GpH7-B3/GpL16-k0. Meanwhile, the homodimerized antibody GpH7-B17/GpL16-k0 whose two H chains were introduced with the same alteration had reduced binding activities to FcγRIIa R and FcγRIIb as compared to GpH7-A5/GpH7-B3/GpL16-k0. These results demonstrate that L234D is an alteration that is effective to increase the binding activities to FcγRIIa R and FcγRIIb when introduced into only one H chain, while it reduces the binding activities to FcγRIIa R and FcγRIIb when introduced into both H chains.

[0422] The above findings demonstrate that, even if a homodimerized antibody whose two H chains have been introduced with the same alteration exhibits reduced FcγR-binding activity, it is possible to increase the FcγR-binding activity by constructing a heterodimerized antibody in which only one of the H chains has been introduced with the alteration.

[0423] Thus, the above findings demonstrate that a superior FcγR-binding property of an antibody Fc domain can be provided by using a heterodimerized antibody whose two H chains have been introduced with different alterations, as compared to a homodimerized antibody made by a conventional method for introducing the same alteration into both H chains.

[0424] As shown in Example 2, it was demonstrated that, using heterodimerized antibodies, higher FcγR-binding activity compared to homodimerized antibodies can be achieved. When the alterations described in Example 2 were introduced into both H chains of an antibody, the FcγR-binding activity was rather reduced as compared to the naturally-occurring antibody. This finding suggests that such alteration results in an increase in the FcγR-binding activity when introduced into one H chain of a heterodimerized antibody; however, when the mutation is introduced into both H chains, upon binding to FcγR, the residue after alteration increases the binding in one chain, while inhibits the interaction with FcγR in the other chain. Herein, the state where FcγR is bound in the direction from the depth side of FIG. 3 is defined as “X-direction binding”, while in an opposite manner, the binding from the front side is defined as “Y-direction binding”. It was speculated that in the heterodimerized antibody only either of the X-direction and Y-direction of FcγR-binding activities is altered, whereas in the homodimerized antibody the FcγR-binding activities in X direction and Y direction are altered in the same manner.

[0425] To prove the hypothesis experimentally, the present inventors found alterations that mainly inhibit FcγR binding in only either of the X-direction and Y-direction, and introduced such alteration into either one of the H chains, and introduced into the same or the other H chain an alteration that enhances the FcγR-binding activity, to verify the inhibition of the FcγR-binding activity. The present inventors aimed to find a method to be used in combination with the above alteration. Conformational information was searched for alterations that are involved in the FcγR binding of an antibody but are only involved in the binding in only either one of the H chains. As a result, P329 was found as a candidate. P329 in the H_A chain forms a hydrophobic core with Trp at positions 87 and 110 in FcγRIII, while P329 in the H_B chain does not directly interact with FcγRIII (Nature, 372: 379-383, 1994) (FIG. 12). For example, it was thought that the substitution of P329 of the H_A chain by an electrically charged residue causes collapse of the hydrophobic core, and results in inhibition of the X-direction binding shown in FIG. 12; however, it was predicted that there is no significant impact on the Y-direction binding, because P329 of the H_B chain, which is not involved in the Y-direction binding on the other side, remains unsubstituted.

[0426] Mutations, electrically charged R, K, D, and E, were introduced into GpH7-B3 at P329 to respectively produce the sequences GpH7-B12, GpH7-B13, GpH7-B14, and GpH7-B15 (SEQ ID NOs: 12 to 15), and expression vectors inserted with these sequences were constructed by the method described in Reference Example 1. The constructed expression vectors were each combined with GpH7-A5 and GpL16-k0 to express heterodimerized antibodies, or they were combined with GpL16-k0 alone but not with other H chains to express homodimerized antibodies. These antibodies were expressed and purified according to the method described in Reference Example 1. The resulting purified antibodies are the following: the heterodimerized antibodies GpH7-A5/GpH7-B12/GpL16-k0, GpH7-A5/GpH7-B13/GpL16-k0, GpH7-A5/GpH7-B14/GpL16-k0, and GpH7-A5/GpH7-B15/GpL16-k0; and homodimerized antibodies GpH7-B12/GpL16-k0, GpH7-B13/GpL16-k0, GpH7-B14/GpL16-k0, and GpH7-B15/GpL16-k0. The prepared antibodies were used to assess the binding activity to each FcγR by the method described in Reference Example 2. The result is shown in FIG. 13.

[0427] The FcγR-binding activity of GpH7-A5/GpH7-B12/GpL16-k0 and GpH7-A5/GpH7-B13/GpL16-k0, which are heterodimerized antibodies introduced with P329R and P329K, respectively, was about ⅕ to ¼ of the binding activity to each FcγR of GpH7-A5/GpH7-B3/GpL16-k0. Meanwhile, the FcγR binding was not observed for the homodimerized antibodies GpH7-B12/GpL16-k0 and GpH7-B13/GpL16-k0. On the other hand, as to FcγRIa, the FcγR-binding activity of GpH7-A5/GpH7-B14/GpL16-k0 and GpH7-A5/GpH7-B15/GpL16-k0, which are the heterodimerized antibodies introduced with P329D and P329E, respectively, was about ⅕ to ¼ of the binding activity to each FcγR of GpH7-A5/GpH7-B3/GpL16-k0; as to FcγR other than FcγRIa, they retained 50% or more of the binding activity. The homodimerized antibodies GpH7-B14/GpL16-k0 and GpH7-B15/GpL16-k0 only retained the FcγRIa-binding activity, and the activity was ⅕ or less the FcγRIa-binding activity of GpH7-A5/GpH7-B3/GpL16-k0. GpH7-B12 and GpH7-B13 are introduced with basic residues, whereas GpH7-B14 and GpH7-B15 are introduced with acidic residues. Thus, it was thought that the FcγR-binding activity is more strongly inhibited by substituting P329 with a basic residue such as Arg and Lys. The result showing that the FcγR-binding activity was maintained when an alteration was introduced into only one H chain, while the binding was almost undetectable when the same alteration was introduced into both H chains, supports the hypothesis that the substitution of a hydrophilic residue for P329 in one H chain inhibits the binding in one direction although the binding in the other direction is retained.

[0428] Then, whether the mutations G237A and L234D that enhance the FcγR-binding activity by heterodimerization, which were found as described in Example 2, enhance the interaction with FcγR exclusively in one direction was assessed by comparing the binding activity to each FcγR of each variant which was prepared to have the alteration P329R in combination with the above alterations. Expression vectors inserted with GpH7-B12 (SEQ ID NO: 12) resulting from the introduction of the mutation P329R into GpH7-B3 such that P329R is introduced into the same H chain as K439E; GpH7-A48 (SEQ ID NO: 16) resulting from the introduction of the mutation P329R into GpH7-A5 such that P329R is introduced into the same H chain as D356K and H435R; GpH7-A45 (SEQ ID NO: 17) resulting from the introduction of the mutations G237A and P329R into GpH7-A5; and GpH7-B41 (SEQ ID NO: 18) resulting from the introduction of the mutations L234D and P329R into GpH7-B5, were constructed by the method described in Reference Example 1. Using these expression vectors and the GpL16-k0 expression vector corresponding to an antibody L chain, antibodies were expressed such that P329R and G237A or L234D are introduced into only either of the H chains according to the method described in Reference Example 1 (Table 1).

[0429]

TABLE 1
		MUTATION		MUTATION
SAMPLE	H1	SITE	H2	SITE
GpH7-A5/GpH7-	A5	—	—	B3	—	—
B3/GpL16-k0
(SEQ ID NO:
3, 4, 5)
GpH7-A5/GpH7-	A5	—	—	B12	—	P329R
B12/GpL16-k0
(SEQ ID NO:
3, 12, 5)
GpH7-A48/GpH7-	A48	—	P329R	B3	—	—
B3/GpL16-k0
(SEQ ID NO:
16, 4, 5)
GpH7-A26/GpH7-	A26	G237A	—	B3	—	—
B3/GpL16-k0
(SEQ ID NO:
6, 4, 5)
GpH7-A26/GpH7-	A26	G237A	—	B12	—	P329R
B12/GpL16-k0
(SEQ ID NO:
6, 12, 5)
GpH7-A45/GpH7-	A45	G237A	P329R	B3	—	—
B3/GpL16-k0
(SEQ ID NO:
17, 4, 5)
GpH7-A5/GpH7-	A5	—	—	B17	L234D	—
B17/GpL16-k0
(SEQ ID NO:
3, 11, 5)
GpH7-A5/GpH7-	A5	—	—	B41	L234D	P329R
B41/GpL16-k0
(SEQ ID NO:
3, 18, 5)
GpH7-A48/GpH7-	A48	—	P329R	B17	L234D	—
B17/GpL16-k0
(SEQ ID NO:
16, 11, 5)

[0430] The column “SAMPLE” indicates antibody names; the columns “H1” and “H2” indicate names of H chain constant regions of respective antibodies; and the column “MUTATION SITE” indicates mutations that are different compared to GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is no particular mutation). The SEQ ID NOs are also shown for the amino acid sequences of the H chain and L chain of each antibody.

[0431] The combination of G237A and P329R was assessed by using GpH7-A5/GpH7-B3/GpL16-k0 as a polypeptide for introduction of alterations; GpH7-A5/GpH7-B12/GpL16-k0 and GpH7-A48/GpH7-B3/GpL16-k0 as variants in which one of the H chains has been introduced with P329R; GpH7-A45/GpH7-B3/GpL16-k0 as a variant in which G237A has been introduced into the same chain as P329R; and GpH7-A26/GpH7-B12/GpL16-k0 as a variant in which G237A has been introduced into the other chain than P329R. They were compared for the binding activity to each FcγR according to the method described in Reference Example 2 (FIG. 14).

[0432] When GpH7-A5/GpH7-B12/GpL16-k0 in which one of the H chains has been introduced with P329R was compared to GpH7-A48/GpH7-B3/GpL16-k0, no significant difference was observed in the pattern of FcγR-binding. Thus, it was thought that P329R has no influence on the FcγR binding even when introduced into an H chain introduced with D356K and H435R or when introduced into an H chain introduced with K439E.

[0433] On the other hand, GpH7-A26/GpH7-B3/GpL16-k0, which is a heterodimerized antibody in which only one of the H chains has been introduced with G237A, showed enhanced binding to FcγRIIa R and IIb as compared to GpH7-A5/GpH7-B3/GpL16-k0. However, as compared to GpH7-A5/GpH7-B12/GpL16-k0 in which only one of the H chains has been introduced with P329R, GpH7-A26/GpH7-B12/GpL16-k0 in which the other H chain has been introduced with G237A exhibited a reduced FcγR binding, and thus the effect of G237A to enhance the binding to FcγRIIa R and IIb was not observed. Meanwhile, as compared to GpH7-A48/GpH7-B3/GpL16-k0 in which only one of the H chains has been introduced with P329R, GpH7-A45/GpH7-B3/GpL16-k0 in which G237A has been introduced with the same H chain as P329R showed an enhanced binding to FcγRIIa R and IIb, and thus the effect of G237A to enhance the binding to FcγRIIa R and IIb was observed. Since the binding between the antibody and FcγR was strongly inhibited when G237A was introduced into the chain other than that introduced with P329R, the above results show that, when G237A introduced into GpH7-A5 is combined with P329R introduced into GpH7-B3, they recognize the binding between the antibody and FcγR in the same direction.

[0434] The alteration L234D was also assessed in the same manner (FIG. 15). GpH7-A5/GpH7-B17/GpL16-k0, which is a heterodimerized antibody in which only one of the H chains has been introduced with L234D, showed an enhanced binding to FcγRIIa R and IIb as compared to GpH7-A5/GpH7-B3/GpL16-k0. However, as compared to GpH7-A48/GpH7-B3/GpL16-k0 in which only one of the H chains has been introduced with P329R, GpH7-A48/GpH7-B17/GpL16-k0 in which L234D has been introduced into the other H chain exhibited a reduced binding to FcγR other than FcγRIa; and thus the effect of L234D to enhance the binding to FcγRIIa R and IIb was not observed. Meanwhile, as compared to GpH7-A5/GpH7-B12/GpL16-k0 in which only one of the H chains has been introduced with P329R, GpH7-A5/GpH7-B41/GpL16-k0 in which L234D has been introduced into the same H chain as P329R showed an enhanced binding to FcγRIIa R and IIb; and thus the effect of L234D to enhance the binding to FcγRIIa R and IIb was observed. Since the binding between the antibody and FcγR was strongly inhibited when L234D was introduced into the chain other than that introduced with P329R, the above results show that, when L234D introduced into GpH7-B3 is combined with P329R introduced into GpH7-A5, they recognize the binding between the antibody and FcγR in the same direction.

[0435] That is, both G237A and L234D increase (/reduce) the FcγR-binding activity of an antibody in the same direction as P329R.

[0436] In this way, the hetero-alteration enhanced the binding to FcγR exclusively in either of the X-direction and Y-direction. This finding demonstrates that the interaction with FcγR is enhanced in an asymmetric manner in the heterodimerized antibodies.

[0437] As described in Example 2, the present inventors revealed that the FcγR binding of an antibody via its Fc domain can be enhanced by introducing different alterations to the two H chains of an antibody rather than introducing the same alteration to both H chains. Thus, to discover alterations with such a property, the present inventors carried out the experiment described below.

[0438] To construct GpH7-B3 variants, amino acids thought to be involved in the FcγR binding and amino acids around them in GpH7-B3 (SEQ ID NO: 4) constructed as described in Example 2, specifically, Leu at position 234 (EU numbering), Leu at position 235 (EU numbering), Gly at position 236 (EU numbering), Gly at position 237 (EU numbering), Pro at position 238 (EU numbering), Ser at position 329 (EU numbering), Asp at position 265 (EU numbering), Val at position 266 (EU numbering), Ser at position 267 (EU numbering), His at position 268 (EU numbering), Glu at position 269 (EU numbering), Asp at position 270 (EU numbering), Pro at position 271 (EU numbering), Gln at position 295 (EU numbering), Tyr at position 296 (EU numbering), Ser at position 298 (EU numbering), Tyr at position 300 (EU numbering), Ser at position 324 (EU numbering), Asn at position 325 (EU numbering), Lys at position 326 (EU numbering), Ala at position 327 (EU numbering), Leu at position 328 (EU numbering), Pro at position 329 (EU numbering), Ala at position 330 (EU numbering), Pro at position 331 (EU numbering), Ile at position 332 (EU numbering), Glu at position 333 (EU numbering), Lys at position 334 (EU numbering), Thr at position 335 (EU numbering), Ile at position 336 (EU numbering), and Ser at position 337 (EU numbering) were each substituted with 18 types of amino acids excluding the original amino acid and cysteine. Each GpH7-B3 variant was named “A_B”, where A represents the information on the amino acid type indicated by one-letter code and the EU numbering of the altered residue, and B indicates the information on the amino acid after substitution. For example, when Gly is substituted for Leu at position 234 (EU numbering), the B3_variant is named L234_01G. For descriptive purposes, in the information on the amino acid after substitution, a numeral specific for the amino acid is added before each one-letter code. Specifically, the symbols used are the following: 01G for Gly; 02A for Ala; 03V for Val, 04F for Phe; 05P for Pro; 06M for Met; 07I for Ile; 08L for Leu; 09D for Asp; 10E for Glu; 11K for Lys; 12R for Arg; 13S for Ser; 14T for Thr; 15Y for Tyr; 16H for His; 18N for Asn; 19Q for Gln; and 20W for Trp.

[0439] Homodimerized antibodies in which both of the H chains have been introduced with a mutation were prepared by the following procedure. Antibodies were expressed using a GpH7-B3 variant as the H chain and GpL16-k0 (SEQ ID NO: 5) as the L chain. The antibodies were prepared according to the method described in Reference Example 1. The homodimerized antibodies in which both of the H chains have been introduced with a mutation thus prepared are referred to as Ho Ab.

[0440] Heterodimerized antibodies in which only one of the H chains has been introduced with a mutation were prepared by the following procedure. Antibodies were expressed using a GpH7-B3 variant and GpH7-A5 (SEQ ID NO: 3) as the H chain and GpL16-k0 (SEQ ID NO: 5) as the L chain. The antibodies were prepared according to the method described in Reference Example 1. The heterodimerized antibodies in which only one of the H chains has been introduced with a mutation thus prepared are referred to as He Ab.

[0441] As a control homodimerized antibody, GpH7-B3/GpL16-k0 that was prepared using GpH7-B3 (SEQ ID NO: 4) as the H chain and GpL16-k0 (SEQ ID NO: 5) as the L chain was prepared according to the method described in Reference Example 1. This control homodimerized antibody is referred to as HoCon Ab. As assessed in Example 2, the binding activity of HoCon Ab to each FcγR is not significantly altered as compared to the native IgG1.

[0442] As a control heterodimerized antibody, GpH7-A5/GpH7-B3/GpL16-k0 that was prepared using GpH7-A5 (SEQ ID NO: 3) and GpH7-B3 (SEQ ID NO: 4) as the H chain and

[0443] GpL16-k0 (SEQ ID NO: 5) as the L chain was prepared according to the method described in Reference Example 1. This control heterodimerized antibody is referred to as HeCon Ab. As assessed in Example 2, the binding activity of HeCon Ab to each FcγR was not significantly altered as compared to the native IgG1.

[0444] Prepared Ho Ab, He Ab, HeCon Ab, and HoCon Ab were assayed for the binding activity to FcγRIa, FcγRIIa(R), FcγRIIa(H), FcγRIIb, and FcγRIIIa according to the method described in Reference Example 2. As to the assay result for each FcγR, a graph was drawn by the following procedure. The binding activity of He Ab to each FcγR was divided by that of HeCon Ab, and then multiplied by 100; this is referred to as He/Con. The binding activity of Ho Ab to each FcγR is divided by that of HoCon Ab, and then multiplied by 100; this is referred to as Ho/Con. Regarding homodimerized and heterodimerized antibodies prepared using a GpH7-B3 variant comprising an alteration of interest, Ho/Con and He/Con values were plotted on the horizontal and vertical axes, respectively. The results for respective FcγRs, i.e., FcγRIa, FcγRIIa(R), FcγRIIa(H), FcγRIIb, and FcγRIIIa, are summarized in FIGS. 16 to 20. Based on the He/Con and Ho/Con values, each alteration can be interpreted as follows.

• [0445] 1. When the He/Con and Ho/Con values are 100: it means that the FcγR-binding activity of the heterodimerized antibody He Ab and homodimerized antibody Ho Ab, which comprise a GpH7-B3 variant introduced with a mutation of interest, are equivalent to the FcγR-binding activity of the control heterodimerized antibody and control homodimerized antibody, respectively.
• [0446] 2. When the He/Con and Ho/Con values are 100 or less: it means that the FcγR-binding activity of the heterodimerized antibody He Ab and homodimerized antibody Ho Ab, which comprise a GpH7-B3 variant introduced with a mutation of interest, are weaker than the FcγR-binding activity of the control heterodimerized antibody and control homodimerized antibody, respectively.
• [0447] 3. When the He/Con and Ho/Con values are 100 or more: it means that the FcγR-binding activity of the heterodimerized antibody He Ab and homodimerized antibody Ho Ab, which comprise a GpH7-B3 variant introduced with a mutation of interest, are stronger than the FcγR-binding activity of the control heterodimerized antibody and control homodimerized antibody, respectively.
• [0448] 4. When the He/Con value is greater than the Ho/Con value: it means that the FcγR-binding activity of the heterodimerized antibody He Ab comprising a GpH7-B3 variant introduced with a mutation of interest is stronger than the activity of the homodimerized antibody Ho Ab.
• [0449] 5. When the He/Con value is smaller than the Ho/Con value: it means that the FcγR-binding activity of the heterodimerized antibody He Ab comprising a GpH7-B3 variant introduced with a mutation of interest is weaker than the activity of the homodimerized antibody Ho Ab.

[0450] Based on the interpretation of items 1 to 5 above, the data points in FIGS. 16 to 20 can be classified as shown in FIG. 21.

[0451] When an alteration is present in Region i in FIG. 21, it means that the alteration, when introduced into both H chains of a homodimerized antibody, reduces its binding to FcγR, whereas the same alteration, when introduced into only one H chain of a heterodimerized antibody, has the effect of enhancing its binding to FcγR. That is, the alteration enhances the FcγR binding of the heterodimerized antibody only. As to each FcγR, alterations comprised in Region i are summarized in Table 2 (Tables 2-1 to 2-3), Table 3 (Tables 3-1 and 3-2), Table 4, Table 5, and Table 6.

[0452] When an alteration is present in Region ii in FIG. 21, it means that the alteration corresponding the point enhancing the FcγR binding, when introduced into both of H chains of a homodimerized antibody, or when introduced into only one H chain of a heterodimerized antibody, and the effect to enhance the binding is stronger in the heterodimerized antibody. Specifically, alterations in this region have a greater effect of enhancing the FcγR binding in the heterodimerized antibody than in the homodimerized antibody. As to each FcγR, alterations comprised in Region ii are summarized in Table 2 (Tables 2-1 to 2-3), Table 3 (Tables 3-1 and 3-2), Table 4, Table 5, and Table 6.

[0453] In each table, He/Con related to the binding activity to FcγRIa, FcγRIIa H, FcγRIIa R, FcγRIIb, and FcγRIIIa is referred to as He/Con_1a, He/Con_2aH, He/Con_2aR, He/Con_2b, and He/Con_3a, respectively; Ho/Con related to the binding activity to FcγRIa, FcγRIIa H, FcγRIIa R, FcγRIIb, and FcγRIIIa is referred to as Ho/Con_1 a, Ho/Con_2aH, Ho/Con_2aR, Ho/Con_2b, and Ho/Con_3a, respectively.

[0454] When an alteration is present in Region iii in FIG. 21, it means that the alteration corresponding to the point, when introduced into both H chains of a homodimerized antibody, or when introduced into only one H chain of a heterodimerized antibody, enhances the FcγR binding, and the binding enhancement effect is stronger in the homodimerized antibody. Specifically, alterations in this region have a greater effect of enhancing the FcγR binding in the homodimerized antibody than in the heterodimerized antibody.

[0455] In the tables below, amino acid alterations are indicated in a way such as A327_03V. The first alphabetical letter (for example, “A” of A327_03V) is a one-letter code representing the amino acid residue before alteration, and the following numeral (for example, “327” of A327_03V) indicates the position of alteration (EU numbering). The last numeral and an alphabetical letter (for example, “03V” of A327_03V) indicate an alphabetical letter where the amino acid residue after alteration is shown by a one-letter code (numeral representing the amino acid type+ alphabetical letter). Such are indicated as follows: 01G(Gly), 02A(Ala), 03V(Val), 04F(Phe), 05P(Pro), 06M(Met), 07I(Ile), 08L(Leu), 09D(Asp), 10E(Glu), 11K(Lys), 12R(Arg), 13S(Ser), 14T(Thr), 15Y(Tyr), 16H(His), 17C(Cys), 18N(Asn), 19Q(Gln), and 20W(Trp). Thus, for example, “A327_03V” means “a substitution of V for A at amino acid position 327 (EU numbering)”.

[0456]

TABLE 2-1
Ia
	NAME	Ho/Con_1a	He/Con_1a
REGION i
	A327_03V	94.4	103.2
	A327_06M	93.1	103.2
	A327_07I	95.5	103.5
	A327_10E	98.8	103.4
	A327_13S	86.1	102.4
	A330_05P	84.4	101.4
	A330_11K	94.1	103.4
	E269_01G	97.4	104.4
	E269_02A	96.2	104.4
	E269_03V	95.4	102.6
	E269_05P	95.7	102.3
	E269_07I	97.1	102.8
	E269_13S	95.9	103.1
	E269_14T	96.2	105.9
	E269_16H	98.4	105.8
	E269_18N	92.7	101.5
	E269_19Q	99.6	105.0
	E269_20W	98.9	105.2
	E333_02A	96.2	103.2
	E333_03V	98.4	105.0
	E333_04F	98.4	102.4
	E333_05P	99.2	102.3
	E333_06M	99.5	102.1
	E333_07I	99.9	102.0
	E333_08L	99.0	102.3
	E333_11K	95.8	102.1
	E333_12R	94.2	102.2
	E333_14T	98.7	102.1
	E333_15Y	100.0	101.2
	E333_16H	99.1	100.8
	G236_02A	92.4	100.2
	I332_12R	93.2	100.1
	L234_03V	98.2	104.4
	L234_04F	93.5	101.0
	L234_06M	96.5	103.8
	L234_07I	99.5	103.0
	L234_10E	79.6	100.5
	L234_15Y	98.9	100.1
	L328_03V	99.2	101.0
	L328_14T	98.6	100.4
	L328_19Q	99.3	101.1
	L328_20W	99.9	102.8
	P238_03V	97.9	100.4
	P238_04F	98.7	101.0
	P238_06M	100.0	102.3
	P238_08L	100.0	100.9
	P238_10E	91.8	101.5
	P238_15Y	99.0	100.9
	P271_14T	99.5	102.3
	P271_15Y	99.4	103.0
	P271_16H	99.4	102.1
	P329_01G	89.5	100.9
	P329_02A	98.2	102.3
	Q295_09D	96.8	100.1
	Q295_11K	99.9	101.3
	Q295_15Y	97.0	101.7
	Q295_16H	99.1	102.5
REGION ii
	A327_09D	101.6	102.8
	A330_08L	106.1	106.9
	A330_09D	108.0	108.6
	A330_14T	104.8	105.1
	A330_16H	103.6	105.8
	E269_04F	102.4	103.1
	E269_06M	101.3	103.6
	E269_08L	101.1	103.6
	E269_09D	106.3	106.6
	E269_15Y	100.2	106.4
	E333_01G	100.4	102.7
	E333_09D	103.2	103.5
	E333_13S	100.6	103.1
	G236_20W	104.4	105.2
	H268_02A	105.7	108.7
	H268_05P	107.6	109.0
	H268_07I	106.4	107.8
	H268_08L	105.1	107.4
	H268_12R	105.3	106.1
	H268_14T	109.0	112.2
	H268_20W	106.2	106.9
	I336_01G	104.3	108.7
	I336_02A	103.1	106.0
	I336_03V	104.8	105.2
	I336_04F	103.9	105.5
	I336_06M	104.0	108.2
	I336_10E	107.2	107.8
	I336_18N	103.6	107.8
	K326_12R	100.1	100.1
	K326_14T	102.9	104.1
	K326_15Y	101.2	105.9
	K326_16H	100.9	103.4
	K326_18N	101.9	105.1
	K334_06M	104.4	104.6
	K334_10E	106.9	109.8
	K334_12R	101.7	103.7
	K334_20W	104.4	105.6
	L328_10E	100.6	101.3
	P271_02A	102.5	103.5
	P271_03V	102.0	102.3
	P271_04F	100.3	101.8
	P271_06M	102.0	102.5
	P271_07I	101.9	102.5
	P271_11K	101.1	102.8
	P271_12R	101.2	102.4
	P271_13S	100.7	102.4
	P271_20W	101.5	102.6
	Q295_02A	104.1	104.3
	Q295_03V	101.6	104.1
	Q295_04F	100.3	102.2
	Q295_05P	101.1	103.4
	Q295_07I	101.2	103.0
	Q295_08L	103.3	104.6
	Q295_13S	101.4	102.0
	Q295_14T	102.5	102.7
	Q295_18N	100.8	102.4
	S239_01G	100.7	101.3

[0457]

	TABLE 2-2
	NAME	Ho/Con_1a	He/Con_1a
REGION i
	S239_18N	97.9	101.2
	S239_19Q	93.4	100.6
	S267_08L	99.9	101.4
	S267_16H	98.2	100.0
	S298_11K	90.4	109.3
	S298_12R	94.5	109.4
	S298_20W	71.5	104.1
	V266_19Q	91.2	100.2
	Y300_12R	96.5	104.6
REGION ii
	S239_09D	100.8	101.0
	S239_14T	100.9	101.1
	S267_01G	102.2	103.2
	S267_02A	108.5	108.9
	S267_03V	102.4	106.0
	S267_09D	105.8	109.7
	S298_01G	102.8	103.7
	S298_02A	106.5	106.6
	S298_03V	106.2	109.7
	S298_04F	106.7	109.0
	S298_06M	106.3	114.5
	S298_07I	103.3	107.0
	S298_08L	104.2	106.7
	S298_10E	106.1	108.2
	S298_14T	106.5	107.1
	S298_15Y	103.1	107.2
	S298_16H	106.8	108.8
	S298_18N	103.9	108.1
	S298_19Q	104.7	109.4
	S324_02A	102.6	102.7
	S324_03V	103.0	103.6
	S324_08L	102.7	103.2
	S324_11K	100.1	102.1
	S324_12R	100.7	101.7
	S324_14T	112.4	120.9
	S324_15Y	111.7	113.8
	S324_16H	110.6	112.6
	S324_18N	110.8	115.5
	S324_19Q	110.7	112.3
	S324_20W	111.5	114.3
	S337_01G	102.3	106.6
	S337_02A	103.1	105.8
	S337_03V	105.1	106.7
	S337_04F	104.7	106.2
	S337_06M	102.1	106.9
	S337_07I	105.3	105.3
	S337_08L	105.3	105.3
	S337_09D	105.5	105.7
	S337_10E	104.2	106.4
	S337_12R	105.4	105.8
	S337_15Y	103.6	103.8
	S337_19Q	104.6	104.6
	S337_20W	104.3	104.9
	T335_04F	104.8	105.0
	T335_05P	106.2	106.4
	T335_06M	106.0	106.7
	T335_07I	105.1	109.1
	T335_08L	105.4	114.6
	T335_09D	106.8	107.5
	T335_10E	107.8	108.8
	T335_11K	102.8	110.8
	T335_12R	106.0	112.8
	T335_15Y	106.3	107.2

[0458]

TABLE 2-3
REGION ii
	NAME	Ho/Con_1a	He/Con_1a
	T335_16H	104.5	105.7
	T335_18N	102.2	105.8
	T335_19Q	104.6	105.0
	T335_20W	104.2	106.2
	Y296_06M	103.8	104.0
	Y296_10E	105.7	105.8
	Y296_11K	102.0	104.7
	Y296_12R	102.9	106.3
	Y296_13S	104.6	105.1
	Y296_14T	104.8	105.1
	Y296_16H	105.3	105.8
	Y296_18N	105.0	105.6
	Y296_19Q	104.3	111.6
	Y296_20W	106.2	110.8
	Y300_01G	109.0	116.1
	Y300_02A	109.8	112.2
	Y300_03V	112.4	117.0
	Y300_06M	111.0	112.2
	Y300_08L	109.3	113.1
	Y300_09D	111.0	112.5
	Y300_10E	110.6	114.4
	Y300_11K	101.2	106.9
	Y300_13S	110.3	111.1
	Y300_14T	109.6	111.7
	Y300_19Q	110.1	112.5
	Y300_20W	110.5	112.4

[0459] With respect to FcγRIa, alterations comprised in Regions i and ii (FIG. 21) are shown.

[0460]

TABLE 3-1
IIa_R
	NAME	Ho/Con_2aR	He/Con_2aR
REGION i
	A330_05P	36.2	114.8
	A330_15Y	87.5	101.9
	A330_19Q	99.1	104.1
	E333_04F	92.4	102.1
	E333_05P	99.9	104.8
	E333_07I	98.3	104.4
	E333_08L	97.1	103.0
	E333_09D	96.5	103.1
	E333_15Y	96.2	100.7
	G236_14T	80.0	102.7
	G237_02A	84.0	126.4
	G237_06M	55.3	133.0
	G237_08L	56.6	133.4
	G237_09D	73.1	155.6
	G237_10E	22.5	116.5
	G237_13S	44.4	120.7
	G237_15Y	76.0	128.6
	G237_18N	73.6	150.1
	G237_19Q	14.3	120.6
	H268_05P	95.9	105.6
	H268_11K	93.4	100.4
	H268_12R	98.5	104.7
	I332_14T	99.6	105.0
	I336_06M	96.4	106.5
	I336_18N	94.5	104.1
	K326_12R	95.7	102.9
	K334_20W	96.4	104.8
	L234_04F	43.3	104.0
	L234_09D	42.8	122.5
	L234_10E	39.9	112.2
	L234_20W	53.8	107.8
	L235_04F	98.9	113.6
	L235_09D	73.0	100.7
	L328_02A	97.0	116.7
	L328_09D	68.8	119.0
	L328_10E	48.1	105.4
	N325_01G	26.7	106.8
	N325_03V	37.8	108.5
	N325_04F	65.7	120.3
	N325_07I	77.0	133.2
	N325_08L	95.0	143.8
	N325_09D	74.8	116.0
	N325_10E	49.9	104.7
	N325_14T	60.2	105.4
	N325_15Y	42.6	100.7
	N325_20W	56.0	133.8
	P238_06M	88.9	139.3
	P238_09D	13.0	148.2
	P238_10E	21.4	151.3
	P238_15Y	95.9	156.2
	P238_16H	25.5	121.5
	P238_19Q	19.4	103.9
	P238_20W	25.7	117.4
	P271_09D	92.4	109.7
	P271_10E	88.8	102.6
	P331_03V	89.1	114.4
REGION ii
	A327_09D	120.0	123.8
	A327_10E	103.7	110.2
	A330_01G	105.9	108.9
	A330_11K	105.7	110.1
	A330_12R	100.8	103.5
	E269_09D	103.2	105.3
	G237_04F	112.8	126.2
	G237_20W	107.1	138.3
	H268_01G	122.0	124.2
	H268_03V	104.9	108.6
	H268_15Y	106.3	109.6
	H268_20W	110.5	113.0
	I332_06M	105.4	108.8
	I336_07I	105.8	108.3
	I336_08L	105.4	110.2
	K326_03V	138.9	140.7
	K326_04F	132.7	135.1
	K326_07I	133.2	147.0
	K326_08L	130.7	136.8
	K326_15Y	133.8	136.4
	K326_18N	113.2	115.4
	K326_20W	119.9	130.8
	K334_02A	100.1	104.9
	K334_04F	109.5	110.4
	K334_05P	101.5	103.1
	K334_08L	105.1	106.2
	K334_10E	105.7	105.7
	K334_12R	110.2	110.6
	K334_13S	105.6	109.1
	K334_19Q	108.1	109.1
	L235_15Y	110.8	129.3
	L235_20W	114.4	130.9
	L328_13S	107.9	116.6
	L328_14T	118.2	118.9
	N325_06M	123.7	152.3
	N325_13S	125.8	143.4
	P238_03V	115.8	120.4
	P238_04F	146.3	174.4
	P271_08L	102.5	103.8
	P331_02A	102.2	106.0
	P331_04F	106.3	110.6
	P331_15Y	105.2	112.4
	P331_16H	100.2	114.0
	P331_20W	101.9	109.8
	S239_08L	105.6	121.5
	S239_10E	121.7	134.9
	S239_18N	103.0	110.7
	S267_03V	114.7	116.5
	S267_06M	102.3	122.5
	S267_09D	167.1	173.0
	S267_19Q	110.3	135.2
	S298_06M	102.5	108.4
	S298_08L	101.4	104.9
	S324_04F	100.6	104.0
	S324_06M	117.9	123.1
	S337_02A	101.5	105.0

[0461]

	TABLE 3-2
	NAME	Ho/Con_2aR	He/Con_2aR
REGION i
	P331_06M	79.8	105.9
	P331_07I	67.0	109.9
	P331_08L	74.4	101.7
	P331_09D	88.8	102.2
	P331_10E	94.6	113.3
	P331_13S	87.8	104.7
	P331_14T	80.8	104.3
	P331_18N	76.9	104.6
	Q295_06M	93.9	101.7
	Q295_10E	99.2	101.6
	S239_05P	51.2	105.7
	S239_06M	82.9	100.6
	S239_07I	85.3	101.9
	S239_14T	92.3	103.4
	S337_04F	98.3	104.6
	S337_08L	99.4	107.4
	S337_11K	94.6	102.9
	T335_01G	97.7	103.8
	T335_12R	94.0	100.2
	Y296_06M	84.0	101.2
	Y296_07I	83.1	101.0
	Y296_09D	94.5	105.7
	Y296_10E	90.2	102.6
	Y296_13S	88.1	101.9
	Y296_14T	90.3	103.1
	Y296_16H	89.5	104.7
	Y296_18N	93.7	104.1
	Y296_19Q	88.9	102.3
	Y300_18N	87.6	100.4
REGION ii
	S337_03V	102.0	106.1
	S337_06M	100.9	105.2
	S337_07I	102.8	108.9
	S337_09D	113.5	114.8
	S337_10E	110.0	111.6
	S337_12R	100.4	107.2
	S337_15Y	100.4	105.4
	S337_16H	106.6	108.6
	S337_18N	103.1	106.6
	S337_19Q	101.8	105.6
	S337_20W	104.7	110.5
	T335_02A	102.3	102.4
	T335_05P	103.6	103.6
	T335_07I	106.7	109.2
	T335_10E	108.7	111.3
	T335_13S	101.6	109.6
	T335_14T	106.0	109.8
	T335_15Y	107.1	111.4
	T335_16H	102.5	108.2
	T335_18N	100.4	106.3
	T335_19Q	102.1	106.0
	T335_20W	101.2	108.7
	V266_06M	145.6	149.9
	Y296_04F	106.4	109.4
	Y296_20W	106.9	109.8

[0462] With respect to FcγRIIa R, alterations comprised in Regions i and ii (FIG. 21) are shown.

[0463]

TABLE 4
IIa H
	NAME	Ho/Con_2aH	He/Con_2aH
REGION i
	A327_09D	90.1	104.7
	A330_19Q	99.2	102.2
	E333_04F	93.6	101.6
	E333_08L	96.5	101.5
	E333_13S	94.6	100.6
	E333_14T	90.4	100.4
	G236_04F	63.2	117.8
	G236_14T	94.2	103.2
	G236_15Y	80.4	136.1
	I336_03V	90.4	102.2
	I336_10E	93.4	101.5
	I336_18N	94.3	103.0
	K326_01G	98.2	100.5
	K326_07I	78.1	100.2
	K326_12R	98.8	100.6
	K334_13S	94.9	101.6
	K334_19Q	97.2	101.3
	L234_04F	54.2	119.8
	L234_15Y	43.8	120.2
	L234_20W	55.3	113.5
	S239_08L	87.0	104.8
	S239_10E	91.4	112.0
	S267_09D	91.5	105.4
	S267_10E	98.5	102.3
	S337_07I	94.0	100.8
	S337_08L	93.1	100.3
	S337_20W	96.6	101.9
	T335_18N	98.0	103.2
	T335_20W	98.7	104.8
	V266_08L	91.5	104.5
	Y296_04F	95.0	100.1
	Y296_09D	99.9	104.1
	Y296_16H	85.7	103.7
REGION ii
	A330_16H	100.6	101.4
	E333_09D	102.7	103.4
	G236_03V	110.7	119.4
	G236_07I	101.0	115.2
	H268_02A	110.9	113.8
	H268_18N	110.1	112.1
	H268_19Q	102.3	102.4
	I336_06M	100.3	106.1
	I336_07I	103.6	104.8
	I336_08L	100.9	105.5
	K326_06M	101.1	103.3
	K326_10E	101.0	101.8
	K326_19Q	100.5	101.4
	K334_04F	103.3	105.7
	K334_08L	103.4	104.2
	K334_16H	101.4	103.9
	L235_04F	100.7	110.6
	L235_15Y	107.1	121.5
	L235_20W	111.8	117.7
	L328_06M	100.2	103.5
	L328_14T	110.3	116.2
	L328_20W	111.8	112.6
	S239_09D	112.4	114.4
	S267_02A	119.5	119.8
	S324_02A	101.5	102.5
	S337_09D	106.9	107.1
	S337_10E	101.3	102.4
	T335_01G	100.9	103.1
	T335_07I	105.4	107.7
	T335_10E	105.2	107.6
	T335_13S	101.4	106.1
	T335_14T	104.3	107.9
	T335_15Y	101.5	106.3
	T335_16H	100.4	105.3
	T335_19Q	102.8	103.1
	V266_07I	104.3	105.5
	Y296_18N	100.1	103.8

[0464] With respect to FcγRIIa H, alterations comprised in Regions i and ii (FIG. 21) are shown.

[0465]

TABLE 5
IIb
	NAME	Ho/Con_2b	He/Con_2b
REGION i
	A327_07I	37.6	126.7
	A330_05P	35.3	136.7
	G237_02A	47.8	123.6
	G237_06M	35.3	129.9
	G237_08L	49.4	148.0
	G237_09D	92.9	222.7
	G237_10E	17.8	114.1
	G237_13S	25.7	110.8
	G237_15Y	83.1	152.5
	G237_18N	63.9	180.2
	G237_19Q	5.3	107.9
	I332_02A	81.8	103.3
	I336_06M	90.1	101.4
	L234_09D	68.5	150.4
	L234_10E	54.9	117.6
	L234_20W	80.6	114.4
	L235_09D	78.7	106.2
	N325_01G	20.2	100.7
	N325_03V	31.8	111.1
	N325_04F	65.7	143.2
	N325_07I	88.2	171.4
	N325_09D	77.0	124.3
	N325_20W	44.0	168.8
	P238_03V	96.6	120.1
	P238_06M	97.1	174.0
	P238_10E	97.9	232.0
	P238_16H	18.8	115.2
	P238_19Q	42.7	110.1
	P238_20W	12.6	122.7
	P271_10E	95.0	109.3
	P331_03V	86.6	118.7
	P331_06M	71.5	101.7
	P331_07I	63.3	114.5
	P331_10E	92.3	115.5
	S239_05P	61.0	101.5
	S239_06M	84.3	100.9
	S267_01G	97.0	104.6
	S337_07I	96.6	104.0
	S337_08L	90.7	100.2
	S337_15Y	93.4	100.2
	S337_18N	98.0	103.3
	S337_20W	99.6	108.8
	T335_13S	95.2	101.7
	T335_16H	97.2	102.4
	T335_18N	96.1	101.6
	T335_19Q	97.3	100.1
	T335_20W	94.4	103.9
REGION ii
	A327_18N	100.9	113.1
	A330_01G	102.1	103.5
	G237_04F	139.8	149.2
	G237_20W	165.7	193.4
	H268_03V	109.1	113.9
	H268_05P	109.0	116.1
	H268_15Y	103.8	106.3
	I332_14T	105.8	106.4
	I336_07I	100.6	103.4
	I336_08L	103.0	107.4
	K326_07I	169.6	198.8
	K326_08L	168.5	176.6
	K326_20W	131.8	148.5
	L235_04F	110.8	118.5
	L235_15Y	126.9	149.1
	L235_20W	116.3	147.3
	L328_02A	116.7	144.5
	L328_09D	158.7	198.8
	L328_10E	146.3	172.2
	L328_13S	146.6	149.2
	N325_06M	159.3	217.0
	N325_08L	105.8	196.7
	N325_13S	159.3	177.2
	P238_04F	209.1	312.0
	P238_09D	206.6	220.9
	P238_15Y	114.2	217.8
	P271_09D	100.1	120.1
	P331_15Y	110.7	114.4
	P331_16H	100.4	116.0
	P331_20W	110.9	111.2
	S239_03V	108.0	108.4
	S239_10E	175.1	180.6
	S267_06M	186.7	209.5
	S267_19Q	184.9	217.5
	S298_06M	101.4	101.8
	S324_06M	103.6	112.1
	S337_16H	102.7	104.6
	T335_07I	104.3	106.1
	T335_10E	107.7	108.4
	T335_14T	101.9	106.6
	T335_15Y	103.9	108.2
	V266_06M	231.3	251.2
	Y296_20W	102.6	103.3

[0466] With respect to FcγRIIb, alterations comprised in Regions i and ii (FIG. 21) are shown.

[0467]

TABLE 6
IIIa
	NAME	Ho/Con_3a	He/Con_3a
REGION i
	A330_05P	56.6	155.1
	A330_07I	93.0	103.1
	E333_02A	93.3	105.4
	E333_03V	86.5	103.0
	E333_04F	87.1	101.1
	E333_13S	95.1	102.7
	E333_20W	90.9	100.9
	G236_15Y	41.6	120.8
	G236_20W	58.3	135.4
	H268_18N	90.3	104.1
	I332_06M	95.4	101.2
	I336_08L	95.8	106.3
	L234_04F	49.9	118.1
	L234_07I	98.2	103.1
	L234_09D	52.8	116.8
	L234_10E	64.7	114.5
	L234_15Y	64.7	130.2
	L234_20W	37.1	106.8
	P238_09D	−0.1	105.8
	P238_10E	0.8	104.7
	P271_01G	88.0	113.2
	S324_02A	99.8	104.1
	S337_04F	95.6	101.4
	S337_14T	97.2	101.0
	S337_15Y	99.0	101.9
	S337_20W	87.8	100.8
	T335_13S	97.6	105.1
	T335_15Y	91.5	104.3
	T335_16H	99.5	103.9
	T335_19Q	97.7	102.2
	T335_20W	91.8	101.8
	V266_08L	90.5	118.0
REGION ii
	E333_09D	109.1	112.1
	H268_02A	118.0	122.2
	I332_01G	103.6	110.7
	I336_02A	103.6	111.0
	I336_03V	110.0	112.2
	I336_06M	107.0	108.9
	I336_07I	100.2	103.1
	I336_18N	100.6	104.0
	K326_07I	133.5	138.6
	K326_08L	113.9	114.2
	K334_01G	103.6	121.4
	K334_09D	149.7	159.7
	Q295_14T	108.2	114.7
	S267_02A	145.3	150.4
	S267_09D	159.7	180.8
	S298_14T	106.7	112.6
	S324_15Y	100.1	101.4
	S324_16H	103.1	103.7
	S337_09D	105.5	109.9
	S337_10E	100.2	105.0
	S337_18N	101.5	104.1
	T335_07I	107.0	107.2
	T335_10E	102.4	106.9
	T335_14T	102.5	108.5

[0468] With respect to FcγRIIIa, alterations comprised in Regions i and ii (FIG. 21) are shown.

[0469] The alterations revealed in this Example, together with Example 2, are thought to support the concept of the improvement of FcγR recognition ability by the heterodimerized antibody described in Example 1. Alterations in Region i in FIG. 21 are thought to reduce the FcγR-binding activity when introduced into both H chains in a conventional way, and have not been recognized as alterations that enhance the binding. The present inventors, however, succeeded in demonstrating that such alterations also improve the FcγR-binding activity by introducing them into only one of the two H chains.

[0470] As described in Example 3, the effect of an alteration on the FcγR binding of a heterodimerized antibody differs in the direction depending on alteration. For this reason, when several alterations are combined to further enhance or reduce the FcγR binding of a heterodimerized antibody, it is necessary to unify the direction of the effect of each alteration on the FcγR binding. If two alterations were introduced in a way that their effects to enhance the FcγR binding of an antibody are different in the direction, the effects of the alterations would annihilate each other and the effect of enhancing the binding would not be observed in spite of the combination of alterations for enhancing the FcγR binding. However, there is no method for predicting in advance which H chain should be introduced with an alteration. Thus, in order to find an appropriate method of combination, generally antibodies in which alterations of interest are introduced into the same chain or different H chains need to be prepared, and they are compared to each other for the FcγR-binding activity. For example, when all of three different types of alterations are introduced into a homodimerized antibody, a single type of antibody in which each alteration has been introduced into both H chains may be prepared. In the case of a heterodimerized antibody, however, it is necessary to determine which H chain should be introduced with each alteration. In this case, as shown in FIG. 22, there are a maximum of four combinations to be tried; i.e., the number of variants that should be prepared for assessment of the alteration combinations is very large, and this is inefficient. Thus, the present inventors tested a method for identifying the direction in which an alteration of interest has an effect on the FcγR binding of an antibody by combining the alteration with P329R that inhibits the FcγR binding of an antibody from only one direction.

[0471] The present inventors examined how the FcγRIIIa binding was altered when alterations L234Y, G236W, and S298A that enhance the FcγRIIIa binding were each introduced into the same H chain as P329R or the other H chain of a heterodimerized antibody found in Example 4. First, variants of interest were expressed and prepared according to the method described in Reference Example 1, using GpH7-A5 as an H chain; and GpL16-k0 as the L chain; and as the other H chain, GpH7-HA5 (SEQ ID NO: 19), GpH7-HA6 (SEQ ID NO: 20), and GpH7-HA 11 (SEQ ID NO: 21) resulting from the introduction of L234Y, G236W, and S298A, respectively, into GpH7-B12 introduced with the alteration P329R. Furthermore, antibodies of interest were expressed and prepared according to the method described in Reference Example 1, using as an H chain, GpH7-A48 (SEQ ID NO: 16) resulting from the introduction of P329R into GpH7-A5; and GpL16-k0 as the L chain; and as the other H chain, GpH7-B3-01-15Y (SEQ ID NO: 22), GpH7-B3-03-20W (SEQ ID NO: 23), and GpH7-B3-15-02A (SEQ ID NO: 24) resulting from the introduction of L234Y, G236W, and S298A, respectively, into GpH7-B3. The antibodies thus obtained were named GpH7-A5/GpH7-HA5/GpL16-k0 (SEQ ID NOs: 3, 19, and 5, respectively), GpH7-A5/GpH7-HA6/GpL16-k0 (SEQ ID NOs: 3, 20, and 5, respectively), GpH7-A5/GpH7-HA11/GpL16-k0 (SEQ ID NOs: 3, 21, and 5, respectively), GpH7-A48/GpH7-B3-01-15Y/GpL16-k0 (SEQ ID NOs: 16, 22, and 5, respectively), GpH7-A48/GpH7-B3-03-20W/GpL16-k0 (SEQ ID NOs: 16, 23, and 5, respectively), and GpH7-A48/GpH7-B3-15-02A/GpL16-k0 (SEQ ID NO: 16, 24, and 5, respectively). The variants were compared to each other for the FcγRIIIa-binding activity according to the method described in Reference Example 2. The effects of the combination of L234Y, G236W, and S298A with P329R are summarized in Table 7.

[0472]

TABLE 7
					FcγRIIIa
		MUTATION		MUTATION	BINDING
SAMPLE	H1	SITE	H2	SITE	ACTIVITY
GpH7-A5/GpH7-B3/GpL16-k0	A5	—	B3	—	—	100
(SEQ ID NO: 3, 4, 5)
GpH7-A48/GpH7-B3-01-15Y/GpL16-k0	A48	P329R	B3-01-15Y	L234Y	—	11
(SEQ ID NO: 16, 22, 5)
GpH7-A5/GpH7-HA5/GpL16-k0	A5	—	HA5	L234Y	P329R	60
(SEQ ID NO: 3, 19, 5)
GpH7-A48/GpH7-B3-03-20W/GpL16-k0	A48	P329R	B3-03-20W	G236W	—	13
(SEQ ID NO: 16, 23, 5)
GpH7-A5/GpH7-HA6/GpL16-k0	A5	—	HA6	G236W	P329R	56
(SEQ ID NO: 3, 20, 5)
GpH7-A48/GpH7-B3-15-02A/GpL16-k0	A48	P329R	B3-15-02A	S298A	—	47
(SEQ ID NO: 16, 24, 5)
GpH7-A5/GpH7-HA11/GpL16-k0	A5	—	HA11	S298A	P329R	84
(SEQ ID NO: 3, 21, 5)

[0473] The column “SAMPLE” indicates antibody names; the columns “H1” and “H2” indicate names of the H chain constant region of respective antibodies; and the column “MUTATION SITE” indicates mutations that are different in comparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is no particular mutation). The FcγRIIIa-binding activity is indicated as a relative binding activity when setting the FcγRIIIa binding of GpH7-A5/GpH7-B3/GpL16-k0 (SEQ ID NO: 3, 4, and 5, respectively) as 100. The SEQ ID NOs are also shown for the amino acid sequences of the H chain and L chain of each antibody.

[0474] Based on the result, the alterations were compared for the FcγRIIIa-binding activity when each alteration has been introduced into the same H chain as P329R or the H chain different from that introduced with P329R. In the case of L234Y, the binding activity of GpH7-A5/GpH7-HA5/GpL16-k0 corresponding to the former was 60, while the binding activity of GpH7-A48/GpH7-B3-01-15Y/GpL16-k0 corresponding to the latter was 11; thus, the FcγR binding was inhibited when the alteration was introduced into the H chain different from that introduced with P329R. In the case of G236W, the binding activity of GpH7-A5/GpH7-HA6/GpL16-k0 corresponding to the former was 56, while the binding activity of GpH7-A48/GpH7-B3-03-20W/GpL16-k0 corresponding to the latter was 13; thus, the FcγR binding was inhibited when the alteration was introduced into the H chain different from that introduced with P329R. In the case of S298A, the binding activity of GpH7-A5/GpH7-HA11/GpL16-k0 corresponding to the former was 84, while the binding activity of GpH7-A48/GpH7-B3-15-02A/GpL16-k0 corresponding to the latter was 47; thus, the FcγR-binding activity was inhibited when the alteration was introduced into the H chain different from that introduced with P329R. For all the alterations, the FcγRIIIa binding was inhibited when an alteration was introduced into the H chain different from that introduced with P329R, as described as the latter case. If the H chain introduced with P329R corresponded to the H_A chain of FIG. 3, P329R is thought to inhibit the binding in the X-direction. Since combinations that resulted in significant inhibition of the binding are those when L234Y, G236W, or S298A were introduced into the H chain different from that introduced with P329R, in this case the alterations would have been introduced into the H_B chain. Since the effect to enhance the FcγRIIIa binding was markedly inhibited when any of the alterations L234Y, G236W, and S298A was introduced into the H chain different from that introduced with P329R, all the alterations, when introduced into the H_B chain, enhanced the FcγRIIIa binding from the X-direction, which is inhibited by P329R. Thus, it was thought that the FcγRIIIa binding can be further enhanced by introducing these alterations into the same H chain.

[0475] The above-described hypothesis was tested by assessing whether the FcγR binding is enhanced by introducing two of L234Y, G236W, and S298A into the same H chain or different H chains. Expression vectors inserted with GpH7-TA1 (SEQ ID NO: 25), GpH7-TA2 (SEQ ID NO: 26), and GpH7-TA3 (SEQ ID NO: 27), resulting from the introduction of L234Y, G236W, and S298A into GpH7-A5, respectively; and expression vectors inserted with GpH7-B3-01-15Y (SEQ ID NO: 22), GpH7-B3-03-20W (SEQ ID NO: 23), and GpH7-B3-15-02A (SEQ ID NO: 24), resulting from the introduction of L234Y, G236W, and S298A into GpH7-B3, respectively, were constructed according to the method described in Reference Example 1. In addition, expression vectors inserted with the following were constructed: GpH7-TA4 (SEQ ID NO: 28) resulting from the introduction of L234Y and G236W into GpH7-A5; GpH7-TA5 (SEQ ID NO: 29) resulting from the introduction of L234Y and S298A into GpH7-A5; and GpH7-TA6 (SEQ ID NO: 30) resulting from the introduction of G236W and S298A into GpH7-A5. These were combined in a manner as shown in Table 8, and GpL16-k0 was added as the L chain to each combination. Antibodies of interest were expressed and prepared according to the method described in Reference Example 1. Regarding the expressed samples, information on the H chains and mutation sites, and assay results on the FcγRIIIa binding of the antibody are summarized in Table 8.

[0476]

TABLE 8
					FcγRIIIa
		MUTATION		MUTATION	BINDING
SAMPLE	H1	SITE	H2	SITE	ACTIVITY
GpH7-A5/GpH7-B3-01-15Y/GpL16-k0	A5	—	—	B3-01-15Y	L234Y	131
(SEQ ID NO: 3, 22, 5)
GpH7-A5/GpH7-B3-03-20W/GpL16-k0	A5	—	—	B3-03-20W	G236W	140
(SEQ ID NO: 3, 23, 5)
GpH7-A5/GpH7-B3-15-02A/GpL16-k0	A5	—	—	B3-15-02A	S298A	163
(SEQ ID NO: 3, 24, 5)
GpH7-TA2/GpH7-B3-01-15Y/GpL16-k0	TA2	—	G236W	B3-01-15Y	L234Y	130
(SEQ ID NO: 26, 22, 5)
GpH7-TA4/GpH7-B3/GpL16-k0	TA4	L234Y	G236W	B3	—	168
(SEQ ID NO: 28, 4, 5)
GpH7-TA1/GpH7-B3-15-02A/GpL16-k0	TA1	L234Y	—	B3-15-02A	S298A	142
(SEQ ID NO: 25, 24, 5)
GpH7-TA5/GpH7-B3/GpL16-k0	TA5	L234Y	S298A	B3	—	208
(SEQ ID NO: 29, 4, 5)
GpH7-TA3/GpH7-B3-03-20W/GpL16-k0	TA3	—	S298A	B3-03-20W	G236W	70
(SEQ ID NO: 27, 23, 5)
GpH7-TA6/GpH7-B3/GpL16-k0	TA6	G236W	S298A	B3	—	228
(SEQ ID NO: 30, 4, 5)

[0477] The column “SAMPLE” indicates antibody names; the columns “H1” and “H2” indicate names of the H chain constant region of respective antibodies; and the column “MUTATION SITE” indicates mutations that are different in comparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is no particular mutation). The FcγRIIIa-binding activity is indicated as a relative binding activity when setting the FcγRIIIa binding of GpH7-A5/GpH7-B3/GpL16-k0 as 100. The SEQ ID NOs are also shown for the amino acid sequences of the H chain and L chain of each antibody.

[0478] The effect of the combination of L234Y and G236W was assessed based on the result shown in Table 8. The FcγRIIIa-binding activity of GpH7-TA2/GpH7-B3-01-15Y/GpL16-k0 in which L234Y and G236W were introduced into different H chains was 130, and was not increased as compared to the binding activity of 131 of GpH7-A5/GpH7-B3-01-15Y/GpL16-k0 introduced with L234Y alone, while it was decreased as compared to the binding activity of 140 of GpH7-A5/GpH7-B3-03-20W/GpL16-k0 introduced with G236W alone. Meanwhile, the binding activity of GpH7-TA4/GpH7-B3/GpL16-k0 in which L234Y and G236W were introduced into the same H chain was 168, and was increased as compared to the binding activity of GpH7-A5/GpH7-B3-01-15Y/GpL16-k0 introduced with L234Y alone and GpH7-A5/GpH7-B3-03-20W/GpL16-k0 introduced with G236W alone. As predicted, this result demonstrates that L234Y and G236W, when introduced into the same H chain, further increase the FcγRIIIa-binding activity.

[0479] Then, the effect of the combination of L234Y and S298A was assessed based on Table 8. The FcγRIIIa-binding activity of GpH7-TA1/GpH7-B3-15-02A/GpL16-k0 in which L234Y and S298A were introduced into different H chains was 142, and was increased as compared to the binding activity of 131 of GpH7-A5/GpH7-B3-01-15Y/GpL16-k0 introduced with L234Y alone, while it was decreased as compared to the binding activity of 163 of GpH7-A5/GpH7-B3-15-02A/GpL16-k0 introduced with S298A alone. That is, since the binding activity of GpH7-TA1/GpH7-B3-15-02A/GpL16-k0 was not increased as compared to when S298A alone was introduced, it can be said that the effect to further increase the FcγRIIIa-binding activity was not provided when S298A and L234Y were introduced into different H chains. Meanwhile, the FcγRIIIa-binding activity of GpH7-TA5/GpH7-B3/GpL16-k0 in which L234Y and S298A were introduced into the same H chain was 208, and was increased as compared to GpH7-A5/GpH7-B3-01-15Y/GpL16-k0 introduced with L234Y alone and GpH7-A5/GpH7-B3-15-02A/GpL16-k0 introduced with S298A alone. As predicted, this result demonstrates that L234Y and S298A, when introduced into the same H chain, further increase the FcγRIIIa-binding activity.

[0480] Next, the effect of the combination of G236W and S298A was assessed based on Table 8. The FcγRIIIa-binding activity of GpH7-TA3/GpH7-B3-03-20W/GpL16-k0 in which G236W and S298A were introduced into different H chains was 70, and was decreased as compared to the binding activity of 140 of GpH7-A5/GpH7-B3-03-20W/GpL16-k0 introduced with G236W alone, and the binding activity of 163 of GpH7-A5/GpH7-B3-15-02A/GpL16-k0 introduced with S298A alone. Meanwhile, the binding activity of GpH7-TA6/GpH7-B3/GpL16-k0 in which G236W and S298A were introduced into the same H chain was 228, and was increased as compared to GpH7-A5/GpH7-B3-03-20W/GpL16-k0 introduced with G236W alone and GpH7-A5/GpH7-B3-15-02A/GpL16-k0 introduced with S298A alone. As predicted, this result demonstrates that G236W and S298A, when introduced into the same H chain, further increase the FcγRIIIa-binding activity.

[0481] As was initially predicted, these results demonstrate that L234Y, G236W, and S298A, only when each is introduced into the same H chain, enhance the binding. This data supports that L234Y, G236W, and S298A, when being present in the same chain, enhance the FcγR binding from the same direction. That is, this shows that one can determine a method for appropriately combining two alterations based on a result predicted from the result of comparison of the FcγRIIIa-binding activity, combining P329R with each of the alterations. In other words, combining with P329R is a useful method for predicting a method of combining alterations in heterodimerized antibodies. This method can be used to reveal other useful combinations of alterations.

[0482] Combinations of two or more alterations were considered based on the result of comparison of the FcγRIIIa-binding activity combining P329R with each of the alterations. It was demonstrated that L234Y and G236W, G236W and S298A, and S298A and L234Y, when introduced into the same H chain, respectively enhance the interaction with FcγRIIIa from the same direction. That is, from this result, it was thought that L234Y, G236W, and S298A all enhance the FcγRIIIa-binding activity from the same direction, and thus these alterations, when introduced into the same H chain, were expected to maximally enhance the FcγRIIIa-binding activity. To assess this hypothesis, GpH7-TA4, GpH7-TA5, and GpH7-TA6 were constructed by introducing into GpH7-A5 each of the alteration groups of L234Y and G236W, L234Y and S298A, and G236W and S298A; GpH7-B3-01-15Y, GpH7-B3-03-20W, and GpH7-B3-15-02A were constructed by introducing L234Y, G236W, and S298A, respectively, into GpH7-B3; expression vectors inserted with the above constructs were prepared according to Reference Example 1. The vectors were combined in a way that the three alterations, L234Y, G236W, and S298A, were introduced into either H chain; and GpL16-k0 was added as the L chain; and antibodies of interest were expressed and prepared according to the method described in Reference Example 1. Furthermore, GpH7-TA7 (SEQ ID NO: 31) resulting from the introduction of three alterations, L234Y, G236W, and S298A into GpH7-A5 was constructed, and combined with GpH7-B3 and GpL16-k0 to express and purify an antibody of interest according to the method described in Reference Example 1. A list of antibodies prepared as described herein and the result of comparison of the FcγRIIIa-binding activity between antibodies are shown in Table 9.

[0483]

TABLE 9
					FcγRIIIa
				MUTATION	BINDING
SAMPLE	H1	MUTATION SITE	H2	SITE	ACTIVITY
GpH7-TA4/GpH7-B3-15-02A/GpL16-k0	TA4	L234Y	G236W	—	B3-15-02A	S298A	151
(SEQ ID NO: 28, 24, 5)
GpH7-TA5/GpH7-B3-03-20W/GpL16-k0	TA5	L234Y	S298A	—	B3-03-20W	G236W	97
(SEQ ID NO: 29, 23, 5)
GpH7-TA6/GpH7-B3-01-15Y/GpL16-k0	TA6	G236W	S298A	—	B3-01-15Y	L234Y	103
(SEQ ID NO: 30, 22, 5)
GpH7-TA7/GpH7-B3/GpL16-k0	TA7	L234Y	G236W	S298A	B3	—	201
(SEQ ID NO: 31, 4, 5)

[0484] The column “SAMPLE” indicates antibody names; the columns “H1” and “H2” indicate names of the H chain constant region of respective antibodies; and the column “MUTATION SITE” indicates mutations that are different in comparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is no particular mutation). The FcγRIIIa-binding activity is indicated as a relative binding activity when setting the FcγRIIIa-binding activity of GpH7-A5/GpH7-B3/GpL16-k0 as 100. The SEQ ID NOs are also shown for the amino acid sequences of the H chain and L chain of each antibody.

[0485] As was predicted from the result of comparison of the FcγRIIIa-binding activity combining P329R with each alteration, GpH7-TA7/GpH7-B3/GpL16-k0 in which L234Y, G236W, and S298A were introduced into the same H chain exhibited most strongly enhanced FcγRIIIa binding. That is, the result demonstrates that one can predict a method for appropriately combining two or more alterations by comparing the FcγRIIIa-binding activity combining P329R with each alteration.

[0486] The results shown in Tables 7, 8, and 9 of Example 5 demonstrate that hetero-alterations which each alone enhances the FcγRIIIa-binding activity, when appropriately combined together, can further enhance the FcγRIIIa binding. Specifically, it was demonstrated that the alterations L234Y, G236W, and S298A, when introduced into the same H chain, further increase the FcγR-binding activity.

[0487] Then, the present inventors examined whether, even multiple alterations are combined, the resulting heterodimerized antibody still had the characteristic of the heterodimerized antibody that the heterodimerized antibody introduced with multiple alterations exhibits more strongly enhanced FcγR binding than the homodimerized antibody introduced with the corresponding multiple alterations. Specifically, GpH7-TA7 and GpH7-TA45 (SEQ ID NO: 32) resulting from the introduction of L234Y, G236W, and S298A into GpH7-A5 and GpH7-B3, respectively, were prepared according to the method described in Reference Example 1. As shown in Table 10, the heterodimerized antibodies GpH7-TA7/GpH7-B3/GpL16-k0 and GpH7-A5/GpH7-TA45/GpL16-k0 in which L234Y, G236W, and S298A were introduced into only one H chain, and the homodimerized antibody GpH7-TA7/GpH7-TA45/GpL16-k0 in which L234Y, G236W, and S298A were introduced into both H chains were expressed and purified according to the method described in Reference Example 1. These antibodies were compared for the FcγRIIIa binding according to the method described in Reference Example 2 (Table 10).

[0488]

TABLE 10
					FcγR
					BINDING
SAMPLE	H1	MUTATION SITE	H2	MUTATION SITE	ACTIVITY
GpH7-A5/GpH7-B3/GpL16-k0	A5	—	—	—	B3	—	—	—	100
(SEQ ID NO: 3, 4, 5)
GpH7-TA7/GpH7-B3/GpL16-k0	TA7	L234Y	G236W	S298A	B3	—	—	—	210
(SEQ ID NO: 31, 4, 5)
GpH7-A5/GpH7-TA45/GpL16-k0	A5	—	—	—	TA45	L234Y	G236W	S298A	225
(SEQ ID NO: 3, 32, 5)
GpH7-TA7/GpH7-TA45/GpL16-k0	TA7	L234Y	G236W	S298A	TA45	L235Y	G237W	S299A	48
(SEQ ID NO: 31, 32, 5)

[0489] The column “SAMPLE” indicates antibody names; the columns “H1” and “H2” indicate names of the H chain constant region of respective antibodies; and the column “MUTATION SITE” indicates mutations that are different in comparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is no mutation). The FcγRIIIa-binding activity is indicated as a relative binding activity when setting the FcγRIIIa binding of GpH7-A5/GpH7-B3/GpL16-k0 as 100. The SEQ ID NOs are also shown for the amino acid sequences of H chain and L chain of each antibody.

[0490] The result shown in Table 10 demonstrates that the homodimerized antibody GpH7-TA7/GpH7-TA45/GpL16-k0 in which both of the H chains were introduced with L234Y, G236W, and S298A exhibited reduced FcγRIIIa binding as compared to the heterodimerized antibodies GpH7-TA7/GpH7-B3/GpL16-k0 and GpH7-A5/GpH7-TA45/GpL16-k0 in which only one of the H chains were introduced with L234Y, G236W, and S298A. This shows that the characteristic of the alterations L234Y, G236W, and S298A that they increase the FcγR-binding activity of a heterodimerized antibody but reduces the FcγR-binding activity of a homodimerized antibody, is retained even when multiple alterations are combined together.

[0491] Then, the present inventors examined whether a heterodimerized antibody in which only one of the H chains was introduced with L234Y, G236W, and S298A retains the directionality of the FcγRIIIa binding discussed in Example 3.

[0492] GpH7-TA8 (SEQ ID NO: 33) and GpH7-B12 (SEQ ID NO: 12) resulting from the introduction of P329R into GpH7-TA7 and GpH7-B3, respectively, were prepared according to the method described in Reference Example 1. As shown in Table 11, the heterodimerized antibody GpH7-TA8/GpH7-B3/GpL16-k0 in which L234Y, G236W, and S298A were introduced into the same H chain as P329R, and the heterodimerized antibody GpH7-TA7/GpH7-B12/GpL16-k0 in which L234Y, G236W, and S298A were introduced into the H chain different from that introduced with P329R were prepared according to the method described in Reference Example 1. As to the FcγRIII-binding activity, these antibodies were compared with the heterodimerized antibody GpH7-TA7/GpH7-B3/GpL16-k0 in which one of the H chains was introduced with L234Y, G236W, and S298A according to the method described in Reference Example 2 (Table 11).

[0493]

TABLE 11
					FcγRIIIa
				MUTATION	BINDING
SAMPLE	H1	MUTATION SITE	H2	SITE	ACTIVITY
GpH7-A5/GpH7-B3/GpL16-k0	A5	—	—	—	—	B3	—	100
(SEQ ID NO: 3, 4, 5)
GpH7-TA7/GpH7-B3/GpL16-k0	TA7	L234Y	G236W	S298A	—	B3	—	210
(SEQ ID NO: 31, 4, 5)
GpH7-TA7/GpH7-B12/GpL16-k0	TA7	L234Y	G236W	S298A	—	B12	P329R	11
(SEQ ID NO: 31, 12, 5)
GpH7-TA8/GpH7-B3/GpL16-k0	TA8	L234Y	G236W	S298A	P329R	B3	—	150
(SEQ ID NO: 33, 4, 5)

[0494] The column “SAMPLE” indicates antibody names; the columns “H1” and “H2” indicate names of the H chain constant region of respective antibodies; and the column “MUTATION SITE” indicates mutations that are different in comparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is no particular mutation). The FcγRIIIa-binding activity is indicated as a relative binding activity when setting the FcγRIIIa binding of GpH7-A5/GpH7-B3/GpL16-k0 as 100. The SEQ ID NOs are also shown for the amino acid sequences of H chain and L chain of each antibody.

[0495] The result shown in Table 11 demonstrates that the heterodimerized antibody GpH7-TA8/GpH7-B3/GpL16-k0 in which the group of alterations L234Y, G236W, and S298A was introduced into the same H chain as P329R, and the heterodimerized antibody GpH7-TA7/GpH7-B12/GpL16-k0 in which the group of alterations L234Y, G236W, and S298A was introduced into the H chain different from that introduced with P329R both exhibited reduced FcγRIIIa-binding activities as compared to GpH7-TA7/GpH7-B3/GpL16-k0. The FcγRIIIa-binding activity of GpH7-TA7/GpH7-B12/GpL16-k0 was 11, and was markedly reduced as compared to the activity of 150 of GpH7-TA8/GpH7-B3/GpL16-k0. This result shows the characteristic that the FcγR-binding activity is markedly reduced if multiple of the alterations L234Y, G236W, and S298A are introduced into an H chain, when they are introduced into the H chain different from that introduced with P329R, which was observed in Example 3 for the alterations L234Y, G236W, and S298A.

[0496] The above findings demonstrate that an appropriate combination of alterations that enhance the FcγR binding can further increase the FcγR-binding activity while keeping the characteristic of a heterodimerized antibody.

[0497] Alterations that increase the ADCC activity by enhancement of the FcγRIIIa binding are already known. For example, the alterations S239D, I332E, and A330L are known as mutations that enhance the FcγRIIIa binding the most when introduced into both H chains of an antibody (Proc. Natl. Acad. Sci. USA, 103, 4005-4010, 2006). The antitumor activity of an antibody was demonstrated to be increased by the enhancement of the antibody-dependent cellular cytotoxic (ADCC) activity. Enhancing the FcγRIIIa-binding of an antibody is an effective means to enhance the utility of antibodies as pharmaceuticals. However, as shown in the Examples above, there are thought to be limitations on the enhancement of the FcγR-binding activity using homodimerized antibodies. Thus, it was thought that the FcγR-binding activity can be further increased by hetero-alteration.

[0498] As mentioned in Example 1, the Fc domain of an antibody interacts with FcγR in an asymmetric manner. In the case of an antibody introduced with the alterations S239D, I332E, and A330L, in view of the three-dimensional structure, it is thought that, in the H_A chain, all of the altered residues of S239D, I332E, and A330L are involved in the enhancement of the interaction with FcγR, while in the H_B chain, residues other than S239D are out of contact with FcγR and do not contribute to the enhancement of the FcγR-binding activity (FIG. 23). That is, in light of the asymmetry of interaction between the Fc domain and FcγR, each alteration introduced by the conventional antibody alteration technology is not sufficiently capable of interaction with FcγR and this is thought to be sufficient for optimizing the antibody/FcγR interaction. For example, in the case of above-described alterations S239D, I332E, and A330L, it is thought that the FcγRIIIa binding can be further enhanced by introducing alterations that enhance the interaction with FcγRIIIa on the H_B chain side, instead of introducing the above alterations into H_B chain. That is, the FcγR binding could be further enhanced by using the technology of the present invention for producing heterodimerized antibodies by introducing different alterations to each antibody H chain (hereinafter referred to as “heterodimerized antibody technology”) than using the technology for introducing the same alteration to both antibody H chain (hereinafter referred to as “prior art” or “homodimerized antibody technology”).

[0499] In view of the three-dimensional structure of the Fc/FcγRIIIa complex, contrary to S239D, I332E, and A330L, it is thought that S298 interacts with FcγR only in the H_B chain as shown in FIG. 23 (JBC, 276: 16469-16477, 2001). Thus, it is thought that, when an alteration is introduced at S298, the residue after substitution mutation also interacts with FcγRIIIa on the H_B chain side. As seen in Example 5, L234Y and G236W are thought to enhance the interaction with FcγR from the same direction as S298A. That is, it is thought that, when S239D, A330L, and I332E are introduced into the same H chain, and L234Y, G236W, and S298A are introduced into the other H chain, all of the introduced alterations can interact with FcγR at the same time, resulting in further enhancement of the interaction with FcγR.

[0500] To assess the above hypothesis, the present inventors carried out the following experiments. The direction of FcγR recognition by each alteration was determined according to the method described in Example 5, using antibodies having an H chain introduced with the alteration L234Y, G236W, S298A, S239D, A330L, or I332E, and P329R introduced into the same or different H chain. Expression vectors inserted with the following were constructed according to the method described in Reference Example 1:

[0501] GpH7-A5; GpH7-A48 (SEQ ID NO: 16) resulting from the introduction of P329R into GpH7-A5; GpH7-HA7 (SEQ ID NO: 34) resulting from the introduction of S239D and P329R into GpH7-B3; GpH7-HA15 (SEQ ID NO: 35) resulting from the introduction of A330L and P329R into GpH7-B3; GpH7-HA18 (SEQ ID NO: 36) resulting from the introduction of I332E and P329R into GpH7-B3; GpH7-HA5 (SEQ ID NO: 19) resulting from the introduction of L234Y and P329R into GpH7-B3; GpH7-HA6 (SEQ ID NO: 20) resulting from the introduction of G236W and P329R into GpH7-B3; GpH7-HA11 (SEQ ID NO: 21) resulting from the introduction of S298A and P329R into GpH7-B3; GpH7-B3-06-09D (SEQ ID NO: 37) resulting from the introduction of S239D into GpH7-B3; GpH7-B3-20-08L (SEQ ID NO: 38) resulting from the introduction of A330L into GpH7-B3; GpH7-B3-22-10E (SEQ ID NO: 39) resulting from the introduction of I332E into GpH7-B3; GpH7-B3-01-15Y (SEQ ID NO: 22) resulting from the introduction of L234Y into GpH7-B3; GpH7-B3-03-20W (SEQ ID NO: 23) resulting from the introduction of G236W into GpH7-B3; and GpH7-B3-15-02A (SEQ ID NO: 24) resulting from the introduction of S298A into GpH7-B3. The expression vectors for the respective H chains were combined in a way that each of the alterations L234Y, G236W, S298A, S239D, A330L, and I332E is present in the same H chain as P329R or in the different H chain from P329R, and the expression vector GpL16-k0 for the L chain was combined with them. Antibodies of interest were expressed and prepared according to the method described in Reference Example 1. The prepared antibodies were used to assay the FcγRIIIa-binding activity. The result of assessment using P329R on the direction of FcγRIIIa recognition by L234Y, G236W, S298A, S239D, A330L, and I332E is summarized in Table 12.

[0502]

TABLE 12
					FcγRIIIa
		MUTATION			BINDING
SAMPLE	H1	SITE	H2	MUTATION SITE	ACTIVITY
GpH7-A5/GpH7-HA7/GpL16-k0	A5	—	HA7	S239D	P329R	3
(SEQ ID NO: 3, 34, 5)
GpH7-A5/GpH7-HA15/GpL16-k0	A5	—	HA15	A330L	P329R	32
(SEQ ID NO: 3, 35, 5)
GpH7-A5/GpH7-HA18/GpL16-k0	A5	—	HA18	I332E	P329R	35
(SEQ ID NO: 3, 36, 5)
GpH7-A5/GpH7-HA5/GpL16-k0	A5	—	HA5	L234Y	P329R	60
(SEQ ID NO: 3, 19, 5)
GpH7-A5/GpH7-HA6/GpL16-k0	A5	—	HA6	G236W	P329R	56
(SEQ ID NO: 3, 20, 5)
GpH7-A5/GpH7-HA11/GpL16-k0	A5	—	HA11	S298A	P329R	84
(SEQ ID NO: 3, 21, 5)
GpH7-A48/GpH7-B3-06-09D/GpL16-k0	A48	P329R	B3-06-09D	S239D	—	123
(SEQ ID NO: 16, 37, 5)
GpH7-A48/GpH7-B3-20-08L/GpL16-k0	A48	P329R	B3-20-08L	A330L	—	60
(SEQ ID NO: 16, 38, 5)
GpH7-A48/GpH7-B3-22-10E/GpL16-k0	A48	P329R	B3-22-10E	I332E	—	189
(SEQ ID NO: 16, 39, 5)
GpH7-A48/GpH7-B3-01-15Y/GpL16-k0	A48	P329R	B3-01-15Y	L234Y	—	11
(SEQ ID NO: 16, 22, 5)
GpH7-A48/GpH7-B3-03-20W/GpL16-k0	A48	P329R	B3-03-20W	G236W	—	13
(SEQ ID NO: 16, 23, 5)
GpH7-A48/6pH7-B3-15-02A/GpL16-k0	A48	P329R	B3-15-02A	S298A	—	47
(SEQ ID NO: 16, 24, 5)

[0503] The column “SAMPLE” indicates antibody names; the columns “H1” and “H2” indicate names of the H chain constant region of respective antibodies; and the column “MUTATION SITE” indicates mutations that are different in comparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is no particular mutation). The FcγRIIIa-binding activity is indicated as a relative binding activity when setting the FcγRIIIa binding of GpH7-A5/GpH7-B3/GpL16-k0 as 100. The SEQ ID NOs are also shown for the amino acid sequences of the H chain and L chain of each antibody.

[0504] Based on the result, as to each altercation, the FcγRIIIa-binding activity is compared between when each alteration is introduced into the same H chain as P329R, and when introduced into the different H chain from P329R. In the case of S239D, the binding activity of GpH7-A5/GpH7-HA7/GpL16-k0 corresponding to the former was 3, while the binding activity of GpH7-A48/GpH7-B3-06-09D/GpL16-k0 corresponding to the latter was 123; thus, the FcγRIIIa binding was inhibited when the alteration was introduced into the same H chain as P329R. In the case of A330L, the binding activity of GpH7-A5/GpH7-HA15/GpL16-k0 corresponding to the former was 32, while the binding activity of GpH7-A48/GpH7-B3-20-08L/GpL16-k0 corresponding to the latter was 60; thus, the FcγRIIIa binding was inhibited when the alteration was introduced into the same H chain as P329R. In the case of I332E, the binding activity of GpH7-A5/GpH7-HA18/GpL16-k0 corresponding to the former was 35, while the binding activity of GpH7-A48/GpH7-B3-22-10E/GpL16-k0 corresponding to the latter was 189; thus, the FcγRIIIa binding was inhibited when the alteration was introduced into the same H chain as P329R. As to all of the alterations, the FcγRIIIa binding was inhibited when the alteration was introduced into the same H chain as P329R, i.e., the former case. If the H chain introduced with P329R corresponded to the H_A chain of FIG. 23, P329R is thought to inhibit the binding from the X-direction. Since combinations that significantly inhibit the binding are those when S239D, A330L, or I332E was introduced into the same H chain as P329R; that is, in this case, the alterations were introduced into the H_A chain. Since the effect to enhance the FcγRIIIa binding was markedly inhibited when any of the alterations S239D, A330L, and I332E was introduced into the same H chain as P329R, all the alterations, when introduced into the H_A chain, enhance the FcγRIIIa binding from the X-direction which is inhibited by P329R. Thus, it is thought that the FcγRIIIa binding can be further enhanced by introducing these alterations into the same H chain. Based on Example 5, it was considered that all of L234Y, G236W, and S298A, when introduced into the H_B chain, enhance the binding from the X-direction. As discussed in Examples 5 and 6, one can find a method for appropriately combining respective alterations by combining them with P329R. Based on the above results, to enhance the FcγRIIIa binding from the X-direction as shown in FIG. 3, S239D, A330L, and I332E need to be introduced into the H_A chain, whereas L234Y, G236W, and S298A need to be introduced into the H_B chain. Thus, it was thought that the FcγRIIIa binding from the X-direction can be further enhanced by introducing the respective alteration groups into different H chains.

[0505] To assess the above hypothesis, expression vectors inserted with the following were constructed according to the method described in Reference Example 1:

GpH7-A57 (SEQ ID NO: 40) resulting from the introduction of all of S239D, A330L, and I332E into GpH7-A5;

GpH7-B78 (SEQ ID NO: 41) resulting from the introduction of all of S239D, A330L, and I332E into GpH7-B3;

GpH7-TA7 (SEQ ID NO: 31) resulting from the introduction of all of L234Y, G236W, and S298A into GpH7-A5; and

GpH7-TA45 (SEQ ID NO: 32) resulting from the introduction of all of L234Y, G236W, and

[0506] S298A into GpH7-B3. Using the expression vectors, and GpH7-A5, GpH7-B3, and GpL16-k0, the following antibodies were expressed and prepared according to the method described in Reference Example 1:

hetero GpH7-TA7/GpH7-B78/GpL16-k0 in which one of the H chains has been introduced with L234Y, G236W, and S298A, and the other has been introduced with S239D, A330L, and I332E;

GpH7-TA7/GpH7-B3/GpL16-k0 in which only one of the H chains has been introduced with L234Y, G236W, and S298A;

GpH7-TA7/GpH7-TA45/GpL16-k0 in which both of the H chains have been introduced with L234Y, G236W, and S298A;

GpH7-A5/GpH7-B78/GpL16-k0 in which only one of the H chains has been introduced with S239D, A330L, and I332E; and

[0507] GpH7-A57/GpH7-B78/GpL16-k0 in which both of the H chains have been introduced with S239D, A330L, and I332E. The prepared antibodies were compared for the FcγRIIIa-binding activity using, as an indicator, KD for FcγRIIIa that was determined according to the method described in Reference Example 2. The assessment result on the effect of the combination of L234Y, G236W, and S298A with S239D, A330L, and I332E is summarized in Table 13.

[0508]

TABLE 13
						KD	KD
SAMPLE	H1	MUTATION SITE	H2	MUTATION SITE	KD (M)	ratio 1	ratio 2
GpH7-G1d/GpL16-k0	G1d				G1d				1.2E−06	1
(SEQ ID NO: 2, 5)
GpH7-A5/GpH7-B3/GpL16-k0	A5	—	—	—	B3	—	—	—	1.6E−06	0.75	1
(SEQ ID NO: 3, 4, 5)
GpH7-TA7/GpH7-B3/GpL16-k0	TA7	L234Y	G236W	S298A	B3	—	—	—	3.2E−07	3.8	5.1
(SEQ ID NO: 31, 4, 5)
GpH7-A5/GpH7-B78/GpL16-k0	A5	—	—	—	B78	S239D	A330L	I332E	5.4E−08	23	30
(SEQ ID NO: 3, 41, 5)
GpH7-A57/GpH7-B78/GpL16-k0	A57	S239D	A330L	I332E	B78	S239D	A330L	I332E	6.2E−09	199	263
(SEQ ID NO: 40, 41, 5)
GpH7-TA7/GpH7-TA45/	TA7	L234Y	G236W	S298A	TA45	L234Y	G236W	S298A	3.3E−06	0.37	0.49
GpL16-k0
(SEQ ID NO: 31, 32, 5)
GpH7-TA7/GpH7-B78/GpL16-k0	TA7	L234Y	G236W	S298A	B78	S239D	A330L	I332E	4.7E−09	261	347
(SEQ ID NO: 31, 41, 5)

[0509] The column “SAMPLE” indicates antibody names; the columns “H1” and “H2” indicate names of the H chain constant region of respective antibodies; and the column “MUTATION SITE” indicates mutations that are different in comparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is no particular mutation). A value obtained by dividing KD of GpH7-G1d/GpL16-k0 for FcγRIIIa by the KD of each antibody is defined as “KD ratio 1”, while a value obtained by dividing KD of GpH7-A5/GpH7-B3/GpL16-k0 for FcγRIIIa by the KD of each antibody was defined as “KD ratio 2”. The SEQ ID NOs are also shown for the amino acid sequences of the H chain and L chain of each antibody.

[0510] In view of the result of Table 13, when GpH7-A5/GpH7-B3/GpL16-k0 in which D356K, H435R, and K439E were each introduced into one H chain is compared to GpH7-G1d/GpL16-k0 which is a native IgG1, the difference in the FcγRIIIa-binding activity is 0.75 times, and no significant difference was observed. Thus, it was thought that the alterations D356K, H435R, and K439E do not affect the FcγRIIIa-binding activity.

[0511] Each alteration was assessed for its effect on homodimerized antibodies that use the prior art. The FcγRIIIa-binding activity of the homodimerized antibody GpH7-A57/GpH7-B78/GpL16-k0 in which both of the H chains were introduced with S239D, A330L, and I332E was increased by about 260 times that of GpH7-A5/GpH7-B3/GpL16-k0. In contrast, the FcγRIIIa-binding activity of the homodimerized antibody GpH7-TA7/GpH7-TA45/GpL16-k0 in which both of the H chains was introduced with L234Y, G236W, and S298A was decreased to 0.49 times. This demonstrates that only the group of the alterations S239D, A330L, and I332E has the effect to enhance the FcγRIIIa-binding activity of homodimerized antibodies.

[0512] Heterodimerized antibodies in which only one of the H chains was introduced with each alteration group were assessed for the effect of each alteration group. The FcγRIIIa-binding activity of the heterodimerized antibody GpH7-A5/GpH7-B78/GpL16-k0 in which one of the H chains was introduced with S239D, A330L, and I332E was increased by about 30 times that of GpH7-A5/GpH7-B3/GpL16-k0, while the FcγRIIIa-binding activity of the heterodimerized antibody GpH7-TA7/GpH7-B3/GpL16-k0 in which one of the H chains was introduced with L234Y, G236W, and S298A was increased by 5.1 times. This result demonstrates that the group of the alterations S239D, A330L, and I332E has the stronger effect to increase the FcγRIIIa-binding activity.

[0513] Each alteration group was assessed for the difference in its effect between the homodimerized antibody and heterodimerized antibody. Regarding S239D, A330L, and I332E, the FcγRIIIa-binding activity of the heterodimerized antibody was increased by 30 times that of GpH7-A5/GpH7-B3/GpL16-k0, while the activity of the homodimerized antibody was increased by about 260 times, demonstrating that the alterations, when introduced into homodimerized antibodies, further increase the FcγRIIIa-binding activity. Meanwhile, regarding L234Y, G236W, and S298A, the FcγRIIIa-binding activity of the heterodimerized antibody was increased by 5.1 times that of GpH7-A5/GpH7-B3/GpL16-k0; nevertheless, the activity of the homodimerized antibody was decreased to 0.49 times. This result shows that the group of the alterations L234Y, G236W, and S298A, only in heterodimerized antibodies, has the effect to enhance the FcγRIIIa-binding activity, as discussed in Example 5.

[0514] It was demonstrated that, in homodimerized antibodies, only the group of the alterations S239D, A330L, and I332E has the effect to enhance the FcγRIIIa binding, and also in heterodimerized antibodies, the group of the alterations S239D, A330L, and I332E more strongly enhances the FcγRIIIa binding. Based on the conventional concept, if the combination of the group of the alterations S239D, A330L, and I332E with the group of the alterations L234Y, G236W is considered, it would be predicted that the effect to enhance the FcγRIIIa binding is the strongest in the homodimerized antibody GpH7-A57/GpH7-B78/GpL16-k0 in which both of the H chains are introduced with only the group of the alterations S239D, A330L, and I332E, which strongly enhance the FcγRIIIa binding of both heterodimerized antibody and homodimerized antibody. However, the FcγRIIIa-binding activity of the heterodimerized antibody GpH7-TA7/GpH7-B78/GpL16-k0 in which S239D, A330L, and I332E were introduced into one H chain and L234Y, G236W, and S298A were introduced into the other H chain was increased by about 350 times that of GpH7-A5/GpH7-B3/GpL16-k0; the effect to enhance the binding was stronger than that for the homodimerized antibody in which both of the H chains were introduced with S239D, A330L, and I332E. This supports the hypothesis that, when the group of the alterations S239D, A330L, and I332E, and the group of the alterations L234Y, G236W, and S298A are introduced into different H chains, all of the introduced alterations enhance the FcγRIIIa-binding activity in both the H_A chain and H_B chain, and the effect is greater than the case when the group of the alterations S239D, A330L, and I332E are introduced into both H chains.

[0515] That is, it is demonstrated that the use of heterodimerized antibodies, instead of conventional homodimerized antibodies, allows finer optimization of the asymmetric interaction between the Fc domain and FcγRIIIa to design Fc domains having stronger binding activity.

[0516] In view of FIG. 23, it was thought that A330L and I332E interact with FcγR only in the H_A chain, while S239D interacts with FcγR in both the H_A chain and H_B chain. Indeed, KD for FcγRIIIa of the heterodimerized antibody GpH7-A5/GpH7-B78/GpL16-k0 in which only one of the H chains was introduced with S239D, A330L, and I332E was 5.4E-8, while the KD of the homodimerized antibody GpH7-A57/GpH7-B78/GpL16-k0 was 6.2E-9; thus, the FcγRIIIa-binding activity was increased by 8.7 times. If it is thought that the difference in the binding activity is due to the involvement of S239D in the enhancement of the binding in both H chains, this difference is thought to be eliminated by introducing S239D into both H chains. To assess this hypothesis, GpH7-A53 (SEQ ID NO: 42) resulting from the introduction of S239D into GpH7-A5 was constructed, and, this is combined with GpH7-B78 introduced with S239D, A330L, and I332E, and the expression and preparation were performed according to the method described in Reference Example 1. The effect of the combination of S239D and S239D, A330L, and I332E was assessed by comparing the FcγRIIIa binding between the heterodimerized and homodimerized antibodies with S239D, A330L, and I332E according to the method described in Reference Example 2 (Table 14).

[0517]

TABLE 14
						KD
Sample	H1	MUTATION SITE	H2	MUTATION SITE	KD (M)	ratio
GpH7-A5/GpH7-B78/GpL16-k0	A5	—	—	—	B78	S239D	A330L	I332E	5.4E−08	1
(SEQ ID NO: 3, 41, 5)
GpH7-A53/GpH7-B78/GpL16-k0	A53	S239D	—	—	B78	S239D	A330L	I332E	1.1E−08	4.9
(SEQ ID NO: 42, 41, 5)
GpH7-A57/GpH7-B78/GpL16-k0	A57	S239D	A330L	I332E	B78	S239D	A330L	I332E	6.2E−09	8.7
(SEQ ID NO: 40, 41, 5)

[0518] The column “SAMPLE” indicates antibody names; the columns “H1” and “H2” indicate names of the H chain constant region of respective antibodies; and the column “MUTATION SITE” indicates mutations that are different in comparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is no particular mutation). A value obtained by dividing KD of GpH7-A5/GpH7-B78/GpL16-k0 for FcγRIIIa by the KD of each antibody was defined as “KD ratio”. The SEQ ID NOs are also shown for the amino acid sequences of the H chain and L chain of each antibody.

[0519] Table 14 shows that the FcγRIIIa-binding activity of GpH7-A53/GpH7-B78/GpL16-k0 in which one of the H chains was introduced with S239D, A330L, and I332E and the other was introduced with S239D was increased by 4.9 times that of GpH7-A5/GpH7-B78/GpL16-k0 in which only one of the H chains was introduced with S239D, A330L, and I332E; while the activity was decreased by only 1.8 times that of GpH7-A57/GpH7-B78/GpL16-k0 in which both of the H chains were introduced with S239D, A330L, and I332E. This result demonstrates that S239D interacts with FcγRIIIa in both H chains, as mentioned above for the hypothesis. It was thought that the interaction with FcγRIIIa can be further enhanced by introducing the above alteration.

[0520] The present inventors tested whether the FcγRIIIa-binding activity can be further increased by introducing S239D into the heterodimerized antibody GpH7-TA7/GpH7-B78/GpL16-k0 in which one of the H chains was introduced with L234Y, G236W, and S298A and the other was introduced with S239D, A330L, and I332E. GpH7-TA22 (SEQ ID NO: 43) was constructed by introducing S239D into GpH7-TA7, and then inserted into an expression vector; and the resulting expression vector was combined with GpL16-k0, and GpH7-B78 obtained by introducing S239D, A330L, and I332E into GpH7-B3; and the antibody of interest was expressed and prepared according to the method described in Reference Example 1. Furthermore, the heterodimerized antibody GpH7-TA22/GpH7-B78/GpL16-k0 was prepared by introducing L234Y, G236W, S239D, and S298A into one H chain, and S239D, A330L, and I332E into the other H chain. To assess the effect of the combination of S239D with S239D, A330L, and I332E, the antibodies were compared for the FcγRIIIa-binding activity according to the method described in Reference Example 2 (Table 15).

[0521]

TABLE 15
						KD
Sample	H1	MUTATION SITE	H2	MUTATION SITE	KD (M)	ratio
GpH7-TA7/GpH7-B78/GpL16-k0	TA7	L234Y/G236W/S298A	—	B78	S239D/A330L/I332E	4.1E−09	1
(SEQ ID NO: 31, 41, 5)
GpH7-TA22/GpH7-B78/GpL16-k0	TA22	L234Y/G236W/S298A	S239D	B78	S239D/A330L/I332E	1.3E−09	3.2
(SEQ ID NO: 43, 41, 5)

[0522] The column “SAMPLE” indicates antibody names; the columns “H1” and “H2” indicate names of the H chain constant region of respective antibodies; and the column “MUTATION SITE” indicates mutations that are different in comparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is no particular mutation). A value obtained by dividing KD of GpH7-TA7/GpH7-B78/GpL16-k0 for FcγRIIIa by the KD of each antibody was defined as “KD ratio”. The SEQ ID NOs are also shown for the amino acid sequences of the H chain and L chain of each antibody.

[0523] Table 15 shows that the binding activity of GpH7-TA22/GpH7-B78/GpL16-k0 resulting from the introduction of S239D into the H chain that does not contain S239D in the heterodimerized antibody GpH7-TA7/GpH7-B78/GpL16-k0 in which one of the H chains was introduced with L234Y, G236W, and S298A and the other H chain with S239D, A330L, and I332E was increased by 3.2 times that of GpH7-TA7/GpH7-B78/GpL16-k0. This result demonstrates that the FcγRIIIa binding can be further enhanced by using S239D.

[0524] Then, the present inventors considered further introducing the alterations Y296W and K334G that enhance the FcγRIIIa binding, which were revealed as described in Example 4.

[0525] First, it was considered which H chain should be introduced with Y296W. GpH7-TA52 (SEQ ID NO: 44) resulting from the introduction of the mutation Y296W into GpH7-TA7 was constructed, and expressed and prepared in combination with GpH7-B78 according to the method described in Reference Example 1. Furthermore, GpH7-TA58 (SEQ ID NO: 45) resulting from the introduction of Y296W into GpH7-B78 was constructed, and expressed and prepared in combination with GpH7-TA22 according to the method described in Reference Example 1. To assess the effects of combinations with Y296W, the prepared antibodies were compared for the FcγRIIIa-binding activity according to the method described in Reference Example 2 (Table 16).

[0526]

TABLE 16
						KD
Sample	H1	MUTATION SITE	H2	MUTATION SITE	KD (M)	ratio
GpH7-TA7/GpH7-B78/GpL16-k0	TA7	L234Y/G236W/S298A	—	B78	S239D/A330L/I332E	—	4.1E−09	1
(SEQ ID NO: 31, 41, 5)
GpH7-TA7/GpH7-TA58/GpL16-k0	TA7	L234Y/G236W/S298A	—	TA58	S239D/A330L/I332E	Y296W	3.3E−09	1.2
(SEQ ID NO: 31, 45, 5)
GpH7-TA52/GpH7-B78/GpL16-k0	TA52	L234Y/G236W/S298A	Y296W	B78	S239D/A330L/I332E	—	2.3E−09	1.8
(SEQ ID NO: 44, 41, 5)

[0527] The column “Sample” indicates antibody names; the columns “H1” and “H2” indicate names of H chain constant region of respective antibodies; and the column “MUTATION SITE” indicates mutations that are different in comparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is no particular mutation). A value obtained by dividing KD of GpH7-TA7/GpH7-B78/GpL16-k0 for FcγRIIIa by the KD of each antibody was defined as “KD ratio”. The SEQ ID NOs are also shown for the amino acid sequences of the H chain and L chain of each antibody.

[0528] The result showed that Y296W, when introduced into the H chain different from that introduced with L234Y, G236W, and S298A, increased the FcγRIIIa-binding activity only by 1.2 times after introduction compared to before; while Y296W, when introduced into the same H chain, increased the FcγRIIIa-binding activity by 1.8 times that of GpH7-TA7/GpH7-B78/GpL16-k0. From this result, it is thought that Y296W, when introduced into the same H chain as L234Y, G236W, and S298A, has the effect to enhance the FcγRIIIa binding.

[0529] Then, GpH7-TA54 (SEQ ID NO: 46) resulting from the introduction of Y296W into GpH7-TA22 was constructed, and expressed and prepared in combination with GpH7-B78 according to the method described in Reference Example 1. Furthermore, GpH7-TA58 (SEQ ID NO: 45) resulting from the introduction of Y296W into GpH7-B78 was constructed, and expressed and prepared in combination with GpH7-TA22 according to the method described in Reference Example 1. To assess the effect of combination with Y296W, the prepared antibodies were compared for the FcγRIIIa-binding activity according to the method described in Reference Example 2 (Table 17).

[0530]

TABLE 17
Sample	H1	MUTATION SITE	H2	MUTATION SITE	KD (M)	KD ratio
GpH7-TA22/GpH7-B78/GpL16-k0	TA22	L234Y/G236W/S239D/S298A	—	B78	S239D/A330L/I332E	—	1.3E−09	1
(SEQ ID NO: 43, 41, 5)
GpH7-TA22/GpH7-TA58/GpL16-k0	TA22	L234Y/G236W/S239D/S298A	—	TA58	S239D/A330L/I332E	Y296W	1.3E−09	1.0
(SEQ ID NO: 43, 45, 5)
GpH7-TA54/GpH7-B78/GpL16-k0	TA54	L234Y/G236W/S239D/S298A	Y296W	B78	S239D/A330L/I332E	—	1.0E−09	1.3
(SEQ ID NO: 46, 41, 5)

[0531] The column “Sample” indicates antibody names; the columns “H1” and “H2” indicate names of the H chain constant region of respective antibodies; and the column “MUTATION SITE” indicates mutations that are different in comparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is no particular mutation). A value obtained by dividing KD of GpH7-TA22/GpH7-B78/GpL16-k0 for FcγRIIIa by the KD of each antibody was defined as “KD ratio”. The SEQ ID NOs are also shown for the amino acid sequences of the H chain and L chain of each antibody.

[0532] The result shown in Table 17 demonstrates that, when Y296W was introduced into the H chain that is different from that introduced with L234Y, G236W, and S298A, there was no difference in the FcγRIIIa-binding activity after the introduction compared to before. When Y296W was introduced into the same H chain as L234Y, G236W, and S298A, the FcγRIIIa-binding activity was increased by 1.3 times that of GpH7-TA22/GpH7-B78/GpL16-k0. From this result, it was thought that Y296W, when introduced into the same H chain as L234Y, G236W, and S298A, has the effect to enhance the FcγRIIIa-binding activity.

[0533] Then, K334G was also assessed in the same manner. GpH7-TA40 (SEQ ID NO: 47) resulting from the introduction of K334G into GpH7-TA7 was constructed, and expressed and prepared in combination with GpH7-B78 according to the method described in Reference Example 1. Furthermore, GpH7-TA50 (SEQ ID NO: 48) resulting from the introduction of K334G into GpH7-B78 was constructed, and expressed and prepared in combination with GpH7-TA7 according to the method described in Reference Example 1. To assess the effect of combinations with K334G, the prepared antibodies were compared for the FcγRIIIa binding according to the method described in Reference Example 2 (Table 18).

[0534]

TABLE 18
						KD
Sample	H1	MUTATION SITE	H2	MUTATION SITE	KD (M)	ratio
GpH7-TA7/GpH7-B78/GpL16-k0	TA7	L234Y/G236W/S298A	—	B78	S239D/A330L/I332E	—	4.1E−09	1.0
(SEQ ID NO: 31, 41, 5)
GpH7-TA7/GpH7-TA50/GpL16-k0	TA7	L234Y/G236W/S298A	—	TA50	S239D/A330L/I332E	K334G	3.4E−09	1.2
(SEQ ID NO: 31, 48, 5)
GpH7-TA40/GpH7-B78/GpL16-k0	TA40	L234Y/G236W/S298A	K334G	B78	S239D/A330L/I332E	—	7.5E−09	0.5
(SEQ ID NO: 47, 41, 5)

[0535] The column “Sample” indicates antibody names; the columns “H1” and “H2” indicate names of the H chain constant regions of each antibody; and the column “MUTATION SITE” indicates mutations that are different in comparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is no particular mutation). A value obtained by dividing KD of GpH7-TA7/GpH7-B78/GpL16-k0 for FcγRIIIa by the KD of each antibody was defined as “KD ratio”. The SEQ ID NOs are also shown for the amino acid sequences of the H chain and L chain of each antibody.

[0536] The result shown in Table 18 demonstrates that, when K334G was introduced into the same H chain as L234Y, G236W, and S298A, the FcγRIIIa-binding activity is decreased by half after the introduction compared to before. When K334G was introduced into the different H chain, the FcγRIIIa-binding activity was increased by 1.2 times that of GpH7-TA7/GpH7-B78/GpL16-k0. From this result, it was thought that K334G, when introduced into the H chain different from that introduced with L234Y, G236W, and S298A, has the effect to enhance the FcγRIIIa-binding activity.

[0537] There are activating FcγR which has ITAM, and inhibitory FcγR which has ITIM. Representative activating FcγRs (activating receptor) include FcγRIa, FcγRIIa, and FcγRIIIa, while representative inhibitory FcγRs (inhibitory receptor) include FcγRIIb. Regarding antibodies targeted to cancer, the ratio of the binding activity to activating FcγR whose action mechanism is based on ADCC activity or antibody-dependent cellular phagocytosis (ADCP) activity against the binding activity to inhibitory FcγR is believed to play an important role (Nature Medicine, 6: 443-446, 2000).

[0538] For antibodies targeted to cancer, it is desirable to increase their binding activity to activating FcγR while reducing their binding activity to inhibitory FcγR. Specifically, desirable alterations are, such as those comprised in Region a shown in FIG. 24, alterations that allow an antibody to bind to activating FcγR more strongly than the native antibody, and bind to inhibitory FcγR more weakly than the native antibody, i.e., alterations that enhance the binding in an activating FcγR-selective manner. Also, the desirable alterations are, such as those in Region b shown in FIG. 25, alterations that allow the ratio between the binding activity to activating FcγR and the binding activity to inhibitory FcγR to be greater than the native antibody. It can be said that such alterations selectively increase the binding activity to activating FcγR as compared to inhibitory FcγR.

[0539] The heterodimerized antibodies He Abs in which one of the H chains was introduced with an alteration were assayed for their binding activities to each activating FcγR and inhibitory FcγR according to the method described in Example 4. The result is summarized in FIGS. 26, 27, 28 and 29 to assess the ratio of the binding activity to each activating FcγR and inhibitory FcγR for each heterodimerized antibody. The activating FcγRs in FIGS. 26, 27, 28, and 29 are FcγRIa, FcγRIIa (R), FcγRIIa (H), and FcγRIIIa, respectively.

[0540] The alterations present in the region in FIG. 26 corresponding to a and b in FIGS. 24 and 25 are listed in Table 19 (Table 19-1 to 19-5). Likewise, as to FcγRIIa (R) (FIG. 27), FcγRIIa (H) (FIG. 28), FcγRIIIa (FIG. 29), alterations present in the region corresponding to a and b are listed in Table 20 (Table 20-1 to 20-3), Table 21 (Table 21-1 to 21-3), and Table 22 (Table 22-1 to 22-3).

[0541]

TABLE 19-1
REGION a
Ia > Con	REGION b
IIb < Con	Ia/Iib=>1.2
	Ia binding	IIb binding		Ia binding	IIb binding	Ia/IIb
NAME	He/Con_1a	He/Con_2b	NAME	He/Con_1a	He/Con_2b	He_1a/2b
L234_03V	104.4	73.1	L234_01G	96.1	50.7	1.90
L234_04F	101.0	99.8	L234_02A	97.5	64.9	1.50
L234_06M	103.8	78.5	L234_03V	104.4	73.1	1.43
L234_07I	102.9	80.9	L234_05P	99.9	72.9	1.37
L234_15Y	100.1	96.3	L234_06M	103.8	78.5	1.32
G236_02A	100.2	96.5	L234_07I	102.9	80.9	1.27
G236_20W	105.2	58.2	L234_11K	91.8	16.2	5.67
S239_14T	101.1	97.3	L234_12R	91.8	18.9	4.86
S239_19Q	100.6	86.1	L234_13S	96.9	59.3	1.63
D265_10E	101.4	38.2	L234_14T	98.6	62.5	1.58
V266_19Q	100.2	89.2	L234_16H	94.0	59.4	1.58
S267_16H	100.0	65.9	L234_19Q	95.7	60.3	1.59
S267_18N	102.4	47.7	L235_01G	87.2	35.4	2.46
H268_04F	106.3	89.6	L235_02A	92.2	63.0	1.46
H268_06M	104.7	65.7	L235_03V	94.7	66.4	1.43
H268_07I	107.8	88.9	L235_05P	94.7	54.7	1.73
H268_08L	107.4	67.8	L235_06M	98.0	81.0	1.21
H268_11K	105.8	83.0	L235_11K	79.9	6.3	12.73
H268_12R	106.1	89.8	L235_12R	78.0	9.6	8.12
H268_14T	112.2	86.0	L235_13S	90.5	48.2	1.88
E269_01G	104.4	51.5	L235_14T	88.7	46.8	1.90
E269_02A	104.4	49.9	L235_18N	92.2	54.9	1.68
E269_03V	102.6	45.1	L235_19Q	94.0	42.6	2.21
E269_04F	103.1	45.0	G236_03V	89.9	35.3	2.55
E269_05P	102.3	52.6	G236_04F	96.4	39.3	2.45
E269_06M	103.6	46.7	G236_05P	77.0	19.0	4.05
E269_07I	102.8	43.9	G236_06M	96.7	45.5	2.13
E269_08L	103.6	42.9	G236_07I	87.5	31.0	2.82
E269_13S	103.1	47.8	G236_08L	79.6	26.8	2.97
E269_14T	105.9	50.6	G236_11K	71.0	49.8	1.42
E269_15Y	106.4	44.7	G236_12R	63.1	6.8	9.30
E269_16H	105.8	38.5	G236_14T	92.2	57.8	1.59
E269_18N	101.5	44.8	G236_15Y	92.5	39.5	2.34
E269_19Q	105.0	44.3	G236_16H	83.5	27.9	2.99
E269_20W	105.2	42.5	G236_19Q	92.1	46.6	1.98
P271_02A	103.5	88.0	G236_20W	105.2	58.2	1.81
P271_03V	102.3	74.8	G237_03V	78.5	61.4	1.28
P271_04F	101.8	59.5	G237_05P	44.5	24.6	1.81
P271_06M	102.5	77.5	G237_11K	63.3	9.0	7.05
P271_07I	102.5	76.8	G237_12R	65.8	9.8	6.71
P271_11K	102.8	90.2	G237_14T	82.3	54.8	1.50
P271_12R	102.4	86.5	G237_16H	72.6	55.2	1.32
P271_13S	102.4	87.6	P238_01G	92.9	56.0	1.66
P271_14T	102.3	95.4	P238_02A	95.2	57.4	1.66
P271_15Y	103.0	52.3	P238_07I	99.1	80.0	1.24
P271_16H	102.1	67.9	P238_11K	77.4	18.1	4.27
P271_18N	103.2	93.3	P238_12R	77.3	51.4	1.50
P271_19Q	103.1	88.6	P238_13S	96.6	70.4	1.37
P271_20W	102.6	58.1	P238_14T	91.1	50.7	1.80
Q295_02A	104.2	79.7	P238_18N	93.3	73.2	1.27
Q295_03V	104.1	75.7	S239_04F	93.8	44.1	2.13
Q295_04F	102.2	64.7	S239_11K	68.0	24.7	2.75
Q295_05P	103.4	67.5	S239_12R	86.5	39.1	2.21
Q295_06M	104.2	90.4	S239_15Y	89.8	34.9	2.57
Q295_07I	103.0	84.3	S239_16H	88.1	49.4	1.78

[0542]

TABLE 19-2
REGION a
Ia > Con	REGION b
IIb < Con	Ia/Iib=>1.2
	Ia binding	IIb binding		Ia binding	IIb binding	Ia/IIb
NAME	He/Con_1a	He/Con_2b	NAME	He/Con_1a	He/Con_2b	He_1a/2b
Q295_09D	100.1	60.8	S239_20W	92.7	45.1	2.06
Q295_10E	103.1	94.8	D265_01G	79.9	16.1	4.95
Q295_11K	101.3	70.4	D265_02A	84.5	19.9	4.25
Q295_12R	100.4	65.2	D265_03V	55.7	3.2	17.30
Q295_13S	102.0	56.9	D265_04F	53.0	3.2	16.77
Q295_14T	102.7	66.8	D265_06M	75.7	10.0	7.57
Q295_15Y	101.7	65.4	D265_07I	54.4	1.3	41.18
Q295_16H	102.5	62.7	D265_08L	56.8	4.6	12.43
Q295_18N	102.4	64.9	D265_10E	101.4	38.2	2.66
Y296_01G	102.6	79.3	D265_11K	43.2	14.9	2.90
Y296_02A	102.9	82.0	D265_12R	44.3	15.0	2.96
Y296_03V	104.0	79.3	D265_13S	68.7	11.3	6.07
Y296_04F	104.1	99.2	D265_14T	78.5	16.0	4.90
Y296_06M	104.0	87.8	D265_15Y	44.3	0.6	73.46
Y296_07I	104.3	84.3	D265_16H	85.4	38.3	2.23
Y296_08L	103.9	82.3	D265_18N	27.7	6.1	4.52
Y296_09D	105.9	98.9	D265_19Q	88.8	33.4	2.66
Y296_10E	105.8	98.0	D265_20W	51.5	1.1	45.61
Y296_11K	104.7	72.8	V266_01G	91.3	51.5	1.77
Y296_12R	106.3	79.5	V266_04F	96.7	57.6	1.68
Y296_13S	105.1	86.3	V266_05P	71.9	0.4	205.21
Y296_14T	105.1	88.2	V266_09D	79.3	10.1	7.88
Y296_16H	105.8	92.2	V266_10E	81.9	15.2	5.37
Y296_18N	105.6	91.5	V266_11K	64.4	1.0	63.53
Y296_19Q	111.6	89.3	V266_13S	90.6	54.4	1.67
S298_01G	103.6	80.1	V266_15Y	93.3	49.4	1.89
S298_02A	106.6	73.4	V266_16H	89.1	57.2	1.56
S298_03V	109.7	64.5	V266_18N	99.5	69.7	1.43
S298_04F	109.0	65.4	V266_20W	91.4	71.9	1.27
S298_07I	107.0	70.6	S267_04F	97.7	48.0	2.04
S298_09D	105.9	48.3	S267_05P	91.8	31.5	2.91
S298_10E	108.2	47.6	S267_11K	93.3	12.2	7.65
S298_11K	109.3	61.3	S267_12R	92.7	11.3	8.21
S298_12R	109.4	57.8	S267_15Y	94.2	37.9	2.48
S298_14T	107.1	77.9	S267_16H	100.0	65.9	1.52
S298_15Y	107.2	61.4	S267_18N	102.4	47.7	2.15
S298_16H	108.8	63.5	S267_20W	99.4	57.2	1.74
S298_18N	108.1	48.1	H268_06M	104.7	65.7	1.59
S298_19Q	109.4	73.3	H268_07I	107.8	88.9	1.21
S298_20W	104.0	41.1	H268_08L	107.4	67.8	1.58
Y300_01G	116.1	48.4	H268_11K	105.8	83.0	1.28
Y300_02A	112.2	69.2	H268_14T	112.2	86.0	1.30
Y300_03V	117.0	73.9	E269_01G	104.4	51.5	2.03
Y300_06M	112.1	84.3	E269_02A	104.4	49.9	2.09
Y300_07I	107.8	74.5	E269_03V	102.6	45.1	2.27
Y300_08L	113.0	73.6	E269_04F	103.1	45.0	2.29
Y300_09D	112.5	93.2	E269_05P	102.3	52.6	1.94
Y300_11K	106.9	51.6	E269_06M	103.6	46.7	2.22
Y300_12R	104.6	41.2	E269_07I	102.8	43.9	2.34
Y300_13S	111.1	53.9	E269_08L	103.6	42.9	2.42
Y300_14T	111.7	55.6	E269_11K	95.2	17.8	5.34
Y300_16H	110.9	87.0	E269_12R	97.1	25.2	3.86
Y300_20W	112.4	84.1	E269_13S	103.1	47.8	2.16
K326_12R	100.1	89.2	E269_14T	105.9	50.6	2.09
S324_02A	102.7	78.3	E269_15Y	106.4	44.7	2.38
S324_03V	103.6	97.8	E269_16H	105.8	38.5	2.75
S324_07I	102.0	95.7	E269_18N	101.5	44.8	2.27

[0543]

TABLE 19-3
REGION a
Ia > Con	REGION b
IIb < Con	Ia/Iib=>1.2
	Ia binding	IIb binding		Ia binding	IIb binding	Ia/IIb
NAME	He/Con_1a	He/Con_2b	NAME	He/Con_1a	He/Con_2b	He_1a/2b
S324_08L	103.2	89.6	E269_19Q	105.0	44.3	2.37
S324_09D	103.5	85.8	E269_20W	105.2	42.5	2.47
S324_10E	104.9	77.4	P271_03V	102.3	74.8	1.37
S324_11K	102.1	68.6	P271_04F	101.8	59.5	1.71
S324_12R	101.7	63.1	P271_06M	102.5	77.5	1.32
S324_14T	120.9	88.4	P271_07I	102.5	76.8	1.33
S324_15Y	113.8	88.2	P271_15Y	103.0	52.3	1.97
S324_16H	112.6	83.5	P271_16H	102.1	67.9	1.50
S324_18N	115.5	81.1	P271_20W	102.6	58.1	1.77
S324_19Q	112.3	66.8	D270_01G	76.3	30.5	2.50
S324_20W	114.3	87.5	D270_02A	71.5	48.3	1.48
A327_03V	103.2	94.7	D270_03V	77.9	39.5	1.97
A327_06M	103.2	59.5	D270_04F	78.1	53.1	1.47
A327_13S	102.4	79.5	D270_05P	67.1	13.9	4.81
P329_01G	100.9	41.7	D270_06M	78.8	48.2	1.64
P329_02A	102.3	40.5	D270_07I	74.6	41.5	1.80
A330_03V	105.5	52.4	D270_08L	73.5	53.6	1.37
A330_04F	106.1	92.8	D270_11K	58.4	20.3	2.88
A330_06M	104.6	80.5	D270_12R	66.3	18.3	3.63
A330_07I	105.2	67.1	D270_13S	75.3	44.2	1.70
A330_08L	106.9	70.5	D270_14T	70.6	57.8	1.22
A330_09D	108.6	55.6	D270_15Y	68.5	44.9	1.53
A330_10E	107.4	70.1	D270_16H	66.0	47.2	1.40
A330_11K	103.4	95.0	D270_18N	67.8	40.3	1.68
A330_14T	105.1	77.9	D270_19Q	78.2	48.0	1.63
A330_15Y	105.0	95.0	D270_20W	68.0	34.7	1.96
A330_16H	105.8	87.6	Q295_01G	97.6	46.0	2.12
A330_18N	106.5	63.7	Q295_02A	104.2	79.7	1.31
A330_19Q	105.2	90.8	Q295_03V	104.1	75.7	1.37
A330_20W	107.6	76.1	Q295_04F	102.2	64.7	1.58
I332_12R	100.1	48.9	Q295_05P	103.4	67.5	1.53
E333_01G	102.6	81.4	Q295_07I	103.0	84.3	1.22
E333_02A	103.2	83.8	Q295_09D	100.1	60.8	1.65
E333_03V	105.0	93.2	Q295_11K	101.3	70.4	1.44
E333_04F	102.4	94.6	Q295_12R	100.4	65.2	1.54
E333_05P	102.3	97.7	Q295_13S	102.0	56.9	1.79
E333_06M	102.1	85.4	Q295_14T	102.7	66.8	1.54
E333_07I	102.0	96.2	Q295_15Y	101.7	65.4	1.56
E333_08L	102.2	95.0	Q295_16H	102.5	62.7	1.64
E333_09D	103.5	94.3	Q295_18N	102.4	64.9	1.58
E333_11K	102.1	80.6	Q295_20W	93.7	44.4	2.11
E333_12R	102.2	80.4	Y296_01G	102.6	79.3	1.29
E333_13S	103.1	81.8	Y296_02A	102.9	82.0	1.26
E333_14T	102.1	86.1	Y296_03V	104.0	79.3	1.31
E333_15Y	101.2	94.4	Y296_05P	95.6	36.6	2.61
E333_16H	100.8	83.3	Y296_07I	104.3	84.3	1.24
K334_01G	103.5	92.5	Y296_08L	103.9	82.3	1.26
K334_09D	106.5	94.8	Y296_11K	104.7	72.8	1.44
T335_01G	104.4	96.7	Y296_12R	106.3	79.5	1.34
T335_02A	103.3	94.0	Y296_13S	105.1	86.3	1.22
T335_05P	106.4	99.9	Y296_19Q	111.6	89.3	1.25
T335_06M	106.7	99.4	S298_01G	103.6	80.1	1.29
T335_11K	110.8	91.0	S298_02A	106.6	73.4	1.45
T335_12R	112.8	91.9	S298_03V	109.7	64.5	1.70
I336_01G	108.7	90.1	S298_04F	109.0	65.4	1.67
I336_02A	106.0	84.9	S298_05P	91.6	11.9	7.70
I336_03V	105.2	90.3	S298_07I	107.0	70.6	1.52