Heterodimeric Fc Polypeptides

Abstract

A non-limiting embodiment of the current invention provides: a polypeptide including a mutant Fc region that includes an amino acid modification in a parent Fc region; and a production method for said polypeptide.

Description

[Technical Field]

[0001] In one non-limiting aspect, the present disclosure relates to polypeptides comprising mutant Fc regions that comprise amino acid modifications in a parent Fc region, and methods for producing the polypeptides. [Background technology]

[0002] Antibodies are attracting attention as pharmaceuticals due to their high stability in the blood and few side effects (Non-Patent Documents 1 and 2). Most antibody drugs currently on the market are antibodies of the human IgG1 subclass. Numerous studies have been conducted on the effector functions of IgG class antibodies, namely, antibody-dependent cellular cytotoxicity (hereinafter referred to as ADCC) and complement-dependent cytotoxicity (hereinafter referred to as CDC), and it has been reported that, among human IgG class antibodies, IgG1 subclass antibodies have the highest ADCC and CDC activities (Non-Patent Document 3). Furthermore, antibody-dependent cell-mediated phagocytosis (ADCP), which is the phagocytosis of target cells mediated by IgG class antibodies, has also been shown to be one of the effector functions of antibodies (Non-Patent Documents 4 and 5).

[0003] The expression of ADCC, CDC, and ADCP by IgG antibodies requires the binding of the antibody Fc region to antibody receptors (hereinafter referred to as FcγR) and various complement components present on the surface of effector cells such as killer cells, natural killer cells, and activated macrophages. In humans, the FcγR protein family has been reported to include isoforms FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb, and each allotype has also been reported (Non-Patent Document 6).

[0004] Enhancement of cytotoxic effector functions, such as ADCC, ADCP, and CDC, has attracted attention as a promising means to enhance the antitumor effects of antibodies. The importance of FcγR-mediated effector functions for the antitumor effects of antibodies has been reported using mouse models (Non-Patent Document 7, Non-Patent Document 8). Furthermore, a correlation was observed between the high-affinity polymorphic allotype (V158) of FcγRIIIa and the low-affinity polymorphic allotype (F158) of FcγRIIIa in humans (Non-Patent Document 9). Similarly, it has been shown that clinical effects differ depending on the FcγRIIa allotype (H131 vs. R131) (Non-Patent Document 10). These reports indicate that antibodies with Fc regions optimized for binding to specific FcγRs mediate stronger effector functions and thereby exert effective antitumor effects.

[0005] The balance of antibody binding activity toward activating receptors (FcγRIa, FcγRIIa, FcγRIIIa, and FcγRIIIb) and toward inhibitory receptors (FcγRIIb) is an important factor in optimizing antibody effector function. Using an Fc region with enhanced binding activity toward activating receptors and reduced binding activity toward inhibitory receptors may confer optimal effector function to an antibody (Non-Patent Document 11). It has been shown that several amino acid residues in the antibody hinge region and CH2 domain, as well as the sugar chain attached to Asn at position 297 (EU numbering) attached to the CH2 domain, are important for the binding between the Fc region and FcγR (Non-Patent Documents 12, 13, and 14). Focusing on this binding site, various Fc region mutants with FcγR binding properties have been studied, and Fc region mutants with enhanced activating FcγR binding activity have been identified (Patent Documents 1 and 2). For example, Lazar et al. succeeded in increasing human FcγRIIIa (V158) binding by approximately 370-fold by substituting Ser at position 239 (EU numbering), Ala at position 330, and Ile at position 332 (EU numbering) with Asp, Leu, and Glu, respectively (Non-Patent Document 15, Patent Document 2). Shinkawa et al. succeeded in increasing FcγRIIIa binding by approximately 100-fold by deleting the fucose attached to Asn at position 297 (EU numbering) (Non-Patent Document 16). These methods introduce the same alterations or the same glycosylation modifications into the Fc regions of both antibody H chains. On the other hand, it has been reported that antibody Fc, despite being a homodimer, binds to FcγR in a 1:1 ratio and asymmetrically recognizes FcγR at the lower hinge and CH2 regions (Non-Patent Document 17). Considering that the Fc region interacts asymmetrically with FcγR, it is thought that introducing different alterations into each H chain will enable more precise optimization of the interaction between IgG and FcγR. Based on this idea, methods have been reported in which different alterations are made to the Fc region of each H chain of an antibody to asymmetrically modify the Fc, thereby optimizing the interaction with FcγR (Patent Documents 3, 4, 5, and 6).In fact, by asymmetrically modifying the Fc region, variants have been obtained that exhibit higher ADCC activity than existing afucosylated antibodies, which are ADCC-enhancing antibodies (Patent Documents 5 and 6).

[0006] In addition to ADCC activity, ADCP activity is also an important effector function of antibodies and has been reported to contribute to antitumor effects (Non-Patent Document 18). ADCP activity can be enhanced by inhibiting "don't eat me" signals, such as those mediated by CD47 (Non-Patent Document 18), or by enhancing FcγRIIa-binding ability (Non-Patent Document 19). However, the amino acid sequences of the activating FcγR FcγRIIa and inhibitory FcγRIIb are highly homologous in their extracellular domains, making it difficult to selectively enhance FcγRIIa-binding ability (Non-Patent Document 20). Therefore, enhancing FcγRIIa-binding ability also enhances FcγRIIb-binding ability, an inhibitory receptor, potentially reducing effector function. In fact, variants with significantly improved FcγRIIa-binding ability also exhibit enhanced FcγRIIb-binding ability compared to native IgG1 (Patent Documents 5 and 6). Therefore, in order to exhibit high ADCC / ADCP activity, it is preferable to enhance the binding to FcγRIIIa and FcγRIIa as much as possible without enhancing the binding ability to FcγRIIb, but such variants have not been reported. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO 2000 / 042072 [Patent Document 2] WO 2006 / 019447 [Patent Document 3] WO 2012 / 058768 [Patent Document 4] WO 2012 / 125850 [Patent Document 5] WO 2013 / 002362

Patent document 6

Non-licensed literature

[0008] [Non-licensed document 1] Nature Biotechnology, 23, 1073-1078 (2005) [Non-licensed document 2] Eur. J. Pharm. Biopharm, 59(3), 389-96 (2005) [Non-licensed document 3] Chemical Immunology, 65, 88 (1997)

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

[0009] The invention of the present disclosure was made in light of these circumstances, and in one aspect, an object of the invention of the present disclosure is to provide a polypeptide having an improved Fc region function (e.g., FcγR-binding ability, ADCC activity, and ADCP activity) compared to conventional polypeptides having an Fc region, as well as a method for producing the polypeptide. [Means for solving the problem]

[0010] In one non-limiting embodiment, the present disclosure provides the following: [1] A polypeptide comprising a mutant Fc region that contains amino acid modifications in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and the mutant Fc region contains amino acid modifications at the following positions: (i) positions 234, 235, 236, 239, 268, 270, and 298, as represented by EU numbering, in the first polypeptide of the parent Fc region; and (ii) positions 270, 298, 326, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region. [2] The polypeptide of [1], wherein the mutant Fc region further comprises an amino acid modification at position 326 (EU numbering) in the first polypeptide of the parent Fc region. [3] The polypeptide of [1] or [2], wherein the mutant Fc region further comprises an amino acid modification at position 236 (EU numbering) in the second polypeptide of the parent Fc region. [4] A polypeptide comprising a mutant Fc region that contains amino acid modifications in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and the mutant Fc region contains amino acid modifications at the following positions: (i) positions 234, 235, 236, 239, 268, 270, 298, and 326, as represented by EU numbering, in the first polypeptide of the parent Fc region; and (ii) positions 236, 270, 298, 326, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region. [5] The polypeptide of any one of [1] to [4], wherein the mutant Fc region further comprises an amino acid modification at position 332 (EU numbering) in the first polypeptide of the parent Fc region. [6] The polypeptide of any one of [1] to [5], wherein the mutant Fc region further comprises an amino acid modification at position 330 (EU numbering) in the first polypeptide of the parent Fc region. [7] The polypeptide of any one of [1] to [6], wherein the mutant Fc region further comprises an amino acid modification at position 332 (EU numbering) in the second polypeptide of the parent Fc region. [8] The polypeptide of any one of [1] to [7], wherein the mutant Fc region further comprises an amino acid modification at position 330 (EU numbering) in the second polypeptide of the parent Fc region. [9] A polypeptide comprising a mutant Fc region that contains amino acid modifications in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and the mutant Fc region contains amino acid modifications at the following positions: (i) positions 234, 235, 236, 239, 268, 270, 298, 330, and 332 (EU numbering) in the first polypeptide of the parent Fc region; and (ii) positions 236, 270, 298, 326, 330, 332, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region.

[10] A polypeptide described in any of [1] to [9], wherein the mutant Fc region further comprises amino acid modifications at positions 250 and 307, as represented by EU numbering, in the first polypeptide of the parent Fc region.

[11] The polypeptide of any one of [1] to

[10] , wherein the mutant Fc region further comprises amino acid modifications at positions 250 and 307, as represented by EU numbering, in a second polypeptide of the parent Fc region.

[12] A polypeptide comprising a mutant Fc region that contains amino acid modifications in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and the mutant Fc region contains amino acid modifications at the following positions: (i) positions 234, 235, 236, 239, 250, 268, 270, 298, and 307, as represented by EU numbering, in the first polypeptide of the parent Fc region; and (ii) positions 250, 270, 298, 307, 326, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region.

[13] A polypeptide comprising a mutant Fc region that contains amino acid modifications in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and the mutant Fc region contains amino acid modifications at the following positions: (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 326 (EU numbering) in the first polypeptide of the parent Fc region; and (ii) positions 236, 250, 270, 298, 307, 326, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region.

[14] A polypeptide comprising a mutant Fc region that contains amino acid modifications in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and the mutant Fc region contains amino acid modifications at the following positions: (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 330, and 332 (EU numbering) in the first polypeptide of the parent Fc region; and (ii) positions 236, 250, 270, 298, 307, 326, 330, 332, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region.

[15] The polypeptide of any one of [1] to

[14] , comprising at least one amino acid modification selected from the amino acid modifications listed below: (i) in a first polypeptide of a parent Fc region, Tyr or Phe at position 234, Gln or Tyr at position 235, Trp at position 236, Met at position 239, Val at position 250, Asp at position 268, Glu at position 270, Ala at position 298, Pro at position 307, Asp at position 326, Met at position 330, and Glu at position 332, as indicated by EU numbering; and (ii) Ala at position 236, Val at position 250, Glu at position 270, Ala at position 298, Pro at position 307, Asp at position 326, Met or Lys at position 330, Asp or Glu at position 332, and Glu at position 334 in the second polypeptide of the parent Fc region, as indicated by EU numbering.

[16] The polypeptide of any one of [1] to

[15] , wherein the mutant Fc region further comprises any one of the following amino acid modifications (a) to (f): (a) a Lys at position 356 (EU numbering) in a first polypeptide of a parent Fc region and a Glu at position 439 (EU numbering) in a second polypeptide of a parent Fc region; (b) Glu at position 439 (EU numbering) in the first polypeptide of the parent Fc region, and Lys at position 356 (EU numbering) in the second polypeptide of the parent Fc region. (c) a Trp at position 366, as indicated by EU numbering, in a first polypeptide of a parent Fc region, and a Ser at position 366, an Ala at position 368, and a Val at position 407, as indicated by EU numbering, in a second polypeptide of a parent Fc region; (d) Ser at position 366, Ala at position 368, and Val at position 407, as indicated by EU numbering, in a first polypeptide of the parent Fc region, and Trp at position 366, as indicated by EU numbering, in a second polypeptide of the parent Fc region; (e) a Cys at position 349 and a Trp at position 366, as indicated by EU numbering, in a first polypeptide of a parent Fc region, and a Cys at position 356, a Ser at position 366, an Ala at position 368, and a Val at position 407, as indicated by EU numbering, in a second polypeptide of a parent Fc region; (f) Cys at position 356, Ser at position 366, Ala at position 368, and Val at position 407, as indicated by EU numbering, in a first polypeptide of the parent Fc region, and Cys at position 349 and Trp at position 366, as indicated by EU numbering, in a second polypeptide of the parent Fc region.

[17] The polypeptide of any one of [1] to

[16] , wherein the mutant Fc region further comprises any one of the following amino acid modifications (a) to (d) in the first polypeptide and / or the second polypeptide of the parent Fc region: (a) Ala at position 434, as expressed in EU numbering; (b) Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440, as expressed in EU numbering; (c) Leu at position 428, Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440, as indicated by EU numbering; (d) Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440, as expressed in EU numbering.

[18] A polypeptide described in any of [1] to

[17] , in which the binding activity of the mutant Fc region to at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa is enhanced compared to the parent Fc region.

[19] The polypeptide of

[18] , wherein the mutant Fc region has enhanced binding activity to FcγRIIa and FcγRIIIa compared to the parent Fc region.

[20] A polypeptide described in any one of [1] to

[19] , wherein the selectivity between activating Fcγ receptors and inhibitory Fcγ receptors is improved in the mutant Fc region compared to the parent Fc region. [20-2] A polypeptide described in any of [1] to

[19] , in which the binding activity of the mutant Fc region to activating Fcγ receptors is selectively enhanced compared to the binding activity of the mutant Fc region to inhibitory Fcγ receptors, compared to the parent Fc region. [20-3] A polypeptide described in any of [1] to

[19] , in which the ratio of binding activity to activating Fcγ receptors to binding activity to inhibitory Fcγ receptors (A / I ratio) is higher in the mutant Fc region than in the parent Fc region. [20-4] The ratio (A / I ratio) of the polypeptide comprising the mutant Fc region is 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 40-fold or more, 50-fold or more, or 60-fold or more, compared to the polypeptide comprising the parent Fc region. The polypeptide described in [20-3], which is 70 times or more, 80 times or more, 90 times or more, 100 times or more, 200 times or more, 300 times or more, 400 times or more, 500 times or more, 600 times or more, 700 times or more, 800 times or more, 900 times or more, 1000 times or more, 2000 times or more, 3000 times or more, 4000 times or more, 5000 times or more, 6000 times or more, 7000 times or more, 8000 times or more, 9000 times or more, or 10000 times or more larger. [20-5] The polypeptide described in [20-3], wherein the value of the ratio (A / I ratio) in the polypeptide comprising a mutant Fc region is 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, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, 2000 or more, 3000 or more, 4000 or more, 5000 or more, 6000 or more, 7000 or more, 8000 or more, 9000 or more, 10000 or more, 11000 or more, 12000 or more, 13000 or more, 14000 or more, or 15000 or more.

[21] The polypeptide of any of

[20] to [20-5], wherein the activating Fcγ receptor is at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, and FcγRIIIa, and the inhibitory Fcγ receptor is FcγRIIb.

[22] The polypeptide of any one of [1] to

[21] , wherein the polypeptide comprising a mutant Fc region is an antibody.

[23] A method for producing a polypeptide comprising a mutant Fc region, comprising the step of introducing amino acid modifications into a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and amino acid modifications are introduced at the following positions: (i) positions 234, 235, 236, 239, 268, 270, and 298, as represented by EU numbering, in the first polypeptide of the parent Fc region; and (ii) positions 270, 298, 326, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region.

[24] A method for producing a polypeptide comprising a mutant Fc region, comprising the step of introducing amino acid modifications into a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains and the amino acid modifications are introduced at the following positions: (i) positions 234, 235, 236, 239, 268, 270, 298, and 326, as represented by EU numbering, in the first polypeptide of the parent Fc region; and (ii) positions 236, 270, 298, 326, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region.

[25] A method for producing a polypeptide comprising a mutant Fc region, comprising the step of introducing amino acid modifications into a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains and the amino acid modifications are introduced at the following positions: (i) positions 234, 235, 236, 239, 268, 270, 298, 330, and 332 (EU numbering) in the first polypeptide of the parent Fc region; and (ii) positions 236, 270, 298, 326, 330, 332, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region.

[0011]

[26] An isolated nucleic acid encoding the polypeptide of any one of [1] to

[22] .

[27] A host cell comprising the nucleic acid according to

[26] .

[28] A method for producing a polypeptide, comprising culturing the host cell of

[27] so that the polypeptide is produced.

[29] The polypeptide of any one of [1] to

[22] for use in treating a tumor.

[30] The polypeptide of any one of [1] to

[22] for use in damaging cells.

[31] The polypeptide of

[30] , wherein the cell damage is caused by ADCC activity, CDC activity, or ADCP activity.

[32] A pharmaceutical composition comprising the polypeptide of any one of [1] to

[22] and a pharmaceutically acceptable carrier.

[33] The pharmaceutical composition according to

[32] , which is a pharmaceutical composition for treating tumors.

[34] The pharmaceutical composition according to

[32] , which is a pharmaceutical composition for cytotoxicity.

[35] The pharmaceutical composition according to

[34] , wherein the cell damage is caused by ADCC activity, CDC activity, or ADCP activity.

[36] A method for treating a tumor, comprising administering the polypeptide of any one of [1] to

[22] or the pharmaceutical composition of

[32] .

[37] A method for damaging cells, comprising administering the polypeptide of any one of [1] to

[22] or the pharmaceutical composition of

[32] .

[38] The method according to

[37] , wherein the cell is injured by ADCC activity, CDC activity, or ADCP activity.

[39] Use of the polypeptide according to any one of [1] to

[22] in the manufacture of a tumor therapeutic agent.

[40] Use of the polypeptide according to any one of [1] to

[22] in the production of a cytotoxic agent.

[41] The use according to

[40] , wherein the cell damage is caused by ADCC activity, CDC activity, or ADCP activity.

[42] A method for modifying the function of a polypeptide comprising an Fc region, comprising the step of introducing amino acid modifications into a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and amino acid modifications are introduced at the following positions: (i) positions 234, 235, 236, 239, 268, 270, and 298, as represented by EU numbering, in the first polypeptide of the parent Fc region; and (ii) positions 270, 298, 326, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region.

[43] A method for modifying the function of a polypeptide comprising an Fc region, comprising the step of introducing amino acid modifications into a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and amino acid modifications are introduced at the following positions: (i) positions 234, 235, 236, 239, 268, 270, 298, and 326, as represented by EU numbering, in the first polypeptide of the parent Fc region; and (ii) positions 236, 270, 298, 326, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region.

[44] A method for modifying the function of a polypeptide comprising an Fc region, comprising the step of introducing amino acid modifications into a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and the amino acid modifications are introduced at the following positions: (i) positions 234, 235, 236, 239, 268, 270, 298, 330, and 332 (EU numbering) in the first polypeptide of the parent Fc region; and (ii) positions 236, 270, 298, 326, 330, 332, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region.

[45] the method according to any one of

[42] to

[44] , wherein the altered function is an enhancement of binding activity to FcγRIIa and FcγRIIIa;

[46] the method of any one of

[42] to

[44] , wherein the functional alteration is an improvement in selectivity between an activating Fcγ receptor and an inhibitory Fcγ receptor;

[47] the method of any of

[42] to

[44] , wherein the functional alteration is a selective enhancement of binding activity to an activating Fcγ receptor compared to binding activity to an inhibitory Fcγ receptor;

[48] ​​The method of any of

[42] to

[44] , wherein the functional alteration is an increase in the ratio of binding activity to activating Fcγ receptors to binding activity to inhibitory Fcγ receptors (A / I ratio).

[49] The ratio (A / I ratio) is 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 40-fold or more, 50-fold or more, 60-fold or more, or 70-fold or more, compared to that of a polypeptide comprising a parent Fc region. , 80-fold or more, 90-fold or more, 100-fold or more, 200-fold or more, 300-fold or more, 400-fold or more, 500-fold or more, 600-fold or more, 700-fold or more, 800-fold or more, 900-fold or more, 1000-fold or more, 2000-fold or more, 3000-fold or more, 4000-fold or more, 5000-fold or more, 6000-fold or more, 7000-fold or more, 8000-fold or more, 9000-fold or more, or 10000-fold or more.

[50] The method of any of

[42] to

[49] , wherein the activating Fcγ receptor is at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, and FcγRIIIa, and the inhibitory Fcγ receptor is FcγRIIb.

[51] The method according to any one of

[42] to

[44] , wherein the altered function is an enhancement of ADCC activity, CDC activity, or ADCP activity. [Brief explanation of the drawings]

[0012] [Figure 1] Figure 1 shows the results of an ADCC reporter gene assay using Hepa1-6 / hEREG cells as target cells and hFcγRIIIaV-expressing Jurkat cells as effector cells. Each point represents the average fold induction value for n=2. [Figure 2] Figure 2 shows the results of an ADCP reporter gene assay using Hepa1-6 / hEREG cells as target cells and hFcγRIIaH-expressing Jurkat cells as effector cells. Each point represents the average fold induction value (n=3). [Figure 3] Figure 3 shows the antitumor effects of EGL-G1d, EGL-afucosyl, and EGL-ART6 in a human FcγR transgenic mouse model transplanted with the Hepa1-6 / hEREG cell line. The antibodies were administered via the tail vein at 10 mg / kg. Each point represents the mean tumor volume for a group of 5 animals. [Figure 4] 4 shows the binding activity of each antibody with a modified Fc to hC1q, where each point represents the average ELISA color development value (n=2). [Figure 5] Figure 5 is a continuation of the graph showing the binding activity of each antibody with a modified Fc to hC1q. Each point represents the average ELISA color development value (n=2). DETAILED DESCRIPTION OF THE INVENTION

[0013] The techniques and procedures described or cited herein are generally well understood and can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd edition (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney), ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DM Weir and C.C.Blackwell, eds.);Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987);PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994);Current Protocols in Immunology (JE Coligan et al., eds., 1991);Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (CA Janeway and P. Travers, 1997);Antibodies (P. Finch, 1997);Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989);Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993), using conventional techniques commonly used by those skilled in the art.

[0014] I. Definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, NY 1992) provide those skilled in the art with general guidance for many of the terms used in this application. All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety.

[0015] For purposes of interpreting this specification, the following definitions will apply, and wherever applicable, terms used in the singular will also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. In the event that any of the definitions below conflict with any document incorporated herein by reference, the definition below shall control.

[0016] As used herein, "first polypeptide" and "second polypeptide" refer to polypeptides that constitute the Fc region of an antibody. "First polypeptide" and "second polypeptide" mean that they have different sequences, and preferably differ in at least the sequence of their CH2 regions. They may also differ in the sequence of their CH3 regions. Such polypeptides may be, for example, polypeptides that constitute the Fc region of native IgG, or may be polypeptides in which modifications have been made to the polypeptides that constitute the Fc region of native IgG.

[0017] Native IgG refers to a polypeptide that includes the same amino acid sequence as IgG found in nature and belongs to the class of antibodies substantially encoded by immunoglobulin gamma genes. For example, native human IgG refers to native human IgG1, native human IgG2, native human IgG3, native human IgG4, etc. Native IgG also includes naturally occurring mutants thereof.

[0018] In the present invention, the term "polypeptide" generally refers to a peptide or protein having a length of about 10 amino acids or more. It is generally a polypeptide derived from a living organism, but is not particularly limited thereto, and may be, for example, a polypeptide consisting of an artificially designed sequence. It may also be a natural polypeptide, a synthetic polypeptide, a recombinant polypeptide, or the like. In addition, the term "protein molecule" in the present invention refers to a molecule containing such a polypeptide.

[0019] A preferred example of the polypeptide of the present invention is an antibody. Further preferred examples include natural IgG and antibodies obtained by modifying natural IgG. An example of natural IgG is, in particular, natural human IgG. Natural IgG refers to a polypeptide that includes the same amino acid sequence as an IgG found in nature and belongs to the class of antibodies substantially encoded by immunoglobulin gamma genes. For example, natural human IgG refers to natural human IgG1, natural human IgG2, natural human IgG3, natural human IgG4, etc. Natural IgG also includes naturally occurring mutants thereof.

[0020] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0021] "Native antibodies" refer to immunoglobulin molecules with various naturally occurring structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant heavy chain domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light chain (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa (κ) or lambda (λ), based on the amino acid sequence of its constant domain.

[0022] The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0023] The term "variable region" or "variable domain" refers to the domains of the heavy and / or light chains of an antibody that are involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR) (see, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated by screening a complementary library of VL or VH domains, respectively, using a VH or VL domain from an antibody that binds to that antigen. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0024] The terms "full length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0025] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain, except that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) residues of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0026] The term "polypeptide comprising an Fc region" is not particularly limited as long as it refers to a polypeptide containing an Fc region, including, for example, an antibody comprising an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) or the C-terminal glycine-lysine (residues 446-447) of the Fc region can be removed, for example, during purification of the polypeptide (e.g., antibody) or by recombinant engineering of a nucleic acid encoding the polypeptide. Thus, a composition comprising a polypeptide having an Fc region of the present invention can include a polypeptide comprising an Fc region with G446-K447, a polypeptide comprising an Fc region with G446 but without K447, a polypeptide with an Fc region from which G446-K447 has been completely removed, or a mixture of the above three types of polypeptides.

[0027] A "native-sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. Native-sequence human Fc regions include native-sequence human IgG1 Fc regions (non-A and A allotypes); native-sequence human IgG2 Fc regions; native-sequence human IgG3 Fc regions; and native-sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0028] A "mutant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by at least one amino acid modification (alteration), preferably one or more amino acid substitutions. Preferably, a mutant Fc region has at least one amino acid substitution, for example, about 1 to about 30 amino acid substitutions, preferably about 1 to about 20 amino acid substitutions, more preferably about 1 to about 10 amino acid substitutions, and most preferably about 1 to about 5 amino acid substitutions, in the native-sequence Fc region or parent Fc region compared to the native-sequence Fc region or parent Fc region. The mutant Fc region herein preferably has at least about 80% homology with the native-sequence Fc region or parent Fc region, preferably at least about 85% homology thereto, more preferably at least about 90% homology thereto, and most preferably at least about 95% homology thereto.

[0029] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps, if necessary, and excluding any conservative substitutions from the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetyx Corporation). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0030] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR binds to IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see, e.g., Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9: 457-492 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med 126: 330-341 (1995). Other FcRs, including those identified in the future, are also encompassed by the term "FcR" herein.

[0031] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for regulating maternal IgG transfer to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)) and immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, for example, Ghetie and Ward, Immunol. Today 18(12): 592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7): 637-640 (1997); Hinton et al., J. Biol. Chem. 279(8): 6213-6216 (2004); WO2004 / 92219 (Hinton et al.)).

[0032] "Effector cell" refers to a leukocyte that expresses one or more FcRs and exerts effector function. In certain embodiments, the cell expresses at least FcγRIII and exerts ADCC effector function. Examples of leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources, e.g., from blood. In certain embodiments, effector cells can be human effector cells.

[0033] "Effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation. A "functional Fc region" comprises an "effector function" of a native sequence Fc region. Such effector function generally requires that the Fc region be combined with a binding domain (e.g., an antibody variable domain) and can be assessed, for example, using various assays disclosed within the definitions herein.

[0034] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" (antibody-dependent cell-mediated cytotoxicity) refers to a form of cytotoxicity in which secreted immunoglobulins bind to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages), thereby enabling these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill them with cytotoxins. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. Nos. 5,500,362 or 5,821,337 or U.S. Pat. No. 6,737,056 (Presta), can be performed. Useful effector cells for such assays include PBMCs and NK cells. Alternatively, or additionally, the ADCC activity of a molecule of interest can be assessed in vivo in an animal model, such as that disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998).

[0035] Examples of "cytotoxic activity" include the above-mentioned antibody-dependent cell-mediated cytotoxicity (ADCC) activity, the below-mentioned complement-dependent cytotoxicity (CDC) activity, and T-cell cytotoxic activity. CDC activity refers to cytotoxic activity mediated by the complement system. On the other hand, ADCC activity refers to the activity of an antibody binding to an antigen present on the surface of a target cell, and then effector cells binding to the antibody, causing the effector cells to injure the target cell. Whether an antibody of interest has ADCC activity or CDC activity can be determined by known methods (e.g., Current Protocols in Immunology, Chapter 7. Immunologic studies in humans, edited by Coligan et al. (1993)).

[0036] The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism for inducing cell death in which the Fc effector domain of a target-bound antibody activates a series of enzymatic reactions that result in the formation of holes in the target cell's membrane. Typically, antigen-antibody complexes formed on the target cell bind to and activate complement component C1q, which then activates the complement cascade, leading to target cell death. Complement activation can also result in the deposition of complement components on the surface of target cells, which promote ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

[0037] The term "antibody-dependent cellular phagocytosis" or "ADCP" refers to the process by which antibody-coated cells, either in whole or in part, are internalized by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the Fc region of immunoglobulin.

[0038] An "isolated" polypeptide is one that has been separated from a component of its original environment. In some embodiments, the polypeptide is purified to greater than 95% or 99% purity, for example, as measured by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing polypeptide purity, see, e.g., Flatman et al., J. Chromatogr. B 848: 79-87 (2007).

[0039] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its original environment. Isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0040] An "isolated nucleic acid encoding a polypeptide" refers to one or more nucleic acid molecules that encode the polypeptide (e.g., an Fc region of an antibody, or the heavy and light chains or fragments thereof of an antibody), and includes nucleic acid molecules carried on a single vector or separate vectors, and nucleic acid molecules present in one or more locations in a host cell.

[0041] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of effecting the expression of nucleic acids to which they are operatively linked. Such vectors are also referred to herein as "expression vectors."

[0042] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived from that cell regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as that for which the original transformed cell was screened or selected are also included herein.

[0043] The terms "pharmaceutical formulation" and "pharmaceutical composition" are used interchangeably and refer to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional components that are unacceptably toxic to the subject to which it is administered.

[0044] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is human.

[0045] A "pharmaceutically acceptable carrier" refers to an ingredient, other than an active ingredient, in a pharmaceutical formulation or composition that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0046] An "effective amount" of an agent (e.g., a pharmaceutical formulation) refers to an amount, at dosages and for periods of time necessary, effective to achieve a desired therapeutic or prophylactic result.

[0047] As used herein, "treatment" (and its grammatical derivatives, such as "treat," "treating," etc.) refers to clinical intervention intended to alter the natural course of the individual being treated and can be performed for prophylaxis or during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, polypeptides comprising the variant Fc regions of the present invention are used to delay the onset of disease or slow the progression of disease.

[0048] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive as used herein.

[0049] The term "tumor tissue" refers to tissue containing at least one tumor cell. Tumor tissue usually consists of a group of tumor cells (parenchyma), which form the main body of the tumor, and the connective tissue and blood vessels (stroma) that exist between them and support the tumor. In some cases, the distinction between the two is clear, while in other cases, the two are mixed. Immune cells and other substances may infiltrate tumor tissue. On the other hand, "non-tumor tissue" refers to tissue other than tumor tissue in the body. Healthy tissue / normal tissue that is not in a diseased state is a typical example of non-tumor tissue.

[0050] <Polypeptides containing mutant Fc regions> In one aspect, the present invention provides an isolated polypeptide comprising a mutant Fc region. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In certain embodiments, the mutant Fc region comprises at least one amino acid residue modification (e.g., substitution) compared to the corresponding sequence in the Fc region of a native sequence or a reference mutant sequence (sometimes collectively referred to herein as a "parent" Fc region). Native sequence Fc regions are typically organized as homodimers consisting of two identical polypeptide chains. The amino acid modification in the mutant Fc region of the present invention may be introduced into either one of the two polypeptide chains of the parent Fc region, or into both of the two polypeptide chains.

[0051] In some aspects, the present invention provides mutant Fc regions with altered function compared to their parent Fc regions. In certain aspects, the mutant Fc regions of the present invention have enhanced binding activity to Fcγ receptors compared to their parent Fc regions. In certain embodiments, the mutant Fc regions of the present invention have enhanced binding activity to at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa compared to their parent Fc regions. In some embodiments, the mutant Fc regions of the present invention have enhanced binding activity to FcγRIIa. In some embodiments, the mutant Fc regions of the present invention have enhanced binding activity to FcγRIIIa. In a further embodiment, the mutant Fc regions of the present invention have enhanced binding activity to FcγRIIa and FcγRIIIa. In another aspect, the mutant Fc regions of the present invention have enhanced ADCC activity, CDC activity, or ADCP activity compared to their parent Fc regions.

[0052] The terms "binding activity" and "binding capacity" are used interchangeably herein and refer to the strength of the sum of noncovalent interactions between one or more binding sites (e.g., variable region or Fc region) of a molecule (e.g., an antibody or other polypeptide) and the molecule's binding partner (e.g., an antigen or Fcγ receptor). Here, "binding activity" is not strictly limited to a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen, or an Fc region and an Fcγ receptor). For example, when members of a binding pair reflect a monovalent 1:1 interaction, binding activity refers to the intrinsic binding affinity ("affinity"). When members of a binding pair are capable of both monovalent and multivalent binding, binding activity is the sum of these avidities. The binding activity of a molecule X for its partner Y can generally be expressed as a dissociation constant (KD) or "amount of analyte bound per unit amount of ligand." Avidity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring avidity are described below.

[0053] In a specific embodiment, the binding activity of the parent Fc region and the mutant Fc region can be expressed as KD (Dissociation constant) values. In one embodiment, the ratio of [KD value of the parent Fc region for FcγRIIa] / [KD value of the mutant Fc region for FcγRIIa] is, for example, 1.5 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, or 50 or more. In a further embodiment, FcγRIIa may be FcγRIIa R or FcγRIIa H, or both. Therefore, the KD value of the Fc region for FcγRIIa may be the KD value of the Fc region for FcγRIIa R or the KD value of the Fc region for FcγRIIa H. Alternatively, it may be the sum or average of both. In one embodiment, the ratio of [binding activity of parent Fc region to FcγRIIIa] / [binding activity of mutant Fc region to FcγRIIIa] is, for example, 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, 50 or more, 100 or more, 200 or more, 300 or more, 500 or more, 1 × 10 3 That's it, 2 x 10 3 That's it, 3 x 10 3 or more, or 5 x 10 3 That is all. In a further embodiment, FcγRIIIa may be FcγRIIIa F or FcγRIIIa V, or both. Therefore, the KD value of the Fc region for FcγRIIIa may be the KD value of the Fc region for FcγRIIIa F or the KD value of the Fc region for FcγRIIa V. Alternatively, it may be the sum or average of both.

[0054] In one embodiment, the KD value of the mutant Fc region for FcγRIIa is, for example, 1.0×10 -6 M or less, 5.0×10 -7 M or less, 3.0×10 -7 M or less, 2.0×10 -7 M or less, 1.0×10 -7 M or less, 5.0×10 -8M or less, 3.0×10 -8 M or less, 2.0×10 -8 M or less, 1.0×10 -8 M or less, 5.0×10 -9 M or less, 3.0×10 -9 M or less, 2.0×10 -9 M or less, or 1.0 x 10 -9 In a further embodiment, FcγRIIa may be FcγRIIa R or FcγRIIa H, or both. In one embodiment, the KD value of the mutant Fc region for FcγRIIIa is, for example, 1.0 × 10 -6 M or less, 5.0×10 -7 M or less, 3.0×10 -7 M or less, 2.0×10 -7 M or less, 1.0×10 -7 M or less, 5.0×10 -8 M or less, 3.0×10 -8 M or less, 2.0×10 -8 M or less, 1.0×10 -8 M or less, 5.0×10 -9 M or less, 3.0×10 -9 M or less, 2.0×10 -9 M, 1.0 × 10 -9 M or less, 5.0×10 -10 M or less, 3.0×10 -10 M or less, 2.0×10 -10 M, or 1.0 x 10 -10 In a further embodiment, FcγRIIIa may be FcγRIIIa F or FcγRIIIa V, or both.

[0055] In another embodiment, the binding activity of the parent Fc region and the mutant Fc region may be expressed as kd (Dissociation rate constant) values ​​instead of KD values.

[0056] In another embodiment, the binding activity of the parent Fc region and the mutant Fc region may be expressed as the amount of Fcγ receptor binding per unit amount. For example, in a surface plasmon resonance assay, the amount of Fc region bound to a sensor chip and the amount of Fcγ receptor binding thereto are each measured in resonance units (RU). The amount of Fcγ receptor binding thereto divided by the amount of Fc region binding can be defined as the amount of Fc region binding to Fcγ receptor per unit amount. Specific methods for measuring and calculating such binding amounts are described in the Examples below. In some embodiments, the ratio of [amount of mutant Fc region binding to FcγRIIa] / [amount of parent Fc region binding to FcγRIIa] is, for example, 1.5 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, or 50 or more. In some embodiments, the ratio of [amount of binding of mutant Fc region to FcγRIIIa] / [amount of binding of parent Fc region to FcγRIIIa] is, for example, 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, 50 or more, 100 or more, 200 or more, 300 or more, 500 or more, 1 × 10 3 That's it, 2 x 10 3 That's it, 3 x 10 3 or more, or 5 x 10 3 That's all.

[0057] In certain embodiments, the KD values, kd values, binding amounts, etc., presented herein are measured or calculated by surface plasmon resonance assays performed at 25°C or 37°C (see, e.g., Example 2 herein).

[0058] In certain aspects, the mutant Fc regions of the present invention have improved selectivity between activating Fcγ receptors and inhibitory Fcγ receptors compared to the parent Fc region. In other words, the mutant Fc regions of the present invention have greater enhanced binding activity to activating Fcγ receptors than to inhibitory Fcγ receptors compared to the parent Fc region. In certain embodiments, the activating Fcγ receptor is at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa R, FcγRIIa H, FcγRIIIa F, and FcγRIIIa V, and the inhibitory Fcγ receptor is FcγRIIb. In some embodiments, the mutant Fc regions of the present invention have improved selectivity between FcγRIIa and FcγRIIb. In some embodiments, the mutant Fc regions of the present invention have improved selectivity between FcγRIIIa and FcγRIIb. In a further aspect, the mutant Fc regions of the present invention have improved selectivity between FcγRIIa and FcγRIIb and between FcγRIIIa and FcγRIIb.

[0059] In certain embodiments, the binding activity of the parent Fc region and the mutant Fc region can be expressed as KD (Dissociation constant) values. The binding activity for FcγRIIa and FcγRIIIa is as described above. In one embodiment, the ratio of [KD value of the parent Fc region for FcγRIIb] / [KD value of the mutant Fc region for FcγRIIb] is, for example, 10 or less, 5 or less, 3 or less, 2 or less, 1 or less, 0.5 or less, 0.3 or less, 0.2 or less, or 0.1 or less. In another embodiment, the binding activity of the parent Fc region and the mutant Fc region may be expressed as kd (Dissociation rate constant) values ​​instead of KD values.

[0060] In another embodiment, the binding activity of the parent Fc region and the mutant Fc region may be expressed as the amount of binding of the Fc region to an Fcγ receptor per unit amount as described above. In some embodiments, the ratio of [amount of binding of the mutant Fc region to FcγRIIb] / [amount of binding of the parent Fc region to FcγRIIb] is, for example, 10 or less, 5 or less, 3 or less, 2 or less, 1 or less, 0.5 or less, 0.3 or less, 0.2 or less, or 0.1 or less. In some embodiments, the amount of binding of the mutant Fc region to FcγRIIb is, for example, 0.5 or less, 0.3 or less, 0.2 or less, 0.1 or less, 0.05 or less, 0.03 or less, 0.02 or less, 0.01 or less, 0.005 or less, 0.003 or less, 0.002 or less, or 0.001 or less.

[0061] In a specific embodiment, improved selectivity between activating Fcγ receptors and inhibitory Fcγ receptors refers to a selective enhancement of binding activity to activating Fcγ receptors compared to binding activity to inhibitory Fcγ receptors; in other words, an increase in the ratio of binding activity to activating Fcγ receptors to binding activity to inhibitory Fcγ receptors (A / I ratio). Such a ratio (A / I ratio) is an indicator of the exertion of superior effector function, and polypeptides with a large A / I ratio can be evaluated as having superior effector function. The binding activity of a parent Fc region and a mutant Fc region to an Fcγ receptor can be expressed as a KD value, kd value, or the amount of Fc region binding to an Fcγ receptor per unit amount. The A / I ratio can be expressed using the KD value, kd value, or binding amount as follows: [KD value for inhibitory Fcγ receptor] / [KD value for activating Fcγ receptor], [kd value for inhibitory Fcγ receptor] / [kd value for activating Fcγ receptor], or [amount of binding to activating Fcγ receptor] / [amount of binding to inhibitory Fcγ receptor].

[0062] In one aspect, the A / I ratio of the mutant Fc region of the present invention is 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, 1.9 times or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, 60 times or more compared to the parent Fc region. or more than 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times, 2000 times, 3000 times, 4000 times, 5000 times, 6000 times, 7000 times, 8000 times, 9000 times, or 10000 times. In one aspect, the A / I ratio of the mutant Fc region of the present invention is 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, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, 2000 or more, 3000 or more, 4000 or more, 5000 or more, 6000 or more, 7000 or more, 8000 or more, 9000 or more, 10000 or more, 11000 or more, 12000 or more, 13000 or more, 14000 or more, or 15000 or more. In one embodiment, the A / I ratio is the ratio of FcγRIa-binding activity to FcγRIIb-binding activity, the ratio of FcγRIIa-binding activity to FcγRIIb-binding activity, the ratio of FcγRIIIa-binding activity to FcγRIIb-binding activity, or the ratio of [the sum or average of two or three of FcγRIa-binding activity, FcγRIIa-binding activity, and FcγRIIIa-binding activity] to FcγRIIb-binding activity. In a specific embodiment, FcγRIIa is FcγRIIa R, FcγRIIa H, or both, and therefore, the binding activity to FcγRIIa is the sum or average of FcγRIIa R-binding activity, FcγRIIa H-binding activity, or both.In a specific embodiment, FcγRIIIa is FcγRIIIa F, FcγRIIIa V, or both, and thus, the binding activity to FcγRIIIa is the sum or average of the binding activity to FcγRIIIa F, the binding activity to FcγRIIIa V, or both.

[0063] In some embodiments, the variant Fc regions of the present invention comprise amino acid modifications at the following positions: (i) positions 234, 235, 236, 239, 268, 270, and 298, as represented by EU numbering, in the first polypeptide of the parent Fc region; and (ii) positions 270, 298, 326, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region. In a specific embodiment, a variant Fc region of the present invention further comprises an amino acid modification at position 326 (EU numbering) in a first polypeptide of the parent Fc region. In a specific embodiment, a variant Fc region of the present invention further comprises an amino acid modification at position 236 (EU numbering) in a second polypeptide of the parent Fc region. In a specific embodiment, a variant Fc region of the present invention further comprises an amino acid modification at position 332 (EU numbering) in a first polypeptide of the parent Fc region. In a specific embodiment, a variant Fc region of the present invention further comprises an amino acid modification at position 330 (EU numbering) in a first polypeptide of the parent Fc region. In a specific embodiment, a variant Fc region of the present invention further comprises an amino acid modification at position 332 (EU numbering) in a second polypeptide of the parent Fc region. In a specific embodiment, a variant Fc region of the present invention further comprises an amino acid modification at position 330 (EU numbering) in a second polypeptide of the parent Fc region. Alternatively, the amino acid modifications described in International Publication Nos. WO2013 / 002362 and WO2014 / 104165 can also be used in the present invention.

[0064] In certain aspects, the mutant Fc regions of the present invention have improved stability compared to the parent Fc region. In certain embodiments, the stability is thermodynamic stability. The thermodynamic stability of a polypeptide can be determined, for example, using the Tm value as an indicator. The Tm value can be measured using techniques known to those skilled in the art, such as CD (circular dichroism), DSC (differential scanning calorimetry), and DSF (differential scanning fluorometry). In one embodiment, the Tm value of the CH2 region of the mutant Fc regions of the present invention is increased by 0.1° or more, 0.2° or more, 0.3° or more, 0.4° or more, 0.5° or more, 1° or more, 2° or more, 3° or more, 4° or more, 5° or more, or 10° or more compared to the parent Fc region.

[0065] In some embodiments, the mutant Fc regions of the present invention comprise at least one amino acid modification in at least one position selected from the group consisting of positions 250 and 307 (EU numbering) in the first polypeptide and / or second polypeptide of the parent Fc region. Alternatively, the amino acid modifications described in International Publication WO2013 / 118858 may also be used in the present invention.

[0066] In a specific aspect, the mutant Fc region of the present invention is composed of two polypeptide chains with different sequences. In a further aspect, the mutant Fc region of the present invention exhibits enhanced heterodimerization between a first polypeptide and a second polypeptide. When producing heterodimeric proteins using recombinant techniques, it is preferable that different peptide chains preferentially associate to form heterodimers, rather than identical polypeptide chains associating to form homodimers. Whether or not heterodimerization of the mutant Fc region has been enhanced can be determined, for example, by separating homodimers and heterodimers from the produced mutant Fc region using techniques such as chromatography and determining the ratio of each component.

[0067] In some embodiments, the mutant Fc regions of the present invention comprise at least one amino acid modification in at least one position selected from the group consisting of positions 349, 356, 366, 368, 407, and 439 (EU numbering) in the first polypeptide and / or second polypeptide of the parent Fc region. Alternatively, the amino acid modifications described in International Publication WO2006 / 106905 and WO1996 / 027011 can also be used in the present invention.

[0068] In certain aspects, the mutant Fc regions of the present invention have enhanced FcRn-binding activity at acidic pH. In some embodiments, acidic pH refers to pH 4.0 to 6.5. In further embodiments, acidic pH is at least one selected from the group consisting of pH 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5. In certain embodiments, the acidic pH is pH 5.8.

[0069] In some embodiments, the mutant Fc region of the present invention comprises at least one amino acid modification in at least one position selected from the group consisting of positions 428, 434, 436, 438, and 440 (EU numbering) in the first polypeptide and / or second polypeptide of the parent Fc region. Alternatively, the amino acid modifications described in International Publication WO2016 / 125495 may also be used in the present invention.

[0070] In one aspect, the mutant Fc region of the present invention comprises at least one amino acid modification at at least one position selected from the group consisting of positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, 334, 349, 356, 366, 368, 407, 428, 434, 436, 438, 439, and 440, as expressed in EU numbering.

[0071] In one embodiment, a variant Fc region of the invention comprises amino acid modifications at positions 234, 235, 236, 239, 268, 270, 298, 326, and 334, as indicated by EU numbering. In a particular embodiment, a variant Fc region of the invention comprises amino acid modifications at (i) positions 234, 235, 236, 239, 268, 270, and 298, as indicated by EU numbering, in a first polypeptide of a parent Fc region, and (ii) positions 270, 298, 326, and 334, as indicated by EU numbering, in a second polypeptide of a parent Fc region. In another specific embodiment, the variant Fc region of the invention comprises amino acid modifications at (i) positions 234, 235, 236, 239, 268, 270, 298, and 326, as indicated by EU numbering, in a first polypeptide of the parent Fc region, and (ii) positions 236, 270, 298, 326, and 334, as indicated by EU numbering, in a second polypeptide of the parent Fc region.

[0072] In one embodiment, a variant Fc region of the invention comprises amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, and 334, as indicated by EU numbering. In a particular embodiment, a variant Fc region of the invention comprises amino acid modifications at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, and 307, as indicated by EU numbering, in a first polypeptide of a parent Fc region, and (ii) positions 250, 270, 298, 307, 326, and 334, as indicated by EU numbering, in a second polypeptide of a parent Fc region. In another specific embodiment, the variant Fc region of the invention comprises amino acid modifications at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 326, as indicated by EU numbering, in a first polypeptide of the parent Fc region, and (ii) positions 236, 250, 270, 298, 307, 326, and 334, as indicated by EU numbering, in a second polypeptide of the parent Fc region.

[0073] In one embodiment, a variant Fc region of the invention comprises amino acid modifications at positions 234, 235, 236, 239, 268, 270, 298, 326, 330, 332, and 334, as indicated by EU numbering. In a particular embodiment, a variant Fc region of the invention comprises amino acid modifications at (i) positions 234, 235, 236, 239, 268, 270, 298, 330, and 332, as indicated by EU numbering, in a first polypeptide of a parent Fc region, and (ii) positions 236, 270, 298, 326, 330, 332, and 334, as indicated by EU numbering, in a second polypeptide of a parent Fc region. In one embodiment, a variant Fc region of the invention comprises amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, and 334, as indicated by EU numbering. In a particular embodiment, a variant Fc region of the invention comprises amino acid modifications at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 330, and 332, as indicated by EU numbering, in a first polypeptide of a parent Fc region, and (ii) positions 236, 250, 270, 298, 307, 326, 330, 332, and 334, as indicated by EU numbering, in a second polypeptide of a parent Fc region. In another specific embodiment, the variant Fc region of the invention comprises amino acid modifications at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 326, as indicated by EU numbering, in a first polypeptide of the parent Fc region, and (ii) positions 236, 250, 270, 298, 307, 326, 330, 332, and 334, as indicated by EU numbering, in a second polypeptide of the parent Fc region.

[0074] In one embodiment, a variant Fc region of the invention comprises amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, and 334, as indicated by EU numbering. In a particular embodiment, a variant Fc region of the invention comprises amino acid modifications at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, and 332, as indicated by EU numbering, in a first polypeptide of a parent Fc region, and (ii) positions 236, 250, 270, 298, 307, 326, 332, and 334, as indicated by EU numbering, in a second polypeptide of a parent Fc region.

[0075] In one embodiment, a variant Fc region of the invention comprises amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, and 334, as indicated by EU numbering. In a particular embodiment, a variant Fc region of the invention comprises amino acid modifications at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 332, as indicated by EU numbering, in a first polypeptide of a parent Fc region, and (ii) positions 250, 270, 298, 307, 326, 332, and 334, as indicated by EU numbering, in a second polypeptide of a parent Fc region.

[0076] In one embodiment, a variant Fc region of the invention comprises amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, and 334, as indicated by EU numbering. In a particular embodiment, a variant Fc region of the invention comprises amino acid modifications at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 330, as indicated by EU numbering, in a first polypeptide of a parent Fc region, and (ii) positions 250, 270, 298, 307, 326, 330, and 334, as indicated by EU numbering, in a second polypeptide of a parent Fc region.

[0077] In a further embodiment, the variant Fc region of the present invention comprises at least one amino acid modification selected from the group consisting of: (i) Tyr or Phe at position 234, Gln or Tyr at position 235, Trp at position 236, Met at position 239, Val at position 250, Asp at position 268, Glu at position 270, Ala at position 298, Pro at position 307, Asp at position 326, Met at position 330, and Glu at position 332, as indicated by EU numbering, in a first polypeptide of the parent Fc region; and (ii) Ala at position 236, Val at position 250, Val at position 270, Glu at position 298, Ala at position 307, Pro at position 326, Asp at position 326, Met or Lys at position 330, Asp or Glu at position 332, and Glu at position 334, as indicated by EU numbering, in a second polypeptide of the parent Fc region.

[0078] In a further embodiment, the mutant Fc region of the present invention further comprises any of the following amino acid modifications (a) to (f): (a) a Lys at position 356 (EU numbering) in a first polypeptide of a parent Fc region and a Glu at position 439 (EU numbering) in a second polypeptide of a parent Fc region; (b) Glu at position 439 (EU numbering) in the first polypeptide of the parent Fc region, and Lys at position 356 (EU numbering) in the second polypeptide of the parent Fc region. (c) a Trp at position 366, as indicated by EU numbering, in a first polypeptide of a parent Fc region, and a Ser at position 366, an Ala at position 368, and a Val at position 407, as indicated by EU numbering, in a second polypeptide of a parent Fc region; (d) Ser at position 366, Ala at position 368, and Val at position 407, as indicated by EU numbering, in a first polypeptide of the parent Fc region, and Trp at position 366, as indicated by EU numbering, in a second polypeptide of the parent Fc region; (e) a Cys at position 349 and a Trp at position 366, as indicated by EU numbering, in a first polypeptide of a parent Fc region, and a Cys at position 356, a Ser at position 366, an Ala at position 368, and a Val at position 407, as indicated by EU numbering, in a second polypeptide of a parent Fc region; (f) Cys at position 356, Ser at position 366, Ala at position 368, and Val at position 407, as indicated by EU numbering, in a first polypeptide of the parent Fc region, and Cys at position 349 and Trp at position 366, as indicated by EU numbering, in a second polypeptide of the parent Fc region.

[0079] In a further aspect, the variant Fc region of the present invention further comprises any of the following amino acid modifications (a) to (d) in the first polypeptide and / or second polypeptide of the parent Fc region: (a) Ala at position 434, as expressed in EU numbering; (b) Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440, as indicated by EU numbering; (c) Leu at position 428, Ala at position 434, Thr at position 436, Arg at position 438, Glu at position 440, as indicated by EU numbering; (d) Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440, as indicated by EU numbering.

[0080] In a further aspect, the present invention provides a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 7-22.

[0081] The term "Fcγ receptor" (herein referred to as Fcγ receptor, FcγR, or FcgR) refers to a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies, and effectively refers to any member of a family of proteins encoded by Fcγ receptor genes. In humans, this family includes FcγRI (CD64), which includes the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 (H type) and R131 (R type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which includes the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any unidentified human FcγR, FcγR isoform, or allotype. FcγRIIb1 and FcγRIIb2 have been reported as splice variants of human FcγRIIb. Furthermore, a splice variant designated FcγRIIb3 has been reported (J Exp Med, 1989, 170: 1369-1385). In addition to these splice variants, human FcγRIIb includes all splice variants registered with NCBI: NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1, and NP_003992.3. Furthermore, human FcγRIIb includes, in addition to FcγRIIb, all genetic polymorphisms that have been reported in the past (Arthritis Rheum. 48: 3242-3252 (2003); Kono et al., Hum. Mol. Genet. 14: 2881-2892 (2005); and Kyogoju et al., Arthritis Rheum. 46: 1242-1254 (2002)), as well as all genetic polymorphisms that will be reported in the future.

[0082] There are two allotypes of FcγRIIa: one in which the amino acid at position 131 of FcγRIIa is histidine (H type), and the other in which the amino acid at position 131 is substituted with arginine (R type) (Warrmerdam, J. Exp. Med. 172: 19-25(1990)).

[0083] FcγRs may be derived from any organism, including, but not limited to, FcγRs from humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any mouse FcγR or FcγR isoform.

[0084] The amino acid sequence of human FcγRI is described in NP_000557.1; the amino acid sequence of human FcγRIIa is described in AAH20823.1, etc.; the amino acid sequence of human FcγRIIb is described in AAI46679.1, etc.; the amino acid sequence of human FcγRIIIa is described in AAH33678.1, etc.; and the amino acid sequence of human FcγRIIIb is described in AAI28563.1.

[0085] Unlike Fcγ receptors, which belong to the immunoglobulin superfamily, human FcRn is structurally similar to major histocompatibility complex (MHC) class I polypeptides, sharing 22–29% sequence identity with class I MHC molecules (Ghetie et al., Immunol. Today (1997) 18(12), 592–598). FcRn is expressed as a heterodimer consisting of a transmembrane α or heavy chain complexed with a soluble β or light chain (β2-microglobulin). Like MHC, the α chain of FcRn consists of three extracellular domains (α1, α2, and α3), and a short cytoplasmic domain anchors the protein to the cell surface. The α1 and α2 domains interact with the FcRn-binding domain in the Fc region of an antibody (Raghavan et al. (Immunity (1994) 1, 303-315). The amino acid sequence of human FcRn is listed in NP_004098.1, and the amino acid sequence of β2-microglobulin is listed in NP_004039.1).

[0086] As used herein, the term "parent Fc region" refers to an Fc region prior to the introduction of the amino acid modifications described herein. In some embodiments, the parent Fc region is a native sequence Fc region (or an Fc region of a native antibody). Examples of antibodies include IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), and IgM. Antibodies may be derived from humans or monkeys (e.g., cynomolgus monkeys, rhesus monkeys, marmosets, chimpanzees, or baboons). Native antibodies may contain naturally occurring mutations. Multiple allotype sequences of IgG due to genetic polymorphisms are described in "Sequences of proteins of immunological interest," NIH Publication No. 91-3242, and any of these sequences may be used in the present invention. In particular, for human IgG1, the amino acid sequence at positions 356-358 (EU numbering) may be either DEL or EEM. Also, for human IgG1, the amino acid at position 214 (EU numbering) can be either K or R. In a specific embodiment, the parent Fc region is an Fc region derived from the heavy chain constant region of human IgG1, human IgG2, human IgG3, or human IgG4. In another embodiment, the parent Fc region is an Fc region derived from the heavy chain constant region of SEQ ID NO: 1 or SEQ ID NO: 28. In a further embodiment, the parent Fc region may be an Fc region created by adding amino acid modifications other than those described herein to a native sequence Fc region (reference variant sequence Fc region). Native sequence Fc regions are usually organized as homodimers consisting of two identical polypeptide chains.

[0087] Additionally, amino acid modifications made for other purposes can be combined in the variant Fc regions described herein. For example, amino acid substitutions that enhance FcRn-binding activity (Hinton et al., J. Immunol. 176(1): 346-356 (2006); Dall'Acqua et al., J. Biol. Chem. 281(33): 23514-23524 (2006); Petkova et al., Intl. Immunol. 18(12): 1759-1769 (2006); Zalevsky et al., Nat. Biotechnol. 28(2): 157-159 (2010); WO2006 / 019447; WO2006 / 053301; and WO2009 / 086320) and amino acid substitutions that improve antibody heterogeneity or stability (WO2009 / 041613) may be made. Alternatively, the mutant Fc regions described herein can be combined with polypeptides having the property of promoting antigen clearance as described in WO2011 / 122011, WO2012 / 132067, WO2013 / 046704, or WO2013 / 180201, polypeptides having the property of specifically binding to target tissues as described in WO2013 / 180200, or polypeptides having the property of repeatedly binding to multiple antigen molecules as described in WO2009 / 125825, WO2012 / 073992, or WO2013 / 047752. Alternatively, the amino acid modifications disclosed in EP1752471 and EP1772465 can be combined in the CH3 of the mutant Fc regions described herein to confer binding ability to other antigens. Alternatively, the mutant Fc region described herein may be combined with amino acid modifications that decrease the pI of the constant region (WO2012 / 016227) to increase plasma retention, or with amino acid modifications that increase the pI of the constant region (WO2014 / 145159) to promote cellular uptake.Alternatively, amino acid modifications that increase the pI of the constant region (WO2016 / 125495) may be combined in the mutant Fc region described herein to promote plasma clearance of the target molecule. In one embodiment, such modifications may include, for example, substitutions at at least one position selected from the group consisting of positions 311, 343, 384, 399, 400, and 413, as represented by EU numbering. In a further embodiment, such substitutions may be substitutions of the amino acid at each position with Lys or Arg.

[0088] Techniques for producing heterodimeric antibodies include, but are not limited to, knob-in-hole technology (see, for example, Nat. Biotechnol., (16); 677-681 (1998) and U.S. Pat. No. 5,731,168) and the manipulation of electrostatic steering effects (WO2006 / 106905, WO2009 / 089004A1, J. Biol. Chem., (285), 19637-19646 (2010), etc.).

[0089] With regard to the binding of heterologous polypeptides containing mutant Fc regions, techniques can be applied that suppress unintended binding of homologous polypeptides containing mutant Fc regions by introducing electrostatic repulsion at the interface of the CH2 or CH3 domain of the Fc region, as described in WO2006 / 106905.

[0090] Examples of amino acid residues in contact with the interface of the CH2 or CH3 domain of the Fc region include residues at positions 356 (EU numbering), 439 (EU numbering), 357 (EU numbering), 370 (EU numbering), 399 (EU numbering), and 409 (EU numbering) in the CH3 domain.

[0091] More specifically, for example, an Fc region can be produced in which one to three pairs of amino acid residues selected from the following (1) to (3) have the same charge: (1) amino acid residues at positions 356 and 439 (EU numbering) in the CH3 domain; (2) amino acid residues at positions 357 and 370 (EU numbering) in the CH3 domain; and (3) amino acid residues at positions 399 and 409 (EU numbering) in the CH3 domain.

[0092] Furthermore, a heterologous polypeptide can be produced comprising a mutant Fc region in which one to three pairs of amino acid residues selected from (1) to (3) above have the same charge in the CH3 domain of a first Fc region, and the pairs of amino acid residues selected in the first Fc region above also have the same charge in the CH3 domain of a second Fc region, but the charges of the first and second Fc regions are opposite.

[0093] In the above-mentioned Fc region, for example, negatively charged amino acid residues are preferably selected from glutamic acid (E) and aspartic acid (D), and positively charged amino acid residues are preferably selected from lysine (K), arginine (R), and histidine (H).

[0094] Other known techniques can also be used to bind heterologous polypeptides containing mutant Fc regions. Specifically, such techniques involve replacing the amino acid side chains present in one Fc region with larger side chains (knobs) and smaller side chains (holes) in the Fc region, thereby locating the knobs within the holes. This promotes efficient binding between Fc region-containing polypeptides with different amino acid sequences (WO1996 / 027011; Ridgway et al., Prot. Eng. 9:617-621(1996); Merchant et al., Nat. Biotech. 16, 677-681(1998)).

[0095] Furthermore, other known techniques can be used for heterologous binding of polypeptides containing mutant Fc regions. Binding of polypeptides containing Fc regions can be efficiently induced using chain-swapped recombinant CH3 domain heterodimers (Davis et al., Prot. Eng. Des. & Sel., 23:195-202(2010)). This technique can also be used to efficiently induce binding between Fc region-containing polypeptides with different amino acid sequences.

[0096] In addition, the heterodimerized antibody production technique described in WO2011 / 028952, which utilizes the combination of antibody CH1 and CL and VH and VL, can also be used.

[0097] Similar to the methods described in WO2008 / 119353 and WO2011 / 131746, it is also possible to use a heterodimeric antibody production technique in which two types of homodimeric antibodies are prepared in advance, the antibodies are incubate under reducing conditions to dissociate them, and then the antibodies are re-bound.

[0098] Furthermore, it is also possible to use techniques for producing heterodimerized antibodies by modifying the CH2 and CH3 domains, similar to the method described in WO2012 / 058768.

[0099] When two polypeptides containing mutant Fc regions with different amino acid sequences are simultaneously expressed to produce a polypeptide containing a heterologous mutant Fc region, a polypeptide containing a homologous mutant Fc region is usually also produced as an impurity. In such cases, the polypeptide containing the heterologous mutant Fc region can be efficiently obtained by separating and purifying it from the polypeptide containing the homologous mutant Fc region using known techniques. A method for efficiently separating and purifying a heterodimerized antibody from a homodimerized antibody using ion exchange chromatography has been reported (WO 2007 / 114325) by introducing amino acid modifications into the variable regions of the two antibody heavy chains that result in a difference in the isoelectric point between the homodimerized antibody and the heterodimerized antibody. Another method for purifying a heterodimerized antibody using protein A chromatography has been reported (WO 1998 / 050431 and WO 1995 / 033844) by constructing a heterodimerized antibody containing two heavy chains derived from mouse IgG2a, which binds to protein A, and rat IgG2b, which does not bind to protein A.

[0100] Furthermore, by substituting amino acids such as Tyr or His for the amino acid residues at positions 435 and 436 (EU numbering) located in the protein A binding site of the antibody heavy chain to confer different protein A binding affinities, heterodimerized antibodies can be efficiently purified using protein A chromatography.

[0101] In the present invention, amino acid modification refers to any substitution, deletion, addition, insertion, and modification, or a combination thereof. In the present invention, amino acid modification can be rephrased as amino acid mutation.

[0102] The number of amino acid modifications introduced into the Fc region is not limited, and in certain embodiments, may be 1, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 8 or less, 10 or less, 12 or less, 14 or less, 16 or less, 18 or less, 20 or less, 22 or less, 24 or less, 26 or less, 28 or less, or 30 or less.

[0103] In one aspect, the present invention provides a method for producing a polypeptide comprising a mutant Fc region. In a further aspect, the present invention provides a method for producing a polypeptide comprising a mutant Fc region with altered function. In a further aspect, the present invention provides a method for altering the function of a polypeptide comprising an Fc region. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In certain embodiments, the methods comprise introducing at least one amino acid modification into a parent Fc region. In certain embodiments, the methods comprise (i) providing a polypeptide comprising a parent Fc region, and (ii) introducing at least one amino acid modification into the parent Fc region. In certain embodiments, the methods may further comprise (iii) measuring the function of the polypeptide comprising the mutant Fc region. A native Fc region is usually composed of two identical polypeptide chains. The amino acid modification to the parent Fc region may be introduced into either or both of the two polypeptide chains of the parent Fc region.

[0104] In another embodiment, a method for producing a polypeptide comprising a mutant Fc region comprises the steps of: (i) providing one or more nucleic acids encoding a polypeptide comprising a parent Fc region; (ii) introducing at least one mutation into the region of the nucleic acid encoding the parent Fc region; (iii) introducing the nucleic acid produced in (ii) into a host cell; and (iv) culturing the cell described in (iii) to express the polypeptide comprising the mutant Fc region. In a specific embodiment, the method may further comprise the step of (v) recovering the polypeptide comprising the mutant Fc region from the host cell culture described in (iv).

[0105] In certain embodiments, the nucleic acids produced in (ii) may be contained in one or more vectors (eg, expression vectors).

[0106] In some embodiments, the amino acid modifications used in this manufacturing method are selected from any single modification, combination of single modifications, or combination modifications listed in Table 1, selected from the amino acid modifications that can be included in the mutant Fc region described above.

[0107] The Fc region may be obtained by partially digesting IgG1, IgG2, IgG3, IgG4 monoclonal antibodies, etc. with a protease such as pepsin, followed by re-elution of the fraction adsorbed to the Protein A column. The protease is not particularly limited, as long as it is capable of digesting full-length antibodies, thereby producing Fab and F(ab')2 in a limiting manner by appropriately setting the enzyme reaction conditions, such as pH, and examples include pepsin and papain.

[0108] In addition to the production methods described above, polypeptides comprising mutant Fc regions of the present invention may also be produced by other methods known in the art. Polypeptides comprising mutant Fc regions produced by the production methods described herein are also included in the present invention.

[0109] In one embodiment, an isolated nucleic acid encoding a polypeptide comprising a mutant Fc region of the present invention is provided. Such a nucleic acid may encode an amino acid sequence comprising a first polypeptide and / or an amino acid sequence comprising a second polypeptide of the mutant Fc region. In a further embodiment, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In a further embodiment, a host cell comprising such a nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., is transformed with) either (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising a first polypeptide of the mutant Fc region and an amino acid sequence comprising a second polypeptide of the mutant Fc region, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising a first polypeptide of the mutant Fc region and a second vector comprising a nucleic acid encoding an amino acid sequence comprising a second polypeptide of the mutant Fc region. In one embodiment, the host cell is eukaryotic (e.g., Chinese hamster ovary (CHO) cell) or lymphoid cell (e.g., Y0, NS0, Sp2 / 0 cell). In one aspect, a method for producing a polypeptide comprising a mutant Fc region of the present invention is provided, comprising culturing a host cell comprising a nucleic acid encoding a polypeptide comprising a mutant Fc region of the present invention under conditions suitable for expression of the polypeptide, and optionally recovering the polypeptide from the host cell (or host cell culture medium).

[0110] For recombinant production of polypeptides comprising a variant Fc region of the invention, nucleic acid encoding the polypeptide is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., using an oligonucleotide probe capable of binding specifically to a gene encoding the Fc region of an antibody).

[0111] Suitable host cells for cloning or expressing vectors encoding polypeptides comprising variant Fc regions of the invention include prokaryotic or eukaryotic cells.

[0112] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast, including fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of antibody Fc regions with partial or fully human glycosylation patterns, are suitable cloning or expression hosts for vectors encoding polypeptides comprising the mutant Fc regions of the invention. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004) and Li et al., Nat. Biotech. 24: 210-215 (2006).

[0113] Host cells derived from multicellular organisms (invertebrates and vertebrates) are also suitable for expressing glycosylated antibody Fc regions. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified for use in conjugation with insect cells, particularly transformation of Spodoptera frugiperda cells.

[0114] Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429.

[0115] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that have been adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7); human embryonic kidney (293 or 293 cells, e.g., as described in Graham et al., J. Gen Virol. 36: 59 (1977)); baby hamster kidney (BHK) cells; mouse Sertoli cells (TM4 cells, e.g., as described in Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney (CV1); African green monkey kidney (VERO-76); human cervical carcinoma (HELA); canine kidney (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (e.g., Mather et al., Annals NY Acad. Sci. 383: 44-68 (1982)). (described in U.S. Pat. No. 6,239,499); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0.

[0116] Various assays described herein or known in the art may be used to identify or screen the mutant Fc regions provided herein, or to characterize their physical or chemical properties or biological activities.

[0117] Assays for determining the binding activity of polypeptides comprising variant Fc regions to one or more FcR family members are described herein or known in the art, including, but not limited to, surface plasmon resonance assays, amplified luminescence proximity homogeneous assay (ALPHA) screening, ELISA, and fluorescence-activated cell sorting (FACS) (Lazar et al., Proc. Natl. Acad. Sci. USA (2006) 103(11): 4005-4010).

[0118] In one embodiment, the binding activity of a polypeptide comprising a mutant Fc region to an FcR family member can be measured using a surface plasmon resonance assay. For example, various FcRs are allowed to interact as analytes with a polypeptide comprising a mutant Fc region immobilized or captured on a sensor chip using known methods and reagents (e.g., Protein A, Protein L, Protein A / G, Protein G, anti-λ chain antibody, anti-κ chain antibody, antigen peptide, antigen protein, etc.). Alternatively, FcRs may be immobilized or captured on a sensor chip, and the polypeptide comprising a mutant Fc region may be used as the analyte. As a result of such interactions, binding sensorgrams are obtained, and by analyzing these, the dissociation constant (KD) of the binding can be calculated. The difference in resonance unit (RU) values ​​in the sensorgrams before and after interaction with the FcR (i.e., the amount of FcR bound) can also be used as an indicator of the binding activity of a polypeptide comprising a mutant Fc region to the FcR. Furthermore, the corrected value (i.e., the amount of FcR binding per unit amount of polypeptide containing a mutant Fc region) obtained by dividing the amount of FcR binding by the difference in RU values ​​in the sensorgram before and after the polypeptide containing a mutant Fc region is immobilized or captured on a sensor chip (i.e., the amount of polypeptide containing a mutant Fc region bound) can be used as an indicator of binding activity.

[0119] Any of the polypeptides comprising the variant Fc regions provided herein may be used in therapeutic methods. In one aspect, a polypeptide comprising a mutated Fc region is provided for use as a pharmaceutical. In a further aspect, a polypeptide comprising a mutated Fc region is provided for use in treating tumors. In a particular embodiment, a polypeptide comprising a mutated Fc region is provided for use in a therapeutic method. In a particular embodiment, the present invention provides a polypeptide comprising a mutated Fc region for use in a method of treating an individual having a tumor, the method comprising administering to the individual an effective amount of a polypeptide comprising a mutated Fc region. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In a further embodiment, the present invention provides a polypeptide comprising a mutated Fc region for use in damaging cells. In a particular embodiment, the present invention provides a polypeptide comprising a mutated Fc region for use in a method of damaging cells in an individual, the method comprising administering to the individual an effective amount of a polypeptide comprising a mutated Fc region to damage the cells. An "individual" according to any of the above embodiments is preferably a human.

[0120] In some embodiments, the tumor is a solid tumor. In solid tumors, tumor cells usually proliferate and form a cluster, which then forms the tumor tissue. In addition, tumor tissue in vivo is often infiltrated with immune cells such as lymphocytes, which also constitute part of the tumor tissue. In one embodiment, cell damage is induced by ADCC activity, CDC activity, or ADCP activity.

[0121] In a further aspect, the present invention provides use of a polypeptide comprising a variant Fc region in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of a tumor. In a further embodiment, the medicament is for use in a method of treating a tumor, the method comprising administering an effective amount of the medicament to an individual having a tumor. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In a further embodiment, the medicament is for cell damage. In a further embodiment, the medicament is for use in a method of cell damage in an individual, the method comprising administering to the individual an effective amount of the medicament to damage the cells. The "individual" according to any of the above embodiments may be a human.

[0122] In a further aspect, the present invention provides a method for treating a tumor. In one embodiment, the method comprises administering to an individual having such a tumor an effective amount of a polypeptide comprising a mutant Fc region. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. The "individual" according to any of the above embodiments may be a human.

[0123] In a further aspect, the present invention provides a method for damaging cells in an individual. In one embodiment, the method comprises administering to the individual an effective amount of a polypeptide comprising a mutant Fc region to damage the cells. In one embodiment, the "individual" is a human.

[0124] In a further aspect, the present invention provides a pharmaceutical formulation (pharmaceutical composition) comprising a polypeptide comprising a mutant Fc region provided herein. In one embodiment, the pharmaceutical formulation (pharmaceutical composition) further comprises a pharmaceutically acceptable carrier. In one embodiment, the present invention provides a pharmaceutical formulation (pharmaceutical composition) for use in treating tumors. In one embodiment, the present invention provides a pharmaceutical formulation (pharmaceutical composition) for use in damaging cells. In another embodiment, the pharmaceutical formulation (pharmaceutical composition) comprises a polypeptide comprising a mutant Fc region provided herein and at least one additional therapeutic agent. [Example]

[0125] [Example 1] Preparation of Fc region variants with enhanced FcγR-binding ability Fc variants that enhance the cytotoxic effector functions ADCC and ADCP have been reported, but both symmetrically engineered CH2 variants and low-fucose antibodies created by glycosylation have left room for further enhancement of FcγR binding. Furthermore, asymmetrically engineered CH2 variants described in WO2013002362 and WO2014104165 have significantly enhanced FcγR binding compared to symmetrically engineered Fc region variants, but there is still room for further improvement. Specifically, the Fc region variant Kn125 / H1076 (abbreviated herein as ART1) described in WO2013002362 and WO2014104165 has strongly enhanced FcγRIIIa binding, but its FcγRIIa binding is only several-fold enhanced compared to IgG1, suggesting that further enhancement is necessary to exhibit strong ADCP activity. Furthermore, Kn120 / H1068 (abbreviated herein as ART2) has enhanced binding to both FcγRIIa and FcγRIIIa, and is expected to have strong ADCC and ADCP activity. However, it also has enhanced binding to inhibitory FcγRIIb, posing the problem of a low A / I ratio, an indicator of superior effector function. In other words, existing methods have not achieved the ideal profile, "antibody engineering technology that binds more strongly to activating FcγRIIa and FcγRIIIa and reduces binding to inhibitory FcγRIIb." Therefore, in the present invention, further combinations of modifications were investigated to create Fc region variants with superior profiles that overcome these challenges.

[0126] Existing Fc region variants for comparison were constructed as follows. First, an antibody heavy chain gene, H240-G1d (SEQ ID NO: 1), was constructed, containing the heavy chain variable region against human epiregulin and the heavy chain constant region sequence of human IgG1, as described in WO2014104165. G1d is a sequence in which the C-terminal Lys and Gly were removed from the heavy chain constant region sequence of native human IgG1. Knobs-into-holes modification (Nat. Biotechnol., 1998, 16, 677), a modification to promote heterodimerization, was introduced into the CH3 region of H240-G1d, and asymmetric modifications to enhance FcγR binding were introduced into the CH2 region to construct the Fc region variants ART1 and ART2 for comparison. ART1, an Fc region variant with enhanced FcγRIIIa binding described in WO2013002362 and WO2014104165, was constructed as follows. H240-Kn125 (SEQ ID NO: 2) was generated by introducing L234Y / L235Q / G236W / S239M / H268D / D270E / S298A, which are FcγR binding-enhancing modifications, into the CH2 region of H240-G1d, and Y349C / T366W into the CH3 region. H240-H1076 (SEQ ID NO: 3) was generated by introducing D270E / K326D / A330M / K334E into the CH2 region of H240-G1d, and D356C / T366S / L368A / Y407V into the CH3 region. H240-Kn125, H240-Hl076, and a plasmid carrying the gene for the light chain of an anti-human epiregulin antibody, L73-k0 (SEQ ID NO: 4), were mixed and transfected into the human fetal kidney cell line Expi 293 (Invitrogen) by lipofection. After 4 days of culture, the supernatant was purified by rProtein A Sepharose. TMAn Fc-modified antibody against human epiregulin (H240-Kn125 / L73-k0 / / H240-Hl076 / L73-k0: antibody abbreviation EGL-ART1) was obtained by purifying the antibody using Fast Flow (Amersham Biosciences) according to a method known to those skilled in the art. The absorbance of the purified antibody solution at 280 nm was measured using a spectrophotometer. The concentration of the purified antibody was calculated from the obtained measurement value using the extinction coefficient calculated by the PACE method (Protein Science (1995) 4, 2411-2423).

[0127] Similarly, EGL-ART2 (H240-Kn120 / L73-k0 / / H240-Hl068 / L73-k0), an Fc region variant with enhanced binding to both FcγRIIa and FcγRIIIa, was produced as described in WO2013002362 and WO2014104165. Furthermore, by symmetrically introducing the FcγR binding-enhancing modifications G236A, S239D, A330L, and I332E into the CH2 region in different combinations, we generated the following antibodies: EGL-SDALIE (H240-Kn032 / L73-k0 / / H240-Hl032 / L73-k0), EGL-GASDIE (H240-Kn037 / L73-k0 / / H240-Hl036 / L73-k0), and EGL-GASDALIE (H240-GASDALIE / L73-k0). In addition to these, we also generated the afucosylated antibody EGL-afucosyl, which has been reported to enhance FcγRIIIa binding (e.g., Glycobiol. Vol. 17 no. 1 pp. 104-118 (2006)). In cells in which the expression of both fucose transporter genes on homologous chromosomes is artificially suppressed, the function of the fucose transporter is inhibited. Using these cells, it is possible to obtain fucose-deficient antibodies (WO2006 / 067913, etc.). Fucose-deficient antibodies can also be obtained by producing antibodies in cells in which beta 1,4-N-acetylglucosaminyltransferase III and Golgi alpha-mannosidae II are forcibly expressed (Biotechnol. Bioeng. (2006) 93 (5), 851-861). EGL-afucosyl (H240-G1d / L73-k_glycomab) was produced using these methods known to those skilled in the art.

[0128] To create variants superior to these existing Fc region variants, the new Fc region variants ART3, ART4, ART5, ART6, ART8, ART10, ART11, and ART12 listed in Table 1 were created. These variants commonly contain L234F, L235Q, G236W, S239M, H268D, D270E, and S298A in one heavy chain and D270E, S298A, K326D, and K334E in the other heavy chain. These variants were created by further incorporating modifications that alter FcγR binding into this core group of asymmetric modifications. Specifically, the introduction of K326D, A330M, and I332E into the chain containing L234F / L235Q / G236W / S239M / H268D / D270E / S298A was investigated. Furthermore, the introduction of G236A, I332E, I332D, and A330M into the chain into which D270E / S298A / K326D / K334E had been introduced was investigated. In addition to these modifications that enhance FcγR binding, T250V and T307P, modifications that improve antibody stability as described in WO2013118858, were introduced into both chains of ART4, ART5, ART6, ART8, ART10, ART11, and ART12.

[0129] (Table 1) Fc region variants created and the modifications introduced TIFF0007731359000001.tif234151TIFF0007731359000002.tif23453

[0130] [Example 2] Evaluation of Fc region variant binding to FcγR The extracellular domains of FcγRs were prepared according to the method described in WO2014104165. Analysis of the interaction between the prepared antibodies and human FcγRs was performed using a Biacore 8K+ as follows. The running buffer was 50 mM Na-Phosphate, 150 mM NaCl, and 0.05% Tween 20 (pH 7.4), and measurements were performed at 25°C. A Series S SA (GE Healthcare) sensor chip with CaptureSelect Human Fab-kappa Kinetics Biotin Conjugate (Thermo Fisher Scientific) immobilized was used. The target antibodies were captured on this chip and allowed to interact with each FcγR diluted in the running buffer. The chip was regenerated with 10 mM Glycine-HCl (pH 1.5), and measurements were performed by repeatedly capturing antibodies. The dissociation constant KD (mol / L) of each antibody for FcγR was calculated using Biacore Insight Evaluation Software. The dissociation constant for FcγRIIb was calculated using a steady state affinity model, and the dissociation constants for other FcγRs were calculated using a 1:1 Langmuir binding model (Table 2).

[0131] (Table 2) Measurement of binding between the prepared variants and human FcγR TIFF0007731359000003.tif234129TIFF0007731359000004.tif23471

[0132] In the table, "relative value for KD of G1d and hFcγRs" refers to the KD value of G1d for each FcγR divided by the KD value of each antibody for each FcγR, indicating the degree to which each antibody is enhanced for G1d. Furthermore, "A / I ratio" refers to the KD of each antibody for FcγRIIb divided by the KD for each FcγR, indicating the degree to which binding to activating FcγRs is selectively enhanced relative to binding to inhibitory FcγRs.

[0133] The ART3, ART4, ART5, ART6, ART8, ART10, ART11, and ART12 constructed in the present invention all showed enhanced binding to FcγRIIIaF and FcγRIIIaV compared to G1d. These variants also showed enhanced binding to both FcγR, FcγRIIIaF and FcγRIIIaV, compared to the existing FcγR-enhancing antibodies GASDALIE, SDALIE, GASDIE, and Afucosyl antibodies, which have symmetrically modified antibodies. Furthermore, ART4 (2519.9-fold), ART6 (986.7-fold), ART8 (1966.7-fold), ART10 (1289.2-fold), and ART12 (577.5-fold) showed enhanced binding to FcγRIIIaF compared to ART2 (459.6-fold) described in WO2014104165. Furthermore, ART4, ART8, and ART10 had enhanced binding to FcγRIIIaF compared to ART1 (1170.2-fold), which had stronger binding to FcγRIIIaF than ART2. Similarly, for FcγRIIIaV, ART4 (462.2-fold), ART6 (321.9-fold), ART8 (694.9-fold), and ART10 (565.5-fold) had enhanced binding compared to ART2 (214.6-fold), and ART4, ART8, and ART10 had even enhanced binding compared to ART1 (322.8-fold). ART6 had similar enhanced binding to FcγRIIIaV as ART1.

[0134] ART3 (32.1-fold), ART4 (6.3-fold), ART5 (33.9-fold), ART6 (118.0-fold), ART8 (15.0-fold), ART10 (2.7-fold), ART11 (4.9-fold), and ART12 (3.1-fold) all had enhanced FcγRIIa activity compared to G1d. In particular, ART3, ART5, ART6, and ART8 were further enhanced compared to ART2 (14.7-fold), an FcγRIIa-enhanced antibody with asymmetrically modified CH2 regions described in WO2014104165. Furthermore, ART3, ART5, and ART6 were more strongly enhanced than GASDIE (16.4-fold), an existing FcγRIIa-enhanced antibody with symmetrical modifications introduced, and are expected to exhibit stronger ADCP activity than any of the existing modified antibodies. ART3 (13.7-fold), ART4 (3.2-fold), ART5 (10.4-fold), ART6 (24.1-fold), ART8 (13.2-fold), ART10 (1.5-fold), and ART11 (1.3-fold) all exhibited enhanced binding to FcγRIIaR compared to G1d. However, the existing variants GASDIE (24.7-fold) and ART2 (49.0-fold) exhibited even greater enhancements than these variants. However, what is noteworthy here is their selectivity for activating FcγRs. In contrast to activating FcγRs, FcγRIIb, an inhibitory receptor, induces intracellular signals that suppress immune responses, and is therefore expected to inhibit signals from activating FcγRs. Indeed, it has been reported that the antitumor effects of antibodies are enhanced in FcγRIIb knockout mice (Nature Medicine 2000, 6, 443-436). Furthermore, a correlation has been observed between differences in antitumor activity among mouse IgG subclasses and the ratio of binding to activating FcγRs to inhibitory FcγRs (A / I ratio) (Science 2005, 310, 1510-1512). Therefore, to exert stronger effector functions, antibodies with reduced binding to FcγRIIb and enhanced binding to activating FcγRs are considered necessary. However, because FcγRIIaR shares high sequence homology with FcγRIIb, it is difficult to confer selectivity, and in fact, the variants reported to date do not appear to have excellent selectivity.The newly created variants ART10 (A / I ratio 6.6), ART11 (A / I ratio 11.5), ART12 (A / I ratio 12.8), ART4 (A / I ratio 22.5), ART8 (A / I ratio 28.1), ART6 (A / I ratio 42.4), ART5 (A / I ratio 49.9), and ART3 (A / I ratio 52.4) were all superior to G1d (A / I ratio 6.1) in terms of the A / I ratio of FcγRIIaR, and among them, ART4, ART8, ART6, ART5, and ART3 were shown to be superior to ART2 (A / I ratio 13.0) and GASDIE (A / I ratio 18.6). Similarly, with regard to the A / I ratio of FcγRIIaH, ART10 (A / I ratio 17.3), ART8 (A / I ratio 47.2), ART12 (A / I ratio 60.3), ART11 (A / I ratio 63.3), ART4 (A / I ratio 65.2), ART3 (A / I ratio 180.7), ART5 (A / I ratio 240.1), and ART6 (A / I ratio 307.0) were superior to G1d (A / I ratio 8.9). Among these, ART8, ART12, ART11, ART4, ART3, ART5, and ART6 showed superior A / I ratios compared to ART2 (A / I ratio 5.8) and GASDIE (A / I ratio 18.2). These results suggest that ART4, ART8, ART3, ART5, and ART6 are antibodies with A / I ratios superior to existing enhanced antibodies against FcγRIIaR. Furthermore, ART3, ART5, and ART6 can be said to be antibodies with superior binding ability and A / I ratio compared to existing enhanced antibodies against FcγRIIaH.

[0135] The A / I ratios of FcγRIIIaF were ART3 (A / I ratio 396.1), ART5 (A / I ratio 398.8), ART11 (A / I ratio 694.1), ART6 (A / I ratio 975.5), ART8 (A / I ratio 2350.3), ART10 (A / I ratio 3159.9), ART12 (A / I ratio 4309.9), and ART4 (A / I ratio 9943.2), all of which were superior to G1d (A / I ratio 3.4). Among these, ART4 showed a superior A / I ratio to ART1 (A / I ratio 4947.2), an FcγRIIIa-specific enhanced variant described in WO2014104165. Similarly, the A / I ratios of FcγRIIIaV were ART3 (A / I ratio 2003.7), ART5 (A / I ratio 2064.8), ART6 (A / I ratio 2625.7), ART11 (A / I ratio 3974.6), ART8 (A / I ratio 6852.0), ART12 (A / I ratio 9721.0), ART10 (A / I ratio 11436.1), and ART4 (A / I ratio 15047.3), all of which were superior to G1d (A / I ratio 28.1) and the existing enhanced variant Afucosyl (A / I ratio 298.1). Among them, ART4 and ART10 showed a better A / I ratio than ART1 (A / I ratio 11261.3), an FcγRIIIa-specific enhanced variant described in WO2014104165. From the above results, it can be said that ART4 is an antibody with even better binding ability and A / I ratio for both FcγRIIIaF and FcγRIIIaV than the existing enhanced antibody ART1.

[0136] [Example 3] Evaluation of antibodies with altered Fc regions by ADCC reporter bioassay (3-1) Generation of human epiregulin-expressing cells (Hepa1-6 / hEREG) The mouse hepatocellular carcinoma line Hepa1-6 cells were purchased from ATCC and transfected with the human EREG (hEREG) gene, followed by selection of clones exhibiting constitutive expression. The hEREG gene was selected using Zeocin. Hepa1-6 / hEREG cells were maintained and passaged in D-MEM (high glucose) medium (SIGMA) containing 10% FBS (SIGMA) and 400 μg / mL Zeocin.

[0137] (3-2) Evaluation by ADCC Reporter Bioassay For in vitro ADCC activity measurement, hFcγRIIIaV ADCC Reporter Bioassay, Effector cells, Propagation Model (Promega) was used. 5 × 10 cells were added to each well of a 384-well plate. 5 The concentration was adjusted to / mL Hepa1-6 / hEREG cells were added as target cells in an assay buffer (96% RPMI, 4% FBS) medium. Next, the antibody prepared in Example 1 and EGL-G4d (heavy chain SEQ ID NO: 28, light chain SEQ ID NO: 4) having a human IgG4 sequence as a negative control were diluted with the assay buffer to a final concentration of 1 μg / mL, giving 11 points with a common ratio of 10, and then added in an assay buffer (10 μL). Finally, 3 × 10 cells were added to the medium as an effector cell solution. 6 10 μL of hFcγRIIIaV-expressing Jurkat cells adjusted to 1 / mL was added, and the mixture was mixed to a total volume of 30 μL. The plate was then incubated overnight at 37°C in a 5% CO2 incubator. The plate was then incubated at room temperature for 15 minutes, and 30 μL of Bio-Glo reagent was added to each well. Bio-Glo Luciferase Assay System (Buffer and Substrate) was used for the Bio-Glo reagent. Luminescence from each well was then measured using a plate reader.

[0138] The luminescence value of each well divided by the luminescence value of the well without antibody was used as the fold induction, which was used as an index to evaluate the ADCC of each antibody. The results are shown in Figure 1. The EC50 value of each sample was calculated using JMP 11.2.1 (SAS Institute Inc.) and is shown in Table 3.

[0139] Table 3: EC50 values ​​of reporter gene induction activity via hFcγRIIIaV for antibodies with each modified Fc TIFF0007731359000005.tif113128

[0140] These results demonstrated that the antibodies with modified Fc produced in this study had stronger reporter gene induction activity in Hepa1-6 / hEREG cells than wild-type human IgG1 constant regions. Furthermore, the results in Table 3 show that all of these variants exhibited activity at lower concentrations than symmetrically engineered CH2 region variants or low-fucose antibodies produced by glycosylation. Of the variants produced in this study, ART3, ART4, ART5, ART6, ART8, ART10, ART11, and ART12 were shown to exhibit activity at concentrations comparable to or lower than ART2. Among these, ART4, ART10, ART11, and ART12 exhibited activity at lower concentrations than ART1, which has even stronger hFcγRIIIaV binding than ART2.

[0141] [Example 4] Evaluation of antibodies with altered Fc regions by ADCP Reporter Bioassay For in vitro ADCP activity measurement, hFcγRIIaH ADCP Reporter Bioassay, Core Kit (Promega) was used. 1 × 10 hFcγRIIaH ADCP Reporter Bioassay, Core Kit (Promega) was used. Each well of a 384-well plate was filled with 1 × 10 hFcγRIIaH ADCP cells in the medium. 6 The concentration was adjusted to / mL 10 μL of Hepa1-6 / hEREG cells were added as target cells to each well, and Assay Buffer (96% RPMI, 4% FBS) was used as the medium. Next, 10 μL of the antibodies prepared in Example 1 were diluted with the assay buffer to final concentrations of 0, 0.001, 0.01, 0.1, 1, and 10 μg / mL, respectively, and then added. Finally, 10 μL of hFcγRIIaH-expressing Jurkat cells provided with the kit were added as the effector cell solution. The mixture was mixed to a total volume of 30 μL, and then allowed to stand at 37°C in a 5% CO2 incubator for 6 hours. The hFcγRIIaH-expressing Jurkat cells had a cell suspension density of 9.68 × 10 5The plate was then left to stand at room temperature for 15 minutes, and 30 μL of Bio-Glo reagent was added to each well. Bio-Glo Luciferase Assay System (Buffer and Substrate) was used for the Bio-Glo reagent. The luminescence of each well was then measured using a plate reader. The luminescence value of each well divided by the luminescence value of the well without antibody was used as the fold induction value, which was used as an index to evaluate the ADCP of each antibody. The results are shown in Figure 2. The EC50 value of each sample was calculated using JMP 11.2.1 (SAS Institute Inc.) and is shown in Table 4.

[0142] Table 4: EC50 values ​​of hFcγRIIaH-mediated reporter gene induction activity of antibodies with each modified Fc TIFF0007731359000006.tif113128

[0143] These results demonstrated that the antibodies with modified Fc produced in this study exhibited stronger reporter gene induction activity in Hepa1-6 / hEREG cells than wild-type human IgG1 constant regions. Furthermore, the results in Table 4 indicated that they exhibited activity at lower concentrations than variants with symmetrically engineered CH2 regions or low-fucose antibodies produced by glycosylation. Furthermore, of the variants produced in this study, ART2, ART3, ART5, ART6, and ART8 were shown to exhibit activity at lower concentrations than ART1. Among these, ART3, ART6, and ART8 exhibited activity at lower concentrations than ART2, which has further enhanced binding to hFcγRIIaH.

[0144] [Example 5] Evaluation of the antitumor effect of antibodies with altered Fc regions in a syngeneic tumor cell transplant model using human FcγR transgenic mice (5-1) Cell line The Hepa1-6 / hEREG cells prepared in Example 3-1 were maintained and passaged in D-MEM (high glucose) medium (SIGMA) containing 10% FBS (SIGMA) and 400 μg / mL Zeocin.

[0145] (5-2) Preparation of a mouse model transplanted with a syngeneic tumor line Human FcγR transgenic mice (Proc Natl Acad Sci USA. 2012 Apr 17; 109(16): 6181-6186) were used for efficacy testing. 16-week-old male mice were intraperitoneally administered 100 μL / head of anti-asialo GM1 antibody (aGM1, WAKO) to improve cell engraftment. The day after aGM1 administration, a cell solution consisting of a 1:1 mixture of Hepa1-6 / hEREG cells and Matrigel (CORNING) was subcutaneously injected at a cell count of 1 × 10 7 The average volume of the transplanted tumor was approximately 300 mm 3 to 500 mm 3 The model was considered to be valid when this was achieved. The volume of the transplanted tumor was calculated using the following formula. Tumor volume = major axis × minor axis × minor axis / 2

[0146] (5-3) Preparation of the administered drug EGL-ART6 produced in the present invention was expected to have the strongest antitumor activity, based on the A / I ratio results in Example 2 and the strength of the reporter gene induction activity in Examples 3 and 4. Therefore, as drugs to be administered to the Hepa1-6 / hEREG cell transplant model, an anti-hEREG control antibody (EGL-G1d) prepared by the same method as in Example 1 and an anti-hEREG antibody with an Fc that has enhanced FcγR binding (EGL-afucosyl, EGL-ART6) were each prepared to 1 mg / mL using His buffer (150 mM NaCl, 20 mM His-HCl buffer pH 6.0).

[0147] (5-4) Drug administration for measuring antitumor effects On day 7 after transplantation, EGL-G1d, EGL-afucosyl, and EGL-ART6 were administered via the tail vein at 10 mg / kg. Details of drug treatments used to measure antitumor effects are shown in Table 5.

[0148] Table 5: Antitumor effect measurement in the Hepa1-6 / hEREG cell transplant model TIFF0007731359000007.tif25128

[0149] (5-5) Evaluation of antitumor effects The antitumor effect was evaluated based on the tumor volume calculated using the formula described in (5-2). The TGI (tumor growth inhibition) value was calculated using the following formula. TGI = (1 - (mean tumor volume of the group of interest at the time of measurement - mean tumor volume before antibody administration) ÷ (mean tumor volume of the control group at the time of measurement - mean tumor volume before antibody administration)) × 100

[0150] As a result, both EGL-afucosyl and EGL-ART6, FcγR binding-enhancing antibodies, showed efficacy of TGI = 80 or higher at 19 days after administration at a dose of 10 mg / kg. In contrast, the control antibody EGL-G1d had a TGI = 31 (Figure 3). This confirmed that EGL-ART6, produced in the present invention, also had an expected enhanced antitumor effect in vivo compared to EGL-afucosyl.

[0151] [Example 6] Evaluation of the binding activity of antibodies with altered Fc regions to C1q While CDC activity of antibodies has been reported to contribute to antitumor effects ( Nat. Immunol., 2017, 18, 889 ), it is also known to cause CDC-related side effects, such as infusion-related reactions ( J. Immunol. 2008, 180, 2294-2298 and Br. J. Haematol. 2001, 115, 807-811 ). Therefore, even when developing antibody drugs with enhanced ADCC or ADCP activity, it is desirable to be able to select the level of CDC activity depending on the target disease. The interaction between complement and Fc is mediated by C1q. Analysis of the interaction between C1q and Fc ( Science, 2018, 359, 794-797 and Molecular Immunology 2012, 51, 66-72 ) suggests that the interaction sites between FcγR and C1q on the Fc region partially overlap. These publications report that residues at positions 329, 330, and 331 (EU numbering) on ​​the Fc domain are important for interaction with C1q, and that residues at positions 268, 270, and 298 (EU numbering) also contribute to C1q binding. Because these positions and their surrounding residues are the sites modified in the present invention to enhance FcγR binding, the resulting Fc domain variants are likely to also enhance or attenuate C1q binding, potentially enabling control of CDC activity in the development of antibody pharmaceuticals. Therefore, the C1q binding of the resulting Fc domain variants was evaluated.

[0152] The anti-human epiregulin antibodies prepared in Examples 1 and 3 were subjected to ELISA. The buffers shown in Table 6 were also prepared appropriately. Human C1q protein (hC1q) was used as the antigen.

[0153] Table 6: Composition of buffer used in human C1q ELISA TIFF0007731359000008.tif27149

[0154] First, a 96-well maxisorp plate (Thermo Fisher) was coated overnight at 4°C with 50 μL of a solution containing each antibody prepared in PBS at concentrations of 30, 10, 3, 1, 0.3, 0.1, or 0.03 μg / mL. Each well of the plate was washed with wash buffer to remove any unbound antibodies, and then the wells were blocked with 200 μL of blocking / dilution buffer for at least 2 hours at room temperature. After removing the blocking / dilution buffer, 50 μL of hC1q (Calbiochem) prepared in blocking / dilution buffer to a final concentration of 3 μg / mL was added to each well. The plate was left at room temperature for 1 hour to allow hC1q to bind to each antibody present in each well. After washing with Wash Buffer, 50 μL of HRP-conjugated anti-hC1q antibody (AbDSerotec) diluted with Blocking / Dilution Buffer was added to each well, and the plate was incubated for 1 hour. After washing with Wash Buffer, TMB single solution (Invitrogen) was added. The color reaction of the solution in each well was stopped by adding Stop Buffer, and the color development was measured by absorbance at 450 nm and 690 nm. The buffer used had the composition listed in Table 6. The measurement results are shown in Figures 4 and 5.

[0155] As shown in Figures 4 and 5, of the variants evaluated, the C1q binding ability of ART3, ART5, and ART11 was enhanced compared to G1d. Furthermore, Afucosyl and ART8 had similar binding ability to G1d. Furthermore, ART1, ART2, ART4, ART6, ART10, ART12, GASDALIE, SDALIE, and GASDIE had reduced C1q binding ability compared to G1d. Among these, the C1q binding ability of ART1, ART2, ART4, ART6, ART12, GASDALIE, SDALIE, and GASDIE was reduced to the same level as G4d, which has the sequence of human IgG4. Because human IgG4 is believed to have almost no CDC activity (J. Immunol. Methods 2005, 306, 151-160), these variants, whose C1q binding was reduced to the same level as G4d, are considered to have almost no CDC activity, similar to IgG4. The amino acid modifications common to these variants that significantly reduced C1q binding include modifications to Ala330 and Ile332. These regions, especially Ala330, are crucial for the interaction with C1q, and it is thought that introducing modifications to this site significantly reduced C1q binding. On the other hand, ART3, ART5, and ART11, which showed enhanced C1q binding compared to G1d, did not have modifications at positions 330 or 332. This is likely due to the effects of modifications to S298A and 326, which are known to improve C1q binding (Science, 2018, 359, 794-797).

[0156] While the foregoing invention has been described in detail by way of illustration and illustration for purposes of clarity of understanding, the descriptions and illustrations herein should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated herein by reference in their entireties. [Industrial Applicability]

[0157] The invention of the present disclosure provides polypeptides comprising Fc region variants that bind more strongly to activating FcγRIIa and FcγRIIIa and exhibit reduced binding to inhibitory FcγRIIb. Such polypeptides of the present disclosure exhibit high ADCC / ADCP activity and are useful in antitumor therapy (e.g., treatment and / or prevention of inflammatory diseases, treatment and / or prevention of various cancers, etc.).

Claims

1. A polypeptide comprising a mutant Fc region that comprises an amino acid modification in a parent Fc region, wherein the parent Fc region is a region from Pro at position 229 to Pro at position 444 in SEQ ID NO: 1, and is composed of two polypeptide chains, and the mutant Fc region has at least 90% amino acid sequence identity with the parent Fc region and comprises the following amino acid modification: (i) a modification in a first polypeptide of a parent Fc region substituting the amino acid at position 234 (EU numbering) with Phe, the amino acid at position 235 with Gln, the amino acid at position 236 with Trp, the amino acid at position 239 with Met, the amino acid at position 268 with Asp, the amino acid at position 270 with Glu, the amino acid at position 298 with Ala, and the amino acid at position 326 with Asp; and (ii) a modification in a second polypeptide of the parent Fc region, in which the amino acid at position 236 (EU numbering) is replaced with Ala, the amino acid at position 270 is replaced with Glu, the amino acid at position 298 is replaced with Ala, the amino acid at position 326 is replaced with Asp, and the amino acid at position 334 is replaced with Glu; The polypeptide has the following characteristics (1) or (2): (1) The mutant Fc region has enhanced binding activity to at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, and FcγRIIIa compared to the parent Fc region; or (2) Compared to the parent Fc region, the mutant Fc region has selectively enhanced binding activity to at least one activating Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa R, FcγRIIa H, FcγRIIIa F, and FcγRIIIa V, compared to binding activity to inhibitory Fcγ receptors.

2. The polypeptide of claim 1, wherein the mutant Fc region further comprises a modification in the first polypeptide of the parent Fc region that replaces the amino acid at position 250 (EU numbering) with Val and the amino acid at position 307 (EU numbering) with Pro, and a modification in the second polypeptide of the parent Fc region that replaces the amino acid at position 250 (EU numbering) with Val and the amino acid at position 307 (EU numbering) with Pro.

3. The polypeptide of claim 1, wherein the mutant Fc region further comprises a modification in the first polypeptide of the parent Fc region that replaces the amino acid at position 250 (EU numbering) with Val and the amino acid at position 307 (EU numbering) with Pro, and a modification in the second polypeptide of the parent Fc region that replaces the amino acid at position 250 (EU numbering) with Val, the amino acid at position 307 (EU numbering) with Pro, the amino acid at position 330 (EU numbering) with Lys, and the amino acid at position 332 (EU numbering) with Asp.

4. The polypeptide of claim 1, wherein the mutant Fc region further comprises modifications in a first polypeptide of the parent Fc region, substituting the amino acid at position 250 (EU numbering) with Val, the amino acid at position 307 with Pro, and the amino acid at position 332 with Glu, as well as modifications in a second polypeptide of the parent Fc region, substituting the amino acid at position 250 (EU numbering) with Val, the amino acid at position 307 with Pro, and the amino acid at position 332 with Glu.

5. A polypeptide according to any one of claims 1 to 4, wherein the polypeptide comprising the mutant Fc region is an antibody.

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