Heterodimer Fc polypeptide

Abstract

The present invention provides a polypeptide in which the function of the Fc region (e.g., binding ability to FcγR, ADCC activity, and ADCP activity) is improved compared to conventional polypeptides having an Fc region, as well as a method for producing the polypeptide. [Solution] A polypeptide is provided comprising a mutant Fc region containing an amino acid modification in the parent Fc region, wherein the parent Fc region is composed of two polypeptide chains containing a specific amino acid sequence, the mutant Fc region has at least 90% amino acid sequence identity with the parent Fc region, and the mutant Fc region contains an amino acid modification at a specific position in the first polypeptide of the parent Fc region and the second polypeptide of the parent Fc region.

Description

[Technical Field]

[0001] In a non-limiting embodiment, the present disclosure relates to a polypeptide comprising a mutant Fc region including an amino acid modification in the parent Fc region, and a method for producing the polypeptide. [Background technology]

[0002] Antibodies are attracting attention as pharmaceuticals due to their high stability in the blood and low incidence of side effects (Non-Patent Literature 1, Non-Patent Literature 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 cell-mediated cytotoxicity (ADCC) and complement-dependent cell-mediated cytotoxicity (CDC), and it has been reported that antibodies of the human IgG class, specifically the IgG1 subclass, have the highest ADCC and CDC activity (Non-Patent Literature 3). Furthermore, antibody-dependent cell-mediated phagocytosis (ADCP), which is phagocytosis of target cells mediated by IgG class antibodies, has also been shown to be one of the effector functions of antibodies (Non-Patent Literature 4, Non-Patent Literature 5).

[0003] The expression of IgG antibodies ADCC, CDC, and ADCP 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 allotypes for each have also been reported (Non-Patent Literature 6).

[0004] The enhancement of cytotoxic effector functions such as ADCC, ADCP, and CDC is attracting attention as a promising means of enhancing the antitumor effects of antibodies. The importance of FcγR-mediated effector function for the antitumor effect of antibodies has been reported using mouse models (Non-Patent Literature 7, Non-Patent Literature 8). Furthermore, a correlation has been observed between clinical efficacy in humans and the high-affinity polymorphic allotype (V158) and low-affinity polymorphic allotype (F158) of FcγRIIIa (Non-Patent Literature 9). Similarly, it has been shown that clinical efficacy differs depending on the allotype (H131 and R131) of FcγRIIa (Non-Patent Literature 10). These reports suggest that antibodies with Fc regions optimized for binding to specific FcγRs mediate more potent effector function, thereby exerting effective antitumor effects.

[0005] The balance of antibody binding activity to the activating receptor (consisting of FcγRIa, FcγRIIa, FcγRIIIa, and FcγRIIIb) and the inhibitory receptor (consisting of FcγRIIb) is a crucial factor in optimizing the effector function of an antibody. By using an Fc region that enhances binding activity to the activating receptor and reduces binding activity to the inhibitory receptor, it may be possible to confer optimal effector function to the antibody (Non-Patent Document 11). Regarding the binding of the Fc region to FcγR, it has been shown that the hinge region of the antibody, several amino acid residues within the CH2 domain, and the glycan attached to the EU numbering 297th Asn bound to the CH2 domain are important (Non-Patent Documents 12, 13, and 14). Focusing on this binding site, various Fc region variants with different FcγR binding characteristics have been studied, and Fc region variants with higher activating FcγR binding activity have been obtained (Patent Documents 1 and 2). For example, Lazar et al. succeeded in increasing the binding to human FcγRIIIa (V158) by approximately 370 times by substituting Ser at EU numbering position 239, Ala at position 330, and Ile at position 332 of human IgG1 with Asp, Leu, and Glu, respectively (Non-Patent Literature 15, Patent Literature 2). Shinkawa et al. succeeded in increasing the binding to FcγRIIIa by approximately 100 times by deleting the fucose in the sugar chain attached to Asn at EU numbering position 297 (Non-Patent Literature 16). These methods introduce the same modification or the same sugar chain modification to the Fc region of both H chains of the antibody. On the other hand, it has been reported that even though the Fc of the antibody is a homodimer, it binds to FcγR in a 1:1 ratio and recognizes FcγR asymmetrically in the lower hinge and CH2 region (Non-Patent Literature 17). Considering that the Fc region interacts asymmetrically with FcγR, it is thought that introducing different modifications to each H chain would allow for more precise optimization of the interaction between IgG and FcγR. Based on this idea, a method has been reported in which different modifications are made to the Fc region of each H chain of the antibody, thereby modifying Fc asymmetrically and optimizing the interaction with FcγR (Patent Documents 3, 4, 5, and 6).In fact, by asymmetrically modifying the Fc region, modified antibodies exhibiting higher ADCC activity compared to existing ADCC-enhancing antibodies such as afucosylated antibodies have been obtained (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 Literature 18). ADCP activity can be enhanced by inhibiting the "Don't eat me" signal represented by CD47 (Non-Patent Literature 18), as well as by strengthening the ability to bind to FcγRIIa (Non-Patent Literature 19). However, the amino acid sequences of the extracellular domains of the active FcγR, FcγRIIa, and the inhibitory FcγRIIb have very high homology, making it difficult to selectively enhance the ability to bind to FcγRIIa (Non-Patent Literature 20). Therefore, enhancing the ability to bind to FcγRIIa may also enhance the ability to bind to the inhibitory receptor FcγRIIb, potentially weakening the effector function. In fact, in modified antibodies with significantly improved ability to bind to FcγRIIa, the ability to bind to FcγRIIb is also enhanced compared to natural IgG1 (Patent Literature 5, Patent Literature 6). Therefore, in order to exhibit high ADCC / ADCP activity, it is preferable to enhance binding to FcγRIIIa and FcγRIIa as much as possible without enhancing binding ability to FcγRIIb, but no such modified compounds have 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 present invention has been made in view of these circumstances, and in one embodiment, the object of the present invention is to provide a polypeptide in which the function of the Fc region (e.g., binding ability to FcγR, ADCC activity, and ADCP activity) is improved compared to a prior art polypeptide 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 containing an amino acid modification in the parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and the mutant Fc region contains an amino acid modification at the following positions: (i) Positions 234, 235, 236, 239, 268, 270, and 298 in the first polypeptide of the parent Fc region, as represented by EU numbering, and (ii) Positions 270, 298, 326, and 334 in the second polypeptide of the parent Fc region, as represented by EU numbering. [2] The polypeptide according to [1], wherein the mutant Fc region further comprises an amino acid modification at position 326, as represented by EU numbering, in the first polypeptide of the parent Fc region. [3] The polypeptide according to [1] or [2], wherein the mutant Fc region further comprises an amino acid modification at position 236, as represented by EU numbering, in the second polypeptide of the parent Fc region. [4] Polypeptides comprising a mutant Fc region containing an amino acid modification in the parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and the mutant Fc region contains an amino acid modification at the following positions: (i) Positions 234, 235, 236, 239, 268, 270, 298, and 326 in the first polypeptide of the parent Fc region, as represented by EU numbering, and (ii) Positions 236, 270, 298, 326, and 334 in the second polypeptide of the parent Fc region, as represented by EU numbering. [5] The polypeptide according to any one of [1] to [4], wherein the mutant Fc region further comprises an amino acid modification at position 332, as represented by EU numbering, in the first polypeptide of the parent Fc region. [6] The polypeptide according to any one of [1] to [5], wherein the mutant Fc region further comprises an amino acid modification at position 330, as represented by EU numbering, in the first polypeptide of the parent Fc region. [7] The polypeptide according to any one of [1] to [6], wherein the mutant Fc region further comprises an amino acid modification at position 332, as represented by EU numbering, in the second polypeptide of the parent Fc region. [8] The polypeptide according to any one of [1] to [7], wherein the mutant Fc region further comprises an amino acid modification at position 330, as represented by EU numbering, in the second polypeptide of the parent Fc region. [9] Polypeptide comprising a mutant Fc region containing an amino acid modification in the parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and the mutant Fc region contains an amino acid modification at the following positions: (i) Positions 234, 235, 236, 239, 268, 270, 298, 330, and 332 in the first polypeptide of the parent Fc region, as represented by EU numbering, and (ii) Positions 236, 270, 298, 326, 330, 332, and 334 in the second polypeptide of the parent Fc region, as represented by EU numbering.

[10] The polypeptide according to any one 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 according to 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 the second polypeptide of the parent Fc region.

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

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

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

[15] A polypeptide according to any one of [1] to

[14] , comprising at least one amino acid modification selected from the amino acid modifications listed below: (i) In the first polypeptide of the 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, Glu at position 332, 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, or Glu at position 334 in the second polypeptide of the parent Fc region, as represented by EU numbering.

[16] The polypeptide described in any of [1] to

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

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

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

[18] A polypeptide according to any one of [1] to

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

[19] The polypeptide described in

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

[20] A polypeptide according to any one of [1] to

[19] , wherein the mutant Fc region exhibits improved selectivity between the active Fcγ receptor and the inhibitory Fcγ receptor compared to the parent Fc region. [20-2] A polypeptide according to any one of [1] to

[19] , wherein, compared to the parent Fc region, the binding activity to the active Fcγ receptor is selectively enhanced in the mutant Fc region compared to the binding activity to the repressive Fcγ receptor. [20-3] A polypeptide according to any one of [1] to

[19] , wherein the ratio of binding activity to the active Fcγ receptor to the binding activity to the inhibitory Fcγ receptor (A / I ratio) is larger in the mutant Fc region compared to the parent Fc region. [20-4] The ratio (A / I ratio) in the polypeptide containing the mutant Fc region 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 polypeptide containing the parent Fc region. The polypeptide described in [20-3] is larger 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. [20-5] The polypeptide according to [20-3], wherein the ratio (A / I ratio) in the polypeptide containing the mutated 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] A polypeptide according to any one of

[20] to [20-5], wherein the active 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 according to any one of [1] to

[21] , wherein the polypeptide containing the mutated Fc region is an antibody.

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

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

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

[0011]

[26] An isolated nucleic acid encoding a polypeptide as described in any of [1] to

[22] .

[27] A host cell containing the nucleic acid described in

[26] .

[28] A method for producing a polypeptide, comprising culturing the host cells described in

[27] so as to produce a polypeptide.

[29] A polypeptide according to any one of [1] to

[22] for use in the treatment of tumors.

[30] A polypeptide according to any one of [1] to

[22] for use in cell injury.

[31] The polypeptide described in

[30] , wherein cell damage is due to ADCC activity, CDC activity, or ADCP activity.

[32] A pharmaceutical composition comprising a polypeptide described in any of [1] to

[22] and a pharmaceutically acceptable carrier.

[33] The pharmaceutical composition according to

[32] , which is a pharmaceutical composition for the treatment of tumors.

[34] The pharmaceutical composition according to

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

[35] The pharmaceutical composition according to

[34] wherein cell damage is due to ADCC activity, CDC activity, or ADCP activity.

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

[22] or a pharmaceutical composition according to

[32] .

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

[22] or a pharmaceutical composition according to

[32] .

[38] The method according to

[37] , wherein the cell damage is due to ADCC activity, CDC activity, or ADCP activity.

[39] Use of a polypeptide described in any of [1] to

[22] in the manufacture of an anti-tumor agent.

[40] Use of a polypeptide described in any of [1] to

[22] in the manufacture of a cytotoxic agent.

[41] Use as described in

[40] , in which cell damage is due to ADCC activity, CDC activity, or ADCP activity.

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

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

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

[45] The method according to any one of

[42] to

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

[46] The method according to any one of

[42] to

[44] , wherein the modification of the function is an improvement in selectivity between the active Fcγ receptor and the inhibitory Fcγ receptor.

[47] The method according to any one of

[42] to

[44] , wherein the modification of the function is a selective enhancement of the binding activity to the active Fcγ receptor compared to the binding activity to the inhibitory Fcγ receptor.

[48] ​​The method according to any one of

[42] to

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

[49] The ratio (A / I ratio) 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, 70 times or more compared to the polypeptide containing the parent Fc region.

[48] ​​The increase of 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.

[50] The method according to any one of

[42] to

[49] , wherein the active 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 modification of the function is an enhancement of ADCC activity, CDC activity, or ADCP activity. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 shows the results of an ADCC reporter gene assay using Hepa1-6 / hEREG cells as target cells and Jurkat cells expressing hFcγRIIIaV 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 Jurkat cells expressing hFcγRIIaH as effector cells. Each point represents the average Fold induction value for 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. Antibodies were administered via tail vein at a dose of 10 mg / kg. Each point represents the mean tumor volume for a group of n=5. [Figure 4] Figure 4 shows the binding activity of each antibody with modified Fc to hC1q. Each point represents the average ELISA colorimetric value for n=2. [Figure 5] Figure 5 is a continuation of the diagram showing the binding activity of each antibody with modified Fc to hC1q. Each point represents the average ELISA colorimetric value for n=2. [Modes for carrying out the invention]

[0013] The methods and procedures described or cited herein are generally well understood, for example, 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 (FM Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE 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. Conventional techniques, such as those widely used by those skilled in the art, are commonly employed by those skilled in the art, as described in Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993).

[0014] I. Definition Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which this invention pertains. 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 general guidance for many of the terms used herein. All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety.

[0015] For the purpose of interpreting this Spec., the following definitions apply, and wherever applicable, a term used in the singular also includes the plural, and vice versa. It should be understood that the terms used herein are intended solely to describe a particular aspect and not to limit it. In the event of any conflict between the following definitions and any document incorporated herein by reference, the following definitions shall prevail.

[0016] In this specification, "first polypeptide" and "second polypeptide" mean polypeptides that constitute the Fc region of an antibody. "First polypeptide" and "second polypeptide" mean that their sequences are different from each other, preferably that at least the CH2 region sequence is different. The CH3 region sequence may also be different. The polypeptide may be, for example, a polypeptide that constitutes the Fc region of natural IgG, or a polypeptide that has been modified from a polypeptide that constitutes the Fc region of natural IgG.

[0017] Natural IgG refers to polypeptides belonging to a class of antibodies that contain the same amino acid sequence as naturally occurring IgG and are substantially encoded by the immunoglobulin gamma gene. 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 variants.

[0018] In this invention, "polypeptide" usually refers to peptides and proteins having a length of about 10 amino acids or more. While it is usually a polypeptide of biological origin, it is not particularly limited; for example, it may be a polypeptide consisting of an artificially designed sequence. It may also be a natural polypeptide, synthetic polypeptide, recombinant polypeptide, etc. In this invention, a protein molecule refers to a molecule containing the polypeptide.

[0019] A preferred example of the polypeptide of the present invention is an antibody. A more preferred example is natural IgG or an antibody modified from natural IgG. A particularly good example of natural IgG is natural human IgG. Natural IgG refers to a polypeptide that contains the same amino acid sequence as naturally occurring IgG and belongs to a class of antibodies substantially encoded by the immunoglobulin gamma gene. 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 variants.

[0020] In this specification, the term “antibody” is used in its broadest sense and encompasses a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0021] "Natural antibodies" refer to immunoglobulin molecules with various structures that occur naturally. For example, a natural IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain 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 chain domain or light chain variable domain, followed by a constant light chain (CL) domain. Based on the amino acid sequence of its constant domain, the light chains of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ).

[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 main 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 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 in the heavy and / or light chains of an antibody that are involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) typically have a similar structure, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs) (see, for example, Kindt et al. Kuby Immunology, 6th ed., WH 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 particular antigen may be isolated by screening complementary libraries of VL or VH domains, respectively, using the VH or VL domains from antibodies that bind to that antigen. See, for example, 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 antibodies having a structure substantially similar to that of a native antibody, or having a heavy chain containing an Fc region as defined herein.

[0025] In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes both the Fc region of the native sequence and mutant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain, provided that the lysine (Lys447) or glycine-lysine (Gly446-Lys447) at the C-terminus of the Fc region is present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

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

[0027] The "natural-type Fc region" contains amino acid sequences identical to those of Fc regions found in nature. The natural-type human Fc region includes the natural-type human IgG1 Fc region (non-A and A allotypes); the natural-type human IgG2 Fc region; the natural-type human IgG3 Fc region; and the natural-type human IgG4 Fc region, as well as naturally occurring variants thereof.

[0028] A "mutant Fc region" includes an amino acid sequence that differs from that of the native sequence Fc region by at least one amino acid modification (alteration), preferably one or more amino acid substitutions. Preferably, the mutant Fc region has at least one amino acid substitution in the native sequence Fc region or the parent Fc region compared to the native sequence Fc region or the parent Fc region, 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. The mutant Fc regions described herein preferably have at least about 80% homology to the native sequence Fc region or the parent Fc region, preferably at least about 85% homology to them, more preferably at least about 90% homology to them, and most preferably at least about 95% homology to them.

[0029] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage ratio of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after the sequences have been aligned to obtain the greatest possible percentage sequence identity and gaps have been introduced as necessary, and no conservative substitutions are considered part of the sequence identity. Alignment for the purpose of determining percentage amino acid sequence identity can be achieved by using various methods within the scope of the art, such as publicly available computer software, including BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetics Co., Ltd.). A person skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm necessary to achieve the greatest possible alignment over the entire length of the sequences being compared.

[0030] An "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is the native human FcR. In some embodiments, the FcR is one that binds to an IgG antibody (gamma receptor) and includes the FcγRI, FcγRII, and FcγRIII subclass receptors, including allelic variants and alternative splicing forms of these receptors. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immune receptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see, e.g., Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). FcRs have been 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 to be identified in the future, are also included in the term "FcR" as used herein.

[0031] The term “Fc receptor” or “FcR” also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)) and the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., 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 cells" refer to leukocytes that express one or more FcRs and exert effector function. In certain embodiments, these cells express at least FcγRIII and exert 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, for example, from blood. In certain embodiments, effector cells may be human effector cells.

[0033] "Effector function" refers to the biological activity that varies depending on the antibody isotype, stemming from the Fc region of the antibody. 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); downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. A "functional Fc region" possesses the "effector function" of a native sequence Fc region. Such effector function generally requires the Fc region to be combined with a binding domain (e.g., an antibody-variable domain) and can be evaluated using various measurement methods disclosed, for example, within the definitions herein.

[0034] Antibody-dependent cell-mediated cytotoxicity (ADCC) is a form of cytotoxicity in which secreted immunoglobulins bind to Fc receptors (FcRs) present on specific cytotoxic cells (e.g., NK cells, neutrophils, and macrophages), thereby enabling these cytotoxic effector cells to specifically bind to target cells containing antigens and subsequently kill those target cells with cytotoxicity. NK cells, the primary cells mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). To evaluate the ADCC activity of the molecule of interest, in vitro ADCC assays, such as those described in U.S. Patent No. 5,500,362, No. 5,821,337, or No. 6,737,056 (Presta), may be performed. Effector cells useful for such assays include PBMCs and NK cells. Alternatively, the ADCC activity of the molecule of interest may be evaluated in vivo in animal models, such as the animal models disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998).

[0035] Examples of "cytotoxic activity" include antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and T cell-mediated cytotoxicity. CDC activity refers to cytotoxic activity mediated by the complement system. ADCC activity, on the other hand, refers to the activity in which an antibody binds to an antigen present on the surface of a target cell, and then effector cells bind to that antibody, causing damage to the target cell by the effector cells. Whether or not a target antibody has ADCC activity or CDC activity can be measured by known methods (e.g., Current Protocols in Immunology, Chapter 7. Immunologic studies in humans, Coligan et al. (1993), etc.).

[0036] The term "complement-dependent cell injury" or "CDC" refers to a mechanism for inducing cell death in which the Fc effector domain of an antibody bound to a target activates a series of enzymatic reactions, resulting in the formation of holes in the membrane of the target cell. Typically, an antigen-antibody complex formed on a target cell binds to and activates complement component C1q, which then activates the complement cascade, leading to the death of the target cell. Complement activation may also result in the deposition of complement components on the surface of the target cell, which in turn promotes ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

[0037] The term "antibody-dependent cell phagocytosis" or "ADCP" refers to the process by which either all or part of an antibody-coated cell is taken up into phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the immunoglobulin Fc region.

[0038] "Isolated" polypeptides are those separated from the components of their original environment. In some embodiments, polypeptides are purified to a purity of over 95% or 99% by, for example, 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, for example, Flatman et al., J. Chromatogr. B 848: 79-87 (2007).

[0039] "Isolated" nucleic acids are nucleic acid molecules that have been separated from the components of their original environment. Isolated nucleic acids include nucleic acid molecules that would normally be found in the cell containing them, but these nucleic acid molecules are located outside the chromosome or in a chromosomal location different from their original chromosomal location.

[0040] "Isolated nucleic acid encoding a polypeptide" means one or more nucleic acid molecules encoding the polypeptide (e.g., the Fc region of an antibody, or the heavy and light chains or fragments thereof of an antibody), and includes nucleic acid molecules mounted on one vector or separate vectors, and nucleic acid molecules present at one or more locations in a host cell.

[0041] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures, and vectors incorporated into the genome of a host cell into which they are introduced. Some vectors can result in the expression of the nucleic acid to which they are operationally ligated. Such vectors are also referred to herein as "expression vectors."

[0042] The terms “host cell,” “host cell line,” and “host cell culture” refer to cells (including their offspring) that are interchangeably used and into which foreign nucleic acids have been introduced. Host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and their offspring, regardless of passage number. Offspring do not have to be completely identical to the parent cells in terms of nucleic acid content and may contain mutations. Mutant offspring that have the same function or biological activity as those used when the original transformed cells were screened or selected are also included herein.

[0043] The terms "pharmaceutical preparation" and "pharmaceutical composition" refer to preparations that are interchangeable, in a form in which the biological activity of the active ingredients contained herein can exert its effect, and that do not contain additional elements that are toxic to an unacceptable degree to the subject to which they are administered.

[0044] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, 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 a human.

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

[0046] The “effective dose” of a drug (for example, a pharmaceutical formulation) refers to the amount in the required dosage and over the required period of time that is effective in achieving the desired therapeutic or prophylactic outcome.

[0047] As used herein, “treatment” (and its grammatical derivatives, e.g., “to treat,” “to treat,” etc.) means a clinical intervention intended to modify the natural course of the individual being treated, and may be carried out for preventive purposes or during the course of a clinical condition. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, symptom reduction, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction of the rate of disease progression, recovery or mitigation of the disease state, and remission or improved prognosis. In some embodiments, polypeptides comprising the mutated Fc region 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, as well as all precancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “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 typically consists of a group of tumor cells (parenchyma) that make up the tumor, and 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 others, the two are intertwined. Immune cells and other organisms may also infiltrate the tumor tissue. On the other hand, "non-tumor tissue" refers to tissue other than tumor tissue in the body. Healthy / normal tissue that is not in a diseased state is a typical example of non-tumor tissue.

[0050] <Polypeptide containing a mutated Fc region> In one aspect, the present invention provides an isolated polypeptide comprising a mutant Fc region. In several aspects, the polypeptide is an antibody. In several 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 the native sequence or a reference mutant sequence (which may be collectively referred to herein as the “parent” Fc region). The Fc region of the native sequence is typically composed of a homodimer 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 polypeptide chains.

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

[0052] The terms “binding activity” and “binding ability” are used interchangeably herein and refer to the total strength of non-covalent interactions between one or more binding sites (e.g., variable regions or Fc regions) of a molecule (e.g., an antibody or other polypeptide) and its 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, if the members of a binding pair reflect a monovalent 1:1 interaction, binding activity refers to the intrinsic binding affinity ("affinity"). If the members of a binding pair are capable of both monovalent and polyvalent binding, binding activity is the sum of these binding forces. The binding activity of molecule X to its partner Y can generally be expressed by its dissociation constant (KD) or “analyte binding per unit amount of ligand.” Binding activity can be measured by conventional methods known in the art, including those described herein. Specific examples and exemplary embodiments for measuring binding activity are described below.

[0053] In certain embodiments, the binding activities of the parental Fc region and the mutant Fc region can be represented by KD (Dissociation constant) values. In one embodiment, the value of the ratio [KD value of the parental 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 may be FcγRIIa H, or may be both. Thus, the KD value of the Fc region for FcγRIIa may be the KD value of the Fc region for FcγRIIa R, or may be the KD value of the Fc region for FcγRIIa H, or may be the sum or average value of both. In one embodiment, the value of the ratio [Binding activity of the parental Fc region for FcγRIIIa] / [Binding activity of the mutant Fc region for 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 or more, 2×10 3 or more, 3×10 3 or more, or 5×10 3 or more. In a further embodiment, FcγRIIIa may be FcγRIIIa F, or may be FcγRIIIa V, or may be both. Thus, the KD value of the Fc region for FcγRIIIa may be the KD value of the Fc region for FcγRIIIa F, or may be the KD value of the Fc region for FcγRIIa V, or may be the sum or average value 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 × 10 -9 It is less than or equal to M. In a further embodiment, FcγRIIa may be FcγRIIa R, FcγRIIa H, or both. In one embodiment, the KD value of the mutant Fc region for FcγRIIIa is, for example, 1.0 × 10⁻⁶. -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 × 10 -10 It is less than or equal to M. In a further embodiment, FcγRIIIa may be FcγRIIIa F, or FcγRIIIa V, or both.

[0055] In another embodiment, the binding activity of the parental Fc region and the mutant Fc region may be expressed in terms of the kd (Dissociation rate constant) value instead of the KD value.

[0056] In another embodiment, the binding activity of the parental Fc region and the mutant Fc region may be expressed as the amount of Fc region binding to the Fcγ receptor per unit amount. For example, in a surface plasmon resonance assay, the amount of Fc region immobilized on a sensor chip and the amount of Fcγ receptor bound to it are each measured as a resonance unit (RU). The amount of Fcγ receptor binding there, divided by the amount of Fc region binding, can be defined as the amount of Fc region binding to the 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 parental Fc region binding to FcγRIIa] is, for example, 1.5 or greater, 2 or greater, 3 or greater, 4 or greater, 5 or greater, 6 or greater, 7 or greater, 8 or greater, 9 or greater, 10 or greater, 15 or greater, 20 or greater, 25 or greater, 30 or greater, 40 or greater, or 50 or greater. In some aspects, the ratio of [binding amount to FcγRIIIa in the mutant Fc region] / [binding amount to FcγRIIIa in the parent Fc region] 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 The above is 2 x 10 3 The above is 3 x 10 3 Above, or 5 x 10 3 That's all.

[0057] In certain embodiments, the KD values, kd values, and binding amounts expressed herein are measured or calculated by performing a surface plasmon resonance assay at 25°C or 37°C (see, for example, Example 2 herein).

[0058] In certain aspects, the mutant Fc region of the present invention exhibits improved selectivity between active and inhibitory Fcγ receptors compared to the parental Fc region. In other words, the mutant Fc region of the present invention exhibits significantly enhanced binding activity to active Fcγ receptors compared to the parental Fc region. In certain embodiments, the active 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 region of the present invention exhibits improved selectivity between FcγRIIa and FcγRIIb. In some embodiments, the mutant Fc region of the present invention exhibits improved selectivity between FcγRIIIa and FcγRIIb. In a further embodiment, the mutant Fc region of the present invention exhibits improved selectivity between FcγRIIa and FcγRIIb, and between FcγRIIIa and FcγRIIb.

[0059] In certain embodiments, the binding activity of the parental Fc region and the mutant Fc region can be expressed by a KD (Dissociation constant) value. The embodiments of the binding activity to FcγRIIa and FcγRIIIa are as described above. In one embodiment, the ratio of [KD value of the parental Fc region to FcγRIIb] / [KD value of the mutant 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 another embodiment, the binding activity of the parental Fc region and the mutant Fc region may be expressed by a kd (Dissociation rate constant) value instead of a KD value.

[0060] In another embodiment, the binding activity of the parental Fc region and the mutant Fc region may be expressed as the amount of the Fc region binding to the Fcγ receptor per unit amount as described above. In some embodiments, the ratio of [binding amount of mutant Fc region to FcγRIIb] / [binding amount of parental 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 binding amount 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 certain embodiments, improved selectivity between active and repressive Fcγ receptors is a selective enhancement of binding activity to active Fcγ receptors compared to binding activity to repressive Fcγ receptors; in other words, an increase in the ratio of binding activity to active Fcγ receptors to binding activity to repressive Fcγ receptors (A / I ratio). Such a ratio (A / I ratio) is an indicator of superior effector function, and polypeptides with a large A / I ratio can be evaluated as having superior effector function. The binding activity of parental and mutant Fc regions to Fcγ receptors can be expressed as KD value, kd value, or the amount of Fc region bound to Fcγ receptors 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 active Fcγ receptor], [kd value for inhibitory Fcγ receptor] / [kd value for active Fcγ receptor], or [binding amount for active Fcγ receptor] / [binding amount for inhibitory Fcγ receptor].

[0062] In one embodiment, the A / I ratio of the mutated 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. The above figures represent increases of 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. In one aspect, the A / I ratio value of the mutated 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 binding activity to FcγRIa to binding activity to FcγRIIb, the ratio of binding activity to FcγRIIa to binding activity to FcγRIIb, the ratio of binding activity to FcγRIIIa to binding activity to FcγRIIb, or the ratio of the sum or average of two or three of the binding activities to FcγRIa, FcγRIIa, and FcγRIIIa to binding activity to FcγRIIb. In a particular embodiment, FcγRIIa is FcγRIIa R, FcγRIIa H, or both, and therefore the binding activity to FcγRIIa is the sum or average of the binding activity to FcγRIIa R, the binding activity to FcγRIIa H, or both.In certain embodiments, FcγRIIIa is FcγRIIIa F, FcγRIIIa V, or both, and therefore the binding activity to FcγRIIIa is the sum or mean of the binding activity to FcγRIIIa F, the binding activity to FcγRIIIa V, or both.

[0063] In some embodiments, the mutant Fc region of the present invention includes amino acid modifications at the following positions: (i) Positions 234, 235, 236, 239, 268, 270, and 298 in the first polypeptide of the parent Fc region, as represented by EU numbering, and (ii) Positions 270, 298, 326, and 334 in the second polypeptide of the parent Fc region, as represented by EU numbering. In certain embodiments, the mutant Fc region of the present invention further includes an amino acid modification at position 326, represented by EU numbering, in the first polypeptide of the parent Fc region. In certain embodiments, the mutant Fc region of the present invention further includes an amino acid modification at position 236, represented by EU numbering, in the second polypeptide of the parent Fc region. In certain embodiments, the mutant Fc region of the present invention further includes an amino acid modification at position 332, represented by EU numbering, in the first polypeptide of the parent Fc region. In certain embodiments, the mutant Fc region of the present invention further includes an amino acid modification at position 330, represented by EU numbering, in the first polypeptide of the parent Fc region. In certain embodiments, the mutant Fc region of the present invention further includes an amino acid modification at position 332, represented by EU numbering, in the second polypeptide of the parent Fc region. In certain embodiments, the mutant Fc region of the present invention further includes an amino acid modification at position 330, represented by EU numbering, in the second polypeptide of the parent Fc region. Alternatively, amino acid modifications described in International Publications WO2013 / 002362 and WO2014 / 104165 can also be used in the present invention.

[0064] In some embodiments, the mutant Fc region of the present invention includes amino acid modifications at the following positions: (i) Positions 234, 235, 236, 239, 268, 270, 298, and 330 in the first polypeptide of the parent Fc region, as represented by EU numbering, and (ii) Positions 270, 298, 326, 330, and 334 in the second polypeptide of the parent Fc region, as represented by EU numbering. In certain embodiments, the mutant Fc region of the present invention further includes an amino acid modification at position 236, represented by EU numbering, in the second polypeptide of the parent Fc region. In certain embodiments, the mutant Fc region of the present invention further includes an amino acid modification at position 332, represented by EU numbering, in the first polypeptide of the parent Fc region. In certain embodiments, the mutant Fc region of the present invention further includes an amino acid modification at position 332, represented by EU numbering, in the second polypeptide of the parent Fc region. In certain embodiments, or the amino acid modifications described in International Publications WO2013 / 002362 and WO2014 / 104165, may also be used in the present invention.

[0065] In certain aspects, the mutant Fc region of the present invention exhibits 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 using indicators such as the Tm value. The Tm value can be measured using methods known to those skilled in the art, such as CD (circular dichroism), DSC (scanning scan calorimeter), and DSF (scanning scan fluorescence quantitative analysis). In one embodiment, the mutant Fc region of the present invention exhibits a Tm value of 0.1 degrees or more, 0.2 degrees or more, 0.3 degrees or more, 0.4 degrees or more, 0.5 degrees or more, 1 degree or more, 2 degrees or more, 3 degrees or more, 4 degrees or more, 5 degrees or more, and 10 degrees or more higher in the CH2 region compared to the parent Fc region.

[0066] In some embodiments, 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 250 and 307, as represented by EU numbering, in the first 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.

[0067] In certain aspects, the mutant Fc region of the present invention is composed of two polypeptide chains with different sequences. In further aspects, heterodimerization between the first polypeptide and the second polypeptide is promoted in the mutant Fc region of the present invention. When producing a heterodimer protein using a recombinant method, it is preferable that different peptide chains preferentially associate to form a heterodimer rather than identical polypeptide chains associating to form a homodimer. Whether heterodimerization of the mutant Fc region is promoted can be determined, for example, by separating the homodimer and heterodimer from the produced mutant Fc region using a method such as chromatography and determining the ratio of each component.

[0068] In some embodiments, the mutant Fc region of the present invention comprises at least one amino acid modification at at least one position in the first and / or second polypeptide of the parent Fc region, selected from the group consisting of positions 349, 356, 366, 368, 407, and 439, as represented by EU numbering. Alternatively, amino acid modifications described in International Publications WO2006 / 106905 and WO1996 / 027011 may also be used in the present invention.

[0069] In certain aspects, the mutant Fc region of the present invention exhibits enhanced binding activity to FcRn under acidic pH conditions. In some embodiments, acidic pH refers to a pH of 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, acidic pH is pH 5.8.

[0070] In some embodiments, 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 428, 434, 436, 438, and 440, as represented by EU numbering, in the first 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.

[0071] In one aspect, the mutant Fc region of the present invention includes 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 represented by EU numbering.

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

[0073] In one embodiment, the mutant Fc region of the present invention includes amino acid modifications at positions 234, 235, 236, 239, 268, 270, 298, 326, 330, and 334 represented by EU numbering. In a particular embodiment, the mutant Fc region of the present invention includes (i) amino acid modifications at positions 234, 235, 236, 239, 268, 270, 298, and 330 represented by EU numbering in the first polypeptide of the parent Fc region, and (ii) amino acid modifications at positions 270, 298, 326, 330, and 334 represented by EU numbering in the second polypeptide of the parent Fc region.

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

[0075] In one embodiment, the mutant Fc region of the present invention comprises amino acid modifications at positions 234, 235, 236, 239, 268, 270, 298, 326, 330, 332, and 334 represented by EU numbering. In a particular embodiment, the mutant Fc region of the present invention comprises (i) amino acid modifications at positions 234, 235, 236, 239, 268, 270, 298, 330, and 332 represented by EU numbering in the first polypeptide of the parent Fc region, and (ii) amino acid modifications at positions 236, 270, 298, 326, 330, 332, and 334 represented by EU numbering in the second polypeptide of the parent Fc region. In one embodiment, the mutant Fc region of the present invention includes amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, and 334 represented by EU numbering. In a particular embodiment, the mutant Fc region of the present invention includes (i) amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 330, and 332 represented by EU numbering in the first polypeptide of the parent Fc region, and (ii) amino acid modifications at positions 236, 250, 270, 298, 307, 326, 330, 332, and 334 represented by EU numbering in the second polypeptide of the parent Fc region. In another specific embodiment, the mutant Fc region of the present invention comprises (i) amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 326, represented by EU numbering, in the first polypeptide of the parent Fc region, and (ii) amino acid modifications at positions 236, 250, 270, 298, 307, 326, 330, 332, and 334, represented by EU numbering, in the second polypeptide of the parent Fc region.

[0076] In one embodiment, the mutant Fc region of the present invention comprises amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, and 334 represented by EU numbering. In a particular embodiment, the mutant Fc region of the present invention comprises (i) amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, and 332 represented by EU numbering in the first polypeptide of the parent Fc region, and (ii) amino acid modifications at positions 236, 250, 270, 298, 307, 326, 332, and 334 represented by EU numbering in the second polypeptide of the parent Fc region.

[0077] In one embodiment, the mutant Fc region of the present invention comprises amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, and 334 represented by EU numbering. In a particular embodiment, the mutant Fc region of the present invention comprises (i) amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 332 represented by EU numbering in the first polypeptide of the parent Fc region, and (ii) amino acid modifications at positions 250, 270, 298, 307, 326, 332, and 334 represented by EU numbering in the second polypeptide of the parent Fc region.

[0078] In one embodiment, the mutant Fc region of the present invention comprises amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, and 334 represented by EU numbering. In a particular embodiment, the mutant Fc region of the present invention comprises (i) amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 330 represented by EU numbering in the first polypeptide of the parent Fc region, and (ii) amino acid modifications at positions 250, 270, 298, 307, 326, 330, and 334 represented by EU numbering in the second polypeptide of the parent Fc region.

[0079] In a further embodiment, the mutant Fc region of the present invention comprises (i) at least one amino acid modification selected from the group consisting of Tyr or Phe at position 234, represented by EU numbering, 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 in the first polypeptide of the parent Fc region, and (ii) at least one amino acid modification selected from the group consisting of 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 at position 330, or Lys, Asp at position 332, or Glu at position 334 in the second polypeptide of the parent Fc region.

[0080] In a further embodiment, the mutant Fc region of the present invention further comprises any of the following amino acid modifications: (a) Lys at position 356 in EU numbering in the first polypeptide of the parent Fc region, and Glu at position 439 in EU numbering in the second polypeptide of the parent Fc region, (b) Glu at position 439 in EU numbering in the first polypeptide of the parent Fc region, and Lys at position 356 in EU numbering in the second polypeptide of the parent Fc region. (c) Trp at position 366 in EU numbering in the first polypeptide of the parent Fc region, and Ser at position 366, Ala at position 368, and Val at position 407 in EU numbering in the second polypeptide of the parent Fc region, (d) Ser at position 366, Ala at position 368, and Val at position 407 in the first polypeptide of the parent Fc region, as represented by EU numbering, and Trp at position 366 in the second polypeptide of the parent Fc region, (e) Cys at position 349 and Trp at position 366 in the first polypeptide of the parent Fc region, as represented by EU numbering, and Cys at position 356, Ser at position 366, Ala at position 368, and Val at position 407 in the second polypeptide of the parent Fc region, as represented by EU numbering, (f) Cys at position 356, Ser at position 366, Ala at position 368, and Val at position 407 in the first polypeptide of the parent Fc region, as represented by EU numbering, and Cys at position 349 and Trp at position 366 in the second polypeptide of the parent Fc region, as represented by EU numbering.

[0081] In a further context, the mutant Fc region of the present invention further comprises any of the following amino acid modifications in the first polypeptide and / or second polypeptide of the parent Fc region: (a) Ala at position 434 as represented by EU numbering, (b) Ala at position 434, Thr at position 436, Arg at position 438, Glu at position 440, as represented by EU numbering. (c) Leu at position 428, Ala at position 434, Thr at position 436, Arg at position 438, Glu at position 440, represented by EU numbering. (d) Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440, as represented by EU numbering.

[0082] In a particular embodiment, the polypeptide comprising the mutated Fc region of the present invention is the constant heavy chain region of an antibody.

[0083] In a further embodiment, the present invention provides a polypeptide comprising any one amino acid sequence from SEQ ID NOs: 7-22 and 35-50.

[0084] In a further embodiment, the present invention provides heavy chain constant regions including the polypeptide chain of SEQ ID NO: 35 and the polypeptide chain of SEQ ID NO: 36, heavy chain constant regions including the polypeptide chain of SEQ ID NO: 37 and the polypeptide chain of SEQ ID NO: 38, heavy chain constant regions including the polypeptide chain of SEQ ID NO: 39 and the polypeptide chain of SEQ ID NO: 40, heavy chain constant regions including the polypeptide chain of SEQ ID NO: 41 and the polypeptide chain of SEQ ID NO: 42, heavy chain constant regions including the polypeptide chain of SEQ ID NO: 43 and the polypeptide chain of SEQ ID NO: 44, heavy chain constant regions including the polypeptide chain of SEQ ID NO: 45 and the polypeptide chain of SEQ ID NO: 46, heavy chain constant regions including the polypeptide chain of SEQ ID NO: 47 and the polypeptide chain of SEQ ID NO: 48, and heavy chain constant regions including the polypeptide chain of SEQ ID NO: 49 and the polypeptide chain of SEQ ID NO: 50.

[0085] "Fcγ receptor" (hereinafter referred to as Fcγ receptor, FcγR, or FcgR) refers to a receptor capable of binding to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies, and effectively means any member of the protein family encoded by the Fcγ receptor gene. In humans, this family includes, but is not limited to, FcγRI(CD64), which contains isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII(CD32), which contains isoforms FcγRIIa (including allotypes H131 (H type) and R131 (R type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII(CD16), which contains isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any undiscovered human FcγR, FcγR isoform, or allotype. FcγRIIb1 and FcγRIIb2 have been reported as splicing variants of human FcγRIIb. Furthermore, a splicing variant called FcγRIIb3 has been reported (J Exp Med, 1989, 170: 1369-1385). In addition to these splicing variants, human FcγRIIb includes all splicing variants registered in 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 previously reported genetic polymorphisms (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)) and all genetic polymorphisms that may be reported in the future.

[0086] FcγRIIa has two allotypes: 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)).

[0087] FcγR includes, but is not limited to, FcγR derived from humans, mice, rats, rabbits, and monkeys, and may be derived from any organism. Mouse FcγR includes, but is not limited to, FcγRI(CD64), FcγRII(CD32), FcγRIII(CD16), and FcγRIII-2(CD16-2), as well as any mouse FcγR or FcγR isoform.

[0088] 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.

[0089] Unlike Fcγ receptors belonging to the immunoglobulin superfamily, human FcRn is structurally similar to major histocompatibility complex (MHC) class I polypeptides and shares 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 chain or heavy chain complexed with a soluble β chain or light chain (β2 microglobulin). Like MHC, the α chain of FcRn consists of three extracellular domains (α1, α2, α3), and a short cytoplasmic domain tethers the protein to the cell surface. The α1 and α2 domains interact with the FcRn-binding domain in the antibody's Fc region (Raghavan et al. (Immunity (1994) 1, 303-315). The amino acid sequence of human FcRn is described in NP_004098.1, and the amino acid sequence of β2 microglobulin is described in NP_004039.1.

[0090] As used herein, the “parent Fc region” refers to the Fc region before the introduction of the amino acid modifications described herein. In some embodiments, the parent Fc region is the Fc region of the native sequence (or the Fc region of a native antibody). Antibodies include, for example, 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. Several 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 them 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. Furthermore, for human IgG1, the amino acid at position 214 (EU numbering) may be either K or R. In certain embodiments, 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 NOs: 1, 28, 29, or 56. In a further embodiment, the parent Fc region may be an Fc region (Fc region of a reference mutant sequence) created by adding amino acid modifications other than those described herein to the Fc region of the native sequence. The Fc region of the native sequence is usually composed of a homodimer consisting of two identical polypeptide chains.

[0091] Furthermore, amino acid modifications performed for other purposes can be combined in the mutant Fc region 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 to improve the heterogeneity or stability of the antibody (WO2009 / 041613) may be added. Alternatively, polypeptides having properties that promote antigen clearance as described in WO2011 / 122011, WO2012 / 132067, WO2013 / 046704, or WO2013 / 180201, polypeptides having properties that specifically bind to target tissues as described in WO2013 / 180200, or polypeptides having properties that repeatedly bind to multiple antigen molecules as described in WO2009 / 125825, WO2012 / 073992, or WO2013 / 047752 may be combined with the mutant Fc region described herein. Alternatively, amino acid modifications disclosed in EP1752471 and EP1772465 may be combined at CH3 of the mutant Fc region described herein for the purpose of conferring binding ability to other antigens. Alternatively, an amino acid modification that lowers the constant region pI (WO2012 / 016227) may be combined with the mutant Fc region described herein for the purpose of increasing plasma retention. Alternatively, an amino acid modification that increases the constant region pI (WO2014 / 145159) may be combined with the mutant Fc region described herein for the purpose of promoting uptake into cells.Alternatively, an amino acid modification (WO2016 / 125495) that increases the constant region pI (for the purpose of promoting the elimination of the target molecule from plasma) may be combined with the mutant Fc region described herein. In one embodiment, such modification may include a substitution 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 substitution may be a Lys or Arg substitution of the amino acid at each position.

[0092] Methods for producing heterodimerized antibodies are not limited to those described above, but may also be used by knob-in-hole techniques (see, for example, Nat. Biotechnol., (16); 677-681 (1998) and U.S. Patent No. 5,731,168) or by manipulating electrostatic steering effects (WO2006 / 106905, WO2009 / 089004A1, J. Biol. Chem., (285), 19637-19646 (2010), etc.).

[0093] Regarding the binding of non-homologous polypeptides containing mutated Fc regions, techniques can be applied to suppress unintended binding of homologous polypeptides containing mutated Fc regions by introducing electrostatic repulsion at the interface of the CH2 or CH3 domain of the Fc region, as described in WO2006 / 106905.

[0094] Examples of amino acid residues in contact with the interface of the CH2 or CH3 domain in the Fc region include the residues at positions 356 (EU numbering), 439 (EU numbering), 357 (EU numbering), 370 (EU numbering), 399 (EU numbering), and 409 (EU numbering) within the CH3 domain.

[0095] More specifically, for example, 1 to 3 pairs of amino acid residues selected from (1) to (3) below can be used to create an Fc region having 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.

[0096] Furthermore, it is possible to create non-homologous polypeptides containing mutant Fc regions in which 1 to 3 pairs of amino acid residues selected from (1) to (3) above have the same charge in the CH3 domain of the first Fc region, and the same pair of amino acid residues selected in the first Fc region also has the same charge in the CH3 domain of the second Fc region, but the charges of the first and second Fc regions are diametrically opposed.

[0097] In the Fc region described above, for example, the negatively charged amino acid residue is preferably selected from glutamic acid (E) and aspartic acid (D), and the positively charged amino acid residue is preferably selected from lysine (K), arginine (R), and histidine (H).

[0098] Further known techniques can be used for the binding of non-homologous polypeptides containing mutant Fc regions. Specifically, such techniques are carried out by substituting an amino acid side chain present in one Fc region with a larger side chain (knob) and substituting an amino acid side chain present in the Fc region with a smaller side chain (hole), thereby placing the knob within the hole. This promotes efficient binding between Fc region-containing polypeptides having different amino acid sequences (WO1996 / 027011; Ridgway et al., Prot. Eng. 9:617-621(1996); Merchant et al., Nat. Biotech. 16, 677-681(1998)).

[0099] Furthermore, other known techniques can be used for non-homologous binding of polypeptides containing mutant Fc regions. Binding of polypeptides containing Fc regions can be efficiently induced using a chain-exchange recombination domain CH3 heterodimer (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 having different amino acid sequences.

[0100] In addition, heterodimerized antibody production techniques using the conjugation of antibody CH1 and CL, and VH and VL, as described in WO2011 / 028952, can also be used.

[0101] It is also possible to use a heterodimerization antibody production technique, similar to the methods described in WO2008 / 119353 and WO2011 / 131746, which involves preparing two types of homodimerizing antibodies in advance, incubating these antibodies under reducing conditions to dissociate them, and then rejoining them.

[0102] Furthermore, it is also possible to use heterodimerized antibody production techniques that involve modifying the CH2 and CH3 domains, similar to the method described in WO2012 / 058768.

[0103] When two polypeptides containing mutant Fc regions with different amino acid sequences are simultaneously expressed to produce polypeptides containing non-homologous mutant Fc regions, polypeptides containing homologous mutant Fc regions are usually also produced as impurities. In such cases, polypeptides containing non-homologous mutant Fc regions can be efficiently obtained by separating and purifying them from polypeptides containing homologous mutant Fc regions using known techniques. A method has been reported for efficiently separating and purifying heterodimerized antibodies from homodimerized antibodies using ion-exchange chromatography by introducing amino acid modifications into the variable regions of the two antibody heavy chains that create a difference in isoelectric points between homodimerized and heterodimerized antibodies (WO2007 / 114325). Another method has been reported for purifying heterodimerized antibodies using protein A chromatography by constructing heterodimerized antibodies containing two heavy chains derived from mouse IgG2a, which binds to protein A, and rat IgG2b, which does not bind to protein A (WO1998 / 050431 and WO1995 / 033844).

[0104] Furthermore, by substituting amino acid residues at positions 435 and 436 (EU numbering) located at the protein A binding site of the antibody heavy chain with amino acids such as Tyr or His, heterodimerized antibodies can be efficiently purified using protein A chromatography to obtain different protein A binding affinities.

[0105] In this invention, amino acid modification means any substitution, deletion, addition, insertion, and modification, or a combination thereof. In this invention, amino acid modification can be rephrased as amino acid mutation.

[0106] The number of amino acid modifications introduced into the Fc region is not limited. In certain embodiments, it 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.

[0107] In one aspect, the present invention provides a method for producing a polypeptide containing a mutant Fc region. In a further aspect, the present invention provides a method for producing a polypeptide containing a functionally modified mutant Fc region. In a further aspect, the present invention provides a method for modifying the function of a polypeptide containing 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 include the step of introducing at least one amino acid modification to a parent Fc region. In certain embodiments, the methods include the steps of (i) providing a polypeptide containing a parent Fc region and (ii) introducing at least one amino acid modification to the parent Fc region. In certain embodiments, the methods may further include the step of (iii) measuring the function of the polypeptide containing the mutant Fc region. The native Fc region is typically composed of two identical polypeptide chains. The amino acid modification to the parent Fc region may be introduced into either one of the two polypeptide chains of the parent Fc region, or into both of the two polypeptide chains.

[0108] In another embodiment, a method for producing a polypeptide containing a mutant Fc region includes: (i) providing one or more nucleic acids encoding a polypeptide containing a parental Fc region; (ii) introducing at least one mutation into the region encoding the parental Fc region of the nucleic acid; (iii) introducing the nucleic acid produced in (ii) into a host cell; and (iv) culturing the cell described in (iii) to express the polypeptide containing the mutant Fc region. In a particular embodiment, the method may further include (v) recovering the polypeptide containing the mutant Fc region from the host cell culture described in (iv).

[0109] In certain embodiments, the nucleic acids produced in (ii) may be contained in one or more vectors (e.g., an expression vector).

[0110] In some embodiments, the amino acid modifications used in this manufacturing method are selected from any single modification, a combination of single modifications, or a combination of modifications listed in Table 1, selected from among the amino acid modifications that may be included in the mutant Fc region described above.

[0111] The Fc region may also be obtained by partially digesting IgG1, IgG2, IgG3, or IgG4 monoclonal antibodies using a protease such as pepsin, and then re-eluting the fraction adsorbed onto a protein A column. The protease is not particularly limited, as long as it can digest the full-length antibody and thereby produce Fab and F(ab')2 in a restrictive manner by appropriately setting the enzymatic reaction conditions such as pH. Examples include pepsin and papain.

[0112] Polypeptides containing the mutant Fc region of the present invention may be produced by methods known in the art, in addition to the production methods described above. Polypeptides containing the mutant Fc region produced by the production methods described herein are also included in the present invention.

[0113] In one embodiment, an isolated nucleic acid is provided that encodes a polypeptide comprising the mutant Fc region of the present invention. Such nucleic acid may encode an amino acid sequence comprising a first polypeptide of the mutant Fc region and / or an amino acid sequence comprising a second polypeptide. In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, the host cell comprises (1) a vector comprising nucleic acids encoding an amino acid sequence comprising 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 nucleic acids encoding an amino acid sequence comprising an amino acid sequence comprising a first polypeptide of the mutant Fc region and a second vector comprising nucleic acids encoding an amino acid sequence comprising a second polypeptide of the mutant Fc region (e.g., transformed). In one embodiment, the host cell is eukaryotic (e.g., Chinese hamster ovary (CHO) cells) or lymphoid cells (e.g., Y0, NS0, Sp2 / 0 cells). In one embodiment, a method is provided for producing a polypeptide containing the mutant Fc region of the present invention, comprising culturing host cells containing a nucleic acid encoding a polypeptide containing the mutant Fc region of the present invention under conditions suitable for polypeptide expression, and optionally recovering the polypeptide from the host cells (or host cell culture medium).

[0114] For the recombinant production of polypeptides containing the mutated Fc region of the present invention, the 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 acids will be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the gene encoding the Fc region of the antibody).

[0115] Suitable host cells for cloning or expressing a vector encoding a polypeptide containing the mutated Fc region of the present invention include prokaryotic cells or eukaryotic cells.

[0116] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts, including strains of fungi and yeasts in which the glycosylation pathway has been "humanized" to produce antibody Fc regions with partial or complete human glycosylation patterns, are suitable cloning or expression hosts for vectors encoding polypeptides containing the mutated Fc region of the present invention. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004) and Li et al., Nat. Biotech. 24: 210-215 (2006).

[0117] Cells derived from multicellular organisms (invertebrates and vertebrates) are also suitable host cells for the expression of the Fc region of glycosylated antibodies. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified for use in conjugation with insect cells, particularly for the transformation of Spodoptera frugiperda cells.

[0118] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429.

[0119] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in a suspension state would be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 cell line (COS-7); human embryonic kidney cell line (293 or 293 cells as described in Graham et al., J. Gen Virol. 36: 59 (1977), etc.); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described in Mather, Biol. Reprod. 23: 243-251 (1980), etc.); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary cancer cells (MMT 060562); and TRI cells (e.g., Mather et al., Annals NY Acad. Sci. 383: 44-68 (1982)). These include MRC5 cells and FS4 cells, as described in [reference]. 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.

[0120] For the purpose of identifying or screening the mutant Fc regions provided herein, or for the purpose of elucidating their physical or chemical properties or biological activity, the measurement methods described herein or various measurement methods known in the art may be used.

[0121] Methods for determining the binding activity of polypeptides containing a mutant Fc region to one or more FcR family members are described herein or known in the art. Such binding assays include, but are 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).

[0122] In one embodiment, the binding activity of a polypeptide containing a mutant Fc region to FcR family members can be measured using a surface plasmon resonance assay. For example, various FcRs are interacted with a polypeptide containing 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.) as an analyte. Alternatively, the FcR may be immobilized or captured on the sensor chip, and the polypeptide containing the mutant Fc region may be used as the analyte. As a result of such interaction, a sensorgram of binding is obtained, and by analyzing these, the dissociation constant (KD) value of the binding can be calculated. Furthermore, the difference in resonance unit (RU) values ​​in the sensorgram before and after interaction with the FcR (i.e., the amount of FcR bound) can be used as an indicator of the binding activity of the polypeptide containing the mutant Fc region to that FcR. Furthermore, a correction value obtained by dividing the amount of FcR bound by the difference in RU values ​​in the sensor gram before and after the polypeptide containing the mutant Fc region is immobilized or captured on the sensor chip (i.e., the amount of polypeptide containing the mutant Fc region bound) may be used as an indicator of binding activity (i.e., the amount of FcR bound per unit amount of polypeptide containing the mutant Fc region).

[0123] Any polypeptide containing a mutated Fc region provided herein may be used in therapeutic methods. In one aspect, a polypeptide containing a mutant Fc region is provided for use as a pharmaceutical. In a further aspect, a polypeptide containing a mutant Fc region is provided for use in the treatment of tumors. In a particular embodiment, a polypeptide containing a mutant Fc region is provided for use in a therapeutic method. In a particular embodiment, the present invention provides a polypeptide containing a mutant Fc region for use in a method for treating an individual having a tumor, comprising the step of administering to the individual an effective amount of the polypeptide containing a mutant Fc region. In one such embodiment, the method further comprises the step of administering to the individual an effective amount of at least one additional therapeutic agent. In a further embodiment, the present invention provides a polypeptide containing a mutant Fc region for use in cell injury. In a particular embodiment, the present invention provides a polypeptide containing a mutant Fc region for use in a method for injuring cells in an individual, comprising the step of administering to the individual an effective amount of the polypeptide containing a mutant Fc region in order to injure cells. In any of the above embodiments, “individual” is preferably a human.

[0124] In some embodiments, tumors are solid tumors. In solid tumors, tumor cells typically proliferate and form a collective, which then constitutes the tumor tissue. In addition, tumor tissue in vivo is often infiltrated by immune cells such as lymphocytes, which also form part of the tumor tissue. In one embodiment, cellular damage is induced by ADCC activity, CDC activity, or ADCP activity.

[0125] In a further aspect, the present invention provides the use of polypeptides comprising a mutated Fc region in the manufacture or preparation of pharmaceuticals. In one embodiment, the pharmaceutical is for the treatment of a tumor. In a further embodiment, the pharmaceutical is for use in a method of treating a tumor, comprising the step of administering an effective amount of the pharmaceutical to an individual having a tumor. In one such embodiment, the method further comprises the step of administering an effective amount of at least one additional therapeutic agent to the individual. In a further embodiment, the pharmaceutical is for cell injury. In a further embodiment, the pharmaceutical is for use in a method of injuring cells in an individual, comprising the step of administering an effective amount of the pharmaceutical to the individual in order to injure cells. The “individual” in any of the above embodiments may be a human.

[0126] In a further aspect, the present invention provides a method for treating a tumor. In one embodiment, the method comprises administering an effective amount of a polypeptide containing a mutated Fc region to an individual having such a tumor. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual. The “individual” in any of the above embodiments may be a human.

[0127] In a further aspect, the present invention provides a method for damaging cells in an organism. In one embodiment, the method comprises administering an effective amount of a polypeptide containing a mutated Fc region to an organism in order to damage cells. In one embodiment, the "organism" is a human.

[0128] In a further aspect, the present invention provides a pharmaceutical formulation (pharmaceutical composition) comprising a polypeptide containing a mutated 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 the treatment of tumors. In one embodiment, the present invention provides a pharmaceutical formulation (pharmaceutical composition) for use in cell injury. In another embodiment, the pharmaceutical formulation (pharmaceutical composition) comprises a polypeptide containing a mutated Fc region provided herein and at least one additional therapeutic agent. [Examples]

[0129] [Example 1] Preparation of an Fc region variant with enhanced binding ability to FcγR While Fc variants that enhance the cytotoxic effector functions of ADCC and ADCP have been reported to date, variants engineered symmetrically in the CH2 region and low-fucose antibodies created by glycosylation all left room for further enhancement of binding to FcγR. Furthermore, the variants described in WO2013002362 and WO2014104165, in which the CH2 region was asymmetrically engineered, showed significantly enhanced binding ability to FcγR compared to symmetrically engineered Fc region variants, but there was still room for further improvement. Specifically, the Fc region variant Kn125 / Hl076 (abbreviated herein: ART1), described in WO2013002362 and WO2014104165, showed strong enhanced binding ability to FcγRIIIa, but its binding ability to FcγRIIa was only a few times greater than that of IgG1, suggesting that further enhancement was necessary to exhibit strong ADCP activity. Furthermore, Kn120 / Hl068 (abbreviated as ART2 in this specification) exhibits enhanced binding to both FcγRIIa and FcγRIIIa, and strong activity is expected for both ADCC and ADCP. However, its binding ability to the inhibitory FcγRIIb is also enhanced, resulting in a small A / I ratio, which is an indicator of excellent effector function. In other words, existing methods have not achieved the most ideal profile: "antibody modification technology that binds more strongly to active FcγRIIa and FcγRIIIa while suppressing binding to inhibitory FcγRIIb." Therefore, in this invention, further combination modifications were considered to create an Fc region modifier with an excellent profile that overcomes these challenges.

[0130] Existing Fc region variants for comparison were prepared as follows. First, the antibody heavy chain gene H240-G1d (SEQ ID NO: 1), which has the heavy chain variable region for human epiregulin described in WO2014104165 and the heavy chain constant region sequence of human IgG1, was prepared. G1d is a sequence obtained by removing the C-terminal Lys and Gly from the heavy chain constant region sequence of natural human IgG1. By introducing the Knobs-into-holes modification (Nat. Biotechnol., 1998, 16, 677), which promotes heterodimerization, to the CH3 region of H240-G1d, and introducing an asymmetric modification to enhance binding to FcγR to the CH2 region, the Fc region variants ART1 and ART2 for comparison were prepared. ART1, an Fc region variant with enhanced binding to FcγRIIIa described in WO2013002362 and WO2014104165, was prepared as follows. H240-Kn125 (SEQ ID NO: 2) was created by introducing L234Y / L235Q / G236W / S239M / H268D / D270E / S298A, which enhances binding to FcγR, into the CH2 region of H240-G1d, and Y349C / T366W into the CH3 region. Furthermore, H240-Hl076 (SEQ ID NO: 3) was created by introducing D270E / K326D / A330M / K334E into the CH2 region of H240-G1d, and D356C / T366S / L368A / Y407V into the CH3 region. Plasmids containing H240-Kn125, H240-Hl076, and the L73-k0 (SEQ ID NO: 4) gene, which is the light chain of the anti-human epiregulin antibody, were mixed and introduced into human embryonic kidney cell-derived Expi 293 strain (Invitrogen) by lipofection. From the supernatant cultured for 4 days, rProtein A Sepharose was extracted. TMBy purifying the antibody against human epiregulin using a Fast Flow system (Amersham Biosciences) and methods known to those skilled in the art, an Fc-modified antibody (H240-Kn125 / L73-k0 / / H240-Hl076 / L73-k0: antibody abbreviation EGL-ART1) was obtained. The absorbance of the purified antibody solution at 280 nm was measured using a spectrophotometer. The concentration of the purified antibody was calculated using the extinction coefficient obtained from the measured values ​​by the PACE method (Protein Science (1995) 4, 2411-2423).

[0131] Similarly, EGL-ART2 (H240-Kn120 / L73-k0 / / H240-Hl068 / L73-k0), an Fc region variant with enhanced binding to both FcγRIIa and FcγRIIIa as described in WO2013002362 and WO2014104165, was created. Furthermore, by symmetrically introducing the FcγR binding-enhancing modified antibodies G236A, S239D, A330L, and I332E in different combinations into the CH2 region, existing FcγR binding-enhancing 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) were created. In addition to these, the afucosylated antibody EGL-afucosyl, which has been reported to enhance binding to FcγRIIIa (Glycobiol. Vol17 no1 pp. 104-118 (2006), etc.), was created as a comparison antibody. In cells where 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 (e.g., WO2006 / 067913). Furthermore, it is also possible to obtain fucose-deficient antibodies by producing antibodies in cells in which beta 1,4-N-acetylglucosaminyltransferaze III and Golgi alpha-mannosidae II are forcibly expressed (Biotechnol, Bioeng. (2006) 93 (5), 851-861). EGL-afucosyl (H240-G1d / L73-k_glycomab) was prepared using these methods known to those skilled in the art.

[0132] 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 fabricated. These variants all share the common introduction of L234F, L235Q, G236W, S239M, H268D, D270E, and S298A into one heavy chain, and D270E, S298A, K326D, and K334E into the other heavy chain. These variants were fabricated by further introducing modifications that alter the binding to FcγR into this core asymmetric variant group. Specifically, the introduction of K326D, A330M, and I332E was considered for the chain containing L234F / L235Q / G236W / S239M / H268D / D270E / S298A. Furthermore, the introduction of G236A, I332E, I332D, and A330M was considered for the strand into which D270E / S298A / K326D / K334E was introduced. In addition to these FcγR binding enhancement modifications, T250V and T307P, which are antibody stability enhancement modifications described in WO2013118858, were introduced into both strands of ART4, ART5, ART6, ART8, ART10, ART11, and ART12.

[0133] The correspondence between the names of each heavy chain constant region used here and their sequence numbers is as follows: G1d (sequence number: 29), Kn125 (sequence number: 30), Hl076 (sequence number: 31), k0 (sequence number: 32), Kn120 (sequence number: 33), Hl068 (sequence number: 34), Kn443 (sequence number: 35), Hl408 (sequence number: 36), Kn456 (sequence number: 37), Hl446 (sequence number: 38), Kn462 (sequence number: 39), Hl441 (sequence number: 40), Kn462 (sequence number: 41). Hl445 (SEQ ID NO: 42), Kn461 (SEQ ID NO: 43), Hl443 (SEQ ID NO: 44), Kn494 (SEQ ID NO: 45), Hl514 (SEQ ID NO: 46), Kn496 (SEQ ID NO: 47), Hl516 (SEQ ID NO: 48), Kn498 (SEQ ID NO: 49), Hl518 (SEQ ID NO: 50), GASDALIE (SEQ ID NO: 51), Kn032 (SEQ ID NO: 52), Hl032 (SEQ ID NO: 53), Kn037 (SEQ ID NO: 54), Hl036 (SEQ ID NO: 55), G4d (SEQ ID NO: 56)

[0134] (Table 1) Created Fc region variants and introduced variants TIFF2026086889000002.tif234151TIFF2026086889000003.tif23453

[0135] [Example 2] Evaluation of binding of Fc region variant to FcγR The extracellular domain of FcγR was prepared using the method described in WO2014104165. Interaction analysis between the prepared antibody and human FcγR was performed using Biacore 8K+ as follows: A running buffer consisting of 50 mM Na-Phosphate, 150 mM NaCl, and 0.05% Tween20 (pH 7.4) was used, and measurements were performed at 25°C. A Series S SA (GE Healthcare) chip immobilized with CaptureSelect Human Fab-kappa Kinetics Biotin Conjugate (Thermo Fisher Scientific) was used. The target antibody was captured onto this chip, and each FcγR diluted in the running buffer was interacted with it. The chip was regenerated using 10 mM Glycine-HCl (pH 1.5), and antibody capture and measurements were repeated. The dissociation constants (KD) (mol / L) for each antibody with respect to FcγR were calculated using Biacore Insight Evaluation Software. The dissociation constant for FcγRIIb was calculated using the Steady State Affinity model, while the dissociation constants for other FcγRs were calculated using the 1:1 Langmuir binding model (Table 2).

[0136] (Table 2) Binding measurement of the prepared modified compounds to human FcγR TIFF2026086889000004.tif234129TIFF2026086889000005.tif23471

[0137] In the table, "Relative value of G1d and hFcγRs relative to KD" refers to the value obtained by dividing the KD value of G1d for each FcγR by the KD value of each antibody for each FcγR, indicating how much each antibody is enhanced relative to G1d. Furthermore, "A / I ratio" refers to the value obtained by dividing the KD of each antibody for FcγRIIb by the KD for each FcγR, indicating how selectively binding to active FcγR is enhanced compared to binding to inhibitory FcγR.

[0138] ART3, ART4, ART5, ART6, ART8, ART10, ART11, and ART12, prepared in this invention, were all enhanced against FcγRIIIaF and FcγRIIIaV compared to G1d. These modified antibodies were also more enhanced against both FcγRIIIaF and FcγRIIIaV compared to existing FcγR-enhancing antibodies such as GASDALIE, SDALIE, GASDIE, and Afucosyl antibodies, which were symmetrically modified. Furthermore, ART4 (2519.9x), ART6 (986.7x), ART8 (1966.7x), ART10 (1289.2x), and ART12 (577.5x) showed enhanced binding to FcγRIIIaF compared to ART2 (459.6x) described in WO2014104165. Furthermore, ART4, ART8, and ART10 showed enhanced binding to FcγRIIIaF even when compared to ART1 (1170.2 times), which had stronger binding to FcγRIIIaF than ART2. Similarly, regarding FcγRIIIaV, ART4 (462.2 times), ART6 (321.9 times), ART8 (694.9 times), and ART10 (565.5 times) showed enhanced binding compared to ART2 (214.6 times), and ART4, ART8, and ART10 showed even stronger binding compared to ART1 (322.8 times). ART6 showed a similar level of enhancement to FcγRIIIaV as ART1.

[0139] ART3 (32.1x), ART4 (6.3x), ART5 (33.9x), ART6 (118.0x), ART8 (15.0x), ART10 (2.7x), ART11 (4.9x), and ART12 (3.1x) were all enhanced against FcγRIIaH compared to G1d. In particular, ART3, ART5, ART6, and ART8 were even more enhanced compared to ART2 (14.7x), an FcγRIIa-enhancing antibody with an asymmetrically modified CH2 region as described in WO2014104165. Furthermore, ART3, ART5, and ART6 were even more strongly enhanced compared to GASDIE (16.4x), an existing FcγRIIa-enhancing antibody with a symmetrically modified region, suggesting that they exhibit stronger ADCP activity than any of the existing modified antibodies. Regarding binding to FcγRIIaR, ART3 (13.7x), ART4 (3.2x), ART5 (10.4x), ART6 (24.1x), ART8 (13.2x), ART10 (1.5x), and ART11 (1.3x) all showed enhanced binding compared to G1d. However, the existing variants GASDIE (24.7x) and ART2 (49.0x) showed even greater enhancement compared to these variants. What is noteworthy here, however, is the selectivity for active FcγR. FcγRIIb, an inhibitory receptor, induces intracellular signals that suppress the immune response, in contrast to active FcγR, and is therefore expected to inhibit signals from active FcγR. In fact, it has been reported that the antitumor effect of antibodies is enhanced in mice in which FcγRIIb is knocked out (Nature Medicine 2000, 6, 443-436). Furthermore, a correlation has been observed between the differences in antitumor effects of mouse IgG subclasses and the binding ratio (A / I ratio) to active FcγR and inhibitory FcγR (Science 2005, 310, 1510-1512). Therefore, in order to exert a stronger effector function, it is thought that an antibody is needed in which binding to active FcγR is enhanced while binding to FcγRIIb is reduced. However, because FcγRIIaR has high sequence homology with FcγRIIb, it is difficult to confer selectivity, and the modified antibodies reported to date cannot be said to have excellent selectivity.In this invention, the newly fabricated modified compounds 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) all showed superiority to G1d (A / I ratio 6.1) in terms of the A / I ratio of FcγRIIaR. Among these, 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, regarding 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 even better A / I ratios compared to ART2 (A / I ratio 5.8) and GASDIE (A / I ratio 18.2). From these results, it can be said that ART4, ART8, ART3, ART5, and ART6 are antibodies with superior A / I ratios compared to existing enhancement antibodies against FcγRIIaH. Furthermore, ART3, ART5, and ART6 can be said to be antibodies that have superior binding affinity and A / I ratio compared to existing enhancement antibodies against FcγRIIaH.

[0140] In terms of A / I ratio for FcγRIIIaF, 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 showed superiority over G1d (A / I ratio 3.4). Among these, ART4 showed a superior A / I ratio compared to ART1 (A / I ratio 4947.2), an FcγRIIIa-specific enhancement variant described in WO2014104165. Similarly, the A / I ratios for 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 enhancement variant Afucosyl (A / I ratio 298.1). Among these, ART4 and ART10 showed better A / I ratios than ART1 (A / I ratio 11261.3), an FcγRIIIa-specific enhancement variant described in WO2014104165. Based on these results, ART4 can be said to be an antibody that has even better binding affinity and A / I ratio than the existing enhancement antibody ART1 for both FcγRIIIaF and FcγRIIIaV.

[0141] [Example 3] Evaluation of an antibody having a modified Fc region by ADCC reporter bioassay (3-1) Generation of human Epiregulin-expressing cells (Hepa1-6 / hEREG) Mouse liver cancer cell line Hepa1-6 was purchased from ATCC, and the human EREG (hEREG) gene was introduced by transfection. Clones exhibiting constitutive expression were selected. 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.

[0142] (3-2) Evaluation using ADCC Reporter Bioassay For in vitro ADCC activity measurement, the hFcγRIIIaV ADCC Reporter Bioassay, Effector cells, and Propagation Model (Promega) were used. Each well of a 384-well plate was filled with 5 × 10⁶ cells in the culture medium. 5 The concentration was adjusted to / mL. Hepa1-6 / hEREG cells were added in 10 μL each as target cells, and Assay Buffer (96% RPMI, 4% FBS) was used as the culture medium. Next, the antibody prepared in Example 1 and EGL-G4d (heavy chain sequence number: 28, light chain sequence number: 4), which has the sequence of human IgG4 as a negative control, were each diluted in assay buffer to a final concentration of 1 μg / mL with a common ratio of 10 to 11 points, and then added in 10 μL each. Finally, 3 × 10⁶ cells were added to the culture medium as the effector cell solution. 6 10 μL of hFcγRIIIaV-expressing Jurkat cells, prepared to 1 / mL, were added to each well, and the mixture was mixed to a total of 30 μL. The plate was then incubated overnight at 37°C in a 5% CO2 incubator. The plate was then left to stand at room temperature for 15 minutes, and 30 μL of Bio-Glo reagent was added to each well. The Bio-glo Luciferase Assay System (Buffer and Substrate) was used as the Bio-Glo reagent. The luminescence in each well was then measured using a plate reader.

[0143] Fold induction was defined as the value of the luminescence in each well divided by the value of the luminescence in the antibody-free well, and this was used as an indicator to evaluate the ADCC of each antibody. The results are shown in Figure 1. The EC50 values ​​for each sample were calculated using JMP 11.2.1 (SAS Institute Inc.) and are shown in Table 3.

[0144] (Table 3) EC50 values ​​of reporter gene induction activity via hFcγRIIIaV for antibodies with each modified Fc. TIFF2026086889000006.tif113128

[0145] These results demonstrate that the reporter gene induction activity of the modified Fc antibodies produced in this study against Hepa1-6 / hEREG cells is potent compared to that of the wild-type human IgG1 constant region. Furthermore, as shown in Table 3, all of these modified antibodies exhibited activity at lower concentrations compared to modified antibodies engineered symmetrically on the CH2 region or low-fucose antibodies produced by glycosylation modification. Among the modified antibodies produced in this study, ART3, ART4, ART5, ART6, ART8, ART10, ART11, and ART12 were shown to exhibit activity at concentrations similar to or lower than ART2. In particular, ART4, ART10, ART11, and ART12 exhibited activity at even lower concentrations than ART1, which has even more enhanced binding to hFcγRIIIaV than ART2.

[0146] [Example 4] Evaluation of an antibody with a modified Fc region using ADCP Reporter Bioassay For in vitro ADCP activity measurement, the hFcγRIIaH ADCP Reporter Bioassay, Core Kit (Promega) was used. 1 × 10¹⁶ samples were added to each well of a 384-well plate using culture medium. 6 The concentration was adjusted to / mL. Hepa1-6 / hEREG cells were added in 10 μL portions as target cells, and Assay Buffer (96% RPMI, 4% FBS) was used as the culture medium. Next, 10 μL of the antibodies prepared in Example 1 were added after being diluted in assay buffer to final concentrations of 0, 0.001, 0.01, 0.1, 1, and 10 μg / mL, respectively. Finally, 10 μL of the hFcγRIIaH-expressing Jurkat cells included in the kit were added as the effector cell solution, and the mixture was mixed to a total of 30 μL. The mixture was then incubated in a 5% CO2 incubator at 37°C for 6 hours. The hFcγRIIaH-expressing Jurkat cells had a cell saturation density of 9.68 × 10⁶. 5The value was / mL. The plate was then left to stand at room temperature for 15 minutes, and 30 μL of Bio-Glo reagent was added to each well. The Bio-glo Luciferase Assay System (Buffer and Substrate) was used as the Bio-Glo reagent. The luminescence of each well was then measured using a plate reader. The value of the luminescence of each well divided by the luminescence of the antibody-free well was defined as Fold induction and used as an index to evaluate the ADCP of each antibody. The results obtained 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.

[0147] (Table 4) EC50 values ​​of reporter gene induction activity via hFcγRIIaH for antibodies with each modified Fc. TIFF2026086889000007.tif113128

[0148] These results demonstrate that the reporter gene induction activity of the modified Fc antibodies produced in this study against Hepa1-6 / hEREG cells is potent compared to that of the wild-type human IgG1 constant region. Furthermore, as shown in Table 4, these antibodies exhibited activity at lower concentrations compared to modified antibodies engineered symmetrically around the CH2 region and low-fucose antibodies produced by glycosylation modifications. In addition, among the modified antibodies produced in this study, ART2, ART3, ART5, ART6, and ART8 showed activity at lower concentrations compared to ART1. Among these, ART3, ART6, and ART8 showed activity at even lower concentrations than ART2, which had further enhanced binding to hFcγRIIaH.

[0149] [Example 5] Evaluation of the antitumor effect of an antibody containing a modified Fc region in a syngeneic tumor cell transplantation 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.

[0150] (5-2) Creation of a mouse model transplanted with a syngeneic tumor cell line Human FcγR transgenic mice (Proc Natl Acad Sci US A. 2012 Apr 17; 109(16): 6181-6186.) were used for the drug efficacy study. Anti-asialoGM1 antibody (aGM1, WAKO) was administered intraperitoneally to 16-week-old male mice at a dose of 100 μL / head to improve cell engraftment. The day after aGM1 administration, a cell solution containing Hepa1-6 / hEREG cells and Matrigel (CORNING) in a 1:1 ratio was subcutaneously administered, resulting in a cell count of 1 × 10⁶. 7 Cells were transplanted, and the average volume of the transplanted tumor was approximately 300 mm². 3 From 500 mm 3 The model was considered successful at that point. The volume of the transplanted tumor was calculated using the following formula. Tumor volume = Long diameter × Short diameter × Short diameter / 2

[0151] (5-3) Preparation of administered drugs Based on the A / I ratio results in Example 2 and the strength of reporter gene induction activity in Examples 3 and 4, EGL-ART6 produced in this invention was expected to exhibit the strongest antitumor activity. Therefore, as the drugs to be administered to the Hepa1-6 / hEREG cell transplantation model, an anti-hEREG control antibody (EGL-G1d) prepared by the same method as in Example 1 and an anti-hEREG antibody (EGL-afucosyl, EGL-ART6) with enhanced Fc binding were prepared using His buffer (150 mM NaCl, 20 mM His-HCl buffer pH 6.0) so that each was at a concentration of 1 mg / mL.

[0152] (5-4) Drug administration when measuring antitumor effect On day 7 post-transplant, EGL-G1d, EGL-afucosyl, and EGL-ART6 were administered via tail vein at a dose of 10 mg / kg. Table 5 shows the details of the drug treatment used when measuring the antitumor effect.

[0153] (Table 5) Measurement of antitumor effect in Hepa1-6 / hEREG cell transplantation model TIFF2026086889000008.tif25128

[0154] (5-5) Evaluation of antitumor effect 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 - (Average tumor volume of the target group at the time of measurement - Average tumor volume before antibody administration) ÷ (Average tumor volume of the control group at the time of measurement - Average tumor volume before antibody administration)) × 100

[0155] As a result, both EGL-afucosyl and EGL-ART6, which enhance FcγR binding, showed therapeutic efficacy of TGI=80 or higher at a dose of 10 mg / kg on day 19 after administration. On the other hand, the control antibody EGL-G1d had a TGI=31 (Figure 3). From this, it was confirmed that EGL-ART6 produced in this invention enhances the antitumor effect against EGL-afucosyl in vivo as expected.

[0156] [Example 6] Evaluation of binding activity of an antibody having a modified Fc region to C1q While some reports suggest that antibody CDC activity contributes to antitumor effects (Nat. Immunol., 2017, 18, 889), it is also known to cause side effects derived from CDC activity, 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 preferable to be able to select the degree of CDC activity depending on the target disease. The interaction between complement and Fc is mediated by C1q. Reports analyzing the interaction between C1q and Fc (Science, 2018, 359, 794-797 and Molecular Immunology 2012, 51, 66-72) suggest that the interaction sites between FcγR and C1q on the Fc region partially overlap. These studies have reported that among the positions on Fc, residues at EU numbering positions 329, 330, and 331 are important for interaction with C1q, and that residues at EU numbering positions 268, 270, and 298 also contribute to binding to C1q. Since these positions and surrounding residues are the sites modified in this invention to enhance binding to FcγR, the created Fc region variants are highly likely to also have enhanced or weakened binding to C1q, potentially allowing for control of CDC activity in the development of antibody drugs. Therefore, the binding of the created Fc region variants to C1q was evaluated.

[0157] The anti-human epiregulin antibodies prepared in Examples 1 and 3 were subjected to ELISA. The buffers shown in Table 6 were prepared as appropriate. Human C1q protein (hC1q) was used as the antigen.

[0158] (Table 6) Composition of buffer used in human C1q ELISA TIFF2026086889000009.tif27149

[0159] First, a 96-well maxisorp plate (Thermo Fisher) was coated overnight at 4°C with 50 μL of solution containing each antibody prepared in PBS at concentrations of 30, 10, 3, 1, 0.3, 0.1, and 0.03 μg / mL. Each well of the plate was washed with wash buffer to remove any antibodies not bound to the plate, and then the well was blocked with 200 μL of Blocking / dilution Buffer at room temperature for at least 2 hours. 50 μL of hC1q (Calbiochem), prepared to a final concentration of 3 μg / mL in Blocking / dilution Buffer, was added to each well from which the Blocking / dilution Buffer had been removed. The plate was allowed to stand 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. After the color reaction of the solution in each well was stopped by the addition of Stop Buffer, the color development was measured by absorbance at 450 nm and 690 nm. The buffer used contained the composition listed in Table 6. The measured results are shown in Figures 4 and 5.

[0160] As shown in Figures 4 and 5, among the evaluated variants, ART3, ART5, and ART11 showed enhanced binding ability to C1q compared to G1d. Afucosyl and ART8 showed binding ability similar to G1d. ART1, ART2, ART4, ART6, ART10, ART12, GASDALIE, SDALIE, and GASDIE showed reduced binding to C1q compared to G1d. Among these, the binding ability to C1q of ART1, ART2, ART4, ART6, ART12, GASDALIE, SDALIE, and GASDIE was attenuated to a level similar to G4d, which has the human IgG4 sequence. Since human IgG4 is considered to have little CDC activity (J. Immunol. Methods 2005, 306, 151-160), these variants, whose binding to C1q is attenuated to a level similar to G4d, are thought to have little CDC activity, similar to IgG4. Common amino acid modifications in these variants with significantly reduced binding to C1q include modifications to Ala330 or Ile332. Since these regions, particularly Ala330, are extremely important for interaction with C1q, it is hypothesized that the introduction of modifications to these sites significantly reduced binding to C1q. On the other hand, ART3, ART5, and ART11, which showed enhanced binding to C1q compared to G1d, did not have modifications to positions 330 or 332, and it is thought that the enhanced binding was due to the effects of modifications to S298A or position 326 (Science, 2018, 359, 794-797), which are known to improve binding to C1q.

[0161] The invention described herein has been described in detail with examples and illustrations for the purpose of aiding clear understanding, but the descriptions and illustrations herein should not be construed as limiting the scope of the invention. All disclosures of patent and scientific documents cited herein are expressly incorporated herein by reference throughout. [Industrial applicability]

[0162] The inventions of this disclosure provide polypeptides comprising Fc domain modifiers that bind more strongly to active FcγRIIa and FcγRIIIa and suppress binding to inhibitory FcγRIIb. Such polypeptides of this disclosure exhibit high ADCC / ADCP activity and are useful in antitumor therapies (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 including an amino acid modification in the parent Fc region, The parent Fc region is the region from the 229th Pro to the 444th Pro in sequence number 1, and is composed of two polypeptide chains. A polypeptide in which the mutated Fc region has at least 90% amino acid sequence identity with the parent Fc region and includes the following amino acid modifications: (i) Amino acid modifications in the first polypeptide of the parent Fc region to Phe at position 234, Gln at position 235, Trp at position 236, Met at position 239, Asp at position 268, Glu at position 270, and Ala at position 298, as represented by EU numbering, and (ii) Amino acid modifications in the second polypeptide of the parent Fc region to Glu at position 270, Ala at position 298, Asp at position 326, and Glu at position 334, as represented by EU numbering. Here, the polypeptide has the characteristics described in (1) or (2) below: (1) Compared to the parent Fc region, the mutant Fc region exhibits enhanced binding activity to at least one active Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa R, FcγRIIa H, FcγRIIIa F, and FcγRIIIa V, or (2) Compared to the parent Fc region, the mutant Fc region selectively enhances the binding activity to at least one active Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa R, FcγRIIa H, FcγRIIIa F, and FcγRIIIa V, compared to the binding activity to the inhibitory Fcγ receptor.

2. The polypeptide according to claim 1, wherein the mutant Fc region further comprises an amino acid modification to Ala at position 236, represented by EU numbering, in the second polypeptide of the parent Fc region.

3. The polypeptide according to claim 1 or 2, wherein the mutant Fc region further comprises an amino acid modification to Glu at position 332, represented by EU numbering, in the first polypeptide of the parent Fc region.

4. The polypeptide according to any one of claims 1 to 3, wherein the mutant Fc region further comprises an amino acid modification to Met at position 330, represented by EU numbering, in the first polypeptide of the parent Fc region.

5. The polypeptide according to any one of claims 1 to 4, wherein the mutant Fc region further comprises an amino acid modification to Glu at position 332, represented by EU numbering, in the second polypeptide of the parent Fc region.

6. The polypeptide according to any one of claims 1 to 5, wherein the mutant Fc region further comprises an amino acid modification to Met at position 330, represented by EU numbering, in the second polypeptide of the parent Fc region.

7. The polypeptide according to any one of claims 1 to 6, wherein the mutant Fc region further comprises amino acid modifications to Val at position 250 and Pro at position 307 in the first polypeptide of the parent Fc region.

8. The polypeptide according to any one of claims 1 to 7, wherein the mutant Fc region further comprises amino acid modifications to Val at position 250 and Pro at position 307 in the second polypeptide of the parent Fc region, as represented by EU numbering.

9. The polypeptide according to any one of claims 1 to 8, wherein the polypeptide containing the mutated Fc region is an antibody.

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