Modified antibody Fc region

Abstract

To provide antibody Fc regions in which an amino acid sequence is modified from a naturally-occurring antibody Fc region, to provide antibodies comprising the Fc regions, to provide pharmaceutical compositions comprising the antibodies, and production methods thereof.SOLUTION: The present invention provides a polypeptide in which stability is improved as compared with its parent polypeptide, by modifying at least one amino acid of a loop site of the Fc region of an antibody. Also provided are a polypeptide in which the binding activity to FcγR is maintained or enhanced while thermal stability is improved as compared with its parent polypeptide, by combining a plurality of amino acid modifications of a loop site; and a polypeptide in which the binding activity to FcγR is reduced while the thermal stability is improved; and additionally a polypeptide in which the binding activity to FcγR is not only adjusted while the thermal stability is improved, but the content of an assembly is also reduced.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention provides antibody Fc regions whose amino acid sequences have been altered from naturally occurring antibody Fc regions, antibodies comprising such Fc regions, pharmaceutical compositions comprising such antibodies, and methods for producing them. [Background technology]

[0002] Antibodies are attracting attention as pharmaceuticals because they are highly stable in plasma and have few side effects. Many IgG-type antibody drugs are on the market, and many antibody drugs are currently being developed (Non-Patent Document 1, Non-Patent Document 2).

[0003] In recent years, research has been actively conducted into enhancing antibody functions by introducing artificial amino acid modifications into the Fc region of an antibody. Specifically, Fc regions with improved pharmacokinetics, Fc regions with enhanced ADCC activity, which is an effector function, and Fc regions with reduced ADCC activity in neutralizing antibodies have been studied (Non-Patent Documents 3-6). However, such modifications of Fc regions are known to have adverse effects on the physical properties of antibodies. For example, it has been reported that modified Fc regions with enhanced ADCC activity have a thermal denaturation midpoint lowered by about 20°C (Non-Patent Document 6). It has also been reported that modified Fc regions with reduced ADCC activity have a thermal denaturation midpoint lowered by about 5°C, are susceptible to degradation by hydrolases, and are susceptible to degradation under acidic conditions (Non-Patent Documents 7-9). Furthermore, it has been reported that modified Fc regions with improved blood retention have reduced thermal stability and storage stability (Patent Document 1). In other words, many of the modified Fc regions discovered so far have lost their good stability, which is one of the advantages of antibodies, along with their enhanced function.

[0004] As an attempt to improve stability, a method of modifying amino acids in the CH2 domain and introducing cysteine ​​has been reported (Non-Patent Document 10). It has been reported that the thermal stability is increased by about 10°C to 20°C by forming a new disulfide bond through the introduction of cysteine. However, since this report evaluated the thermal stability only in the CH2 domain, the thermal stability in the form of IgG is unknown. In addition, an increase in heterogeneity is expected due to the formation of a new disulfide bond. As described above, no Fc region that is excellent in both activity and stability has been reported to date.

[0005] Prior art literature related to the invention of this application is shown below. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2007 / 092772 [Patent Document 2] WO2010 / 085682 [Non-patent literature]

[0007] [Non-Patent Document 1] Monoclonal antibody successes in the clinic, Janice M Reichert, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz, Nature Biotechnology 23, 1073-1078 (2005) [Non-Patent Document 2] Pavlou AK, Belsey MJ., The therapeutic antibodies market to 2008., Eur J Pharm Biopharm. 2005 Apr;59(3):389-96. [Non-Patent Document 3] Hinton PR, Xiong JM, Johlfs MG, Tang MT, Keller S, Tsurushita N., An engineered human IgG1 antibody with longer serum half-life., J Immunol. 2006 Jan 1;176(1):346-56

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[0008] The present invention has been made in light of these circumstances, and an object of the present invention is to provide a polypeptide having improved stability by modifying amino acids in the antibody Fc region. [Means for solving the problem]

[0009] The present inventors considered that in creating a modified Fc region, it would be desirable to have one that has enhanced function while maintaining antibody stability, or one that restores the stability that has been reduced as a result of enhanced function. As a result of intensive research, the present inventors have succeeded in obtaining a polypeptide having an antibody Fc region, which has improved stability compared to a parent polypeptide by modifying at least one amino acid in a loop region of the Fc region. Furthermore, by combining multiple amino acid modifications in the loop region, it was possible to obtain a polypeptide with improved thermal stability while maintaining or enhancing FcγR-binding activity, and a polypeptide with improved thermal stability while decreasing FcγR-binding activity, as compared to the parent polypeptide. Furthermore, it was possible to obtain a polypeptide with improved thermal stability, adjusted FcγR-binding activity, and a reduced aggregate content.

[0010] That is, the present invention relates to the following. [1] A polypeptide having an antibody Fc region, in which at least one amino acid in a loop region of the Fc region has been modified, thereby improving stability compared to a parent polypeptide. [2] The polypeptide according to [1], wherein the stability is evaluated or determined using the thermal denaturation midpoint (Tm) as an index. [3] position 234 (EU numbering), position 235 (EU numbering), position 236 (EU numbering), position 237 (EU numbering), position 238 (EU numbering), position 239 (EU numbering), position 247 (EU numbering), position 250 (EU numbering), position 265 (EU numbering), position 266 (EU numbering), position 267 (EU numbering), position 268 (EU numbering), position 269 (EU numbering), position 270 (EU numbering), position 271 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), position 300 (EU numbering), position 307 (EU numbering), or position 309 (EU numbering) in the loop region of the Fc region. The polypeptide according to [1] or [2], wherein at least one amino acid mutation has been introduced into an amino acid site selected from the group consisting of: 315th (EU numbering), 324th (EU numbering), 325th (EU numbering), 326th (EU numbering), 327th (EU numbering), 329th (EU numbering), 330th (EU numbering), 333rd (EU numbering), 335th (EU numbering), 337th (EU numbering), 360th (EU numbering), 385th (EU numbering), 386th (EU numbering), 387th (EU numbering), 389th (EU numbering), 428th (EU numbering), and 433rd (EU numbering). [4] The polypeptide according to any one of [1] to [3], which further maintains or enhances FcγR-binding activity compared to the parent polypeptide. [5] The polypeptide according to any one of [1] to [3], which further has reduced FcγR-binding activity as compared to the parent polypeptide. [6] The polypeptide according to any one of [1] to [5], wherein at least one amino acid mutation has been introduced into an amino acid site selected from the group consisting of: position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 266 (EU numbering), position 267 (EU numbering), position 268 (EU numbering), position 269 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), position 300 (EU numbering), position 324 (EU numbering), position 325 (EU numbering), position 326 (EU numbering), and position 330 (EU numbering) in the loop region of the Fc region. [7] The polypeptide according to any one of [1] to [4] and [6], wherein at least one amino acid mutation has been introduced into an amino acid site selected from the group consisting of position 234 (EU numbering), position 266 (EU numbering), position 268 (EU numbering), position 269 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 300 (EU numbering), position 324 (EU numbering), position 326 (EU numbering), and position 330 (EU numbering) in the loop region of the Fc region. [8] the polypeptide of any one of [1] to [4], [6], and [7], wherein the amino acid modification in the loop site of the Fc region is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Ile, substitution of the amino acid at position 266 (EU numbering) with Ile, substitution of the amino acid at position 268 (EU numbering) with Gln, substitution of the amino acid at position 269 (EU numbering) with Asp, substitution of the amino acid at position 270 (EU numbering) with Glu, substitution of the amino acid at position 295 (EU numbering) with Met or Leu, substitution of the amino acid at position 300 (EU numbering) with Glu, substitution of the amino acid at position 324 (EU numbering) with His, substitution of the amino acid at position 326 (EU numbering) with Ser or Ala, and substitution of the amino acid at position 330 (EU numbering) with His or Tyr; [9] The polypeptide of any one of [1] to [3], [5], and [6], wherein at least one amino acid mutation has been introduced into an amino acid site selected from the group consisting of: position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 267 (EU numbering), position 268 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering) in the loop region of the Fc region.

[10] The polypeptide of any one of [1] to [3], [5], [6], and [9], wherein the amino acid modification in the loop site of the Fc region is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys or Arg, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys, substitution of the amino acid at position 267 (EU numbering) with Pro, substitution of the amino acid at position 268 (EU numbering) with Met or Lys, substitution of the amino acid at position 270 (EU numbering) with Phe, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with any of Gly, His, or Met.

[11] The polypeptide according to any one of [1] to [4], wherein at least one amino acid mutation has been introduced into an amino acid site selected from the group consisting of positions 295, 326, and 330 (EU numbering) in the loop region of the Fc region.

[12] The polypeptide of any one of [1] to [4] and

[11] , wherein the amino acid modification in the loop region of the Fc region is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 295 (EU numbering) with Met or Leu, substitution of the amino acid at position 326 (EU numbering) with Ser or Ala, and substitution of the amino acid at position 330 (EU numbering) with His or Tyr.

[13] The polypeptide of any one of [1] to [3] and [5], wherein at least one amino acid mutation has been introduced into an amino acid site selected from the group consisting of: position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 268 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering) in the loop region of the Fc region.

[14] The polypeptide of any one of [1] to [3], [5], and

[13] , wherein the amino acid modification in the loop site of the Fc region is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys or Ser, substitution of the amino acid at position 268 (EU numbering) with Lys or His, substitution of the amino acid at position 270 (EU numbering) with Phe or Asp, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

[15] The polypeptide of any one of [1] to [3] and [5], wherein at least one amino acid mutation has been introduced into an amino acid sequence selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 268 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering) in the loop region of the Fc region.

[16] The polypeptide of any one of [1] to [3], [5], and

[15] , wherein the amino acid modification in the loop site of the Fc region is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys, substitution of the amino acid at position 268 (EU numbering) with Lys, substitution of the amino acid at position 270 (EU numbering) with Phe, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

[17] The polypeptide of any one of [1] to [3] and [5], wherein at least one amino acid mutation has been introduced into an amino acid sequence selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering) in the loop region of the Fc region.

[18] The polypeptide of any one of [1] to [3], [5], and

[17] , wherein the amino acid modification in the loop site of the Fc region is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

[19] The polypeptide of any one of [1] to [3] and [5], wherein at least one amino acid mutation has been introduced into an amino acid site selected from the group consisting of: position 234 (EU numbering), position 235 (EU numbering), position 268 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering) in the loop region of the Fc region.

[20] The polypeptide of any one of [1] to [3], [5], and

[19] , wherein the amino acid modification in the loop site of the Fc region is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 268 (EU numbering) with Lys, substitution of the amino acid at position 270 (EU numbering) with Phe, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

[21] The polypeptide of any one of [1] to [3] and [5], wherein the amino acid modification site in the loop region of the Fc region comprises at least one amino acid mutation introduced into an amino acid site selected from the group consisting of: position 234 (EU numbering), position 235 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering).

[22] The polypeptide of any one of [1] to [3], [5], and

[21] , wherein the amino acid modification in the loop region of the Fc region is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

[23] The polypeptide of any one of [1] to [4], wherein at least one amino acid mutation has been introduced into an amino acid site selected from the group consisting of: position 247 (EU numbering), position 250 (EU numbering), position 307 (EU numbering), position 309 (EU numbering), position 315 (EU numbering), position 360 (EU numbering), position 385 (EU numbering), position 386 (EU numbering), position 387 (EU numbering), position 389 (EU numbering), position 428 (EU numbering), and position 433 (EU numbering) in the loop region of the Fc region.

[24] the amino acid modification in the loop region of the Fc region includes substitution of the amino acid at position 247 (EU numbering) with Val, substitution of the amino acid at position 250 (EU numbering) with any of Phe, Ile, Met, Val, Trp, or Tyr, substitution of the amino acid at position 307 (EU numbering) with any of Ala, Gln, or Pro, substitution of the amino acid at position 309 (EU numbering) with any of Ala, Arg, or Pro, substitution of the amino acid at position 315 (EU numbering) with Ala, or substitution of the amino acid at position 360 (EU numbering) with His; The polypeptide of any one of [1] to [4] and

[23] , wherein the amino acid modification is at least one selected from the group consisting of substitution of the amino acid at position 385 (EU numbering) with Asp, substitution of the amino acid at position 386 (EU numbering) with Pro, substitution of the amino acid at position 387 (EU numbering) with Glu, substitution of the amino acid at position 389 (EU numbering) with Ser, substitution of the amino acid at position 428 (EU numbering) with any of His, Trp, Tyr, or Phe, and substitution of the amino acid at position 433 (EU numbering) with Lys.

[25] The polypeptide according to any one of [1] to [3] and [5], wherein at least one amino acid mutation has been introduced into the loop region of the Fc region at amino acid position 298 (EU numbering) or amino acid position 309 (EU numbering).

[26] The polypeptide of any one of [1] to [3], [5], and

[25] , wherein the amino acid modification in the loop region of the Fc region is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 298 (EU numbering) with Gly and substitution of the amino acid at position 309 (EU numbering) with Asp.

[27] A method for improving the stability of a polypeptide having an antibody Fc region, as compared to a parent polypeptide, by introducing at least one amino acid modification into a loop region of the Fc region.

[28] The method according to

[27] , wherein the stability is evaluated or determined using a thermal denaturation midpoint (Tm) as an index.

[29] A method for producing a polypeptide having an antibody Fc region, in which at least one amino acid in a loop region of the Fc region has been modified, and thus the stability is improved as compared with that of a parent polypeptide, the method comprising the steps of: (a) modifying at least one amino acid in a loop region of an antibody Fc region of a polypeptide having the Fc region; (b) measuring the stability of the polypeptide modified in step (a); and (c) selecting a polypeptide having improved stability compared to the parent polypeptide.

[30] A method for producing a polypeptide having an antibody Fc region, in which at least one amino acid in a loop region of the Fc region has been modified, and thus the stability is improved as compared with that of a parent polypeptide, the method comprising the steps of: (a) modifying a nucleic acid encoding the polypeptide such that it has enhanced stability compared to a parent polypeptide; (b) introducing the modified nucleic acid into a host cell and culturing it so as to express the modified nucleic acid; (c) recovering the polypeptide from the host cell culture.

[31] the following in the loop region of the Fc region: position 234 (EU numbering), position 235 (EU numbering), position 236 (EU numbering), position 237 (EU numbering), position 238 (EU numbering), position 239 (EU numbering), position 247 (EU numbering), position 250 (EU numbering), position 265 (EU numbering), position 266 (EU numbering), position 267 (EU numbering), position 268 (EU numbering), position 269 (EU numbering), position 270 (EU numbering), position 271 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), position 300 (EU numbering), position 307 (EU numbering), or position 309 (EU numbering); The method according to any one of

[27] to

[30] , wherein at least one amino acid mutation is introduced into an amino acid site selected from the group consisting of: 315th (EU numbering), 324th (EU numbering), 325th (EU numbering), 326th (EU numbering), 327th (EU numbering), 329th (EU numbering), 330th (EU numbering), 333rd (EU numbering), 335th (EU numbering), 337th (EU numbering), 360th (EU numbering), 385th (EU numbering), 386th (EU numbering), 387th (EU numbering), 389th (EU numbering), 428th (EU numbering), and 433rd (EU numbering).

[32] The method according to any one of

[27] to

[31] , further comprising a step of modifying the polypeptide so as to maintain or enhance its FcγR-binding activity as compared with that of the parent polypeptide.

[33] The method according to any one of

[27] to

[31] , further comprising the step of modifying the parent polypeptide so that it has a reduced FcγR-binding activity as compared to that of the parent polypeptide.

[34] The method according to any one of

[27] to

[33] , wherein at least one amino acid mutation is introduced into an amino acid site selected from the group consisting of: position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 266 (EU numbering), position 267 (EU numbering), position 268 (EU numbering), position 269 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), position 300 (EU numbering), position 324 (EU numbering), position 325 (EU numbering), position 326 (EU numbering), and position 330 (EU numbering) in the loop region of the Fc region.

[35] The method according to any one of

[27] to

[32] and

[34] , wherein at least one amino acid mutation is introduced into an amino acid site selected from the group consisting of position 234 (EU numbering), position 266 (EU numbering), position 268 (EU numbering), position 269 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 300 (EU numbering), position 324 (EU numbering), position 326 (EU numbering), and position 330 (EU numbering) in the loop region of the Fc region.

[36] The method of any one of

[27] to

[32] ,

[34] , and

[35] , wherein the amino acid modification in the loop site of the Fc region is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Ile, substitution of the amino acid at position 266 (EU numbering) with Ile, substitution of the amino acid at position 268 (EU numbering) with Gln, substitution of the amino acid at position 269 (EU numbering) with Asp, substitution of the amino acid at position 270 (EU numbering) with Glu, substitution of the amino acid at position 295 (EU numbering) with Met or Leu, substitution of the amino acid at position 300 (EU numbering) with Glu, substitution of the amino acid at position 324 (EU numbering) with His, substitution of the amino acid at position 326 (EU numbering) with Ser or Ala, and substitution of the amino acid at position 330 (EU numbering) with His or Tyr.

[37] The method of any one of

[27] to

[31] ,

[33] , and

[34] , wherein at least one amino acid mutation is introduced into an amino acid site selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 267 (EU numbering), position 268 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering) in the loop region of the Fc region.

[38] The method of any one of

[27] to

[31] ,

[33] ,

[34] , and

[37] , wherein the amino acid modification in the loop site of the Fc region is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys or Arg, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys, substitution of the amino acid at position 267 (EU numbering) with Pro, substitution of the amino acid at position 268 (EU numbering) with Met or Lys, substitution of the amino acid at position 270 (EU numbering) with Phe, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with any of Gly, His, or Met.

[39] The method according to any one of

[27] to

[32] , wherein at least one amino acid mutation is introduced into an amino acid site selected from the group consisting of positions 295, 326, and 330 (EU numbering) in the loop region of the Fc region.

[40] The method according to any one of

[27] to

[32] and

[39] , wherein the amino acid modification in the loop region of the Fc region is at least one amino acid modification selected from the group consisting of substitution with Met or Leu at position 295 (EU numbering), substitution with Ser or Ala at position 326 (EU numbering), and substitution with His or Tyr at position 330 (EU numbering).

[41] The method of any one of

[27] to

[31] and

[33] , wherein at least one amino acid mutation is introduced into an amino acid site selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 268 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering) in the loop region of the Fc region.

[42] The method of any one of

[27] to

[31] ,

[33] , and

[41] , wherein the amino acid modification in the loop site of the Fc region is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys or Ser, substitution of the amino acid at position 268 (EU numbering) with Lys or His, substitution of the amino acid at position 270 (EU numbering) with Phe or Asp, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

[43] The method of any one of

[27] to

[31] and

[33] , wherein at least one amino acid mutation is introduced into an amino acid site selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 239 (EU numbering), position 268 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering) in the loop region of the Fc region.

[44] The method of any one of

[27] to

[31] ,

[33] , and

[43] , wherein the amino acid modification in the loop site of the Fc region is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys, substitution of the amino acid at position 268 (EU numbering) with Lys, substitution of the amino acid at position 270 (EU numbering) with Phe, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

[45] The method according to any one of

[27] to

[31] and

[33] , wherein at least one amino acid mutation is introduced into an amino acid site selected from the group consisting of positions 234 (EU numbering), 235 (EU numbering), 239 (EU numbering), 295 (EU numbering), 296 (EU numbering), 298 (EU numbering), and 325 (EU numbering) in the loop region of the Fc region.

[46] The method of any one of

[27] to

[31] ,

[33] , and

[45] , wherein the amino acid modification in the loop site of the Fc region is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

[47] The method of any one of

[27] to

[31] and

[33] , wherein at least one amino acid mutation is introduced into an amino acid site selected from the group consisting of position 234 (EU numbering), position 235 (EU numbering), position 268 (EU numbering), position 270 (EU numbering), position 295 (EU numbering), position 296 (EU numbering), position 298 (EU numbering), and position 325 (EU numbering) in the loop region of the Fc region.

[48] ​​The method of any one of

[27] to

[31] ,

[33] , and

[47] , wherein the amino acid modification in the loop site of the Fc region is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 268 (EU numbering) with Lys, substitution of the amino acid at position 270 (EU numbering) with Phe, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

[49] The method of any one of

[27] to

[31] and

[33] , wherein at least one amino acid mutation is introduced into an amino acid site selected from the group consisting of positions 234 (EU numbering), 235 (EU numbering), 295 (EU numbering), 296 (EU numbering), 298 (EU numbering), and 325 (EU numbering) in the loop region of the Fc region.

[50] The method of any one of

[27] to

[31] ,

[33] , and

[49] , wherein the amino acid modification in the loop region of the Fc region is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

[51] The method according to any one of

[27] to

[32] , wherein at least one amino acid mutation is introduced into an amino acid site selected from the group consisting of: position 247 (EU numbering), position 250 (EU numbering), position 307 (EU numbering), position 309 (EU numbering), position 315 (EU numbering), position 360 (EU numbering), position 385 (EU numbering), position 386 (EU numbering), position 387 (EU numbering), position 389 (EU numbering), position 428 (EU numbering), and position 433 (EU numbering) in the loop region of the Fc region.

[52] the amino acid modification in the loop region of the Fc region includes substitution of the amino acid at position 247 (EU numbering) with Val, substitution of the amino acid at position 250 (EU numbering) with any of Phe, Ile, Met, Val, Trp, or Tyr, substitution of the amino acid at position 307 (EU numbering) with any of Ala, Gln, or Pro, substitution of the amino acid at position 309 (EU numbering) with any of Ala, Arg, or Pro, substitution of the amino acid at position 315 (EU numbering) with Ala, or substitution of the amino acid at position 360 (EU numbering) with His; The method according to any one of

[27] to

[32] and

[51] , wherein the amino acid modification is at least one selected from the group consisting of substitution of the amino acid at position 385 (EU numbering) with Asp, substitution of the amino acid at position 386 (EU numbering) with Pro, substitution of the amino acid at position 387 (EU numbering) with Glu, substitution of the amino acid at position 389 (EU numbering) with Ser, substitution of the amino acid at position 428 (EU numbering) with any of His, Trp, Tyr, or Phe, and substitution of the amino acid at position 433 (EU numbering) with Lys.

[53] The method according to any one of

[27] to

[31] and

[33] , wherein at least one amino acid mutation is introduced into the loop region of the Fc region at amino acid position 298 (EU numbering) or amino acid position 309 (EU numbering).

[54] The method according to any one of

[27] to

[31] ,

[33] , and

[53] , wherein the amino acid modification in the loop region of the Fc region is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 298 (EU numbering) with Gly and substitution of the amino acid at position 309 (EU numbering) with Asp.

[55] The method according to any one of

[27] to

[54] , wherein the modification is in a polypeptide having an Fc region of human IgG.

[56] A nucleic acid encoding a polypeptide having an antibody Fc region, in which at least one amino acid in a loop region of the Fc region has been modified, thereby improving stability compared to the parent polypeptide.

[57] A vector comprising the nucleic acid according to

[56] .

[58] A host cell transformed with the vector according to

[57] .

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

[26] , or a polypeptide produced by the method according to any one of

[27] to

[55] .

[60] A therapeutic or preventive agent for immune inflammatory diseases or cancer, comprising the pharmaceutical composition according to

[59] .

[61] Immune inflammatory diseases include rheumatoid arthritis, autoimmune hepatitis, autoimmune thyroiditis, autoimmune bullous disease, autoimmune adrenocorticitis, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, megalocytic anemia, autoimmune atrophic gastritis, autoimmune neutropenia, autoimmune orchitis, autoimmune encephalomyelitis, autoimmune receptor disease, autoimmune infertility, chronic active hepatitis, glomerulonephritis, interstitial pulmonary fibrosis, multiple sclerosis, Pajut's disease, osteoporosis, multiple myeloma, uveitis, acute and chronic spondylitis, gouty arthritis, inflammatory bowel disease, adult respiratory distress syndrome (ARDS), psoriasis, Crohn's disease, Graves' disease, juvenile diabetes mellitus, Addison's disease, myasthenia gravis, uveitis lenticulata, systemic lupus erythematosus, allergic rhinitis, allergic The treatment or prevention agent according to

[60] , wherein the disease is selected from the group consisting of dermatitis, ulcerative colitis, hypersensitivity, asthma, muscle degeneration, cachexia, systemic sclerosis, localized scleroderma, Sjogren's syndrome, Behcet's disease, Reiter's syndrome, type I and type II diabetes mellitus, bone resorption diseases, graft vs. host reaction, ischemia-reperfusion injury, atherosclerosis, brain trauma, multiple sclerosis, cerebral malaria, sepsis, septic shock, toxic shock syndrome, fever, and malgias due to staining, aplastic anemia, hemolytic anemia, idiopathic thrombocytopenia, Goodpasture's syndrome, Guillain-Barré syndrome, Hashimoto's disease, pemphigus, IgA nephropathy, hay fever, antiphospholipid syndrome, polymyositis, Wegener's sarcoma, arteritis nodosa, mixed connective tissue disease, and fibromyalgia.

[62] The therapeutic or preventive agent according to

[60] , wherein the cancer is pancreatic cancer, prostate cancer, breast cancer, skin cancer, gastrointestinal cancer, lung cancer, hepatocellular carcinoma, cervical cancer, uterine cancer, ovarian cancer, fallopian tube cancer, vaginal cancer, liver cancer, bile duct cancer, bladder cancer, ureter cancer, thyroid cancer, adrenal cancer, kidney cancer, other glandular tissue cancer, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, synovial sarcoma, angiosarcoma, fibrosarcoma, malignant peripheral nerve tumor, gastrointestinal stromal tumor, desmoid tumor, Ewing's sarcoma, osteosarcoma, chondrosarcoma, leukemia, lymphoma, myeloma, or other solid organ tumor.

[63] A method for treating or preventing an immune inflammatory disease or cancer, comprising the step of administering to a subject the polypeptide according to any one of [1] to

[26] , or a polypeptide produced by the method according to any one of

[27] to

[55] .

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

[26] or a polypeptide produced by the method according to any one of

[27] to

[55] , for use in the treatment or prevention of an immunoinflammatory disease or cancer.

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

[26] or the polypeptide produced by the method according to any one of

[27] to

[55] in the manufacture of an agent for the treatment or prevention of an immunoinflammatory disease or cancer.

[66] A method for producing a therapeutic or preventive agent for an immunoinflammatory disease or cancer, comprising a step of using the polypeptide according to any one of [1] to

[26] , or a polypeptide produced by the method according to any one of

[27] to

[55] . [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows the thermal denaturation midpoints (Tm) of the antibodies prepared. [Figure 2-1] FIG. 1 shows chromatograms measuring the aggregate content of TS20 to TS27, which are combinations of multiple modifications. [Figure 2-2] FIG. 1 shows chromatograms measuring the aggregate content of TS28 to TS39, which are combinations of multiple modifications. [Figure 2-3] FIG. 1 shows chromatograms measuring the aggregate content of TS40 to TS43, which are combinations of multiple modifications. [Diagram 3] The relationship between the amino acid residues constituting the Fc region of IgG1, IgG2, IgG3, and IgG4 and the EU numbering of Kabat (herein also referred to as EU INDEX) is shown. [Figure 4] FIG. 1 shows the amino acid modifications (L234-S239, D265-P271, Q295, Y296, S298, Y300, S324-S337; shaded areas) in the loop portion of the CH2 domain of Fc region B3 (sequence number: 16). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention provides polypeptides having an antibody Fc region whose stability is improved as compared to that of a parent polypeptide by introducing amino acid substitutions into the loop region of the Fc region. The present invention further provides a method for improving the stability of a polypeptide having an antibody Fc domain, as compared to a parent polypeptide, by introducing amino acid substitutions into the loop region of the antibody Fc domain.The present invention further provides a method for producing a polypeptide having an antibody Fc domain, the stability of which is improved, as compared to a parent polypeptide, by introducing amino acid substitutions into the loop region of the antibody Fc domain.

[0013] The polypeptide in the present invention generally refers to a peptide or protein having a length of about 10 amino acids or more. Generally, the polypeptide is derived from an organism, but is not particularly limited, and may be, for example, a polypeptide consisting of an artificially designed sequence. The polypeptide may be a natural polypeptide, a synthetic polypeptide, a recombinant polypeptide, or the like. The polypeptide may be an antibody. A preferred example of the polypeptide of the present invention is human IgG. When human IgG is used as an antibody, the type of its constant region is not limited, and human IgG of isotypes (subclasses) such as IgG1, IgG2, IgG3, and IgG4 can be used.

[0014] As used herein, the term "parent polypeptide" refers to a polypeptide that serves as a basis or standard for the preparation of a polypeptide having an Fc region of an antibody of the present invention. That is, it may be a polypeptide having an Fc region of an antibody before at least one amino acid in the Fc region is modified. The parent polypeptide of the present invention may be, for example, a polypeptide having an Fc region of a native IgG, or a polypeptide having an Fc region of an IgG in which a modification other than the amino acid modification of the present invention has been added to a native IgG.

[0015] Native IgG refers to a polypeptide that includes the same amino acid sequence as IgG found in nature and belongs to the class of antibodies substantially encoded by immunoglobulin gamma genes. For example, native human IgG refers to native human IgG1, native human IgG2, native human IgG3, native human IgG4, etc. Native IgG also includes naturally occurring mutants thereof. The Fc region of native IgG refers to an Fc region that includes the same amino acid sequence as an Fc region originating from naturally occurring IgG. The Fc region of native IgG is shown in FIG. 3 (SEQ ID NOs: 11 to 14), and refers to, for example, an Fc region originating from native human IgG1, an Fc region originating from native human IgG2, an Fc region originating from native human IgG3, or an Fc region originating from native human IgG4. The Fc region of native IgG also includes naturally occurring mutants thereof.

[0016] The amino acid positions of the present invention are defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991)). In this specification, amino acid modification sites in the Fc region of an antibody are represented according to EU numbering based on the Kabat amino acid positions.

[0017] In the present invention, a portion connecting α-helices and β-sheets is called a loop, and there is no restriction on its length or structure. In the present invention, a portion connecting β-sheets in the CH2 domain is called a loop, loop site, loop portion, or loop structure. Specific amino acid positions of loop sites subjected to modification in the present invention are 234th EU numbering, 235th EU numbering, 236th EU numbering, 237th EU numbering, 238th EU numbering, 239th EU numbering, 247th EU numbering, 250th EU numbering, 265th EU numbering, 266th EU numbering, 267th EU numbering, 268th EU numbering, 269th EU numbering, 270th EU numbering, 271st EU numbering, 295th EU numbering, 296th EU numbering, 298th EU numbering, 299th EU numbering, 300th EU numbering, 301st EU numbering, 302nd EU numbering, 303rd EU numbering, 304th EU numbering, 305th EU numbering, 306th EU numbering, 307th EU numbering, 308th EU numbering, 309th EU numbering, 310th EU numbering, 311th EU numbering, 312th EU numbering, 313th EU numbering, 314th EU numbering, 315th EU numbering, 316th EU numbering, 317th EU numbering, 318th EU numbering, 319th EU numbering, 320th EU numbering, 321st EU numbering, 322nd EU numbering, 323rd EU numbering, 324th EU numbering, 325th EU numbering, 326th EU numbering, 327th EU numbering, 32 numbering, 300th EU numbering, 307th EU numbering, 309th EU numbering, 315th EU numbering, 324th EU numbering, 325th EU numbering, 326th EU numbering, 327th EU numbering, 329th EU numbering, 330th EU numbering, 333rd EU numbering, 335th EU numbering, 337th EU numbering, 360th EU numbering, 385th EU numbering, 386th EU numbering, 387th EU numbering, 389th EU numbering, 428th EU numbering and 433rd EU numbering.

[0018] The term "Fc region" refers to a region in an antibody molecule that includes a fragment consisting of the hinge region or a part thereof, and the CH2 and CH3 domains. The Fc region of an IgG class refers to, for example, but is not limited to, the region from cysteine ​​at position 226 to the C-terminus, or from proline at position 230 to the C-terminus in Kabat EU numbering (also referred to as EU INDEX herein) (see FIG. 3). The Fc region can be suitably obtained by partially digesting IgG1, IgG2, IgG3, IgG4 monoclonal antibodies, etc. with a protease such as pepsin, and then re-eluting the fraction adsorbed to a Protein A column. There are no particular limitations on such a protease, so long as it can digest full-length antibodies so as to produce Fab or F(ab')2 in a limited manner by appropriately setting the enzyme reaction conditions, such as pH, and examples of such an enzyme include pepsin and papain.

[0019] The polypeptides of the present invention include polypeptides having an Fc region with improved stability compared to a parent polypeptide. A preferred embodiment of the polypeptide having an Fc region with improved stability compared to a parent polypeptide is, for example, a polypeptide in which at least one amino acid is modified in the loop region of the Fc region.

[0020] The present invention relates to a loop region of an Fc region of an antibody (e.g., human IgG (IgG1, IgG2, IgG3, IgG4)), and to a position at EU numbering 234, EU numbering 235, EU numbering 236, EU numbering 237, EU numbering 238, EU numbering 239, EU numbering 247, EU numbering 250, EU numbering 265, EU numbering 266, EU numbering 267, EU numbering 268, EU numbering 269, EU numbering 270, EU numbering 271, EU numbering 295, EU numbering 296, EU numbering 298, EU numbering 300, EU numbering 311, EU numbering 312, EU numbering 313, EU numbering 314, EU numbering 315, EU numbering 316, EU numbering 317, EU numbering 318, EU numbering 319, EU numbering 320, EU numbering 321, EU numbering 322, EU numbering 323, EU numbering 324, EU numbering 325, EU numbering 326, EU numbering 327, EU numbering 328, EU numbering 329, EU numbering 330, EU numbering 331, EU numbering 332, EU numbering 333, EU numbering 334, EU numbering 335, EU numbering 336, EU numbering 337, EU numbering 338, EU numbering 339, EU numbering 347, EU numbering 350, EU numbering 351, EU numbering 352, EU numbering 353, EU numbering 354, EU numbering 355, EU numbering 356, EU numbering 357, EU numbering 358, The present invention provides an antibody Fc region comprising an Fc region in which at least one or more amino acid modifications have been introduced at an amino acid site selected from the group consisting of: 307 (EU numbering), 309 (EU numbering), 315 (EU numbering), 324 (EU numbering), 325 (EU numbering), 326 (EU numbering), 327 (EU numbering), 329 (EU numbering), 330 (EU numbering), 333 (EU numbering), 335 (EU numbering), 337 (EU numbering), 360 (EU numbering), 385 (EU numbering), 386 (EU numbering), 387 (EU numbering), 389 (EU numbering), 428 (EU numbering), and 433 (EU numbering). For example, by introducing the above modifications into human IgG, it is possible to provide a polypeptide with improved stability compared to the parent polypeptide.

[0021] Furthermore, the present invention provides antibody Fc regions comprising an Fc region in which at least one amino acid mutation has been introduced into a loop region of the Fc region of an antibody (e.g., human IgG (IgG1, IgG2, IgG3, IgG4)) at an amino acid site selected from the group consisting of EU numbering 234, EU numbering 235, EU numbering 239, EU numbering 266, EU numbering 267, EU numbering 268, EU numbering 269, EU numbering 270, EU numbering 295, EU numbering 296, EU numbering 298, EU numbering 300, EU numbering 324, EU numbering 325, EU numbering 326, and EU numbering 330 of the IgG.

[0022] As used herein, "stability" refers to, for example, the thermodynamic stability of a polypeptide, but is not limited thereto. The thermodynamic stability of a polypeptide can be evaluated or judged, for example, using the thermal denaturation midpoint (Tm) of the CH2 region as an indicator. That is, it is preferable to evaluate or judge the polypeptide of the present invention using the thermal denaturation midpoint (Tm) as an indicator. Tm can be measured by CD (circular dichroism), DSC (differential scanning calorimetry), or DSF (differential scanning fluorometry).

[0023] The above-mentioned method used for evaluating thermal stability makes it possible to independently evaluate the thermal stability of the three regions, CH2, CH3, and Fab, when a sample in the form of IgG is subjected to measurement. Since CH2 has a lower thermal stability than CH2 and CH3 of the Fc region, it is believed that improving the thermal stability of CH2 will lead to improving the thermal stability of the Fc region.

[0024] In addition, IgG maintains a highly controlled three-dimensional structure, and the three-dimensional structure and physical stability of each region affect each other. In other words, a modification introduced into one region may affect other regions, changing the three-dimensional structure and physical stability of the entire IgG. For this reason, when evaluating the effect of introducing a modification, it is desirable to perform the evaluation in the form of IgG. For these reasons, in this specification, the thermal stability of the CH2 region of a modified antibody produced in the form of IgG was evaluated.

[0025] CD calculates Tm by observing the change in mean residue molar ellipticity (θ) with increasing temperature. An example of a CD measuring device is a circular dichroism spectrometer (JASCO). When the CD spectrum is measured at an appropriate wavelength (e.g., 208 nm or 222 nm) while increasing the temperature, θ increases at a certain temperature and remains constant at subsequent temperatures. The temperature at which the midpoint between θ at low temperature and θ at high temperature is taken as Tm. For measurement, a protein solution prepared with, for example, citric acid, Tris, or phosphoric acid solution can be used, and can be used at a concentration of several hundred μg / mL.

[0026] DSC calculates Tm by observing the change in heat quantity with increasing temperature. Examples of DSC measuring instruments include MicroCal VP-DSC and Micro Cal Capillary DSC (both from GE Healthcare). When a protein solution and a buffer solution are sealed in a measuring cell and the temperature difference between the cells is measured while increasing the temperature, the reaction changes to an endothermic reaction at a certain temperature. The temperature at this point is called Tm. For example, protein solutions prepared with citrate buffer, TBS, PBS, histidine buffer, etc. can be used for the measurement, and can be used at concentrations from tens of μg / mL to hundreds of μg / mL.

[0027] DSF uses a fluorescent reagent (e.g., SYPRO Orange) that specifically binds to hydrophobic residues, and calculates Tm by observing the exposure of hydrophobic residues as the temperature increases. When a protein solution and a fluorescent reagent are mixed in an appropriate ratio and the fluorescence intensity is measured while increasing the temperature using an RT-PCR device, an increase in the fluorescence intensity is observed at a certain temperature. The temperature at this point is called Tm. Examples of equipment for measuring DSF include Rotor-Gene Q (QIAGEN) and CFX96 Real-Time PCR Analysis System (Bio-Rad). For example, a protein solution prepared with PBS or histidine buffer can be used for the measurement, and a concentration of several tens to several hundreds of μg / mL can be used for the measurement.

[0028] As used herein, improved stability of a polypeptide means that the Tm of the CH2 region in the Fc region of a test polypeptide is improved by 0.1 degrees or more, preferably 0.2 degrees or more, 0.3 degrees or more, 0.4 degrees or more, 0.5 degrees or more, 1 degree or more, 2 degrees or more, 3 degrees or more, 4 degrees or more, 5 degrees or more, 10 degrees or more, or 20 degrees or more, compared to the Tm of the CH2 region in the Fc region of a parent polypeptide serving as a control, determined based on the above-mentioned measurement method.

[0029] Furthermore, the polypeptide of the present invention may be a polypeptide having an Fc region that not only has improved stability compared to a parent polypeptide but also maintains or enhances binding activity to an Fcγ receptor (sometimes referred to as Fcγ receptor or FcγR herein) compared to a parent polypeptide. Examples of polypeptides of the present invention having an Fc region that not only has improved stability compared to a parent polypeptide but also maintains or enhances binding activity to FcγR compared to a parent polypeptide include polypeptides having an amino acid site described in any of TS1-TS8, TS20-TS27, TS44-TS50, TS52-TS55, and TS57-TS67 shown in the Examples below.

[0030] (TS1-TS8) Examples of polypeptides that maintain or enhance FcγR-binding activity compared to a parent polypeptide include polypeptides in which at least one amino acid mutation has been introduced into an amino acid site selected from the group consisting of EU numbering 234, EU numbering 266, EU numbering 268, EU numbering 269, EU numbering 270, EU numbering 295, EU numbering 300, EU numbering 324, EU numbering 326, and EU numbering 330 in the loop region of the Fc region.

[0031] Preferred polypeptides in this embodiment include polypeptides in which the amino acid modification site in the loop region of the Fc region is at EU numbering position 234, EU numbering position 266, EU numbering position 268, EU numbering position 269, EU numbering position 270, EU numbering position 295, EU numbering position 300, EU numbering position 324, EU numbering position 326, and EU numbering position 330.

[0032] Further preferred polypeptides in this embodiment include polypeptides in which the amino acid modification in the loop region of the Fc region is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Ile, substitution of the amino acid at position 266 (EU numbering) with Ile, substitution of the amino acid at position 268 (EU numbering) with Gln, substitution of the amino acid at position 269 (EU numbering) with Asp, substitution of the amino acid at position 270 (EU numbering) with Glu, substitution of the amino acid at position 295 (EU numbering) with Met or Leu, substitution of the amino acid at position 300 (EU numbering) with Glu, substitution of the amino acid at position 324 (EU numbering) with His, substitution of the amino acid at position 326 (EU numbering) with Ser or Ala, and substitution of the amino acid at position 330 (EU numbering) with His or Tyr.

[0033] Further preferred polypeptides in this embodiment are those in which the amino acid modification in the loop region of the Fc region is substitution of the amino acid at EU numbering position 234 with Ile, substitution of the amino acid at EU numbering position 266 with Ile, substitution of the amino acid at EU numbering position 268 with Gln, substitution of the amino acid at EU numbering position 269 with Asp, substitution of the amino acid at EU numbering position 270 with Glu, substitution of the amino acid at EU numbering position 295 with Met or Leu, substitution of the amino acid at EU numbering position 300 with Glu, substitution of the amino acid at EU numbering position 324 with His, substitution of the amino acid at EU numbering position 326 with Ser or Ala, and substitution of the amino acid at EU numbering position 330 with His or Tyr.

[0034] (TS20-TS27) Examples of polypeptides that maintain or enhance FcγR-binding activity compared to a parent polypeptide include polypeptides in which at least one amino acid mutation has been introduced into an amino acid site selected from the group consisting of positions 295, 326, and 330 (EU numbering) in the loop region of the Fc region.

[0035] In this embodiment, preferred polypeptides are those in which the amino acid modification sites in the loop region of the Fc region are at positions 295, 326, and 330 (EU numbering).

[0036] Further preferred polypeptides in this embodiment include polypeptides in which the amino acid modification in the loop region of the Fc region is at least one amino acid modification selected from the group consisting of substitution of amino acid position 295 (EU numbering) with Met or Leu, substitution of amino acid position 326 (EU numbering) with Ser or Ala, and substitution of amino acid position 330 (EU numbering) with His or Tyr.

[0037] A further preferred polypeptide in this embodiment is a polypeptide in which the amino acid modification in the loop region of the Fc region is a substitution of amino acid position 295 (EU numbering) with Met or Leu, amino acid position 326 (EU numbering) with Ser or Ala, and amino acid position 330 (EU numbering) with His or Tyr.

[0038] (TS44~TS50, TS52~TS55, TS57~TS67) Examples of polypeptides that maintain or enhance FcγR-binding activity compared to a parent polypeptide include polypeptides in which at least one amino acid mutation has been introduced into an amino acid site selected from the group consisting of EU numbering position 247, EU numbering position 250, EU numbering position 307, EU numbering position 309, EU numbering position 315, EU numbering position 360, EU numbering position 385, EU numbering position 386, EU numbering position 387, EU numbering position 389, EU numbering position 428, and EU numbering position 433 in the loop region of the Fc region.

[0039] In this embodiment, preferred polypeptides are those in which the amino acid modification site in the loop region of the Fc region is at EU numbering position 247, EU numbering position 250, EU numbering position 307, EU numbering position 309, EU numbering position 315, EU numbering position 360, EU numbering position 385, EU numbering position 386, EU numbering position 387, EU numbering position 389, EU numbering position 428, or EU numbering position 433.

[0040] In further preferred polypeptides in this embodiment, the amino acid modification in the loop region of the Fc region is a substitution of the amino acid at position 247 (EU numbering) with Val, a substitution of the amino acid at position 250 (EU numbering) with any of Phe, Ile, Met, Val, Trp, or Tyr, a substitution of the amino acid at position 307 (EU numbering) with any of Ala, Gln, or Pro, a substitution of the amino acid at position 309 (EU numbering) with any of Ala, Arg, or Pro, a substitution of the amino acid at position 315 (EU numbering) with Ala, or a substitution of the amino acid at position 320 (EU numbering) with any of Ala, Arg, or Pro. and polypeptides having at least one or more amino acid modifications selected from the group consisting of substitution of the amino acid at position 360 (EU numbering) with His, substitution of the amino acid at position 385 (EU numbering) with Asp, substitution of the amino acid at position 386 (EU numbering) with Pro, substitution of the amino acid at position 387 (EU numbering) with Glu, substitution of the amino acid at position 389 (EU numbering) with Ser, substitution of the amino acid at position 428 (EU numbering) with either His, Trp, Tyr, or Phe, and substitution of the amino acid at position 433 (EU numbering) with Lys.

[0041] Further preferred polypeptides in this embodiment are those in which the amino acid modifications in the loop site of the Fc region are substitution of the amino acid at EU numbering position 247 with Val, substitution of the amino acid at EU numbering position 250 with any of Phe, Ile, Met, Val, Trp, or Tyr, substitution of the amino acid at EU numbering position 307 with any of Ala, Gln, or Pro, substitution of the amino acid at EU numbering position 309 with any of Ala, Arg, or Pro, substitution of the amino acid at EU numbering position 315 with Ala, substitution of the amino acid at EU numbering position 360 with His, substitution of the amino acid at EU numbering position 385 with Asp, substitution of the amino acid at EU numbering position 386 with Pro, substitution of the amino acid at EU numbering position 387 with Glu, substitution of the amino acid at EU numbering position 389 with Ser, substitution of the amino acid at EU numbering position 428 with any of His, Trp, Tyr, or Phe, and substitution of the amino acid at EU numbering position 433 with Lys.

[0042] For example, when the polypeptide is an antibody, the antibody is preferably used as a cancer antibody, in which effector function is important.

[0043] Furthermore, the polypeptide of the present invention may be a polypeptide having an Fc region that not only has improved stability compared to a parent polypeptide but also has reduced Fcγ receptor-binding activity compared to the parent polypeptide. Examples of polypeptides of the present invention that have an Fc region that not only has improved stability compared to a parent polypeptide but also has reduced FcγR-binding activity compared to the parent polypeptide include polypeptides having amino acid alterations in TS9-TS19, TS28-TS43, TS51, or TS56 shown in the Examples below.

[0044] (TS9-TS19) Examples of polypeptides that have reduced binding activity to FcγR compared to a parent polypeptide include polypeptides in which at least one amino acid mutation has been introduced into an amino acid site selected from the group consisting of EU numbering 234, EU numbering 235, EU numbering 239, EU numbering 267, EU numbering 268, EU numbering 270, EU numbering 295, EU numbering 296, EU numbering 298, and EU numbering 325 in the loop region of the Fc region.

[0045] Preferred polypeptides in this embodiment are those in which the amino acid modification sites in the loop region of the Fc region are at position 234 (EU numbering), 235 (EU numbering), 239 (EU numbering), 267 (EU numbering), 268 (EU numbering), 270 (EU numbering), 295 (EU numbering), 296 (EU numbering), 298 (EU numbering), and 325 (EU numbering).

[0046] Further preferred polypeptides in this embodiment include polypeptides in which the amino acid modification in the loop region of the Fc region is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys or Arg, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys, substitution of the amino acid at position 267 (EU numbering) with Pro, substitution of the amino acid at position 268 (EU numbering) with Met or Lys, substitution of the amino acid at position 270 (EU numbering) with Phe, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with any of Gly, His, or Met.

[0047] Further preferred polypeptides in this embodiment are those in which the amino acid modification in the loop site of the Fc region is substitution of the amino acid at position 234 (EU numbering) with Lys or Arg, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys, substitution of the amino acid at position 267 (EU numbering) with Pro, substitution of the amino acid at position 268 (EU numbering) with Met or Lys, substitution of the amino acid at position 270 (EU numbering) with Phe, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, or substitution of the amino acid at position 325 (EU numbering) with any of Gly, His, or Met.

[0048] (TS28-TS34) Examples of polypeptides whose binding activity to FcγR is reduced compared to that of a parent polypeptide include polypeptides in which at least one amino acid mutation has been introduced into an amino acid site selected from the group consisting of EU numbering 234, EU numbering 235, EU numbering 239, EU numbering 268, EU numbering 270, EU numbering 295, EU numbering 296, EU numbering 298, and EU numbering 325 in the loop region of the Fc region.

[0049] In this embodiment, preferred polypeptides are those in which the amino acid modification sites in the loop region of the Fc region are at EU numbering position 234, EU numbering position 235, EU numbering position 239, EU numbering position 268, EU numbering position 270, EU numbering position 295, EU numbering position 296, EU numbering position 298, and EU numbering position 325.

[0050] Further preferred polypeptides in this embodiment include polypeptides in which the amino acid modification in the loop region of the Fc region is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys or Ser, substitution of the amino acid at position 268 (EU numbering) with Lys or His, substitution of the amino acid at position 270 (EU numbering) with Phe or Asp, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

[0051] Further preferred polypeptides in this embodiment are those in which the amino acid modification in the loop region of the Fc region is substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys or Ser, substitution of the amino acid at position 268 (EU numbering) with Lys or His, substitution of the amino acid at position 270 (EU numbering) with Phe or Asp, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

[0052] (TS28, TS29, TS36, TS37) Examples of polypeptides whose binding activity to FcγR is reduced compared to that of a parent polypeptide include polypeptides in which at least one amino acid mutation has been introduced into an amino acid site selected from the group consisting of EU numbering 234, EU numbering 235, EU numbering 239, EU numbering 268, EU numbering 270, EU numbering 295, EU numbering 296, EU numbering 298, and EU numbering 325 in the loop region of the Fc region.

[0053] Preferred polypeptides in this embodiment are those in which the amino acid modification sites in the loop region of the Fc region are at EU numbering position 234, EU numbering position 235, EU numbering position 239, EU numbering position 268, EU numbering position 270, EU numbering position 295, EU numbering position 296, EU numbering position 298, and EU numbering position 325.

[0054] Further preferred polypeptides in this embodiment include polypeptides in which the amino acid modification in the loop region of the Fc region is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys, substitution of the amino acid at position 268 (EU numbering) with Lys, substitution of the amino acid at position 270 (EU numbering) with Phe, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

[0055] Further preferred polypeptides in this embodiment are those in which the amino acid modification in the loop region of the Fc region is substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys, substitution of the amino acid at position 268 (EU numbering) with Lys, substitution of the amino acid at position 270 (EU numbering) with Phe, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

[0056] (TS30, TS31, TS38, TS39) Examples of polypeptides whose binding activity to FcγR is reduced compared to that of a parent polypeptide include polypeptides in which at least one amino acid mutation has been introduced into an amino acid site selected from the group consisting of EU numbering 234, EU numbering 235, EU numbering 239, EU numbering 295, EU numbering 296, EU numbering 298, and EU numbering 325 in the loop region of the Fc region.

[0057] Preferred polypeptides in this embodiment are those in which the amino acid modification sites in the loop region of the Fc region are at EU numbering position 234, EU numbering position 235, EU numbering position 239, EU numbering position 295, EU numbering position 296, EU numbering position 298, and EU numbering position 325.

[0058] Further preferred polypeptides in this embodiment include polypeptides in which the amino acid modification in the loop region of the Fc region is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

[0059] Further preferred polypeptides in this embodiment are those in which the amino acid modification in the loop region of the Fc region is substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 239 (EU numbering) with Lys, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

[0060] (TS32, TS33, TS40, TS41) Examples of polypeptides whose binding activity to FcγR is reduced compared to that of a parent polypeptide include polypeptides in which at least one amino acid mutation has been introduced into an amino acid site selected from the group consisting of EU numbering 234, EU numbering 235, EU numbering 268, EU numbering 270, EU numbering 295, EU numbering 296, EU numbering 298, and EU numbering 325 in the loop region of the Fc region.

[0061] Preferred polypeptides in this embodiment are those in which the amino acid modification sites in the loop region of the Fc region are at EU numbering position 234, EU numbering position 235, EU numbering position 268, EU numbering position 270, EU numbering position 295, EU numbering position 296, EU numbering position 298, and EU numbering position 325.

[0062] Further preferred polypeptides in this embodiment include polypeptides in which the amino acid modification in the loop region of the Fc region is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 268 (EU numbering) with Lys, substitution of the amino acid at position 270 (EU numbering) with Phe, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

[0063] Further preferred polypeptides in this embodiment are those in which the amino acid modification in the loop region of the Fc region is substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 268 (EU numbering) with Lys, substitution of the amino acid at position 270 (EU numbering) with Phe, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

[0064] (TS34, TS35, TS42, TS43) Examples of polypeptides whose binding activity to FcγR is reduced compared to that of a parent polypeptide include polypeptides in which at least one amino acid mutation has been introduced into an amino acid site selected from the group consisting of positions 234 (EU numbering), 235 (EU numbering), 295 (EU numbering), 296 (EU numbering), 298 (EU numbering), and 325 (EU numbering) in the loop region of the Fc region.

[0065] Preferred polypeptides in this embodiment are those in which the amino acid modification sites in the loop region of the Fc region are at EU numbering position 234, EU numbering position 235, EU numbering position 295, EU numbering position 296, EU numbering position 298, and EU numbering position 325.

[0066] Further preferred polypeptides in this embodiment include polypeptides in which the amino acid modification in the loop region of the Fc region is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

[0067] Further preferred polypeptides in this embodiment are those in which the amino acid modification in the loop region of the Fc region is substitution of the amino acid at position 234 (EU numbering) with Lys, substitution of the amino acid at position 235 (EU numbering) with Lys or Arg, substitution of the amino acid at position 295 (EU numbering) with Met, substitution of the amino acid at position 296 (EU numbering) with Gly, substitution of the amino acid at position 298 (EU numbering) with Gly, and substitution of the amino acid at position 325 (EU numbering) with His or Gly.

[0068] (TS51, TS56) An example of a polypeptide whose binding activity to FcγR is reduced compared to a parent polypeptide is a polypeptide in which at least one amino acid mutation has been introduced into the amino acid positions 298 and 309 (EU numbering) in the loop region of the Fc region.

[0069] Preferred polypeptides in this embodiment include polypeptides in which the amino acid modification in the loop region of the Fc region is at position 298 or 309 (EU numbering).

[0070] Further preferred polypeptides in this embodiment include polypeptides in which the amino acid modification in the loop region of the Fc region is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 298 (EU numbering) with Gly and substitution of the amino acid at position 309 (EU numbering) with Asp.

[0071] Further preferred polypeptides in this embodiment include polypeptides in which the amino acid modification in the loop region of the Fc region is a substitution of the amino acid at position 298 (EU numbering) with Gly and a substitution of the amino acid at position 309 (EU numbering) with Asp.

[0072] For example, when the polypeptide is an antibody, the antibody is preferably used as a neutralizing antibody.

[0073] The term "Fcγ receptor" refers to a receptor capable of binding to the Fc region of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody, and means any member of a family of proteins substantially encoded by the Fcγ receptor gene. In humans, this family includes FcγRI (CD64) including isoforms FcγRIa, FcγRIb and FcγRIc; FcγRII (CD32) including isoforms FcγRIIa (including allotypes H131 (H type) and R131 (R type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) and FcγRIIc; and FcγRIII (CD16) including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any undiscovered human FcγRs or FcγR isoforms or allotypes, but are not limited to these. The FcγR of the present invention includes, but is not limited to, those derived from humans as described above, as well as those derived from mice, rats, rabbits and monkeys, and may be derived from any organism. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16) and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRs or FcγR isoforms or allotypes. Suitable examples of Fcγ receptors in the present invention include human FcγI (CD64), FcγIIA (CD32), FcγIIB (CD32), FcγIIIA (CD16) and / or FcγIIIB (CD16).

[0074] The polynucleotide and amino acid sequences of FcγI are set forth in SEQ ID NOs: 1 (NM_000566.3) and 2 (NP_000557.1), respectively. The polynucleotide and amino acid sequences of FcγIIA are set forth in SEQ ID NOs: 3 (BC020823.1) and 4 (AAH20823.1), respectively. The polynucleotide and amino acid sequences of FcγIIB are set forth in SEQ ID NOs: 5 (BC146678.1) and 6 (AAI46679.1), respectively. The polynucleotide and amino acid sequences of FcγIIIA are set forth in SEQ ID NOs: 7 (BC033678.1) and 8 (AAH33678.1), respectively. The polynucleotide sequence and amino acid sequence of FcγIIIB are set forth in SEQ ID NOs: 9 (BC128562.1) and 10 (AAI28563.1), respectively (RefSeq accession numbers are indicated in parentheses). Furthermore, there are two types of genetic polymorphisms in FcγRIIa, in which the 131st amino acid of FcγRIIa is substituted with histidine (H type) or arginine (R type) (J. Exp. Med, 172, 19-25, 1990).

[0075] In the present invention, whether the binding activity of the polypeptide or Fc region of the present invention to various FcγRs has decreased or whether the binding activity has been maintained or enhanced can be determined, for example, as shown in the present Example. That is, using BIACORE, an interaction analysis device utilizing the surface plasmon resonance (SPR) phenomenon, it is possible to determine whether the value of the dissociation constant (KD) obtained by flowing various FcγRs as analytes through a sensor chip on which a polypeptide (antibody) is immobilized or captured with Protein A, an antigen peptide, or the like has decreased or increased, or whether the amount of change in the sensorgram value before and after flowing various FcγRs as analytes through a sensor chip on which a polypeptide (antibody) is immobilized or captured with Protein A, an antigen peptide, or the like has decreased or increased.

[0076] Specifically, the binding activity of the Fc region to the Fcγ receptor can be measured by ELISA, FACS (fluorescence activated cell sorting), ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), BIACORE method utilizing the surface plasmon resonance (SPR) phenomenon, etc. (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0077] The ALPHA screen is carried out by ALPHA technology using two beads, donor and acceptor, based on the following principle: A luminescence signal is detected only when a molecule bound to a donor bead biologically interacts with a molecule bound to an acceptor bead and the two beads are in close proximity. A photosensitizer in the donor bead excited by a laser converts surrounding oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and, when it reaches a nearby acceptor bead, triggers a chemiluminescence reaction in the bead, which ultimately emits light. If there is no interaction between the molecules bound to the donor bead and the acceptor bead, the singlet oxygen produced by the donor bead does not reach the acceptor bead, and no chemiluminescence reaction occurs.

[0078] For example, a biotin-labeled polypeptide complex binds to donor beads, and an Fcγ receptor tagged with glutathione S-transferase (GST) binds to acceptor beads. In the absence of a competing polypeptide complex having a mutant Fc region, a polypeptide complex having a wild-type Fc region interacts with the Fcγ receptor and generates a signal at 520-620 nm. A polypeptide complex having an untagged mutant Fc region competes with the interaction between a polypeptide complex having a wild-type Fc region and the Fcγ receptor. Relative binding affinity can be determined by quantitating the decrease in fluorescence that occurs as a result of competition.

[0079] It is known that polypeptide complexes such as antibodies can be biotinylated using Sulfo-NHS-biotin or the like. Methods for tagging Fcγ receptors with GST include expressing a fusion gene in which a polynucleotide encoding an Fcγ receptor is fused in frame with a polynucleotide encoding GST in a cell or the like carrying an expression vector, and purifying the gene using a glutathione column. The obtained signals are suitably analyzed, for example, by fitting them to a one-site competition model using nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).

[0080] One of the substances (ligand) for which interaction is to be observed is fixed on the gold thin film of the sensor chip, and light is applied from the back of the sensor chip so that it is totally reflected at the interface between the gold thin film and the glass. When the other substance (analyte) for which interaction is to be observed is poured onto the surface of the sensor chip and the ligand and analyte bind, the mass of the immobilized ligand molecule increases, and the refractive index of the solvent on the sensor chip surface changes. This change in refractive index shifts the position of the SPR signal (conversely, when the bond is dissociated, the position of the signal returns). The Biacore system takes the amount of shift mentioned above, that is, the change in mass on the sensor chip surface, as the vertical axis, and displays the change in mass over time as measurement data (sensorgram). The amount of binding of the analyte to the ligand captured on the sensor chip surface can be obtained from the sensorgram. In addition, the kinetics: binding rate constant (ka) and dissociation rate constant (kd) can be obtained from the curve of the sensorgram, and the dissociation constant (KD) can be obtained from the ratio of these constants. Inhibition measurement methods are also preferably used in the BIACORE method. An example of an inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

[0081] In the present invention, a polypeptide with reduced binding activity to FcγR (a polypeptide with reduced binding activity to FcγR) refers to a polypeptide that binds to at least one type of FcγR with essentially weaker binding affinity than the parent polypeptide when assayed using essentially the same amounts of the parent polypeptide and a polypeptide (also referred to as a polypeptide mutant) that contains at least one amino acid modification in the Fc region of the parent polypeptide.

[0082] For example, in the binding amount measured by the above-mentioned measurement method, when the binding amount of the parent polypeptide to each FcγR is taken as 100, the binding amount of the polypeptide mutant to each FcγR (hereinafter referred to as binding amount ratio) is preferably reduced to 80 or less, preferably 50 or less, 40 or less, 30 or less, or 20 or less, and particularly preferably 10 or less, 5 or less, 4 or less, 3 or less, 2 or less, 1 or less, or 0.1 or less.

[0083] In the present invention, a polypeptide with enhanced binding activity to FcγR (a polypeptide with enhanced binding activity to FcγR) refers to a polypeptide that binds to at least one type of FcγR with substantially stronger binding affinity than the parent polypeptide when assayed using essentially the same amount of the parent polypeptide and the polypeptide mutant. For example, in terms of the amount of binding measured by the above-mentioned measurement method, the amount of binding of the polypeptide mutant to each FcγR (hereinafter referred to as binding amount ratio) is preferably improved to 120 or more, 150 or more, 200 or more, or 300 or more when the binding amount of the parent polypeptide to each FcγR is set to 100.

[0084] A polypeptide that maintains (has maintained) binding activity to FcγR (a polypeptide that maintains (has maintained) binding activity to Fcγr) refers to a polypeptide that binds to FcγR with essentially the same binding affinity as the parent polypeptide when assayed using essentially the same amounts of the parent polypeptide and a polypeptide (also called a polypeptide variant) that contains at least one amino acid modification in the Fc region of the parent polypeptide. For example, in terms of the amount of binding measured by the above-mentioned measurement method, the amount of binding of the polypeptide mutant to each FcγR (hereinafter referred to as binding amount ratio) is preferably 80 or more and 120 or less, assuming that the binding amount of the parent polypeptide to each FcγR is 100.

[0085] The present invention makes it possible to further modify the Fc region of a polypeptide of the present invention (for example, human IgG) that includes the above-mentioned modifications. For example, another modification to the Fc region can be exemplified by substituting Pro at position 238 (EU numbering) with Asp and / or substituting Leu at position 328 (EU numbering) with Glu in human IgG (IgG1, IgG2, IgG3, IgG4). By introducing the modification of substituting Pro at position 238 (EU numbering) with Asp and / or substituting Leu at position 328 (EU numbering) with Glu into human IgG, it is possible to provide a polypeptide that maintains or decreases the binding activity to any of the R-type and H-type genetic polymorphisms of FcγRI, FcγRIIIa, and FcγRIIa, and has enhanced binding activity to FcγRIIb, as compared to the parent polypeptide.

[0086] It is possible to add another modification to the Fc region of human IgG containing the modification of substituting Pro at position 238 (EU numbering) with Asp and / or substituting Leu at position 328 (EU numbering) with Glu. Here, the modification means any of amino acid substitution, deletion, addition, and insertion, or a combination thereof. In addition to these modifications, it may also include an additional modification. The additional modification may be selected from, for example, any of amino acid substitution, deletion, and modification, or a combination thereof. For example, it is also possible to add a modification that further reduces the binding activity to any of FcγRI, FcγRIIa (H type), FcγRIIa (R type), and FcγRIIIa.

[0087] Among these, preferred are modifications that reduce only the binding activity to any one of FcγRI, FcγRIIa (H type), FcγRIIa (R type), and FcγRIIIa, without reducing the binding activity to FcγRIIb. Preferred amino acid substitutions for such modifications include, for example, substitution of Gly at position 237 (EU numbering) with Trp, substitution of Gly at position 237 (EU numbering) with Phe, substitution of Pro at position 238 (EU numbering) with Phe, substitution of Asn at position 325 (EU numbering) with Met, substitution of Ser at position 267 (EU numbering) with Met, and substitution of Ser at position 267 (EU numbering) with Met. substitution of Ile, substitution of Leu at position 328 (EU numbering) by Asp, substitution of Ser at position 267 (EU numbering) by Val, substitution of Leu at position 328 (EU numbering) by Trp, substitution of Ser at position 267 (EU numbering) by Gln, substitution of Ser at position 267 (EU numbering) by Met, substitution of Gly at position 236 (EU numbering) by Asp, substitution of Ala at position 327 (EU numbering) by Asn, substitution of Asn at position 325 (EU numbering) by Ser, substitution of Leu at position 235 (EU numbering) by Tyr, substitution of Val at position 266 of the ring is substituted by Met, Leu at position 328 (EU numbering) is substituted by Tyr, Leu at position 235 (EU numbering) is substituted by Trp, Leu at position 235 (EU numbering) is substituted by Phe, Ser at position 239 (EU numbering) is substituted by Gly, Ala at position 327 (EU numbering) is substituted by Glu, Ala at position 327 (EU numbering) is substituted by Gly, Pro at position 238 (EU numbering) is substituted by Leu, Ser at position 239 (EU numbering) is substituted by Leu, Leu at position 328 (EU numbering) at position 327 (EU numbering) to Thr; replacement of Leu at position 328 (EU numbering) to Ser; replacement of Leu at position 328 (EU numbering) to Met; replacement of Pro at position 331 (EU numbering) to Trp; replacement of Pro at position 331 (EU numbering) to Tyr; replacement of Pro at position 331 (EU numbering) to Phe; replacement of Ala at position 327 (EU numbering) to Asp; replacement of Leu at position 328 (EU numbering) to Phe; replacement of Pro at position 271 (EU numbering) to Leu; replacement of Ser at position 267 (EU numbering) to Glu;Leu at position 328 (EU numbering) by Ala, Leu at position 328 (EU numbering) by Ile, Leu at position 328 (EU numbering) by Gln, Leu at position 328 (EU numbering) by Val, Lys at position 326 (EU numbering) by Trp, Lys at position 334 (EU numbering) by Arg, His at position 268 (EU numbering) by Gly, His at position 268 (EU numbering) by Asn, Ser at position 324 (EU numbering) by Val, Val substitution by Leu, Pro substitution by Gly at position 271 (EU numbering), Ile substitution by Phe at position 332 (EU numbering), Ser substitution by Ile at position 324 (EU numbering), Glu substitution by Pro at position 333 (EU numbering), Tyr substitution by Asp at position 300 (EU numbering), Ser substitution by Asp at position 337 (EU numbering), Tyr substitution by Gln at position 300 (EU numbering), Thr substitution by Asp at position 335 (EU numbering), Ser substitution by Asn at position 239 (EU numbering), E Substitution of Lys at position 326 (EU numbering) by Leu, substitution of Lys at position 326 (EU numbering) by Ile, substitution of Ser at position 239 (EU numbering) by Glu, substitution of Lys at position 326 (EU numbering) by Phe, substitution of Lys at position 326 (EU numbering) by Val, substitution of Lys at position 326 (EU numbering) by Tyr, substitution of Ser at position 267 (EU numbering) by Asp, substitution of Lys at position 326 (EU numbering) by Pro, substitution of Lys at position 326 (EU numbering) by His, substitution of Lys at position 334 (EU numbering) substitution of Lys at position 334 (EU numbering) by Ala, substitution of Lys at position 334 (EU numbering) by Trp, substitution of His at position 268 (EU numbering) by Gln, substitution of Lys at position 326 (EU numbering) by Gln, substitution of Lys at position 326 (EU numbering) by Glu, substitution of Lys at position 326 (EU numbering) by Met, substitution of Val at position 266 (EU numbering) by Ile, substitution of Lys at position 334 (EU numbering) by Glu, substitution of Tyr at position 300 (EU numbering) by Glu, substitution of Lys at position 334 (EU numbering) by Met,Substitution of Lys at position 334 (EU numbering) by Val, substitution of Lys at position 334 (EU numbering) by Thr, substitution of Lys at position 334 (EU numbering) by Ser, substitution of Lys at position 334 (EU numbering) by His, substitution of Lys at position 334 (EU numbering) by Phe, substitution of Lys at position 334 (EU numbering) by Gln, substitution of Lys at position 334 (EU numbering) by Pro, substitution of Ly at position 334 (EU numbering) Examples of such substitutions include substitution of Lys at position 334 (EU numbering) with Tyr, substitution of Lys at position 334 (EU numbering) with Ile, substitution of Gln at position 295 (EU numbering) with Leu, substitution of Lys at position 334 (EU numbering) with Leu, substitution of Lys at position 334 (EU numbering) with Asn, substitution of His at position 268 (EU numbering) with Ala, substitution of Ser at position 239 (EU numbering) with Asp, and substitution of Ser at position 267 (EU numbering) with Ala.

[0088] Furthermore, among these modifications, modifications that reduce the binding activity to FcγRIIa (R type) without reducing the binding activity to FcγRIIb are preferred. Preferred amino acid substitutions as such modifications include, for example, substitution of Gly at position 237 (EU numbering) with Trp, substitution of Leu at position 328 (EU numbering) with Asp, substitution of Gly at position 236 (EU numbering) with Asp, substitution of Ala at position 327 (EU numbering) with Asn, and substitution of Ala at position 327 (EU numbering) with Gly. substitution of Ser at position 239 (EU numbering) with Leu; substitution of Pro at position 331 (EU numbering) with Trp; substitution of Pro at position 331 (EU numbering) with Tyr; substitution of Pro at position 331 (EU numbering) with Phe; substitution of Pro at position 271 (EU numbering) with Leu; substitution of Leu at position 328 (EU numbering) with Gln; substitution of Tyr at position 300 (EU numbering) with Asp; and substitution of Ser at position 239 (EU numbering) with Asn.

[0089] Furthermore, it is also possible to add a modification to the Fc region of human IgG containing a substitution of Pro at position 238 (EU numbering) with Asp and / or a substitution of Leu at position 328 (EU numbering) with Glu to enhance the binding activity to FcγRIIb. Among these modifications, modifications that enhance only the binding activity to FcγRIIb without enhancing the binding activity to any of FcγRI, FcγRIIa (H type), FcγRIIa (R type), and FcγRIIIa are preferred, and preferred amino acid substitutions for such modifications include, for example, substitution of Gly at position 237 (EU numbering) with Trp, substitution of Gly at position 237 (EU numbering) with Phe, substitution of Pro at position 238 ... Asn at position 325 replaced by Met, Ser at position 267 (EU numbering) replaced by Ile, Leu at position 328 (EU numbering) replaced by Asp, Ser at position 267 (EU numbering) replaced by Val, Leu at position 328 (EU numbering) replaced by Trp, Ser at position 267 (EU numbering) replaced by Gln, Ser at position 267 (EU numbering) replaced by Met, Gly at position 236 (EU numbering) replaced by Asp, Ala to Asn, Asn at position 325 (EU numbering) to Ser, Leu at position 235 (EU numbering) to Tyr, Val at position 266 (EU numbering) to Met, Leu at position 328 (EU numbering) to Tyr, Leu at position 235 (EU numbering) to Trp, Leu at position 235 (EU numbering) to Phe, Ser at position 239 (EU numbering) to Gly, Ala at position 327 (EU numbering) to Gl substitution of u at position 327 (EU numbering), substitution of Ala at position 327 (EU numbering) by Gly, substitution of Pro at position 238 (EU numbering) by Leu, substitution of Ser at position 239 (EU numbering) by Leu, substitution of Leu at position 328 (EU numbering) by Thr, substitution of Leu at position 328 (EU numbering) by Ser, substitution of Leu at position 328 (EU numbering) by Met, substitution of Pro at position 331 (EU numbering) by Trp, substitution of Pro at position 331 (EU numbering) by Tyr,Pro at position 331 (EU numbering) replaced by Phe, Ala at position 327 (EU numbering) replaced by Asp, Leu at position 328 (EU numbering) replaced by Phe, Pro at position 271 (EU numbering) replaced by Leu, Ser at position 267 (EU numbering) replaced by Glu, Leu at position 328 (EU numbering) replaced by Ala, Leu at position 328 (EU numbering) replaced by Ile, Leu at position 328 (EU numbering) replaced by Gln, Leu at position 328 (EU numbering) replaced by Val, Lys at position 334 (EU numbering) replaced by Trp, Lys at position 334 (EU numbering) replaced by Arg, His at position 268 (EU numbering) replaced by Gly, His at position 268 (EU numbering) replaced by Asn, Ser at position 324 (EU numbering) replaced by Val, Val at position 266 (EU numbering) replaced by Leu, Pro at position 271 (EU numbering) replaced by Gly, Ile at position 332 (EU numbering) replaced by Phe, Ser at position 324 (EU numbering) replaced by Ile, Glu at position 333 (EU numbering) replaced by Pro, Tyr at position 300 (EU numbering) by Asp, Ser at position 337 (EU numbering) by Asp, Tyr at position 300 (EU numbering) by Gln, Thr at position 335 (EU numbering) by Asp, Ser at position 239 (EU numbering) by Asn, Lys at position 326 (EU numbering) by Leu, Lys at position 326 (EU numbering) by Ile, Ser at position 239 (EU numbering) by Glu, Lys at position 326 (EU numbering) by Phe, substitution of Lys at position 326 (EU numbering) by Val, substitution of Lys at position 326 (EU numbering) by Tyr, substitution of Ser at position 267 (EU numbering) by Asp, substitution of Lys at position 326 (EU numbering) by Pro, substitution of Lys at position 326 (EU numbering) by His, substitution of Lys at position 334 (EU numbering) by Ala, substitution of Lys at position 334 (EU numbering) by Trp, substitution of His at position 268 (EU numbering) by Gln, substitution of Lys at position 326 (EU numbering) by Gln, substitution of Lys at position 326 (EU numbering) by Glu,Lys at position 326 (EU numbering) replaced by Met, Val at position 266 (EU numbering) replaced by Ile, Lys at position 334 (EU numbering) replaced by Glu, Tyr at position 300 (EU numbering) replaced by Glu, Lys at position 334 (EU numbering) replaced by Met, Lys at position 334 (EU numbering) replaced by Val, Lys at position 334 (EU numbering) replaced by Thr, Lys at position 334 (EU numbering) replaced by Ser, Lys at position 334 (EU numbering) replaced by His, Lys at position 334 (EU numbering) replaced by Phe, Examples of such substitutions include substitution of Lys at position 334 (EU numbering) with Gln, substitution of Lys at position 334 (EU numbering) with Pro, substitution of Lys at position 334 (EU numbering) with Tyr, substitution of Lys at position 334 (EU numbering) with Ile, substitution of Gln at position 295 (EU numbering) with Leu, substitution of Lys at position 334 (EU numbering) with Leu, substitution of Lys at position 334 (EU numbering) with Asn, substitution of His at position 268 (EU numbering) with Ala, substitution of Ser at position 239 (EU numbering) with Asp, and substitution of Ser at position 267 (EU numbering) with Ala.

[0090] Furthermore, among these modifications, modifications that reduce the binding activity to FcγRIIa (R type) without reducing the binding activity to FcγRIIb are preferred. Preferred amino acid substitutions for such modifications include, for example, substitution of Gly at position 237 (EU numbering) with Trp, substitution of Leu at position 328 (EU numbering) with Asp, substitution of Gly at position 236 (EU numbering) with Asp, substitution of Ala at position 327 (EU numbering) with Asn, substitution of Ala at position 327 (EU numbering) with Gl, and substitution of Ala at position 327 (EU numbering) with Gl. These include substitution of y at position 239 (EU numbering) with Leu, substitution of Pro at position 331 (EU numbering) with Trp, substitution of Pro at position 331 (EU numbering) with Tyr, substitution of Pro at position 331 (EU numbering) with Phe, substitution of Pro at position 271 (EU numbering) with Leu, substitution of Leu at position 328 (EU numbering) with Gln, substitution of Tyr at position 300 (EU numbering) with Asp, and substitution of Ser at position 239 (EU numbering) with Asn.

[0091] Furthermore, amino acid substitutions may be made in the Fc region of the antibody to improve, for example, the binding activity to FcRn (J Immunol. 2006 Jan 1;176(1):346-56, J Biol Chem. 2006 Aug 18;281(33):23514-24, Int Immunol. 2006 Dec;18(12):1759-69, Nat Biotechnol. 2010 Feb;28(2):157-9, WO / 2006 / 019447, WO / 2006 / 053301, WO / 2009 / 086320), or to improve the heterogeneity or stability of the antibody (WO / 2009 / 041613).

[0092] Furthermore, the following modifications can be appropriately made to the amino acid modifications of the present invention. In order to control the plasma retention of an antibody, the amino acid modification of the present invention can be combined with an amino acid modification for modifying the isoelectric point (pI value) of the antibody. As for the modification of the constant region, for example, the modification of amino acids at EU numbering positions 250 and 428 described in known documents (J. Immunol. 2006, 176(1):346-356 and Nat. Biotechnol. 1997 15(7):637-640, etc.) can be mentioned. In addition, as the modification of the variable region, the modification of amino acids described in WO2007 / 114319 and WO2009 / 041643 can be mentioned. The amino acid to be modified is preferably an amino acid exposed on the surface of a polypeptide having antigen-binding activity. For example, when the polypeptide of the present invention has a heavy chain constant region, the substitution of the amino acid at EU numbering position 196 in the amino acid sequence of the heavy chain constant region can be mentioned. When the heavy chain constant region is IgG4, for example, the pI value can be lowered by substituting lysine at position 196 with glutamine, thereby increasing plasma retention. Plasma retention can also be controlled by modifying the binding ability to FcRn. Examples of amino acid modifications for modifying the binding ability to FcRn include amino acid substitutions in the antibody heavy chain constant region described in known literature (The Journal of Biological Chemistry vol.276, No.9 6591-6604, 2001 and Molecular Cell, Vol.7, 867-877, 2001).

[0093] Modifications for improved stability under acidic conditions For example, when the polypeptide of the present invention has an IgG4 heavy chain constant region, it is preferable to suppress the formation of half molecules of IgG4 under acidic conditions and maintain a stable four-chain structure (H2L2 structure). Therefore, it is preferable to replace arginine (Immunology 2002, 105, 9-19), which is the amino acid at position 409 of the EU numbering system and plays an important role in maintaining the four-chain structure, with lysine of the IgG1 type, which maintains a stable four-chain structure even under acidic conditions. Such modifications can be used in combination with the amino acid modifications of the present invention.

[0094] Modifications to reduce heterogeneity It is possible to combine the amino acid modification of the present invention with the method described in WO2009 / 041613. Specifically, for example, when the polypeptide of the present invention has an IgG1 heavy chain constant region, it is possible to reduce heterogeneity by combining a modification that deletes two amino acids at the C-terminus of the IgG1 heavy chain constant region, i.e., glycine at position 446 and lysine at position 447 (EU numbering), with an amino acid modification based on the Examples of the present invention.

[0095] Modifications to suppress deamidation The amino acid modification of the present invention can be combined with an amino acid modification for suppressing a deamidation reaction. It has been reported that deamidation is particularly likely to occur at a site (···NG···) where asparagine (N) and glycine (G) are adjacent to each other (Geiger et al. J. Bio. Chem. 1987; 262:785-794). When a site where asparagine and glycine are adjacent exists in the polypeptide of the present invention, the deamidation reaction can be suppressed by modifying the amino acid sequence. Specifically, for example, either one or both of the amino acid sequences of asparagine and glycine are substituted with other amino acids. More specifically, for example, asparagine is substituted with aspartic acid.

[0096] A preferred example of the polypeptide of the present invention is an IgG antibody. When an IgG antibody is used as the antibody, the type of its Fc region is not limited, and IgG isotypes (subclasses) such as IgG1, IgG2, IgG3, and IgG4 can be used. The IgG antibody of the present invention is preferably human IgG, more preferably human IgG1 or human IgG4, and the amino acid sequences of the heavy chain Fc regions of human IgG1 and human IgG4 are known. As for the human IgG1 Fc region, a number of allotype sequences due to genetic polymorphism are described in Sequences of proteins of immunological interest, NIH Publication No. 91-3242, and any of these may be used in the present invention.

[0097] <replacement> For example, an amino acid residue can be substituted with another amino acid residue for the purpose of modifying the following aspects (a) to (c): (a) the backbone structure of the polypeptide in the sheet or helix regions; (b) the charge or hydrophobicity at the target site, or (c) Side chain size.

[0098] Amino acid residues are classified into the following groups based on common side chain properties: (1) Hydrophobic: norleucine, met, ala, val, leu, ile; (2) Neutral hydrophilic: cys, ser, thr, asn, gln; (3) Acidic: asp, glu; (4) Basic: his, lys, arg; (5) Residues that affect chain orientation: gly, pro; and (6) Aromaticity: trp, tyr, phe.

[0099] Substitutions of amino acid residues within each of these groups are called conservative substitutions, while substitutions of amino acid residues between other groups are called non-conservative substitutions. Substitutions in the present invention may be conservative substitutions, non-conservative substitutions, or a combination of conservative and non-conservative substitutions.

[0100] Amino acid sequence modifications are prepared by a variety of methods known in the art. These methods include, but are not limited to, site-directed mutagenesis (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis. Gene 152, 271-275; Zoller, MJ, and Smith, M.(1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors.Methods Enzymol. 100, 468-500; Kramer, W, Drutsa, V, Jansen, HW, Kramer, B, Pflugfelder, M, and Fritz, HJ(1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456; Kramer W, and Fritz Mutations can be performed by methods such as PCR mutagenesis and cassette mutagenesis, etc. (HJ (1987) Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154, 350-367; Kunkel, TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci US A. 82, 488-492), PCR mutagenesis, and cassette mutagenesis.

[0101] The amino acid modification of the present invention includes post-translational modification. Specific examples of post-translational modification include the addition or deletion of a sugar chain. For example, in the IgG1 Fc region consisting of the amino acid sequence set forth in SEQ ID NO: 11, the 297th amino acid residue in EU numbering can be modified with a sugar chain. The sugar chain structure to be modified is not limited. In general, antibodies expressed in eukaryotic cells include sugar chain modifications in the Fc region. Therefore, antibodies expressed in the following cells are usually modified with some kind of sugar chain. Mammalian antibody-producing cells A eukaryotic cell transformed with an expression vector containing DNA encoding the antibody.

[0102] The eukaryotic cells shown here include yeast and animal cells. For example, CHO cells and HEK293H cells are representative animal cells for transformation with an expression vector containing DNA encoding an antibody. On the other hand, the Fc region of the present invention also includes those that are not glycosylated at the relevant position. Antibodies whose Fc region is not glycosylated can be obtained by expressing a gene encoding the antibody in a prokaryotic cell such as E. coli. More specifically, for example, sialic acid may be added to the sugar chain of the Fc region (MAbs. 2010 Sep-Oct;2(5):519-27.).

[0103] <Antibody> Furthermore, the present invention provides antibodies having an Fc region in which any of the above-mentioned amino acid sequences has been altered.

[0104] The term "antibody" in the present invention is used in the broadest sense and includes any antibody, such as a monoclonal antibody (including a full-length monoclonal antibody), a polyclonal antibody, an antibody mutant, an antibody fragment, a multispecific antibody (e.g., a bispecific antibody (sometimes called a dual-specific antibody)), a chimeric antibody, or a humanized antibody, so long as it exhibits the desired biological activity. The antibody of the present invention is not limited by the type of antigen, the origin of the antibody, etc., and may be any antibody. The origin of the antibody is not particularly limited, but examples thereof include human antibody, mouse antibody, rat antibody, rabbit antibody, etc.

[0105] Methods for producing antibodies are well known to those skilled in the art. For example, monoclonal antibodies may be produced by the hybridoma method (Kohler and Milstein, Nature 256:495 (1975)) or recombinant method (U.S. Patent No. 4,816,567). Alternatively, they may be isolated from a phage antibody library (Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1991)). In the present invention, monoclonal antibodies include humanized antibodies and chimeric antibodies. As a method for obtaining antibody genes, in addition to the above, techniques for B cell cloning (identification and cloning of the coding sequence of each antibody, isolation thereof, and use for constructing expression vectors for producing each antibody (particularly IgG1, IgG2, IgG3, or IgG4), etc.) as described in Bernasconi et al. (Science (2002) 298, 2199-2202) or WO2008 / 081008 can be used as appropriate.

[0106] Furthermore, the antibodies of the present invention may have modified sugar chains. Examples of antibodies with modified sugar chains include antibodies with modified glycosylation (WO99 / 54342, etc.), antibodies lacking fucose added to the sugar chain (WO00 / 61739, WO02 / 31140, WO2006 / 067847, WO2006 / 067913, etc.), and antibodies with sugar chains containing bisecting GlcNAc (WO02 / 79255, etc.).

[0107] A humanized antibody is also called a reshaped human antibody. Specifically, humanized antibodies in which the CDR of an antibody from an animal other than human, for example, a mouse antibody, is grafted onto a human antibody are known. General gene recombination techniques for obtaining humanized antibodies are also known. Specifically, overlap extension PCR is known as a method for grafting the CDR of a mouse antibody onto a human FR. Furthermore, it is also possible to design amino acids in the variable region in which mouse CDRs are grafted onto the human FR region, and synthesize genes for those amino acids. Examples of companies that undertake gene synthesis include Life Technologies and GenScript.

[0108] A vector for expressing a humanized antibody can be prepared by inserting a DNA encoding an antibody variable region in which three CDRs and four FRs are linked with a DNA encoding a human antibody Fc region into an expression vector so that they are fused in frame. After introducing the integration vector into a host to establish a recombinant cell, the recombinant cell is cultured to express the DNA encoding the humanized antibody, and the humanized antibody is produced in the culture of the cultured cell (see European Patent Publication EP 239400 and International Publication WO1996 / 002576).

[0109] If necessary, amino acid residues in the FR can be substituted so that the CDRs of the reshaped human antibody form an appropriate antigen-binding site. For example, amino acid sequence mutations can be introduced into the FR by applying the PCR method used for grafting mouse CDRs to human FRs.

[0110] Transgenic animals carrying the entire repertoire of human antibody genes (see International Publications WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, WO1996 / 033735) are used as immunized animals, and desired human antibodies can be obtained by DNA immunization.

[0111] Furthermore, a technique for obtaining a human antibody by panning using a human antibody library is also known. For example, the V region of a human antibody is expressed on the surface of a phage as a single chain antibody (scFv) by phage display. A phage expressing an scFv that binds to an antigen can be selected. By analyzing the gene of the selected phage, the DNA sequence encoding the V region of a human antibody that binds to an antigen can be determined. After determining the DNA sequence of the scFv that binds to an antigen, the V region sequence can be fused in frame with the sequence of the C region of a desired human antibody and then inserted into an appropriate expression vector to prepare an expression vector. The expression vector is introduced into a suitable expression cell as listed above, and the gene encoding the human antibody is expressed to obtain the human antibody. These methods are already known (see International Publications WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, and WO1995 / 015388).

[0112] In the present specification, the antigen is not particularly limited and may be any antigen. Examples of the antigen include ligands (cytokines, chemokines, etc.), receptors, cancer antigens, MHC antigens, differentiation antigens, immunoglobulins, and immune complexes that contain immunoglobulins as a part.

[0113] Examples of cytokines include interleukins 1 to 18, colony-stimulating factors (G-CSF, M-CSF, GM-CSF, etc.), interferons (IFN-α, IFN-β, IFN-γ, etc.), growth factors (EGF, FGF, IGF, NGF, PDGF, TGF, HGF, etc.), tumor necrosis factors (TNF-α, TNF-β), lymphotoxin, erythropoietin, leptin, SCF, TPO, MCAF, and BMP.

[0114] Examples of chemokines include CC chemokines such as CCL1 to CCL28, CXC chemokines such as CXCL1 to CXCL17, C chemokines such as XCL1 to XCL2, and CX3C chemokines such as CX3CL1.

[0115] Examples of receptors include receptors belonging to receptor families such as the hematopoietic factor receptor family, cytokine receptor family, tyrosine kinase receptor family, serine / threonine kinase receptor family, TNF receptor family, G protein-coupled receptor family, GPI-anchored receptor family, tyrosine phosphatase receptor family, adhesion factor family, and hormone receptor family. The receptors belonging to these receptor families and their characteristics are described in many publications, for example, Cooke BA., King RJB., van der Molen HJ. ed. New Comprehesive Biochemistry Vol.18B "Hormones and their Actions Part II" pp.1-46 (1988) Elsevier Science Publishers BV., Patthy (Cell (1990) 61 (1), 13-14), Ullrich et al. (Cell (1990) 61 (2), 203-212), Massague (e has an acute accent mark) (Cell (1992) 69 (6), 1067-1070), Miyajima et al. (Annu. Rev. Immunol. (1992) 10, 295-331), Taga et al. (FASEB J. (1992) 6, (Annu. Rev. Biochem. (1993), 62, 453-481), Smith et al. (Cell (1994) 76 (6) 959-962), Flower DR. Biochim. Biophys. Acta, Flower (Biochim. Biophys. Acta (1999) 1422 (3) 207-234, etc.

[0116] Specific examples of receptors belonging to the above receptor families include human or mouse erythropoietin (EPO) receptors (Blood (1990) 76 (1), 31-35; Cell (1989) 57 (2), 277-285), human or mouse granulocyte colony-stimulating factor (G-CSF) receptors (Proc. Natl. Acad. Sci. USA. (1990) 87 (22), 8702-8706; mG-CSFR, Cell (1990) 61 (2), 341-350), human or mouse thrombopoietin (TPO) receptors (Proc Natl Acad Sci USA. (1992) 89 (12), 5640-5644; EMBO J. (1993) 12(7), 2645-53), human or mouse insulin receptors (Nature (1985) 313 (6005), 756-761), human or mouse Flt-3 ligand receptor (Proc. Natl. Acad. Sci. USA. (1994) 91 (2), 459-463), human or mouse platelet-derived growth factor (PDGF) receptor (Proc. Natl. Acad. Sci. USA. (1988) 85 (10) 3435-3439), human or mouse interferon (IFN)-α, β receptor (Cell (1990) 60 (2), 225-234. and Cell (1994) 77 (3), Suitable examples of such an antibody include human or mouse leptin receptor, human or mouse growth hormone (GH) receptor, human or mouse interleukin (IL)-10 receptor, human or mouse insulin-like growth factor (IGF)-I receptor, human or mouse leukemia inhibitory factor (LIF) receptor, and human or mouse ciliary neurotrophic factor (CNTF) receptor.

[0117] Cancer antigens are antigens that are expressed in association with the malignant transformation of cells, and are also called tumor-specific antigens. In addition, abnormal sugar chains that appear on the cell surface or protein molecules when cells become cancerous are also cancer antigens, and are also called cancer sugar chain antigens. Examples of cancer antigens include, for example, GPC3 (Int J Cancer. (2003) 103 (4), 455-65), which belongs to the GPI-anchored receptor family as the above-mentioned receptor and is expressed in several cancers including liver cancer, EpCAM (Proc Natl Acad Sci US A. (1989) 86 (1), 27-31), CA19-9, CA15-3, cereal SSEA-1 (SLX), and the like, which are expressed in several cancers including lung cancer.

[0118] MHC antigens are mainly classified into MHC class I antigens and MHC class II antigens. MHC class I antigens include HLA-A, -B, -C, -E, -F, -G, and -H, while MHC class II antigens include HLA-DR, -DQ, and -DP.

[0119] Differentiation antigens include CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15s, CD16, CD18, CD19, CD20, CD21, CD23, CD25, CD28, CD29, CD30, CD32, CD33, CD34, CD35, CD38, CD40, CD41a, CD41b, CD42a, CD42b, These may include CD43, CD44, CD45, CD45RO, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD51, CD54, CD55, CD56, CD57, CD58, CD61, CD62E, CD62L, CD62P, CD64, CD69, CD71, CD73, CD95, CD102, CD106, CD122, CD126, and CDw130.

[0120] Immunoglobulins include IgA, IgM, IgD, IgG, and IgE, and immune complexes contain at least one of the immunoglobulin components.

[0121] The variable region constituting the antibody of the present invention can be a variable region that recognizes any antigen. The amino acid sequence constituting the antibody variable region is permissible to be modified in one or more amino acid residues as long as the antigen-binding activity is maintained. When modifying the amino acid sequence of the variable region, the site to be modified and the number of amino acids to be modified are not particularly limited. For example, amino acids present in CDR and / or FR can be modified as appropriate. When modifying amino acids in the variable region, although not particularly limited, it is preferable that the binding activity is maintained, for example, it is preferable that the binding activity is 50% or more, preferably 80% or more, and more preferably 100% or more compared to before modification. Furthermore, the binding activity may be increased by amino acid modification, for example, the binding activity may be 2-fold, 5-fold, 10-fold or more compared to before modification.

[0122] As the value of the antigen-binding activity of the antibody of the present invention, when the antigen is a soluble antigen, the KD (dissociation constant) can be used, whereas when the antigen is a membrane antigen, the apparent KD (apparent dissociation constant) can be used. The KD (dissociation constant) and the apparent KD (apparent dissociation constant) can be measured by methods known to those skilled in the art, for example, Biacore (GE Healthcare), Scatchard plot, flow cytometer, etc. can be used.

[0123] In addition, other indices for comparing the antigen-binding activity of the antibodies of the present invention include, for example, k d (dissociation rate constant) can be used, and when the antigen is a membrane antigen, the apparent k d It is possible to use the apparent dissociation rate constant k d (dissociation rate constant), and apparent k d The (apparent dissociation rate constant) can be measured by a method known to those skilled in the art, for example, using Biacore (GE healthcare), a flow cytometer, or the like.

[0124] In the antibody of the present invention, the modification of the amino acid sequence can be at least one of substitution, addition, deletion, and modification of amino acid residues. The site to be modified and the number of amino acids to be modified are not particularly limited, and generally, the modification is within 50 amino acids, preferably within 30 amino acids, and more preferably within 10 amino acids (e.g., within 5 amino acids, within 3 amino acids). Alternatively, modification of, for example, 20% or less, specifically 10% or less (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% or less) of the amino acid residues in the entire amino acid sequence is permitted. In other words, the antibody of the present invention also includes an antibody containing an amino acid sequence having a homology (identity) of preferably 80% or more, more preferably 90% or more (e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more) with the amino acid sequence before modification.

[0125] For example, modification of the N-terminus of a variable region from glutamine to pyroglutamic acid by pyroglutamylation is a modification well known to those skilled in the art. Thus, the antibody of the present invention also includes a variable region in which the N-terminus of the heavy chain is modified to pyroglutamic acid when the N-terminus is glutamine.

[0126] The variable region of the antibody of the present invention may have any sequence, and may be the variable region of an antibody of any origin, such as a mouse antibody, a rat antibody, a rabbit antibody, a goat antibody, a camel antibody, a humanized antibody obtained by humanizing these non-human antibodies, and a human antibody. A "humanized antibody", also called a reshaped human antibody, is an antibody derived from a mammal other than a human, for example, an antibody in which the complementarity determining region (CDR) of a mouse antibody is grafted onto the CDR of a human antibody. Methods for identifying CDRs are known (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al., Nature (1989) 342: 877). General gene recombination techniques are also known (see European Patent Application Publication No. EP 125023 and WO 96 / 02576). Furthermore, various amino acid substitutions may be introduced into the variable regions of these antibodies in order to improve antigen binding, pharmacokinetics, stability, and antigenicity. The variable regions of the antibodies of the present invention may have pH-dependency in antigen binding, and thus may be capable of repeatedly binding to antigens (WO / 2009 / 125825).

[0127] The light chain constant region of an antibody may be either of the κ-chain and λ-chain types. Furthermore, the light chain constant region of the present invention may be a light chain constant region that has been modified by amino acid substitution, deletion, addition, and / or insertion.

[0128] The heavy chain Fc region of the antibody of the present invention can be, for example, the heavy chain Fc region of a human IgG antibody, preferably the heavy chain Fc region of a human IgG1 antibody or a human IgG4 antibody.

[0129] Furthermore, the polypeptides of the present invention can be bound to other proteins, physiologically active peptides, etc. to form Fc fusion protein molecules. Examples of other proteins and biologically active peptides include, but are not limited to, receptors, adhesion molecules, ligands, and enzymes.

[0130] Preferred examples of the Fc fusion protein molecule of the present invention include proteins in which an Fc domain is fused to a receptor protein that binds to a target, such as TNFR-Fc fusion protein, IL1R-Fc fusion protein, VEGFR-Fc fusion protein, and CTLA4-Fc fusion protein (Nat Med. 2003 Jan;9(1):47-52, BioDrugs. 2006;20(3):151-60). Furthermore, the protein fused to the polypeptide of the present invention may be any molecule as long as it binds to a target molecule, and examples of such molecules include scFv molecules (WO2005 / 037989), single domain antibody molecules (WO2004 / 058821, WO2003 / 002609), antibody-like molecules (Current Opinion in Biotechnology 2006, 17:653-658, Current Opinion in Biotechnology 2007, 18:1-10, Current Opinion in Structural Biology 1997, 7:463-469, Protein Science 2006, 15:14-27), DARPins (WO2002 / 020565), Affibodies (WO1995 / 001937), Avimers (WO2004 / 044011, (WO2005 / 040229), Adnectin (WO2002 / 032925), etc. Furthermore, the antibody and Fc fusion protein molecule may be a multispecific antibody that binds to multiple types of target molecules or epitopes.

[0131] The antibody of the present invention also includes modified antibodies. Examples of modified antibodies include antibodies bound to various molecules such as polyethylene glycol (PEG) and cytotoxic substances. Such modified antibodies can be obtained by chemically modifying the antibody of the present invention. Methods for modifying antibodies have already been established in this field.

[0132] Furthermore, the antibody of the present invention may be a bispecific antibody. A bispecific antibody is an antibody having variable regions that recognize different epitopes in the same antibody molecule, and the epitopes may be present in different molecules or may be present in the same molecule.

[0133] The polypeptide of the present invention can be produced by a method known to those skilled in the art. For example, the antibody can be produced by the following method, but is not limited thereto.

[0134] A DNA encoding an antibody heavy chain in which one or more amino acid residues in the Fc region have been substituted with other amino acids of interest, and a DNA encoding an antibody light chain are expressed. A DNA encoding a heavy chain in which one or more amino acid residues in the Fc region have been substituted with other amino acids of interest can be obtained, for example, by obtaining the Fc region portion of DNA encoding a natural heavy chain and introducing appropriate substitutions so that codons encoding specific amino acids in the Fc region code for other amino acids of interest.

[0135] It is also possible to obtain DNA encoding a heavy chain in which one or more amino acid residues in the Fc region of a native heavy chain have been substituted with other amino acids of interest by designing a DNA encoding a protein in which one or more amino acid residues in the Fc region have been substituted with other amino acids of interest in advance and chemically synthesizing the DNA. The amino acid substitution site and type of substitution are not particularly limited. Furthermore, the substitution is not limited to substitution, and may be any of deletion, addition, insertion, or a combination of these.

[0136] Furthermore, a DNA encoding a heavy chain in which one or more amino acid residues in the Fc region have been substituted with other amino acids of interest can be produced by dividing it into partial DNAs. Examples of combinations of partial DNAs include, but are not limited to, DNA encoding a variable region and DNA encoding an Fc region, or DNA encoding a Fab region and DNA encoding an Fc region. DNA encoding a light chain can also be produced by dividing it into partial DNAs in a similar manner.

[0137] Methods for expressing the above DNA include the following methods. For example, a DNA encoding a heavy chain variable region is incorporated into an expression vector together with a DNA encoding a heavy chain Fc region to construct a heavy chain expression vector. Similarly, a DNA encoding a light chain variable region is incorporated into an expression vector together with a DNA encoding a light chain Fc region to construct a light chain expression vector. These heavy and light chain genes can also be incorporated into a single vector.

[0138] When DNA encoding the antibody of interest is incorporated into an expression vector, it is incorporated into the expression vector so that it is expressed under the control of an expression control region, for example, an enhancer or a promoter. Next, a host cell is transformed with this expression vector to express the antibody. In this case, a suitable combination of a host and an expression vector can be used.

[0139] Examples of vectors include M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script, etc. Furthermore, for the purpose of subcloning or excision of cDNA, in addition to the above vectors, for example, pGEM-T, pDIRECT, pT7, etc. can be used.

[0140] When a vector is used for the purpose of producing the polypeptide of the present invention, an expression vector is particularly useful. For example, when the host is Escherichia coli such as JM109, DH5α, HB101, or XL1-Blue, it is essential that the expression vector has a promoter capable of efficient expression in Escherichia coli, such as the lacZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427, the entirety of which is incorporated herein by reference), the araB promoter (Better et al., Science (1988) 240, 1041-1043, the entirety of which is incorporated herein by reference), or the T7 promoter. In addition to the above vectors, such vectors include pGEX-5X-1 (Pharmacia), "QIAexpress system" (QIAGEN), pEGFP, or pET (in this case, the host is preferably BL21, which expresses T7 RNA polymerase).

[0141] The vector may also contain a signal sequence for polypeptide secretion. When the polypeptide is produced in the periplasm of E. coli, the signal sequence for polypeptide secretion may be the pelB signal sequence (Lei, SP et al. J. Bacteriol. (1987) 169, 4397, the entirety of which is incorporated herein by reference). The vector can be introduced into the host cell by, for example, the lipofectin method, the calcium phosphate method, or the DEAE-Dextran method.

[0142] In addition to E. coli expression vectors, examples of vectors for producing the polypeptides of the present invention include mammalian expression vectors (e.g., pcDNA3 (Invitrogen), pEGF-BOS (Nucleic Acids. Res. 1990, 18(17), p5322, the entirety of which is incorporated herein by reference), pEF, and pCDM8), insect cell expression vectors (e.g., "Bac-to-BAC baculovairus expression system" (GIBCO BRL), and pBacPAK8), plant expression vectors (e.g., pMH1 and pMH2), animal virus expression vectors (e.g., pHSV, pMV, and pAdexLcw), retrovirus expression vectors (e.g., pZIPneo), yeast expression vectors (e.g., "Pichia Expression Kit" (Invitrogen), pNV11, and SP-Q01), and Bacillus subtilis expression vectors (e.g., pPL608 and pKTH50).

[0143] When the purpose is to express in animal cells such as CHO cells, COS cells, and NIH3T3 cells, it is essential to have a promoter necessary for expression in the cells, such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108, the entirety of which is incorporated herein by reference), the MMTV-LTR promoter, the EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322, the entirety of which is incorporated herein by reference), the CAG promoter (Gene. (1991) 108, 193, the entirety of which is incorporated herein by reference), the CMV promoter, etc., and it is even more preferable if it has a gene for selecting transformed cells (for example, a drug resistance gene that can be distinguished by a drug (neomycin, G418, etc.)). Examples of vectors having such characteristics include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0144] Furthermore, when the aim is to stably express a gene and to amplify the copy number of the gene in the cell, a method can be used in which a vector (e.g., pCHOI, etc.) having a DHFR gene that complements the nucleic acid synthesis pathway is introduced into a CHO cell lacking the nucleic acid synthesis pathway and the vector is amplified with methotrexate (MTX). When the aim is to transiently express a gene, a method can be used in which a COS cell having a gene expressing SV40 T antigen on its chromosome is transformed with a vector (pcD, etc.) having an SV40 replication origin. Replication origins derived from polyoma virus, adenovirus, bovine papilloma virus (BPV), etc. can also be used. Furthermore, in order to amplify the copy number of the gene in the host cell system, the expression vector can contain, as a selection marker, an aminoglycoside transferase (APH) gene, a thymidine kinase (TK) gene, an Escherichia coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, a dihydrofolate reductase (dhfr) gene, etc.

[0145] The antibody can be recovered, for example, by culturing the transformed cells and then isolating the antibody from the cells or culture medium of the molecularly transformed cells. The antibody can be separated and purified by an appropriate combination of methods such as centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, 1q, FcRn, protein A, protein G column, affinity chromatography, ion exchange chromatography, and gel filtration chromatography.

[0146] The present invention also provides a method for modifying a polypeptide to create a polypeptide with improved stability compared to a parent polypeptide. That is, the present invention relates to a method for improving the stability of a polypeptide having an antibody Fc region compared to a parent polypeptide by modifying at least one amino acid in a loop region of the Fc region. In this method, it is preferable to use the thermal midpoint (Tm) as an index for evaluating or diagnosing stability.

[0147] The present invention also provides methods for producing polypeptides that have improved stability compared to the parent polypeptide. One embodiment of the production method of the present invention provides a method for producing a polypeptide having, for example, an antibody Fc region, in which at least one amino acid in a loop region of the Fc region has been modified, thereby improving stability compared to a parent polypeptide. For example, the production method may include the following steps: (a) modifying at least one amino acid in a loop region of an antibody Fc region of a polypeptide having the Fc region; (b) measuring the stability of the polypeptide modified in step (a); and (c) selecting a polypeptide having improved stability compared to the parent polypeptide.

[0148] A preferred embodiment is a method for producing a polypeptide having an antibody Fc region, in which at least one amino acid in a loop region of the Fc region has been modified, and thus the stability is improved compared to that of a parent polypeptide, the method comprising the steps of: (a) modifying a nucleic acid encoding the polypeptide such that it has enhanced stability compared to a parent polypeptide; (b) introducing the nucleic acid into a host cell and culturing it so as to express the nucleic acid; (c) recovering the polypeptide from the host cell culture.

[0149] Furthermore, the present invention also includes polypeptides (antibodies) and Fc fusion protein molecules produced by this production method.

[0150] In a preferred embodiment of the above method, for example, in a polypeptide having an Fc region of an antibody (e.g., human IgG), the polypeptide is selected from the group consisting of: EU numbering 234, EU numbering 235, EU numbering 236, EU numbering 237, EU numbering 238, EU numbering 239, EU numbering 247, EU numbering 250, EU numbering 265, EU numbering 266, EU numbering 267, EU numbering 268, EU numbering 269, EU numbering 270, EU numbering 271, EU numbering 295, EU numbering 296, EU numbering 298, EU numbering 300, and EU numbering 307. The nucleic acid encoding the polypeptide is modified so that at least one or more amino acids are modified in an amino acid site in the loop portion of the Fc region selected from the group consisting of: position 309 (EU numbering), position 315 (EU numbering), position 324 (EU numbering), position 325 (EU numbering), position 326 (EU numbering), position 327 (EU numbering), position 329 (EU numbering), position 330 (EU numbering), position 333 (EU numbering), position 335 (EU numbering), position 337 (EU numbering), position 360 (EU numbering), position 385 (EU numbering), position 386 (EU numbering), position 387 (EU numbering), position 389 (EU numbering), position 428 (EU numbering), and position 433 (EU numbering).

[0151] In the above-mentioned method or production method (hereinafter, sometimes simply referred to as "method"), it is preferable to further reduce the binding activity to FcγR. To modify the polypeptide so as to maintain or enhance the binding activity to FcγR compared to the parent polypeptide, for example, a step of modifying the amino acid modification site of TS1-TS8, TS20-TS27, TS44-TS50, TS52-TS55, or TS57-TS67 shown in the Examples below may be included.

[0152] Alternatively, in the above-mentioned method, it is preferable to further maintain or enhance the binding activity to FcγR. Modification to decrease the binding activity to FcγR compared to the parent polypeptide may include, for example, a step of modifying the amino acid modification sites of TS9-19, TS28-43, TS51, and TS56 shown in the Examples below. The alteration sites of TS1 to TS67 are as described above.

[0153] In the above-mentioned method, it is preferable to carry out amino acid modification in a polypeptide having the Fc region of an antibody (eg, human IgG).

[0154] Furthermore, the present invention provides a nucleic acid encoding a polypeptide having an antibody Fc region, in which at least one amino acid in a loop region of the Fc region has been modified, thereby improving stability compared to a parent polypeptide. The nucleic acid of the present invention may be in any form, such as DNA or RNA.

[0155] Furthermore, the present invention provides a vector comprising the nucleic acid of the present invention. The type of vector can be appropriately selected by those skilled in the art depending on the host cell into which the vector is to be introduced, and for example, the vectors described above can be used.

[0156] Furthermore, the present invention relates to a host cell transformed with the vector of the present invention. The host cell can be appropriately selected by those skilled in the art, and for example, the host cells described above can be used.

[0157] <Pharmaceutical Composition> The present invention provides a pharmaceutical composition comprising a polypeptide or Fc fusion protein molecule of the present invention. The pharmaceutical composition of the present invention can be formulated by a known method by introducing a pharma- ceutical acceptable carrier in addition to the above-mentioned polypeptide or Fc fusion protein molecule of the present invention. For example, it can be used parenterally in the form of a sterile solution or suspension injection with water or other pharma- ceutical acceptable liquid. For example, it can be formulated by appropriately combining with a pharmacologically acceptable carrier or vehicle, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc., and mixing in a unit dose form required for generally accepted pharmaceutical practice. Specific examples of carriers include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl acetal diethyl amino acetate, polyvinyl pyrrolidone, gelatin, medium chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts, etc. The amount of active ingredient in these preparations is such that an appropriate dose within the indicated range can be obtained.

[0158] Sterile compositions for injection can be formulated according to standard pharmaceutical practice using vehicles such as distilled water for injection.

[0159] Examples of aqueous solutions for injection include isotonic solutions containing physiological saline, glucose or other auxiliary drugs, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, and suitable solubilizing agents, such as alcohol, specifically ethanol, polyalcohols, such as propylene glycol and polyethylene glycol, and nonionic surfactants, such as polysorbate 80. (TM) , may be used in combination with HCO-50.

[0160] The oily liquid may be sesame oil or soybean oil, and may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. Buffers such as phosphate buffer, sodium acetate buffer, soothing agents such as procaine hydrochloride, stabilizers such as benzyl alcohol, phenol, and antioxidants may also be used. The prepared injection solution is usually filled into a suitable ampule.

[0161] The administration is preferably parenteral, and specific examples of the dosage form include injections, nasal administration, pulmonary administration, transdermal administration, etc. Examples of injections include intravenous injections, intramuscular injections, intraperitoneal injections, subcutaneous injections, etc., and the drug can be administered systemically or locally.

[0162] In addition, the administration method can be appropriately selected depending on the age and symptoms of the patient. The dosage of the pharmaceutical composition containing the antibody or the polynucleotide encoding the antibody can be selected, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight per administration. Alternatively, the dosage can be selected, for example, in the range of 0.001 to 100,000 mg / body per patient, but is not necessarily limited to these values. The dosage and administration method vary depending on the patient's body weight, age, symptoms, etc., but can be appropriately selected by those skilled in the art.

[0163] In the present invention, the pharmaceutical composition containing the above-mentioned polypeptide of the present invention is useful, for example, as an active ingredient of a therapeutic or preventive agent for immunoinflammatory diseases, cancer, and the like. "Immune inflammatory diseases" include, but are not limited to, rheumatoid arthritis, autoimmune hepatitis, autoimmune thyroiditis, autoimmune bullous disease, autoimmune adrenocorticitis, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, megalocytic anemia, autoimmune atrophic gastritis, autoimmune neutropenia, autoimmune orchitis, autoimmune encephalomyelitis, autoimmune receptor disease, autoimmune infertility, chronic active hepatitis, glomerulonephritis, interstitial pulmonary fibrosis, multiple sclerosis, Paget's disease, osteoporosis, multiple myeloma, uveitis, acute and chronic spondylitis, gouty arthritis, inflammatory bowel disease, adult respiratory distress syndrome (ARDS), psoriasis, Crohn's disease, Graves' disease, juvenile diabetes mellitus, Addison's disease, myasthenia gravis, phacoemulsification, Systemic lupus erythematosus, allergic rhinitis, allergic dermatitis, ulcerative colitis, hypersensitivity, asthma, muscle degeneration, cachexia, systemic sclerosis, localized scleroderma, Sjogren's syndrome, Behcet's disease, Reiter's syndrome, diabetes mellitus type I and II, bone resorption diseases, graft vs. host reaction, ischemia-reperfusion injury, atherosclerosis, brain trauma, multiple sclerosis, cerebral malaria, sepsis, septic shock, toxic shock syndrome, fever, and malgias due to staining, aplastic anemia, hemolytic anemia, idiopathic thrombocytopenia, Goodpasture's syndrome, Guillain-Barre syndrome, Hashimoto's disease, pemphigus, IgA nephropathy, hay fever, antiphospholipid syndrome, polymyositis, Wegener's sarcoma, arteritis nodosa, mixed connective tissue disease, fibromyalgia.

[0164] In the present invention, the term "cancer" refers to a physiological state of a mammal that is typically characterized by uncontrolled cell proliferation, or refers to such a physiological state. In the present invention, the types of cancer are not particularly limited, but include the following. Carcinomas (epithelial cancers) include pancreatic cancer, prostate cancer, breast cancer, skin cancer, gastrointestinal cancer, lung cancer, hepatocellular carcinoma, cervical cancer, uterine cancer, ovarian cancer, fallopian tube cancer, vaginal cancer, liver cancer, bile duct cancer, bladder cancer, ureter cancer, thyroid cancer, adrenal cancer, kidney cancer, or other glandular tissue cancer. Sarcomas (non-epithelial cancers) include liposarcoma, leiomyosarcoma, rhabdomyosarcoma, synovial sarcoma, angiosarcoma, fibrosarcoma, malignant peripheral nerve tumor, gastrointestinal stromal tumor, desmoid tumor, Ewing's sarcoma, osteosarcoma, chondrosarcoma, leukemia, lymphoma, myeloma, and other solid organ tumors, such as melanoma or brain tumor.

[0165] The correspondence between the three-letter and one-letter amino acid codes used in this specification is as follows: Alanine: Ala: A Arginine:Arg:R Asparagine:Asn:N Aspartic acid:Asp:D Cysteine:Cys:C Glutamine:Gln:Q Glutamic acid: Glu:E Glycine:Gly:G Histidine:His:H Isoleucine:Ile:I Leucine:Leu:L Lysine:Lys:K Methionine:Met:M Phenylalanine:Phe:F Proline: Pro:P Serine:Ser:S Threonine: Thr:T Tryptophan:Trp:W Tyrosine:Tyr:Y Valin:Val:V

[0166] All prior art documents cited in this specification are hereby incorporated by reference. EXAMPLES

[0167] The present invention will be described in more detail below with reference to examples, but is not limited to these examples. In these examples, the amino acid sites modified in the Fc region were determined according to the EU numbering system (see Sequences of proteins of immunological interest, NIH Publication No. 91-3242).

[0168] [Example 1] Improvement of thermostability by modifying the loop region of the CH2 domain It is generally known that β-sheet structures are susceptible to structural changes due to amino acid modifications, and that modifications are likely to result in decreased thermal stability (Biochemistry 1994;33:5510-5517, Nature 1994;367:660-663). Therefore, in the present invention, we attempted to improve thermal stability by modifying the loop portion of the Fc region.

[0169] The amino acids L234-S239, D265-P271, Q295, Y296, S298, Y300, and S324-S337, which are loop portions of the CH2 domain of the Fc region B3 (SEQ ID NO: 16), were replaced with 18 kinds of amino acids excluding the original amino acids and cysteine, to prepare an Fc mutant (FIG. 4). This is called an Fc variant. An anti-GPC3 antibody was prepared using this Fc variant. An antibody consisting of the variable region GpH7 (SEQ ID NO: 17) as the H chain, the Fc variant as the Fc region, and the variable region GpL16 (SEQ ID NO: 18) and the constant region k0 (SEQ ID NO: 19) as the L chain (hereinafter referred to as GpH7-Fc variant / GpL16-k0) was prepared according to the method of Reference Example 1.

[0170] The thermal denaturation midpoint (Tm) of the prepared antibodies was evaluated according to the method of Reference Example 2. In the following, unless otherwise specified, Tm refers to the Tm of the CH2 region when a sample in the form of IgG was subjected to the measurement. The obtained Tm data is shown in Figure 1. The Tm of unmutated B3 (GpH7-B3 / GpL16-k0: GpH7 (sequence number: 17), B3 (sequence number: 16), GpL16 (sequence number: 18), k0 (sequence number: 19)) was approximately 68°C, and mutants that showed a Tm higher than this value are summarized in Tables 1-1 and 1-2.

[0171] [Table 1-1] Table 1-2 is a continuation of Table 1-1.

[0172] [Table 1-2]

[0173] [Example 2] Preparation and evaluation of a novel IgG1 Fc region with improved thermal stability Based on the data in Example 1 and structural information (Nature 2000; 406: 267-273), several modifications that were thought to be particularly effective in improving thermostability were selected. The selected single modification or multiple modifications were introduced into the H chains of anti-GPC3 antibody (GpH7-G1d / GpL16-k0: GpH7 (SEQ ID NO: 17), G1d (SEQ ID NO: 15), GpL16 (SEQ ID NO: 18), k0 (SEQ ID NO: 19) and anti-IL6R antibody (MH0-G1d / ML0-k0: GpH7 (SEQ ID NO: 17), G1d (SEQ ID NO: 15), ML0 (SEQ ID NO: 21), k0 (SEQ ID NO: 19) to produce TS1 to TS19 (SEQ ID NO: 26 to 44) shown in Table 2. At the same time, in order to evaluate the effect of improving the thermal stability in the form of IgG by introducing a disulfide bond, the modifications of m01 and m02 reported in a previous paper (J Biol Chem. 2009;284:14203-14210) was introduced into the H chain of anti-IL6R to produce modified TSm01 and TSm02 (SEQ ID NOs: 24 and 25). Expression and purification of each antibody were performed by the method described in Reference Example 1.

[0174] [Table 2]

[0175] Using the prepared antibodies, Tm was compared by the method shown in Reference Example 2. The results of Tm measurement are shown in Table 3. [Table 3]

[0176] In addition, in these results, samples that were not evaluated are indicated as "nt" (not tested), and samples in which it was difficult to detect Tm are indicated as "nd" (not detected).

[0177] Among the modifications of TS1 to TS19, TS1 to 3 and TS5 to 19 were observed to improve Tm when the H chain sequence was either anti-GPC3 or anti-IL6R. On the other hand, TS4 decreased Tm for both H chain sequences. The Q295M modification introduced into TS4 alone was found to improve Tm by about 2°C (TS3), but it was revealed that the simultaneous introduction of the Y300E modification rather decreased Tm. According to the structural information, Q295 and Y300 are present in a position where they are paired on the same loop. The Q295M modification may enhance the interaction of the amino acid side chain with Y300. However, it was presumed that the simultaneous introduction of the Q295M and Y300E modifications eliminated the interaction between the amino acid side chains.

[0178] Thus, it was revealed that, except for modifications that have a negative effect on each other structurally, combining modifications that improve Tm further improves Tm. Based on the above, it is possible to create multiple combinations of modifications that improve Tm based on structural information other than TS1-3 and TS5-19 shown here.

[0179] TSm01 and TSm02, which were evaluated at the same time, expressed only the CH2 domain in a previous paper (J Biol Chem. 2009;284:14203-14210), and the Tm was evaluated, and the Tm improvement effect of about 10 to 20 ° C was observed. However, when the Tm was evaluated in the form of IgG in this study, multiple thermal denaturation points were detected in TSm01, and it was difficult to clearly grasp the thermal denaturation of the CH2 domain. This suggests that TSm01 is likely to be structurally heterogeneous.

[0180] Furthermore, the Tm of TSm02 was increased by only about 4° C. compared to IgG1. This demonstrated that the effect of introducing a disulfide bond in improving the Tm of IgG was small, and that the modification discovered by the present inventors was more effective in improving Tm.

[0181] The anti-GPC3 antibody used in this study has a Fab Tm of 74.7°C. For this reason, it was inferred that for TS13 to TS19, in which modifications were introduced into the anti-GPC3 H chain, the fluorescence transition curves of CH2 and Fab overlap, making it difficult to accurately calculate the Tm. For this reason, it was considered inappropriate to use the anti-GPC3 H chain to evaluate the effect of further improving Tm by combining modified residues, and the anti-IL6R H chain was used for the subsequent studies.

[0182] [Example 3] Evaluation of binding of novel Fc regions to human Fcγ receptor In the modification of antibody Fc region, Fc region with enhanced effector function such as ADCC activity, or Fc region with reduced effector function such as ADCC activity have been reported (Reference: Current Opinion, 2009, 20, 685-691). Antibodies with enhanced effector function are mainly useful for cancer antibodies, while antibodies with reduced effector function are useful for neutralizing antibodies and receptor-Fc fusion such as Enbrel and Orencis, and it is important to use them according to their purpose.

[0183] The CH2 domain modified in this study is known to be involved in interactions with several hFcγ receptors (hereinafter referred to as hFcgRs) that affect effector functions. Thus, the binding of TS1 to TS19 to hFcgRs using the H chain of anti-IL6R prepared in Example 2 was measured by the method described in Reference Example 3. The binding of each antibody to each hFcgRs was calculated when the binding of G1 to each hFcgRs was set to 100, and the results are summarized in Table 4.

[0184] [Table 4]

[0185] As a result, the binding of TS1 to TS8 to hFcgRs was equivalent to that of G1, whereas the binding of TS9 to 19 to hFcgRs was weaker than that of G1.

[0186] [Example 4] Construction and evaluation of novel Fc regions with combinations of alterations that maintain binding to hFcgRs When introducing modifications aimed at improving stability, it is important for cancer antibodies, for which effector function is important, to maintain binding to hFcgRs. In Example 3, modifications of TS1 to TS8 that maintain binding to hFcgRs were combined to newly prepare modified TS20 to TS27 shown in Table 5 (SEQ ID NOs: 45 to 52). Expression and purification of each antibody were performed by the method described in Reference Example 1. The Tm of the prepared antibodies was evaluated by the method described in Reference Example 2, and the results are shown in Table 6.

[0187] [Table 5]

[0188] [Table 6]

[0189] Furthermore, binding to hFcgRs was measured by the method described in Reference Example 3. The binding of each antibody to each hFcgRs was calculated assuming the binding of G1 to each hFcgRs to be 100, and is shown in Table 7.

[0190] [Table 7]

[0191] By combining the modifications, the Tm was improved in all of the modified variants TS20 to TS27 while maintaining the binding ability to hFcgRs. The modified variants with the greatest Tm improvement effect were TS20 and TS24, which had a Tm approximately 3°C higher than that of G1.

[0192] [Example 5] Construction and evaluation of novel Fc regions with combinations of alterations that reduce binding to hFcgRs When introducing modifications aimed at improving stability, it is desirable to reduce the effector function of the neutralizing antibody as much as possible. Modifications of TS9 to TS19 that reduced the binding to hFcgRs in Example 3 were combined to newly prepare modified antibodies TS28 to TS43 shown in Table 8 (SEQ ID NOs: 53 to 68). Expression and purification of each antibody were performed by the method described in Reference Example 1. The Tm of the prepared antibodies was evaluated by the method described in Reference Example 2, and the results are shown in Table 9.

[0193] [Table 8]

[0194] [Table 9]

[0195] Furthermore, binding to hFcgRs was measured by the method described in Reference Example 3. The binding of each antibody to each hFcgRs was calculated assuming the binding of G1 to each hFcgRs to be 100, and is shown in Table 10.

[0196] [Table 10]

[0197] By combining the modifications, the Tm was improved in all of the modified antibodies TS28 to TS43 by increasing by 13° C. or more. Furthermore, the binding ability to FcgRs was also significantly reduced by combining the modifications.

[0198] [Example 6] Evaluation of aggregate content of novel IgG1 Fc regions with improved thermal stability Since aggregates affect storage stability and immunogenicity, it is desirable that modifications that improve thermal stability do not increase the aggregate content as much as possible. Therefore, the aggregate contents of TS20 to TS43, which combine multiple modifications, and TSm01 and TSm02 were evaluated by the method shown in Reference Example 4. The measurement chromatograms are shown in FIG. 2, and the aggregate contents are shown in Table 11.

[0199] [Table 11]

[0200] The aggregate content of TS20 to TS27, which maintained the binding ability to hFcgRs, was comparable to that of G1, demonstrating that these modifications to improve thermal stability do not significantly affect the aggregate content. On the other hand, the aggregate content of TS28 to TS43, which showed a significant decrease in the binding ability to hFcgRs, increased slightly in TS29, 33, and 35, but the aggregate content of the other variants was comparable to that of G1. In contrast, the aggregate content of TSm01 and TSm02, which are disulfide bond-introduced variants, was significantly increased to approximately 5-fold and 3-fold that of G1, respectively.

[0201] The above results demonstrate that the modification discovered by the present inventors in this study is the first modification that improves thermal stability while maintaining physical properties.

[0202] [Example 7] Preparation and evaluation of a novel IgG1 Fc region with improved thermal stability It was clear from the above that Tm is improved by introducing a mutation into the CH2 domain. Therefore, for regions not examined for modification in Example 1, whether there is a mutation that improves Tm was examined, and new variants of TS44 to TS67 shown in Table 12 were prepared (SEQ ID NOs: 69 to 92). Expression and purification of each antibody were performed by the method described in Reference Example 1, and the results are shown in Table 13. Furthermore, binding to hFcgRs was measured by the method described in Reference Example 3. The binding of each antibody to each hFcgRs was calculated when the binding of G1 to each hFcgRs was set to 100, and is shown in Table 14.

[0203] [Table 12]

[0204] [Table 13]

[0205] [Table 14]

[0206] The variant that showed the greatest Tm improvement in this study was TS48, which showed an improvement of about 5°C, and the other variants also showed improvement in Tm. Regarding the binding ability to hFcgRs, TS51 and TS56 showed a slight decrease in the binding to hFcgRs, while all variants except TS51 and TS56 maintained their binding to hFcgRs.

[0207] [Reference Example 1] Construction of an expression vector for an antibody and expression and purification of the antibody The amino acid substitution was introduced by a method known to those skilled in the art using QuikChange Site-Directed Mutagenesis Kit (Stratagene), PCR, etc., or Infusion Advantage PCR cloning kit (TAKARA), etc., to construct an expression vector. The nucleotide sequence of the obtained expression vector was determined by a method known to those skilled in the art. The prepared plasmid was transiently introduced into human fetal renal carcinoma cell-derived HEK293H strain (Invitrogen) or FreeStyle293 cells (Invitrogen) to express the antibody. The antibody was purified from the obtained culture supernatant using rProtein A SepharoseTM Fast Flow (GE Healthcare) by a method known to those skilled in the art. The purified antibody concentration was calculated by measuring the absorbance at 280 nm using a spectrophotometer, and the antibody concentration was calculated using the extinction coefficient calculated from the obtained value by the PACE method (Protein Science 1995; 4: 2411-2423).

[0208] [Reference Example 2] Evaluation of thermal denaturation midpoint (Tm) of modified antibodies by differential scanning fluorimetry In this study, the thermal stability of the antibody was evaluated by measuring the thermal denaturation midpoint (Tm) using differential scanning fluorimetry with Rotor-Gene Q (QIAGEN). This method has already been reported to show a good correlation with Tm evaluation using a differential scanning calorimeter, which is widely known as a method for evaluating the thermal stability of antibodies (Journal of Pharmaceutical Science 2010; 4: 1707-1720).

[0209] Measurement samples were prepared by diluting 5000-fold concentration of SYPRO orange (Molecular Probes) with PBS (Sigma) and mixing with the antibody solution. 20 μL of each sample was placed in a measurement tube and the temperature was raised from 30°C to 99°C. After raising the temperature by 0.4°C and leaving it for about 6 seconds, the fluorescence intensity was detected at 470 nm (excitation wavelength) / 555 nm (fluorescence wavelength). The temperature at which fluorescence transition was observed was calculated using the data by Rotor-Gene Q Series Software (QIAGEN), and this value was taken as Tm.

[0210] [Reference Example 3] Evaluation of binding to hFcgRs Using Biacore T100 (GE Healthcare), the interaction between the antibody and hFcgRs (hFcgRIa, hFcgRIIa(R), hFcgRIIa(H), hFcgRIIb, hFcgRIIIa(F), hFcgRIIIa(V)) was analyzed. HBS-EP+ (GE Healthcare) was used as the running buffer, and the measurement temperature was 25°C. Protein A (Invitrogen) was immobilized by the amine coupling method, and the target antibody was captured thereon. After the antibody was captured, hFcγR diluted with the running buffer was allowed to interact with hFcgRIa at a flow rate of 30μL / min for 5 minutes and other hFcγR at a flow rate of 5μL / min for 1 minute, and the amount of binding to the antibody was measured and compared between the antibodies. However, because the amount of hFcγR binding depends on the amount of captured antibody, the amount of FcgR binding was corrected so that the amount of each antibody captured was 200 RU (resonance unit) for hFcgRIa and 1000 RU (resonance unit) for the other hFcgRs. The antibody captured on the chip was washed by reacting with 10 mM glycine-HCl, pH 1.5 at a flow rate of 30 μL / min for 30 seconds, and the chip was regenerated and used repeatedly.

[0211] [Reference Example 4] Evaluation of aggregate content The aggregate content in the purified antibody was evaluated by SEC analysis using the Alliance system (Waters). The mobile phase was 50 mM phosphate buffer, pH 7.0 (Isekyu) containing 300 mM sodium chloride, and the analytical column was G3000SW. XLMeasurements were performed at a wavelength of 215 nm using a chromatograph (TOSOH). Data analysis was performed using Empower2 (Waters), and the components eluted on the higher molecular weight side than the monomer were collectively considered as aggregates, and their content was calculated. [Industrial Applicability]

[0212] In many of the reported antibody Fc region variants, the stability of the Fc region has been reduced. Furthermore, the effector function of the Fc portion of an antibody, i.e., binding to FcγR, is important. The present invention makes it possible to provide a highly stable Fc that maintains FcγR binding, or a highly stable Fc region variant with reduced FcγR binding.

Claims

1. A method for improving the thermal stability of a polypeptide comprising an Fc region of a human IgG1 antibody, comprising introducing at least one amino acid modification into the Fc region, including a substitution of amino acid position 239 (EU numbering) with Lys, compared to a parent polypeptide that does not contain the amino acid modification.

2. The method described in claim 1, wherein the polypeptide into which the modification has been introduced has reduced binding activity to FcγR compared to the parent polypeptide.

3. The method according to claim 1 or 2, wherein the thermal stability is evaluated or determined using a thermal denaturation midpoint (Tm) as an index.

4. 234th EU numbering, 236th EU numbering, 237th EU numbering, 238th EU numbering, 247th EU numbering, 250th EU numbering, 265th EU numbering, 266th EU numbering, 267th EU numbering, 268th EU numbering, 269th EU numbering, 270th EU numbering, 271st EU numbering, 295th EU numbering, 296th EU numbering, 298th EU numbering, 300th EU numbering, 307th EU numbering, 309th EU numbering, 315th EU numbering, 4. The method according to claim 1 , further comprising introducing at least one amino acid mutation at an amino acid site selected from the group consisting of: 324 (EU numbering), 325 (EU numbering), 326 (EU numbering), 327 (EU numbering), 329 (EU numbering), 330 (EU numbering), 333 (EU numbering), 335 (EU numbering), 337 (EU numbering), 360 (EU numbering), 385 (EU numbering), 386 (EU numbering), 387 (EU numbering), 389 (EU numbering), 428 (EU numbering), and 433 (EU numbering).

5. The method according to any one of claims 1 to 4, wherein the Fc region does not have a glycosylation modification at amino acid position 297 (EU numbering).

6. The method according to any one of claims 1 to 5, wherein the polypeptide is an antibody.

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