Method for altering plasma retention and immunogenicity of antigen-binding molecule

• US 10,618,965 B2
• Filed: 03/30/2012
• Issued: 04/14/2020
• Est. Priority Date: 02/25/2011
• Status: Active Grant

• Current Assignee: Chugai Seiyaku Kabushiki Kaisha (Roche Holding AG)
• Original Assignee: Chugai Seiyaku Kabushiki Kaisha (Roche Holding AG)
• Inventors: Igawa, Tomoyuki, Maeda, Atsuhiko, Haraya, Kenta, Iwayanagi, Yuki, Tachibana, Tatsuhiko, Mimoto, Futa, Kuramochi, Taichi, Katada, Hitoshi, Kadono, Shojiro
• Primary Examiner(s): Huynh, Phuong
• Application Number:
  US14/007,947
• Publication Number:
  US 20140105889A1
• Time in Patent Office: 2,937 Days
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Abstract

The present invention demonstrated that the modification of the Fc region of an antigen-binding molecule into an Fc region that does not form in a neutral pH range a heterotetramer complex containing two molecules of FcRn and an active Fcγ receptor improved the pharmacokinetics of the antigen-binding molecule and reduced the immune response to the antigen-binding molecule. The present invention also revealed methods for producing antigen-binding molecules having the properties described above, and successfully demonstrated that pharmaceutical compositions containing as an active ingredient such an antigen-binding molecule or an antigen-binding molecule produced by a production method of the present invention have excellent features over conventional antigen-binding molecules in that when administered, they exhibit improved pharmacokinetics and reduced in vivo immune response.

Claims

1. A method for reducing immunogenicity of an antibody, the method comprising:

identifying a first antibody comprising (a) an Fc region with a human FcRn-binding activity at pH 7.0 that is stronger than the human FcRn-binding activity at pH 7.0 of a native human IgG1 Fc region, and (b) an antigen-binding domain whose antigen-binding activity varies with pH as described in (i) below, or with calcium ion concentration as described in (ii) below;

(i) the antigen-binding activity is lower at pH 5.8 than at pH 7.4, wherein the ratio of the KD value for antigen-binding activity at pH 5.8 to the KD value for antigen-binding activity at pH 7.4 (KD (pH 5.8)/KD (pH 7.4)) is 2 or more when the KD (pH 5.8) and KD (pH 7.4) values are determined using a surface plasmon resonance technique in which the antibody is immobilized, the antigen serves as analyte, and the following conditions are used;

10 mM 2-(N-morpholino)ethanesulfonic acid (MES) pH 5.8 or pH 7.4, 150 mM NaCl, 0.05% polysorbate 20, at 37°

C.;

(ii) the antigen-binding activity is lower at a calcium ion concentration of 3 μ

M than at a calcium ion concentration of 2 mM, wherein the ratio of the KD value for antigen-binding activity at a calcium ion concentration of 3 μ

M to the KD value for antigen-binding activity at a calcium ion concentration of 2 mM (KD (3 μ

M)/ KD (2 mM)) is 2 or more when the KD (3 μ

M) and KD (2 mM) values are determined using a surface plasmon resonance technique in which the antibody is immobilized, the antigen serves as analyte, and the following conditions are used;

10 mM N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) pH 7.4, 150 mM NaCl, 0.05% polysorbate 20, and either 2 mM CaCl₂ or 3 μ

M CaCl₂, at 37°

C.;

producing a second antibody whose ability to form a heterocomplex with two molecules of the human FcRn and one molecule of an activating Fcγ

receptor at pH 7.4 is reduced compared to the ability of the first antibody to form such a heterocomplex at pH 7.4, and whose ability to bind to the activating Fcγ

receptor is decreased compared to the ability of the native human IgG1 Fc region to bind to the activating Fcγ

receptor, the second antibody being identical to the first antibody except for one or more amino acids in the Fc region, wherein the activating Fcγ

receptor is human Fcγ

RIa, human Fcγ

RIIa(R), human Fcγ

RIIa(H), human Fcγ

RIIIa(V), or human Fcγ

RIIIa(F), and wherein at least one of the following positions in the Fc region of the second antibody is occupied by one of the amino acid residues listed for that position (by EU numbering);

Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Met, Phe, Pro, Ser, Thr, or Trp at position 234;

Ala, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Pro, Ser, Thr, Val, or Arg at position 235;

Arg, Asn, Gln, His, Leu, Lys, Met, Phe, Pro, or Tyr at position 236;

Ala, Asn, Asp, Gln, Glu, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Val, Tyr, or Arg at position 237;

Ala, Asn, Gln, Glu, Gly, His, Ile, Lys, Thr, Trp, or Arg at position 238;

Gln, His, Lys, Phe, Pro, Trp, Tyr, or Arg at position 239;

Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val at position 265;

Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Phe, Pro, Ser, Thr, Trp, or Tyr at position 266;

Arg, His, Lys, Phe, Pro, Trp, or Tyr at position 267;

Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val at position 269;

Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val at position 270;

Arg, His, Phe, Ser, Thr, Trp, or Tyr at position 271;

Arg, Asn, Asp, Gly, His, Phe, Ser, Trp, or Tyr at position 295;

Arg, Gly, Lys, or Pro at position 296;

Ala at position 297;

Arg, Gly, Lys, Pro, Trp, or Tyr at position 298;

Arg, Lys, or Pro at position 300;

Lys or Pro at position 324;

Ala, Arg, Gly, His, Ile, Lys, Phe, Pro, Thr, Trp, Tyr, or Val at position 325;

Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val at position 327;

Arg, Asn, Gly, His, Lys, or Pro at position 328;

Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, Val, or Arg at position 329;

Pro or Ser at position 330;

Arg, Gly, or Lys at position 331;

Arg, Lys, or Pro at position 332; and

conducting an assay to confirm that the second antibody has decreased immunogenicity compared to the first antibody.

4. The method of claim 1, wherein the Fc region of the second antibody differs from the Fc region of the first antibody by amino acid substitution at one or more positions, including at least one of the following positions:

235, 237, 238, 239, 270, 298, 325, 329 (by EU numbering).

7. The method of claim 1, wherein the Fc region of the first antibody and the Fc region of the second antibody differ from the native IgG1 Fc region by amino acid substitution at one or more positions, including at least one of the following positions (by EU numbering):

237, 248, 250, 252, 254, 255, 256, 257, 258, 265, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434, 436.

8. The method of claim 1, wherein at least one of the following positions (by EU numbering) in the Fc region of the second antibody is occupied by one of the amino acid residues listed for that position:

Met at position 237;

Ile at position 248;

Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr at position 250;

Phe, Trp, or Tyr at position 252;

Thr at position 254;

Glu at position 255;

Asn, Asp, Glu, or Gln at position 256;

Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val at position 257;

His at position 258;

Ala at position 265;

Ala or Glu at position 286;

His at position 289;

Ala at position 297;

Gly at position 298;

Ala at position 303;

Ala at position 305;

Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr at position 307;

Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr at position 308;

Ala, Asp, Glu, Pro, or Arg at position 309;

Ala, His, or Ile at position 311;

Ala or His at position 312;

Lys or Arg at position 314;

Ala, Asp, or His at position 315;

Ala at position 317;

Val at position 332;

Leu at position 334;

His at position 360;

Ala at position 376;

Ala at position 380;

Ala at position 382;

Ala at position 384;

Asp or His at position 385;

Pro at position 386;

Glu at position 387;

Ala or Ser at position 389;

Ala at position 424;

Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr at position 428;

Lys at position 433;

Ala, Phe, His, Ser, Trp, or Tyr at position 434;

His, Ile, Leu, Phe, Thr, or Val at position 436.

12. The method of claim 1, comprising assaying, at a pH that is in the range from pH 6.7 to pH 10.0, the second antibody'"'"'s ability to form the heterocomplex with two molecules of FcRn and one molecule of an activating Fcγ

R.

14. The method of claim 1, comprising assaying the second antibody'"'"'s ability to bind to the activating Fcγ

receptor and thereby determining that the second antibody'"'"'s ability to bind to the activating Fcγ

receptor is decreased compared to the first antibody'"'"'s ability to bind to the activating Fcγ

receptor.

15. The method of claim 1, wherein the antigen-binding activity of the antigen-binding domain varies with pH as described in (i), and the antigen-binding domain comprises a histidine residue at each of one or more positions selected from the group consisting of H chain variable region positions 27, 31, 32, 33, 35, 50, 58, 59, 61, 62, 99, 100b and 102, and L chain variable region positions 24, 27, 28, 31, 32, 50, 52, 53, 54, 55, 56, 89, 90, 91, 92, 93, and 94 (by Kabat numbering).

18. The method of claim 1, wherein the antigen-binding activity of the antigen-binding domain varies with calcium ion concentration as described in (ii), and at least one position of the antigen-binding domain selected from the following list of H and L chain positions is occupied by an amino acid residue having metal-chelating activity:

H chain variable region positions 95, 96, 100a and 101, and L chain variable region positions 30, 31, 32, 50, and 92 (by Kabat numbering).

21. The method of claim 1, wherein the one or more amino acids include the amino acid at position 235 (EU numbering), which is Arg in the second antibody.

22. The method of claim 1, wherein the Fc region of the first antibody and the Fc region of the second antibody differ from the native human IgG1 Fc region by amino acid substitution at one or more positions, including at least one of the following positions:

248, 289, 314, 315, 360, 384, 386, 387, 389, 424 (by EU numbering).

26. The method of claim 1, further comprising assaying the second antibody'"'"'s ability to form a heterocomplex with two molecules of the human FcRn and one molecule of the activating Fcγ

receptor at pH 7.4, and thereby determining that the second antibody'"'"'s ability to form such a heterocomplex is reduced compared to the ability of the first antibody to form such a heterocomplex at pH 7.4.

2. A method for reducing immunogenicity of an antibody, the method comprising:

identifying a first antibody comprising (a) an Fc region with a human FcRn-binding activity at pH 7.0 that is stronger than the human FcRn-binding activity at pH 7.0 of a native human IgG1 Fc region, and (b) an antigen-binding domain whose antigen-binding activity varies with pH as described in (i) below, or with calcium ion concentration as described in (ii) below;

(i) the antigen-binding activity is lower at pH 5.8 than at pH 7.4, wherein the ratio of the KD value for antigen-binding activity at pH 5.8 to the KD value for antigen-binding activity at pH 7.4 (KD (pH 5.8)/ KD (pH 7.4)) is 2 or more when the KD values for KD (pH 5.8) and KD (pH 7.4) are determined using a surface plasmon resonance technique in which the antibody is immobilized, the antigen serves as analyte, and the following conditions are used;

10 mM 2-(N-morpholino)ethanesulfonic acid (MES) at pH 5.8 or pH 7.4, 150 mM NaCl, 0.05% polysorbate 20, at 37°

C.;

(ii) the antigen-binding activity is lower at a calcium ion concentration of 3 μ

M than at a calcium ion concentration of 2 mM, wherein the ratio of the KD value for antigen-binding activity at a calcium ion concentration of 3 μ

M to the KD value for antigen-binding activity at a calcium ion concentration of 2 mM (KD (3 μ

M) / KD (2 mM)) is 2 or more when the KD values for KD (3 μ

M) and KD (2 mM) are determined using a surface plasmon resonance technique in which the antibody is immobilized, the antigen serves as analyte, and the following conditions are used;

10 mM N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) at pH 7.4, 150 mM NaCl, 0.05% polysorbate 20, and either 2mM CaCl₂ or 3 μ

M CaCl₂, at 37°

C.;

producing a second antibody whose ability to form a heterocomplex with two molecules of the human FcRn and one molecule of an activating Fcγ

receptor at pH 7.4 is reduced compared to the ability of the first antibody to form such a heterocomplex at pH 7.4, the second antibody being identical to the first antibody except at one or more positions in the Fc region, including

(a) position 238 (EU numbering), which in the second antibody is Asp and in the first antibody is not Asp, or

(b) position 328 (EU numbering), which in the second antibody is Glu and in the first antibody is not Glu,

wherein the second antibody binds more strongly to human Fcγ

RIIb than to the activating Fcγ

receptor, wherein the activating Fcγ

receptor is human Fcγ

RIa, human Fcγ

RIIa(R), human Fcγ

RIIa(H), human Fcγ

RIIIa(V), or human Fcγ

RIIIa(F); and

conducting an assay to confirm that the second antibody has decreased immunogenicity compared to the first antibody.

5. The method of claim 2, wherein the Fc region of the second antibody differs from the Fc region of the first antibody by amino acid substitution at one or more positions including position 238 or 328 of a heavy chain (by EU numbering).

6. The method of claim 2, wherein the Fc region of the second antibody differs from the Fc region of the first antibody by substitution at one or more positions, including position 238 (EU numbering), which is Asp in the second antibody, and at least one additional position selected from the list below, wherein the substitution at any of the listed positions is a substitution with an amino acid residue listed for that position below (by EU numbering):

Asp at position 233;

Trp or Tyr at position 234;

Ala, Asp, Glu, Leu, Met, Phe, Trp, or Tyr at position 237;

Asp at position 239;

Ala, Gln, or Val at position 267;

Asn, Asp, or Glu at position 268;

Gly at position 271;

Ala, Asn, Asp, Gln, Glu, Leu, Met, Ser, or Thr at position 326;

Arg, Lys, or Met at position 330;

Ile, Leu, or Met at position 323; and

Asp at position 296.

13. The method of claim 2, comprising assaying the second antibody'"'"'s binding to the human Fcγ

RIIb and to the activating Fcγ

receptor, thereby determining that the second antibody'"'"'s ability to bind to the human Fcγ

RIIb is greater than its ability to bind to the activating Fcγ

receptor.

16. The method of claim 2, wherein the antigen-binding activity of the antigen-binding domain varies with pH as described in (i), and the antigen-binding domain comprises a histidine residue at each of one or more positions selected from the group consisting of H chain variable region positions 27, 31, 32, 33, 35, 50, 58, 59, 61, 62, 99, 100b and 102, and L chain variable region positions 24, 27, 28, 31, 32, 50, 52, 53, 54, 55, 56, 89, 90, 91, 92, 93, and 94 (by Kabat numbering).

19. The method of claim 2, wherein the antigen-binding activity of the antigen-binding domain varies with calcium ion concentration as described in (ii), and at least one position of the antigen-binding domain selected from the following list of H and L chain positions is occupied by an amino acid residue having metal-chelating activity:

H chain variable region positions 95, 96, 100a and 101, and L chain variable region positions 30, 31, 32, 50, and 92 (by Kabat numbering).

23. The method of claim 2, wherein the Fc region of the first antibody and the Fc region of the second antibody differ from the native human IgG1 Fc region by amino acid substitution at one or more positions, including at least one of the following positions:

248, 289, 314, 315, 360, 384, 386, 387, 389, 424 (by EU numbering).

27. The method of claim 2, further comprising assaying the second antibody'"'"'s ability to form a heterocomplex with two molecules of the human FcRn and one molecule of the activating Fcγ

receptor at pH 7.4, and thereby determining that the second antibody'"'"'s ability to form such a heterocomplex is reduced compared to the ability of the first antibody to form such a hetero complex at pH 7.4.

29. The method of claim 2, wherein the Fc region of the first antibody and the Fc region of the second antibody differ from the native human IgG1 Fc region by amino acid substitution at one or more positions, including at least one of the following positions (by EU numbering):

237, 248, 250, 252, 254, 255, 256, 257, 258, 265, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434, 436.

30. The method of claim 2, wherein at least one of the following positions (by EU numbering) in the Fc region of the second antibody is occupied by one of the amino acid residues listed for that position:

Met at position 237;

Ile at position 248;

Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr at position 250;

Phe, Trp, or Tyr at position 252;

Thr at position 254;

Glu at position 255;

Asn, Asp, Glu, or Gln at position 256;

Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val at position 257;

His at position 258;

Ala at position 265;

Ala or Glu at position 286;

His at position 289;

Ala at position 297;

Gly at position 298;

Ala at position 303;

Ala at position 305;

Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr at position 307;

Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr at position 308;

Ala, Asp, Glu, Pro, or Arg at position 309;

Ala, His, or Ile at position 311;

Ala or His at position 312;

Lys or Arg at position 314;

Ala, Asp, or His at position 315;

Ala at position 317;

Val at position 332;

Leu at position 334;

His at position 360;

Ala at position 376;

Ala at position 380;

Ala at position 382;

Ala at position 384;

Asp or His at position 385;

Pro at position 386;

Glu at position 387;

Ala or Ser at position 389;

Ala at position 424;

Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr at position 428;

Lys at position 433;

Ala, Phe, His, Ser, Trp, or Tyr at position 434;

His, Ile, Leu, Phe, Thr, or Val at position 436.

31. The method of claim 2, comprising assaying the second antibody'"'"'s ability to form the heterocomplex with two molecules of the human FcRn and one molecule of the activating Fcγ

R at a pH that is in the range from pH 6.7 to pH 10.0.

33. The method of claim 2, wherein the heavy chain of the second antibody comprises either (1) Asp at position 238 (by EU numbering), or (2) Glu at position 328 (by EU numbering), but does not comprise both (1) and (2).

3. A method for reducing immunogenicity of an antibody, the method comprising:

identifying a first antibody comprising

(a) a first Fc region that binds to a human FcRn at pH 7.0 more strongly than a native human IgG1 Fc region binds to the human FcRn at pH 7.0, and that is able to form a heterocomplex with two molecules of the human FcRn and one molecule of an activating Fcγ

receptor at pH 7.4, and

(b) an antigen-binding domain whose antigen-binding activity varies with pH as described in (i) below, or with calcium ion concentration as described in (ii) below;

(i) the antigen-binding activity is lower at pH 5.8 than at pH 7.4, wherein the ratio of the KD value for antigen-binding activity at pH 5.8 to the KD value for antigen-binding activity at pH 7.4 (KD (pH 5.8)/ KD (pH 7.4)) is 2 or more when the KD (pH 5.8) and KD (pH 7.4) values are determined using a surface plasmon resonance technique in which the antibody is immobilized, the antigen serves as analyte, and the following conditions are used;

10 mM 2-(N-morpholino)ethanesulfonic acid (MES) pH 5.8 or pH 7.4, 150 mM NaCl, 0.05% polysorbate 20, at 37°

C.;

(ii) the antigen-binding activity is lower at a calcium ion concentration of 3μ

M than at a calcium ion concentration of 2 mM, wherein the ratio of the KD value for antigen-binding activity at a calcium ion concentration of 3 μ

M to the KD value for antigen-binding activity at a calcium ion concentration of 2 mM (KD (3 μ

M)/ KD (2 mM)) is 2 or more when the KD (3 μ

M) and KD (2 mM) values are determined using a surface plasmon resonance technique in which the antibody is immobilized, the antigen serves as analyte, and the following conditions are used;

10 mM N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) pH 7.4, 150 mM NaCl, 0.05% polysorbate 20, and either 2mM CaCl₂ or 3 μ

M CaCl₂, at 37°

C.;

producing a second antibody comprising

(A) a second Fc region comprising two Fc polypeptides with different amino acid sequences, wherein one of the two Fc polypeptides has detectable human FcRn-binding activity at pH 7.4 and the other Fc polypeptide does not, and wherein the ability of the second Fc region to form a heterocomplex with two molecules of the human FcRn and one molecule of the activating Fcγ

receptor at pH 7.4 is reduced compared to the ability of the first antibody to form such a heterocomplex at pH 7.4, and

(B) an antigen-binding domain identical to the antigen-binding domain of the first antibody; and

conducting an assay to confirm that the second antibody has decreased immunogenicity compared to the first antibody, wherein the activating Fcγ

receptor is human Fcγ

RIa, human Fcγ

RIIa(R), human Fcγ

RIIa(H), human Fcγ

RIIIa(V), or human Fcγ

RIIIa(F).

9. The method of claim 3, wherein the two Fc polypeptides differ from each other at one or more positions including at least one of the following positions:

237, 248, 250, 252, 254, 255, 256, 257, 258, 265, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434, 436 (by EU numbering).

10. The method of claim 3, wherein the second antibody'"'"'s Fc polypeptide that has detectable FcRn-binding activity at pH 7.4 differs from the native human IgG1 Fc region by amino acid substitution at one or more positions, including at least one of the following positions:

237, 248, 250, 252, 254, 255, 256, 257, 258, 265, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434, 436 (by EU numbering).

11. The method of claim 3, wherein at least one of the following positions in the second antibody'"'"'s Fc polypeptide that has detectable FcRn-binding activity at pH 7.4 is occupied by one of the amino acid residues listed for that position below (by EU numbering):

Met at position 237;

Ile at position 248;

Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr at position 250;

Phe, Trp, or Tyr at position 252;

Thr at position 254;

Glu at position 255;

Asn, Asp, Glu, or Gln at position 256;

Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val at position 257;

His at position 258;

Ala at position 265;

Ala or Glu at position 286;

His at position 289;

Ala at position 297;

Gly at position 298;

Ala at position 303;

Ala at position 305;

Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr position 307;

Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr at position 308;

Ala, Asp, Glu, Pro, or Arg at position 309;

Ala, His, or Ile at position 311;

Ala or His at position 312;

Lys or Arg at position 314;

Ala, Asp, or His at position 315;

Ala at position 317;

Val at position 332;

Leu at position 334;

His at position 360;

Ala at position 376;

Ala at position 380;

Ala at position 382;

Ala at position 384;

Asp or His at position 385;

Pro at position 386;

Glu at position 387;

Ala or Ser at position 389;

Ala at position 424;

Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr at position 428;

Lys at position 433;

Ala, Phe, His, Ser, Trp, or Tyr at position 434;

His, Ile, Leu, Phe, Thr, or Val at position 436.

17. The method of claim 3, wherein the antigen-binding activity of the antigen-binding domain varies with pH as described in (i), and the antigen-binding domain comprises a histidine residue at each of one or more positions selected from the group consisting of H chain variable region positions 27, 31, 32, 33, 35, 50, 58, 59, 61, 62, 99, 100b and 102, and L chain variable region positions 24, 27, 28, 31, 32, 50, 52, 53, 54, 55, 56, 89, 90, 91, 92, 93, and 94 (by Kabat numbering).

20. The method of claim 3, wherein the antigen-binding activity of the antigen-binding domain varies with calcium ion concentration as described in (ii), and at least one position of the antigen-binding domain selected from the following list of H and L chain positions is occupied by an amino acid residue having metal-chelating activity:

H chain variable region positions 95, 96, 100a and 101, and L chain variable region positions 30, 31, 32, 50, and 92 (by Kabat numbering).

24. The method of claim 3, wherein the two Fc polypeptides of the second Fc region differ from each other at one or more amino acid positions, including at least one of the following positions:

248, 289, 314, 315, 360, 384, 386, 387, 389, 424 (by EU numbering).

25. The method of claim 3, wherein the second antibody'"'"'s Fc polypeptide that has detectable human FcRn-binding activity at pH 7.4 differs from the native human IgG1 Fc region by amino acid substitution at one or more of the following positions:

248, 289, 314, 315, 360, 384, 386, 387, 389, 424 (by EU numbering).

28. The method of claim 3, further comprising assaying the second antibody'"'"'s ability to form a heterocomplex with two molecules of the human FcRn and one molecule of the activating Fcγ

receptor at pH 7.4, and thereby determining that the second antibody'"'"'s ability to form such a heterocomplex is reduced compared to the ability of the first antibody to form such a heterocomplex at pH 7.4.

32. The method of claim 3, comprising assaying the second antibody'"'"'s ability to form the heterocomplex with two molecules of the human FcRn and one molecule of the activating Fcγ

R at a pH that is in the range from pH 6.7 to pH 10.0.