Fc domain transformation

Abstract

Provided is a polypeptide comprising a modified antibody Fc region that, compared to a polypeptide comprising the Fc region of native IgG, maintains its binding activity to FcγRIIb while reducing its binding activity to all activating FcγRs, particularly FcγRIIa (R type). [Solution] A polypeptide is provided that contains an antibody Fc region variant that includes an amino acid sequence in which an amino acid modification at EU numbering position 238 is combined with other specific amino acid modifications.

Description

[Technical Field]

[0001] The present invention relates to Fc region variants that have been produced by introducing amino acid modifications into the Fc region of an antibody, which can reduce binding activity to all activating FcγRs, particularly FcγRIIa (R type), while maintaining FcγRIIb-binding activity, when compared to polypeptides comprising the Fc region of native human IgG; polypeptides comprising the Fc region variants; and pharmaceutical compositions containing the polypeptides. [Background technology]

[0002] Antibodies have attracted attention as pharmaceuticals due to their high stability in plasma and minimal side effects. Many IgG-type antibody drugs are currently on the market, and numerous antibody drugs are currently being developed (Non-Patent Documents 1 and 2). Meanwhile, various technologies applicable to second-generation antibody drugs have been developed, including those that improve effector function, antigen-binding ability, pharmacokinetics, and stability, or reduce the risk of immunogenicity (Non-Patent Document 3). Because antibody drugs generally require very high dosages, challenges include the difficulty of preparing subcutaneous formulations and high manufacturing costs. Potential methods for reducing the dosage of antibody drugs include improving the pharmacokinetics of antibodies and improving the affinity between antibodies and antigens.

[0003] Artificial amino acid substitution in the constant region has been reported as a method for improving the pharmacokinetics of antibodies (Non-Patent Document 4 and Non-Patent Document 5). Affinity maturation technology (Non-Patent Document 6) has been reported as a technique for enhancing antigen-binding ability and antigen-neutralizing ability, and it is possible to enhance antigen-binding activity by introducing mutations into amino acids in the CDR region of the variable region, etc. By enhancing antigen-binding ability, it is possible to improve in vitro biological activity or reduce dosage, and it is also possible to improve in vivo (living organism) efficacy (Non-Patent Document 7).

[0004] On the other hand, the amount of antigen that can be neutralized per antibody molecule depends on affinity, and by increasing affinity, it is possible to neutralize an antigen with a small amount of antibody. Various methods can increase antibody affinity (Non-Patent Document 6). Furthermore, if an antibody can be covalently bound to an antigen and affinity can be increased infinitely, it would be possible for a single antibody molecule to neutralize a single antigen molecule (two antigens in the case of a bivalent antibody). However, previous methods have limited the ability of a single antibody molecule to bind to a single antigen molecule (two antigens in the case of a bivalent antibody). On the other hand, it has recently been reported that by using an antigen-binding molecule that binds to an antigen in a pH-dependent manner, a single antigen-binding molecule can bind to multiple antigen molecules (Patent Document 1, Non-Patent Document 8). pH-dependent antigen-binding molecules bind strongly to antigens under neutral conditions in plasma and dissociate from the antigen under acidic conditions in endosomes. Furthermore, after dissociation, the antigen-binding molecule is recycled into plasma by FcRn and can re-bind to the antigen, enabling a single pH-dependent antigen-binding molecule to repeatedly bind to multiple antigens.

[0005] Furthermore, it has been reported that pH-dependent antigen-binding molecules modified to enhance FcRn binding under neutral conditions (pH 7.4) have the effect of repeatedly binding to antigens and eliminating antigens from plasma, and therefore, administration of such antigen-binding molecules can remove antigens from plasma (Patent Document 2). pH-dependent antigen-binding molecules containing the Fc region of a conventional IgG antibody show almost no binding to FcRn under neutral conditions. Therefore, intracellular uptake of the antigen-binding molecule complex is thought to be mainly due to nonspecific uptake. According to this report, pH-dependent antigen-binding molecules modified to enhance FcRn binding under neutral conditions (pH 7.4) can further accelerate antigen elimination compared to pH-dependent antigen-binding molecules containing the Fc region of a conventional IgG antibody (Patent Document 2).

[0006] Because antigens have a much shorter plasma retention time than antibodies that have an FcRn-mediated recycling mechanism, binding of antigens to antibodies that have this recycling mechanism (i.e., whose binding is not pH-dependent) in plasma typically prolongs their plasma retention time and increases the plasma antigen concentration. For example, if an antigen in plasma has multiple physiological functions, even if one physiological activity is blocked by antibody binding, the plasma concentration of the antigen may increase, exacerbating symptoms caused by other physiological functions due to antibody binding. From this perspective, it is sometimes preferable to eliminate antigens from plasma. While methods have been reported in which modifications to the Fc region, such as those described above, that enhance FcRn binding, are used to accelerate antigen elimination, no other methods for accelerating antigen elimination have been reported.

[0007] In addition, some antibody drugs have been reported to have side effects resulting from the interaction between IgG and FcγR. For example, it is known that the frequency of thromboembolism increases in patients administered bevacizumab, an antibody against VEGF (Non-Patent Document 9). Similarly, thromboembolism was observed in clinical development trials of an antibody against CD40 ligand, leading to the discontinuation of the clinical trial (Non-Patent Document 10). The activating Fcγ receptor FcγRIIa is expressed on platelets (Non-Patent Document 11). Subsequent studies using animal models have suggested that all administered antibodies induce platelet aggregation via binding to FcγRIIa on platelets, resulting in thrombus formation (Non-Patent Documents 12 and 13). It has been reported that platelets are activated by an FcγRIIa-dependent mechanism in patients with systemic lupus erythematosus, an autoimmune disease, and that platelet activation correlates with the severity of the disease (Non-Patent Document 14). Furthermore, previous studies using animal models have reported that immune complexes of antibodies and multivalent antigens induce anaphylaxis via activating FcγR (Non-Patent Document 15). In addition, it has been reported that the uptake of immune complexes between polyvalent antigens and antibodies via activated FcγR increases the production of antibody titers against those antigens (Non-Patent Documents 16 and 17). This result suggests that antibody drugs that recognize polyvalent antigens may be more likely to produce antibodies against the antibody drug itself. If antibodies against antibody drugs are produced, their blood dynamics may worsen, or neutralizing antibodies may weaken the drug's efficacy. In this way, antibodies bind to multivalent antigens to form immune complexes, and these complexes may interact with activating FcγRs to induce various side effects, reducing the value of antibodies as pharmaceuticals. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. WO2009 / 125825 [Patent Document 2] International Publication No. WO2011 / 122011 [Non-patent literature]

[0009] [Non-Patent Document 1] Monoclonal antibody successes in the clinic, Janice M Reichert, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz, Nat. Biotechnol. (2005) 23, 1073 - 1078 [Non-patent document 2] Pavlou AK, Belsey MJ., The therapeutic antibodies market to 2008., Eur. J. Pharm. Biopharm. (2005) 59 (3), 389-396 [Non-patent document 3] Kim SJ, Park Y, Hong HJ., Antibody engineering for the development of therapeutic antibodies., Mol. Cells. (2005) 20 (1), 17-29

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[0010] The present invention was made in light of these circumstances, and its object is to provide molecules that accelerate antigen elimination while overcoming the drawbacks associated with binding to activating FcγRs by introducing amino acid modifications into the Fc region of an antibody. Specifically, the present invention provides Fc region variants that, compared to polypeptides comprising the Fc region of a native IgG antibody, maintain FcγRIIb-binding activity but can reduce binding activity to all activating FcγRs, particularly FcγRIIa (R type), as well as polypeptides comprising the Fc region variants and pharmaceutical compositions containing the polypeptides. [Means for solving the problem]

[0011] The present inventors have conducted extensive research into Fc region variants that, by introducing amino acid modifications into the Fc region, can reduce binding activity to all activating FcγRs, particularly FcγRIIa (R type), while maintaining FcγRIIb binding activity compared to polypeptides containing the Fc region of native IgG. As a result, the present inventors have found that by combining an Fc region variant in which the amino acid at EU numbering position 238 in the Fc region has been modified with other amino acid modifications, it is possible to reduce binding activity to all activating FcγRs, particularly FcγRIIa (R type), while maintaining FcγRIIb binding activity.

[0012] That is, the present invention relates to the following. [1] An Fc region variant comprising an amino acid alteration at position 238 (EU numbering) in the Fc region and any of the amino acid alterations (a) to (k) below, wherein the variant maintains its FcγRIIb-binding activity and has reduced binding activity to all activating FcγRs when compared to the Fc region of a native IgG. (a) Amino acid 235 (EU numbering) in the Fc region (b) Amino acid 237 (EU numbering) in the Fc region (c) Amino acid 241 in the Fc region (EU numbering) (d) Amino acid 268 (EU numbering) in the Fc region (e) Amino acid 295 (EU numbering) in the Fc region (f) Amino acid 296 (EU numbering) in the Fc region (g) Amino acid 298 (EU numbering) in the Fc region (h) Amino acid 323 (EU numbering) in the Fc region (i) Amino acid 324 (EU numbering) in the Fc region (j) Amino acid 330 (EU numbering) in the Fc region (k) at least two amino acids selected from (a) to (j) [2] The variant according to [1] above, wherein at least two amino acids selected in (k) of [1] above are a combination of amino acids set forth in any one of (1) to (3) below: (1) amino acids at positions 241, 268, 296, and 324 (EU numbering) in the Fc region (2) amino acids 237, 241, 296, and 330 (EU numbering) in the Fc region (3) amino acids at positions 235, 237, 241, and 296 (EU numbering) in the Fc region [3] A variant Fc region in which the amino acid at position 238 (EU numbering) in the Fc region is Asp and which has any of the amino acids (a) to (k) below, wherein the variant maintains its FcγRIIb-binding activity and has reduced binding activity to all activating FcγRs when compared to the Fc region of a native IgG. (a) Amino acid 235 in the Fc region (EU numbering) is Phe (b) the amino acid at position 237 (EU numbering) in the Fc region is Gln or Asp (c) Amino acid 241 (EU numbering) in the Fc region is Met or Leu (d) The amino acid at position 268 (EU numbering) in the Fc region is Pro (e) the amino acid at position 295 (EU numbering) in the Fc region is Met or Val; (f) the amino acid at position 296 (EU numbering) of the Fc region is Glu, His, Asn, or Asp; (g) the amino acid at position 298 (EU numbering) of the Fc region is Ala or Met; (h) the amino acid at position 323 (EU numbering) in the Fc region is Ile; (i) The amino acid at position 324 (EU numbering) of the Fc region is Asn or His (j) The amino acid at position 330 (EU numbering) of the Fc region is His or Tyr (k) at least two amino acids selected from (a) to (j) [4] A variant Fc region in which the amino acid at position 238 (EU numbering) in the Fc region is Asp and which has an amino acid selected from any of the following (1) to (3): (1) In the Fc region, the amino acid at position 241 (EU numbering) is Met, the amino acid at position 268 is Pro, the amino acid at position 296 is Glu, and the amino acid at position 324 is His. (2) The Fc region contains Gln or Asp at amino acid position 237, Met at amino acid position 241, Glu at amino acid position 296, and His at amino acid position 330 (EU numbering). (3) In the Fc region, the amino acid at position 235 (EU numbering) is Phe, the amino acid at position 237 is Gln or Asp, the amino acid at position 241 is Met, and the amino acid at position 296 is Glu [5] A variant Fc region comprising amino acid alterations at amino acids 238 and 271 (EU numbering) in the Fc region, and any of the amino acid alterations (a) to (h) below, wherein the variant maintains its FcγRIIb-binding activity and has reduced binding activity to all activating FcγRs, when compared to the Fc region of a native IgG. (a) Amino acid 234 (EU numbering) in the Fc region (b) Amino acid 235 (EU numbering) in the Fc region (c) Amino acid 236 (EU numbering) in the Fc region (d) Amino acid 237 (EU numbering) in the Fc region (e) Amino acid 239 (EU numbering) in the Fc region (f) Amino acid 265 (EU numbering) in the Fc region (g) Amino acid 267 (EU numbering) in the Fc region (h) Amino acid 297 (EU numbering) in the Fc region [6] The variant according to [5], wherein the amino acid modifications are a combination of amino acid modifications according to any one of (1) to (3) below: (1) amino acids at positions 233, 238, 264, 267, 268, and 271 (EU numbering) in the Fc region (2) amino acids at positions 233, 237, 238, 264, 267, 268, 271, 296, 297, 330, and 396 (EU numbering) in the Fc region; (3) amino acids at positions 233, 238, 264, 267, 268, 271, and 296 (EU numbering) in the Fc region [7] An Fc region variant in which the amino acid at position 238 (EU numbering) is Asp and the amino acid at position 271 is Gly, and which has any of the amino acids (a) to (h) below, wherein the variant maintains its FcγRIIb-binding activity and has reduced binding activity to all activating FcγRs, when compared to the Fc region of a native IgG. (a) the amino acid at position 234 (EU numbering) of the Fc region is Ala, His, Asn, Lys, or Arg (b) Ala at amino acid position 235 (EU numbering) in the Fc region (c) The amino acid at position 236 (EU numbering) in the Fc region is Gln (d) the amino acid at position 237 (EU numbering) of the Fc region is Arg or Lys (e) The amino acid at position 239 (EU numbering) in the Fc region is Lys (f) the amino acid at position 265 (EU numbering) in the Fc region is Lys, Asn, Arg, Ser, or Val (g) the amino acid at position 267 (EU numbering) in the Fc region is Lys, Arg, or Tyr (h) Ala at amino acid position 297 (EU numbering) in the Fc region [8] A variant Fc region, in which the amino acid at position 238 (EU numbering) is Asp and the amino acid at position 271 (EU numbering) is Gly in the Fc region, and which contains any of the amino acids (1) to (3) below: (1) In the Fc region, the amino acid at position 233 (EU numbering) is Asp, the amino acid at position 238 is Asp, the amino acid at position 264 is Ile, the amino acid at position 267 is Arg, the amino acid at position 268 is Glu, and the amino acid at position 271 is Gly (2) In the Fc region, the amino acid at position 233 is Asp, the amino acid at position 237 is Asp, the amino acid at position 238 is Asp, the amino acid at position 264 is Ile, the amino acid at position 267 is Ala, the amino acid at position 268 is Glu, the amino acid at position 271 is Gly, the amino acid at position 296 is Asp, the amino acid at position 297 is Ala, the amino acid at position 330 is Arg, and the amino acid at position 396 is Met (EU numbering). (3) In the Fc region, the amino acid at position 233 (EU numbering) is Asp, the amino acid at position 238 is Asp, the amino acid at position 264 is Ile, the amino acid at position 267 is Arg, the amino acid at position 268 is Pro, the amino acid at position 271 is Gly, and the amino acid at position 296 is Glu [9] An Fc region variant described in any of [1] to [8] above, further having reduced complement binding.

[10] The Fc region variant described in [9], wherein the Fc region variant with reduced complement binding comprises an amino acid modification at EU numbering position 322 in the Fc region, or amino acid modifications at EU numbering positions 327, 330, and 331 in the Fc region.

[11] The Fc region variant according to [9], wherein the amino acid at position 322 (EU numbering) of the Fc region is Ala or Glu, or the amino acid at position 327 (EU numbering) of the Fc region is Gly, the amino acid at position 330 is Ser, and the amino acid at position 331 is Ser, all of which are amino acids according to EU numbering.

[12] A variant Fc region comprising amino acid alterations at amino acids 238, 271, 327, 330, and 331 (EU numbering) in the Fc region, wherein the variant maintains its FcγRIIb-binding activity and has reduced binding activity to all activating FcγRs when compared to the Fc region of a native IgG.

[13] The variant according to

[12] above, further comprising any one of the following amino acid modifications (a) to (e): (a) Amino acid 233 in the Fc region (EU numbering) (b) Amino acid 237 (EU numbering) in the Fc region (c) Amino acid 264 (EU numbering) in the Fc region (d) Amino acid 267 (EU numbering) in the Fc region (e) Amino acid 268 (EU numbering) in the Fc region

[14] The variant according to

[13] , wherein the amino acid modification is a combination of amino acid modifications according to any one of (1) to (4) below: (1) amino acids at positions 237, 238, 268, 271, 327, 330, and 331 (EU numbering) in the Fc region (2) amino acids at positions 233, 237, 238, 268, 271, 327, 330, and 331 (EU numbering) of the Fc region; (3) amino acids at positions 238, 267, 268, 271, 327, 330, and 331 (EU numbering) in the Fc region (4) amino acids at positions 238, 264, 267, 271, 327, 330, and 331 (EU numbering) in the Fc region

[15] A variant Fc region in which the amino acid at position 238 (EU numbering) is Asp, the amino acid at position 271 is Gly, the amino acid at position 327 is Gly, the amino acid at position 330 is Ser, and the amino acid at position 331 is Ser (EU numbering), wherein the variant maintains its FcγRIIb-binding activity and has reduced binding activity to all activating FcγRs when compared to the Fc region of a native IgG.

[16] The variant according to

[15] above, further comprising any one of the following amino acids (a) to (h): (a) The amino acid at position 233 (EU numbering) in the Fc region is Asp (b) Asp at amino acid position 237 (EU numbering) in the Fc region (c) Amino acid 264 (EU numbering) in the Fc region is Ile (d) Ala at amino acid position 267 (EU numbering) in the Fc region (e) the amino acid at position 268 (EU numbering) of the Fc region is Asp or Glu

[17] A variant Fc region, in which the amino acid at position 238 (EU numbering) is Asp and the amino acid at position 271 (EU numbering) is Gly in the Fc region, and which contains any of the amino acids (1) to (4) below: (1) In the Fc region, the amino acid at position 237 (EU numbering) is Asp, the amino acid at position 238 is Asp, the amino acid at position 268 is Asp or Glu, the amino acid at position 271 is Gly, the amino acid at position 327 is Gly, the amino acid at position 330 is Ser, and the amino acid at position 331 is Ser (EU numbering). (2) In the Fc region, the amino acid at position 233 is Asp, the amino acid at position 237 is Asp, the amino acid at position 238 is Asp, the amino acid at position 268 is Asp, the amino acid at position 271 is Gly, the amino acid at position 327 is Gly, the amino acid at position 330 is Ser, and the amino acid at position 331 is Ser (EU numbering). (3) In the Fc region, the amino acid at position 238 (EU numbering) is Asp, the amino acid at position 267 is Ala, the amino acid at position 268 is Glu, the amino acid at position 271 is Gly, the amino acid at position 327 is Gly, the amino acid at position 330 is Ser, and the amino acid at position 331 is Ser (EU numbering). (4) In the Fc region, the amino acid at position 238 (EU numbering) is Asp, the amino acid at position 264 is Ile, the amino acid at position 267 is Ala, the amino acid at position 271 is Gly, the amino acid at position 327 is Gly, the amino acid at position 330 is Ser, and the amino acid at position 331 is Ser (EU numbering).

[18] The Fc region variant of any of [1] to

[17] above, wherein the binding activity to FcγRIIb is at least 80% of the binding amount of the Fc region of native IgG, and the binding activity to FcγRIIaR is 30% or less of the binding amount of the Fc region of native IgG.

[19] An Fc region variant described in any of [1] to

[18] above, which has a relative binding activity ratio of at least 0.75 compared to the binding activity to FcγRIIb of a polypeptide comprising the Fc region of native IgG, and a binding activity ratio to all activating FcγRs of 0.2 or less.

[20] The Fc region variant according to

[19] above, further comprising a relative binding activity of 0.1 or less compared to the binding activity of a polypeptide comprising the Fc region of native IgG to FcγRIIa R .

[21] A polypeptide comprising a variant Fc region described in any of [1] to

[20] .

[22] The polypeptide described in

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

[23] The polypeptide described in

[21] , wherein the polypeptide containing the Fc region variant is an Fc fusion protein molecule.

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

[21] to

[23] above and a medically acceptable carrier.

[25] The polypeptide according to

[21] , further comprising an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions.

[26] The polypeptide described in

[25] above, wherein the ion concentration conditions are calcium ion concentration conditions.

[27] The polypeptide of

[26] , wherein the antigen-binding domain has lower antigen-binding activity under a low calcium ion concentration condition than under a high calcium ion concentration condition.

[28] The polypeptide according to any one of

[25] to

[27] , wherein the ion concentration conditions are pH conditions.

[29] The polypeptide of

[28] , wherein the antigen-binding domain has lower antigen-binding activity in an acidic pH range than in a neutral pH range.

[30] The polypeptide described in any one of

[25] to

[29] , wherein the polypeptide containing the Fc region variant is an IgG antibody.

[31] A polypeptide described in any of

[25] to

[29] , wherein the polypeptide containing the Fc region variant is an Fc fusion protein molecule.

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

[25] to

[31] above and a medically acceptable carrier.

[33] The pharmaceutical composition according to

[32] , wherein the pharmaceutical composition is an antigen in plasma that binds to the antigen-binding domain of the polypeptide according to any one of

[25] to

[31] , and the pharmaceutical composition is for promoting elimination of the antigen from plasma.

[34] Use of the polypeptide to promote the elimination from plasma of an antigen in plasma that binds to the antigen-binding domain of the polypeptide described in any one of

[25] to

[31] .

[35] A method for reducing the binding of a polypeptide comprising an Fc region to all activating FcγRs while maintaining the FcγRIIb-binding activity of the polypeptide, by altering at least one amino acid selected from amino acid 238 (EU numbering) of the Fc region, and amino acids 235, 237, 241, 268, 295, 296, 298, 323, 324, and 330 (EU numbering) of the Fc region to another amino acid.

[36] The method according to

[35] above, wherein the amino acid modification in the Fc region is substitution of amino acid 238 (EU numbering) with Asp, substitution of amino acid 235 with Phe, substitution of amino acid 237 with Gln, substitution of amino acid 241 with Met or Leu, substitution of amino acid 268 with Pro, substitution of amino acid 295 with Met or Val, substitution of amino acid 296 with Glu, His, Asn, or Asp, substitution of amino acid 298 with Ala or Met, substitution of amino acid 323 with Ile, substitution of amino acid 324 with Asn or His, or substitution of amino acid 330 with His or Tyr.

[37] A method for producing a polypeptide comprising an Fc region variant in which at least one amino acid selected from amino acid 238 (EU numbering), and amino acids 235, 237, 241, 268, 295, 296, 298, 323, 324, and 330 (EU numbering) in the Fc region is altered to another amino acid, thereby maintaining FcγRIIb-binding activity while reducing binding to all activating FcγRs compared to before the alteration.

[38] The method according to

[37] above, wherein the amino acid modification in the Fc region is substitution of amino acid 238 with Asp (EU numbering), substitution of amino acid 235 with Phe, substitution of amino acid 237 with Gln, substitution of amino acid 241 with Met or Leu, substitution of amino acid 268 with Pro, substitution of amino acid 295 with Met or Val, substitution of amino acid 296 with Glu, His, Asn, or Asp, substitution of amino acid 298 with Ala or Met, substitution of amino acid 323 with Ile, substitution of amino acid 324 with Asn or His, or substitution of amino acid 330 with His or Tyr.

[39] A method for reducing the binding activity to all activating FcγRs while maintaining the same level of FcγRIIb-binding activity as native IgG, by introducing into a polypeptide containing an Fc region a combination of amino acid alterations that increase the FcγRIIb-binding activity by at least two times that of the Fc region of native IgG, and amino acid alterations that reduce the binding activity to all FcγRs.

[40] The method according to

[39] , wherein the amino acid modification that results in FcγRIIb-binding activity that is at least twice as high as that of the Fc region of native IgG is an amino acid modification listed in Table 11.

[41] The method of

[39] or

[40] , wherein the amino acid modification that reduces the binding activity to all FcγRs is a modification of at least one amino acid selected from amino acids 234, 235, 236, 237, 239, 265, 267, and 297 (EU numbering) in the Fc region with another amino acid.

[42] The method of any of

[39] to

[41] above, wherein the amino acid modification in the Fc region is substitution of amino acid 234 (EU numbering) with Ala, His, Asn, Lys, or Arg, substitution of amino acid 235 with Ala, substitution of amino acid 236 with Gln, substitution of amino acid 237 with Arg or Lys, substitution of amino acid 239 with Lys, substitution of amino acid 265 with Lys, Asn, Arg, Ser, or Val, substitution of amino acid 267 with Lys, Arg, or Tyr, or substitution of amino acid 297 with Ala.

[43] The method of any one of

[35] ,

[36] , and

[39] to

[42] above, wherein the binding activity to FcγRIIb is maintained at at least 80% of the binding amount of the Fc region of native IgG, and the binding activity to FcγRIIaR is reduced to 30% or less of the binding amount of the Fc region of native IgG.

[44] The method of any of

[35] ,

[36] , and

[39] to

[43] , wherein the ratio of relative binding activity compared to the binding activity to FcγRIIb of a polypeptide comprising the Fc region of native IgG is maintained at least 0.75, and the ratio of binding activity to all activating FcγRs is reduced to 0.2 or less.

[45] The method according to

[44] , wherein the ratio of the relative binding activity to FcγRIIa R compared with the binding activity of a polypeptide comprising the Fc region of native IgG is further reduced to 0.05 or less.

[46] A method for producing a polypeptide containing an Fc region variant that maintains the same level of FcγRIIb-binding activity as native IgG but reduces its binding activity to all activating FcγRs, by introducing a combination of amino acid alterations that increase the FcγRIIb-binding activity by at least two times that of the Fc region of native IgG, and amino acid alterations that reduce its binding activity to all FcγRs.

[47] The method according to

[46] above, wherein the amino acid modification that results in FcγRIIb-binding activity that is at least twice as high as that of the Fc region of native IgG is an amino acid modification listed in Table 11.

[48] ​​The method of

[46] or

[47] , wherein the amino acid modification that reduces the binding activity to all FcγRs is a modification of at least one amino acid selected from amino acids 234, 235, 236, 237, 239, 265, 267, and 297 (EU numbering) in the Fc region with another amino acid.

[49] The method of any of

[46] to

[48] above, wherein the amino acid modification in the Fc region is substitution of amino acid 234 (EU numbering) with Ala, His, Asn, Lys, or Arg, substitution of amino acid 235 with Ala, substitution of amino acid 236 with Gln, substitution of amino acid 237 with Arg or Lys, substitution of amino acid 239 with Lys, substitution of amino acid 265 with Lys, Asn, Arg, Ser, or Val, substitution of amino acid 267 with Lys, Arg, or Tyr, or substitution of amino acid 297 with Ala.

[50] The method of any one of

[37] ,

[38] , and

[46] to

[49] above, wherein the binding activity to FcγRIIb is maintained at at least 80% of the binding amount of the Fc region of native IgG, and the binding activity to all activating FcγRs is reduced to 30% or less of the binding amount of the Fc region of native IgG.

[51] The method of any of

[37] ,

[38] , and

[46] to

[50] , wherein the ratio of relative binding activity compared to the binding activity to FcγRIIb of a polypeptide comprising the Fc region of native IgG is maintained at least 0.75, and the ratio of binding activity to all activating FcγRs is reduced to 0.2 or less.

[52] The method according to

[51] above, wherein the ratio of the relative binding activity compared to the binding activity of a polypeptide comprising the Fc region of native IgG to FcγRIIa R is reduced to 0.1 or less.

[53] A method according to any one of

[37] ,

[38] , and

[46] to

[52] , further comprising introducing a modification that reduces complement binding in combination.

[54] The method described in

[53] , wherein the modification that reduces complement binding is an amino acid modification at EU numbering position 322 in the Fc region, or an amino acid modification at EU numbering positions 327, 330, and 331 in the Fc region.

[55] The method according to

[53] , wherein the modification that reduces complement binding is a substitution of amino acid 322 (EU numbering) in the Fc region with Ala or Glu, or a substitution of amino acid 327 (EU numbering) in the Fc region with Gly, a substitution of amino acid 330 with Ser, and a substitution of amino acid 331 with Ser.

[56] A method for reducing the binding of a polypeptide comprising an Fc region to all activating FcγRs while maintaining the FcγRIIb-binding activity of the polypeptide, by altering at least one amino acid selected from amino acids 238, 271, 327, 330, and 331 (EU numbering) of the Fc region, or further selected from amino acids 233, 237, 264, 267, and 268 (EU numbering) of the Fc region to another amino acid.

[57] The method according to

[56] above, wherein the amino acid modification in the Fc region is substitution of amino acid 238 (EU numbering) in the Fc region with Asp, substitution of amino acid 271 with Gly, substitution of amino acid 327 with Gly, substitution of amino acid 330 with Ser, substitution of amino acid 331 with Ser, substitution of amino acid 233 with Asp, substitution of amino acid 237 with Asp, substitution of amino acid 264 with Ile, substitution of amino acid 267 with Ala, or substitution of amino acid 268 with Asp or Glu.

[58] A method for producing a polypeptide containing an Fc region variant, in which at least one amino acid selected from amino acids 238, 271, 327, 330, and 331 (EU numbering) of the Fc region, or further amino acids 233, 237, 264, 267, and 268 (EU numbering) of the Fc region is altered to another amino acid, thereby maintaining FcγRIIb-binding activity, reducing binding to all activating FcγRs, and reducing complement binding compared to before the alteration.

[59] The method according to

[58] above, wherein the amino acid modification in the Fc region is substitution of amino acid 238 (EU numbering) in the Fc region with Asp, substitution of amino acid 271 with Gly, substitution of amino acid 327 with Gly, substitution of amino acid 330 with Ser, substitution of amino acid 331 with Ser, substitution of amino acid 233 with Asp, substitution of amino acid 237 with Asp, substitution of amino acid 264 with Ile, substitution of amino acid 267 with Ala, or substitution of amino acid 268 with Asp or Glu.

[0013] The present invention also relates to methods for reducing the binding activity of an Fc region to all activating FcγRs, particularly FcγRIIa (R type), while maintaining the FcγRIIb-binding activity of the Fc region by introducing amino acid alterations into the Fc region of the present invention.The present invention also relates to methods for suppressing the production of antibodies against a polypeptide comprising the Fc region of the present invention by introducing amino acid alterations into the Fc region of the present invention. The present invention further relates to methods for promoting elimination of an antigen from plasma using an Fc region variant of the present invention incorporating amino acid modifications in the Fc region, and a polypeptide comprising an antigen-binding domain that is present in soluble form in plasma and has binding activity for a pathogenic antigen, the binding activity of which changes depending on ion concentration conditions. The present invention also relates to the use of an Fc region variant of the present invention incorporating amino acid modifications in the Fc region, and a polypeptide that is present in soluble form in plasma and has binding activity for a pathogenic antigen, the binding activity of which changes depending on ion concentration conditions, for promoting elimination of the antigen from plasma.

[0014] The present invention also relates to a therapeutic or preventive agent for immunoinflammatory diseases, which comprises the polypeptide of the present invention. It also relates to a therapeutic or preventive method for immunoinflammatory diseases, which comprises the step of administering the polypeptide of the present invention to a subject. The present invention also relates to a kit for use in the therapeutic or preventive method for immunoinflammatory diseases, which comprises the polypeptide of the present invention. The present invention also relates to use of the polypeptide of the present invention in the production of a therapeutic or preventive agent for immunoinflammatory diseases. The present invention also relates to the polypeptide of the present invention for use in the therapeutic or preventive method for immunoinflammatory diseases.

[0015] The present invention also relates to an agent for inhibiting the activation of B cells, mast cells, dendritic cells, and / or basophils, which comprises a polypeptide of the present invention. The present invention also relates to a method for inhibiting the activation of B cells, mast cells, dendritic cells, and / or basophils, which comprises the step of administering a polypeptide of the present invention to a subject. The present invention also relates to a kit for use in the method for inhibiting the activation of B cells, mast cells, dendritic cells, and / or basophils, which comprises a polypeptide of the present invention. The present invention also relates to use of a polypeptide of the present invention in the production of an agent for inhibiting the activation of B cells, mast cells, dendritic cells, and / or basophils. The present invention also relates to a polypeptide of the present invention for use in the method for inhibiting the activation of B cells, mast cells, dendritic cells, and / or basophils.

[0016] The present invention also relates to a therapeutic agent for a disease caused by a deficiency in a protein necessary for the body, comprising the polypeptide of the present invention. The present invention also relates to a method for treating a disease caused by a deficiency in a protein necessary for the body, comprising the step of administering the polypeptide of the present invention to a subject. The present invention also relates to a kit for use in the method for treating a disease caused by a deficiency in a protein necessary for the body, comprising the polypeptide of the present invention. The present invention also relates to the use of the polypeptide of the present invention in the production of a therapeutic agent for a disease caused by a deficiency in a protein necessary for the body. The present invention also relates to the polypeptide of the present invention for use in the method for treating a disease caused by a deficiency in a protein necessary for the body.

[0017] The present invention also relates to a viral growth inhibitor comprising the polypeptide of the present invention. The present invention also relates to a method for inhibiting viral growth comprising the step of administering the polypeptide of the present invention to a subject. The present invention also relates to a kit for use in the method for inhibiting viral growth of the present invention, comprising the polypeptide of the present invention. The present invention also relates to use of the polypeptide of the present invention in the production of an agent for inhibiting viral growth. The present invention also relates to the polypeptide of the present invention for use in the method for inhibiting viral growth of the present invention. [Effects of the Invention]

[0018] The present invention provides Fc region variants that maintain FcγRIIb-binding activity and exhibit reduced binding activity to all activating FcγRs, particularly FcγRIIa (R type), compared to the Fc region of native IgG. Use of polypeptides containing these Fc region variants makes it possible to enhance the inhibitory signal of inflammatory immune responses mediated by ITIM phosphorylation of FcγRIIb, while maintaining the ability to eliminate immune complexes mediated by FcγRIIb to a similar extent as native IgG. Furthermore, conferring the ability to selectively bind to FcγRIIb to an Fc region may enable the suppression of anti-antibody production. Furthermore, reduced binding to activating FcγRs makes it possible to avoid platelet activation mediated by the interaction of immune complexes with FcγRIIa on platelets and dendritic cell activation due to crosslinking of activating FcγRs. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 shows the results of gel filtration chromatography analysis confirming that human IgE and the pH-dependent anti-IgE antibody clone 278 form large immune complexes in a pH-dependent manner. [Figure 2] FIG. 1 shows the time course of human IgE concentration in plasma of normal mice in a group administered with human IgE alone, a group administered with human IgE + clone 278 antibody, and a group administered with human IgE + clone 278 antibody. [Figure 3]FIG. 1 shows the time course of antibody concentrations in plasma of normal mice in the human IgE+ clone 278-administered group and the human IgE+Xolair antibody-administered group. [Figure 4] FIG. 1 shows the time course of human IgE concentration in plasma of normal mice in the human IgE alone administration group, the human IgE+278-IgG1 antibody administration group, and the human IgE+278-F760 antibody administration group. [Figure 5] FIG. 1 shows the time course of normal mouse antibody concentrations in the human IgE+278-IgG1 antibody administration group and the human IgE+278-F760 antibody administration group. [Figure 6] FIG. 1 shows the results of evaluating DC activation by Fc variants using the expression level of IL-8 as an index. [Figure 7] This figure confirms the expression of CD62p (p-selectin) on the membrane surface of washed platelets when Fc variants were added. The solid line represents the result when 5c8-F648 was added and ADP stimulation was performed, and the solid line represents the result when 5c8-P600 was added and ADP stimulation was performed. [Figure 8] This figure confirms the expression of activated integrin (PAC-1) on the membrane surface of washed platelets when Fc variants were added. The solid line represents the result when 5c8-F648 was added and ADP stimulation was performed, and the solid line represents the result when 5c8-P600 was added and ADP stimulation was performed. [Figure 9] This figure shows that a pH-dependent binding antibody repeatedly binds to a soluble antigen. (i) The antibody binds to a soluble antigen, (ii) it is nonspecifically taken up into cells by pinocytosis, (iii) the antibody binds to FcRn in the endosome, and the soluble antigen dissociates from the antibody, (iv) the soluble antigen is transported to the lysosome and degraded, (v) the antibody from which the soluble antigen has dissociated is recycled into plasma by FcRn, and (vi) the recycled antibody becomes able to bind to the soluble antigen again. [Figure 10]This figure shows that enhancing FcRn binding under neutral conditions further improves the repeatable antigen binding of a pH-dependent binding antibody. (i) The antibody binds to a soluble antigen, (ii) it is internalized into cells by pinocytosis via FcRn, (iii) the soluble antigen dissociates from the antibody in endosomes, (iv) the soluble antigen is translocated to lysosomes and degraded, (v) the antibody from which the soluble antigen has dissociated is recycled into plasma by FcRn, and (vi) the recycled antibody is able to bind to the soluble antigen again. [Figure 11] FIG. 1 shows sensorgrams using Biacore showing the interaction of anti-human IgA antibody with human IgA at pH 7.4 and pH 5.8, Ca 2+ 1.2 mM and Ca 2+ 3 μM. [Figure 12] FIG. 1 shows the time course of plasma antibody concentrations of GA2-IgG1 and GA2-F1087 in normal mice. [Figure 13] FIG. 1 shows the time course of plasma hIgA concentration in normal mice administered with hIgA alone, GA2-IgG1, or GA2-F1087. [Figure 14] FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of normal mice administered with Fv4-mIgG1, Fv4-mIgG1-mF44, a variant of Fv4-mIgG1 with enhanced binding to mouse FcγRIIb and mouse FcγRIII, or Fv4-mIgG1-mF46, a variant of Fv4-mIgG1 with further enhanced binding to mouse FcγRIIb and mouse FcγRIII. [Figure 15] FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of FcγRIII-deficient mice administered with Fv4-mIgG1, Fv4-mIgG1-mF44, a variant of Fv4-mIgG1 with enhanced binding to mouse FcγRIIb and mouse FcγRIII, or Fv4-mIgG1-mF46, a variant of Fv4-mIgG1 with further enhanced binding to mouse FcγRIIb and mouse FcγRIII. [Figure 16] FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of Fc receptor γ chain-deficient mice when the mice were administered Fv4-mIgG1, Fv4-mIgG1-mF44, a variant of Fv4-mIgG1 with enhanced binding to mouse FcγRIIb and mouse FcγRIII, or Fv4-mIgG1-mF46, a variant of Fv4-mIgG1 with further enhanced binding to mouse FcγRIIb and mouse FcγRIII. [Figure 17] FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of FcγRIIb-deficient mice administered with Fv4-mIgG1, Fv4-mIgG1-mF44, a variant of Fv4-mIgG1 with enhanced binding to mouse FcγRIIb and mouse FcγRIII, or Fv4-mIgG1-mF46, a variant of Fv4-mIgG1 with further enhanced binding to mouse FcγRIIb and mouse FcγRIII. [Figure 18] FIG. 1 shows the relationship between the amino acid residues constituting the constant regions of IgG1, IgG2, IgG3, and IgG4 and the EU numbering system (also referred to herein as EU INDEX). [Figure 19] FIG. 1 illustrates the efficiency of antigen elimination per antibody molecule of a multispecific pH / Ca-dependent antibody that recognizes two or more epitopes present in a monomeric antigen and is suitable for forming large immune complexes. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention provides Fc region variants that maintain binding activity to FcγRIIb when compared to polypeptides comprising the Fc region of a native IgG antibody, but can reduce binding activity to all activating FcγRs, particularly FcγRIIa (R type), and polypeptides comprising the Fc region variants.

[0021] More specifically, the present invention provides Fc region variants comprising an amino acid sequence in which the amino acid alteration at EU numbering position 238 is combined with other specific amino acid alterations, and polypeptides comprising the Fc region variants. Furthermore, the present invention provides methods for reducing binding activity to all activating FcγRs, particularly FcγRIIa (R type), while maintaining FcγRIIb-binding activity, compared to polypeptides comprising the Fc region of a native IgG antibody, by introducing the amino acid alterations into the Fc region, and methods for producing polypeptides comprising Fc region variants that, by introducing the amino acid alterations into the Fc region, have reduced binding activity to all activating FcγRs, particularly FcγRIIa (R type), while maintaining FcγRIIb-binding activity, compared to polypeptides comprising the Fc region of a native IgG antibody. The present invention also provides an Fc region variant in which the amino acid modification has been introduced into the Fc region, a polypeptide containing an antigen-binding domain that is present in a soluble form in plasma and has binding activity to a pathogenic antigen, and whose binding activity to the antigen changes depending on ion concentration conditions, and a method for promoting the elimination of the antigen from plasma using the polypeptide.

[0022] In the present invention, the term "polypeptide" generally refers to a peptide or protein having a length of about 10 amino acids or more. It is generally a polypeptide derived from a living organism, but is not particularly limited thereto, and may be, for example, a polypeptide consisting of an artificially designed sequence. It may also be a natural polypeptide, a synthetic polypeptide, a recombinant polypeptide, or the like.

[0023] A preferred example of the polypeptide of the present invention is an antibody. A more preferred example is native IgG, particularly native human IgG. Native IgG refers to a polypeptide that includes the same amino acid sequence as an IgG found in nature and belongs to the class of antibodies substantially encoded by immunoglobulin gamma genes. For example, 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.

[0024] The light chain constant region of an antibody includes IgK (Kappa, κ chain), IgL1, IgL2, IgL3, IgL6, and IgL7 (Lambda, λ chain) types, but any of these light chain constant regions may be used. Multiple allotype sequences due to genetic polymorphisms for the human IgK (Kappa) constant region and the human IgL7 (Lambda) constant region 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. Furthermore, the light chain constant region of the present invention may be a light chain constant region that has been modified, for example, by amino acid substitution, addition, deletion, insertion, and / or modification. Examples of antibody Fc regions include IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM types. The Fc region of the antibody of the present invention may be, for example, that of a human IgG antibody, preferably that of a human IgG1 antibody. The Fc region of the present invention can be, for example, an Fc region derived from a constant region of native IgG, specifically, a constant region derived from native human IgG1 (SEQ ID NO: 31), a constant region derived from native human IgG2 (SEQ ID NO: 32), a constant region derived from native human IgG3 (SEQ ID NO: 33), or a constant region derived from native human IgG4 (SEQ ID NO: 34). Figure 18 shows the sequences of the constant regions of native IgG1, IgG2, IgG3, and IgG4. The constant regions of native IgG also include naturally occurring mutants thereof. For the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, multiple allotype sequences due to genetic polymorphisms 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. In particular, for the human IgG1 sequence, the amino acid sequence at positions 356-358 (EU numbering) may be either DEL or EEM.

[0025] An Fcγ receptor (sometimes referred to herein as Fcγ receptor, FcγR, or FcgR) refers to a receptor that can bind to the Fc region of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody, and refers to any member of a family of proteins substantially encoded by the Fcγ receptor gene. In humans, this family includes FcγRI (CD64) including the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32) including the isoforms FcγRIIa (including allotypes H131 (H type) and R131 (R type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16) including the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any unidentified human FcγRs or FcγR isoforms or allotypes, but are not limited to these. Human FcγRIIb has been reported to have two splicing variants, FcγRIIb1 and FcγRIIb2. Another splicing variant, FcγRIIb3, has also been reported (J. Exp. Med., 1989, 170: 1369). Human FcγRIIb includes these splicing variants as well as the splicing variants NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1, and NP_003992.3 registered with NCBI. Furthermore, human FcγRIIb encompasses all previously reported genetic polymorphisms, including FcγRIIb (Arthritis Rheum, 2003, 48: 3242-52, Hum Mol Genet, 2005, 14: 2881-92, Arthritis Rheum. 2002 May;46(5):1242-54), as well as any genetic polymorphisms that will be reported in the future. FcγR may be derived from any organism, including, but not limited to, those derived from humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any unidentified mouse FcγRs or FcγR isoforms or allotypes. Preferred examples of such Fcγ receptors include human FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16), and / or FcγRIIIB (CD16).

[0026] The polynucleotide and amino acid sequences of FcγRI are set forth in SEQ ID NOs: 35 (NM_000566.3) and 36 (NP_000557.1), respectively. The polynucleotide and amino acid sequences of FcγRIIA are set forth in SEQ ID NOs: 37 (BC020823.1) and 38 (AAH20823.1), respectively. The polynucleotide and amino acid sequences of FcγRIIB are set forth in SEQ ID NOs: 39 (BC146678.1) and 40 (AAI46679.1), respectively. The polynucleotide and amino acid sequences of FcγRIIIA are set forth in SEQ ID NOs: 41 (BC033678.1) and 42 (AAH33678.1), respectively. The polynucleotide sequence and amino acid sequence of FcγRIIIB are set forth in SEQ ID NOs: 43 (BC128562.1) and 44 (AAI28563.1), respectively (RefSeq accession numbers are shown in parentheses).

[0027] 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). FcγRI (CD64), which includes FcγRIa, FcγRIb, and FcγRIc, and FcγRIII (CD16), which includes the isoform FcγRIIIa (including allotypes V158 and F158), are composed of an α chain that binds the Fc portion of IgG and a common γ chain containing ITAMs that transduce activation signals intracellularly. FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) is a GPI-anchored protein. On the other hand, FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 and R131) and FcγRIIc, contains ITAMs in its cytoplasmic domain. These receptors are expressed on many immune cells, including macrophages, mast cells, and antigen-presenting cells. Binding of these receptors to the Fc portion of IgG transmits activation signals that promote the phagocytic activity of macrophages, the production of inflammatory cytokines, mast cell degranulation, and enhanced function of antigen-presenting cells. Fcγ receptors capable of transmitting activation signals as described above are also referred to as activating Fcγ receptors in the present invention. On the other hand, the cytoplasmic domain of FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) contains ITIM, which transmits inhibitory signals. In B cells, cross-linking of FcγRIIb with the B cell receptor (BCR) suppresses activation signals from the BCR, resulting in the suppression of antibody production by the BCR. In macrophages, cross-linking of FcγRIII with FcγRIIb suppresses phagocytic ability and the ability to produce inflammatory cytokines. Fcγ receptors that have the ability to transmit inhibitory signals as described above are also referred to as inhibitory Fcγ receptors in the present invention.

[0028] In the present invention, the term "variant Fc region" refers to an Fc region in which at least one amino acid of the present invention has been modified with another amino acid, rather than an Fc region in which an amino acid modification of the present invention has not been introduced. Here, "at least one amino acid has been modified with another amino acid" includes an Fc region in which the amino acid modification has been introduced and an Fc region consisting of the same amino acid sequence.

[0029] Native IgG refers to a polypeptide that includes the same amino acid sequence as that of naturally occurring IgG 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 and IgGs into which modifications that do not substantially affect FcγR-binding activity have been introduced.

[0030] The Fc region of native IgG refers to an Fc region comprising the same amino acid sequence as an Fc region derived from naturally occurring IgG. The heavy chain constant region of native IgG is shown in Figure 18 (SEQ ID NOs: 31 to 34), and refers to, for example, the Fc region in the heavy chain constant region derived from native human IgG1, the Fc region in the heavy chain constant region derived from native human IgG2, the Fc region in the heavy chain constant region derived from native human IgG3, and the Fc region in the heavy chain constant region derived from native human IgG4 in Figure 18. The Fc region of native IgG also includes naturally occurring mutants and Fc regions into which modifications that do not substantially affect FcγR-binding activity have been introduced.

[0031] In the present invention, whether the binding activity of a polypeptide or Fc region variant comprising an Fc region variant of the present invention to various FcγRs has been enhanced, or whether the binding activity has been maintained or decreased can be determined by determining whether the dissociation constant (KD) value has decreased or increased, obtained from the analysis of sensorgrams obtained by immobilizing antibodies on a sensor chip or capturing them with Protein A, Protein L, Protein A / G, Protein G, anti-lamda chain antibodies, anti-kappa chain antibodies, antigen peptides, antigen proteins, etc., using BIACORE, an interaction analysis device that utilizes the surface plasmon resonance (SPR) phenomenon, as shown in the present Examples or Reference Examples. The sensorgrams are obtained by analyzing various FcγRs as analytes and then allowing them to interact with the sensor chip. Alternatively, it can be determined by whether the change in resonance unit (RU) value on a sensorgram before and after interaction of various FcγRs as an analyte with antibodies on a sensor chip immobilized on a sensor chip or captured with Protein A, Protein L, Protein A / G, Protein G, anti-lamda chain antibodies, anti-kappa chain antibodies, antigen peptides, antigen proteins, etc., divided by the change in resonance unit (RU) value before and after immobilization or capture of the antibody on the sensor chip, decreased or increased. It can also be determined by whether the dissociation constant (KD) value obtained from analyzing a sensorgram obtained by interacting a sample such as an antibody to be evaluated as an analyte with a sensor chip on which FcγRs are immobilized directly or via an anti-tag antibody, etc., decreased or increased. It can also be determined by whether the change in sensorgram value decreased or increased before and after interaction of a sample such as an antibody to be evaluated as an analyte with a sensor chip on which FcγRs are immobilized directly or via an anti-tag antibody, etc.

[0032] Specifically, the binding activity of Fc region variants to Fcγ receptors can be measured by ELISA, FACS (fluorescence activated cell sorting), ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), BIACORE method utilizing surface plasmon resonance (SPR) phenomenon, etc. (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0033] The ALPHA screen is performed using ALPHA technology, which uses two beads, donor and acceptor, based on the following principle: A luminescent signal is detected only when a molecule bound to the donor bead biologically interacts with a molecule bound to the acceptor bead and the two beads are in close proximity. A photosensitizer inside 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 chemiluminescent reaction within the bead, ultimately emitting light. If the molecules bound to the donor bead and the molecules bound to the acceptor bead do not interact, the singlet oxygen produced by the donor bead does not reach the acceptor bead, and no chemiluminescent reaction occurs.

[0034] For example, a biotin-labeled polypeptide complex is bound to donor beads, and a glutathione S-transferase (GST)-tagged Fcγ receptor is bound to acceptor beads. In the absence of a competing polypeptide complex containing a variant Fc domain, a polypeptide complex containing a wild-type Fc domain interacts with the Fcγ receptor, generating a signal at 520-620 nm. A polypeptide complex containing an untagged mutant Fc domain competes with the interaction between the polypeptide complex containing the wild-type Fc domain and the Fcγ receptor. Relative binding activity can be determined by quantifying the decrease in fluorescence that occurs as a result of competition. It is known that polypeptide complexes such as antibodies can be biotinylated using sulfo-NHS-biotin or similar. An Fcγ receptor can be tagged with GST by expressing a fusion gene in-frame fusing a polynucleotide encoding the Fcγ receptor with a polynucleotide encoding GST in cells harboring an expression vector, followed by purification using a glutathione column. The resulting signals are suitably analyzed by fitting to a one-site competition model using non-linear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).

[0035] One of the substances (ligand) whose interaction is to be observed is immobilized on a thin gold film on a sensor chip. When light is shone from the back of the sensor chip so that it is totally reflected at the interface between the gold film and the glass, a portion of the reflected light exhibits a reduced reflection intensity (SPR signal). When the other substance (analyte) whose interaction is to be observed is flowed over the surface of the sensor chip, binding occurs between the ligand and the analyte. The mass of the immobilized ligand molecule increases, causing a change in the refractive index of the solvent on the sensor chip surface. This change in refractive index shifts the position of the SPR signal (conversely, dissociation returns the signal position). The Biacore system plots the amount of this shift, i.e., the change in mass on the sensor chip surface, on the vertical axis, and displays the change in mass over time as measurement data (sensorgram). The amount of analyte bound to the ligand captured on the sensor chip surface can be determined from the sensorgram curve. Furthermore, the kinetics (association rate constant (ka) and dissociation rate constant (kd)) can be calculated from the ratio of these constants to determine the dissociation constant (KD). Inhibition assays are also suitable for use with the BIACORE method. An example of an inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

[0036] An Fc region or polypeptide comprising said Fc region that maintains its FcγRIIb-binding activity refers to a polypeptide that binds to FcγRIIb with essentially the same binding activity as that of the parent polypeptide, when assayed using essentially the same amounts of a polypeptide comprising the Fc region of a native IgG (also referred to as a polypeptide comprising a parent Fc region or a parent polypeptide) and a polypeptide comprising the Fc region containing an amino acid modification of the present invention (a polypeptide comprising an Fc region variant). Specifically, this refers to an Fc region variant that maintains at least 55.5% of the FcγRIIb binding of a polypeptide comprising the parent Fc region.

[0037] Furthermore, an Fc region or a polypeptide comprising such an Fc region with reduced, diminished, or weakened binding activity to activating FcγR refers to an Fc region variant or a polypeptide comprising such an Fc region variant that binds to activating FcγR with essentially weaker binding activity than a polypeptide comprising the parent Fc region, when an assay is performed using essentially the same amounts of a polypeptide comprising the Fc region of a native IgG (also referred to as a polypeptide comprising a parent Fc region or a parent polypeptide) and a polypeptide comprising the Fc region in which an amino acid modification of the present invention is present (a polypeptide comprising an Fc region variant). Whether or not Fc region variants of the present invention maintain the FcγRIIb-binding activity of the Fc region of native IgG can be determined, for example, by comparing the KD value for FcγRIIb of a polypeptide comprising an Fc region variant of the present invention, determined according to the above example, with the KD value for FcγRIIb of a polypeptide comprising the Fc region of native IgG. Specifically, if the KD value of a polypeptide comprising an Fc region variant of the present invention is equivalent to or lower than that of a polypeptide comprising the parent Fc region, it can be determined that the polypeptide comprising an Fc region variant of the present invention maintains FcγRIIb-binding activity compared to the polypeptide comprising the parent Fc region variant. Furthermore, whether or not the Fc region variants of the present invention have reduced binding activity to activating FcγR compared to that of the Fc region of native IgG can be determined, for example, by comparing the KD value for activating FcγR of a polypeptide comprising an Fc region variant of the present invention, determined according to the above example, with the KD value for activating FcγR of a polypeptide comprising the Fc region of native IgG. Specifically, when the KD value of a polypeptide comprising an Fc region variant of the present invention is increased compared to a polypeptide comprising the parent Fc region, the polypeptide comprising an Fc region variant of the present invention can be determined to have reduced binding activity to activating FcγR compared to a polypeptide comprising the parent Fc region variant. In particular, because binding activity to FcγRIIa (R type) is more likely to correlate with binding activity to FcγRIIb than to other activating FcγRs, the most difficult challenge in selectively reducing binding activity to activating FcγRs other than FcγRIIb is to find amino acid modifications that can reduce binding activity to FcγRIIa (R type) while maintaining binding activity to FcγRIIb.

[0038] "Equivalent or maintained binding activity to FcγRIIb" refers to, for example, the KD value ratio (KD value for FcγRIIb of polypeptide comprising parent Fc region) / (KD value for FcγRIIb of polypeptide comprising Fc region variant) measured by the above-mentioned method, which is preferably at least 0.75, more preferably at least 0.8, and even more preferably at least 0.9. Furthermore, a KD value ratio of about 5 is sufficient, and does not need to be higher in order to say that binding activity to FcγRIIb is equivalent or maintained.

[0039] A decrease, reduction, or attenuation in binding activity to activating FcγR refers to, for example, a KD value ratio measured by the above-mentioned method, [KD value of polypeptide comprising a parent Fc region for activating FcγR] / [KD value of polypeptide comprising an Fc region variant for activating FcγR], of preferably 0.2 or less, more preferably 0.15 or less, and even more preferably 0.1 or less.

[0040] In particular, since the sequence of the extracellular region of FcγRIIa is 93% identical to that of FcγRIIb and the structures are extremely similar, it is difficult to decrease the binding activity of FcγRIIaR while maintaining the binding activity to FcγRIIb. Therefore, with regard to binding to FcγRIIaR, the KD value ratio (KD value for FcγRIIaR of a polypeptide comprising a parent Fc region) / (KD value for FcγRIIaR of a polypeptide comprising a variant Fc region) is preferably 0.1 or less, and more preferably 0.05.

[0041] Furthermore, whether the binding activity of the polypeptides of the present invention to various FcγRs has been maintained, enhanced, or decreased can also be determined from the increase or decrease in the amount of binding of various FcγRs to the polypeptides of the present invention determined according to the above examples. Here, the amount of binding of various FcγRs to a polypeptide means the difference in RU values ​​in sensorgrams showing changes before and after allowing various FcγRs (analytes) to interact with each polypeptide, divided by the difference in RU values ​​in sensorgrams showing changes before and after capturing the polypeptide on a sensor chip. Furthermore, an Fc region with improved selectivity for FcγRIIb or a polypeptide comprising such an Fc region, or an Fc region with selectively reduced binding activity to activating FcγR or a polypeptide comprising such an Fc region, refers to an Fc region or a polypeptide comprising such an Fc region that maintains binding activity to FcγRIIb but has reduced, diminished, or weakened binding activity to activating FcγR.

[0042] Furthermore, the KD values ​​(mol / L) of the Fc region variants of the present invention for FcγRIIb and activating FcγR are not particularly limited. For example, the value for FcγRIIb is 7.0 × 10 -6 may be equal to or less than 6.0×10 -6 Less than or equal to 5.0 × 10 -6 or less, and the value for activating FcγR is 2.5 × 10 -9 or more, preferably 3.0×10 -9 More preferably, 3.5 × 10 -9 Above, especially the value for FcγRIIa (R type) was 2.0 × 10 -5 It is preferable that this is equal to or greater than this.

[0043] The term "Fc region" refers to a fragment of an antibody molecule consisting of the hinge region or 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, according to EU numbering (also referred to herein as EU INDEX) (see Figure 18).

[0044] 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 or Protein G column. There are no particular limitations on the protease used, as long as it can digest full-length antibodies so as to produce Fab and F(ab')2 in a limited manner by appropriately setting the enzyme reaction conditions, such as pH, and examples thereof include pepsin and papain.

[0045] The present invention provides Fc region variants of human IgG (IgG1, IgG2, IgG3, IgG4) that contain a combination of amino acid alterations in the Fc region of human IgG, combining alteration of the amino acid at EU numbering position 238 with another amino acid and alteration of any of the amino acids listed in (a) to (k) below with another amino acid. By introducing these alterations into the Fc region, it is possible to provide polypeptides comprising Fc region variants that maintain FcγRIIb-binding activity while reducing binding activity to all activating FcγRs, in particular FcγRIIa (R type), compared to polypeptides comprising the Fc region of native IgG. (a) Amino acid 235 (EU numbering) in the Fc region (b) Amino acid 237 (EU numbering) in the Fc region (c) Amino acid 241 in the Fc region (EU numbering) (d) Amino acid 268 (EU numbering) in the Fc region (e) Amino acid 295 (EU numbering) in the Fc region (f) Amino acid 296 (EU numbering) in the Fc region (g) Amino acid 298 (EU numbering) in the Fc region (h) Amino acid 323 (EU numbering) in the Fc region (i) Amino acid 324 (EU numbering) in the Fc region (j) Amino acid 330 (EU numbering) in the Fc region (k) at least two amino acids selected from (a) to (j)

[0046] The combination of at least two amino acids selected in (k) above is not particularly limited, as long as it maintains FcγRIIb-binding activity while reducing binding activity to all activating FcγRs, compared to a polypeptide comprising the Fc region of native IgG. However, the following combinations (1) to (3) are preferred: (1) amino acids at positions 241, 268, 296, and 324 (EU numbering) in the Fc region (2) amino acids 237, 241, 296, and 330 (EU numbering) in the Fc region (3) amino acids at positions 235, 237, 241, and 296 (EU numbering) in the Fc region

[0047] The amino acids selected as amino acids after modification are not particularly limited, as long as they maintain binding activity to FcγRIIb compared to a polypeptide comprising the Fc region of native IgG while reducing binding activity to all activating FcγRs. However, preferred amino acids are Asp at amino acid position 238 (EU numbering), Phe at amino acid position 235, Gln or Asp at amino acid position 237, Met or Leu at amino acid position 241, Pro at amino acid position 268, Met or Val at amino acid position 295, Glu, His, Asn or Asp at amino acid position 296, Ala or Met at amino acid position 298, Ile at amino acid position 323, Asn or His at amino acid position 324, and His or Tyr at amino acid position 330. Furthermore, the amino acids selected as amino acids after modification in (1) to (3) above include: (1) In the Fc region, the amino acid at position 241 (EU numbering) is Met, the amino acid at position 268 is Pro, the amino acid at position 296 is Glu, and the amino acid at position 324 is His. (2) The Fc region contains Gln or Asp at amino acid position 237, Met at amino acid position 241, Glu at amino acid position 296, and His at amino acid position 330 (EU numbering). (3) In the Fc region, the amino acid at position 235 (EU numbering) is Phe, the amino acid at position 237 is Gln or Asp, the amino acid at position 241 is Met, and the amino acid at position 296 is Glu is preferred.

[0048] The present invention also provides Fc region variants containing a combination of amino acid modifications in the Fc region of human IgG, including modifications of amino acids 238 and 271 (EU numbering) to other amino acids and modifications of any of the amino acids listed below in (a) to (h) to other amino acids. By introducing these modifications into the Fc region, it is possible to provide polypeptides containing Fc region variants that maintain FcγRIIb-binding activity while reducing binding activity to all activating FcγRs, in particular FcγRIIa (R type), compared to polypeptides comprising the Fc region of native IgG. (a) Amino acid 234 (EU numbering) in the Fc region (b) Amino acid 235 (EU numbering) in the Fc region (c) Amino acid 236 (EU numbering) in the Fc region (d) Amino acid 237 (EU numbering) in the Fc region (e) Amino acid 239 (EU numbering) in the Fc region (f) Amino acid 265 (EU numbering) in the Fc region (g) Amino acid 267 (EU numbering) in the Fc region (h) Amino acid 297 (EU numbering) in the Fc region

[0049] In addition to the amino acids listed in (a) to (h) above, other amino acids may be combined with the amino acid modifications at positions 238 and 271 (EU numbering) to other amino acids. Such amino acid combinations are not particularly limited, but preferred are combinations of modifications selected from the following (1) to (3): (1) amino acids at positions 233, 238, 264, 267, 268, and 271 (EU numbering) in the Fc region (2) amino acids at positions 233, 237, 238, 264, 267, 268, 271, 296, 297, 330, and 396 (EU numbering) in the Fc region; (3) amino acids at positions 233, 238, 264, 267, 268, 271, and 296 (EU numbering) in the Fc region

[0050] The amino acids selected as the modified amino acids are not particularly limited, as long as they maintain binding activity to FcγRIIb compared to a polypeptide comprising the Fc region of native IgG while reducing binding activity to all activating FcγRs. However, it is preferred that the amino acid at position 238 (EU numbering) is Asp, the amino acid at position 271 is Gly, the amino acid at position 234 is Ala, His, Asn, Lys, or Arg, the amino acid at position 235 is Ala, the amino acid at position 236 is Gln, the amino acid at position 237 is Arg or Lys, the amino acid at position 239 is Lys, the amino acid at position 265 is Lys, Asn, Arg, Ser, or Val, the amino acid at position 267 is Lys, Arg, or Tyr, and the amino acid at position 297 is Ala.

[0051] In addition, the amino acids selected as the amino acids after modification in the above (1) to (3) include: (1) In the Fc region, the amino acid at position 233 (EU numbering) is Asp, the amino acid at position 238 is Asp, the amino acid at position 264 is Ile, the amino acid at position 267 is Arg, the amino acid at position 268 is Glu, and the amino acid at position 271 is Gly (2) In the Fc region, the amino acid at position 233 is Asp, the amino acid at position 237 is Asp, the amino acid at position 238 is Asp, the amino acid at position 264 is Ile, the amino acid at position 267 is Ala, the amino acid at position 268 is Glu, the amino acid at position 271 is Gly, the amino acid at position 296 is Asp, the amino acid at position 297 is Ala, the amino acid at position 330 is Arg, and the amino acid at position 396 is Met (EU numbering). (3) In the Fc region, the amino acid at position 233 (EU numbering) is Asp, the amino acid at position 238 is Asp, the amino acid at position 264 is Ile, the amino acid at position 267 is Arg, the amino acid at position 268 is Pro, the amino acid at position 271 is Gly, and the amino acid at position 296 is Glu is preferred.

[0052] The present invention also provides Fc region variants comprising alterations of the amino acids at positions 238, 271, 327, 330, and 331 (EU numbering) to other amino acids in the Fc region of human IgG. These variants also include Fc region variants comprising a combination of amino acid alterations of any of the amino acids listed below (a) to (e) to other amino acids. Introducing these alterations into the Fc region makes it possible to provide polypeptides comprising Fc region variants that maintain FcγRIIb-binding activity while reducing binding activity to all activating FcγRs, in particular FcγRIIa (R type), compared to polypeptides comprising the Fc region of native IgG. (a) Amino acid 233 in the Fc region (EU numbering) (b) Amino acid 237 (EU numbering) in the Fc region (c) Amino acid 264 (EU numbering) in the Fc region (d) Amino acid 267 (EU numbering) in the Fc region (e) Amino acid 268 (EU numbering) in the Fc region

[0053] In addition to the amino acids listed in (a) to (e) above, other amino acids may be combined with the amino acid modifications at positions 238 and 271 (EU numbering) to other amino acids. Such amino acid combinations are not particularly limited, but preferred are combinations of modifications selected from the following (1) to (4): (1) amino acids at positions 237, 238, 268, 271, 327, 330, and 331 (EU numbering) in the Fc region (2) amino acids at positions 233, 237, 238, 268, 271, 327, 330, and 331 (EU numbering) of the Fc region; (3) amino acids at positions 238, 267, 268, 271, 327, 330, and 331 (EU numbering) in the Fc region (4) amino acids at positions 238, 264, 267, 271, 327, 330, and 331 (EU numbering) in the Fc region

[0054] The amino acids selected as the modified amino acids are not particularly limited, as long as they maintain binding activity to FcγRIIb while reducing binding activity to all activating FcγRs compared to a polypeptide comprising the Fc region of native IgG. However, it is preferred that the amino acid at position 238 (EU numbering) is Asp, the amino acid at position 271 is Gly, the amino acid at position 327 is Gly, the amino acid at position 330 is Ser, the amino acid at position 331 is Ser, the amino acid at position 233 is Asp, the amino acid at position 237 is Asp, the amino acid at position 264 is Ile, the amino acid at position 267 is Ala, and the amino acid at position 268 is Asp or Glu.

[0055] In addition, the amino acids selected as the amino acids after modification in the above (1) to (4) include: (1) In the Fc region, the amino acid at position 237 (EU numbering) is Asp, the amino acid at position 238 is Asp, the amino acid at position 268 is Asp or Glu, the amino acid at position 271 is Gly, the amino acid at position 327 is Gly, the amino acid at position 330 is Ser, and the amino acid at position 331 is Ser (EU numbering). (2) In the Fc region, the amino acid at position 233 is Asp, the amino acid at position 237 is Asp, the amino acid at position 238 is Asp, the amino acid at position 268 is Asp, the amino acid at position 271 is Gly, the amino acid at position 327 is Gly, the amino acid at position 330 is Ser, and the amino acid at position 331 is Ser (EU numbering). (3) In the Fc region, the amino acid at position 238 (EU numbering) is Asp, the amino acid at position 267 is Ala, the amino acid at position 268 is Glu, the amino acid at position 271 is Gly, the amino acid at position 327 is Gly, the amino acid at position 330 is Ser, and the amino acid at position 331 is Ser (EU numbering). (4) In the Fc region, the amino acid at position 238 (EU numbering) is Asp, the amino acid at position 264 is Ile, the amino acid at position 267 is Ala, the amino acid at position 271 is Gly, the amino acid at position 327 is Gly, the amino acid at position 330 is Ser, and the amino acid at position 331 is Ser (EU numbering). is preferred.

[0056] In addition to these alterations, the present invention also allows for at least one other alteration to be made to the Fc region, without particular limitation, as long as it reduces the binding activity to activating FcγR while maintaining the binding activity to FcγRIIb. Such modifications include, for example, modifications that reduce complement-binding activity. Specific examples include an amino acid modification at EU numbering position 322 in the Fc region, or a combination of amino acid modifications at EU numbering positions 327, 330, and 331 in the Fc region. The amino acids selected as the modified amino acids are not particularly limited, as long as they maintain FcγRIIb-binding activity, reduce binding activity to all activating FcγRs, and reduce complement-binding activity compared to a polypeptide comprising the Fc region of native IgG. However, preferred amino acids are Ala or Glu at amino acid position 322 (EU numbering), Gly at amino acid position 327, Ser at amino acid position 330, and Ser at amino acid position 331.

[0057] In the present invention, whether or not a polypeptide or Fc region variant comprising an Fc region variant of the present invention has reduced complement-binding activity can be confirmed by methods similar to those used to confirm whether or not binding activity to FcγR has been reduced as described above. Specifically, as shown in this Example, for example, using BIACORE, an interaction analysis device that utilizes the surface plasmon resonance (SPR) phenomenon, an antibody to be evaluated is immobilized on a sensor chip or captured with Protein A, Protein L, Protein A / G, Protein G, an anti-lamda chain antibody, an anti-kappa chain antibody, an antigen peptide, an antigen protein, or the like, and the antibody is allowed to interact with complement as an analyte. This can be determined by examining whether or not the dissociation constant (KD) value obtained from the analysis of the sensorgram is increased. Alternatively, it can be determined by whether or not the change in resonance unit (RU) value on a sensorgram before and after interacting complement as an analyte with an antibody of interest immobilized on a sensor chip or captured with Protein A, Protein L, Protein A / G, Protein G, anti-lamda chain antibody, anti-kappa chain antibody, antigen peptide, antigen protein, etc., is increased when divided by the change in resonance unit (RU) value before and after immobilizing or capturing the antibody on the sensor chip. It can also be determined by whether or not the dissociation constant (KD) value obtained from analyzing a sensorgram obtained by interacting a sample such as an antibody of interest as an analyte with a sensor chip on which complement is immobilized directly or via an anti-tag antibody, etc., is increased. It can also be determined by whether or not the change in sensorgram value is increased before and after interacting a sample such as an antibody of interest as an analyte with a sensor chip on which complement is immobilized directly or via an anti-tag antibody, etc. Alternatively, the amount of binding can be evaluated by ELISA, in which complement is added to a plate on which the antibody to be evaluated is immobilized, either via an antigen or directly, and then an anti-human C1q antibody labeled with peroxidase or the like is added.

[0058] Furthermore, polypeptides containing Fc region variants of the present invention can also be combined with amino acid modifications made for other purposes, such as amino acid substitutions that improve FcRn-binding activity (J Immunol. 2006 Jan 1;176(1):346-56, J Biol Chem. 2006 Aug 18;281(33):23514-24, Int Immunol. 2006 Dec;18(12):1759-69, Nat Biotechnol. 2010 Feb;28(2):157-9, WO / 2006 / 019447, WO / 2006 / 053301, WO / 2009 / 086320) or amino acid substitutions that improve antibody heterogeneity or stability (WO / 2009 / 041613). Alternatively, the present invention also includes polypeptides comprising Fc region variants of the present invention that have been conferred with the property of promoting antigen elimination as described in WO2011 / 122011 and PCT / JP2011 / 072550, or polypeptides comprising the property of repeatedly binding to multiple antigen molecules as described in WO2009 / 125825 and PCT / JP2011 / 077619. Alternatively, polypeptides comprising Fc region variants of the present invention may be combined with amino acid modifications (WO / 2012 / 016227) that lower the pI of the constant region in order to increase blood retention. Alternatively, polypeptides comprising Fc region variants of the present invention may be combined with amino acid modifications in CH3 as described in EP1752471 and EP1772465 in order to confer binding ability to other antigens.

[0059] When a polypeptide comprising an Fc region variant of the present invention contains an antigen-binding domain such as an antibody, amino acid modifications can be combined to change the antigen-binding activity depending on ion concentration conditions in order to enhance the antigen elimination effect of the polypeptide from plasma.

[0060] As used herein, the "antigen-binding domain" may be any domain with any structure as long as it binds to the target antigen. Examples of such domains include the variable regions of the heavy and light chains of antibodies, a module called an A domain of about 35 amino acids contained in Avimer, a cell membrane protein present in living organisms (International Publication Nos. WO2004 / 044011 and WO2005 / 040229), Adnectin (International Publication No. WO2002 / 032925) containing the 10Fn3 domain, which is a domain that binds to proteins in fibronectin, a glycoprotein expressed on cell membranes, Affibody (International Publication No. WO1995 / 001937) using an IgG binding domain consisting of a 58-amino acid three-helix bundle of Protein A as a scaffold, and DARPins (Designed Ankyrin Repeat (AR)) which are regions exposed on the molecular surface of ankyrin repeats (AR) with a structure in which a 33-amino acid turn, two antiparallel helices, and a loop subunit are repeatedly stacked. Preferred examples of the antigen-binding domain of the present invention include an anticalin molecule, which is a four-loop region supporting one side of a barrel structure in which eight highly conserved antiparallel strands twist toward the center, found in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) (International Publication WO 2003 / 029462), and a concave region of a parallel sheet structure within a horseshoe-shaped structure in which leucine-rich repeat (LRR) modules of the variable lymphocyte receptor (VLR), which does not have an immunoglobulin structure and is part of the adaptive immune system of jawless fish such as lampreys and hagfish, are repeatedly stacked (International Publication WO 2008 / 016854). Preferred examples of the antigen-binding domain of the present invention include antigen-binding domains comprising the variable regions of the heavy and light chains of antibodies.Suitable examples of such antigen-binding domains include "scFv (single chain Fv)," "single chain antibody," "Fv," "scFv2 (single chain Fv 2)," "Fab," and "F(ab')2."

[0061] As used herein, "ion concentration" refers to, for example, metal ion concentration. "Metal ions" refer to ions of elements belonging to Group I, such as alkali metals and copper group elements excluding hydrogen, Group II, such as alkaline earth metals and zinc group elements, Group III, such as boron, Group IV, such as carbon and silicon, Group VIII, such as iron group elements and platinum group elements, and each of the A subgroups of Groups V, VI, and VII, as well as metal elements such as antimony, bismuth, and polonium. Metal atoms have the property of releasing valence electrons to become cations, which is called ionization tendency. Metals with a high ionization tendency are considered to be chemically active.

[0062] An example of a suitable metal ion in the present invention is calcium ion. Calcium ions are involved in the regulation of many biological phenomena, including muscle contraction (e.g., skeletal muscle, smooth muscle, cardiac muscle), activation of leukocytes (e.g., motility and phagocytosis), activation of platelets (e.g., deformation and secretion), activation of lymphocytes, activation of mast cells (e.g., histamine secretion), cell responses mediated by catecholamine α receptors and acetylcholine receptors, exocytosis, release of transmitters from neuronal terminals, and axonal flow in neurons. Known intracellular calcium ion receptors include troponin C, calmodulin, parvalbumin, and myosin light chain, which have multiple calcium ion-binding sites and are thought to have originated from a common molecular evolutionary origin, and many of their binding motifs are also known. Well-known examples include the cadherin domain, the EF hand found in calmodulin, the C2 domain found in protein kinase C, the Gla domain found in the blood coagulation protein Factor IX, the C-type lectins found in the asialoglycoprotein receptor and mannose-binding receptor, the A domain found in the LDL receptor, annexin, the thrombospondin type 3 domain, and the EGF-like domain.

[0063] In the present invention, when the metal ion is a calcium ion, calcium ion concentration conditions include low and high calcium ion concentrations. "The binding activity changes depending on the calcium ion concentration" refers to the change in antigen-binding activity of an antigen-binding molecule due to the difference between low and high calcium ion concentrations. For example, this may be the case where the antigen-binding activity of an antigen-binding molecule is higher under high calcium ion concentrations than under low calcium ion concentrations. Another example is where the antigen-binding activity of an antigen-binding molecule is higher under low calcium ion concentrations than under high calcium ion concentrations.

[0064] As used herein, a high calcium ion concentration is not limited to a specific numerical value, but may preferably be a concentration selected from the range of 100 μM to 10 mM. In another embodiment, it may be a concentration selected from the range of 200 μM to 5 mM. In a different embodiment, it may be a concentration selected from the range of 400 μM to 3 mM, and in another embodiment, it may be a concentration selected from the range of 200 μM to 2 mM. It may also be a concentration selected from the range of 400 μM to 1 mM. In particular, a concentration selected from the range of 500 μM to 2.5 mM, which is close to the calcium ion concentration in plasma (blood) in vivo, is preferred.

[0065] As used herein, a low calcium ion concentration is not limited to a specific numerical value, but may preferably be a concentration selected from the range of 0.1 μM to 30 μM. In another embodiment, it may be a concentration selected from the range of 0.2 μM to 20 μM. In a different embodiment, it may be a concentration selected from the range of 0.5 μM to 10 μM, and in another embodiment, it may be a concentration selected from the range of 1 μM to 5 μM. Furthermore, it may be a concentration selected from the range of 2 μM to 4 μM. In particular, a concentration selected from the range of 1 μM to 5 μM, which is close to the ionized calcium concentration in early endosomes in vivo, is preferred.

[0066] In the present invention, "the antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration" means that the antigen-binding activity of an antigen-binding molecule at a calcium ion concentration selected from the range of 0.1 μM to 30 μM is weaker than that at a calcium ion concentration selected from the range of 100 μM to 10 mM. Preferably, the antigen-binding activity of an antigen-binding molecule at a calcium ion concentration selected from the range of 0.5 μM to 10 μM is weaker than that at a calcium ion concentration selected from the range of 200 μM to 5 mM. Particularly preferably, the antigen-binding activity at a calcium ion concentration in early endosomes in vivo is weaker than that at a calcium ion concentration in plasma in vivo. Specifically, the antigen-binding activity of an antigen-binding molecule at a calcium ion concentration selected from the range of 1 μM to 5 μM is weaker than that at a calcium ion concentration selected from the range of 500 μM to 2.5 mM.

[0067] Whether or not the antigen-binding activity of an antigen-binding molecule changes depending on the metal ion concentration can be determined by using known measurement methods, such as those described above in the section on binding activity. For example, to confirm that the antigen-binding activity of an antigen-binding molecule changes more significantly under high calcium ion concentrations than under low calcium ion concentrations, the antigen-binding activities of the antigen-binding molecule under low and high calcium ion concentrations are compared.

[0068] Furthermore, in the present invention, the expression "the antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration" can also be expressed as "the antigen-binding activity of an antigen-binding molecule at a high calcium ion concentration is higher than that at a low calcium ion concentration." In the present invention, "the antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration" can also be expressed as "the antigen-binding ability at a low calcium ion concentration is weaker than that at a high calcium ion concentration," and "the antigen-binding activity at a low calcium ion concentration is reduced compared to that at a high calcium ion concentration" can also be expressed as "the antigen-binding ability at a low calcium ion concentration is weakened compared to that at a high calcium ion concentration."

[0069] Conditions other than calcium ion concentration when measuring antigen-binding activity can be appropriately selected by those skilled in the art and are not particularly limited. For example, measurement can be performed under conditions of HEPES buffer and 37°C. For example, measurement can be performed using Biacore (GE Healthcare). When the antigen is a soluble antigen, the binding activity to the soluble antigen can be evaluated by passing the antigen as an analyte through a chip on which the antigen-binding molecule is immobilized. When the antigen is a membrane-type antigen, the binding activity to the membrane-type antigen can be evaluated by passing the antigen-binding molecule as an analyte through a chip on which the antigen is immobilized.

[0070] In the antigen-binding molecules of the present invention, the ratio of antigen-binding activity under low calcium ion concentrations to that under high calcium ion concentrations is not particularly limited, as long as the antigen-binding activity under low calcium ion concentrations is weaker than that under high calcium ion concentrations, but preferably the ratio of the KD (Dissociation constant) for the antigen under low calcium ion concentrations to the KD under high calcium ion concentrations, KD (3 μM Ca) / KD (2 mM Ca), is 2 or greater, more preferably 10 or greater, and even more preferably 40 or greater. The upper limit of KD (3 μM Ca) / KD (2 mM Ca) is not particularly limited, and may be any value, such as 400, 1,000, or 10,000, as long as it can be produced by those skilled in the art.

[0071] As a value of antigen-binding activity, KD (dissociation constant) can be used when the antigen is a soluble antigen, but apparent KD (apparent dissociation constant) can be used when the antigen is a membrane-type antigen. KD (dissociation constant) and apparent KD (apparent dissociation constant) can be measured by methods known to those skilled in the art, such as Biacore (GE Healthcare), Scatchard plot, flow cytometer, etc.

[0072] Alternatively, the dissociation rate constant kd (dissociation rate constant) can also be suitably used as an indicator of the ratio between the antigen-binding activity of an antigen-binding molecule of the present invention under low calcium concentration conditions and under high calcium concentration conditions. When kd (dissociation rate constant) is used instead of KD (dissociation constant) as an indicator of the binding activity ratio, the ratio of kd (dissociation rate constant) for the antigen under low calcium concentration conditions to kd (dissociation rate constant) under high calcium concentration conditions, kd (low calcium concentration condition) / kd (high calcium concentration condition), is preferably 2 or more, more preferably 5 or more, even more preferably 10 or more, and even more preferably 30 or more. There are no particular upper limits to the value of Kd (low calcium concentration condition) / kd (high calcium concentration condition), and any value, such as 50, 100, or 200, may be used as long as it can be produced within the technical common sense of a person skilled in the art.

[0073] When the antigen is a soluble antigen, kd (dissociation rate constant) can be used as the value of antigen-binding activity, whereas when the antigen is a membrane-type antigen, apparent kd (apparent dissociation rate constant) can be used. kd (dissociation rate constant) and apparent kd (apparent dissociation rate constant) can be measured by methods known to those skilled in the art, for example, using Biacore (GE Healthcare) or a flow cytometer. In the present invention, when measuring the antigen-binding activity of an antigen-binding molecule at different calcium ion concentrations, it is preferable to keep all conditions other than the calcium concentration the same.

[0074] For example, an antigen-binding domain or antigen-binding molecule whose antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by screening for antigen-binding domains or antibodies, comprising the following steps (a) to (c): (a) determining the antigen-binding activity of an antigen-binding domain or antigen-binding molecule under a low calcium concentration condition; (b) determining the antigen-binding activity of the antigen-binding domain or antigen-binding molecule under a high calcium concentration condition; (c) selecting antigen-binding domains or antigen-binding molecules whose antigen-binding activity at a low calcium concentration is lower than that at a high calcium concentration. Furthermore, an embodiment of the present invention, an antigen-binding domain or antigen-binding molecule whose antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration, can be obtained by screening antigen-binding domains or antigen-binding molecules or a library thereof, comprising the following steps (a) to (c): (a) contacting an antigen with an antigen-binding domain or antigen-binding molecule, or a library thereof, under conditions of a high calcium concentration; (b) placing the antigen-binding domain or antigen-binding molecule bound to the antigen in step (a) under a low calcium concentration condition; (c) isolating the antigen-binding domain or antigen-binding molecule dissociated in step (b).

[0075] Furthermore, an antigen-binding domain or antigen-binding molecule whose antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by screening antigen-binding domains or antigen-binding molecules, or a library thereof, comprising the following steps (a) to (d): (a) contacting a library of antigen-binding domains or antigen-binding molecules with an antigen under low calcium concentration conditions; (b) selecting antigen-binding domains or antigen-binding molecules that do not bind to the antigen in step (a); (c) allowing the antigen-binding domain or antigen-binding molecule selected in step (b) to bind to an antigen under high calcium concentration conditions; (d) isolating the antigen-binding domain or antigen-binding molecule that bound to the antigen in step (c).

[0076] Furthermore, an antigen-binding domain or antigen-binding molecule whose antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by a screening method comprising the following steps (a) to (c): (a) contacting a library of antigen-binding domains or antigen-binding molecules with an antigen-immobilized column under high calcium concentration conditions; (b) eluting the antigen-binding domain or antigen-binding molecule bound to the column in step (a) from the column under low calcium concentration conditions; (c) isolating the antigen-binding domain or antigen-binding molecule eluted in step (b).

[0077] Furthermore, an antigen-binding domain or antigen-binding molecule whose antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by a screening method comprising the following steps (a) to (d): (a) passing a library of antigen-binding domains or antigen-binding molecules through an antigen-immobilized column under low calcium concentration conditions; (b) recovering the antigen-binding domain or antigen-binding molecule that did not bind to the column and was eluted in step (a); (c) allowing the antigen-binding domain or antigen-binding molecule recovered in step (b) to bind to an antigen under high calcium concentration conditions; (d) isolating the antigen-binding domain or antigen-binding molecule that bound to the antigen in step (c).

[0078] Furthermore, an antigen-binding domain or antigen-binding molecule whose antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by a screening method comprising the following steps (a) to (d): (a) contacting a library of antigen-binding domains or antigen-binding molecules with an antigen under high calcium concentration conditions; (b) obtaining the antigen-binding domain or antigen-binding molecule bound to the antigen in step (a); (c) placing the antigen-binding domain or antigen-binding molecule obtained in step (b) under a low calcium concentration condition; (d) isolating antigen-binding domains or antigen-binding molecules selected in step (c) whose antigen-binding activity is weaker than the criterion selected in step (b).

[0079] The above steps may be repeated two or more times. Thus, the present invention provides antigen-binding domains or antigen-binding molecules whose antigen-binding activity at low calcium ion concentrations is lower than that at high calcium ion concentrations, obtained by the above-mentioned screening methods, which further comprise repeating steps (a) to (c) or (a) to (d) two or more times. The number of times steps (a) to (c) or (a) to (d) are repeated is not particularly limited, but is typically no more than 10 times.

[0080] In the above-mentioned screening methods, the antigen-binding activity of an antigen-binding domain or antigen-binding molecule under low calcium concentration conditions is not particularly limited, as long as it is an antigen-binding activity at an ionized calcium concentration of 0.1 μM to 30 μM, with preferred ionized calcium concentrations including 0.5 μM to 10 μM. A more preferred ionized calcium concentration includes the ionized calcium concentration in early endosomes in vivo, specifically 1 μM to 5 μM. Furthermore, the antigen-binding activity of an antigen-binding domain or antigen-binding molecule under high calcium concentration conditions is not particularly limited, as long as it is an antigen-binding activity at an ionized calcium concentration of 100 μM to 10 mM, with preferred ionized calcium concentrations including 200 μM to 5 mM. A more preferred ionized calcium concentration includes the ionized calcium concentration in plasma in vivo, specifically 0.5 mM to 2.5 mM.

[0081] Another example of a screening method for antigen-binding domains or antigen-binding molecules whose antigen-binding activity at low calcium ion concentrations is lower than that at high calcium ion concentrations, which is one embodiment of the present invention, is the method described in WO2012 / 073992 etc. (e.g., paragraphs 0200-0213).

[0082] The antigen-binding activity of an antigen-binding domain or antigen-binding molecule can be measured by methods known to those skilled in the art, and conditions other than ionized calcium concentration can be appropriately determined by those skilled in the art. The antigen-binding activity of an antigen-binding domain or antigen-binding molecule can be evaluated as KD (Dissociation constant), apparent KD (Apparent dissociation constant), kd (Dissociation rate constant), or apparent kd (Apparent dissociation rate constant), etc. These can be measured by methods known to those skilled in the art, such as Biacore (GE Healthcare), Scatchard plots, FACS, etc.

[0083] In the present invention, the step of selecting antigen-binding domains or antigen-binding molecules whose antigen-binding activity under a high calcium concentration condition is higher than that under a low calcium concentration condition is the same as the step of selecting antigen-binding domains or antigen-binding molecules whose antigen-binding activity under a low calcium concentration condition is lower than that under a high calcium concentration condition.

[0084] As long as the antigen-binding activity under a high calcium concentration is higher than that under a low calcium concentration, the difference between the antigen-binding activity under a high calcium concentration and that under a low calcium concentration is not particularly limited; however, the antigen-binding activity under a high calcium concentration is preferably at least 2 times, more preferably at least 10 times, and even more preferably at least 40 times that under a low calcium concentration.

[0085] The antigen-binding domains or antigen-binding molecules of the present invention to be screened by the above-mentioned screening methods may be any antigen-binding domains or antigen-binding molecules, and for example, the above-mentioned antigen-binding domains or antigen-binding molecules can be screened. For example, antigen-binding domains or antigen-binding molecules having native sequences may be screened, or antigen-binding domains or antigen-binding molecules with substituted amino acid sequences may be screened.

[0086] For example, an example of a screening method for antigen-binding domains or antigen-binding molecules whose antigen-binding activity at low calcium ion concentrations is lower than that at high calcium ion concentrations is the method described in WO2012 / 073992 etc. (e.g., paragraphs 0200-0213), which is one embodiment of the present invention.

[0087] Antigen-binding domains or antigen-binding molecules of the present invention whose antigen-binding activity changes depending on calcium ion concentration conditions and are screened by the above-mentioned screening methods may be prepared in any manner. For example, when the metal ion is calcium ion concentration, it is possible to use pre-existing antigen-binding domains or antigen-binding molecules, pre-existing libraries (such as phage libraries), antibodies or libraries prepared from hybridomas obtained by immunizing animals or B cells from immunized animals, or antibodies or libraries in which amino acids capable of chelating calcium (e.g., aspartic acid and glutamic acid) or unnatural amino acid mutations have been introduced into these antibodies or libraries (e.g., libraries with an increased content of amino acids capable of chelating calcium (e.g., aspartic acid and glutamic acid) or unnatural amino acids, or libraries in which amino acids capable of chelating calcium (e.g., aspartic acid and glutamic acid) or unnatural amino acid mutations have been introduced at specific sites).

[0088] As described above, when the metal ion is a calcium ion, examples of amino acids that change the antigen-binding activity of an antigen-binding molecule depending on ion concentration conditions include any amino acid that forms a calcium-binding motif. Calcium-binding motifs are well known to those skilled in the art and have been described in detail (e.g., Springer et al. (Cell (2000) 102, 275-277), Kawasaki and Kretsinger (Protein Prof. (1995) 2, 305-490), Moncrief et al. (J. Mol. Evol. (1990) 30, 522-562), Chauvaux et al. (Biochem. J. (1990) 265, 261-265), Bairoch and Cox (FEBS Lett. (1990) 269, 454-456), Davis (New Biol. (1990) 2, 410-419), Schaefer et al. (Genomics (1995) 25, 638-643), Economou et al. (EMBO J. (1990) 9, 349-354), Wurzburg et al. (Structure. (2006) 14, 6, 1049-1058)). That is, any known calcium-binding motif, such as that of ASGPR, CD23, MBR, or C-type lectins such as DC-SIGN, can be contained in the antigen-binding molecules of the present invention. In addition to the above, preferred examples of such calcium-binding motifs include the calcium-binding motif contained in the antigen-binding domain of SEQ ID NO: 45.

[0089] Furthermore, amino acids with metal chelating activity can also be suitably used as amino acids that change the antigen-binding activity of the antigen-binding domain contained in the antigen-binding molecule of the present invention depending on the calcium ion concentration. Suitable examples of amino acids with metal chelating activity include serine (Ser(S)), threonine (Thr(T)), asparagine (Asn(N)), glutamine (Gln(Q)), aspartic acid (Asp(D)), and glutamic acid (Glu(E)).

[0090] The position of the antigen-binding domain containing the amino acid is not limited to a specific position and can be any position in the heavy chain variable region or light chain variable region that forms the antigen-binding domain, as long as the antigen-binding activity of the antigen-binding molecule is changed depending on the calcium ion concentration. In a non-limiting embodiment, the antigen-binding domain of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences, in which the heavy chain antigen-binding domain contains an amino acid that changes the antigen-binding activity of the antigen-binding molecule depending on the calcium ion concentration. In another non-limiting embodiment, the antigen-binding domain of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences, in which the heavy chain CDR3 contains the amino acid. In another embodiment, the antigen-binding domain of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences, in which the heavy chain CDR3 contains the amino acid at positions 95, 96, 100a, and / or 101, as defined by the Kabat numbering system, in the heavy chain CDR3.

[0091] In one non-limiting embodiment of the present invention, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences, each of which contains an amino acid in its light chain antigen-binding domain that changes the antigen-binding activity of the antigen-binding molecule depending on the calcium ion concentration. In another non-limiting embodiment, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences, each of which contains the amino acid in its light chain CDR1. In other embodiments, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences, each of which contains the amino acid at positions 30, 31, and / or 32 according to the Kabat numbering system in light chain CDR1.

[0092] In another non-limiting embodiment, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences that contain the amino acid residue in light chain CDR2. In another non-limiting embodiment, a library is provided that mainly consists of antigen-binding molecules with different sequences that contain the amino acid residue at position 50 according to the Kabat numbering system in light chain CDR2.

[0093] In yet another embodiment, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences containing the amino acid residue at position 92 (Kabat numbering) in the light chain CDR3.

[0094] In another embodiment of the present invention, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences in which the amino acid residue is contained in two or three CDRs selected from the above-described light chain CDR1, CDR2, and CDR3. Furthermore, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences in which the amino acid residue is contained in any one or more of positions 30, 31, 32, 50, and / or 92 according to the Kabat numbering system in the light chain.

[0095] In a particularly preferred embodiment, it is desirable that the framework sequences of the light chain and / or heavy chain variable regions of the antigen-binding molecule comprise human germline framework sequences. Therefore, in one aspect of the present invention, if the framework sequences are completely human sequences, the antigen-binding molecules of the present invention are expected to induce little or no immunogenic response when administered to humans (e.g., for the treatment of a disease). In this sense, "comprising a germline sequence" in the present invention means that a portion of the framework sequence of the present invention is identical to a portion of any human germline framework sequence. For example, an antigen-binding molecule of the present invention in which the heavy chain FR2 sequence is a combination of heavy chain FR2 sequences from multiple different human germline framework sequences is also considered to be an antigen-binding molecule "comprising a germline sequence" in the present invention.

[0096] Suitable examples of frameworks include currently known fully human framework region sequences included on websites such as V-Base (http: / / vbase.mrc-cpe.cam.ac.uk / ). These framework region sequences can be appropriately used as germline sequences contained in the antigen-binding molecules of the present invention. Germline sequences can be classified based on their similarity (Tomlinson et al. (J. Mol. Biol. (1992) 227, 776-798), Williams and Winter (Eur. J. Immunol. (1993) 23, 1456-1461), and Cox et al. (Nat. Genetics (1994) 7, 162-168)). Suitable germline sequences can be appropriately selected from Vκ, which are classified into seven subgroups, Vλ, which are classified into ten subgroups, and VH, which are classified into seven subgroups.

[0097] Fully human VH sequences include, but are not limited to, sequences from the VH1 subgroup (e.g., VH1-2, VH1-3, VH1-8, VH1-18, VH1-24, VH1-45, VH1-46, VH1-58, VH1-69), the VH2 subgroup (e.g., VH2-5, VH2-26, VH2-70), the VH3 subgroup (e.g., VH3-7, VH3-9, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-3), and the VH4 subgroup (e.g., VH4-5, VH4-6, VH4-70, VH4-8, VH4-9, VH4-10, VH4-11, VH4-13, VH4-15, VH4-16, VH4-20, VH4-21, VH4-23, VH4-3). Suitable examples of VH sequences include those of the VH4 subgroup (VH4-0, VH3-33, VH3-35, VH3-38, VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66, VH3-72, VH3-73, VH3-74), the VH4 subgroup (VH4-4, VH4-28, VH4-31, VH4-34, VH4-39, VH4-59, VH4-61), the VH5 subgroup (VH5-51), the VH6 subgroup (VH6-1), and the VH7 subgroup (VH7-4, VH7-81). These are also described in known literature (Matsuda et al. (J. Exp. Med. (1998) 188, 1973-1975)), and those skilled in the art can appropriately design antigen-binding molecules of the present invention based on this sequence information. Completely human frameworks or framework subregions other than these can also be suitably used.

[0098] Completely human Vκ sequences include, but are not limited to, A20, A30, L1, L4, L5, L8, L9, L11, L12, L14, L15, L18, L19, L22, L23, L24, O2, O4, O8, O12, O14, and O18, which are classified into the Vk1 subgroup, and A1, A2, A3, A5, A7, A17, and A19, which are classified into the Vk2 subgroup. Preferred examples thereof include 18, A19, A23, O1, O11, A11, A27, L2, L6, L10, L16, L20, and L25, which are classified into the Vk3 subgroup, B3, which is classified into the Vk4 subgroup, B2, which is classified into the Vk5 subgroup (also referred to as Vk5-2 in this specification), and A10, A14, and A26, which are classified into the Vk6 subgroup (Kawasaki et al. (Eur. J. Immunol. (2001) 31, 1017-1028), Schable and Zachau (Biol. Chem. Hoppe Seyler (1993) 374, 1001-1022), and Brensing-Kuppers et al. (Gene (1997) 191, 173-181)).

[0099] Fully human Vλ sequences include, but are not limited to, V1-2, V1-3, V1-4, V1-5, V1-7, V1-9, V1-11, V1-13, V1-16, V1-17, V1-18, V1-19, V1-20, and V1-22, which are classified into the VL1 subgroup; V2-1, V2-6, V2-7, and V2-8, which are classified into the VL1 subgroup; Preferred examples thereof include V2-8, V2-11, V2-13, V2-14, V2-15, V2-17, and V2-19; ​​V3-2, V3-3, and V3-4, which are classified into the VL3 subgroup; V4-1, V4-2, V4-3, V4-4, and V4-6, which are classified into the VL4 subgroup; and V5-1, V5-2, V5-4, and V5-6, which are classified into the VL5 subgroup (Kawasaki et al., Genome Res. (1997) 7, 250-261).

[0100] Typically, these framework sequences differ from each other by one or more amino acid residues. These framework sequences can be used together with "at least one amino acid residue that changes the antigen-binding activity of an antigen-binding molecule depending on ion concentration conditions" of the present invention. Other examples of fully human frameworks that can be used together with "at least one amino acid residue that changes the antigen-binding activity of an antigen-binding molecule depending on ion concentration conditions" of the present invention include, but are not limited to, KOL, NEWM, REI, EU, TUR, TEI, LAY, and POM (see, for example, Kabat et al. (1991) and Wu et al. (J. Exp. Med. (1970) 132, 211-250)).

[0101] Although the present invention is not bound by any particular theory, it is believed that one reason the use of germline sequences is expected to eliminate adverse immune responses in most individuals is that somatic mutations frequently occur in the variable regions of immunoglobulins as a result of the affinity maturation step that occurs during a normal immune response. These mutations occur primarily around the CDRs, whose sequences are hypervariable, but also affect residues in the framework regions. These framework mutations are absent from germline genes and are unlikely to be immunogenic in patients. However, the normal human population is exposed to the majority of framework sequences expressed by germline genes, and as a result of immune tolerance, these germline frameworks are expected to be less immunogenic or non-immunogenic in patients. To maximize the likelihood of immune tolerance, genes encoding the variable regions can be selected from a commonly present, functional set of germline genes.

[0102] To prepare antigen-binding molecules of the present invention in which amino acids that change the antigen-binding activity of the antigen-binding molecule depending on calcium ion concentration conditions are contained in the variable region sequence, heavy chain variable region sequence, light chain variable region sequence, CDR sequence, or framework sequence, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately used.

[0103] For example, a library containing multiple antigen-binding molecules of the present invention with different sequences can be prepared by combining a light chain variable region selected as a framework sequence that previously contains at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on the calcium ion concentration with a heavy chain variable region prepared as a randomized variable region sequence library. A non-limiting example of such a library, when the ion concentration is calcium ion concentration, is a library that combines the light chain variable region sequence set forth in SEQ ID NO: 45 (Vk5-2) with a heavy chain variable region prepared as a randomized variable region sequence library.

[0104] Furthermore, the light chain variable region sequence selected as a framework sequence already containing at least one amino acid residue that changes the antigen-binding activity of the antigen-binding domain or antigen-binding molecule depending on the calcium ion concentration can be designed to contain various amino acids as residues other than the amino acid residue. In the present invention, such residues are referred to as "flexible residues." As long as the antigen-binding activity of the antigen-binding domain or antigen-binding molecule of the present invention changes depending on the ion concentration, the number and location of the flexible residues are not limited to any particular embodiment. That is, one or more flexible residues may be contained in the heavy chain and / or light chain CDR sequences and / or FR sequences. For example, when the ion concentration is calcium ion concentration, non-limiting examples of flexible residues to be introduced into the light chain variable region sequence of SEQ ID NO: 45 (Vk5-2) include the amino acid residues listed in Table 1 or Table 2.

[0105] [Table 1]

[0106] [Table 2]

[0107] As used herein, "flexible residues" refers to amino acid residue variations present at positions in the light and heavy chain variable regions where amino acids are highly diverse when comparing the amino acid sequences of known and / or natural antibodies or antigen-binding domains, with several different amino acids present at that position. Highly diverse positions are typically present in the CDR regions. In one embodiment, data provided by Kabat, Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md.) (1987 and 1991) are useful for determining highly diverse positions in known and / or natural antibodies. Additionally, several databases on the Internet (http: / / vbase.mrc-cpe.cam.ac.uk / , http: / / www.bioinf.org.uk / abs / index.html) provide collected sequences and their arrangements of numerous human light and heavy chains. Information on these sequences and their arrangements is useful for determining highly diverse positions in the present invention. According to the present invention, an amino acid position is said to be highly diverse if it has a diversity of preferably about 2 to about 20, preferably about 3 to about 19, preferably about 4 to about 18, preferably 5 to 17, preferably 6 to 16, preferably 7 to 15, preferably 8 to 14, preferably 9 to 13, preferably 10 to 12 possible different amino acid residues at that position. In some embodiments, an amino acid position may have a diversity of preferably at least about 2, preferably at least about 4, preferably at least about 6, preferably at least about 8, preferably about 10, preferably about 12 possible different amino acid residues.

[0108] Furthermore, a library containing a plurality of antigen-binding molecules of the present invention with different sequences can also be prepared by combining a light chain variable region into which at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on the ion concentration conditions has been introduced with a heavy chain variable region prepared as a randomized variable region sequence library. A non-limiting example of such a library, when the ion concentration is calcium ion concentration, is a library that combines a light chain variable region sequence in which a specific germline residue, such as SEQ ID NO: 46 (Vk1), SEQ ID NO: 47 (Vk2), SEQ ID NO: 48 (Vk3), or SEQ ID NO: 49 (Vk4), has been substituted with at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on the calcium ion concentration, with a heavy chain variable region prepared as a randomized variable region sequence library. Non-limiting examples of such amino acid residues include amino acid residues contained in the light chain CDR1. Other non-limiting examples of such amino acid residues include amino acid residues contained in the light chain CDR2. Further, other non-limiting examples of such amino acid residues include amino acid residues contained in the CDR3 of the light chain.

[0109] As described above, non-limiting examples of amino acid residues contained in the light chain CDR1 include the amino acid residues at positions 30, 31, and / or 32 according to EU numbering in the CDR1 of the light chain variable region. A non-limiting example of an amino acid residue contained in the light chain CDR2 includes the amino acid residue at position 50 according to Kabat numbering in the CDR2 of the light chain variable region. A non-limiting example of an amino acid residue contained in the light chain CDR3 includes the amino acid residue at position 92 according to Kabat numbering in the CDR3 of the light chain variable region. These amino acid residues may be contained alone or in combination, as long as they form a calcium-binding motif and / or the antigen-binding activity of the antigen-binding molecule changes depending on calcium ion concentration. Troponin C, calmodulin, parvalbumin, myosin light chain, and the like are known to have multiple calcium ion-binding sites and are thought to have originated from a common origin in molecular evolution, and it is also possible to design light chain CDR1, CDR2, and / or CDR3 to contain these binding motifs. For example, for the above purpose, a cadherin domain, an EF hand contained in calmodulin, a C2 domain contained in protein kinase C, a Gla domain contained in the blood coagulation protein Factor IX, C-type lectins contained in asialoglycoprotein receptors and mannose-binding receptors, an A domain contained in LDL receptors, annexins, thrombospondin type 3 domains, and EGF-like domains can be appropriately used.

[0110] Even when a light chain variable region into which at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on the ion concentration conditions is introduced is combined with a heavy chain variable region prepared as a randomized variable region sequence library, flexible residues can be designed to be included in the sequence of the light chain variable region, as described above. As long as the antigen-binding activity of the antigen-binding molecule of the present invention changes depending on the ion concentration conditions, the number and position of the flexible residues are not limited to a specific embodiment. That is, one or more flexible residues can be included in the CDR sequences and / or FR sequences of the heavy and / or light chains. For example, when the ion concentration is calcium ion concentration, non-limiting examples of flexible residues to be introduced into the light chain variable region sequence include the amino acid residues listed in Table 1 or Table 2.

[0111] A suitable example of a heavy chain variable region to be combined is a randomized variable region library. A randomized variable region library can be prepared by appropriately combining known methods. In a non-limiting aspect of the present invention, an immune library constructed from antibody genes derived from lymphocytes of animals immunized with a specific antigen, patients with infectious diseases or humans with increased blood antibody titers after vaccination, cancer patients, or patients with autoimmune diseases can be suitably used as a randomized variable region library.

[0112] In a non-limiting embodiment of the present invention, a synthetic library in which the CDR sequences of V genes in genomic DNA or reconstructed functional V genes are replaced with a synthetic oligonucleotide set containing a sequence encoding a codon set of appropriate length can also be used as a randomized variable region library. In this case, since diversity in the gene sequences of heavy chain CDR3 is observed, it is also possible to replace only the CDR3 sequence. The criterion for generating amino acid diversity in the variable regions of antigen-binding molecules is to provide diversity to amino acid residues at surface-exposed positions of the antigen-binding molecule. A surface-exposed position refers to a position that is determined to be surface-exposed and / or capable of contacting an antigen based on the structure, structural ensemble, and / or modeled structure of the antigen-binding molecule, and is generally the CDR. Preferably, the surface-exposed position is determined using coordinates from a three-dimensional model of the antigen-binding molecule using a computer program such as the InsightII program (Accelrys). Surface-exposed positions can be determined using algorithms known in the art (e.g., Lee and Richards (J. Mol. Biol. (1971) 55, 379-400); Connolly (J. Appl. Cryst. (1983) 16, 548-558)). Determination of surface-exposed positions can be performed using software suitable for protein modeling and three-dimensional structural information obtained from antibodies. Suitable software available for this purpose includes the SYBYL Biopolymer Module software (Tripos Associates). Generally, and preferably, when an algorithm requires user-input size parameters, the "size" of the probe used in the calculation is set to a radius of about 1.4 angstroms or less. Furthermore, methods for determining surface exposed regions and areas using software for personal computers are described by Pacios (Comput. Chem. (1994) 18 (4), 377-386 and J. Mol. Model. (1995) 1, 46-53).

[0113] Furthermore, in a non-limiting embodiment of the present invention, a naive library consisting of naive sequences, which are antibody sequences constructed from antibody genes derived from lymphocytes of healthy individuals and whose repertoire is unbiased, can also be particularly preferably used as a randomized variable region library (Gejima et al., Human Antibodies (2002) 11, 121-129, and Cardoso et al., Scand. J. Immunol. (2000) 51, 337-344). The amino acid sequence comprising a naive sequence described in the present invention refers to an amino acid sequence obtained from such a naive library.

[0114] In one embodiment of the present invention, antigen-binding domains of the present invention can be obtained from a library containing multiple antigen-binding molecules of the present invention with different sequences by combining a heavy chain variable region selected as a framework sequence that previously contains "at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on ion concentration conditions" with a light chain variable region prepared as a randomized variable region sequence library. A non-limiting example of such a library, in which the ion concentration is calcium ion concentration, is a library that combines the heavy chain variable region sequence set forth in SEQ ID NO: 50 (6RL#9-IgG1) or SEQ ID NO: 51 (6KC4-1#85-IgG1) with a light chain variable region prepared as a randomized variable region sequence library. Alternatively, instead of the light chain variable region prepared as a randomized variable region sequence library, a library can be prepared by appropriately selecting from light chain variable regions with germline sequences. A suitable example is a library that combines the heavy chain variable region sequence set forth in SEQ ID NO: 50 (6RL#9-IgG1) or SEQ ID NO: 51 (6KC4-1#85-IgG1) with a light chain variable region having a germline sequence.

[0115] Furthermore, flexible residues can be included in the heavy chain variable region sequence selected as a framework sequence that already contains the aforementioned "at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on ion concentration conditions." The number and location of the flexible residues are not limited to a specific embodiment, as long as the antigen-binding activity of the antigen-binding molecule of the present invention changes depending on ion concentration conditions. That is, one or more flexible residues can be included in the heavy chain and / or light chain CDR sequences and / or FR sequences. For example, when the ion concentration is calcium ion concentration, non-limiting examples of flexible residues to be introduced into the heavy chain variable region sequence of SEQ ID NO: 50 (6RL#9-IgG1) include all amino acid residues in heavy chain CDR1 and CDR2, as well as amino acid residues in heavy chain CDR3 other than positions 95, 96, and / or 100a. Alternatively, non-limiting examples of flexible residues that may be introduced into the heavy chain variable region sequence set forth in SEQ ID NO: 51 (6KC4-1#85-IgG1) include all amino acid residues in heavy chain CDR1 and CDR2, as well as amino acid residues in CDR3 other than positions 95 and / or 101 of heavy chain CDR3.

[0116] Furthermore, a library containing multiple antigen-binding molecules with different sequences can also be prepared by combining a heavy chain variable region into which the aforementioned "at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on ion concentration conditions" has been introduced with a light chain variable region prepared as a randomized variable region sequence library or a light chain variable region having a germline sequence. A non-limiting example of such a library, when the ion concentration is calcium ion concentration, is a library that combines a heavy chain variable region sequence in which a specific residue in the heavy chain variable region has been substituted with at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on calcium ion concentration conditions, with a light chain variable region prepared as a randomized variable region sequence library or a light chain variable region having a germline sequence. Non-limiting examples of such amino acid residues include amino acid residues contained in the heavy chain CDR1. Other non-limiting examples of such amino acid residues include amino acid residues contained in the heavy chain CDR2. Another non-limiting example of such amino acid residues includes amino acid residues contained in the heavy chain CDR3. Non-limiting examples of such amino acid residues contained in the heavy chain CDR3 include amino acids at positions 95, 96, 100a, and / or 101 according to the Kabat numbering system in the CDR3 of the heavy chain variable region. Furthermore, these amino acid residues may be contained alone or in combination of two or more, as long as they form a calcium-binding motif and / or the antigen-binding activity of the antigen-binding molecule changes depending on calcium ion concentration.

[0117] Even when combining a heavy chain variable region into which at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on ion concentration conditions is introduced with a light chain variable region prepared as a randomized variable region sequence library or a light chain variable region having a germline sequence, as described above, it is possible to design the heavy chain variable region sequence to contain flexible residues. As long as the antigen-binding activity of the antigen-binding molecule of the present invention changes depending on ion concentration conditions, the number and position of the flexible residues are not limited to a specific embodiment. That is, one or more flexible residues may be contained in the heavy chain CDR sequence and / or FR sequence. Furthermore, a randomized variable region library can also be suitably used as the amino acid sequence of CDR1, CDR2, and / or CDR3 of the heavy chain variable region other than the amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on ion concentration conditions. When a germline sequence is used as the light chain variable region, non-limiting examples include germline sequences such as SEQ ID NO: 46 (Vk1), SEQ ID NO: 47 (Vk2), SEQ ID NO: 48 (Vk3), and SEQ ID NO: 49 (Vk4).

[0118] As the amino acid that changes the antigen-binding activity of an antigen-binding molecule depending on calcium ion concentration conditions, any amino acid can be suitably used as long as it forms a calcium-binding motif, and specific examples of such amino acids include electron-donating amino acids, such as serine, threonine, asparagine, glutamine, aspartic acid, and glutamic acid.

[0119] Furthermore, the "ion concentration condition" of the present invention can also be exemplified by the "pH condition." The pH condition can also be referred to as the hydrogen ion concentration condition. In the present invention, the condition of the concentration of protons, i.e., the atomic nuclei of hydrogen atoms, is treated synonymously with the hydrogen exponent (pH) condition. When the activity of hydrogen ions in an aqueous solution is expressed as aH+, pH is defined as -log10aH+. When the ionic strength in an aqueous solution is (for example, 10 -3If the temperature is lower than 25°C, aH+ is approximately equal to the hydrogen ionic strength. For example, the ionic product of water at 25°C and 1 atmosphere is Kw = aH + aOH = 10 -14 Therefore, in pure water, aH+ = aOH = 10 -7 In this case, a pH of 7 is neutral, an aqueous solution with a pH of less than 7 is acidic, and an aqueous solution with a pH of more than 7 is alkaline.

[0120] In the present invention, when pH conditions are used as ion concentration conditions, pH conditions include high proton concentration or low pH, i.e., acidic pH conditions, and low proton concentration or high pH, ​​i.e., neutral pH conditions. The phrase "the antigen-binding activity of an antigen-binding domain contained in an antigen-binding molecule of the present invention changes depending on pH conditions" refers to a change in the antigen-binding activity of the antigen-binding domain contained in the antigen-binding molecule due to the difference between high proton concentration or low pH (acidic pH range) and low proton concentration or high pH (neutral pH range). For example, this may be the case when the antigen-binding activity of an antigen-binding molecule is higher under neutral pH conditions than under acidic pH conditions. Another example may be the case when the antigen-binding activity of an antigen-binding molecule is higher under acidic pH conditions than under neutral pH conditions.

[0121] As used herein, the neutral pH range is not limited to a specific numerical value, but may preferably be selected from the range of pH 6.7 to pH 10.0. In another embodiment, it may be selected from the range of pH 6.7 to pH 9.5. In a different embodiment, it may be selected from the range of pH 7.0 to pH 9.0, and in another embodiment, it may be selected from the range of pH 7.0 to pH 8.0. In particular, pH 7.4, which is close to the pH in plasma (blood) in vivo, is preferred.

[0122] As used herein, the acidic pH range is not limited to a specific numerical value, but may preferably be selected from the range of pH 4.0 to pH 6.5. In another embodiment, it may be selected from the range of pH 4.5 to pH 6.5. In a different embodiment, it may be selected from the range of pH 5.0 to pH 6.5, and in another embodiment, it may be selected from the range of pH 5.5 to pH 6.5. In particular, a pH of 5.8, which is close to the ionized calcium concentration in early endosomes in vivo, is preferred.

[0123] In the present invention, "antigen-binding activity at a high proton concentration or low pH (acidic pH range) is lower than that at a low proton concentration or high pH (neutral pH range)" means that the antigen-binding activity of an antigen-binding domain of the present invention or an antigen-binding molecule comprising said domain at a pH selected from the range of pH 4.0 to pH 6.5 is weaker than that at a pH selected from the range of pH 6.7 to pH 10.0. Preferably, this means that the antigen-binding activity of an antigen-binding domain of the present invention or an antigen-binding molecule comprising said domain at a pH selected from the range of pH 4.5 to pH 6.5 is weaker than that at a pH selected from the range of pH 6.7 to pH 9.5, and more preferably, it means that the antigen-binding activity of an antigen-binding molecule at a pH selected from the range of pH 5.0 to pH 6.5 is weaker than that at a pH selected from the range of pH 7.0 to pH 9.0. Preferably, it means that the antigen-binding activity of an antigen-binding molecule at a pH selected from the range of pH 5.5 to pH 6.5 is weaker than that at a pH selected from the range of pH 7.0 to pH 8.0. Particularly preferably, it means that the antigen-binding activity at the pH in early endosomes in vivo is weaker than that at the pH in plasma in vivo, and specifically, it means that the antigen-binding activity of an antigen-binding molecule at pH 5.8 is weaker than that at pH 7.4.

[0124] Whether the antigen-binding activity of an antigen-binding domain or an antigen-binding molecule containing said domain changes depending on pH conditions can be determined by using known measurement methods, for example, as described above in the section on binding activity. For example, these measurement methods measure binding activity under different pH conditions. For example, to confirm that the antigen-binding activity of an antigen-binding domain or an antigen-binding molecule containing said domain changes more significantly under a neutral pH range than under an acidic pH range, the antigen-binding activities of the domain or molecule under acidic and neutral pH ranges are compared.

[0125] Furthermore, in the present invention, the expression "the antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than the antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range" can also be expressed as "the antigen-binding activity of an antigen-binding domain or an antigen-binding molecule comprising said domain at a low proton concentration or high pH, ​​i.e., in a neutral pH range, is higher than the antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range." In the present invention, "antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than that at a low proton concentration or high pH, ​​i.e., in a neutral pH range" may also be expressed as "antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is weaker than that at a low proton concentration or high pH, ​​i.e., in a neutral pH range". Similarly, "antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is reduced compared to that at a low proton concentration or high pH, ​​i.e., in a neutral pH range" may also be expressed as "antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is weakened than that at a low proton concentration or high pH, ​​i.e., in a neutral pH range".

[0126] Conditions other than hydrogen ion concentration or pH when measuring antigen-binding activity can be appropriately selected by those skilled in the art and are not particularly limited. For example, measurements can be performed in HEPES buffer at 37°C. Measurements can be performed using, for example, Biacore (GE Healthcare). When measuring the binding activity of an antigen-binding domain or an antigen-binding molecule containing the domain to an antigen, if the antigen is a soluble antigen, the binding activity to the soluble antigen can be evaluated by passing the antigen as an analyte through a chip on which the antigen-binding domain or an antigen-binding molecule containing the domain is immobilized. When the antigen is a membrane-type antigen, the binding activity to the membrane-type antigen can be evaluated by passing the antigen-binding domain or an antigen-binding molecule containing the domain as an analyte through a chip on which the antigen is immobilized. In the antigen-binding molecules of the present invention, as long as the antigen-binding activity at a high proton concentration or low pH, i.e., an acidic pH range, is weaker than the antigen-binding activity at a low proton concentration or high pH, ​​i.e., a neutral pH range, the ratio of the antigen-binding activity at a high proton concentration or low pH, i.e., an acidic pH range, to the antigen-binding activity at a low proton concentration or high pH, ​​i.e., a neutral pH range, is not particularly limited. Preferably, the ratio of the KD (Dissociation constant) for the antigen at a high proton concentration or low pH, i.e., an acidic pH range, to the KD at a low proton concentration or high pH, ​​i.e., a neutral pH range, KD (pH5.8) / KD (pH7.4), is 2 or greater, more preferably 10 or greater, and even more preferably 40 or greater. The upper limit of the KD (pH 5.8) / KD (pH 7.4) value is not particularly limited, and may be any value, such as 400, 1000, or 10000, as long as it can be produced by those skilled in the art.

[0127] Alternatively, the dissociation rate constant kd (dissociation rate constant) can also be suitably used as an indicator of the ratio of antigen-binding activity of an antigen-binding domain of the present invention or an antigen-binding molecule comprising said domain at a high proton concentration or low pH, i.e., an acidic pH range, to that at a low proton concentration or high pH, ​​i.e., a neutral pH range. When kd (dissociation rate constant) is used instead of KD (dissociation constant) as an indicator of binding activity ratio, the ratio of kd (dissociation rate constant) at a high proton concentration or low pH, i.e., an acidic pH range, to kd (dissociation rate constant) at a low proton concentration or high pH, ​​i.e., a neutral pH range, i.e., kd (at an acidic pH range) / kd (at a neutral pH range), is preferably 2 or more, more preferably 5 or more, even more preferably 10 or more, and more preferably 30 or more. The upper limit of the value of Kd (in the acidic pH range) / kd (in the neutral pH range) is not particularly limited, and may be any value, such as 50, 100, or 200, as long as it can be produced within the technical common sense of a person skilled in the art.

[0128] When the antigen is a soluble antigen, kd (dissociation rate constant) can be used as the value of antigen-binding activity, whereas when the antigen is a membrane-type antigen, apparent kd (apparent dissociation rate constant) can be used. kd (dissociation rate constant) and apparent kd (apparent dissociation rate constant) can be measured by methods known to those skilled in the art, such as using Biacore (GE Healthcare) or a flow cytometer. In the present invention, when measuring the antigen-binding activity of an antigen-binding domain or an antigen-binding molecule containing the domain at different hydrogen ion concentrations, i.e., pH, it is preferable to keep all conditions other than hydrogen ion concentration, i.e., pH, the same.

[0129] For example, an antigen-binding domain or antigen-binding molecule whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than its antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, which is one embodiment of the present invention, can be obtained by screening for antigen-binding domains or antigen-binding molecules, comprising the following steps (a) to (c): (a) determining the antigen-binding activity of an antigen-binding domain or antigen-binding molecule under an acidic pH range condition; (b) determining the antigen-binding activity of the antigen-binding domain or antigen-binding molecule under a neutral pH condition; (c) selecting antigen-binding domains or antigen-binding molecules whose antigen-binding activity in an acidic pH range is lower than that in a neutral pH range.

[0130] Furthermore, an embodiment of the present invention, an antigen-binding domain or antigen-binding molecule whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than its antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, can be obtained by screening antigen-binding domains or antigen-binding molecules or a library thereof, which comprises the following steps (a) to (c): (a) contacting an antigen with an antigen-binding domain or antigen-binding molecule, or a library thereof, under a neutral pH range; (b) placing the antigen-binding domain or antigen-binding molecule bound to the antigen in step (a) under an acidic pH range; (c) isolating the antigen-binding domain or antigen-binding molecule dissociated in step (b).

[0131] Furthermore, an antigen-binding domain or antigen-binding molecule whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than its antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, which is one embodiment of the present invention, can be obtained by screening antigen-binding domains or antigen-binding molecules, or a library thereof, comprising the following steps (a) to (d): (a) contacting a library of antigen-binding domains or antigen-binding molecules with an antigen under an acidic pH range; (b) selecting antigen-binding domains or antigen-binding molecules that do not bind to the antigen in step (a); (c) allowing the antigen-binding domain or antigen-binding molecule selected in step (b) to bind to an antigen in a neutral pH range; (d) isolating the antigen-binding domain or antigen-binding molecule that bound to the antigen in step (c).

[0132] Furthermore, an antigen-binding domain or antigen-binding molecule whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than its antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, which is one embodiment of the present invention, can be obtained by a screening method comprising the following steps (a) to (c): (a) contacting a library of antigen-binding domains or antigen-binding molecules with an antigen-immobilized column at a neutral pH; (b) eluting the antigen-binding domain or antigen-binding molecule bound to the column in step (a) from the column under an acidic pH condition; (c) isolating the antigen-binding domain or antigen-binding molecule eluted in step (b).

[0133] Furthermore, an antigen-binding domain or antigen-binding molecule whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than its antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, which is one embodiment of the present invention, can be obtained by a screening method comprising the following steps (a) to (d): (a) passing a library of antigen-binding domains or antigen-binding molecules through an antigen-immobilized column under an acidic pH condition; (b) recovering the antigen-binding domain or antigen-binding molecule that did not bind to the column and was eluted in step (a); (c) allowing the antigen-binding domain or antigen-binding molecule recovered in step (b) to bind to an antigen in a neutral pH range; (d) isolating the antigen-binding domain or antigen-binding molecule that bound to the antigen in step (c).

[0134] Furthermore, an antigen-binding domain or antigen-binding molecule whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than its antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, which is one embodiment of the present invention, can be obtained by a screening method comprising the following steps (a) to (d): (a) contacting a library of antigen-binding domains or antigen-binding molecules with an antigen under a neutral pH range; (b) obtaining the antigen-binding domain or antigen-binding molecule bound to the antigen in step (a); (c) placing the antigen-binding domain or antigen-binding molecule obtained in step (b) under an acidic pH condition; (d) isolating antigen-binding domains or antigen-binding molecules selected in step (c) whose antigen-binding activity is weaker than the criterion selected in step (b).

[0135] The above steps may be repeated two or more times. Thus, the present invention provides antigen-binding domains or antigen-binding molecules whose antigen-binding activity in an acidic pH range is lower than that in a neutral pH range, obtained by the above-mentioned screening method, which further comprises repeating steps (a) to (c) or (a) to (d) two or more times. The number of times steps (a) to (c) or (a) to (d) are repeated is not particularly limited, but is typically no more than 10 times.

[0136] In the screening methods of the present invention, the antigen-binding activity of an antigen-binding domain or antigen-binding molecule under high proton concentration conditions or low pH, i.e., in the acidic pH range, is not particularly limited as long as it is antigen-binding activity between pH 4.0 and 6.5, with a preferred pH being between pH 4.5 and 6.6. Another preferred pH is antigen-binding activity between pH 5.0 and 6.5, with a further preferred pH being between pH 5.5 and 6.5. A more preferred pH is the pH within early endosomes in vivo, specifically, antigen-binding activity at pH 5.8. Furthermore, the antigen-binding activity of an antigen-binding domain or antigen-binding molecule under low proton concentration conditions or high pH, ​​i.e., in the neutral pH range, is not particularly limited as long as it is antigen-binding activity between pH 6.7 and 10, with a preferred pH being between pH 6.7 and 9.5. Another preferred pH is antigen-binding activity between pH 7.0 and 9.5, with a further preferred pH being between pH 7.0 and 8.0. A more preferable pH is the pH in plasma in vivo, specifically, the antigen-binding activity at pH 7.4.

[0137] The antigen-binding activity of an antigen-binding domain or antigen-binding molecule can be measured by methods known to those skilled in the art, and conditions other than ionized calcium concentration can be appropriately determined by those skilled in the art. The antigen-binding activity of an antigen-binding domain or antigen-binding molecule can be evaluated as KD (Dissociation constant), apparent KD (Apparent dissociation constant), kd (Dissociation rate constant), or apparent kd (Apparent dissociation rate constant), etc. These can be measured by methods known to those skilled in the art, such as Biacore (GE Healthcare), Scatchard plots, FACS, etc.

[0138] In the present invention, the step of selecting antigen-binding domains or antigen-binding molecules whose antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, is higher than that at a high proton concentration or low pH, i.e., in an acidic pH range, means the same as the step of selecting antigen-binding domains or antigen-binding molecules whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than that at a low proton concentration or high pH, ​​i.e., in a neutral pH range.

[0139] As long as the antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, is higher than that at a high proton concentration or low pH, i.e., in an acidic pH range, the difference in antigen-binding activity between a low proton concentration or high pH, ​​i.e., in a neutral pH range, and a high proton concentration or low pH, i.e., in an acidic pH range, is not particularly limited; however, the antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, is preferably at least 2-fold, more preferably at least 10-fold, and even more preferably at least 40-fold, that at a high proton concentration or low pH, i.e., in an acidic pH range.

[0140] Antigen-binding domains or antigen-binding molecules of the present invention whose antigen-binding activity changes depending on hydrogen ion concentration conditions and which are screened by the above-mentioned screening methods may be prepared in any manner. For example, pre-existing antigen-binding molecules, pre-existing libraries (such as phage libraries), antibodies or libraries prepared from hybridomas obtained by immunizing animals or B cells from immunized animals, antibodies or libraries in which amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acid mutations have been introduced into these antibodies or libraries (libraries with an increased content of amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids, libraries in which amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acid mutations have been introduced at specific sites, etc., can be used.

[0141] Antigen-binding domains or antigen-binding molecules whose antigen-binding activity at low proton concentrations or high pH, ​​i.e., in the neutral pH range, is higher than that at high proton concentrations or low pH, i.e., in the acidic pH range, can be obtained from antigen-binding domains or antigen-binding molecules prepared from hybridomas obtained by immunizing animals or B cells from immunized animals. Suitable methods include, for example, antigen-binding molecules in which at least one amino acid in the antigen-binding domain or antigen-binding molecule is substituted with an amino acid with a side chain pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or a non-natural amino acid mutation, or in which an amino acid with a side chain pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or a non-natural amino acid is inserted into the antigen-binding domain or antigen-binding molecule, as described in International Publication WO 2009 / 125825.

[0142] The position at which the mutation of an amino acid with a side chain pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid is introduced is not particularly limited, and may be any site as long as the antigen-binding activity in the acidic pH range is weaker than that in the neutral pH range compared to before the substitution or insertion (the value of KD(acidic pH range) / KD(neutral pH range) is increased, or the value of kd(acidic pH range) / kd(neutral pH range) is increased). For example, when the antigen-binding molecule is an antibody, suitable sites include the variable region and CDRs of the antibody. The number of amino acids to be substituted with amino acids (e.g., histidine and glutamic acid) or unnatural amino acids having a side chain pKa of 4.0-8.0, or the number of amino acids to be inserted, can be determined appropriately by those skilled in the art. A single amino acid (e.g., histidine and glutamic acid) or unnatural amino acid having a side chain pKa of 4.0-8.0 may be substituted, a single amino acid (e.g., histidine and glutamic acid) or unnatural amino acid having a side chain pKa of 4.0-8.0 may be inserted, two or more amino acids (e.g., histidine and glutamic acid) or unnatural amino acids having a side chain pKa of 4.0-8.0 may be substituted, or two or more amino acids (e.g., histidine and glutamic acid) or unnatural amino acids having a side chain pKa of 4.0-8.0 may be inserted. In addition to substitution with or insertion of an amino acid with a side chain pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid, deletion, addition, insertion, and / or substitution of other amino acids may also be performed simultaneously.Substitution with or insertion of amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids can be carried out randomly by methods known to those skilled in the art, such as histidine scanning, in which alanine in alanine scanning is replaced with histidine, and antigen-binding molecules with increased KD (acidic pH range) / KD (neutral pH range) or kd (acidic pH range) / kd (neutral pH range) values ​​compared to before mutation can be selected from antigen-binding domains or antibodies into which mutations such as substitution or insertion of amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids have been randomly introduced.

[0143] Preferred examples of antigen-binding molecules that have been mutated to amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids and have lower antigen-binding activity in the acidic pH range than in the neutral pH range include antigen-binding molecules whose antigen-binding activity in the neutral pH range after mutation to amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids is equivalent to the antigen-binding activity in the neutral pH range before mutation to amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids. In the present invention, an antigen-binding molecule after mutation with an amino acid having a side chain pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or a non-natural amino acid has antigen-binding activity equivalent to that of an antigen-binding molecule before mutation with an amino acid having a side chain pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or a non-natural amino acid means that, when the antigen-binding activity of the antigen-binding molecule before mutation with an amino acid having a side chain pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or a non-natural amino acid is taken as 100%, the antigen-binding activity of the antigen-binding molecule after mutation with an amino acid having a side chain pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or a non-natural amino acid is at least 10%, preferably 50% or more, more preferably 80% or more, and even more preferably 90% or more. The antigen-binding activity at pH 7.4 after mutation with an amino acid whose side chain has a pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid may be higher than the antigen-binding activity at pH 7.4 before mutation with an amino acid whose side chain has a pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid. If the antigen-binding activity of an antigen-binding molecule is reduced by substitution with or insertion of an amino acid whose side chain has a pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid, the antigen-binding activity can be made equivalent to the antigen-binding activity before substitution or insertion of an amino acid whose side chain has a pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid by substituting, deleting, adding, and / or inserting one or more amino acids in the antigen-binding molecule.The present invention also encompasses antigen-binding molecules whose binding activity is equivalent to that of the antigen-binding molecules obtained by substituting or inserting an amino acid with a side chain pKa of 4.0 to 8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid, followed by substituting, deleting, adding, and / or inserting one or more amino acids.

[0144] In one embodiment of the present invention, a library comprising multiple antigen-binding domains or antigen-binding molecules of the present invention with different sequences can also be prepared by combining a light chain variable region into which "at least one amino acid residue that changes the antigen-binding activity of an antigen-binding domain or antigen-binding molecule depending on hydrogen ion concentration conditions" has been introduced with a heavy chain variable region prepared as a randomized variable region sequence library.

[0145] Non-limiting examples of such amino acid residues include those contained in light chain CDR1. Other non-limiting examples of such amino acid residues include those contained in light chain CDR2. Further non-limiting examples of such amino acid residues include those contained in light chain CDR3.

[0146] As described above, non-limiting examples of amino acid residues contained in the light chain CDR1 include those at positions 24, 27, 28, 31, 32, and / or 34 according to the Kabat numbering system in the CDR1 of the light chain variable region. Non-limiting examples of amino acid residues contained in the light chain CDR2 include those at positions 50, 51, 52, 53, 54, 55, and / or 56 according to the Kabat numbering system in the CDR2 of the light chain variable region. Non-limiting examples of amino acid residues contained in the light chain CDR3 include those at positions 89, 90, 91, 92, 93, 94, and / or 95A according to the Kabat numbering system in the CDR3 of the light chain variable region. Furthermore, these amino acid residues may be contained alone or in combination of two or more, as long as the binding activity of the antigen-binding molecule to an antigen changes depending on the hydrogen ion concentration.

[0147] Even when combining a light chain variable region into which the aforementioned "at least one amino acid residue that changes the antigen-binding activity of an antigen-binding molecule depending on hydrogen ion concentration conditions" has been introduced with a heavy chain variable region prepared as a randomized variable region sequence library, it is possible to design the sequence of the light chain variable region to contain flexible residues, as described above. As long as the antigen-binding activity of the antigen-binding domain or antigen-binding molecule of the present invention changes depending on hydrogen ion concentration conditions, the number and position of the flexible residues are not limited to any particular embodiment. That is, one or more flexible residues may be contained in the CDR sequences and / or FR sequences of the heavy and / or light chains. For example, non-limiting examples of flexible residues that can be introduced into the light chain variable region sequence include the amino acid residues listed in Table 3 or Table 4. Furthermore, germline sequences such as, but not limited to, Vk1 (SEQ ID NO: 46), Vk2 (SEQ ID NO: 47), Vk3 (SEQ ID NO: 48), and Vk4 (SEQ ID NO: 49) can be suitably used as the amino acid sequence of the light chain variable region excluding flexible residues and amino acid residues that change the antigen-binding activity of the antigen-binding domain or antigen-binding molecule depending on hydrogen ion concentration conditions.

[0148] [Table 3]

[0149] [Table 4]

[0150] Any amino acid residue can be suitably used as the amino acid residue that changes the antigen-binding activity of an antigen-binding domain or antigen-binding molecule depending on hydrogen ion concentration conditions. Specific examples of such amino acid residues include amino acids with a side chain pKa of 4.0 to 8.0. Suitable examples of such electron-donating amino acids include natural amino acids such as histidine and glutamic acid, as well as histidine analogs (US2009 / 0035836) and unnatural amino acids such as m-NO2-Tyr (pKa 7.45), 3,5-Br2-Tyr (pKa 7.21), and 3,5-I2-Tyr (pKa 7.38) (Bioorg. Med. Chem. (2003) 11 (17), 3761-3768). Particularly suitable examples of such amino acid residues include amino acids whose side chain pKa is 6.0 to 7.0. Suitable examples of such electron-donating amino acids include histidine.

[0151] A suitable example of a heavy chain variable region to be combined is a randomized variable region library. A randomized variable region library can be prepared by appropriately combining known methods. In a non-limiting aspect of the present invention, an immune library constructed from antibody genes derived from lymphocytes of animals immunized with a specific antigen, patients with infectious diseases or humans with increased blood antibody titers after vaccination, cancer patients, or patients with autoimmune diseases can be suitably used as a randomized variable region library.

[0152] In a non-limiting embodiment of the present invention, a synthetic library in which the CDR sequences of V genes in genomic DNA or reconstructed functional V genes are replaced with a synthetic oligonucleotide set containing a sequence encoding a codon set of appropriate length can also be used as a randomized variable region library, as described above. In this case, since diversity in the gene sequences of heavy chain CDR3 is observed, it is also possible to replace only the CDR3 sequence. The criterion for generating amino acid diversity in the variable regions of antigen-binding molecules is to provide diversity to amino acid residues at surface-exposed positions of the antigen-binding molecule. A surface-exposed position refers to a position that is determined to be surface-exposed and / or capable of contacting an antigen based on the structure, structural ensemble, and / or modeled structure of the antigen-binding molecule, and is generally the CDR. Preferably, the surface-exposed position is determined using coordinates from a three-dimensional model of the antigen-binding molecule using a computer program such as the InsightII program (Accelrys). Surface-exposed positions can be determined using algorithms known in the art (e.g., Lee and Richards (J. Mol. Biol. (1971) 55, 379-400); Connolly (J. Appl. Cryst. (1983) 16, 548-558)). Determination of surface-exposed positions can be performed using software suitable for protein modeling and three-dimensional structural information obtained from antibodies. Suitable software available for this purpose includes the SYBYL Biopolymer Module software (Tripos Associates). Generally, and preferably, when an algorithm requires a user-input size parameter, the "size" of the probe used in the calculation is set to a radius of about 1.4 angstroms or less. Furthermore, methods for determining surface exposed regions and areas using software for personal computers are described by Pacios (Comput. Chem. (1994) 18 (4), 377-386 and J. Mol. Model. (1995) 1, 46-53).

[0153] Furthermore, in a non-limiting embodiment of the present invention, a naive library consisting of naive sequences, which are antibody sequences constructed from antibody genes derived from lymphocytes of healthy individuals and whose repertoire is unbiased, can also be particularly preferably used as a randomized variable region library (Gejima et al., Human Antibodies (2002) 11, 121-129, and Cardoso et al., Scand. J. Immunol. (2000) 51, 337-344).

[0154] Furthermore, amino acid modifications can be combined to further enhance the human FcRn-binding activity under acidic pH conditions. More specifically, for example, modifications used to enhance human FcRn-binding activity under acidic pH conditions include substituting Met at position 428 (EU numbering) with Leu and Asn at position 434 with Ser (Nat Biotechnol, 2010 28:157-159), substituting Asn at position 434 with Ala (Drug Metab Dispos. 2010 Apr;38(4):600-5), substituting Met at position 252 with Tyr, Ser at position 254 with Thr, and Thr at position 256 with Glu (J Biol Chem, 2006, 281:23514-23524), and substituting Thr at position 250 with Gln and Met at position 428 with Leu (J Immunol. 2006, 176(1):346-56), a method of substituting Asn at position 434 with His (Clinical Pharmacology & Therapeutics (2011) 89(2):283-290.), and modifications such as those described in WO2010106180, WO2010045193, WO2009058492, WO2008022152, WO2006050166, WO2006053301, WO2006031370, WO2005123780, WO2005047327, WO2005037867, WO2004035752, WO2002060919, etc.

[0155] Recently, it has been reported that a humanized anti-CD4 antibody in which Asn at position 434 (EU numbering) was substituted with His to enhance its binding activity to human FcRn under acidic pH conditions and improve plasma retention binds to rheumatoid factor (RF) (Clin Pharmacol Ther. 2011 Feb;89(2):283-90). This antibody has the Fc region of human IgG1, and the substitution of Asn at position 434, which is located in the FcRn-binding site, with His allows rheumatoid factor, which recognizes the substitution, to bind.

[0156] As mentioned above, various modifications have been reported to enhance the binding activity to human FcRn under acidic pH conditions. However, introducing these modifications into the FcRn-binding site in the Fc region may potentially enhance the binding activity to rheumatoid factors that recognize this site. However, by introducing a modification into the corresponding site in the Fc region that reduces only the rheumatoid factor-binding activity without reducing the FcRn-binding activity, it is possible to prepare an antigen-binding molecule that has enhanced human FcRn-binding activity in the acidic pH range but no rheumatoid factor-binding activity.

[0157] Such modifications that reduce the binding activity to rheumatoid factors include modifications at positions 248-257, 305-314, 342-352, 380-386, 388, 414-421, 423, 425-437, 439, and 441-444 (EU numbering), preferably at positions 387, 422, 424, 426, 433, 436, 438, and 440. Particularly preferred are modifications that substitute Val at position 422 with Glu or Ser, Ser at position 424 with Arg, His at position 433 with Asp, Tyr at position 436 with Thr, Gln at position 438 with Arg or Lys, and Ser at position 440 with Glu or Asp. These modifications may be used alone or in combination.

[0158] Alternatively, an N-glycosylation sequence may be introduced into the site in order to reduce the binding activity to rheumatoid factor. Specifically, Asn-Xxx-Ser / Thr (Xxx represents any amino acid except Pro) is known as an N-glycosylation sequence. Introducing this sequence into the site in the Fc region allows addition of an N-glycan, which can inhibit binding to RF due to steric hindrance of the N-glycan. Preferred modifications for adding an N-glycan include substituting Lys at position 248 with Asn, substituting Ser at position 424 with Asn, substituting Tyr at position 436 with Asn and Gln at position 438 with Thr, and substituting Gln at position 438 with Asn. Particularly preferred is substituting Ser at position 424 with Asn.

[0159] Preferred examples of polypeptides of the present invention comprising an Fc region variant include polypeptides comprising at least two associated Fc region variants, such as IgG antibodies. When an IgG antibody is used as the antibody, the type of constant region is not limited, and IgG isotypes (subclasses) such as IgG1, IgG2, IgG3, and IgG4 can be used. The IgG antibodies of the present invention are preferably human IgG, more preferably human IgG1 or human IgG4, and the amino acid sequences of the heavy chain constant regions of human IgG1 and human IgG4 are known. For the human IgG1 constant region, several allotype sequences due to genetic polymorphisms 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.

[0160] In the present invention, an amino acid modification refers to any one of substitution, deletion, addition, insertion, or modification, or a combination thereof. In the present invention, an amino acid modification can be referred to as an amino acid mutation, and the terms are used interchangeably. When an amino acid residue is substituted, the purpose is to achieve modifications in, for example, the following points (a) to (c) by substituting another amino acid residue. (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.

[0161] 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 influence chain orientation: gly, pro; and (6) Aromaticity: trp, tyr, phe.

[0162] 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. The substitutions in the present invention may be conservative substitutions, non-conservative substitutions, or a combination of conservative and non-conservative substitutions.

[0163] 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, HJ (1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456). Mutations can be performed by methods such as 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 U S A. 82, 488-492), PCR mutagenesis, cassette mutagenesis, etc.

[0164] The amino acid modifications of the present invention include post-translational modifications. Specific examples of post-translational modifications include the addition or deletion of a glycosylation chain. For example, in the IgG1 constant region consisting of the amino acid sequence set forth in SEQ ID NO: 31, the amino acid residue at position 297 (EU numbering) can be modified with a glycosylation chain. The glycosylation structure to be modified is not limited. In general, antibodies expressed in eukaryotic cells contain glycosylation modifications in the constant region. Therefore, antibodies expressed in the following cells are usually modified with some form of glycosylation. Mammalian antibody-producing cells A eukaryotic cell transformed with an expression vector containing DNA encoding the antibody

[0165] The eukaryotic cells shown here include yeast and animal cells. For example, CHO cells and HEK293H cells are typical animal cells for transformation with an expression vector containing antibody-encoding DNA. On the other hand, constant regions of the present invention also include those that are not glycosylated at the relevant positions. Antibodies whose constant regions are not glycosylated can be obtained by expressing a gene encoding the antibody in prokaryotic cells such as Escherichia coli.

[0166] 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.).

[0167] Furthermore, the present invention provides antibodies comprising any of the above-described Fc region variants.

[0168] 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), a chimeric antibody, or a humanized antibody, as long as it exhibits the desired biological activity.

[0169] 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 include human antibody, mouse antibody, rat antibody, and rabbit antibody.

[0170] Methods for producing antibodies are well known to those skilled in the art, and include, for example, monoclonal antibodies, which may be produced by hybridoma techniques (Kohler and Milstein, Nature 256:495 (1975)), recombinant techniques (U.S. Patent No. 4,816,567), or isolated from phage antibody libraries (Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1991)).

[0171] Humanized antibodies are also called reshaped human antibodies. Specifically, humanized antibodies in which the CDRs of a non-human animal, such as a mouse antibody, are grafted onto a human antibody are well known. Common genetic recombination techniques for obtaining humanized antibodies are also known. Specifically, overlap extension PCR is a well-known method for grafting the CDRs of a mouse antibody onto human FRs.

[0172] A vector for expressing a humanized antibody can be prepared by inserting DNA encoding an antibody variable region in which three CDRs and four FRs are linked together with DNA encoding a human antibody constant region into an expression vector so that they are fused in frame. After introducing the integration vector into a host to establish recombinant cells, the recombinant cells are cultured to express the DNA encoding the humanized antibody, and the humanized antibody is produced in the cultured cells (see European Patent Publication EP 239400 and International Publication WO1996 / 002576).

[0173] 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 to graft mouse CDRs onto human FRs.

[0174] Transgenic animals carrying the entire repertoire of human antibody genes (see International Publications WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, and WO1996 / 033735) can be used as immunized animals, and desired human antibodies can be obtained by DNA immunization.

[0175] Furthermore, techniques for obtaining human antibodies by panning using a human antibody library are 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. Phages expressing scFvs that bind to an antigen can be selected. The DNA sequence encoding the V region of a human antibody that binds to an antigen can be determined by analyzing the genes of the selected phage. After determining the DNA sequence of the scFv that binds to the 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 such as those 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).

[0176] The variable regions constituting the antibodies of the present invention can be variable regions that recognize any antigen.

[0177] The antigen used herein is not particularly limited and may be any antigen. Suitable examples of antigens include ligands (cytokines, chemokines, etc.), receptors, cancer antigens, MHC antigens, differentiation antigens, immunoglobulins, and immune complexes containing immunoglobulins as a part.

[0178] 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. 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.

[0179] 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, 3387-3396), Fantl et al. (Annu. Rev. Biochem. (1993), 62, 453-481), Smith et al. (Cell (1994) 76 (6) 959-962), Flower DR. (Biochim. Biophys. Acta (1999) 1422 (3) 207-234), etc.

[0180] Specific examples of receptors belonging to the above receptor families include human or mouse erythropoietin (EPO) receptor (Blood (1990) 76 (1), 31-35, Cell (1989) 57 (2), 277-285), human or mouse granulocyte colony-stimulating factor (G-CSF) receptor (Proc. Natl. Acad. Sci. USA. (1990) 87 (22), 8702-8706, mG-CSFR, Cell (1990) 61 (2), 341-350), human or mouse thrombopoietin (TPO) receptor (Proc Natl Acad Sci USA. (1992) 89 (12), 5640-5644, EMBO J. (1993) 12 (7), 2645-53), and human or mouse insulin receptor (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), 391-400), 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, human or mouse ciliary neurotrophic factor (CNTF) receptor, etc.

[0181] Cancer antigens are antigens that are expressed in association with the malignant transformation of cells and are also called tumor-specific antigens. Abnormal sugar chains that appear on cell surfaces or protein molecules when cells become cancerous are also cancer antigens and are also called cancer sugar chain antigens. Suitable examples of cancer antigens include GPC3 (Int J Cancer. (2003) 103 (4), 455-65), which belongs to the GPI-anchored receptor family and is expressed in several cancers including liver cancer, EpCAM (Proc Natl Acad Sci U S A. (1989) 86 (1), 27-31), CA19-9, CA15-3, and cereal SSEA-1 (SLX), among others.

[0182] 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.

[0183] 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.

[0184] Immunoglobulins include IgA, IgM, IgD, IgG, and IgE, and immune complexes contain at least one immunoglobulin component. Other antigens include 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, and activin RIB. ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, A RC, ART, Artemin, Anti-Id, ASPARTIC, Atrial Natriuretic Factor, av / b3 Integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD 8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD3 3 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, Cytokine-related antigen, DAN, DCC, DCR3, DC-SIGN, Complement-accelerating factor (Decay accelerating)factor), des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2 / E phB4, EPO, ERCC, E-selectin, ET-1, Factor IIa, Factor VII, Factor VIIIc, Factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalcohol In, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GD F-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut4, glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GRO, growth hormone-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high-molecular-weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFGPEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, Tegrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta 1, integrin alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta 1, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta 1, integrin beta 2, interferon gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1bp1, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES , MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Müllerian inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, NC adherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDK-1, P ECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PlGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSVFgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, thrombin, thymic Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B(OPG OCIF, TR1), TNFRSF12(TWEAK R FN14), TNFRSF13B(TACI), TNFRSF13C(BAFF R), TNFRSF14(HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16(NGFR p75NTR), TNFRSF17(BCMA), TNFRSF18(GITR AITR), TNFRSF19(TROY TAJ, TRADE), TNFRSF19L(RELT), TNFRSF1A(TNF RI CD120a, p55-60), TNFRSF1B(TNF RIICD120b, p75-80), TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1 R), TNFRSF5(CD40 p50), TNFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B(DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB CD137, ILA), TNFRSF21(DR6), TNFRSF22(DcTRAIL R2 TNFRH2), TNFRST23(DcTRAIL R1 TNFRH1), TNFRSF25(DR3) Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-α connectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand) CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand, Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand, CD70), TNFSF8 (CD30 ligand, CD153), TNFSF9 (4-1BB ligand, CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAILR, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expressed Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, virus Rux antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, Aβ, CD81 CD97, CD98, DDR1, DKK1, EREG, Hsp90, IL-17 / IL-17R, IL-20 / IL-20R, oxidized LDL, PCSK9, prekallikrein, RON, TMEM16F, SOD1, Chromogranin A, Chromogranin B, tau, VAP1, polymeric kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, factor B, factor D, factor H, properdin, sclerostin, fibrinogen, fibrin, prothrombin, thrombin, tissue factor, factor V, factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X, factor Xa,Examples include factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, syndecan-1, syndecan-2, syndecan-3, syndecan-4, LPA, S1P, and receptors for hormones and growth factors.

[0185] The amino acid sequence constituting the variable region can be modified by one or more amino acid residues as long as its antigen-binding activity is maintained. When modifying the amino acid sequence of the variable region, the site of modification and the number of amino acids to be modified are not particularly limited. For example, amino acids present in the 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, the binding activity is 50% or more, preferably 80% or more, and more preferably 100% or more compared to before modification. Furthermore, the amino acid modification may increase the binding activity, for example, by 2-fold, 5-fold, 10-fold, etc. compared to before modification. In the antibodies 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.

[0186] For example, modification of the N-terminus of a variable region from glutamine to pyroglutamic acid by pyroglutamylation is well known to those skilled in the art. Thus, when the N-terminus of the heavy chain of an antibody of the present invention is glutamine, it comprises a variable region in which the glutamine has been modified to pyroglutamic acid.

[0187] The variable regions of the antibodies of the present invention may have any sequence, including those of any origin, such as mouse, rat, rabbit, goat, or camel antibodies, as well as humanized antibodies derived from these non-human antibodies and human antibodies. A "humanized antibody," also known as a reshaped human antibody, is an antibody derived from a non-human mammal, such as a mouse, in which the complementarity-determining regions (CDRs) of the antibody are grafted onto the CDRs of a human antibody. Methods for identifying CDRs are known (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al., Nature (1989) 342:877). General genetic 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 to improve antigen binding, pharmacokinetics, stability, and antigenicity. The variable regions of the antibodies of the present invention may exhibit pH-dependence in antigen binding, thereby enabling repeated binding to the antigen (WO2009 / 125825).

[0188] The light chain constant region of an antibody may be either a κ chain or a λ chain type. Furthermore, in the present invention, the light chain constant region may be a light chain constant region that has been modified by amino acid substitution, deletion, addition, and / or insertion.

[0189] The heavy chain constant region of the antibody of the present invention can be, for example, the heavy chain constant region of a human IgG antibody, preferably the heavy chain constant region of a human IgG1 antibody or a human IgG4 antibody.

[0190] Furthermore, the Fc region variants of the present invention can be conjugated to other proteins, physiologically active peptides, etc. to form Fc fusion protein molecules. Here, a fusion protein refers to a chimeric polypeptide containing at least two different polypeptides that are not naturally linked in nature. Examples of other proteins and physiologically active peptides include, but are not limited to, receptors, adhesion molecules, ligands, and enzymes.

[0191] Preferred examples of the Fc fusion protein molecules of the present invention include proteins in which an Fc region 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 thereof 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, Examples include antibodies and Fc fusion proteins (WO2005 / 040229), Adnectin (WO2002 / 032925), etc. Furthermore, antibodies and Fc fusion protein molecules may be multispecific antibodies that bind to multiple types of target molecules or epitopes.

[0192] The antibodies of the present invention also include modified antibodies. Examples of modified antibodies include antibodies conjugated to various molecules such as polyethylene glycol (PEG) or cytotoxic substances. Such modified antibodies can be obtained by chemically modifying the antibodies of the present invention. Methods for modifying antibodies have already been established in this field.

[0193] Furthermore, the antibody of the present invention may be a bispecific antibody, which refers to an antibody having variable regions that recognize different epitopes within the same antibody molecule, and the epitopes may be present in different molecules or in the same molecule.

[0194] The polypeptides of the present invention can be produced by methods known to those skilled in the art. For example, antibodies can be produced by the following method, but the method is not limited thereto.

[0195] DNA encoding an antibody heavy chain in which one or more amino acid residues in the Fc region have been substituted with other amino acids of interest, and DNA encoding an antibody light chain are expressed. 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 native heavy chain and introducing appropriate substitutions so that codons encoding specific amino acids in the F region now encode other amino acids of interest.

[0196] Alternatively, 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 can be obtained by first designing 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 and then chemically synthesizing the DNA. The amino acid substitution site and type are not particularly limited. Furthermore, the substitution is not limited to substitution, and may be deletion, addition, insertion, or a combination thereof.

[0197] Furthermore, 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 as partial DNAs. Examples of combinations of partial DNAs include, but are not limited to, DNA encoding a variable region and DNA encoding a constant region, or DNA encoding a Fab region and DNA encoding an Fc region. DNA encoding a light chain can also be produced as partial DNAs in a similar manner.

[0198] Methods for expressing the above DNA include the following. For example, a heavy chain expression vector is constructed by incorporating DNA encoding a heavy chain variable region into an expression vector together with DNA encoding a heavy chain constant region. Similarly, a light chain expression vector is constructed by incorporating DNA encoding a light chain variable region into an expression vector together with DNA encoding a light chain constant region. These heavy and light chain genes can also be incorporated into a single vector.

[0199] When DNA encoding the antibody of interest is incorporated into an expression vector, it is incorporated into the expression vector so that expression is under the control of an expression control region, such as an enhancer or promoter. Next, host cells are transformed with this expression vector to express the antibody. In this case, an appropriate combination of host and expression vector can be used.

[0200] 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.

[0201] When vectors are used to produce the polypeptides of the present invention, expression vectors are 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 contain a promoter that enables efficient expression in E. coli, such as the lacZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427, incorporated herein by reference in its entirety), the araB promoter (Better et al., Science (1988) 240, 1041-1043, incorporated herein by reference in its entirety), or the T7 promoter. In addition to the above vectors, other such vectors include pGEX-5X-1 (Pharmacia), the "QIAexpress system" (QIAGEN), pEGFP, and pET (in this case, the host is preferably BL21, which expresses T7 RNA polymerase).

[0202] The vector may also contain a signal sequence for polypeptide secretion. When producing a polypeptide in the periplasm of E. coli, the signal sequence used may be the pelB signal sequence (Lei, SP et al., J. Bacteriol. (1987) 169, 4397, the entire contents of which are incorporated herein by reference). The vector can be introduced into host cells using, for example, the lipofectin method, the calcium phosphate method, or the DEAE-Dextran method.

[0203] In addition to E. coli expression vectors, examples of vectors for producing the polypeptides of the present invention include mammalian-derived expression vectors (e.g., pcDNA3 (Invitrogen), pEGF-BOS (Nucleic Acids. Res. 1990, 18(17), p5322, the entire contents of which are incorporated herein by reference), pEF, and pCDM8), insect cell-derived expression vectors (e.g., the "Bac-to-BAC baculovairus expression system" (GIBCO BRL), and pBacPAK8), plant-derived expression vectors (e.g., pMH1 and pMH2), animal virus-derived expression vectors (e.g., pHSV, pMV, and pAdexLcw), retrovirus-derived expression vectors (e.g., pZIPneo), yeast-derived expression vectors (e.g., the "Pichia Expression Kit" (Invitrogen), pNV11, and SP-Q01), and Bacillus subtilis-derived expression vectors (e.g., pPL608 and pKTH50).

[0204] For expression in animal cells such as CHO cells, COS cells, and NIH3T3 cells, it is essential to have a promoter necessary for intracellular expression, such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108, incorporated herein by reference in its entirety), the MMTV-LTR promoter, the EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322, incorporated herein by reference in its entirety), the CAG promoter (Gene. (1991) 108, 193, incorporated herein by reference in its entirety), or the CMV promoter. It is even more preferable if the vector contains a gene for selecting transformed cells (e.g., a drug resistance gene that can be distinguished by a drug (e.g., neomycin, G418, etc.)). Examples of vectors with such properties include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0205] Furthermore, to achieve stable gene expression and increase the intracellular copy number of a gene, one method involves introducing a vector (e.g., pCHOI) containing a complementary DHFR gene into CHO cells deficient in the nucleic acid synthesis pathway and amplifying the gene with methotrexate (MTX). Another method involves transforming COS cells carrying a gene expressing SV40 T antigen on their chromosomes with a vector (e.g., pcD) containing an SV40 replication origin. Replication origins derived from polyomavirus, adenovirus, bovine papillomavirus (BPV), etc. can also be used. Furthermore, to increase the gene copy number in a host cell system, the expression vector can contain a selection marker such as the aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, or dihydrofolate reductase (dhfr) gene.

[0206] Antibodies can be recovered, for example, by culturing the transformed cells and then isolating the antibodies from the transformed cells or from the culture medium. Antibody isolation and purification can be carried out 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.

[0207] The present invention also provides a method for promoting the elimination of an antigen from plasma by using an Fc region variant of the present invention and a polypeptide comprising an antigen-binding domain that has binding activity to a pathogenic antigen present in a soluble form in plasma and whose binding activity to the antigen changes depending on ion concentration conditions. As described in International Publication No. WO 2011 / 122011, a pH-dependent antigen-binding molecule that has been further modified to enhance FcRn binding under neutral conditions (pH 7.4) has been reported to have the effect of repeatedly binding to antigens and eliminating antigens from plasma, and therefore, it has been reported that antigens can be removed from plasma by administering a polypeptide having such an antigen-binding domain (International Publication No. WO 2011 / 122011). However, no method for accelerating antigen clearance has been reported to date other than by enhancing FcRn binding under neutral conditions.

[0208] In this example, it was confirmed that a polypeptide containing an antigen-binding domain whose antigen-binding activity changes depending on pH conditions accelerates antigen elimination from plasma through binding to FcγR more than the antigen alone, despite containing an Fc region derived from native IgG1 that does not have enhanced FcRn binding in the neutral pH range. Without being bound by any particular theory, the following mechanism is exemplified as why this occurs in clone 278 and the like.

[0209] In the case of antibodies with a single binding site for the antigen-binding domain (i.e., a homomonomer), such as sIL-6R, two molecules of antigen bind to one molecule of an antibody containing a bivalent antigen-binding domain, forming a complex with one molecule of the anti-sIL-6R antibody and two molecules of the antigen molecule containing two antigen-binding units. Therefore, as shown in Figure 9, such an antigen-antibody complex has only one Fc region (the Fc region of native IgG1). Because the complex binds to one molecule of FcγR or two molecules of FcRn via one Fc region, its affinity for these receptors is similar to that of a normal IgG antibody, and it is thought that intracellular uptake occurs mainly nonspecifically.

[0210] On the other hand, when an antigen is a dimer of a heterocomplex of heavy and light chains, such as human IgE, and there are two epitopes to which the antigen-binding domain binds, it is thought that it would be difficult for each of the bivalent antigen-binding domains contained in a single anti-IgE antibody molecule to bind to each of the two epitopes present in a single IgE molecule, due to the arrangement of the epitopes, etc. As a result, it is thought that an antigen-antibody complex (immune complex) containing at least four molecules (i.e., two molecules of IgE, which are antigen molecules, and two molecules of anti-IgE antibody, which are polypeptides containing the antigen-binding domain) is formed by binding to two antigen-binding units present in two IgE molecules that bind to the bivalent antigen-binding domain present in a single anti-IgE antibody molecule.

[0211] Therefore, when a polypeptide containing an antigen-binding domain, such as an antibody that binds to an antigen molecule containing two or more sites to which the antigen-binding domain can bind, forms a large immune complex of at least tetramer, the immune complex can bind strongly with avidity to FcγR, FcRn, complement receptors, etc. via at least two or more multivalent Fc regions. However, when an antigen molecule has only one site to which the antigen-binding domain can bind, the affinity of the immune complex between the polypeptide containing the antigen-binding domain and the antigen molecule for these receptors via the Fc region is insufficient compared to when the above immune complex is formed. As a result, the immune complex is taken up with high efficiency by cells expressing these receptors.

[0212] When an antigen molecule contains a site that binds to two or more antigen-binding domains, the polypeptide of the present invention may have an antigen-binding domain whose antigen binding activity changes depending on ion concentration conditions, such as pH-dependent binding. For example, if the polypeptide is an antibody, it will form an antigen-antibody complex (immune complex) consisting of at least four molecules (two antigen molecules and two antibody molecules) in plasma. When the immune complex is taken up into cells, the ion concentration conditions are different from those in plasma, causing the antigen to dissociate from the antibody in the endosome. Therefore, the formation of the immune complex is dissolved in the endosome of the cell into which the immune complex has been taken up. The dissociated antigen cannot bind to FcRn in the endosome and is therefore translocated to the lysosome and then degraded. Meanwhile, the antibody that has dissociated from the antigen is thought to be recycled back into plasma after binding to FcRn in the endosome. Similar recycling is possible under ion concentration conditions other than the pH conditions used in this example. Reference Examples 3 to 6 demonstrate that the elimination of antigens from plasma can be accelerated by using an antigen-binding domain whose antigen-binding activity changes depending on calcium concentration conditions, instead of pH-dependent conditions.

[0213] Therefore, the elimination of an antigen can be accelerated by having a complex formed between an antigen and a polypeptide having an antigen-binding domain for that antigen, and the immune complex has two or more multivalent Fc regions that are directed against FcγR, FcRn, complement receptors, etc.

[0214] In addition, the studies in Reference Examples 7 to 9 show that, among FcγRs, FcγRIIB, an inhibitory FcγR, makes the greatest contribution to FcγR-mediated antigen elimination. In other words, even if FcγR binding is reduced, the antibody's ability to eliminate antigens via FcγR can be maintained as long as FcγRIIB binding can be maintained.

[0215] To date, several antibody drugs have been reported to have side effects resulting from the interaction between IgG and FcγR. For example, it is known that the frequency of thromboembolism increases in patients receiving bevacizumab, an antibody against VEGF (J. Natl. Cancer Inst. (2007) 99 (16), 1232-1239). Furthermore, thromboembolism was also observed in clinical development trials of an antibody against CD40 ligand, leading to the discontinuation of the clinical trial (Arthritis. Rheum. (2003) 48 (3), 719-727). Platelets express the activating Fcγ receptor FcγRIIa, not the inhibitory Fcγ receptor FcγRIIb (J. Exp. Med. (2006) 203 (9), 2157-2164). However, subsequent studies using animal models have suggested that all of the administered antibodies induce platelet aggregation via binding to FcγRIIa on platelets, resulting in thrombus formation (J. Thromb. Haemost. (2009) 7 (1), 171-181, J. Immunol. (2010) 185 (3), 1577-1583). It has been reported that in patients with systemic lupus erythematosus, an autoimmune disease, platelets are activated by an FcγRIIa-dependent mechanism, and that platelet activation correlates with the severity of the disease (Sci. Transl. Med. (2010) 2 (47), 47-63).

[0216] Previous studies using animal models have also shown that immune complexes of antibodies and multivalent antigens induce anaphylaxis via activating FcγR (Bruhns P., Blood. (2012) 119(24):5640-9.).

[0217] In addition, it has been reported that the uptake of immune complexes between polyvalent antigens and antibodies via activated FcγR increases the production of antibody titers against those antigens (Scand J Immunol. (2006)64(3):177-84.; J Immunol. (1999) 163:618-22.). This suggests that antibody drugs that recognize polyvalent antigens may be more likely to produce antibodies against the antibody drug itself. If antibodies against the antibody drug are produced, their blood dynamics may deteriorate, potentially reducing their effectiveness.

[0218] In this way, antibodies bind to multivalent antigens to form immune complexes, and these immune complexes may interact with activating FcγRs to induce various side effects, reducing the value of antibodies as pharmaceuticals. Multivalent antigens (multimeric antigens) include GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), TNF, TNF-alpha, TNF-alphabeta, TNF-beta2, TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), and TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand, AITR ligand, TL6), TNFSF1A (TNF-α connectin, DIF, TNFSF2), TNFSF1B (TNF-β LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand Examples include CD137 ligand), VEGF, IgE, IgA, IgG, IgM, RANKL, TGF-alpha, TGF-beta, TGF-beta pan specific, or IL-8.

[0219] One possible way to solve these problems is to attenuate FcγR binding, but if binding to all FcγRs is reduced, it is unlikely that the antibody will be able to accelerate FcγR-mediated antigen elimination.

[0220] As mentioned above, among the FcγRs, FcγRIIB plays a major role in the FcγR-mediated antigen removal by antibodies, and side effects resulting from interaction with FcγR are caused by interaction with activating FcγR. Therefore, by selectively attenuating binding to other activating FcγRs while maintaining binding to FcγRIIB, it is possible to produce excellent antibodies that have reduced side effects resulting from activating FcγRs without losing their antigen removal ability.

[0221] Therefore, by using Fc region variants of the present invention and polypeptides comprising an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions, it is possible to obtain an excellent promoting effect on the elimination from plasma of pathogenic antigens that are present in soluble form in plasma.

[0222] Furthermore, by using the polypeptides of the present invention, similar effects can be obtained even when the antigen to which the polypeptide binds is an antigen (monomeric antigen) that has only one site to which the antigen-binding domain can bind. An example of such a method is the promotion of elimination of monomeric antigens from plasma using a cocktail of polypeptides having antigen-binding domains.

[0223] As described above, when the antigen is a multimeric antigen (for example, but not limited to, immunoglobulins such as IgA and IgE, or TNF superfamily members such as TNF or CD154), a large immune complex containing two or more antigen-binding molecules and two or more antigen-binding units may be formed. On the other hand, even when the antigen is a monomeric antigen, a mixture of polypeptides containing two or more appropriate antigen-binding domains that each bind to a different epitope present in the monomeric antigen and whose binding to the epitope changes depending on ion concentration conditions (such as pH or Ca) is also considered to be capable of forming a large immune complex containing a polypeptide containing two or more antigen-binding domains and binding sites (monomeric antigens) for the two or more antigen-binding domains. Herein, a mixture of polypeptides containing two or more appropriate antigen-binding domains that each bind to a different epitope present in the monomeric antigen and whose binding to the epitope changes depending on ion concentration conditions (such as pH or Ca) is referred to as an antigen-binding molecule cocktail. Among these polypeptides containing an antigen-binding domain, at least one polypeptide (contained in an antigen-binding domain) that forms an immune complex may be an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions. Another example is a method for promoting the elimination of monomeric antigens from plasma using polypeptides containing multispecific or multiparatopic antigen-binding domains.

[0224] Furthermore, even when the antigen is a monomeric antigen, antigen-binding molecules comprising antigen-binding domains in which each antigen-binding domain contained in a polypeptide containing an antigen-binding domain has the characteristic of binding to a different epitope present in the monomeric antigen, and the epitope binding of each antigen-binding domain changes depending on ion concentration conditions (such as pH or Ca), are also considered to be capable of forming large immune complexes comprising polypeptides comprising two or more antigen-binding domains and two or more antigen-binding units (monomeric antigens). Non-limiting examples of such polypeptides include multispecific antibodies or multiparatopic antibodies comprising appropriate variable regions that bind to different epitopes present in the monomeric antigen. As a non-limiting embodiment of such multispecific or multiparatopic antibodies, antibodies whose variable regions exhibit pH- or Ca-dependent binding (bispecific or biparatopic antibodies comprising a right-arm variable region recognizing epitope A and a left-arm variable region recognizing epitope B as shown in Figure 19) are also believed to be capable of forming large immune complexes comprising two or more antibodies and two or more antigen-binding units (monomeric antigens).

[0225] By screening for combinations of antigen-binding domains for different epitopes of a monomeric antigen, where the binding activity for each epitope changes depending on ion concentration conditions and the antigen-binding domains are capable of binding to the above-described receptors with avidity, it is possible to obtain antigen-binding molecules that further accelerate the elimination of monomeric antigens from plasma. The ion concentration conditions that change the binding activity for each epitope of a multispecific or multiparatopic antigen-binding domain may be the same or different. For example, an antigen-binding molecule comprising a bispecific or biparatopic antigen-binding domain in which the epitope-binding activity of one of the bispecific or biparatopic antigen-binding domains changes depending on pH conditions or metal ion concentrations such as Ca ion concentration is exemplified as a non-limiting embodiment of the antigen-binding molecule of the present invention. Furthermore, for example, antigen-binding molecules comprising bispecific or biparatopic antigen-binding domains in which the epitope-binding activity of one antigen-binding domain changes depending on pH conditions and the epitope-binding activity of the other antigen-binding domain changes depending on metal ion concentrations such as Ca ion concentrations are exemplified as non-limiting embodiments of the antigen-binding molecules of the present invention. Furthermore, antigen-binding molecules comprising bispecific or biparatopic antigen-binding domains in which the epitope-binding activity of one antigen-binding domain changes depending on pH conditions and the epitope-binding activity of the other antigen-binding domain also changes depending on pH conditions are exemplified as non-limiting embodiments of the antigen-binding molecules of the present invention. Furthermore, a non-limiting embodiment of the antigen-binding molecules of the present invention also includes polypeptide antigen-binding molecules comprising bispecific or biparatopic antigen-binding domains, in which the epitope-binding activity of one antigen-binding domain changes depending on metal ion concentrations such as Ca ion concentrations, and the epitope-binding activity of the other antigen-binding domain changes depending on metal ion concentrations such as Ca ion concentrations.

[0226] As for the polypeptides of the present invention comprising multispecific antigen-binding domains or polypeptide molecules comprising multiple paratopic antigen-binding domains, polypeptides comprising at least two antigen-binding domains, in which at least one antigen-binding domain binds to a first epitope in an antigen molecule and at least one other antigen-binding domain binds to a second epitope in the antigen molecule, are referred to as multispecific antigen-binding molecules from the perspective of their reaction specificity. When a single antigen-binding molecule binds to two different epitopes via two different antigen-binding domains contained in the single antigen-binding molecule, the antigen-binding molecule is referred to as a bispecific antigen-binding molecule. Furthermore, when a single antigen-binding molecule binds to three different epitopes via three different antigen-binding domains contained in the single antigen-binding molecule, the antigen-binding molecule is referred to as a trispecific antigen-binding molecule.

[0227] The paratope in the antigen-binding domain that binds to a first epitope in an antigen molecule and the paratope in the antigen-binding domain that binds to a second epitope structurally different from the first epitope are structurally different from each other. Therefore, an antigen-binding molecule that contains at least two antigen-binding domains, in which at least one antigen-binding domain binds to a first epitope in an antigen molecule and at least one other antigen-binding domain binds to a second epitope in the antigen molecule, is called a multiparatopic antigen-binding molecule from the perspective of its structural specificity. When an antigen-binding molecule binds to two different epitopes via two types of antigen-binding domains contained in a single antigen-binding molecule, the antigen-binding molecule is called a biparatopic antigen-binding molecule. Furthermore, when an antigen-binding molecule binds to three different epitopes via three types of antigen-binding domains contained in a single antigen-binding molecule, the antigen-binding molecule is called a tripleparatopic antigen-binding molecule.

[0228] Multivalent, multispecific or multiparatopic antigen-binding molecules containing one or more antigen-binding domains and methods for their preparation are also described in non-patent literature such as Conrath et al. (J. Biol. Chem. (2001) 276 (10) 7346-7350), Muyldermans (Rev. Mol. Biotech. (2001) 74, 277-302) and Kontermann RE (2011) Bispecific Antibodies (Springer-Verlag), as well as in patent literature such as International Publication No. WO1996 / 034103 or WO1999 / 023221. The antigen-binding molecules of the present invention can be produced using the multispecific or multiparatopic antigen-binding molecules and methods for their preparation described therein.

[0229] As one embodiment of the above-mentioned multispecific or multiparatopic antigen-binding molecules and methods for preparing them, bispecific antibodies and methods for producing them are exemplified below. Bispecific antibodies are antibodies that contain two types of variable regions that specifically bind to different epitopes. IgG-type bispecific antibodies can be secreted by hybrid hybridomas (quadromas) generated by fusing two types of IgG antibody-producing hybridomas (Milstein et al., Nature (1983) 305, 537-540).

[0230] When producing bispecific antibodies using recombinant techniques such as those described above in the antibody section, a method can be used in which genes encoding heavy chains containing two desired variable regions are introduced into cells and co-expressed. However, even considering the heavy chain combinations in such co-expression methods, the resulting mixture contains (i) a heavy chain combination in which one heavy chain containing a variable region that binds to a first epitope is paired with another heavy chain containing a variable region that binds to a second epitope, (ii) a heavy chain combination in which only heavy chains containing a variable region that binds to the first epitope are paired, and (iii) a heavy chain combination in which only heavy chains containing a variable region that binds to the second epitope are paired, in a molecular ratio of 2:1:1. It is difficult to purify antigen-binding molecules containing the desired heavy chain combination from a mixture of these three types of heavy chain combinations.

[0231] When producing bispecific antibodies using such recombinant techniques, bispecific antibodies containing heterogeneous heavy chain combinations can be preferentially secreted by modifying the CH3 domains of the heavy chains with appropriate amino acid substitutions. Specifically, the amino acid side chains in the CH3 domain of one heavy chain are replaced with larger side chains (knobs) and the amino acid side chains in the CH3 domain of the other heavy chain are replaced with smaller side chains (holes) so that the knobs can be positioned in the holes, promoting heterologous heavy chain formation and inhibiting homologous heavy chain formation (International Publication WO 1996 / 027011; Ridgway et al., Protein Engineering (1996) 9, 617-621; Merchant et al., Nat. Biotech. (1998) 16, 677-681).

[0232] Furthermore, a technique for producing bispecific antibodies is also known in which a method for controlling the association of polypeptides or heteromultimers composed of polypeptides is applied to the association of heavy chains. That is, a method for controlling the association of heavy chains having the same sequence by modifying amino acid residues that form an interface within the heavy chains, thereby inhibiting the association of heavy chains having the same sequence and forming two heavy chains with different sequences, can be employed for producing bispecific antibodies (International Publication No. WO2006 / 106905). Furthermore, a technique for obtaining bispecific antibodies by obtaining two types of monoclonal antibodies and mixing them in vitro in the presence of a reducing agent has also been reported (International Publication No. WO 2008 / 119353). In this method, two types of monoclonal antibodies are cleaved into half molecules by a reducing agent, and these half molecules are reassembled to obtain bispecific antibodies at a certain rate. A method for more efficiently obtaining bispecific antibodies by controlling the reassembly of the half molecules through the substitution of amino acids in the CH3 domain has also been reported (International Publication No. WO 2011 / 131746). Such methods can also be used to produce bispecific antibodies.

[0233] In the present invention, "promoting antigen elimination from plasma" refers to an improved ability to eliminate antigens present in plasma when a polypeptide containing an antigen-binding domain (hereinafter also referred to as an antigen-binding molecule) is administered to a living body or when the antigen-binding molecule is secreted into the body. Therefore, it is sufficient that the rate of antigen elimination from plasma is faster when an antigen-binding molecule is administered compared to when an antigen-binding molecule containing an antigen-binding domain whose antigen-binding activity does not change with ion concentration, an antigen-binding molecule containing an FcRn-binding domain that does not have FcRn-binding activity under acidic pH conditions, or an antigen-binding molecule containing an Fcγ receptor-binding domain that does not have selective binding activity to Fcγ receptors is administered. Whether an antigen-binding molecule has an increased ability to eliminate antigens from plasma can be determined, for example, by administering a soluble antigen and the antigen-binding molecule to a living body and measuring the plasma concentration of the soluble antigen after administration. If the plasma soluble antigen concentration is reduced after administration of a soluble antigen and an antigen-binding molecule containing an antigen-binding domain whose antigen-binding activity changes depending on ion concentration, an FcRn-binding domain that has FcRn-binding activity under acidic pH conditions, or an Fcγ receptor-binding domain that has selective binding activity to Fcγ receptors (selective FcγR-binding domain), the antigen elimination ability of the antigen-binding molecule in plasma can be determined to be increased. Here, a selective FcγR-binding domain refers to a domain that maintains binding to FcγRIIb but has reduced binding to activating FcγR. Soluble antigens may be antigens that bind to antigen-binding molecules in plasma or antigens that do not bind to antigen-binding molecules, and their concentrations can be determined as "plasma antigen-binding molecule-bound antigen concentration" and "plasma antigen-unbound antigen concentration," respectively (the latter is synonymous with "plasma free antigen concentration"). The "total antigen concentration in plasma" refers to the combined concentration of antigen-binding molecule-bound antigen and antigen-unbound antigen, or the "free antigen concentration in plasma," which is the concentration of antigen-unbound antigen. Therefore, the soluble antigen concentration can be determined as the "total antigen concentration in plasma."Various methods for measuring "total plasma antigen concentration" or "free plasma antigen concentration" are well known in the art, as described herein below.

[0234] In the present invention, "improved pharmacokinetics," "improved pharmacokinetics," and "excellent pharmacokinetics" can be rephrased as "improved plasma (blood) retention," "improved plasma (blood) retention," "excellent plasma (blood) retention," and "prolonged plasma (blood) retention," and these terms are used interchangeably.

[0235] In the present invention, "improved pharmacokinetics" refers not only to an increase in the time from administration of an antigen-binding molecule to humans or non-human animals such as mice, rats, monkeys, rabbits, and dogs until the antigen-binding molecule disappears from plasma (e.g., until the antigen-binding molecule becomes unable to return to plasma due to intracellular degradation, etc.), but also to an increase in the time the antigen-binding molecule remains in plasma in an antigen-binding state (e.g., in a state where the antigen-binding molecule is not bound to an antigen) between administration and degradation until disappearance. Human IgG having a native Fc region can bind to FcRn derived from non-human animals. For example, human IgG having a native Fc region can bind more strongly to mouse FcRn than to human FcRn (Int. Immunol. (2001) 13(12), 1551-1559). Therefore, for the purpose of confirming the properties of the antigen-binding molecules of the present invention, they can be preferably administered using mice. As another example, mice in which the native FcRn gene has been disrupted and which have and express a transgene related to the human FcRn gene (Methods Mol. Biol. (2010) 602, 93-104) can also be used for administration to confirm the properties of the antigen-binding molecules of the present invention described below. Specifically, "improved pharmacokinetics" also includes extending the time until an antigen-binding molecule that is not bound to an antigen (antigen-unbound antigen-binding molecule) is degraded and eliminated. Even if an antigen-binding molecule is present in plasma, if an antigen is already bound to that antigen-binding molecule, that antigen-binding molecule cannot bind to a new antigen. Therefore, if the time that an antigen-binding molecule is not bound to an antigen is extended, the time that it can bind to a new antigen is extended (increasing the opportunity to bind to a new antigen), thereby reducing the time that an antigen is not bound to an antigen-binding molecule in vivo and enabling the time that an antigen is bound to an antigen-binding molecule to be extended. If the elimination of an antigen from plasma can be accelerated by administration of an antigen-binding molecule, the plasma concentration of the antigen-unbound antigen-binding molecule will increase, and the time that the antigen remains bound to the antigen-binding molecule will be extended.In other words, "improved pharmacokinetics of an antigen-binding molecule" in the present invention includes an improvement in any pharmacokinetic parameter of the antigen-free antigen-binding molecule (an increase in plasma half-life, an increase in mean plasma residence time, or a decrease in plasma clearance), an extension of the time that the antigen remains bound to the antigen-binding molecule after administration of the antigen-binding molecule, or an acceleration of antigen elimination from plasma by the antigen-binding molecule. This can be determined by measuring any parameter, such as the plasma half-life, mean plasma residence time, or plasma clearance, of the antigen-binding molecule or the antigen-free antigen-binding molecule (Understanding through Pharmacokinetics Exercises (Nanzando)). For example, when an antigen-binding molecule is administered to mice, rats, monkeys, rabbits, dogs, humans, etc., the plasma concentration of the antigen-binding molecule or the antigen-free antigen-binding molecule is measured, and each parameter is calculated. If the plasma half-life or mean plasma residence time is prolonged, the pharmacokinetics of the antigen-binding molecule can be said to be improved. These parameters can be measured by methods known to those skilled in the art, and can be appropriately evaluated, for example, by noncompartmental analysis using the pharmacokinetic analysis software WinNonlin (Pharsight) in accordance with the accompanying instructions. The plasma concentration of antigen-binding molecules that are not bound to antigen can be measured by methods known to those skilled in the art, and can be, for example, the method described in Clin. Pharmacol. (2008) 48(4), 406-417.

[0236] In the present invention, "improved pharmacokinetics" also includes an extension of the time during which an antigen remains bound to an antigen-binding molecule after administration of the antigen-binding molecule. Whether or not the time during which an antigen remains bound to an antigen-binding molecule after administration of the antigen-binding molecule has been extended can be determined by measuring the plasma concentration of free antigen and observing the time until the plasma concentration of free antigen or the ratio of the free antigen concentration to the total antigen concentration increases.

[0237] The plasma concentration of free antigen not bound to antigen-binding molecules, or the ratio of free antigen concentration to total antigen concentration, can be determined by methods known to those skilled in the art. For example, it can be determined using the method described in Pharm. Res. (2006) 23 (1), 95-103. Furthermore, when an antigen exhibits some function in vivo, whether the antigen binds to an antigen-binding molecule that neutralizes the antigen function (antagonist molecule) can also be evaluated by determining whether the antigen function is neutralized. Whether the antigen function is neutralized can be evaluated by measuring some in vivo marker that reflects the antigen function. Whether the antigen binds to an antigen-binding molecule that activates the antigen function (agonist molecule) can be evaluated by measuring some in vivo marker that reflects the antigen function.

[0238] Measurements such as measurement of plasma free antigen concentration, measurement of the ratio of the amount of free antigen in plasma to the total amount of antigen in plasma, and measurement of in vivo markers are not particularly limited, but are preferably performed after a certain time has passed since administration of the antigen-binding molecule. In the present invention, the period after a certain time has passed since administration of the antigen-binding molecule is not particularly limited and can be determined appropriately by a person skilled in the art depending on the properties of the administered antigen-binding molecule, etc. Examples include one day after administration of the antigen-binding molecule, three days after administration of the antigen-binding molecule, seven days after administration of the antigen-binding molecule, 14 days after administration of the antigen-binding molecule, and 28 days after administration of the antigen-binding molecule. In the present invention, the term "plasma antigen concentration" encompasses both the "total plasma antigen concentration," which is the combined concentration of antigen bound to the antigen-binding molecule and antigen unbound to the antigen-binding molecule, and the "free plasma antigen concentration," which is the concentration of antigen unbound to the antigen-binding molecule.

[0239] The total plasma antigen concentration can be reduced by 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold or more by administration of an antigen-binding molecule of the present invention, compared to administration of an antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity is independent of ion concentration, or an antigen-binding molecule comprising an Fc region with impaired FcγR-binding activity, or compared to administration of no antigen-binding molecule of the present invention.

[0240] The antigen / antigen-binding molecule molar ratio can be calculated as follows: A value = molar concentration of antigen at each time point B value = molar concentration of antigen-binding molecules at each time point C value = molar concentration of antigen per molar concentration of antigen-binding molecules at each time point (antigen / antigen-binding molecule molar ratio) C=A / B.

[0241] A smaller C value indicates a higher antigen elimination efficiency per antigen-binding molecule, whereas a larger C value indicates a lower antigen elimination efficiency per antigen-binding molecule.

[0242] The antigen / antigen-binding molecule molar ratio can be calculated as described above.

[0243] The antigen / antigen-binding molecule molar ratio can be reduced by 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold or more by administration of the antigen-binding molecule of the present invention compared to administration of an antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity does not change depending on ion concentration, an antigen-binding molecule comprising an FcRn-binding domain that does not have FcRn-binding activity in an acidic pH range, or an antigen-binding molecule comprising an Fcγ receptor-binding domain that does not have selective binding activity to Fcγ receptor.

[0244] In the present invention, reference antigen-binding molecules used for comparison with antigen-binding molecules of the present invention include antigen-binding molecules comprising an antigen-binding domain whose antigen-binding activity does not change depending on ion concentration, antigen-binding molecules comprising an FcRn-binding domain that does not have FcRn-binding activity under acidic pH conditions, and antigen-binding molecules comprising an Fcγ receptor-binding domain that does not have selective binding activity to Fcγ receptors.

[0245] When evaluating the effect of an FcRn-binding domain with FcRn-binding activity under acidic pH conditions, the reduction in plasma total antigen concentration or antigen / antibody molar ratio can be evaluated using either an antigen-antibody co-injection model or a steady-state antigen infusion model using human FcRn transgenic mouse strain 32 or strain 276 (Jackson Laboratories, Methods Mol. Biol. (2010) 602, 93-104) if the antigen-binding molecule does not cross-react with the mouse counterpart antigen. If the antigen-binding molecule cross-reacts with the mouse counterpart, it can also be evaluated by simply injecting the antigen-binding molecule into human FcRn transgenic mouse strain 32 or strain 276 (Jackson Laboratories). In the co-injection model, a mixture of the antigen-binding molecule and antigen is administered to mice. In the steady-state antigen infusion model, an infusion pump filled with antigen solution is implanted in mice to achieve a constant plasma antigen concentration, and then the antigen-binding molecule is injected into the mice. The test antigen-binding molecule is administered at the same dose. Plasma total antigen concentration, plasma free antigen concentration, and plasma antigen-binding molecule concentration are measured at appropriate time points using methods known to those skilled in the art.

[0246] When evaluating the effect of an Fcγ receptor-binding domain with selective binding activity to an Fcγ receptor, the reduction in plasma total antigen concentration or antigen / antibody molar ratio can be evaluated using either an antigen-antibody simultaneous injection model or a steady-state antigen infusion model using commonly used C57BL / 6J mice (Charles River Japan) if the antigen-binding molecule does not cross-react with the mouse counterpart antigen. If the antigen-binding molecule cross-reacts with the mouse counterpart, it can also be evaluated by simply injecting the antigen-binding molecule into commonly used C57BL / 6J mice (Charles River Japan).

[0247] In the simultaneous injection model, a mixture of antigen-binding molecules and antigens is administered to mice.In the steady-state antigen infusion model, mice are implanted with an infusion pump filled with antigen solution to achieve a constant plasma antigen concentration, and then the antigen-binding molecules are injected into the mice.The test antigen-binding molecules are administered at the same dose.The total plasma antigen concentration, the free plasma antigen concentration, and the plasma antigen-binding molecule concentration are measured at appropriate time points using methods known to those skilled in the art.

[0248] The long-term effects of the present invention can be evaluated by measuring the total or free antigen concentration in plasma and the antigen / antigen-binding molecule molar ratio 2, 4, 7, 14, 28, 56, or 84 days after administration. In other words, for the purpose of evaluating the properties of the antigen-binding molecules of the present invention, the long-term plasma antigen concentration is determined by measuring the total or free antigen concentration in plasma and the antigen / antigen-binding molecule molar ratio 2, 4, 7, 14, 28, 56, or 84 days after administration of the antigen-binding molecule. Whether or not a reduction in plasma antigen concentration or antigen / antigen-binding molecule molar ratio is achieved by the antigen-binding molecules described in the present invention can be determined by evaluating the reduction at any one or more of the time points described above.

[0249] The short-term effects of the present invention can be evaluated by measuring the total or free antigen concentration in plasma and the antigen / antigen-binding molecule molar ratio 15 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, or 24 hours after administration. In other words, for the purpose of evaluating the properties of the antigen-binding molecules of the present invention, the short-term plasma antigen concentration is determined by measuring the total or free antigen concentration in plasma and the antigen / antigen-binding molecule molar ratio 15 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, or 24 hours after administration of the antigen-binding molecule.

[0250] The route of administration of the antigen-binding molecules of the present invention can be selected from intradermal injection, intravenous injection, intravitreal injection, subcutaneous injection, intraperitoneal injection, parenteral injection, and intramuscular injection.

[0251] In the present invention, it is preferable that the pharmacokinetics of an antigen-binding molecule in humans is improved. When it is difficult to measure plasma retention in humans, plasma retention in humans can be predicted based on plasma retention in mice (e.g., normal mice, human antigen-expressing transgenic mice, human FcRn-expressing transgenic mice, etc.) or monkeys (e.g., cynomolgus monkeys, etc.).

[0252] In the present invention, "improved pharmacokinetics and plasma retention of an antigen-binding molecule" means that any pharmacokinetic parameter upon administration of an antigen-binding molecule to a living body is improved (any of an increased plasma half-life, an increased mean plasma retention time, a decreased plasma clearance, or bioavailability), or that the plasma concentration of the antigen-binding molecule at an appropriate time after administration is improved. This can be determined by measuring any parameter of the antigen-binding molecule, such as the plasma half-life, mean plasma retention time, plasma clearance, or bioavailability (Understanding Pharmacokinetics Through Exercises (Nanzando)). For example, when an antigen-binding molecule is administered to mice (normal mice and human FcRn transgenic mice), rats, monkeys, rabbits, dogs, or humans, the plasma concentration of the antigen-binding molecule is measured, and each parameter is calculated. A prolonged plasma half-life or a prolonged mean plasma retention time can be considered to have improved pharmacokinetics of the antigen-binding molecule. These parameters can be measured by methods known to those skilled in the art, and can be appropriately evaluated, for example, by noncompartmental analysis using the pharmacokinetic analysis software WinNonlin (Pharsight) in accordance with the accompanying instructions.

[0253] Four types of FcγR have been identified in mice: FcγRI, FcγRIIb, FcγRIII, and FcγRIV. Human counterparts, FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIa, and FcγRIIIb, have also been identified. Among these FcγRs, only FcγRIIb, which is considered to be inhibitory, is conserved in both humans and mice. While the other FcγRs, except for FcγRIIIb, transduce activating signals via an immunoreceptor tyrosine-based activating motif (ITAM), FcγRIIb transduces inhibitory signals via an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM) (Nat. Rev. Immunol. (2008) 8, 34-47).

[0254] FcγRIIb1 and FcγRIIb2 have been reported as splicing variants of FcγRIIb. In both humans and mice, FcγRIIb1 has a longer intracellular domain than FcγRIIb2, and it has been confirmed that FcγRIIb1 is expressed in B cells, while FcγRIIb2 is expressed in macrophages, mast cells, dendritic cells, basophils, neutrophils, and eosinophils (J. Clin. Immunol. (2005) 25 (1), 1-18).

[0255] It has been reported that dysfunction and decreased expression of FcγRIIb are correlated with the onset of autoimmune diseases in humans. For example, some SLE patients have been reported to have reduced FcγRIIb expression due to a genetic polymorphism in the FcγRIIb promoter region, which weakens transcriptional activator binding (Hum. Genet. (2005) 117, 220-227; J. Immunol. (2004) 172, 7192-7199; J. Immunol. (2004) 172, 7186-7191). Furthermore, two genetic polymorphisms have been reported in SLE patients, where the 233rd amino acid of FcγRIIb is either Ile or Thr. This site is located in the transmembrane domain of FcγRIIb, and it has been reported that when the 233rd amino acid is Thr, FcγRIIb is less likely to reside in lipid rafts than when it is Ile, resulting in a decrease in the signaling function of FcγRIIb (Nat. Med. (2005) 11, 1056-1058, Hum. Mol. Genet., (2005) 14, 2881-2892). It has also been reported that knockout mice in which the FcγRIIb gene of C57BL / 6 mice was disrupted exhibit SLE-like symptoms, such as autoantibody production and glomerulonephritis (Immunity 13 (2000) 277-285, J. Exp. Med. (2002) 195, 1167-1174). Furthermore, decreased expression of FcγRIIb has been reported in mice, which have been considered a model of spontaneous SLE (Immunogenetics (2000) 51, 429-435, Int. Immunol. (1999) 11, 1685-1691, Curr. Biol. (2000) 10, 227-230, J. Immunol. (2002) 169, 4340-4346). These findings suggest that FcγRIIb regulates humoral immunity in mice, as in humans.

[0256] When antibodies containing the Fc of the present invention eliminate antigens via FcγRIIb, the endocytic function of FcγRIIb is thought to be the most important factor. As mentioned above, there are two splicing variants of FcγRIIb, FcγRIIb1 and FcγRIIb2, and it has been reported that the latter is primarily involved in the endocytosis of antibody-antigen immune complexes (J. Immunol. (1994), 152, 574-585; Science (1992) 256, 1808-1812; Cell (1989) 58, 317-327). It has previously been reported that mouse FcγRIIb2 is incorporated into clathrin-coated pits and undergoes endocytosis (Cell (1989) 58, 317-327). Furthermore, it has been reported that the dileucine motif is required for FcγRIIb2-mediated endocytosis, and this motif is conserved in both humans and mice (EMBO J. (1994) 13 (13), 2963-2969). This suggests that FcγRIIb2 has endocytic activity in humans as well as in mice.

[0257] On the other hand, it has been reported that FcγRIIb1, unlike FcγRIIb2, does not undergo endocytosis. FcγRIIb1 contains an insertion sequence in the intracellular domain that is not found in FcγRIIb2. This sequence is thought to inhibit the incorporation of FcγRIIb1 into clathrin-coated pits, thereby inhibiting endocytosis (J. Cell. Biol. (1992) 116, 875-888, J. Cell. Biol. (1989) 109, 3291-3302). In humans, as in mice, FcγRIIb1 contains an insertion sequence in the same region as FcγRIIb2, and therefore, it is predicted that a similar mechanism is responsible for the difference in endocytic ability between FcγRIIb1 and FcγRIIb2. It has also been reported that approximately 40% of immune complexes on the cell surface are internalized within 20 minutes in both humans and mice (Mol. Immunol. (2011) 49, 329-337, Science (1992) 256, 1808-1812). Based on this, it is predicted that FcγRIIb2 also internalizes immune complexes into cells in humans at a rate similar to that observed in mice.

[0258] Among the FcγR family, FcγRIIb is the only one that has intracellular ITIM in both humans and mice, and the distribution of expressing cells is the same, suggesting that their functions in immune regulation are similar. Furthermore, considering the fact that immune complexes are internalized into cells at similar rates in both humans and mice, it is believed that the effect of antibody-mediated antigen elimination via FcγRIIb in humans can be predicted using mice. Indeed, in Reference Example 7, it was shown that, compared with mIgG1, an antigen-binding molecule that binds to soluble antigens in a pH-dependent manner, mF44 and mF46, which are antigen-binding molecules that bind to soluble antigens in a pH-dependent manner and have enhanced affinity for mouse FcγRIIb and FcγRIII, increased antigen clearance when administered to normal mice compared to mIgG1.

[0259] Furthermore, in Reference Example 8 described below, a similar experiment was performed using Fc receptor γ chain-deficient mice. It has been reported that in mice, FcγRs other than FcγRIIb are expressed only in the presence of gamma chain; therefore, only FcγRIIb is expressed in Fc receptor γ chain-deficient mice. By administering mF44 and mF46, antigen-binding molecules that bind to soluble antigens in a pH-dependent manner, to Fc receptor γ chain-deficient mice, it was possible to examine the effect of accelerating antigen clearance when selectively enhancing FcγRIIb binding. The results of Reference Example 8 showed that mF44 and mF46, antigen-binding molecules that bind to soluble antigens in a pH-dependent manner administered to Fc receptor γ chain-deficient mice, increased antigen clearance compared to mIgG1, an antigen-binding molecule that binds to soluble antigens in a pH-dependent manner administered to the same mice. Furthermore, the results of Reference Example 8 revealed that mF44 and mF46 eliminated antigens to approximately the same extent when administered to Fc receptor γ chain-deficient mice as when administered to normal mice.

[0260] In Reference Example 8, a similar experiment was performed using FcγRIII-deficient mice. Since mIgG1, mF44, and mF46 bind only to FcγRIIb and FcγRIII among the mFcγRs, it was possible to examine the effect of accelerating antigen clearance when these antibodies were administered to FcγRIII-deficient mice, selectively enhancing FcγRIIb binding. The results of Reference Example 8 showed that mF44 and mF46 administered to FcγRIII-deficient mice increased antigen clearance compared to mIgG1 administered to the same mice. Furthermore, the results of Reference Example 8 revealed that mF44 and mF46, when administered to FcγRIII-deficient mice, eliminated antigens to approximately the same extent as when administered to normal mice and when administered to Fc receptor γ-chain-deficient mice.

[0261] These results demonstrate that selectively enhancing FcγRIIb binding, without enhancing binding to activating FcγR, can accelerate antigen elimination. This suggests that FcγRIIb is primarily involved in FcγR-mediated immune complex clearance, and suggests that maintaining FcγR-mediated immune complex clearance efficiency of an antibody can be maintained as long as the antibody's FcγR binding to FcγRIIb is maintained.

[0262] In addition to the literature reports discussed above, the above-mentioned mouse verification results suggest that FcγRIIb-mediated internalization of immune complexes occurs in the human body as well, as in mice, and that antibodies with Fc that selectively enhance binding to human FcγRIIb can accelerate the elimination of their antigens. Furthermore, as discussed above, since FcγRIIb-mediated internalization of immune complexes occurs at similar rates in mice and humans, it is thought that the same effect in accelerating antigen elimination as that of antibodies with Fc that have enhanced affinity for mouse FcγRIIb can be achieved in the human body by using Fc with similar enhanced affinity for human FcγRIIb.

[0263] The present invention also provides methods for producing polypeptides comprising antibody Fc region variants that maintain their FcγRIIb-binding activity while reducing their binding activity to activating FcγRs compared to polypeptides comprising the parent Fc region, the methods comprising adding at least one amino acid modification to the Fc region variants. For example, the production method may include the following steps: (a) modifying at least one amino acid in a polypeptide comprising an Fc region; (b) measuring the binding activity of the polypeptide altered in step (a) to FcγRIIb and to activating FcγR; and (c) selecting a polypeptide comprising an Fc region variant that maintains binding activity to FcγRIIb while having reduced binding activity to activating FcγR compared to a polypeptide comprising a parent Fc region.

[0264] A preferred embodiment is a method for producing a polypeptide comprising a variant Fc region, comprising the steps of: (a) modifying a nucleic acid encoding a polypeptide comprising a parent Fc region so that the polypeptide has reduced binding activity to activating FcγR while maintaining its FcγRIIb-binding activity; (b) introducing the nucleic acid into a host cell and culturing it so that it is expressed; (c) recovering the polypeptide from the host cell culture. Furthermore, antibodies and Fc fusion protein molecules produced by this production method are also included in the present invention.

[0265] The present invention also provides a method for reducing the binding activity of a polypeptide comprising an antibody Fc region variant to all activating FcγRs, particularly FcγRIIa (R type), while maintaining the binding activity to FcγRIIb compared to a polypeptide comprising a parent Fc region, which method comprises adding at least one amino acid modification to the Fc region variant, or a method for producing a polypeptide comprising an Fc region variant of the present invention. For example, the method may include the following steps: (a) modifying at least one amino acid in a polypeptide comprising an Fc region; (b) measuring the FcγRIIa-binding activity and FcγRIIb-binding activity of the polypeptide altered in step (a); and (c) A step of selecting a polypeptide containing an Fc region variant that maintains binding activity to FcγRIIb while having reduced binding activity to FcγRIIa (R type) compared to a polypeptide containing a parent Fc region.

[0266] A preferred embodiment is a method for reducing the binding activity of a polypeptide comprising a parent Fc region to all activating FcγRs, particularly FcγRIIa (R type), while maintaining the FcγRIIb-binding activity, or a method for producing a polypeptide comprising a variant Fc region, comprising: (a) modifying a nucleic acid encoding a polypeptide comprising a parent Fc region so that the polypeptide has reduced FcγRIIa (R type)-binding activity while maintaining FcγRIIb-binding activity, compared to a polypeptide comprising the parent Fc region; (b) introducing the nucleic acid into a host cell and culturing it so that it is expressed; (c) recovering the polypeptide from the host cell culture. Furthermore, antibodies and Fc fusion protein molecules produced by this production method are also included in the present invention.

[0267] The present invention also provides a method for suppressing the production of antibodies against a polypeptide comprising an Fc region when administered to a living body, compared to a polypeptide comprising a parent Fc region, comprising modifying at least one amino acid in the Fc region, or a method for producing a polypeptide in which the production of antibodies against the polypeptide is suppressed. For example, the method may include the following steps: (a) modifying at least one amino acid in a polypeptide comprising an Fc region; and (b) A step of confirming that when a polypeptide containing the Fc region modified in step (a) is administered to a living body, antibody production is suppressed compared to a polypeptide containing the parent Fc region.

[0268] Such polypeptides are thought to be useful as pharmaceuticals because they can suppress antibody production without activating activating FcγR.

[0269] In the above-mentioned methods, it is preferable to reduce the binding activity to all activating FcγRs, particularly FcγRIIa (R type), while maintaining the binding activity to FcγRIIb.

[0270] In a preferred embodiment of the above-mentioned method, for example, the Fc region of human IgG is modified so that the amino acid at EU numbering position 238 is altered to another amino acid, and at least one amino acid selected from amino acids at EU numbering positions 235, 237, 241, 268, 295, 296, 298, 323, 324, and 330 is altered to another amino acid. Other amino acid alterations to be combined with the amino acid alteration at EU numbering position 238 may be combinations of two or more amino acids selected from the above. Preferred combinations include (1) to (3) below. (1) amino acids at positions 241, 268, 296, and 324 (EU numbering) in the Fc region (2) amino acids 237, 241, 296, and 330 (EU numbering) in the Fc region (3) amino acids at positions 235, 237, 241, and 296 (EU numbering) in the Fc region

[0271] The amino acids selected as the modified amino acids are not particularly limited, as long as they maintain FcγRIIb-binding activity compared to before modification while reducing binding selectivity for all activating FcγRs, in particular FcγRIIa(R). However, it is preferred that the amino acid at position 238 (EU numbering) is Asp, the amino acid at position 235 (EU numbering) is Phe, the amino acid at position 237 is Gln or Asp, the amino acid at position 241 is Met or Leu, the amino acid at position 268 is Pro, the amino acid at position 295 is Met or Val, the amino acid at position 296 is Glu, His, Asn, or Asp, the amino acid at position 298 is Ala or Met, the amino acid at position 323 is Ile, the amino acid at position 324 is Asn or His, and the amino acid at position 330 is His or Tyr.

[0272] Furthermore, in a preferred embodiment of the above-mentioned method, for example, the Fc region of human IgG is modified so that a variant Fc region containing amino acid alterations that increase the FcγRIIb-binding activity by at least two times that of the Fc region of native IgG is introduced in combination with amino acid alterations that reduce the binding activity to all FcγRs. In the present invention, "amino acid modifications that result in FcγRIIb-binding activity that is at least twice that of the Fc region of native IgG" are not particularly limited, but include, for example, the amino acid modifications listed in Table 11.

[0273] Furthermore, in the present invention, "amino acid modifications that reduce binding activity to all FcγRs" are not particularly limited, and examples include at least one amino acid selected from amino acids 234, 235, 236, 237, 239, 265, 267, and 297 (EU numbering) in the Fc region. Preferred combinations include, for example, amino acid alterations that increase the FcγRIIb-binding activity by at least two times compared to the Fc region of native IgG to amino acids 238 and 271 (EU numbering) in the Fc region, and amino acid alterations that decrease the binding activity to all FcγRs to at least one amino acid selected from amino acids 234, 235, 236, 237, 239, 265, 267, and 297 (EU numbering) in the Fc region.

[0274] Specifically, the combinations of amino acid modifications described in (1) to (3) below can be given as preferred combinations of modifications. (1) amino acids at positions 233, 238, 264, 267, 268, and 271 (EU numbering) in the Fc region (2) amino acids at positions 233, 237, 238, 264, 267, 268, 271, 296, 297, 330, and 396 (EU numbering) in the Fc region; (3) amino acids at positions 233, 238, 264, 267, 268, 271, and 296 (EU numbering) in the Fc region

[0275] The amino acids selected as the modified amino acids are not particularly limited, as long as they maintain binding activity to FcγRIIb compared to before modification while reducing binding selectivity to all activating FcγRs, in particular FcγRIIa(R). However, it is preferred that the amino acid at position 238 (EU numbering) is Asp, the amino acid at position 271 is Gly, the amino acid at position 234 (EU numbering) is Ala, His, Asn, Lys, or Arg, the amino acid at position 235 is Ala, the amino acid at position 236 is Gln, the amino acid at position 237 is Arg or Lys, the amino acid at position 239 is Lys, the amino acid at position 265 is Lys, Asn, Arg, Ser, or Val, the amino acid at position 267 is Lys, Arg, or Tyr, and the amino acid at position 297 is Ala.

[0276] In a preferred embodiment of the above-mentioned method, for example, the Fc region of human IgG is modified so that amino acids at positions 238, 271, 327, 330, and 331 (EU numbering) are modified to another amino acid. Furthermore, the Fc region is modified so that at least one amino acid selected from amino acids 233, 237, 264, 267, and 268 is modified to another amino acid. The other amino acid modifications to be combined may be two or more amino acids selected from the above. Preferred combinations include (1) to (4) below. (1) amino acids at positions 237, 238, 268, 271, 327, 330, and 331 (EU numbering) in the Fc region (2) amino acids at positions 233, 237, 238, 268, 271, 327, 330, and 331 (EU numbering) of the Fc region; (3) amino acids at positions 238, 267, 268, 271, 327, 330, and 331 (EU numbering) in the Fc region (4) amino acids at positions 238, 264, 267, 271, 327, 330, and 331 (EU numbering) in the Fc region

[0277] The amino acids selected as the modified amino acids are not particularly limited, as long as they maintain binding activity to FcγRIIb compared to before modification while reducing binding selectivity to all activating FcγRs, in particular FcγRIIa(R). However, it is preferred that the amino acid at position 238 (EU numbering) is Asp, the amino acid at position 271 is Gly, the amino acid at position 327 is Gly, the amino acid at position 330 is Ser, the amino acid at position 331 is Ser, the amino acid at position 233 is Asp, the amino acid at position 237 is Asp, the amino acid at position 264 is Ile, the amino acid at position 267 is Ala, and the amino acid at position 268 is Asp or Glu.

[0278] Furthermore, the present invention provides methods for modifying polypeptides to prepare polypeptides that have reduced binding activity to activating FcγRs, particularly FcγRIIa (R type), while maintaining FcγRIIb-binding activity, compared to polypeptides comprising a parent Fc region.The present invention also provides methods for modifying polypeptides to prepare polypeptides that have reduced binding activity to activating FcγRs, particularly FcγRIIa (R type), while maintaining FcγRIIb-binding activity, compared to polypeptides comprising a parent Fc region. The present invention also provides methods for modifying polypeptides to produce polypeptides that, when administered to a living body, suppress antibody production compared to polypeptides containing the parent Fc region.

[0279] Preferred embodiments include, for example, the combinations of amino acid modifications described in the methods for producing polypeptides comprising Fc region variants that maintain the above-mentioned FcγRIIb-binding activity while reducing the binding activity to activating FcγR, particularly FcγRIIa (R type). Furthermore, the various methods described above can be used in combination with other amino acid modifications, as long as the FcγRIIb-binding activity is maintained and the binding activity to all activating FcγRs is reduced compared to a polypeptide comprising the Fc region of native IgG. Such modifications include, for example, modifications that reduce complement-binding activity. Specific examples include an amino acid modification at EU numbering position 322 in the Fc region, or a combination of amino acid modifications at EU numbering positions 327, 330, and 331 in the Fc region. The amino acids selected for the modified amino acids are not particularly limited, as long as they maintain FcγRIIb-binding activity, reduce binding activity to all activating FcγRs, and reduce complement-binding activity compared to a polypeptide comprising the Fc region of native IgG. However, preferred amino acids are Ala or Glu at EU numbering position 322, Gly at EU numbering position 327, Ser at EU numbering position 330, and Ser at EU numbering position 331.

[0280] The present invention further provides nucleic acids encoding polypeptides comprising an Fc region, wherein at least one amino acid has been altered, and wherein the polypeptide comprises an Fc region variant that maintains FcγRIIb-binding activity while decreasing its binding activity to activating FcγRs, particularly FcγRIIa (R type), compared to a polypeptide comprising the parent Fc region. The present invention also provides nucleic acids encoding polypeptides comprising an Fc region, wherein at least one amino acid has been altered, and wherein the polypeptide comprises an Fc region variant that maintains FcγRIIb-binding activity while decreasing its binding activity to activating FcγRs, particularly FcγRIIa (R type), compared to a polypeptide comprising the parent Fc region. The nucleic acids of the present invention may be in any form, such as DNA or RNA.

[0281] 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.

[0282] The present invention also relates to host cells transformed with the vectors of the present invention. Host cells can be appropriately selected by those skilled in the art, and the host cells described above can be used, for example. Specific examples of host cells include the following: When eukaryotic cells are used as host cells, animal cells, plant cells, or fungal cells can be used as appropriate. Specific examples of animal cells include the following: (1) Mammalian cells: CHO (Chinese hamster ovary cell line), COS (Monkey kidney cell line), myeloma (Sp2 / O, NS0, etc.), BHK (baby hamster kidney cell line), Hela, Vero, HEK293 (human embryonic kidney cell line with sheared adenovirus (Ad)5 DNA), Freestyle 293, PER.C6 cells (human embryonic retinal cell line transformed with the Adenovirus Type 5 (Ad5) E1A and E1B genes), etc. (Current Protocols in Protein Science (May 2001, Unit 5.9, Table 5.9.1)) (2) Amphibian cells: Xenopus oocytes, etc. (3) Insect cells: sf9, sf21, Tn5, etc.

[0283] Alternatively, an antibody gene expression system using plant cells derived from the genus Nicotiana, such as Nicotiana tabacum, is known. Callus cultured cells can be appropriately used for transformation of plant cells.

[0284] Furthermore, the following fungal cells can be used: - Yeast: Saccharomyces genus such as Saccharomyces cerevisiae, Pichia genus such as Pichia pastoris -Filamentous fungi: Aspergillus genus, such as Aspergillus niger

[0285] Furthermore, the present invention provides a method for reducing the binding activity of a polypeptide comprising an Fc region to activating FcγR, particularly FcγRIIa (R type), while maintaining the binding activity to FcγRIIb, compared to a polypeptide comprising a parent Fc region, which method comprises adding at least one amino acid modification to the Fc region. The present invention also provides a method for suppressing the production of antibodies against a polypeptide comprising an Fc region when administered to a living body, compared to a polypeptide comprising the parent Fc region, comprising adding at least one amino acid modification to the Fc region.

[0286] Preferred embodiments include, for example, the combinations of amino acid modifications described in the methods for producing polypeptides comprising Fc region variants that maintain the above-mentioned FcγRIIb-binding activity while reducing the binding activity to activating FcγR, particularly FcγRIIa (R type). Furthermore, the present invention also includes polypeptides produced by any of the above methods.

[0287] The present invention provides pharmaceutical compositions containing polypeptides comprising the Fc region variants of the present invention. Pharmaceutical compositions of the present invention can be formulated by known methods by incorporating a pharmaceutically acceptable carrier in addition to the above-described antibodies or Fc fusion protein molecules of the present invention. For example, they can be administered parenterally in the form of a sterile solution or suspension injection in water or other pharmaceutically acceptable liquid. For example, they can be formulated by appropriately combining them with pharmacologically acceptable carriers or vehicles, specifically, sterile water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, etc., and blending them in a unit dosage 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 methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglycerides, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salts, etc. The amount of the active ingredient in these preparations is such that an appropriate dose within the indicated range can be obtained. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice using a vehicle such as distilled water for injection.

[0288] Examples of aqueous solutions for injection include isotonic solutions containing physiological saline, glucose or other auxiliary agents, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, and may be used in combination with an appropriate solubilizing agent, such as alcohol, specifically ethanol, polyalcohols such as propylene glycol and polyethylene glycol, and nonionic surfactants such as polysorbate 80™ and HCO-50.

[0289] Examples of oily liquids include sesame oil and soybean oil, and they may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. They may also contain buffers such as phosphate buffer and sodium acetate buffer, soothing agents such as procaine hydrochloride, stabilizers such as benzyl alcohol, phenol, and antioxidants. The prepared injection solution is usually filled into suitable ampoules.

[0290] The administration is preferably parenteral, and specific examples include injection dosage forms, intranasal administration dosage forms, pulmonary administration dosage forms, transdermal administration dosage forms, etc. Injection dosage forms can be administered systemically or locally by, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.

[0291] Furthermore, the administration method for the pharmaceutical composition of the present invention can be selected appropriately depending on the patient's age and symptoms. The dosage of a pharmaceutical composition containing an antibody or a polynucleotide encoding the antibody can be selected, for example, from the range of 0.0001 mg to 1,000 mg per kg of body weight per administration. Alternatively, the dosage can be selected, for example, from the range of 0.001 to 100,000 mg / body weight per patient, but is not necessarily limited to these values. The dosage and administration method vary depending on the patient's weight, age, symptoms, etc., but can be appropriately selected by those skilled in the art.

[0292] The above-mentioned polypeptides containing Fc region variants of the present invention are useful as active ingredients of drugs that suppress the activation of B cells, mast cells, dendritic cells, and / or basophils. Polypeptides containing Fc region variants of the present invention selectively act on FcγRIIb without activating activating FcγR, thereby suppressing the activation of B cells, mast cells, dendritic cells, and / or basophils. B cell activation includes proliferation, IgE production, IgM production, IgA production, and the like. The above-mentioned polypeptides containing Fc region variants of the present invention suppress IgE production by B cells by crosslinking FcγRIIb with IgE, suppress IgM production by crosslinking IgM with B cells, and suppress IgA production by crosslinking IgA with IgA. In addition, similar inhibitory effects can be achieved by directly or indirectly crosslinking FcγRIIb with molecules expressed on B cells, such as BCR, CD19, and CD79b, which contain ITAM domains intracellularly or interact with ITAM domains. Mast cell activation includes proliferation, activation by IgE, etc., degranulation, etc. In mast cells, the polypeptides containing the Fc region variants of the present invention can inhibit proliferation, activation by IgE, etc., and degranulation by directly or indirectly crosslinking FcγRIIb with molecules that contain ITAM domains or interact with ITAM domains expressed on mast cells, such as IgE receptors FcεRI, DAP12, and CD200R3. Basophil activation also includes proliferation and degranulation. In basophils, the polypeptides containing the Fc region variants of the present invention can inhibit activation, degranulation, and proliferation by directly or indirectly crosslinking FcγRIIb with molecules on the cell membrane that contain an ITAM domain intracellularly or interact with an ITAM domain. Dendritic cell activation also includes proliferation and degranulation. The polypeptides containing the above-mentioned Fc region variants of the present invention can also inhibit activation, degranulation, and proliferation in dendritic cells by directly or indirectly cross-linking FcγRIIb with molecules on the cell membrane that contain ITAM domains intracellularly or that interact with ITAM domains.

[0293] In the present invention, polypeptides comprising the above-mentioned Fc region variants of the present invention are useful as active ingredients in therapeutic or preventive agents for immunoinflammatory diseases. As described above, polypeptides comprising the Fc region variants of the present invention can suppress the activation of B cells, mast cells, dendritic cells, and / or basophils. As a result, immunoinflammatory diseases can be treated or prevented by administering polypeptides comprising the Fc region variants of the present invention. "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, and 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, Addison's disease, myasthenia gravis, uveitis lentis, systemic lupus erythematosus, allergic rhinitis, allergic dermatitis, ulcerative colitis, hypersensitivity, muscle degeneration, cachexia, systemic sclerosis, localized scleroderma, Sjogren's syndrome, Chet's disease, Reiter's syndrome, type I and type II diabetes, bone resorption disorders, graft-versus-host reaction, ischemia-reperfusion injury, atherosclerosis, brain trauma, cerebral malaria, sepsis, septic shock, toxic shock syndrome, fever, 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 granulomatosis, arteritis nodosa, mixed connective tissue disease, fibromyalgia, asthma, atopic dermatitis, chronic atrophic gastritis, primary biliary cirrhosis, primary sclerosing cholangitis, autoimmune pancreatitis, aortitis syndrome, rapidly progressive glomerulonephritis, megaloblastic anemia, idiopathic thrombocytopenic purpura, primary hypothyroidism, idiopathic Addison's disease, insulin-dependent diabetes mellitus, chronic discoid lupus erythematosus, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, epidermolysis bullosa acquisita,Alopecia areata, vitiligo vulgaris, Sutton's acquired centrifugal vitiligo, Harada's disease, autoimmune optic neuropathy, idiopathic azoospermia, habitual abortion, hypoglycemia, chronic urticaria, ankylosing spondylitis, psoriatic arthritis, enteropathic arthritis, reactive arthritis, spondyloarthropathy, enthesitis, irritable bowel syndrome, chronic fatigue syndrome, dermatomyositis, inclusion body myositis, Schmidt's syndrome, Graves' disease, pernicious anemia, lupoid hepatitis, presenile dementia, Alzheimer's disease, demyelinating diseases, amyotrophic lateral sclerosis, hypoparathyroidism, Dressler's syndrome, Eaton-Lambert syndrome, dermatitis herpetiformis, alopecia, progressive systemic sclerosis CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly and telangiectasia), sarcoidosis, rheumatic fever, erythema multiforme, Cushing's syndrome, transfusion reactions, leprosy, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, giant cell arteritis, eczema, lymphomatoid granulomatosis, Kawasaki disease, endocarditis, endomyocardial fibrosis, endophthalmitis, erythroblastosis fetalis, eosinophilic fasciitis, Felty's syndrome, Henoch-Schönlein purpura, transplant rejection, mumps, cardiomyopathy, septic arthritis, familial Mediterranean fever, Muckle-Wells syndrome, hyper-IgD syndrome.

[0294] Furthermore, polypeptides containing the above-described Fc region variants of the present invention are useful as active ingredients in drugs for treating or preventing autoimmune diseases in which the production of antibodies against autoantigens (autoantibodies) is thought to be the cause of the disease, by suppressing the production of the autoantibodies. It has been reported that the use of a molecule fusing AchR, an autoantigen in myasthenia gravis, with the Fc portion of an antibody can suppress the proliferation of B cells expressing BCRs that recognize AchR and induce apoptosis (J Neuroimmunol, 227, 35-43, 2010). The use of a fusion protein of an antigen recognized by an autoantibody with the antibody Fc region described in the present invention can crosslink the BCR of B cells expressing a BCR against the autoantigen with FcγRIIb, thereby suppressing the proliferation of B cells expressing a BCR against the autoantigen and inducing apoptosis. These autoimmune diseases include Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, autoimmune pancreatitis, aortitis syndrome, Goodpasture's syndrome, rapidly progressive glomerulonephritis, megaloblastic anemia, autoimmune hemolytic anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Graves' disease, Hashimoto's disease, and primary hypothyroidism. These conditions include, but are not limited to, idiopathic Addison's disease, insulin-dependent diabetes mellitus, chronic discoid lupus erythematosus, localized scleroderma, pemphigus, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, epidermolysis bullosa acquisita, alopecia areata, vitiligo vulgaris, Sutton's acquired vitiligo centrifugally, Harada's disease, autoimmune optic neuropathy, idiopathic azoospermia, recurrent abortion, type 2 diabetes mellitus, hypoglycemia, and chronic urticaria.

[0295] Furthermore, polypeptides containing the above-described Fc region variants of the present invention are useful as active ingredients in therapeutic agents for diseases in which a vital protein is missing. Treatments for diseases in which a vital protein is missing are typically administered as a drug to replenish the protein. However, because patients are originally deficient in the protein, the exogenously replenished protein is recognized as a foreign substance, leading to the production of antibodies against the protein. As a result, the protein becomes more susceptible to elimination, reducing its efficacy as a drug. Fusion proteins of such proteins with antibody Fc regions described in the present invention can crosslink BCR and FcγRIIb on B cells that recognize the protein, thereby suppressing antibody production against the protein. Proteins to be supplemented include Factor VIII, Factor IX, TPO, EPO, α-iduronidase, iduronate sulfatase, type A heparan N-sulfatase, type B α-N-acetylglucosaminidase, type C acetyl CoA:α-glucosaminidase acetyltransferase, type D N-acetylglucosamine 6-sulfatase, galactose 6-sulfatase, N-acetylgalactosamine 4-sulfatase, β-glucuronidase, α-galactosidase, acidic α-galactosidase, and glucocerebrosidase. Diseases requiring supplementation of these proteins include, but are not limited to, hemophilia, idiopathic thrombocytopenic purpura, renal anemia, and lysosomal diseases (mucopolysaccharidosis, Fabry disease, Pompe disease, and Gaucher disease).

[0296] Furthermore, polypeptides containing the above-described Fc region variants of the present invention are useful as active ingredients in antiviral agents. Antibodies against viruses that contain the Fc region described in the present invention can suppress antibody-dependent enhancement (ADE) observed in antibodies against viruses. ADE is a phenomenon in which a virus is phagocytosed via activating FcγRs by neutralizing antibodies against that virus, infecting FcγR-expressing cells and thereby spreading the infection. It has been reported that the binding of neutralizing antibodies against dengue virus to FcγRIIb plays an important role in suppressing ADE (Proc Natl Acad Sci USA, 108, 12479-12484, 2011). Dengue virus-specific immune complexes formed by neutralizing antibodies against dengue virus cross-link FcγRIIb, thereby inhibiting FcγR-mediated phagocytosis and thereby suppressing ADE. Examples of viruses include, but are not limited to, dengue viruses (DENV1, DENV2, and DENV4) and HIV.

[0297] Furthermore, polypeptides containing the above-described Fc region variants of the present invention are useful as active ingredients in agents for preventing or treating arteriosclerosis. Antibodies against oxidized LDL, a cause of arteriosclerosis, that contain the Fc region described in the present invention can prevent FcγRIIa-dependent adhesion of inflammatory cells. Antioxidized LDL antibodies inhibit the interaction between oxidized LDL and CD36, but it has been reported that antioxidized LDL antibodies bind to endothelial cells, and their Fc moiety is recognized and adhered by monocytes in an FcγRIIa- or FcγRI-dependent manner (Immunol Lett, 108, 52-61, 2007). By using such antibodies containing the Fc region described in the present invention, FcγRIIa-dependent binding is inhibited and monocyte adhesion is suppressed by FcγRIIb-mediated inhibitory signaling.

[0298] In the present invention, polypeptides comprising the above-described Fc region variants of the present invention are useful as active ingredients in therapeutic or preventive agents for cancer. As described above, by maintaining binding to FcγRIIb and reducing binding to all activating FcγRs, it is possible to suppress platelet activation via an FcγRIIa-dependent mechanism while maintaining the agonistic activity of agonist antibodies, thereby reducing the risk of thromboembolism and the like. Therefore, agonist antibodies using the Fc region variants described in the present invention are useful for treating or preventing cancer. Specifically, the Fc region variants described in the present invention enhance the agonistic activity of agonistic antibodies against the TNF receptor family, such as Aliases, CD120a, CD120b, Lymphotoxin β receptor, CD134, CD40, FAS, TNFRSF6B, CD27, CD30, CD137, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, RANK, Osteoprotegerin, TNFRSF12A, TNFRSF13B, TNFRSF13C, TNFRSF14, Nerve growth factor receptor, TNFRSF17, TNFRSF18, TNFRSF19, TNFRSF21, ...

Claims

[Claim 1] The invention described herein.

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