FcγRIIb-specific Fc region variant

Abstract

To provide Fc region variants having enhanced binding activity to FcγRIIb and / or enhanced binding selectivity to FcγRIIb in comparison with FcγRIIa (type R), polypeptides comprising the Fc region variant, pharmaceutical compositions comprising the polypeptides, therapeutic or prophylactic agents for immuno-inflammatory diseases comprising the pharmaceutical compositions, and methods for manufacturing them, as well as methods for enhancing binding selectivity to FcγRIIb as compared with FcγRIIa (type R).SOLUTION: Disclosed herein is a polypeptide comprising an antibody Fc region variant comprising an amino acid sequence having an amino acid modification at position 238 according to EU numbering in combination with other specific amino acid modifications.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an Fc region variant in which an amino acid modification is introduced into the Fc region of an antibody, and compared with the Fc region into which no amino acid modification is introduced, the binding activity to FcγRIIb is enhanced and / or the binding selectivity to FcγRIIb compared with FcγRIIa (R type) is enhanced, a polypeptide containing the Fc region variant, and a pharmaceutical composition containing the polypeptide.

Background Art

[0002] Antibodies have attracted attention as pharmaceuticals because of their high stability in blood and few side effects (Non-Patent Document 1, Non-Patent Document 2). Most of the currently marketed antibody drugs are antibodies of the human IgG1 subclass. As one of the functions of IgG class antibodies, antibody-dependent cell-mediated cytotoxicity activity (hereinafter referred to as ADCC activity) is known (Non-Patent Document 3). In order for an antibody to exhibit ADCC activity, binding between the Fc region of the antibody and an Fcγ receptor (hereinafter referred to as FcγR), which is an antibody-binding receptor present on the surface of effector cells such as killer cells, natural killer cells, and activated macrophages, is required.

[0003] In humans, isoforms of FcγRIa (CD64A), FcγRIIa (CD32A), FcγRIIb (CD32B), FcγRIIIa (CD16A), and FcγRIIIb (CD16B) have been reported in the FcγR protein family, and their respective allotypes have also been reported (Non-Patent Document 7). FcγRIa, FcγRIIa, and FcγRIIIa are called activating FcγRs because they have immunologically active functions, and FcγRIIb has an immunosuppressive function and is called an inhibitory FcγR (Non-Patent Document 8).

[0004] Regarding the binding between the Fc region and FcγR, it has been shown that several amino acid residues in the hinge region and CH2 domain of the antibody and the sugar chain added to Asn at position 297 in the EU numbering that binds to the CH2 domain are important (Non-Patent Document 4, Non-Patent Document 5, Non-Patent Document 6). Focusing on antibodies with mutations introduced into these sites, various mutants with different FcγR binding properties have been studied so far, and Fc region mutants with higher binding activity to activated FcγR have been obtained (Patent Document 1, Patent Document 2, Patent Document 3, Patent Document 4).

[0005] When cross-linked by immune complexes, activated FcγR causes phosphorylation of immunoreceptor tyrosine-based activating motifs (ITAMs) contained in the intracellular domain or the FcR common γ-chain, which is the interaction partner, activates SYK, which is a signaling substance, and initiates an activation signal cascade to cause an inflammatory immune response (Non-Patent Document 9).

[0006] FcγRIIb is the only FcγR expressed on B cells (Non-Patent Document 10). It has been reported that the first immune response of B cells is suppressed by the interaction of the Fc region of an antibody with FcγRIIb (Non-Patent Document 11). It has also been reported that when FcγRIIb on B cells and the B cell receptor (BCR) are cross-linked via immune complexes in the blood, the activation of B cells is suppressed and the antibody production of B cells is suppressed (Non-Patent Document 12). The transmission of the immunosuppressive signal via this BCR and FcγRIIb requires an immunoreceptor tyrosine-based inhibitory motif (ITIM) contained in the intracellular domain of FcγRIIb (Non-Patent Document 13, Non-Patent Document 14). When a signal enters and ITIM is phosphorylated, SH2-containing inositol polyphosphate 5-phosphatase (SHIP) is recruited, inhibiting the transmission of the signal cascade of other activated FcγRs and suppressing the inflammatory immune response (Non-Patent Document 15). It has also been reported that by simply aggregating FcγRIIb, the proliferation of B cells and the calcium influx due to the cross-linking of BCR can be transiently suppressed without apoptosis of IgM-producing B cells in a BCR-independent manner (Non-Patent Document 16).

[0007] Furthermore, FcγRIIb is also expressed on dendritic cells, macrophages, activated neutrophils, mast cells, and basophils. Also in these cells, FcγRIIb inhibits the functions of activated FcγRs such as phagocytosis and the release of inflammatory cytokines, suppressing the inflammatory immune response (Non-Patent Document 8).

[0008] The importance of the immunosuppressive function of FcγRIIb has been clarified by studies using FcγRIIb knockout mice. In FcγRIIb knockout mice, humoral immunity is not properly regulated (Non-Patent Document 17), and the susceptibility to collagen-induced arthritis (CIA) increases (Non-Patent Document 18), and lupus-like symptoms and Goodpasture syndrome-like symptoms are reported (Non-Patent Document 19).

[0009] In addition, dysregulation of FcγRIIb has also been reported to be associated with human autoimmune diseases. For example, the association between gene polymorphisms in the promoter region and transmembrane region of FcγRIIb and the incidence of systemic lupus erythematosus (SLE) (Non-Patent Documents 20, 21, 22, 23, 24), and the decreased expression of FcγRIIb on the surface of B cells in SLE patients have been reported (Non-Patent Documents 25, 26).

[0010] Thus, from the mouse model and clinical findings, FcγRIIb is considered to play a role in controlling autoimmune diseases and inflammatory diseases, particularly through its involvement with B cells, and is a promising target molecule for controlling autoimmune diseases and inflammatory diseases.

[0011] IgG1, which is mainly used as a commercially available antibody drug, is known to strongly bind not only to FcγRIIb but also to activated FcγRs (Non-Patent Document 27). By using an Fc region with enhanced binding to FcγRIIb or improved selectivity for binding to FcγRIIb compared to activated FcγRs, it is possible to develop an antibody drug having immunosuppressive properties compared to IgG1. For example, it has been suggested that the activation of B cells can be inhibited by using an antibody having a variable region that binds to BCR and an Fc that enhances the binding to FcγRIIb (Non-Patent Document 28). It has been reported that the cross-linking of FcγRIIb on B cells and IgE bound to B-cell receptor suppresses the differentiation of B cells into plasma cells and, as a result, the production of IgE, and that the human IgG and IgM concentrations are maintained while the human IgE concentration decreases in mice transplanted with human PBMC (Non-Patent Document 29). It has been reported that not only IgE but also the cross-linking of CD79b, a constituent molecule of the B-cell receptor complex, and FcγRIIB with an antibody suppresses the proliferation of B cells in vitro and alleviates the symptoms in a collagen arthritis model (Non-Patent Document 30). In addition to B cells, by using a molecule in which the Fc portion of IgE that binds to FcεRI, the receptor of IgE, and the Fc portion of IgG with enhanced binding to FcγRIIb are fused to cross-link FcεRI and FcγRIIb on mast cells, it has been reported that phosphorylation of FcγRIIb is induced and the FcεRI-dependent calcium influx is suppressed, suggesting that the inhibition of degranulation via the stimulation of FcγRIIb is possible by enhancing the binding to FcγRIIb (Non-Patent Document 31). From these facts, it is suggested that an antibody having an Fc with improved binding activity to FcγRIIb is promising as a therapeutic agent for inflammatory diseases such as autoimmune diseases.

[0012] In addition, in the presence of an immune complex of an antibody and an antigen, it has been reported that the activation of dendritic cells and macrophages via Toll-like receptor 4 by LPS stimulation is suppressed, and it has been suggested that this effect is also mediated by FcγRIIb of the immune complex (Non-Patent Documents 32 and 33). From this, it is expected that by using an antibody with enhanced binding to FcγRIIb, it is possible to enhance the effect of suppressing the activation signal via TLR, and it is suggested that it is promising as a therapeutic agent for inflammatory diseases such as autoimmune diseases.

[0013] In addition, mutants with enhanced binding to FcγRIIb have been suggested to be promising not only as therapeutic agents for inflammatory diseases such as autoimmune diseases but also as cancer therapeutic agents. So far, it has been clarified that FcγRIIb plays an important role in the agonist activity of agonist antibodies against the anti-TNF receptor superfamily. Specifically, it has been suggested that the interaction with FcγRIIb is necessary for the agonist activity of antibodies against CD40, DR4, DR5, CD30, and CD137 contained in the TNF receptor family (Non-Patent Documents 34, 35, 36, 37, 38, 39, and 40). In Non-Patent Document 34, it has been shown that the anti-tumor effect of an anti-CD40 antibody is enhanced by using an antibody with enhanced binding to FcγRIIb. From this, it is expected that an antibody with enhanced binding to FcγRIIb has the effect of enhancing the agonist action of agonist antibodies including antibodies against the anti-TNF receptor superfamily. In addition, it has been shown that the proliferation of cells is suppressed by cross-linking Kit and FcγRIIb on cells expressing Kit using an antibody that recognizes Kit, which is one of the Receptor Tyrosine kinases (RTKs). The same effect has been reported even when this Kit is constitutively activated and has a mutation that promotes carcinogenesis (Non-Patent Document 41). From this, it is expected that by using an antibody with enhanced binding to FcγRIIb, it is possible to enhance the inhibitory effect on cells expressing RTK with a constitutively activated mutation.

[0014] Previously, antibodies having an Fc with improved binding activity to FcγRIIb have been reported (Non-Patent Document 28). In this document, modifications such as S267E / L328F, G236D / S267E, and S239D / S267E were introduced into the Fc region of the antibody to improve the binding activity to FcγRIIb. Among these, the antibody into which the S267E / L328F mutation was introduced bound most strongly to FcγRIIb, and the binding to FcγRIIa H-type in which the 131st residue of FcγRIa and FcγRIIa is His was maintained at the same level as that of natural IgG1. However, according to another report, this modification enhanced the binding to FcγRIIa R-type in which the 131st residue of FcγRIIa is His by several hundred times to the same extent as the binding to FcγRIIb, and the selectivity of the binding to FcγRIIb was not improved when compared with FcγRIIa R-type (Patent Document 5).

[0015] Regarding cells such as platelets that express FcγRIIa but do not express FcγRIIb (Non-Patent Document 8), it is considered that only the effect of enhancing the binding to FcγRIIa, rather than the enhancement of the binding to FcγRIIb, is affected. For example, it is known that the risk of thromboembolism increases in a patient group administered bevacizumab, an antibody against VEGF (Non-Patent Document 42). Similarly, thromboembolism was also observed in the clinical development trial of an antibody against CD40 ligand, and the clinical trial was terminated (Non-Patent Document 43). In the case of any of these antibodies, subsequent studies using animal models and the like have suggested that the administered antibody aggregates platelets via binding to FcγRIIa on platelets and forms thrombi (Non-Patent Documents 44 and 45). In systemic lupus erythematosus, which is one of the autoimmune diseases, there is a report that platelets are activated by an FcγRIIa-dependent mechanism and that the activation of platelets correlates with the severity (Non-Patent Document 46). Administering an antibody with enhanced binding to FcγRIIa to patients who originally have a high risk of developing such thromboembolism would further increase the risk of developing thromboembolism and is extremely dangerous.

[0016] In addition, it has been reported that antibodies with enhanced binding to FcγRIIa enhance antibody-dependent cell phagocytosis activity (ADCP) mediated by macrophages (Non-Patent Document 47). It is considered that when the antigen to which the antibody binds is phagocytosed by macrophages, the antibody itself is also phagocytosed. When the antibody is administered as a pharmaceutical, it is assumed that peptide fragments derived from the administered antibody are also likely to be antigen-presented, which is thought to increase the risk of producing antibodies against the antibody pharmaceutical (anti-drug antibodies). That is, enhancing the binding to FcγRIIa increases the risk of anti-drug antibody production and significantly reduces the value as a pharmaceutical. In addition, it has been suggested that FcγRIIb on dendritic cells contributes to peripheral tolerance by suppressing the activation of dendritic cells by immune complexes composed of antigens and antibodies or by suppressing antigen presentation to T cells via activated Fcγ receptors (Non-Patent Document 48). Since FcγRIIa is also expressed on dendritic cells, when an antibody having an Fc that selectively enhances binding to FcγRIIb is used as a pharmaceutical, it is less likely to be antigen-presented to dendritic cells, etc. due to the selectively enhanced binding to FcγRIIb, and the risk of anti-drug antibody production can also be relatively attenuated, which is considered useful in this regard. That is, enhancing the binding to FcγRIIa significantly reduces the value as a pharmaceutical in terms of increasing the risk of thrombosis via platelet aggregation and increasing immunogenicity and the risk of anti-drug antibody production.

[0017] From such a perspective, looking at the above-mentioned Fc variant with enhanced binding to FcγRIIb, the binding to the R type of FcγRIIa is significantly enhanced compared to natural IgG1, so its value as a pharmaceutical for patients having the R type of FcγRIIa is significantly reduced. The H type and R type of FcγRIIa are observed at approximately the same frequency in Caucasians and African-Americans (Non-Patent Document 49, Non-Patent Document 50). From this, when this Fc variant is used for the treatment of autoimmune diseases, the number of patients who can be safely used while enjoying the effect as a pharmaceutical is limited.

[0018] In addition, it has been reported that dendritic cells lacking FcγRIIb or dendritic cells in which the interaction between FcγRIIb and the Fc portion of the antibody is inhibited by an anti-FcγRIIb antibody undergo dendritic cell maturation (Non-Patent Document 51, Non-Patent Document 52). From this report, it is suggested that FcγRIIb suppresses dendritic cell maturation in a steady state where inflammation or the like is not occurring and the cells are not activated. Since FcγRIIa is also expressed on the surface of dendritic cells in addition to FcγRIIb, even if the binding to inhibitory FcγRIIb is enhanced, if the binding to activating FcγRIIa or the like is also enhanced, it is considered that dendritic cell maturation will be promoted as a result. That is, it is considered important for an antibody to have an immunosuppressive effect to improve the ratio of the binding activity to FcγRIIb to the binding activity to FcγRIIa, rather than just the binding activity to FcγRIIb. Therefore, when considering the creation of pharmaceuticals that utilize the immunosuppressive effect mediated by the binding of FcγRIIb, there is a need for Fc variants that not only enhance the binding activity to FcγRIIb but also maintain the binding to both the H-type and R-type gene polymorphisms of FcγRIIa at the same level as natural IgG1 or attenuate it below that level.

[0019] On the other hand, there have been reports of examples in which the selectivity of binding to FcγRIIb has been increased by introducing amino acid modifications into the Fc region (Non-Patent Document 53). However, in any of the mutants reported in this document with improved selectivity for FcγRIIb, the binding to FcγRIIb was decreased compared to natural IgG1. Therefore, it is considered difficult for these mutants to actually induce an immunosuppressive reaction via FcγRIIb more than IgG1.

[0020] Also, in the agonist antibodies described above, since FcγRIIb plays an important role, enhancing its binding activity is also expected to enhance agonist activity. However, if the binding to FcγRIIa is enhanced in the same way, it may exhibit unwanted ADCC activity, ADCP activity, etc., and there is a risk of side effects. From such a perspective, it is preferable to be able to selectively enhance the binding activity to FcγRIIb.

[0021] From these results, when creating antibody drugs for the treatment of autoimmune diseases and cancer using FcγRIIb, it is important that, compared to natural IgG, the binding activity is maintained or decreased for any gene polymorphism of FcγRIIa, and the binding activity to FcγRIIb is enhanced. However, FcγRIIb is one of the activating FcγRs, and its extracellular region sequence is 93% identical to that of FcγRIIa, with extremely similar structures. Furthermore, FcγRIIa has two gene polymorphisms: the 131st amino acid is His in the H type (H-type) and Arg in the R type (R-type), and the interaction with antibodies is different for each (Non-Patent Document 54). Therefore, it is considered a difficult task to create an Fc region variant that selectively enhances binding to FcγRIIb while distinguishing the extremely similar sequences of FcγRIIa and FcγRIIb. In fact, mutants with sufficient binding activity and selectivity for FcγRIIb have not been obtained so far. Patent Document 5 also reports mutants with enhanced binding activity to FcγRIIb, but the degree is weak, and the development of mutants with the above-mentioned properties has been demanded.

Prior Art Documents

Patent Documents

[0022]

Patent Document 1

Patent Document 2

Patent Document 3

[0023] [Non-Patent Document 1] Nat Biotechnol, 23, 1073-1078, 2005 [Non-Patent Document 2] Eur J Pharm Biopharm, 59(3), 389-96. 2005 [Non-Patent Document 3] Chem Immunol, 65, 88-110, 1997 [Non-Patent Document 4] J Biol Chem, 276, 16478-16483, 2001 [Non-Patent Document 5] Eur J Immunol 23, 1098-1104, 1993 [Non-Patent Document 6] Immunology, 86, 319-324, 1995 [Non-Patent Document 7] Immunol Lett, 82, 57-65, 2002 [Non-Patent Document 8] Nat Rev Immunol, 10, 328-343, 2010 [Non-Patent Document 9] Nat Rev Immunol, 8, 34-47, 2008 [Non-Patent Document 10] Eur J Immunol 19, 1379-1385, 1989 [Non-Patent Document 11] J Exp Med 129, 1183-1201, 1969 [Non-Patent Document 12] Immunol Lett 88, 157-161, 2003 [Non-Patent Document 13] Science, 256, 1808 - 1812, 1992

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Summary of the Invention

Problems to be Solved by the Invention

[0024] The present invention has been made in view of such circumstances, and an object thereof is to introduce an amino acid modification into the Fc region of an antibody, and compared with the Fc region into which no amino acid modification has been introduced, the binding activity to FcγRIIb is enhanced, and / or the binding selectivity to FcγRIIb compared with FcγRIIa (R type) is enhanced. An Fc region variant, a polypeptide containing the Fc region variant, and a pharmaceutical composition containing the polypeptide are provided.

Means for Solving the Problems

[0025] The present inventors introduced an amino acid modification into the Fc region, and compared with the Fc region into which no amino acid modification was introduced, the binding to FcγRIIb was enhanced, and the binding selectivity to FcγRIIb compared with FcγRIIa (R type) was enhanced. Intensive research was conducted on the Fc region variant and the polypeptide containing the Fc region variant. As a result, the present inventors combined other amino acid modifications with the Fc region variant in which the 238th amino acid in the EU numbering of the Fc region was modified, and found that the binding activity to FcγRIIb was enhanced and / or the binding selectivity to FcγRIIb compared with FcγRIIa (R type) was enhanced.

[0026] That is, the present invention relates to the following. 〔1〕 An Fc region variant in which at least one amino acid selected from the 238th amino acid of EU numbering, and the 233rd amino acid, 234th amino acid, 235th amino acid, 237th amino acid, 264th amino acid, 265th amino acid, 266th amino acid, 267th amino acid, 268th amino acid, 269th amino acid, 271st amino acid, 272nd amino acid, 274th amino acid, 296th amino acid, 326th amino acid, 327th amino acid, 330th amino acid, 331st amino acid, 332nd amino acid, 333rd amino acid, 334th amino acid, 355th amino acid, 356th amino acid, 358th amino acid, 396th amino acid, 409th amino acid, and 419th amino acid of EU numbering is modified to another amino acid, and the binding activity of the variant to the Fcγ receptor is such that the value of [KD value of the polypeptide containing the Fc region without amino acid modification for FcγRIIb] / [KD value of the polypeptide containing the Fc region variant for FcγRIIb] is 15.0 or more. 〔2〕 The Fc region variant according to 〔1〕, wherein the 238th amino acid, 268th amino acid, and 271st amino acid of EU numbering are modified to other amino acids, and further, at least one amino acid selected from the 233rd amino acid, 237th amino acid, 264th amino acid, 267th amino acid, 272nd amino acid, 296th amino acid, 327th amino acid, 330th amino acid, 332nd amino acid, and 396th amino acid of EU numbering is modified to another amino acid. 〔3〕 An Fc region variant having at least one amino acid selected from the group of amino acids wherein the 238th amino acid of the EU numbering is Asp, and the 233rd amino acid is Asp, the 234th amino acid is Tyr, the 235th amino acid is Phe, the 237th amino acid is Asp, the 264th amino acid is Ile, the 265th amino acid is Glu, the 266th amino acid is Phe, Leu or Met, the 267th amino acid is Ala, Glu, Gly or Gln, the 268th amino acid is Asp, Gln or Glu, the 269th amino acid is Asp, the 271st amino acid is Gly, the 272nd amino acid is Asp, Phe, Ile, Met, Asn, Pro or Gln, the 274th amino acid is Gln, the 296th amino acid is Asp or Phe, the 326th amino acid is Ala or Asp, the 327th amino acid is Gly, the 330th amino acid is Lys, Arg or Ser, the 331st amino acid is Ser, the 332nd amino acid is Lys, Arg, Ser or Thr, the 333rd amino acid is Lys, Arg, Ser or Thr, the 334th amino acid is Arg, Ser or Thr, the 355th amino acid is Ala, Gln, the 356th amino acid is Glu, the 358th amino acid is Met, the 396th amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr, the 409th amino acid is Arg and the 419th amino acid is Glu, and wherein the binding activity of the variant to the Fcγ receptor is such that the value of [KD value of the polypeptide containing the Fc region without amino acid modification for FcγRIIb] / [KD value of the polypeptide containing the Fc region variant for FcγRIIb] is 15.0 or more. 〔4〕 The Fc region variant according to 〔3〕, which has at least one amino acid selected from the group of amino acids where the 238th amino acid of the EU numbering in the Fc region is Asp, the 268th amino acid is Asp or Glu, and the 271st amino acid is Gly, and further, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala or Gly, the 272nd amino acid is Asp or Pro, the 296th amino acid is Asp, the 327th amino acid is Gly, the 330th amino acid is Arg, the 332nd amino acid is Thr, and the 396th amino acid is Leu or Met. 〔5〕 The Fc region variant according to any one of 〔1〕 to 〔4〕, where the value of 〔KD value of the polypeptide containing the Fc region without amino acid modification for FcγRIIb〕 / 〔KD value of the polypeptide containing the Fc region variant for FcγRIIb〕 is 50.0 or more. 〔6〕 The Fc region variant according to any one of 〔1〕 to 〔4〕, where the value of 〔KD value of the polypeptide containing the Fc region without amino acid modification for FcγRIIb〕 / 〔KD value of the polypeptide containing the Fc region variant for FcγRIIb〕 is 100.0 or more. 〔7〕 The Fc region variant according to any one of 〔1〕 to 〔6〕, where the value of 〔KD value of the polypeptide containing the Fc region variant for FcγRIIa (R type)〕 / 〔KD value of the polypeptide containing the Fc region variant for FcγRIIb〕 is 10.0 or more. 〔8〕 The Fc region variant according to any one of 〔1〕 to 〔6〕, where the value of 〔KD value of the polypeptide containing the Fc region variant for FcγRIIa (R type)〕 / 〔KD value of the polypeptide containing the Fc region variant for FcγRIIb〕 is 20.0 or more. 〔9〕 The Fc region variant according to any one of 〔1〕 to 〔8〕, where the Fc region variant contains the amino acid modification described in any one of the following (a) to (x). (a) Amino acid modifications at the 238th, 233rd, 237th, 268th, 271st, 296th, and 330th positions of the EU numbering in the Fc region (b) Amino acid modifications at positions 238, 237, 268, 271, 296, and 330 of the EU numbering in the Fc region (c) Amino acid modifications at positions 238, 233, 237, 268, 271, 296, 330, and 332 of the EU numbering in the Fc region (d) Amino acid modifications at positions 238, 233, 237, 264, 267, 268, 271, and 330 of the EU numbering in the Fc region (e) Amino acid modifications at positions 238, 233, 237, 267, 268, 271, 296, 330, and 332 of the EU numbering in the Fc region (f) Amino acid modifications at positions 238, 237, 267, 268, 271, 296, 330, and 332 of the EU numbering in the Fc region (g) Amino acid modifications at positions 238, 233, 237, 268, 271, 296, 327, and 330 of the EU numbering in the Fc region (h) Amino acid modifications at positions 238, 233, 237, 264, 267, 268, and 271 of the EU numbering in the Fc region (i) Amino acid modifications at positions 238, 233, 237, 264, 267, 268, 271, 296, and 330 of the EU numbering in the Fc region (j) Amino acid modifications at positions 238, 233, 237, 264, 267, 268, 271, 296, 330, and 396 of the EU numbering in the Fc region (k) Amino acid modifications at positions 238, 237, 264, 267, 268, 271, and 330 of the EU numbering in the Fc region (l) Amino acid modifications at positions 238, 237, 264, 267, 268, 271, 296, and 330 of the EU numbering in the Fc region (m) Amino acid modifications at positions 238, 264, 267, 268, and 271 of the EU numbering in the Fc region (n) Amino acid modifications at positions 238, 264, 267, 268, 271, and 296 of the EU numbering in the Fc region (o) Amino acid modifications at positions 238, 237, 267, 268, 271, 296, and 330 of the EU numbering in the Fc region (p) Amino acid modifications at positions 238, 233, 237, 264, 267, 268, 271, 330, and 396 of the EU numbering in the Fc region (q) Amino acid modifications at positions 238, 233, 237, 264, 267, 268, 271, 296, 327, 330, and 396 of the EU numbering in the Fc region (r) Amino acid modifications at positions 238, 233, 237, 264, 267, 268, 271, 272, and 296 of the EU numbering in the Fc region (s) Amino acid modifications at positions 238, 237, 264, 267, 268, 271, 272, and 330 of the EU numbering in the Fc region (t) Amino acid modifications at positions 238, 237, 264, 267, 268, 271, 272, 296, and 330 of the EU numbering in the Fc region (u) Amino acid modifications at positions 238, 233, 264, 267, 268, and 271 of the EU numbering in the Fc region (v) Amino acid modifications at positions 238, 237, 267, 268, 271, 296, and 330 of the EU numbering in the Fc region (w) Amino acid modifications at positions 238, 264, 267, 268, 271, 272, and 296 of the EU numbering in the Fc region (x) Amino acid modifications at positions 238, 233, 264, 267, 268, 271, and 296 of the EU numbering in the Fc region 〔10〕 The Fc region variant according to any one of 〔1〕 to 〔8〕, wherein the Fc region variant has the amino acid sequence described in any one of the following (a) to (x). (a) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 268th amino acid is Asp, the 271st amino acid is Gly, the 296th amino acid is Asp, and the 330th amino acid is Arg (b) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 237th amino acid is Asp, the 268th amino acid is Asp or Glu, the 271st amino acid is Gly, the 296th amino acid is Asp, and the 330th amino acid is Arg (c) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 268th amino acid is Asp, the 271st amino acid is Gly, the 296th amino acid is Asp, the 330th amino acid is Arg, and the 332nd amino acid is Thr (d) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Gly or Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, and the 330th amino acid is Arg (e) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 296th amino acid is Asp, the 330th amino acid is Arg, and the 332nd amino acid is Thr (f) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 237th amino acid is Asp, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 296th amino acid is Asp, the 330th amino acid is Arg, and the 332nd amino acid is Thr (g) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 268th amino acid is Asp, the 271st amino acid is Gly, the 296th amino acid is Asp, the 327th amino acid is Gly, and the 330th amino acid is Arg (h) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, and the 271st amino acid is Gly (i) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 296th amino acid is Asp, and the 330th amino acid is Arg (j) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 296th amino acid is Asp, the 330th amino acid is Arg, and the 396th amino acid is Met or Leu (k) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, and the 330th amino acid is Arg (l) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 296th amino acid is Asp, and the 330th amino acid is Arg (m) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, and the 271st amino acid is Gly (n) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, and the 296th amino acid is Asp (o) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 237th amino acid is Asp, the 267th amino acid is Ala or Gly, the 268th amino acid is Glu, the 271st amino acid is Gly, the 296th amino acid is Asp, and the 330th amino acid is Arg (p) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 330th amino acid is Arg, and the 396th amino acid is Met or Leu (q) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 296th amino acid is Asp, the 327th amino acid is Gly, the 330th amino acid is Arg, and the 396th amino acid is Met (r) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 272nd amino acid is Asp, and the 296th amino acid is Asp (s) An amino acid sequence in which the 238th amino acid of the EU numbering in the Fc region is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 272nd amino acid is Pro, and the 330th amino acid is Arg (t) An amino acid sequence in which the 238th amino acid of the EU numbering in the Fc region is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 272nd amino acid is Pro, the 296th amino acid is Asp, and the 330th amino acid is Arg (u) An amino acid sequence in which the 238th amino acid of the EU numbering in the Fc region is Asp, the 233rd amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, and the 271st amino acid is Gly (v) An amino acid sequence in which the 238th amino acid of the EU numbering in the Fc region is Asp, the 237th amino acid is Asp, the 267th amino acid is Gly, the 268th amino acid is Asp, the 271st amino acid is Gly, the 296th amino acid is Asp, and the 330th amino acid is Arg (w) An amino acid sequence in which the 238th amino acid of the EU numbering in the Fc region is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 272nd amino acid is Asp, and the 296th amino acid is Asp (x) An amino acid sequence in which the 238th amino acid of the EU numbering in the Fc region is Asp, the 233rd amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, and the 296th amino acid is Asp 〔11〕 An Fc region variant consisting of any amino acid sequence selected from SEQ ID NOs: 43 to 68, SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NOs: 75 to 77. A polypeptide comprising at least two Fc region variants according to any one of [1] to

[11] , wherein the two Fc region variants are associated with each other. 〔13〕 The polypeptide according to

[12] , wherein the two associated Fc region variants contained in the polypeptide have the same amino acid sequence. 〔14〕 The polypeptide according to

[12] , wherein the two associated Fc region variants contained in the polypeptide have different amino acid sequences. 〔15〕 The polypeptide according to

[14] , wherein the amino acid sequences of the two associated Fc region variants are such that at least one amino acid selected from the 235th, 236th, 237th, 238th, and 239th amino acids of the EU numbering of the Fc region variant is different. 〔16〕 The polypeptide according to

[15] , wherein the amino acid sequence of either one of the two associated Fc region variants has at least one amino acid selected from the group consisting of Asp, Gln, Glu, or Thr for the 235th amino acid, Asn for the 236th amino acid, Phe or Trp for the 237th amino acid, Glu, Gly, or Asn for the 238th amino acid, and Asp or Glu for the 239th amino acid. 〔17〕 The polypeptide according to any one of

[12] to

[16] , wherein the polypeptide containing the Fc region variant is an IgG antibody. 〔18〕 The polypeptide according to any one of

[12] to

[16] , wherein the polypeptide containing the Fc region variant is an Fc fusion protein molecule. 〔19〕 A pharmaceutical composition containing the polypeptide according to any one of

[12] to

[18] .

[0027] The present invention also relates to a method for enhancing the binding activity of the Fc region to FcγRIIb and enhancing the binding selectivity for FcγRIIb as compared to FcγRIIa (R type) by introducing an amino acid modification in the Fc region of the present invention. The present invention also relates to a method for suppressing the production of an antibody against a polypeptide containing the Fc region by introducing an amino acid modification in the Fc region of the present invention.

[0028] The present invention also relates to a therapeutic or prophylactic agent for an immune-inflammatory disease containing the polypeptide of the present invention. The present invention also relates to a method for treating or preventing an immune-inflammatory disease, which comprises a 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 or preventing an immune-inflammatory disease of the present invention, which contains the polypeptide of the present invention. The present invention also relates to the use of the polypeptide of the present invention in the manufacture of a therapeutic or prophylactic agent for an immune-inflammatory disease. The present invention also relates to the polypeptide of the present invention for use in the method for treating or preventing an immune-inflammatory disease of the present invention.

[0029] The present invention also relates to an inhibitor for activating B cells, mast cells, dendritic cells and / or basophils, which contains the 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 a 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 the activation of B cells, mast cells, dendritic cells and / or basophils of the present invention, which contains the polypeptide of the present invention. The present invention also relates to the use of the polypeptide of the present invention in the manufacture of an inhibitor for activating B cells, mast cells, dendritic cells and / or basophils. The present invention also relates to the polypeptide of the present invention for use in the method for inhibiting the activation of B cells, mast cells, dendritic cells and / or basophils of the present invention.

[0030] Furthermore, the present invention relates to a therapeutic agent for a disease lacking a protein necessary for a living body, which contains the polypeptide of the present invention. The present invention also relates to a method for treating a disease lacking a protein necessary for a living body, which includes the step of administering the polypeptide of the present invention to a subject. The present invention also relates to a kit for use in a method for treating a disease lacking a protein necessary for a living body, which contains the polypeptide of the present invention. The present invention also relates to the use of the polypeptide of the present invention in the manufacture of a therapeutic agent for a disease lacking a protein necessary for a living body. The present invention also relates to the polypeptide of the present invention for use in a method for treating a disease lacking a protein necessary for a living body.

[0031] Furthermore, the present invention relates to a virus growth inhibitor containing the polypeptide of the present invention. The present invention also relates to a method for inhibiting virus growth, which includes the step of administering the polypeptide of the present invention to a subject. The present invention also relates to a kit for use in a method for inhibiting virus growth, which contains the polypeptide of the present invention. The present invention also relates to the use of the polypeptide of the present invention in the manufacture of a virus growth inhibitor. The present invention also relates to the polypeptide of the present invention for use in a method for inhibiting virus growth.

Advantages of the Invention

[0032] The present invention provides an Fc region variant in which the binding activity to FcγRIIb is enhanced compared to the Fc region without amino acid modification, and / or the binding selectivity to FcγRIIb is enhanced compared to FcγRIIa (R type). By using a polypeptide containing the Fc region variant, it becomes possible to enhance the inhibitory signal of the inflammatory immune response via phosphorylation of the ITIM of FcγRIIb. In addition, by imparting the property of selectively binding to FcγRIIb to the Fc region, it may be possible to suppress the production of anti-drug antibodies. Further, by using the Fc region variant of the present invention for a polypeptide having a binding activity to human FcRn under conditions of an acidic pH range and having an antigen-binding domain in which the binding activity of the antigen-binding molecule to the antigen changes depending on the ionic concentration conditions, it is possible to promote the disappearance of the antigen bound by the polypeptide present in the plasma.

Brief Description of Drawings

[0033]

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Mode for Carrying Out the Invention

[0034] The present invention provides an Fc region variant in which the binding activity to FcγRIIb is enhanced and / or the binding selectivity to FcγRIIb is enhanced compared to FcγRIIa (R type), as compared to an Fc region into which no amino acid modification has been introduced, and a polypeptide containing the Fc region variant. More specifically, the present invention provides an Fc region variant comprising an amino acid sequence in which the 238th amino acid modification of EU numbering is combined with other specific amino acid modifications, and a polypeptide comprising the Fc region variant. Further, the present invention provides a method for enhancing the binding activity to FcγRIIb and / or enhancing the binding selectivity to FcγRIIb compared to FcγRIIa (R type) by introducing the amino acid modification into the Fc region as compared to the Fc region into which no amino acid modification has been introduced. The present invention also provides a method for suppressing the production of an antibody against the Fc region when administered to a living body by introducing the amino acid modification into the Fc region as compared to the Fc region into which no amino acid modification has been introduced.

[0035] The "polypeptide" in the present invention generally refers to a peptide and a protein having a length of about 10 amino acids or more. Further, it is generally a polypeptide derived from a living organism, but is not particularly limited, and may be, for example, a polypeptide consisting of an artificially designed sequence. Further, it may be any of a natural polypeptide, a synthetic polypeptide, a recombinant polypeptide, or the like. Preferred examples of the polypeptide of the present invention include antibodies. More preferred examples include native IgG, particularly native human IgG. Native IgG means a polypeptide that includes the same amino acid sequence as that found in nature and belongs to the class of antibodies substantially encoded by the immunoglobulin gamma gene. For example, native human IgG means native human IgG1, native human IgG2, native human IgG3, native human IgG4, and the like. Native IgG also includes naturally occurring variants and the like. The light chain constant regions of antibodies include IgK (Kappa, κ chain), IgL1, IgL2, IgL3, IgL6, IgL7 (Lambda, λ chain) type constant regions, and any of the light chain constant regions may be used. As the human IgK (Kappa) constant region and the human IgL7 (Lambda) constant region, multiple allotype sequences due to genetic polymorphisms are described in Sequences of proteins of immunological interest, NIH Publication No.91-3242, and any of them may be used in the present invention. 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, addition, deletion, insertion and / or modification, etc. As the Fc region of an antibody, for example, Fc regions of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, IgM types exist. The Fc region of the antibody of the present invention can use, for example, the Fc region of a human IgG antibody, preferably the Fc region of a human IgG1 antibody. As the Fc region of the present invention, for example, the constant region of natural IgG, specifically, the constant region derived from natural human IgG1 (SEQ ID NO: 11), the constant region derived from natural human IgG2 (SEQ ID NO: 12), the constant region derived from natural human IgG3 (SEQ ID NO: 13), the constant region derived from natural human IgG4 (SEQ ID NO: 14) can be used. Figure 21 shows the sequences of the constant regions of natural IgG1, IgG2, IgG3, IgG4. The constant region of natural IgG also includes naturally occurring variants and the like. As the constant regions of human IgG1, human IgG2, human IgG3, 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 them may be used in the present invention. In particular, as the sequence of human IgG1, the amino acid sequence at positions 356-358 of EU numbering may be DEL or EEM.

[0036] The Fcγ receptor (which may be referred to herein as the Fcγ receptor, FcγR or FcgR) refers to a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, IgG4 monoclonal antibodies, and means any member of the 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 undiscovered human FcγRs or FcγR isoforms or allotypes, but is not limited thereto. Also, FcγRIIb1 and FcγRIIb2 have been reported as splicing variants of human FcγRIIb. In addition, a splicing variant called FcγRIIb3 has also been reported (J. Exp. Med, 1989, 170: 1369). Human FcγRIIb includes all of its splicing variants, in addition to the splicing variants of NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1, NP_003992.3 registered in NCBI. Also, human FcγRIIb includes all existing reported gene polymorphisms, including FcγRIIb (Arthritis Rheum, 2003, 48: 3242-52, Hum Mol Genet, 2005, 14: 2881-92, Arthritis Rheum. 2002 May;46(5):1242-54.), and also includes any gene polymorphisms to be reported in the future. FcγRs include, but are not limited to, those derived from humans, mice, rats, rabbits, and monkeys, and may be from any organism. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRs or FcγR isoforms or allotypes. Preferred examples of such Fcγ receptors include human FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16), and / or FcγRIIIB (CD16).

[0037] The polynucleotide sequence and amino acid sequence of FcγRI are shown in SEQ ID NO: 1 (NM_000566.3) and SEQ ID NO: 2 (NP_000557.1), respectively, The polynucleotide sequence and amino acid sequence of FcγRIIA are shown in SEQ ID NO: 3 (BC020823.1) and SEQ ID NO: 4 (AAH20823.1), respectively, The polynucleotide sequence and amino acid sequence of FcγRIIB are shown in SEQ ID NO: 5 (BC146678.1) and SEQ ID NO: 6 (AAI46679.1), respectively, The polynucleotide sequence and amino acid sequence of FcγRIIIA are shown in SEQ ID NO: 7 (BC033678.1) and SEQ ID NO: 8 (AAH33678.1), respectively, and The polynucleotide sequence and amino acid sequence of FcγRIIIB are described in SEQ ID NO: 9 (BC128562.1) and SEQ ID NO: 10 (AAI28563.1), respectively (the numbers in parentheses indicate the RefSeq accession numbers).

[0038] In addition, there are two 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).

[0039] As used herein, the term "Fc region without amino acid modification" refers to the Fc region before the introduction of the amino acid modifications of the present invention. The "Fc region without amino acid modification" in the present invention may be, for example, the Fc region of native IgG, or the Fc region of IgG in which modifications other than the amino acid modifications of the present invention have been made to native IgG. In the present invention, the "Fc region variant" means an Fc region in which at least one amino acid of the present invention has been modified to another amino acid in the Fc region without the amino acid modification of the present invention. Here, the phrase "at least one amino acid has been modified to another amino acid" includes the Fc region into which the amino acid modification has been introduced and the Fc region consisting of the same amino acid sequence as that thereof.

[0040] Native IgG refers to a polypeptide belonging to the class of antibodies substantially encoded by the immunoglobulin gamma gene, including the same amino acid sequence as that of IgG found in nature. 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 variants thereof.

[0041] The Fc region of native IgG refers to an Fc region including the same amino acid sequence as the Fc region originating from IgG found in nature. The heavy chain constant region of native IgG is shown in Figure 21 (SEQ ID NOs: 11-14). For example, it means the Fc region in the heavy chain constant region originating from native human IgG1 in Figure 21, the Fc region in the heavy chain constant region originating from native human IgG2, the Fc region in the heavy chain constant region originating from native human IgG3, and the Fc region in the heavy chain constant region originating from native human IgG4. The Fc region of native IgG also includes naturally occurring variants thereof.

[0042] In the present invention, whether the binding activity of the polypeptide or Fc region variant containing the Fc region variant of the present invention to various FcγRs is enhanced, or the binding activity is maintained or decreased can be determined, for example, as shown in this example, by using BIACORE, an interaction analysis instrument utilizing the surface plasmon resonance (SPR) phenomenon. The dissociation constant (KD) value obtained from the analysis results of the sensorgram in which various FcγRs are allowed to interact with a sensor chip on which an antibody is immobilized or a sensor chip captured with ProteinA, ProteinL, ProteinA / G, ProteinG, anti-lambda chain antibody, anti-kappa chain antibody, antigen peptide, antigen protein, etc. as the analyte. It can also be determined whether the value has decreased or increased. Alternatively, for the antibody on the sensor chip immobilized on the sensor chip or captured with ProteinA, ProteinL, ProteinA / G, ProteinG, anti-lambda chain antibody, anti-kappa chain antibody, antigen peptide, antigen protein, etc., it can also be determined whether the value obtained by dividing the change amount of the resonance unit (RU) value on the sensorgram before and after allowing various FcγRs to interact as the analyte by the change amount of the resonance unit (RU) before and after immobilizing or capturing the antibody on the sensor chip has decreased or increased. Further, it can be determined whether the dissociation constant (KD) value obtained from the analysis of the sensorgram in which a sample such as an antibody to be evaluated is allowed to interact as the analyte using a sensor chip on which FcγR is directly immobilized or immobilized via an anti-tag antibody, etc. has decreased or increased. Alternatively, it can also be determined whether the change amount of the sensorgram value before and after allowing a sample such as an antibody to be evaluated to interact as the analyte with a sensor chip on which FcγR is directly immobilized or immobilized via an anti-tag antibody, etc. has decreased or increased.

[0043] Specifically, the binding activity of the Fc region variant to the Fcγ receptor can be measured by, in addition to ELISA and FACS (fluorescence activated cell sorting), the ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) and the BIACORE method using the surface plasmon resonance (SPR) phenomenon (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0044] The ALPHA screen is carried out based on the following principle by the ALPHA technology using two beads, a donor bead and an acceptor bead. When a molecule bound to the donor bead biologically interacts with a molecule bound to the acceptor bead, a luminescence signal is detected only when the two beads are in proximity. The photosensitizer in the donor bead excited by a laser converts the surrounding oxygen into singlet oxygen in an excited state. The singlet oxygen diffuses around the donor bead and, when it reaches the nearby acceptor bead, causes a chemiluminescence reaction in the bead, and finally light is emitted. When the molecule bound to the donor bead and the molecule bound to the acceptor bead do not interact, the singlet oxygen produced by the donor bead does not reach the acceptor bead, so the chemiluminescence reaction does not occur.

[0045] For example, a polypeptide conjugate labeled with biotin is bound to donor beads, and an Fcγ receptor tagged with glutathione S-transferase (GST) is bound to acceptor beads. In the absence of a polypeptide conjugate containing a competing Fc region variant, the polypeptide conjugate containing the wild-type Fc region and the Fcγ receptor interact to produce a signal at 520 - 620 nm. A polypeptide conjugate containing a mutated Fc region without a tag competes with the interaction between the polypeptide conjugate containing the wild-type Fc region and the Fcγ receptor. The relative binding activity can be determined by quantifying the decrease in fluorescence resulting from the competition. Biotinylation of a polypeptide conjugate such as an antibody using Sulfo-NHS-biotin or the like is known. As a method for tagging the Fcγ receptor with GST, a fusion gene in which a polynucleotide encoding the Fcγ receptor and a polynucleotide encoding GST are fused in-frame is expressed in cells or the like held in a vector capable of expressing the fusion gene, and a method of purifying using a glutathione column or the like can be appropriately employed. The obtained signal is preferably analyzed by fitting it to a one-site competition model using non-linear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).

[0046] One of the substances to observe the interaction (ligand) is immobilized on the gold thin film of the sensor chip. When light is applied from the back side of the sensor chip so as to totally reflect at the interface between the gold thin film and the glass, a part of the reflected light with a reduced reflection intensity (SPR signal) is formed. When the other substance to observe the interaction (analyte) is flowed onto the surface of the sensor chip and the ligand and the analyte bind to each other, the mass of the immobilized ligand molecules increases, and the refractive index of the solvent on the surface of the sensor chip changes. Due to this change in the refractive index, the position of the SPR signal shifts (conversely, when the binding dissociates, the position of the signal returns). The Biacore system takes the amount of the above shift, that is, the mass change on the surface of the sensor chip, on the vertical axis and displays the time change of the mass as measurement data (sensorgram). The binding amount of the analyte to the ligand captured on the surface of the sensor chip can be determined from the sensorgram. Also, the kinetics: association rate constant (ka) and dissociation rate constant (kd) can be determined from the curve of the sensorgram, and the dissociation constant (KD) can be determined from the ratio of the constants. The inhibition measurement method is also preferably used in the BIACORE method. Examples of the inhibition measurement method are described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005 - 4010.

[0047] The Fc region or the polypeptide containing the Fc region with reduced binding activity to FcγR refers to a polypeptide containing an Fc region into which no amino acid modification has been introduced (also referred to as a polypeptide containing the parental Fc region or a parental polypeptide) and a polypeptide containing at least one amino acid modification in the Fc region (also referred to as a polypeptide containing an Fc region variant or a modified polypeptide). When an assay is performed with the amounts of the two polypeptides being essentially the same, it refers to an Fc region variant or a polypeptide containing the Fc region variant that binds to FcγR with essentially weaker binding activity than the polypeptide containing the parental Fc region.

[0048] The Fc region or polypeptide containing the Fc region with enhanced binding activity to FcγR refers to an Fc region variant or a polypeptide containing the Fc region variant that binds to FcγR with essentially stronger binding activity than the polypeptide containing the parental Fc region when an assay is performed with the amount of the polypeptide containing the parental Fc region being essentially the same as the amount of the polypeptide containing the Fc region variant.

[0049] The polypeptide with maintained binding activity to FcγR refers to a polypeptide that binds to FcγR with equivalent binding activity that is essentially unchanged from the parental polypeptide when an assay is performed with the amount of the polypeptide having the parental Fc region being essentially the same as the amount of the polypeptide containing the Fc region variant.

[0050] In the present invention, when it is said that the binding activity to FcγRIIb is enhanced, for example, in the KD value measured by the above measurement method, the KD value ratio of [KD value of the polypeptide containing the parental Fc region to FcγRIIb] / [KD value of the polypeptide containing the Fc region variant to FcγRIIb] is preferably 15.0 or more, 20.0 or more, 25.0 or more, 30.0 or more, 35.0 or more, 40.0 or more, 45.0 or more, further 50.0 or more, 55.0 or more, 60.0 or more, 65.0 or more, 70.0 or more, 75.0 or more, 80.0 or more, 85.0 or more, 90.0 or more, 95.0 or more, 100.0 or more.

[0051] Also, when it is said that the Fc region variant of the present invention has enhanced binding selectivity for FcγRIIb over FcγRIIa, (i) the binding activity to FcγRIIb is enhanced and the binding activity to FcγRIIa is maintained or decreased, (ii) the binding activity to FcγRIIb is enhanced and the binding activity to FcγRIIa is also enhanced, but the degree of enhancement of the binding activity to FcγRIIa is lower than the degree of enhancement of the binding activity to FcγRIIb, or (iii) the binding activity to FcγRIIb is decreased, but the degree of decrease of the binding activity is lower than the degree of decrease of the binding activity to FcγRIIa. This means that whether the Fc region variant of the present invention has enhanced binding selectivity for FcγRIIb compared to FcγRIIa can be determined, for example, by comparing the ratio of the KD value for FcγRIIa to the KD value for FcγRIIb (KD value for FcγRIIa / KD value for FcγRIIb) of the polypeptide containing the Fc region variant of the present invention, obtained according to the above example, with the ratio of the KD value for FcγRIIa to the KD value for FcγRIIb (KD value for FcγRIIa / KD value for FcγRIIb) of the polypeptide containing the parental Fc region. Specifically, when the value of the above KD value ratio is larger in the polypeptide containing the Fc region variant of the present invention than in the polypeptide containing the parental Fc region, it can be determined that the polypeptide containing the Fc region variant of the present invention has enhanced binding selectivity for FcγRIIb compared to FcγRIIa. In particular, since the binding activity for FcγRIIa (R type) is more likely to be correlated with the binding activity for FcγRIIb than that for FcγRIIa (H type), finding an amino acid modification that can enhance the binding selectivity for FcγRIIb compared to FcγRIIa (R type) is important for enhancing the binding selectivity for FcγRIIb compared to other FcγRs other than FcγRIIb. As the binding selectivity between FcγRIIa (R type) and FcγRIIb, for example, in the KD value measured by the above measurement method, the KD value ratio of [KD value for FcγRIIa (R type) of the polypeptide containing the Fc region variant] / [KD value for FcγRIIb of the polypeptide containing the Fc region variant] is preferably 10.0 or more, more preferably 20.0 or more. As the binding selectivity between FcγRIIa (H type) and FcγRIIb, for example, in the KD value measured by the above measurement method, the KD value ratio of [KD value for FcγRIIa (H type) of the polypeptide containing the Fc region variant] / [KD value for FcγRIIb of the polypeptide containing the Fc region variant] is preferably 100.0 or more, 200 or more, 300 or more, 400 or more, 500 or more, more preferably 600 or more, 700 or more, 800 or more, 900 or more.

[0052] Whether the binding activity of the polypeptide of the present invention to various FcγRs is maintained, enhanced, or decreased can also be determined by the increase or decrease in the amount of binding of various FcγRs to the polypeptide of the present invention as determined according to the above examples. Here, the amount of binding of various FcγRs to the polypeptide means a value obtained by dividing the difference in RU values in the sensorgram that changes before and after interacting various FcγRs, which are analytes, with each polypeptide, by the difference in RU values in the sensorgram that changes before and after capturing the polypeptide on the sensor chip.

[0053] In addition, as the Fc region variant of the present invention, there is no particular limitation on the KD value (mol / L) for FcγRIIb. For example, it may be -7 9×10 or less, preferably -7 5×10 or less, more preferably -7 3×10 or less, even more preferably -7 1×10 or less, even more preferably -8 5×10 or less.

[0054] The "Fc region" refers to a fragment consisting of the hinge region or a part thereof, CH2, and CH3 domains in an antibody molecule. The Fc region of the IgG class, in EU numbering (also referred to as EU INDEX in this specification) (see Figure 21), means, for example, from the 226th cysteine to the C-terminus, or from the 230th proline to the C-terminus, but is not limited thereto.

[0055] The Fc region can be preferably obtained by subjecting IgG1, IgG2, IgG3, IgG4 monoclonal antibodies, etc. to partial digestion with a proteolytic enzyme such as pepsin, and then re-eluting the fraction adsorbed on a protein A column. Such a proteolytic enzyme is not particularly limited as long as it can digest the full-length antibody to produce Fab or F(ab')2 in a limited manner by appropriately setting the reaction conditions of the enzyme such as pH. Examples include pepsin and papain.

[0056] The present invention provides an Fc region variant comprising a modification in the Fc region of human IgG (IgG1, IgG2, IgG3, IgG4), which is a modification of the amino acid at position 238 according to EU numbering to another amino acid, and a modification of at least one amino acid selected from the amino acids at positions 233, 234, 235, 237, 264, 265, 266, 267, 268, 269, 271, 272, 274, 296, 326, 327, 330, 331, 332, 333, 334, 355, 356, 358, 396, 409, and 419 according to EU numbering to another amino acid. By combining a modification of the amino acid at position 238 according to EU numbering to another amino acid in the Fc region of human IgG and a modification of at least one amino acid selected from the amino acids at positions 233, 234, 237, 264, 265, 266, 267, 268, 269, 271, 272, 274, 296, 326, 327, 330, 331, 332, 333, 334, 355, 356, 358, 396, 409, and 419 according to EU numbering to another amino acid, compared with a polypeptide comprising an Fc region into which no such amino acid modification has been introduced, the binding activity to FcγRIIb is enhanced and / or the binding selectivity to FcγRIIb, particularly the selectivity to FcγRIIa (R type), is enhanced compared with FcγRIIa. It is possible to provide a polypeptide comprising an Fc region variant with such enhanced properties.Other amino acid modifications combined with the 238th amino acid modification according to the EU numbering include the 233rd, 237th, 264th, 267th, 268th, 271st, 272nd, 296th, 327th, 330th, 332nd, 333rd, and 396th amino acids according to the EU numbering, and particularly, the 233rd, 237th, 264th, 267th, 268th, 271st, 296th, 330th, and 396th amino acids according to the EU numbering are preferred. In particular, a modification combining the 238th, 268th, and 271st amino acids according to the EU numbering with at least one amino acid selected from the 233rd, 237th, 264th, 267th, 272nd, 296th, 327th, 330th, 332nd, and 396th amino acids according to the EU numbering is mentioned as a preferred combination of amino acid modifications from the viewpoint of enhancing the binding activity to FcγRIIb or enhancing the binding selectivity to FcγRIIb compared to FcγRIIa.

[0057] The amino acids to be modified are not particularly limited as long as their binding activity to FcγRIIb is enhanced compared to that before modification, or their binding selectivity for FcγRIIb is enhanced compared to that of FcγRIIa. However, it is preferable that the 238th amino acid according to the EU numbering is Asp, the 233rd amino acid is Asp, the 234th amino acid is Tyr, the 235th amino acid is Phe, the 237th amino acid is Asp, the 264th amino acid is Ile, the 265th amino acid is Glu, the 266th amino acid is Phe, Leu or Met, the 267th amino acid is Ala, Glu, Gly or Gln, the 268th amino acid is Asp, Gln or Glu, the 269th amino acid is Asp, the 271st amino acid is Gly, the 272nd amino acid is Asp, Phe, Ile, Met, Asn, Pro or Gln, the 274th amino acid is Gln, the 296th amino acid is Asp or Phe, the 326th amino acid is Ala or Asp, the 327th amino acid is Gly, the 330th amino acid is Lys, Arg or Ser, the 331st amino acid is Ser, the 332nd amino acid is Lys, Arg, Ser or Thr, the 333rd amino acid is Lys, Arg, Ser or Thr, the 334th amino acid is Arg, Ser or Thr, the 355th amino acid is Ala, Gln, the 356th amino acid is Glu, the 358th amino acid is Met, the 396th amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr, the 409th amino acid is Arg, and the 419th amino acid is Glu.In particular, when combining the 238th amino acid, 268th amino acid, and 271st amino acid according to EU numbering with at least one amino acid selected from the 233rd amino acid, 264th amino acid, 267th amino acid, 272nd amino acid, 296th amino acid, 327th amino acid, 330th amino acid, 332nd amino acid, and 396th amino acid according to EU numbering, it is preferable that the 238th amino acid according to EU numbering is Asp, the 268th amino acid is Asp or Glu, the 271st amino acid is Gly, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala or Gly, the 272nd amino acid is Asp or Pro, the 296th amino acid is Asp, the 327th amino acid is Gly, the 330th amino acid is Arg, the 332nd amino acid is Thr, and the 396th amino acid is Leu or Met.

[0058] In addition to these modifications, the present invention can further add at least one other modification to the Fc region. There is no particular limitation as long as it enhances the binding activity to FcγRIIb and / or enhances the binding selectivity to FcγRIIb rather than FcγRIIa. The modification can also be carried out in combination with a modification that replaces a part of the Fc region with the corresponding site of another Fc region having a different isotype. For example, by combining the substitution of the amino acid sequence from the 118th Ala to the 225th Thr according to EU numbering of the Fc region derived from IgG1 with the amino acid sequence from the 118th Ala to the 222nd Pro according to EU numbering of the Fc region derived from IgG4 with the above-mentioned amino acid modification, it is also possible to enhance the binding activity to FcγRIIb and / or the binding selectivity to FcγRIIb. Specifically, for example, like IL6R - BP478 / IL6R - L described in Example 7, a combination of the amino acid modification introduced by IL6R - BP230 and the modification of substituting the amino acid sequence from the 118th Ala to the 225th Thr according to EU numbering of G1d with the amino acid sequence from the 118th Ala to the 222nd Pro according to EU numbering of G4d can be mentioned.

[0059] Among these, modifications that enhance the binding activity to FcγRIIb, or modifications that enhance the binding selectivity to FcγRIIb over FcγRIIa (R type) are preferred. Examples of such preferred combinations of amino acid modifications include the following combinations (a) to (x). (a) Amino acid modifications at positions 238, 233, 237, 268, 271, 296, and 330 in the EU numbering of the Fc region (b) Amino acid modifications at positions 238, 237, 268, 271, 296, and 330 in the EU numbering of the Fc region (c) Amino acid modifications at positions 238, 233, 237, 268, 271, 296, 330, and 332 in the EU numbering of the Fc region (d) Amino acid modifications at positions 238, 233, 237, 264, 267, 268, 271, and 330 in the EU numbering of the Fc region (e) Amino acid modifications at positions 238, 233, 237, 267, 268, 271, 296, 330, and 332 in the EU numbering of the Fc region (f) Amino acid modifications at positions 238, 237, 267, 268, 271, 296, 330, and 332 in the EU numbering of the Fc region (g) Amino acid modifications at positions 238, 233, 237, 268, 271, 296, 327, and 330 in the EU numbering of the Fc region (h) Amino acid modifications at positions 238, 233, 237, 264, 267, 268, and 271 in the EU numbering of the Fc region (i) Amino acid modifications at positions 238, 233, 237, 264, 267, 268, 271, 296, and 330 in the EU numbering of the Fc region (j) Amino acid modifications at positions 238, 233, 237, 264, 267, 268, 271, 296, 330, and 396 in the EU numbering of the Fc region (k) Amino acid modifications at positions 238, 237, 264, 267, 268, 271, and 330 in the EU numbering of the Fc region (l) Amino acid modifications at positions 238, 237, 264, 267, 268, 271, 296, and 330 in the EU numbering of the Fc region (m) Amino acid modifications at positions 238, 264, 267, 268, and 271 in the EU numbering of the Fc region (n) Amino acid modifications at positions 238, 264, 267, 268, 271, and 296 in the EU numbering of the Fc region (o) Amino acid modifications at positions 238, 237, 267, 268, 271, 296, and 330 in the EU numbering of the Fc region (p) Amino acid modifications at positions 238, 233, 237, 264, 267, 268, 271, 330, and 396 in the EU numbering of the Fc region (q) Amino acid modifications at positions 238, 233, 237, 264, 267, 268, 271, 296, 327, 330, and 396 in the EU numbering of the Fc region (r) Amino acid modifications at positions 238, 233, 237, 264, 267, 268, 271, 272, and 296 in the EU numbering of the Fc region (s) Amino acid modifications at positions 238, 237, 264, 267, 268, 271, 272, and 330 in the EU numbering of the Fc region (t) Amino acid modifications at positions 238, 237, 264, 267, 268, 271, 272, 296, and 330 in the EU numbering of the Fc region (u) Amino acid modifications at positions 238, 233, 264, 267, 268, and 271 in the EU numbering of the Fc region (v) Amino acid modifications at positions 238, 237, 267, 268, 271, 296, and 330 of the EU numbering in the Fc region (w) Amino acid modifications at positions 238, 264, 267, 268, 271, 272, and 296 of the EU numbering in the Fc region (x) Amino acid modifications at positions 238, 233, 264, 267, 268, 271, and 296 of the EU numbering in the Fc region

[0060] Furthermore, among these combinations of modifications, the following combinations of amino acid modifications (a) to (x) are listed as more preferred combinations. (a) An amino acid sequence in which the amino acid at position 238 of the EU numbering in the Fc region is Asp, the amino acid at position 233 is Asp, the amino acid at position 237 is Asp, the amino acid at position 268 is Asp, the amino acid at position 271 is Gly, the amino acid at position 296 is Asp, and the amino acid at position 330 is Arg (b) An amino acid sequence in which the amino acid at position 238 of the EU numbering in the Fc region is Asp, the amino acid at position 237 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 296 is Asp, and the amino acid at position 330 is Arg (c) An amino acid sequence in which the amino acid at position 238 of the EU numbering in the Fc region is Asp, the amino acid at position 233 is Asp, the amino acid at position 237 is Asp, the amino acid at position 268 is Asp, the amino acid at position 271 is Gly, the amino acid at position 296 is Asp, the amino acid at position 330 is Arg, and the amino acid at position 332 is Thr (d) An amino acid sequence in which the amino acid at position 238 of the EU numbering in the Fc region is Asp, 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 Gly or Ala, the amino acid at position 268 is Glu, the amino acid at position 271 is Gly, and the amino acid at position 330 is Arg (e) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 296th amino acid is Asp, the 330th amino acid is Arg, and the 332nd amino acid is Thr (f) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 237th amino acid is Asp, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 296th amino acid is Asp, the 330th amino acid is Arg, and the 332nd amino acid is Thr (g) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 268th amino acid is Asp, the 271st amino acid is Gly, the 296th amino acid is Asp, the 327th amino acid is Gly, and the 330th amino acid is Arg (h) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, and the 271st amino acid is Gly (i) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 296th amino acid is Asp, and the 330th amino acid is Arg (j) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 296th amino acid is Asp, the 330th amino acid is Arg, and the 396th amino acid is Met or Leu (k) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, and the 330th amino acid is Arg (l) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 296th amino acid is Asp, and the 330th amino acid is Arg (m) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, and the 271st amino acid is Gly (n) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, and the 296th amino acid is Asp (o) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 237th amino acid is Asp, the 267th amino acid is Ala or Gly, the 268th amino acid is Glu, the 271st amino acid is Gly, the 296th amino acid is Asp, and the 330th amino acid is Arg (p) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 330th amino acid is Arg, and the 396th amino acid is Met or Leu (q) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 296th amino acid is Asp, the 327th amino acid is Gly, the 330th amino acid is Arg, and the 396th amino acid is Met (r) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 272nd amino acid is Asp, and the 296th amino acid is Asp (s) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 272nd amino acid is Pro, and the 330th amino acid is Arg (t) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 272nd amino acid is Pro, the 296th amino acid is Asp, and the 330th amino acid is Arg (u) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, and the 271st amino acid is Gly (v) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 237th amino acid is Asp, the 267th amino acid is Gly, the 268th amino acid is Asp, the 271st amino acid is Gly, the 296th amino acid is Asp, and the 330th amino acid is Arg (w) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 272nd amino acid is Asp, and the 296th amino acid is Asp (x) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, and the 296th amino acid is Asp

[0061] In addition, amino acid modifications carried out for other purposes can also be combined with the polypeptide containing the Fc region variant of the present invention. For example, amino acid substitutions that improve the binding activity to FcRn (J Immunol. 2006 Jan 1;176(1):346-56, J Biol Chem. 2006 Aug 18;281(33):23514-24., Int Immunol. 2006 Dec;18(12):1759-69., Nat Biotechnol. 2010 Feb;28(2):157-9., WO / 2006 / 019447, WO / 2006 / 053301, WO / 2009 / 086320), amino acid substitutions for improving antibody heterogeneity and stability (WO / 2009 / 041613) may be added. Alternatively, a polypeptide having a property for promoting antigen disappearance described in WO2011 / 122011, PCT / JP2011 / 072550, or a polypeptide having a property for repeatedly binding to antigens of multiple molecules described in WO2009 / 125825, WO2012 / 073992, WO2013 / 047752 are also included in the present invention. Alternatively, an amino acid modification (WO / 2012 / 016227) that lowers the pI of the constant region may be combined with the polypeptide containing the Fc region variant of the present invention for the purpose of enhancing blood retention. Alternatively, amino acid modifications described in EP1752471, EP1772465 may be combined with the polypeptide containing the Fc region variant of the present invention for the purpose of conferring binding ability to other antigens when used.

[0062] When the polypeptide containing the Fc region variant of the present invention is an antigen-binding molecule such as an antibody, in order to enhance the antigen disappearance effect from the plasma of the antigen-binding molecule, amino acid modifications can be combined to enhance the human FcRn binding activity under conditions in the acidic pH range. More specifically, for example, as modifications used to enhance the human FcRn binding activity under conditions in the acidic pH range, methods include substituting Met at position 428 with Leu and Asn at position 434 with Ser, represented by the EU numbering of IgG antibodies (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), substituting Thr at position 250 with Gln and Met at position 428 with Leu (J Immunol. 2006, 176(1):346-56), substituting Asn at position 434 with His (Clinical Pharmacology & Therapeutics (2011) 89(2):283-290.), and it is also considered possible to carry out by using modifications as described in WO2010106180, WO2010045193, WO2009058492, WO2008022152, WO2006050166, WO2006053301, WO2006031370, WO2005123780, WO2005047327, WO2005037867, WO2004035752, WO2002060919, etc.

[0063] In recent years, in order to enhance the binding activity to human FcRn under conditions of an acidic pH range and improve the retention in plasma for humanized anti-CD4 antibodies, an antibody molecule in which Asn at position 434 represented by EU numbering is substituted with His has been reported to bind to rheumatoid factor (RF) (Clin Pharmacol Ther. 2011 Feb;89(2):283-90). Although this antibody has the Fc region of human IgG1, it has been shown that by substituting Asn at position 434 located in the binding site for FcRn with His, rheumatoid factor that recognizes the substitution site binds.

[0064] As described above, various modifications have been reported as modifications for enhancing the binding activity to human FcRn under conditions of an acidic pH range. However, by introducing these modifications into the FcRn binding site in the Fc region, there is a possibility of enhancing the binding to rheumatoid factor that recognizes the site. However, by introducing a modification that only reduces the binding activity to rheumatoid factor without reducing the binding activity to FcRn in the said site of the Fc region, it is possible to produce an antigen-binding molecule that does not have binding to rheumatoid factor and enhances the binding activity to human FcRn under conditions of an acidic pH range.

[0065] As modifications for reducing the binding activity to rheumatoid factor, modifications at positions 248-257, 305-314, 342-352, 380-386, 388, 414-421, 423, 425-437, 439, 441-444 represented by EU numbering are used. Preferably, modifications at positions 387, 422, 424, 426, 433, 436, 438, 440 are preferably used. Particularly preferably, modifications including substitution of Val at position 422 with Glu or Ser, substitution of Ser at position 424 with Arg, substitution of His at position 433 with Asp, substitution of Tyr at position 436 with Thr, substitution of Gln at position 438 with Arg or Lys, and substitution of Ser at position 440 with Glu or Asp are used. These modifications may be used alone or in combination at multiple sites.

[0066] Alternatively, in order to reduce the binding activity to rheumatoid factor, an N-glycan addition sequence may be introduced into the site. Specifically, Asn-Xxx-Ser / Thr (Xxx is any amino acid except Pro) is known as an N-glycan addition sequence. By introducing this sequence into the site in the Fc region, an N-glycan can be added, and it is possible to inhibit the binding to RF due to the steric hindrance of the N-glycan. As modifications for adding an N-glycan, preferably, modifications including substitution of Lys at position 248 with Asn, substitution of Ser at position 424 with Asn, substitution of Tyr at position 436 with Asn and substitution of Gln at position 438 with Thr, and substitution of Gln at position 438 with Asn are used. Particularly preferably, the modification of substituting Ser at position 424 with Asn is used.

[0067] As a preferred example of the polypeptide containing the Fc region variant of the present invention, a polypeptide containing at least two Fc region variants, such as an IgG antibody, in which the two Fc region variants are associated, can be mentioned. When an IgG antibody is used as the polypeptide of the present invention, the type of its constant region is not limited, and IgG of isotypes (subclasses) such as IgG1, IgG2, IgG3, and IgG4 can be used. The IgG antibody of the present invention is preferably human IgG, more preferably human IgG1 and human IgG4, and the amino acid sequences of the heavy chain constant regions of human IgG1 and human IgG4 are known. As the human IgG1 constant region, multiple allotype sequences due to genetic polymorphisms are described in Sequences of proteins of immunological interest, NIH Publication No.91-3242, and any of them may be used in the present invention.

[0068] The two associated Fc region variants contained in the polypeptide may be Fc region variants into which the same amino acid modification has been introduced (hereinafter referred to as a polypeptide containing a homomeric Fc region variant), or may be Fc region variants consisting of different amino acid sequences into which different amino acid modifications have been introduced, or may be Fc region variants consisting of different amino acid sequences into which an amino acid modification has been introduced only into one of the Fc regions (hereinafter referred to as a polypeptide containing a heteromeric Fc region variant). As the amino acid modification introduced only into one of the Fc regions, the loop structure site at positions 233 to 239 of the CH2 domain of the Fc region involved in binding to FcγRIIb and FcγRIIa is preferred. A modification is introduced into the loop structure of the CH2 region of one Fc region to enhance the binding activity to FcγRIIb and / or enhance the binding selectivity to FcγRIIb compared to FcγRIIa (R type), and an amino acid modification is preferably introduced into the loop structure of the CH2 region of the other Fc region to destabilize it. As the amino acid modification for destabilizing the loop structure of the CH2 region, for example, it is possible to destabilize the loop structure by substituting at least one amino acid selected from the amino acids at positions 235, 236, 237, 238, and 239 of the EU numbering with another amino acid. Specifically, for example, the loop structure of the CH2 region can be destabilized by modifying the amino acid at position 235 of the EU numbering to Asp, Gln, Glu, or Thr, modifying the amino acid at position 236 to Asn, modifying the amino acid at position 237 to Phe or Trp, modifying the amino acid at position 238 to Glu, Gly, or Asn, and modifying the amino acid at position 239 to Asp or Glu.

[0069] To produce the polypeptide containing the heteromeric Fc region variant of the present invention, it is necessary to associate Fc region variants having different amino acids with each other, or to separate the polypeptide containing the target heteromeric Fc region variant from the polypeptide containing other homomeric Fc region variants.

[0070] For the association of polypeptides having different amino acids, a technique can be applied that introduces a charge repulsion at the interface of the second constant region (CH2) of the antibody heavy chain or the third constant region (CH3) of the heavy chain to suppress the association of unwanted heavy chains (WO2006 / 106905).

[0071] In the technique of introducing a charge repulsion at the interface of CH2 or CH3 to suppress the unintended association of heavy chains, examples of the amino acid residues that contact at the interface of other constant regions of the heavy chain include regions corresponding to the 356th residue, 439th residue, 357th residue, 370th residue, 399th residue, and 409th residue in the EU numbering in the CH3 region.

[0072] More specifically, for example, in an antibody containing two heavy chain CH3 regions, one to three sets of amino acid residues selected from the following sets of amino acid residues (1) to (3) in the first heavy chain CH3 region can be made into an antibody in which the amino acid residues have the same charge; (1) Amino acid residues contained in the heavy chain CH3 region, which are the 356th and 439th amino acid residues in the EU numbering, (2) Amino acid residues contained in the heavy chain CH3 region, which are the 357th and 370th amino acid residues in the EU numbering, (3) Amino acid residues contained in the heavy chain CH3 region, which are the 399th and 409th amino acid residues in the EU numbering.

[0073] Furthermore, a set of amino acid residues selected from the sets of amino acid residues (1) to (3) shown above in the second heavy chain CH3 region different from the first heavy chain CH3 region, and one to three sets of amino acid residues corresponding to the sets of amino acid residues (1) to (3) having the same charge in the first heavy chain CH3 region can be made into an antibody in which the amino acid residues have a charge opposite to that of the corresponding amino acid residues in the first heavy chain CH3 region.

[0074] Each of the amino acid residues described in the above (1) to (3) is close to each other when they associate. A person skilled in the art can find the sites corresponding to the amino acid residues described in the above (1) to (3) for a desired H-chain CH3 region or H-chain constant region by homology modeling or the like using commercially available software, and can appropriately subject the amino acid residues at these sites to modification.

[0075] In the above antibody, the "amino acid residue having a charge" is preferably selected from amino acid residues included in any one of the following groups (X) or (Y); (X) Glutamic acid (E), Aspartic acid (D), (Y) Lysine (K), Arginine (R), Histidine (H).

[0076] In the above antibody, "having the same charge" means, for example, that any of two or more amino acid residues has an amino acid residue included in any one of the above groups (X) or (Y). "Having opposite charges" means, for example, when at least one of two or more amino acid residues has an amino acid residue included in any one of the above groups (X) or (Y), the remaining amino acid residues have amino acid residues included in different groups.

[0077] In a preferred embodiment, in the above antibody, the first H-chain CH3 region and the second H-chain CH3 region may be crosslinked by a disulfide bond.

[0078] In the present invention, the amino acid residues to be subjected to modification are not limited to the amino acid residues in the variable region or constant region of the above-described antibody. A person skilled in the art can find the amino acid residues forming an interface for a polypeptide variant or heteromultimer by homology modeling or the like using commercially available software, and can subject the amino acid residues at these sites to modification so as to control the association.

[0079] Other known techniques can also be used for the association of the heterologous Fc region variants of the present invention. By substituting the amino acid side chains present in the variable region of one H chain of the antibody with larger side chains (knobs; protrusions), and substituting the amino acid side chains present in the corresponding variable region of the other H chain with smaller side chains (holes; voids), the protrusions can be arranged in the holes, thereby enabling efficient association of polypeptides having different amino acids with an Fc region (WO1996 / 027011, Ridgway JB et al., Protein Engineering (1996) 9, 617-621, Merchant AM et al. Nature Biotechnology (1998) 16, 677-681).

[0080] In addition to this, other known techniques can also be used for the association of the heterologous Fc region variants. By using strand-exchange engineered domain CH3 in which a part of CH3 of one H chain of the antibody is replaced with a sequence derived from IgA corresponding to that part, and a sequence derived from IgA corresponding to that part is introduced into the complementary part of CH3 of the other H chain, the association of polypeptides having different sequences can be efficiently induced by the complementary association of CH3 (Protein Engineering Design & Selection, 23; 195-202, 2010). Using this known technique, it is also possible to efficiently cause the association of polypeptides having different amino acids with an Fc region. The technique for producing heterodimeric antibodies using the association of CH1 and CL, and the association of VH and VL of the antibodies described in WO2011 / 028952 can also be used. Techniques for producing heterodimeric antibodies can also be used, such as the methods described in WO2008 / 119353 and WO2011 / 131746, in which two types of homodimeric antibodies are prepared in advance, incubated under reducing conditions to dissociate them, and then reassociated. In addition, like the method described in J. Mol. (2012) 420, 204-219, a technique for producing a heterodimeric antibody can also be used by introducing residues having a charge such as Lys, Arg, Glu, and Asp into CH3 of IgG1 and IgG2 so as to be electrostatically repulsive. In addition, like the method described in WO2012 / 058768, a technique for producing a heterodimeric antibody can also be used by modifying the CH2 and CH3 regions.

[0081] In addition, even when a polypeptide containing a hetero Fc region variant cannot be efficiently formed, a polypeptide containing a hetero Fc region variant can also be obtained by separating and purifying the polypeptide containing a hetero Fc region variant from a polypeptide containing a homo Fc region variant. When producing a polypeptide containing a hetero Fc region variant composed of a first polypeptide and a second polypeptide having different sequences, a polypeptide containing a homo Fc region composed of only two first polypeptides and a polypeptide containing a homo Fc region composed of only two second polypeptides are mixed as impurities. As a method for efficiently removing these two types of polypeptides containing a homo Fc region, known techniques can be used. A method has been reported in which amino acid substitutions are introduced into the variable regions of two types of H chains to give a difference in isoelectric point, enabling purification of the two types of homomers and the target heterodimeric antibody by ion exchange chromatography (WO2007114325). As a method for purifying a heterodimeric antibody, a method for purifying a heterodimeric antibody composed of an H chain of mouse IgG2a that binds to protein A and an H chain of rat IgG2b that does not bind to protein A using protein A has been reported (WO98050431, WO95033844).

[0082] In addition, by using a heavy chain in which the 435th and 436th amino acid residues, which are the binding sites of IgG and Protein A, are substituted with amino acids having different binding affinities for Protein A, such as Tyr and His, the interaction between each heavy chain and Protein A can be changed, and by using a Protein A column, only the heterodimeric antibody can be efficiently purified.

[0083] A plurality of these substitutions and techniques, for example, two or more, can be used in combination. In addition, these modifications can be appropriately added to the first polypeptide and the second polypeptide separately. Note that the polypeptide of the present invention may be produced based on the one to which the above modifications are added.

[0084] In the present invention, the modification of an amino acid means any one of substitution, deletion, addition, insertion or modification, or a combination thereof. In the present invention, the modification of an amino acid can be rephrased as a mutation of an amino acid and is used in the same meaning. When substituting an amino acid residue, it is intended to modify the following points (a) to (c) by substituting with another amino acid residue. (a) The backbone structure of the polypeptide in the region of the sheet structure or the helical structure; (b) The charge or hydrophobicity at the target site, or (c) The size of the side chain.

[0085] Amino acid residues are classified into the following groups based on the characteristics of general side chains: (1) Hydrophobic: norleucine, met, ala, val, leu, ile; (2) Neutral hydrophilic: cys, ser, thr, asn, gln; (3) Acidic: asp, glu; (4) Basic: his, lys, arg; (5) Residues that affect the orientation of the chain: gly, pro; and (6) Aromatic: trp, tyr, phe.

[0086] 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 combinations of conservative and non-conservative substitutions.

[0087] Modifications of amino acid sequences are prepared by various 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(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, and other methods.

[0088] The modification of the amino acids of the present invention includes post-translational modifications. As specific post-translational modifications, addition or deletion of sugar chains can be indicated. For example, in the IgG1 constant region consisting of the amino acid sequence set forth in SEQ ID NO: 11, the 297th amino acid residue in EU numbering can be modified with a sugar chain. The sugar chain structure to be modified is not limited. Generally, antibodies expressed in eukaryotic cells contain sugar chain modifications in the constant region. Therefore, antibodies expressed in the following cells are usually modified with some sugar chain. · Antibody-producing cells of mammals · Eukaryotic cells transformed with an expression vector containing DNA encoding an antibody

[0089] The eukaryotic cells shown here include yeast and animal cells. For example, CHO cells and HEK293H cells are representative animal cells for transformation with an expression vector containing DNA encoding an antibody. On the other hand, those without sugar chain modification at this position are also included in the constant region of the present invention. Antibodies whose constant region is not modified with a sugar chain can be obtained by expressing a gene encoding an antibody in prokaryotic cells such as Escherichia coli.

[0090] More specifically, for example, it may be one in which sialic acid is added to the sugar chain of the Fc region (MAbs. 2010 Sep - Oct;2(5):519 - 27.).

[0091] Furthermore, the present invention provides an antibody comprising an Fc region variant described in any of the above.

[0092] The term "antibody" in the present invention is used in the broadest sense and includes any antibody such as monoclonal antibodies (including full - length monoclonal antibodies), polyclonal antibodies, antibody variants, antibody fragments, multispecific antibodies (bispecific antibodies) (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, etc., as long as they exhibit the desired biological activity.

[0093] The antibody of the present invention is not limited by the type of antigen, the origin of the antibody, etc., and any antibody may be used. The origin of the antibody is not particularly limited, and examples thereof include human antibodies, mouse antibodies, rat antibodies, rabbit antibodies, and the like.

[0094] Methods for producing antibodies are well known to those skilled in the art. For example, in the case of monoclonal antibodies, they may be produced by the hybridoma method (Kohler and Milstein, Nature 256:495 (1975)), the recombinant method (U.S. Patent No. 4,816,567). They may also be isolated from phage antibody libraries (Clackson et al., Nature 352:624-628 (1991); Marks et al., J.Mol.Biol. 222:581-597 (1991)).

[0095] Humanized antibodies are also referred to as reshaped human antibodies. Specifically, humanized antibodies in which the CDRs of non-human animals, such as mouse antibodies, are transplanted into human antibodies are known. General genetic recombination techniques for obtaining humanized antibodies are also known. Specifically, for example, Overlap Extension PCR is known as a method for transplanting the CDRs of mouse antibodies into human FRs.

[0096] A vector for expressing a humanized antibody can be prepared by inserting into an expression vector a DNA encoding an antibody variable region in which three CDRs and four FRs are linked and a DNA encoding a human antibody constant region so as to be fused in-frame. After introducing the integration vector into a host to establish recombinant cells, the recombinant cells are cultured, and the DNA encoding the humanized antibody is expressed, whereby the humanized antibody is produced in the culture of the cultured cells (see European Patent Publication EP 239400, International Publication WO1996 / 002576).

[0097] If necessary, the amino acid residues of the FR can also be replaced so that the CDRs of the reconstructed human antibody form an appropriate antigen-binding site. For example, the PCR method used for transplanting mouse CDRs into human FR can be applied to introduce mutations in the amino acid sequence of the FR.

[0098] Transgenic animals having all repertoires of human antibody genes (see International Publications WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, WO1996 / 033735) can be used as immunized animals, and desired human antibodies can be obtained by DNA immunization.

[0099] 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 by the phage display method as a single-chain antibody (scFv). Phages expressing scFv that binds to an antigen can be selected. By analyzing the gene of the selected phage, the DNA sequence encoding the V region of the human antibody that binds to the antigen can be determined. After determining the DNA sequence of the scFv that binds to the antigen, the V region sequence is fused in-frame with the sequence of the desired human antibody C region and then inserted into an appropriate expression vector to produce an expression vector. The expression vector is introduced into a suitable expression cell as described above, and the human antibody is obtained by expressing the gene encoding the human antibody. These methods are already known (see International Publications WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, WO1995 / 015388).

[0100] The variable region constituting the antibody of the present invention can be a variable region that recognizes any antigen.

[0101] In this specification, the antigen is not particularly limited and can be any antigen. Examples of antigens include, for example, ligands (such as cytokines, chemokines, etc.), receptors, cancer antigens, MHC antigens, differentiation antigens, immunoglobulins, and immune complexes partially containing immunoglobulins.

[0102] Examples of cytokines include interleukin 1 to 18, colony stimulating factors (such as G-CSF, M-CSF, GM-CSF, etc.), interferons (such as IFN-α, IFN-β, IFN-γ, etc.), growth factors (such as EGF, FGF, IGF, NGF, PDGF, TGF, HGF, etc.), tumor necrosis factors (TNF-α, TNF-β), lymphotoxins, erythropoietin, leptin, SCF, TPO, MCAF, 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.

[0103] Examples of receptors include, for example, receptors belonging to receptor families such as the hematopoietic factor receptor family, the cytokine receptor family, the tyrosine kinase type receptor family, the serine / threonine kinase type receptor family, the TNF receptor family, the G protein-coupled receptor family, the GPI anchor type receptor family, the tyrosine phosphatase type receptor family, the adhesion factor family, the hormone receptor family, etc. Regarding the receptors belonging to these receptor families and their characteristics, they are described in a number of literatures, 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(italicized)(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.

[0104] Specific receptors belonging to the above receptor family include, for example, 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 U S A. (1992) 89 (12), 5640-5644, EMBO J. (1993) 12(7), 2645-53), 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. are preferably exemplified.

[0105] Cancer antigens are antigens that are expressed with the malignant transformation of cells and are also called tumor-specific antigens. In addition, abnormal sugar chains that appear on the cell surface or protein molecules when cells become cancerous are also cancer antigens and are also called cancer sugar chain antigens. Examples of cancer antigens include, for example, GPC3 (Int J Cancer. (2003) 103 (4), 455-65), which belongs to the GPI-anchor type receptor family as the above-mentioned receptor and is expressed in several cancers including liver cancer, and EpCAM (Proc Natl Acad Sci U S A. (1989) 86 (1), 27-31), which is expressed in multiple cancers including lung cancer, CA19-9, CA15-3, serial SSEA-1 (SLX), etc.

[0106] 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, -H, and MHC class II antigens include HLA-DR, -DQ, -DP.

[0107] Differentiation antigens may 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, 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, CDw130.

[0108] Immunoglobulins include IgA, IgM, IgD, IgG, IgE. In addition, immune complexes contain at least one component of immunoglobulins. Other antigens include molecules such as the following: 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, 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, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulator (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, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (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, welchii 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, Cytokeratin tumor-related antigen, DAN, DCC, DcR3, DC-SIGN, Complement regulatory factor (Decay acceleratingfactor), 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 / EphB4, 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, fractalkine, 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), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha 1, GFR-alpha 2, GFR-alpha 3, 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, integrin alpha2, integrin alpha3, integrin alpha4, integrin alpha4 / beta1, integrin alpha4 / beta7, integrin alpha5 (alphaV), integrin alpha5 / beta1, integrin alpha5 / beta3, integrin alpha6, integrin beta1, integrin beta2, 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, Leftin, 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 inhibiting substance, Mug, MuSK, NAIP, NAP, NCAD, N-Cadherin, 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, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PlGF, PLP, PP14, proinsulin, prolactin, protein C, PS, PSA, PSCA, prostate specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, 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-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, Thrombin, Thymic Ck-1, Thyroid-stimulating hormone, Tie, TIMP, TIQ, Tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta2, 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-a connectin (Conectin), 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, Viral 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, High Molecular 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 thereof may 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.

[0109] The amino acid sequence constituting the variable region is allowed to have modifications of 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 to be modified and the number of amino acids to be modified are not particularly limited. For example, the amino acids present in the CDR and / or FR can be appropriately modified. When modifying the amino acids of the variable region, although not particularly limited, it is preferably maintained its binding activity. For example, it preferably has a binding activity of 50% or more, preferably 80% or more, more preferably 100% or more compared to before the modification. Also, the binding activity may be increased by amino acid modification. For example, the binding activity may be 2-fold, 5-fold, 10-fold, etc. compared to before the modification. In the antibody of the present invention, the modification of the amino acid sequence can be at least one of substitution, addition, deletion, and modification of amino acid residues.

[0110] For example, the modification to pyroglutamic acid by pyroglutamylation of glutamine at the N-terminus of the variable region is a modification well known to those skilled in the art. Therefore, when the N-terminus of the heavy chain of the antibody of the present invention is glutamine, it includes a variable region modified to pyroglutamic acid.

[0111] The variable region of the antibody of the present invention may have any sequence, and may be the variable region of an antibody derived from any source, such as a mouse antibody, a rat antibody, a rabbit antibody, a goat antibody, a camel antibody, a humanized antibody obtained by humanizing these non-human antibodies, and a human antibody. A "humanized antibody" is also referred to as a reshaped human antibody, and is an antibody derived from a non-human mammal, for example, an antibody in which the complementarity determining regions (CDRs) of a mouse antibody are transplanted into 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). Also, general gene recombination techniques thereof are known (see European Patent Application Publication No. EP 125023, WO 96 / 02576). Further, various amino acid substitutions may be introduced into the variable regions of these antibodies to improve antigen binding, pharmacokinetics, stability, and immunogenicity. The variable region of the antibody of the present invention may be capable of repeatedly binding to an antigen by having pH-dependence in binding to the antigen (WO2009 / 125825).

[0112] The light chain constant region of the antibody has κ-chain and λ-chain type constant regions, and either light chain constant region may be used. Further, 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, etc.

[0113] As the heavy chain constant region of the antibody of the present invention, for example, the heavy chain constant region of a human IgG antibody can be used, and preferably the heavy chain constant regions of human IgG1 antibody and human IgG4 antibody.

[0114] In addition, the Fc region variant of the present invention can be combined with other proteins, bioactive peptides, etc. to form an Fc fusion protein molecule. Here, the fusion protein refers to a chimeric polypeptide containing at least two different polypeptides that are not naturally linked. Examples of other proteins and bioactive peptides include, but are not limited to, receptors, adhesion molecules, ligands, and enzymes.

[0115] Preferred examples of the Fc fusion protein molecule of the present invention include proteins in which an Fc region is fused to a receptor protein that binds to a target. For example, TNFR-Fc fusion protein, IL1R-Fc fusion protein, VEGFR-Fc fusion protein, CTLA4-Fc fusion protein, etc. (Nat Med. 2003 Jan;9(1):47-52, BioDrugs. 2006;20(3):151-60.) can be mentioned. In addition, the protein to be fused to the polypeptide of the present invention may be any molecule as long as it binds to the target molecule. For example, 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), for example, DARPins (WO2002 / 020565), Affibody (WO1995 / 001937), Avimer (WO2004 / 044011, WO2005 / 040229), Adnectin (WO2002 / 032925), etc. can be mentioned. In addition, the antibody and the Fc fusion protein molecule may be a multispecific antibody that binds to multiple types of target molecules or epitopes.

[0116] The antibody of the present invention also includes modified antibodies. Examples of modified antibodies include, for example, antibodies conjugated with various molecules such as polyethylene glycol (PEG) and cytotoxic substances. Such antibody modifications can be obtained by chemically modifying the antibody of the present invention. Methods for modifying antibodies have already been established in this field.

[0117] Furthermore, the antibody of the present invention may be a bispecific antibody. A bispecific antibody 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.

[0118] The polypeptide 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 methods, but are not limited thereto.

[0119] DNA encoding the heavy chain of the antibody, DNA encoding the heavy chain in which one or more amino acid residues in the Fc region are substituted with other amino acids of interest, and DNA encoding the light chain of the antibody are expressed. The DNA encoding the heavy chain in which one or more amino acid residues in the Fc region are substituted with other amino acids of interest can be obtained, for example, by obtaining the Fc region portion of the DNA encoding the natural heavy chain and appropriately introducing substitutions so that the codon encoding a specific amino acid in the Fc region encodes the other amino acid of interest.

[0120] Alternatively, DNA encoding a protein in which one or more amino acid residues in the Fc region of the natural heavy chain are substituted with other amino acids of interest can be designed in advance, and the DNA can be chemically synthesized to obtain DNA encoding the heavy chain in which one or more amino acid residues in the Fc region are substituted with other amino acids of interest. The substitution sites and types of amino acid substitutions are not particularly limited. Also, it is not limited to substitution, and may be any of deletion, addition, insertion, or a combination thereof.

[0121] In addition, DNA encoding a heavy chain in which one or more amino acid residues are substituted with other amino acids of interest can be produced in divided 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 in divided partial DNAs in the same manner.

[0122] Examples of methods for expressing the above DNA include the following. For example, DNA encoding a heavy chain variable region is incorporated into an expression vector together with DNA encoding a heavy chain constant region to construct a heavy chain expression vector. Similarly, DNA encoding a light chain variable region is incorporated into an expression vector together with DNA encoding a light chain constant region to construct a light chain expression vector. The genes for these heavy and light chains can also be incorporated into a single vector.

[0123] When incorporating DNA encoding the target antibody into an expression vector, it is incorporated into the expression vector so as to be expressed under the control of an expression control region, for example, an enhancer or a promoter. Next, a host cell is transformed with this expression vector to express the antibody. In that case, an appropriate combination of host and expression vector can be used.

[0124] Examples of vectors include M13-based vectors, pUC-based vectors, pBR322, pBluescript, pCR-Script, etc. Also, when the purpose is cDNA subcloning or excision, in addition to the above vectors, for example, pGEM-T, pDIRECT, pT7, etc. can be used.

[0125] When using a vector for the purpose of producing the polypeptide of the present invention, in particular, an expression vector is useful. As the expression vector, for example, when the host is Escherichia coli such as JM109, DH5α, HB101, or XL1-Blue, a promoter capable of efficient expression in Escherichia coli, for example, the lacZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427, the entire content of which is incorporated herein by reference), the araB promoter (Better et al., Science (1988) 240, 1041-1043, the entire content of which is incorporated herein by reference), or the T7 promoter, etc. is essential. As such vectors, in addition to the above vectors, pGEX-5X-1 (manufactured by Pharmacia), "QIAexpress system" (manufactured by QIAGEN), pEGFP, or pET (in this case, the host is preferably BL21 expressing T7 RNA polymerase), etc. can be mentioned.

[0126] In addition, the vector may contain a signal sequence for polypeptide secretion. As the signal sequence for polypeptide secretion, when producing in the periplasm of Escherichia coli, the pelB signal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169, 4397, the entire content of which is incorporated herein by reference) can be used. Introduction of the vector into the host cell can be carried out using, for example, the lipofectin method, the calcium phosphate method, or the DEAE-Dextran method.

[0127] In addition to the E. coli expression vector, for example, as vectors for producing the polypeptide of the present invention, expression vectors derived from mammals (e.g., pcDNA3 (manufactured by Invitrogen), pEGF-BOS (Nucleic Acids. Res. 1990, 18(17), p5322, which is hereby incorporated by reference in its entirety), pEF, pCDM8), expression vectors derived from insect cells (e.g., "Bac-to-BAC baculovairus expression system" (manufactured by GIBCO BRL), pBacPAK8), expression vectors derived from plants (e.g., pMH1, pMH2), expression vectors derived from animal viruses (e.g., pHSV, pMV, pAdexLcw), expression vectors derived from retroviruses (e.g., pZIPneo), expression vectors derived from yeast (e.g., "Pichia Expression Kit" (manufactured by Invitrogen), pNV11, SP-Q01), and expression vectors derived from Bacillus subtilis (e.g., pPL608, pKTH50) can be mentioned.

[0128] When aiming for expression in animal cells such as CHO cells, COS cells, and NIH3T3 cells, it is essential to have promoters necessary for intracellular expression, such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108, which is hereby incorporated by reference in its entirety), MMTV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322, which is hereby incorporated by reference in its entirety), CAG promoter (Gene. (1991) 108, 193, which is hereby incorporated by reference in its entirety), CMV promoter, etc. It is more preferable to have a gene for selecting transformed cells (e.g., a drug resistance gene that can be discriminated by a drug (such as neomycin, G418, etc.)). Examples of vectors having such characteristics include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOP13, etc.

[0129] Furthermore, when the aim is to stably express a gene and amplify the copy number of the gene in cells, a method can be used in which a vector having a DHFR gene (for example, pCHOI, etc.) that complements a CHO cell lacking a nucleic acid synthesis pathway is introduced and amplified with methotrexate (MTX). When the aim is transient expression of a gene, a method can be used in which COS cells having a gene expressing SV40 T antigen on the chromosome are transformed with a vector (such as pcD) having an SV40 origin of replication. As the origin of replication, those derived from polyomavirus, adenovirus, bovine papillomavirus (BPV), etc. can also be used. Furthermore, for gene copy number amplification in a host cell line, the expression vector can contain, as a selection marker, an aminoglycoside transferase (APH) gene, a thymidine kinase (TK) gene, an Escherichia coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, a dihydrofolate reductase (dhfr) gene, etc.

[0130] Recovery of the antibody can be carried out, for example, by culturing the transformed cells and then separating them from inside the molecularly transformed cells or from the culture broth. For separation and purification of the antibody, methods such as centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, 1q, FcRn, protein A, protein G columns, affinity chromatography, ion exchange chromatography, gel filtration chromatography, etc. can be appropriately combined and carried out.

[0131] The present invention also provides a method for producing a polypeptide containing an Fc region variant of an antibody, which comprises adding at least one amino acid modification to the Fc region variant, and which contains an Fc region variant having enhanced binding activity to FcγRIIb as compared to a polypeptide containing a parental Fc region. For example, a production method including the following steps can be mentioned; (a) A step of adding at least one amino acid modification to the Fc region in a polypeptide containing an Fc region, (b) Measuring the binding activity of the polypeptide modified in step (a) to FcγRIIb, and (c) Selecting a polypeptide comprising an Fc region variant with enhanced binding activity to FcγRIIb as compared to a polypeptide comprising the parental Fc region.

[0132] Preferably, a method for producing a polypeptide comprising an Fc region variant, comprising: (a) Modifying the nucleic acid encoding the polypeptide so that the binding activity to FcγRIIb is enhanced as compared to a polypeptide comprising the parental Fc region; (b) Introducing the nucleic acid into a host cell and culturing the host cell for expression; (c) Recovering the polypeptide from the host cell culture. Also included in the present invention are antibodies and Fc fusion protein molecules produced by the production method.

[0133] The present invention also provides a method for producing a polypeptide comprising an Fc region variant with enhanced binding selectivity to FcγRIIb as compared to FcγRIIa (R type), compared to a polypeptide comprising the parental Fc region, comprising adding at least one amino acid modification to the Fc region variant in the polypeptide comprising the antibody Fc region variant. For example, a production method including the following steps can be mentioned: (a) Adding at least one amino acid modification to the Fc region in a polypeptide comprising the Fc region; (b) Measuring the binding activity of the polypeptide modified in step (a) to FcγRIIa and the binding activity to FcγRIIb, and (c) Selecting a polypeptide comprising an Fc region variant with enhanced binding selectivity to FcγRIIb as compared to FcγRIIa (R type), compared to a polypeptide comprising the parental Fc region.

[0134] Preferably, a method for producing a polypeptide comprising an Fc region variant, comprising: (a) Modifying the nucleic acid encoding the polypeptide so that the binding selectivity for FcγRIIb compared to FcγRIIa (R-type) is enhanced as compared to a polypeptide containing the parental Fc region; (b) Culturing the host cell into which the nucleic acid has been introduced so as to express it; (c) Recovering the polypeptide from the host cell culture. The method includes these steps. Further included in the present invention are antibodies and Fc fusion protein molecules produced by the production method.

[0135] Further, the present invention provides a method for producing a polypeptide containing an antibody Fc region variant, which includes adding at least one amino acid modification to the Fc region variant. The binding activity for FcγRIIb is enhanced as compared to a polypeptide containing the parental Fc region, and the polypeptide contains an Fc region variant with enhanced binding selectivity for FcγRIIb compared to FcγRIIa (R-type). For example, a production method including the following steps can be mentioned; (a) Adding at least one amino acid modification to the Fc region in a polypeptide containing the Fc region; (b) Measuring the binding activity of the polypeptide modified in step (a) for FcγRIIa and the binding activity for FcγRIIb, and (c) Selecting a polypeptide containing an Fc region variant in which the binding activity for FcγRIIb is enhanced and the binding selectivity for FcγRIIb compared to FcγRIIa (R-type) is enhanced as compared to a polypeptide containing the parental Fc region.

[0136] Preferably, it is a method for producing a polypeptide containing an Fc region variant, (a) Modifying the nucleic acid encoding the polypeptide so that the binding activity for FcγRIIb is enhanced and the binding selectivity for FcγRIIb compared to FcγRIIa (R-type) is enhanced as compared to a polypeptide containing the parental Fc region; (b) Culturing the host cell into which the nucleic acid has been introduced so as to express it; (c) Recovering the polypeptide from the host cell culture. The method includes this step. Furthermore, the antibodies and Fc fusion protein molecules produced by the production method are also included in the present invention.

[0137] The present invention also provides a method for producing a polypeptide containing an Fc region, which includes adding at least one amino acid modification to the Fc region. When the polypeptide is administered to a living body, compared with the polypeptide containing the parental Fc region, the production of antibodies against the polypeptide is suppressed. For example, a production method including the following steps can be mentioned: (a) Adding at least one amino acid modification to the Fc region in a polypeptide containing the Fc region, and (b) Confirming that when the polypeptide containing the Fc region modified in step (a) is administered to a living body, the production of antibodies is suppressed compared with the polypeptide containing the parental Fc region.

[0138] Whether the production of antibodies against the polypeptide is suppressed can be confirmed by methods such as actually administering the polypeptide to an animal. Alternatively, the binding activities to FcγRIIa and FcγRIIb are measured, and it can also be determined that the production of antibodies is suppressed when the value obtained by dividing the KD value for FcγRIIa by the KD value for FcγRIIb increases. Such a polypeptide can suppress the production of antibodies without activating the active FcγR, so it is considered useful as a pharmaceutical.

[0139] In the above production method, it is preferable to enhance the binding activity to FcγRIIb and enhance the binding selectivity of FcγRIIb compared with FcγRIIa (R type). As a preferred embodiment in the above manufacturing method, for example, in the Fc region of human IgG, in addition to the modification of the amino acid at position 238 in the EU numbering to another amino acid, the amino acids at positions 233, 234, 235, 237, 264, 265, 266, 267, 268, 269, 271, 272, 274, 296, 326, 327, 330, 331, 332, 333, 334, 355, 356, 358, 396, 409, and 419 in the EU numbering are selected, and at least one amino acid modification to another amino acid is introduced, and the Fc region is modified accordingly. As other amino acid modifications combined with the amino acid modification at position 238 in the EU numbering, the amino acids at positions 233, 237, 264, 267, 268, 271, 272, 296, 327, 330, 332, 333, and 396 in the EU numbering are preferred. In particular, the amino acids at positions 233, 237, 264, 267, 268, 271, 296, 330, and 396 in the EU numbering are preferred.In particular, a modification combining the 238th amino acid, 268th amino acid, and 271st amino acid in EU numbering with at least one amino acid selected from the 233rd amino acid, 237th amino acid, 264th amino acid, 267th amino acid, 272nd amino acid, 296th amino acid, 327th amino acid, 330th amino acid, 332nd amino acid, and 396th amino acid in EU numbering is mentioned as a preferred combination of amino acid modifications from the viewpoint of enhancing the binding activity to FcγRIIb or enhancing the binding selectivity to FcγRIIb compared to FcγRIIa.

[0140] The amino acids to be modified are not particularly limited as long as their binding activity to FcγRIIb is enhanced compared to before modification, or their binding selectivity for FcγRIIb is enhanced compared to FcγRIIa. However, it is preferable that the 238th amino acid in EU numbering is Asp, the 233rd amino acid is Asp, the 234th amino acid is Tyr, the 237th amino acid is Asp, the 264th amino acid is Ile, the 265th amino acid is Glu, the 266th amino acid is Phe, Leu or Met, the 267th amino acid is Ala, Glu, Gly or Gln, the 268th amino acid is Asp, Gln or Glu, the 269th amino acid is Asp, the 271st amino acid is Gly, the 272nd amino acid is Asp, Phe, Ile, Met, Asn, Pro or Gln, the 274th amino acid is Gln, the 296th amino acid is Asp or Phe, the 326th amino acid is Ala or Asp, the 327th amino acid is Gly, the 330th amino acid is Lys, Arg or Ser, the 331st amino acid is Ser, the 332nd amino acid is Lys, Arg, Ser or Thr, the 333rd amino acid is Lys, Arg, Ser or Thr, the 334th amino acid is Arg, Ser or Thr, the 355th amino acid is Ala, Gln, the 356th amino acid is Glu, the 358th amino acid is Met, the 396th amino acid is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr, the 409th amino acid is Arg, and the 419th amino acid is Glu. As the amino acids to be modified, those with enhanced binding activity to FcγRIIb compared to before modification and enhanced binding selectivity for FcγRIIb compared to FcγRIIa are more preferable.In particular, when combining the 238th amino acid, 268th amino acid, and 271st amino acid of the EU numbering with at least one amino acid selected from the 233rd amino acid, 264th amino acid, 267th amino acid, 272nd amino acid, 296th amino acid, 327th amino acid, 330th amino acid, 332nd amino acid, and 396th amino acid of the EU numbering, it is preferable that the 238th amino acid of the EU numbering is Asp, the 268th amino acid is Asp or Glu, the 271st amino acid is Gly, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala or Gly, the 272nd amino acid is Asp or Pro, the 296th amino acid is Asp, the 327th amino acid is Gly, the 330th amino acid is Arg, the 332nd amino acid is Thr, and the 396th amino acid is Leu or Met.

[0141] Among these combinations, it is preferable to introduce modifications that enhance the binding activity to FcγRIIb, or modifications that enhance the binding selectivity to FcγRIIb rather than FcγRIIa (R type). Preferred combinations of amino acid substitutions as such modifications include, for example, the combinations of (a) to (x) below. (a) Amino acid modifications at the 238th, 233rd, 237th, 268th, 271st, 296th, and 330th positions of the EU numbering in the Fc region (b) Amino acid modifications at the 238th, 237th, 268th, 271st, 296th, and 330th positions of the EU numbering in the Fc region (c) Amino acid modifications at the 238th, 233rd, 237th, 268th, 271st, 296th, 330th, and 332nd positions of the EU numbering in the Fc region (d) Amino acid modifications at the 238th, 233rd, 237th, 264th, 267th, 268th, 271st, and 330th positions of the EU numbering in the Fc region (e) Amino acid modifications at positions 238, 233, 237, 267, 268, 271, 296, 330, and 332 of the EU numbering in the Fc region (f) Amino acid modifications at positions 238, 237, 267, 268, 271, 296, 330, and 332 of the EU numbering in the Fc region (g) Amino acid modifications at positions 238, 233, 237, 268, 271, 296, 327, and 330 of the EU numbering in the Fc region (h) Amino acid modifications at positions 238, 233, 237, 264, 267, 268, and 271 of the EU numbering in the Fc region (i) Amino acid modifications at positions 238, 233, 237, 264, 267, 268, 271, 296, and 330 of the EU numbering in the Fc region (j) Amino acid modifications at positions 238, 233, 237, 264, 267, 268, 271, 296, 330, and 396 of the EU numbering in the Fc region (k) Amino acid modifications at positions 238, 237, 264, 267, 268, 271, and 330 of the EU numbering in the Fc region (l) Amino acid modifications at positions 238, 237, 264, 267, 268, 271, 296, and 330 of the EU numbering in the Fc region (m) Amino acid modifications at positions 238, 264, 267, 268, and 271 of the EU numbering in the Fc region (n) Amino acid modifications at positions 238, 264, 267, 268, 271, and 296 of the EU numbering in the Fc region (o) Amino acid modifications at positions 238, 237, 267, 268, 271, 296, and 330 of the EU numbering in the Fc region (p) Amino acid modifications at positions 238, 233, 237, 264, 267, 268, 271, 330, and 396 of the EU numbering in the Fc region (q) Amino acid modifications at positions 238th, 233rd, 237th, 264th, 267th, 268th, 271st, 296th, 327th, 330th, and 396th in the EU numbering of the Fc region (r) Amino acid modifications at positions 238th, 233rd, 237th, 264th, 267th, 268th, 271st, 272nd, and 296th in the EU numbering of the Fc region (s) Amino acid modifications at positions 238th, 237th, 264th, 267th, 268th, 271st, 272nd, and 330th in the EU numbering of the Fc region (t) Amino acid modifications at positions 238th, 237th, 264th, 267th, 268th, 271st, 272nd, 296th, and 330th in the EU numbering of the Fc region (u) Amino acid modifications at positions 238th, 233rd, 264th, 267th, 268th, and 271st in the EU numbering of the Fc region (v) Amino acid modifications at positions 238th, 237th, 267th, 268th, 271st, 296th, and 330th in the EU numbering of the Fc region (w) Amino acid modifications at positions 238th, 264th, 267th, 268th, 271st, 272nd, and 296th in the EU numbering of the Fc region (x) Amino acid modifications at positions 238th, 233rd, 264th, 267th, 268th, 271st, and 296th in the EU numbering of the Fc region

[0142] Furthermore, among these combinations of modifications, the following combinations of amino acid modifications (a) to (x) are listed as more preferred combinations. (a) An amino acid sequence in which the amino acid at position 238th in the EU numbering of the Fc region is Asp, the amino acid at position 233rd is Asp, the amino acid at position 237th is Asp, the amino acid at position 268th is Asp, the amino acid at position 271st is Gly, the amino acid at position 296th is Asp, and the amino acid at position 330th is Arg (b) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 237th amino acid is Asp, the 268th amino acid is Asp or Glu, the 271st amino acid is Gly, the 296th amino acid is Asp, and the 330th amino acid is Arg (c) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 268th amino acid is Asp, the 271st amino acid is Gly, the 296th amino acid is Asp, the 330th amino acid is Arg, and the 332nd amino acid is Thr (d) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Gly or Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, and the 330th amino acid is Arg (e) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 296th amino acid is Asp, the 330th amino acid is Arg, and the 332nd amino acid is Thr (f) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 237th amino acid is Asp, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 296th amino acid is Asp, the 330th amino acid is Arg, and the 332nd amino acid is Thr (g) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 268th amino acid is Asp, the 271st amino acid is Gly, the 296th amino acid is Asp, the 327th amino acid is Gly, and the 330th amino acid is Arg (h) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, and the 271st amino acid is Gly (i) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 296th amino acid is Asp, and the 330th amino acid is Arg (j) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 296th amino acid is Asp, the 330th amino acid is Arg, and the 396th amino acid is Met or Leu (k) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, and the 330th amino acid is Arg (l) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 296th amino acid is Asp, and the 330th amino acid is Arg (m) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, and the 271st amino acid is Gly (n) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, and the 296th amino acid is Asp (o) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 237th amino acid is Asp, the 267th amino acid is Ala or Gly, the 268th amino acid is Glu, the 271st amino acid is Gly, the 296th amino acid is Asp, and the 330th amino acid is Arg (p) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 330th amino acid is Arg, and the 396th amino acid is Met or Leu (q) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 296th amino acid is Asp, the 327th amino acid is Gly, the 330th amino acid is Arg, and the 396th amino acid is Met (r) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 272nd amino acid is Asp, and the 296th amino acid is Asp (s) An amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 272nd amino acid is Pro, and the 330th amino acid is Arg The amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 237th amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 272nd amino acid is Pro, the 296th amino acid is Asp, and the 330th amino acid is Arg The amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, and the 271st amino acid is Gly The amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 237th amino acid is Asp, the 267th amino acid is Gly, the 268th amino acid is Asp, the 271st amino acid is Gly, the 296th amino acid is Asp, and the 330th amino acid is Arg The amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, the 272nd amino acid is Asp, and the 296th amino acid is Asp The amino acid sequence in which the 238th amino acid in the EU numbering of the Fc region is Asp, the 233rd amino acid is Asp, the 264th amino acid is Ile, the 267th amino acid is Ala, the 268th amino acid is Glu, the 271st amino acid is Gly, and the 296th amino acid is Asp

[0143] Furthermore, the present invention provides a method for modifying a polypeptide for producing a polypeptide having enhanced binding activity to FcγRIIb or enhanced binding selectivity for FcγRIIb compared to FcγRIIa (R type), as compared to a polypeptide containing a parental Fc region. The present invention also provides a method for modifying a polypeptide for producing a polypeptide having enhanced binding activity to FcγRIIb and enhanced binding selectivity for FcγRIIb as compared to FcγRIIa (R type), as compared to a polypeptide containing a parental Fc region. The present invention also provides a method for modifying a polypeptide for producing a polypeptide in which antibody production is suppressed when administered to a living body, as compared to a polypeptide containing a parental Fc region.

[0144] As a preferred embodiment, for example, a combination of amino acid modifications described in the method for producing a polypeptide containing an Fc region variant having enhanced binding activity to FcγRIIb or enhanced binding selectivity for FcγRIIb compared to FcγRIIa (R type) as described above can be mentioned. As a more preferred embodiment, for example, a combination of amino acid modifications described in the method for producing a polypeptide containing an Fc region variant having enhanced binding activity to FcγRIIb and enhanced binding selectivity for FcγRIIb as compared to FcγRIIa (R type) as described above can be mentioned.

[0145] Furthermore, the present invention provides a nucleic acid encoding a polypeptide comprising an Fc region, wherein at least one amino acid is modified, and the binding activity to FcγRIIb is enhanced as compared with a polypeptide comprising a parental Fc region, or the binding selectivity to FcγRIIb as compared with the binding activity to FcγRIIa (R type) is enhanced. The present invention also provides a nucleic acid encoding a polypeptide comprising an Fc region, wherein at least one amino acid is modified, and the binding activity to FcγRIIb is enhanced as compared with a polypeptide comprising a parental Fc region, and the binding selectivity to FcγRIIb as compared with the binding activity to FcγRIIa (R type) is enhanced. The nucleic acid of the present invention may be in any form such as DNA, RNA, etc.

[0146] Furthermore, the present invention provides a vector containing the nucleic acid of the present invention described above. The type of the vector can be appropriately selected by those skilled in the art according to the host cell into which the vector is introduced. For example, the above-described vector can be used.

[0147] Furthermore, the present invention relates to a host cell transformed with the vector of the present invention described above. The host cell can be appropriately selected by those skilled in the art. For example, the above-described host cell can be used. Specifically, for example, the following host cells can be mentioned. When a eukaryotic cell is used as the host cell, an animal cell, a plant cell, or a fungal cell can be appropriately used. Specifically, as the animal cell, the following cells can be exemplified. (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 cell (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: African clawed frog oocytes, etc. (3) Insect cells: sf9, sf21, Tn5, etc.

[0148] Alternatively, as plant cells, an antibody gene expression system using cells derived from the genus Nicotiana such as Nicotiana tabacum is known. For the transformation of plant cells, callus-cultured cells can be appropriately used.

[0149] Furthermore, as fungal cells, the following cells can be used. - Yeast: Genus Saccharomyces such as Saccharomyces serevisiae, Genus Pichia such as Pichia pastoris - Filamentous fungi: Genus Aspergillus such as Aspergillus niger

[0150] Furthermore, the present invention provides a method for enhancing the binding activity to FcγRIIb and / or enhancing the binding selectivity to FcγRIIb as compared to the binding activity to FcγRIIa (R type) in a polypeptide containing an Fc region, which comprises adding at least one amino acid modification to the Fc region. The present invention also provides a method for suppressing the production of an antibody against a polypeptide when administered to a living body, in a polypeptide containing an Fc region, which comprises adding at least one amino acid modification to the Fc region.

[0151] As a preferred embodiment, for example, there may be mentioned the combination of amino acid modifications described in the method for producing a polypeptide containing an Fc region variant in which the binding activity to FcγRIIb as described above is enhanced and / or the binding selectivity to FcγRIIb as compared to FcγRIIa (R type) is enhanced. The present invention also includes a polypeptide produced by any of the above methods.

[0152] The present invention provides a pharmaceutical composition containing a polypeptide containing an Fc region variant of the present invention. In addition to the above-mentioned antibody or Fc fusion protein molecule of the present invention, the pharmaceutical composition of the present invention can be formulated by introducing a pharmaceutically acceptable carrier and using a known method. For example, it can be used parenterally in the form of a sterile solution or suspension in water or other pharmaceutically acceptable liquids. For example, it can be formulated by appropriately combining a pharmacologically acceptable carrier or medium, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc., and mixing in a unit dosage form required for generally recognized pharmaceutical practice. Specifically, examples of carriers include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, calcium carmellose, sodium carmellose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinyl pyrrolidone, gelatin, medium-chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts, etc. The amount of the active ingredient in these formulations is such that an appropriate dosage within the indicated range can be obtained. A sterile composition for injection can be formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection.

[0153] Examples of the aqueous solution for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and may be used in combination with a suitable solubilizing agent, such as alcohol, specifically ethanol, polyalcohol, such as propylene glycol, polyethylene glycol, nonionic surfactant, such as polysorbate 80 (TM), HCO-50.

[0154] Examples of the oily liquid include sesame oil and soybean oil, and they may be used in combination with benzyl benzoate and benzyl alcohol as solubilizing agents. Further, they may be formulated with a buffer such as phosphate buffer or sodium acetate buffer, a soothing agent such as procaine hydrochloride, a stabilizer such as benzyl alcohol or phenol, and an antioxidant. The prepared injection solution is usually filled into a suitable ampoule.

[0155] Administration is preferably parenteral administration, and specifically, examples include injection dosage forms, nasal administration dosage forms, pulmonary administration dosage forms, transdermal administration dosage forms, etc. The injection dosage form can be administered systemically or locally, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.

[0156] Also, the administration method of the pharmaceutical composition of the present invention can be appropriately selected according to the age and symptoms of the patient. As the dosage of the pharmaceutical composition containing an antibody or a polynucleotide encoding an antibody, for example, it can be selected in the range of 0.0001 mg to 1000 mg per 1 kg of body weight per administration. Alternatively, for example, the dosage can be selected in the range of 0.001 to 100000 mg / body per patient, but these numerical values are not necessarily limited thereto. The dosage and administration method vary depending on the body weight, age, symptoms, etc. of the patient, but those skilled in the art can appropriately select them.

[0157] The polypeptide containing the Fc region variant of the present invention is useful as an active ingredient of a drug that suppresses the activation of B cells, mast cells, dendritic cells and / or basophils. The polypeptide containing the Fc region variant of the present invention can selectively act on FcγRIIb without activating the active form of FcγR, and suppress the activation of B cells, mast cells, dendritic cells and / or basophils. The activation of B cells includes proliferation, IgE production, IgM production, IgA production, etc. The polypeptide containing the Fc region variant of the present invention suppresses the IgE production of B cells by cross-linking FcγRIIb and IgE, suppresses the IgM production of B cells by cross-linking with IgM, and suppresses the IgA production by cross-linking with IgA. In addition, by directly or indirectly cross-linking molecules containing an ITAM domain intracellularly or interacting with an ITAM domain among molecules expressed on B cells such as BCR, CD19, CD79b, etc. and FcγRIIb, the same inhibitory effect as above is exerted. The activation of mast cells includes proliferation, activation by IgE, etc., degranulation, etc. The polypeptide containing the Fc region variant of the present invention can suppress proliferation, activation by IgE, etc., and degranulation in mast cells by directly or indirectly cross-linking molecules containing an ITAM domain expressed on mast cells such as FcεRI, DAP12, CD200R3, which are IgE receptors, and FcγRIIb. The activation of basophils includes proliferation, degranulation, etc. The polypeptide containing the Fc region variant of the present invention can also suppress activation, degranulation, and proliferation in basophils by directly or indirectly cross-linking FcγRIIb and a molecule containing an ITAM domain intracellularly or interacting with an ITAM domain among molecules on the cell membrane. The activation of dendritic cells includes proliferation, degranulation, etc. The polypeptide containing the Fc region variant of the present invention can also suppress activation, degranulation, and proliferation in dendritic cells by directly or indirectly cross-linking a molecule containing an ITAM domain intracellularly or interacting with an ITAM domain among molecules on the cell membrane and FcγRIIb.

[0158] In the present invention, the polypeptide containing the Fc region variant of the present invention is useful as an active ingredient of a therapeutic or prophylactic agent for immune-inflammatory diseases. As described above, since the polypeptide containing the Fc region variant of the present invention can suppress the activation of B cells, mast cells, dendritic cells and / or basophils, as a result, by administering the polypeptide containing the Fc region variant of the present invention, it is possible to treat or prevent immune-inflammatory diseases. "Immune-inflammatory diseases" include, but are not limited to, the following: rheumatoid arthritis, autoimmune hepatitis, autoimmune thyroiditis, autoimmune bullous disease, autoimmune adrenalitis, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, megaloblastic anemia, autoimmune atrophic gastritis, autoimmune neutropenia, autoimmune orchitis, autoimmune encephalomyelitis, autoimmune receptor disease, autoimmune infertility, chronic active hepatitis, glomerulonephritis, interstitial pulmonary fibrosis, multiple sclerosis, Paget's disease, osteoporosis, multiple myeloma, uveitis, acute and chronic spondylitis, gouty arthritis, inflammatory bowel disease, adult respiratory distress syndrome (ARDS), psoriasis, Crohn's disease, Graves' disease, juvenile diabetes, Addison's disease, myasthenia gravis, crystalline uveitis, systemic lupus erythematosus, allergic rhinitis, allergic dermatitis, ulcerative colitis, hypersensitivity, muscle degeneration, cachexia, systemic scleroderma, localized scleroderma, Sjogren's syndrome, Behcet's disease, Reiter's syndrome, type I and type II diabetes, bone resorption diseases, graft-versus-host reaction, ischemic 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 antibody syndrome, polymyositis, Wegener's granulomatosis, polyarteritis nodosa, mixed connective tissue disease, fibromyalgia, asthma, atopic dermatitis, chronic atrophic gastritis, primary biliary cirrhosis, primary sclerosing cholangitis, autoimmune pancreatitis, Takayasu arteritis, rapidly progressive glomerulonephritis, megaloblastic anemia, idiopathic thrombocytopenic purpura, primary hypothyroidism, idiopathic Addison's disease, insulin-dependent diabetes, chronic discoid lupus erythematosus, pemphigoid, gestational herpes, linear IgA bullous dermatosis, acquired epidermolysis bullosa,Alopecia areata, vitiligo vulgaris, Sutton's post-traumatic leukoderma, Harada's disease, autoimmune optic neuropathy, idiopathic azoospermia, habitual abortion, hypoglycemia, chronic urticaria, ankylosing spondylitis, psoriatic arthritis, enteropathic arthritis, reactive arthritis, spondyloarthritis, enthesitis, irritable bowel syndrome, chronic fatigue syndrome, dermatomyositis, inclusion body myositis, Schmidt syndrome, Graves' disease, pernicious anemia, lupoid hepatitis, presenile dementia, Alzheimer's disease, demyelinating diseases, amyotrophic lateral sclerosis, hypoparathyroidism, Dressler syndrome, Eaton-Lambert syndrome, dermatitis herpetiformis, alopecia, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal motility disorder, sclerodactyly and telangiectasia), sarcoidosis, rheumatic fever, erythema multiforme, Cushing's syndrome, transfusion reaction, leprosy, Takayasu arteritis, polymyalgia rheumatica, temporal arteritis, giant cell arteritis, eczema, lymphomatoid granulomatosis, Kawasaki disease, endocarditis, endomyocardial fibrosis, endophthalmitis, erythroblastosis fetalis, eosinophilic fasciitis, Felty syndrome, Henoch-Schönlein purpura, transplant rejection, mumps, cardiomyopathy, suppurative arthritis, familial Mediterranean fever, Muckle-Wells syndrome, hyper IgD syndrome.

[0159] In addition, the polypeptide containing the Fc region variant of the present invention is useful as an active ingredient of a drug that suppresses the production of autoantibodies against self-antigens and treats or prevents the autoimmune disease in autoimmune diseases where the production of antibodies (autoantibodies) against self-antigens is considered to be the cause of the disease. By using a molecule in which AchR, an autoantigen of myasthenia gravis, is fused with the Fc portion of an antibody, it has been reported that the proliferation of B cells expressing BCR that recognizes AchR is suppressed and apoptosis is induced (J Neuroimmunol, 227, 35-43, 2010). By using a fusion protein of an antigen recognized by an autoantibody and the antibody Fc region described in the present invention, it is possible to crosslink BCR and FcγRIIb of B cells expressing BCR against the self-antigen, suppress the proliferation of B cells expressing BCR against the self-antigen, and induce apoptosis. Such autoimmune diseases include Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, autoimmune pancreatitis, Takayasu arteritis, Goodpasture syndrome, rapidly progressive glomerulonephritis, megaloblastic anemia, autoimmune hemolytic anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Graves' disease, Hashimoto's disease, primary hypothyroidism, idiopathic Addison's disease, insulin-dependent diabetes, chronic discoid lupus erythematosus, localized scleroderma, pemphigus, pemphigoid, gestational herpes, linear IgA bullous dermatosis, acquired epidermolysis bullosa, alopecia areata, vitiligo vulgaris, Sutton's post-traumatic leukoderma centrifugum, Vogt-Koyanagi-Harada disease, autoimmune optic neuropathy, idiopathic azoospermia, habitual abortion, type 2 diabetes, hypoglycemia, chronic urticaria, but are not limited to these.

[0160] Furthermore, the polypeptide containing the Fc region variant of the present invention is useful as an active ingredient of a therapeutic agent for diseases lacking a protein necessary for the living body. For diseases lacking a protein necessary for the living body, a treatment method of administering and supplementing the protein as a drug is used. However, since the patient originally lacks the protein, the protein supplemented from the outside is recognized as a foreign substance, and an antibody against the protein is produced. As a result, the protein is easily removed, and the effect as a drug is attenuated. By using a fusion protein of such a protein and the antibody Fc region described in the present invention, it is possible to crosslink BCR and FcγRIIb on B cells that recognize the protein and suppress antibody production against the protein. Examples of the protein to be supplemented include Factor VIII, Factor IX, TPO, EPO, α-iduronidase, iduronate sulfatase, A-type heparan N-sulfatase, B-type α-N-acetylglucosaminidase, C-type acetyl CoA:α-glucosaminidase acetyltransferase, D-type N-acetylglucosamine 6-sulfatase, galactose 6-sulfatase, N-acetylgalactosamine 4-sulfatase, β-glucuronidase, α-galactosidase, acidic α-galactosidase, glucocerebrosidase. Examples of the diseases to be supplemented with these proteins include hemophilia, idiopathic thrombocytopenic purpura, renal anemia, lysosomal diseases (mucopolysaccharidosis, Fabry disease, Pompe disease, Gaucher disease), etc. However, it is not limited thereto.

[0161] Furthermore, the polypeptide containing the Fc region variant of the present invention is useful as an active ingredient of an antiviral agent. An antibody against a virus that contains the Fc region described in the present invention can suppress antibody-dependent enhancement found in antibodies against viruses. Antibody-dependent enhancement is a phenomenon in which a virus uses a neutralizing antibody against the virus to be phagocytosed via activated FcγR and infect FcγR-expressing cells, thereby expanding the infection. It has been reported that the binding of a neutralizing antibody against dengue virus to FcγRIIb plays an important role in suppressing antibody-dependent enhancement (Proc Natl Acad Sci USA, 108, 12479-12484, 2011). The immune complex formed by a neutralizing antibody against dengue virus and the dengue virus crosslinks FcγRIIb, thereby inhibiting phagocytosis via FcγR, and as a result, suppressing antibody-dependent enhancement. Viruses include dengue virus (DENV1, DENV2, DENV4) and HIV. However, it is not limited to these only.

[0162] Furthermore, the polypeptide containing the Fc region variant of the present invention is useful as an active ingredient of an arteriosclerosis preventive or therapeutic agent. An antibody against oxidized LDL, which is a cause of arteriosclerosis, and which contains the Fc region described in the present invention can prevent the adhesion of FcγRIIa-dependent inflammatory cells. An anti-oxidized LDL antibody inhibits the interaction between oxidized LDL and CD36, but it has been reported that an anti-oxidized LDL antibody binds to endothelial cells, and its Fc portion is recognized and adhered to by monocytes in an FcγRIIa- and FcγRI-dependent manner (Immunol Lett, 108, 52-61, 2007). By using the antibody containing the Fc region described in the present invention for such an antibody, it is considered that FcγRIIa-dependent binding is inhibited and the adhesion of monocytes is suppressed by an inhibitory signal via FcγRIIb.

[0163] In the present invention, the polypeptide containing the Fc region variant of the present invention is useful as an active ingredient of a therapeutic or prophylactic agent for cancer. As described above, by enhancing the binding to FcγRIIb, the agonist activity of the agonist antibody is enhanced, and it is known that the antitumor effect of the antibody is also enhanced. Therefore, the agonist antibody using the Fc region variant described in the present invention is useful for the treatment or prevention of cancer. Specifically, the Fc region variant described in the present invention enhances the agonist activity of agonist antibodies against, for example, TNF receptor families 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, TNFRSF25, Ectodysplasin A2 receptor, and can be used for the treatment or prevention of cancer. In addition to the above, the agonist activity of agonist antibodies against molecules that require interaction with FcγRIIb for their agonist activity is also enhanced. In addition, by incorporating the Fc region variant of the present invention into a polypeptide having a binding activity to a molecule such as Kit, which is one of the Receptor Tyrosine kinases (RTK), and whose cell proliferation is suppressed by cross-linking with FcγRIIb, it becomes possible to enhance the inhibitory effect on cells expressing the molecule.Cancer includes, but is not limited to: lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung), colorectal cancer, rectal cancer, colon cancer, breast cancer, liver cancer, gastric cancer, pancreatic cancer, kidney cancer, prostate cancer, ovarian cancer, thyroid cancer, bile duct cancer, peritoneal cancer, mesothelioma, squamous cell carcinoma, cervical cancer, endometrial cancer, bladder cancer, esophageal cancer, head and neck cancer, nasopharyngeal cancer, salivary gland tumor, thymoma, skin cancer, basal cell tumor, malignant melanoma, anal cancer, penile cancer, testicular cancer, Wilms tumor, acute myeloid leukemia (including acute myeloblastic leukemia, acute myelogenous leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, and acute monocytic leukemia), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma (including Burkitt lymphoma, chronic lymphocytic leukemia, mycosis fungoides, mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, hairy cell leukemia / plasmacytoma, peripheral T-cell lymphoma, and adult T-cell leukemia / lymphoma), Langerhans cell histiocytosis, multiple myeloma, myelodysplastic syndrome, brain tumor (including glioma, astrocytoma, glioblastoma, meningioma, and ependymoma), neuroblastoma, retinoblastoma, osteosarcoma, Kaposi sarcoma, Ewing sarcoma, angiosarcoma, hemangiopericytoma.

[0164] The present invention also relates to a method for treating or preventing an immune-inflammatory disease, which comprises a step of administering to a subject (patient) a polypeptide comprising a modified Fc region of the present invention or a polypeptide comprising a modified Fc region produced by the production method of the present invention.

[0165] The present invention also provides a kit for use in the treatment method or prevention method of the present invention, which comprises at least a polypeptide containing the Fc region variant of the present invention or a polypeptide containing the Fc region variant produced by the production method of the present invention, or a pharmaceutical composition of the present invention. In addition, other pharmaceutically acceptable carriers, media, instructions describing the usage method, etc. can also be packaged in the kit. The present invention also relates to the use of a polypeptide containing the Fc region variant of the present invention or a polypeptide containing the Fc region variant produced by the production method of the present invention in the manufacture of a therapeutic or prophylactic agent for immune-inflammatory diseases. The present invention also relates to a polypeptide containing the Fc region variant of the present invention or a polypeptide containing the Fc region variant produced by the production method of the present invention for use in the treatment method or prevention method of the present invention.

[0166] The correspondence between the three-letter notation and one-letter notation of amino acids used in the present specification is as follows. Alanine: Ala: A Arginine: Arg: R Asparagine: Asn: N Aspartic acid: Asp: D Cysteine: Cys: C Glutamine: Gln: Q Glutamic acid: Glu: E Glycine: Gly: G Histidine: His: H Isoleucine: Ile: I Leucine: Leu: L Lysine: Lys: K Methionine: Met: M Phenylalanine: Phe: F Proline: Pro: P Serine: Ser: S Threonine: Thr: T Tryptophan: Trp: W Tyrosine: Tyr: Y Valine: Val: V

[0167] All prior art documents cited in this specification are incorporated herein by reference.

Example

[0168] The present invention is further illustrated by the following examples, but is not limited to the following examples.

[0169] 〔Example 1〕Evaluation of the platelet aggregation ability of an antibody having an Fc that enhances the binding to existing FcgRIIb As shown in Table 16 of Reference Example 4, an existing FcgRIIb enhancement technique (Non-Patent Document 28) that introduces a modification to substitute Ser at position 267 and Leu at position 328 of the EU numbering of human native IgG1 with Glu and Phe, respectively, enhances the binding to FcgRIIb by 408-fold compared to IgG1, weakens the binding to FcgRIIaH to 0.51-fold, while enhancing the binding to FcgRIIaR by 522-fold. As described in the "Background Art", even if the binding to FcgRIIb is enhanced, for cells such as platelets that express only FcgRIIa, only the enhancement effect on FcgRIIa is considered to have an impact. That is, existing techniques with enhanced binding to FcgRIIaR have a risk of enhancing platelet aggregation activity and increasing the risk of developing thrombosis. To confirm this, it was verified whether platelet aggregation activity is enhanced when the binding of the antibody to FcgRIIa is actually enhanced.

[0170] As the heavy chain of a human IgG1 antibody that binds to IgE, omalizumab_VH-G1d (SEQ ID NO: 25) and as the light chain, omalizumab_VL-CK (SEQ ID NO: 26) were prepared using the method of Reference Example 1. Further, for omalizumab_VH-G1d, in order to enhance the binding activity to human FcγRIIb, omalizumab_VH-G1d-v3 in which Ser at position 267 represented by EU numbering was replaced with Glu and Leu at position 328 was replaced with Phe was prepared. Using the method of Reference Example 1, omalizumab-G1d-v3 containing omalizumab_VH-G1d-v3 as the heavy chain and omalizumab_VL-CK as the light chain was prepared. Using this antibody, the platelet aggregation ability was evaluated.

[0171] Platelet aggregation was measured using a platelet aggregation measuring device, Hemotrace 712 (LMS Co., Ltd.). First, approximately 50 mL of whole blood was collected in 4.5 mL vacuum blood collection tubes containing 0.5 mL of 3.8% sodium citrate in aliquots. The blood was centrifuged at 200 g for 15 minutes, and the supernatant was collected to obtain Platelet Rich Plasma (PRP). The obtained PRP was washed with Buffer 1 (137 mM NaCl, 2.7 mM KCl, 12 mM NaHCO3, 0.42 mM NaH2PO4, 2 mM MgCl2, 5 mM HEPES, 5.55 mM dextrose, 1.5 U / mL apyrase, 0.35% BSA), and then further replaced with Buffer 2 (137 mM NaCl, 2.7 mM KCl, 12 mM NaHCO3, 0.42 mM NaH2PO4, 2 mM MgCl2, 5 mM HEPES, 5.55 mM dextrose, 2 mM CaCl2, 0.35% BSA) to prepare washed platelets at approximately 300,000 cells per 1 μL. A measurement cuvette containing a stir bar was set in the platelet aggregation measuring device, and 156 μL of the washed platelets was dispensed therein. Inside the instrument, the cuvette was maintained at 37.0°C, and the stir bar stirred the platelets at 1000 rpm. 44 μL of an immunocomplex of omalizumab-G1d-v3 and IgE at a molar ratio of 1:1 (prepared so that the final concentrations were 600 μg / mL and 686 μg / mL, respectively) was added thereto and reacted for 5 minutes. Further, adenosine diphosphate (ADP, SIGMA) at a concentration that does not cause secondary aggregation was added to confirm whether aggregation was enhanced.

[0172] The results for each donor of the gene polymorphism (H / H or R / H) of FcγRIIa obtained in this assay are shown in FIGS. 1 and 2. From the results in FIG. 1, it was shown that in the polymorphism (R / H) of FcγRIIa, platelet aggregation was enhanced when the immunocomplex was added. On the other hand, as shown in FIG. 2, in the FcγRIIa polymorphism (H / H), platelet aggregation was not enhanced.

[0173] Next, platelet activation was evaluated using activation markers. Platelet activation can be measured by an increase in the expression of activation markers such as CD62p (p-selectin) or activated integrin on the platelet membrane surface. After adding 2.3 μL of immune complex to 7.7 μL of washed platelets adjusted by the method described above and reacting at room temperature for 5 minutes, ADP was further added to a final concentration of 30 μM to induce activation, and it was confirmed whether the activation by ADP was enhanced by the immune complex. As a negative control, a sample to which phosphate buffer (pH 7.4, Gibco) was added instead of the immune complex was used. Each sample after the reaction was stained with a PE-labeled anti-CD62 antibody (BECTON DICKINSON), a PerCP-labeled anti-CD61 antibody, and a FITC-labeled PAC-1 antibody (BD bioscience), and the fluorescence intensity of each was measured using a flow cytometer (FACS CantoII, BD bioscience).

[0174] The results of CD62p expression obtained by this assay method are shown in Figure 3, and the results of activated integrin expression are shown in Figure 4. The washed platelets used were obtained from one healthy subject with the R / H polymorphism of FcγRIIa. Both CD62p and activated integrin induced to be expressed on the platelet membrane surface by ADP stimulation were enhanced in the presence of the immune complex.

[0175] From these results, in an antibody having an Fc that enhances the binding to existing human FcγRIIb with Ser at position 267 and Leu at position 328 replaced with Glu and Phe, respectively, represented by EU numbering in the Fc of IgG1, when the 131st amino acid among the genetic polymorphisms of FcγRIIa is R, it was revealed that the platelet aggregation activity is enhanced compared to the case where the 131st amino acid is H. That is, it was suggested that an antibody having an Fc that enhances the binding to existing human FcγRIIb has a risk of increasing the risk of thrombosis onset due to platelet aggregation in humans having the FcγRIIa R type, and the usefulness of an Fc that more selectively enhances the binding to FcγRIIb and overcomes this problem was revealed.

[0176] [Example 2] Preparation of a Variant with Enhanced Binding to FcγRIIb As shown in Example 1, when enhancing the binding to FcγRIIb, it is necessary to enhance the binding to FcγRIIb while suppressing the binding to other active FcγRs as much as possible. Therefore, modifications that have the effect of enhancing the binding to FcγRIIb or increasing selectivity were combined, and the preparation of a variant with further enhanced binding or selectivity to FcγRIIb was investigated. Specifically, based on the P238D modification, which shows excellent effects in both enhancing the binding to FcγRIIb and improving selectivity, the modifications that showed effects when combined with P238D in Reference Example 6, Reference Example 8, and Reference Example 9 were further combined. The variable region of IL6R-H (SEQ ID NO: 18), which is the variable region of an antibody against human interleukin 6 receptor disclosed in WO2009 / 125825, was used as the antibody H-chain variable region. As the antibody H-chain constant region, IL6R-G1d (SEQ ID NO: 19) having G1d with the C-terminal Gly and Lys of human IgG1 removed was prepared. Furthermore, IL6R-B3 (SEQ ID NO: 23) with K439E introduced into IL6R-G1d was prepared. On the other hand, a variant was prepared by combining E233D, L234Y, G237D, S267Q, H268D, P271G, Y296D, K326D, K326A, A330R, A330K, which are modifications that showed effects when combined with P238D in Reference Example 6, Reference Example 8, and Reference Example 9. IL6R-L (SEQ ID NO: 21) was commonly used as the antibody L-chain. According to the method of Reference Example 1, antibodies were expressed and purified from these variants, and the binding to each FcγR (FcγRIa, FcγRIIa H-type, FcγRIIa R-type, FcγRIIb, FcγRIIIa V-type) was evaluated by the method of Reference Example 2.

[0177] The KD values for each FcγR of each variant are shown in Table 1. The modifications in the table indicate the modifications introduced to IL6R-B3 (SEQ ID NO: 23). However, IL6R-B3 / IL6R-L used as the template when preparing each variant is indicated as *. In the table, "KD(IIaR) / KD(IIb)" is the value obtained by dividing the KD value for FcγRIIaR of each variant by the KD value for FcγRIIb of each variant, and the larger this value, the higher the selectivity for FcγRIIb. "KD(IIb) of the parental polypeptide / KD(IIb) of the modified polypeptide" refers to the value obtained by dividing the KD value for FcγRIIb of IL6R-B3 / IL6R-L by the KD value for FcγRIIb of each variant. Also, "KD(IIaR) of the parental polypeptide / KD(IIaR) of the modified polypeptide" refers to the value obtained by dividing the KD value for FcγRIIaR of IL6R-B3 / IL6R-L by the KD value for FcγRIIaR of each variant. Note that the cells shaded in gray in Table 1 had weak binding of FcγR to IgG and were judged to be unable to be correctly analyzed by kinetic analysis. Therefore, the value calculated using the formula of 〔Formula 2〕 TIFF0007698004000001.tif9156 is shown.

[0178]

Table 1

[0179] IL6R-G1d / IL6R-L having the sequence of human native IgG1, when the binding to each FcgR of IL6R-B3 / IL6R-L with K439E introduced therein was set to 1, the binding to FcgRIa was 1.3-fold, the binding to FcgRIIaR was 1.1-fold, the binding to FcgRIIaH was 1.1-fold, the binding to FcgRIIb was 1.2-fold, and the binding to FcgRIIIaV was 0.9-fold. The binding to all these FcgRs was equivalent to that of IL6R-G1d / IL6R-L. Therefore, comparing the binding of each variant with IL6R-B3 / IL6R-L before the introduction of the modification is considered to be equivalent to comparing each variant with IL6R-G1d / IL6R-L having the sequence of human native IgG1. Thus, in the following examples, the binding activity of each variant was compared with IL6R-B3 / IL6R-L before the introduction of the modification.

[0180] From Table 1, for all variants, the affinity for FcgRIIb was improved compared to IL6R-B3 before the introduction of the modification, being 2.6-fold for the lowest IL6R-BF648 / IL6R-L and 147.6-fold for the highest IL6R-BP230 / IL6R-L. Also, the value of KD(IIaR) / KD(IIb) indicating selectivity was 10.0 for the lowest IL6R-BP234 / IL6R-L and 32.2 for the highest IL6R-BP231 / IL6R-L, and the selectivity of all variants was improved compared to 0.3 of IL6R-B3 / IL6R-L before the introduction of the modification. In addition, for all variants, the binding to FcgRIa, FcgRIIaH, and FcgRIIIaV was lower than that of IL6R-B3 / IL6R-L before the introduction of the modification.

[0181] Example 3: X-ray crystallographic analysis of the complex of Fc with enhanced binding to FcγRIIb and the extracellular region of FcγRIIb and the complex with the extracellular region of FcγRIIaR In Example 2, the variant IL6R-BP230 / IL6R-L with the most enhanced binding to FcgRIIb had its binding to FcgRIIb enhanced approximately 150-fold compared to IL6R-B3 / IL6R-L before the introduction of the modification, and the binding to the FcgRIIaR type was also suppressed to about 1.9-fold. Therefore, IL6R-BP230 / IL6R-L is a variant excellent in both binding to FcgRIIb and selectivity. However, in order to produce an even more excellent variant, it is preferable that the binding to FcgRIIb can be further enhanced after suppressing the binding to FcgRIIaR as much as possible.

[0182] As shown in FIG. 28 of Reference Example 7, in the Fc containing the P238D modification, a strong electrostatic interaction is formed between Asp at position 270 of the EU numbering of CH2 domain B and Arg at position 131 of FcγRIIb. However, while this 131st residue is His in FcγRIIIa and FcγRIIaH type, it is the same Arg as FcγRIIb in the FcγRIIaR type. As a result, there is no difference in the interaction at this part, which is a factor making it difficult to achieve selectivity with the FcγRIIaR type.

[0183] On the other hand, FcγRIIa and FcγRIIb have 93% identity in the amino acid sequence of the extracellular region and have very high homology. Analyzing the crystal structure of the complex of the extracellular region of natural type IgG1 Fc (hereinafter Fc (WT)) and FcγRIIaR type (J. Imunol. 2011, 187, 3208 - 3217), only three amino acids (Gln127, Leu132, Phe160) were found to be different between the FcγRIIaR type and FcγRIIb near the interaction interface, and considerable difficulty was expected in improving the selectivity with the FcγRIIaR type. Therefore, in order to further enhance the binding activity to FcγRIIb and improve selectivity, it is necessary to simultaneously obtain the three-dimensional structures of the complex of Fc with enhanced binding to FcγRIIb and the extracellular region of FcγRIIb, as well as the three-dimensional structure of the complex with the extracellular region of FcγRIIaR type, which is expected to be the most difficult to improve selectivity. After clarifying the subtle differences in interactions due to receptor differences in three-dimensional structure, it was considered necessary to carefully examine the amino acid mutations to be introduced. Therefore, X-ray crystallographic analysis of the complex of Fc(P208), which is a variant that was the basis for the preparation of IL6R-BP230 / IL6R-L and is the Fc of IL6R-BP208 / IL6R-L (prepared in Reference Example 9) excluding the modification of K439E, with the extracellular region of FcγRIIb and the extracellular region of FcγRIIaR type was performed.

[0184] 3-1. X-ray Crystallographic Analysis of the Complex of Fc(P208) and the Extracellular Region of FcγRIIb As a result of the structural analysis, the three-dimensional structure of the Fc(P208) / FcγRIIb extracellular region complex was determined at a resolution of 2.81 Å, and the structure obtained as a result of the analysis is shown in Figure 5. The extracellular region of FcγRIIb is sandwiched and bound between the two Fc CH2 domains, which is similar to the three-dimensional structures of the complexes of the natural-type IgG Fc, Fc(WT), with the extracellular regions of FcγRIIIa (Proc. Natl. Acad. Sci. USA, 2011, 108, 12669-126674), FcγRIIIb (Nature, 2000, 400, 267-273; J. Biol. Chem. 2011, 276, 16469-16477), and FcγRIIa that have been analyzed so far.

[0185] However, upon closer inspection, in the Fc(P208) / FcγRIIb extracellular domain complex, due to the effects of the introduction of the G237D and P238D mutations, the loop structure from position 233 to 239 following the hinge region of Fc CH2 domain A changed compared to the Fc(WT) / FcγRIIa R-type extracellular domain complex (Figure 6). As a result, a strong hydrogen bond was observed to form between the main chain of Asp at position 237 of Fc(P208) in EU numbering and the side chain of Tyr at position 160 of FcγRIIb (Figure 7). Since this Tyr160 is Phe in both the H-type and R-type of FcγRIIa and hydrogen bond formation is impossible, this hydrogen bond was considered to make an important contribution to the acquisition of selectivity in terms of improving the binding activity to FcγRIIb and reducing the binding activity to FcγRIIa.

[0186] On the other hand, the side chain of Asp at position 237 of Fc(P208) in EU numbering itself did not form any particularly prominent interaction with FcγRIIb, nor was any interaction with residues inside Fc observed. Around this Asp at position 237 in EU numbering, Ile at position 332, Glu at position 333, and Lys at position 334 in EU numbering within Fc were located in the vicinity (Figure 8). By substituting these sites with hydrophilic residues to form an interaction with the side chain of Asp at position 237 in EU numbering and stabilizing this loop structure, it was considered possible to lead to a reduction in the entropic energy loss associated with the formation of the hydrogen bond with Tyr at position 160 of FcγRIIb, resulting in an increase in the binding free energy, that is, an improvement in the binding activity.

[0187] When comparing the X-ray crystal structure of the Fc(P238D)-FcγRIIb extracellular region complex with the P238D modification shown in Reference Example 7 and the X-ray crystal structure of the Fc(P208)-FcγRIIb extracellular region complex, Fc(P208) contains five new mutations compared to Fc(P238D), but most of them are limited to changes at the side chain level. However, in the CH2 domain B of Fc, the modification of Pro at position 271 in the EU numbering to Gly resulted in a positional change at the main chain level, and at the same time, a structural change occurred in the loop of EU numbering 266-270 in front (Figure 9). As shown in Reference Example 8, in Fc(P238D), when Asp at position 270 in the EU numbering forms a strong electrostatic interaction with Arg at position 131 of FcγRIIb, it has been suggested that there may be steric chemical stress on this Pro part at position 271 in the EU numbering. The structural change observed by the introduction of Gly at position 271 in the EU numbering this time is considered to be the result of the elimination of the structural strain accumulated in the Pro part before the modification, and it is speculated that the eliminated part led to an improvement in the binding free energy with FcγRIIb, that is, an improvement in the binding activity.

[0188] Furthermore, due to the structural change in the loop of EU numbering 266-271, it was confirmed that Arg at position 292 in the EU numbering was undergoing a structural change while taking two states. At that time, Arg at position 292 in the EU numbering forms an electrostatic interaction with Asp at position 268 in the EU numbering, which is one of the other modified residues in Fc(P208) (Figure 9), and it is considered possible that it contributes to the stabilization of this loop structure. The electrostatic interaction formed between Asp at position 270 in the EU numbering in this loop and Arg at position 131 of FcγRIIb greatly contributes to the binding activity with FcγRIIb. Therefore, the introduction of the H268D modification may have led to an increase in the binding free energy, that is, an improvement in the binding activity, by stabilizing this loop structure in the conformation during the binding of FcγRIIb and reducing the entropic energy loss associated with the binding.

[0189] Furthermore, based on the results of this structural analysis, when examining the possibility of further modification aiming at improving the activity, Ser at EU numbering 239 was found as one of the candidates for the modification introduction site. As shown in Fig. 10, Ser at EU numbering 239 of this CH2 domain B is located in the direction in which Lys at position 117 of FcγRIIb extends in the most natural form structurally. However, in this analysis, the electron density of Lys at position 117 of FcγRIIb was not confirmed, and since this Lys residue does not have a certain structure, at present, the involvement of this Lys residue in the interaction with Fc (P208) is considered limited. However, when Ser at EU numbering 239 of this CH2 domain B is modified to Asp or Glu having a negative charge, an electrostatic interaction can be expected between Lys at position 117 of FcγRIIb having a positive charge, and as a result, an improvement in the binding activity to FcγRIIb was expected.

[0190] On the other hand, looking at the structure of Ser at EU numbering 239 in CH2 domain A, this amino acid side chain forms a hydrogen bond with the main chain of Gly at EU numbering 236, continues from the hinge region, and was considered to stabilize the loop structure from position 233 to 239 including Asp at EU numbering 237 that forms a hydrogen bond with the side chain of FcγRIIb Tyr160 (Fig. 7). Stabilizing this loop structure in the conformation at the time of binding reduces the entropic energy loss associated with binding, and as a result, leads to an increase in the binding free energy, that is, an improvement in the binding activity. On the other hand, when Ser at EU numbering 239 of this CH2 domain A is modified to Asp or Glu, the hydrogen bond with the Gly main chain at EU numbering 239 is lost, and there is also a possibility of causing an electrostatic repulsion with Asp at EU numbering 265 existing immediately nearby, and it was considered that a large destabilization of the loop structure might occur. The energy of this destabilization acts to reduce the binding free energy with FcγRIIb, and as a result, there is a possibility of causing a decrease in the binding activity.

[0191] [Expression and Purification of Fc(P208)] Fc(P208) was prepared as follows. First, IL6R-P208 was prepared by changing Glu at the 439th position of EU numbering of IL6R-BP208 (SEQ ID NO: 24) to Lys, which is the sequence of natural human IgG1. Next, Cys at the 220th position of EU numbering was replaced with Ser, and the gene sequence Fc(P208) obtained by cloning its C-terminus from Glu at the 216th position of EU numbering by PCR was used to prepare, express, and purify an expression vector according to the method described in Reference Example 1. Note that Cys at the 220th position of EU numbering forms a disulfide bond with the Cys of the L chain in normal IgG1. However, when preparing only Fc, the L chain is not co-expressed, so it was replaced with Ser to avoid unnecessary disulfide bond formation.

[0192] [Expression and purification of the extracellular region of FcγRIIb] It was prepared according to the method of Reference Example 2.

[0193] [Purification of the complex of Fc(P208) and the extracellular region of FcγRIIb] To 1.5 mg of the FcγRIIb extracellular region sample obtained for crystallization, 0.15 mg of Endo F1 (Protein Science 1996, 5, 2617-2622) expressed and purified by Escherichia coli as a fusion protein with glutathione S-transferase was added, and the mixture was allowed to stand at room temperature for 3 days under buffer conditions of 0.1 M Bis-Tris pH 6.5 to cleave, leaving N-acetylglucosamine directly bound to Asn in the N-type sugar chain. Next, the FcγRIIb extracellular region sample subjected to this sugar chain cleavage treatment was concentrated using an ultrafiltration membrane with a molecular weight cut-off of 5000 MWCO and purified by gel filtration column chromatography (Superdex200 10 / 300) equilibrated with 20 mM HEPES pH 7.5, 0.1 M NaCl. Further, Fc (P208) was added to the obtained sugar chain-cleaved FcγRIIb extracellular region fraction in a molar ratio such that FcγRIIb extracellular region was slightly in excess, and after concentration using an ultrafiltration membrane with a molecular weight cut-off of 10000 MWCO, it was purified by gel filtration column chromatography (Superdex200 10 / 300) equilibrated with 25 mM HEPES pH 7.5, 0.1 M NaCl to obtain a sample of the Fc(P208) / FcγRIIb extracellular region complex.

[0194] [Crystallization of the Fc(P208) / FcγRIIb Complex Extracellular Region Complex] A sample of the Fc(P208) / FcγRIIb extracellular domain complex was concentrated to approximately 10 mg / ml using a 10,000 MWCO ultrafiltration membrane, and crystallization was performed using the hanging drop vapor diffusion method in combination with the Seeding method. For crystallization, a VDXm plate (Hampton Research) was used. A crystallization drop was prepared by mixing the reservoir solution (0.1 M Bis-Tris pH 6.5, 19% (w / v) PEG3350, 0.2 M Potassium Phosphate dibasic) with the crystallization sample at a ratio of reservoir solution:crystallization sample = 0.85 μl:0.85 μl. Then, 0.15 μl of a dilution solution prepared from a seed crystal solution obtained by crushing crystals of the same complex under the same conditions using a Seed Bead (Hampton Research) was added thereto. The well containing the reservoir was sealed and left standing at 20°C, and plate-like crystals were successfully obtained.

[0195] [Measurement of X-ray diffraction data from crystals of the Fc(P208) / FcγRIIb extracellular domain complex] One single crystal of the obtained Fc(P208) / FcγRIIb extracellular domain complex was immersed in a solution of 0.1 M Bis-Tris pH 6.5, 24% (w / v) PEG3350, 0.2 M dibasic potassium phosphate, 20% (v / v) ethylene glycol. Then, it was scooped up with a pin with a tiny nylon loop, frozen in liquid nitrogen, and X-ray diffraction data were measured at BL32XU of Spring-8. During the measurement, the frozen state was maintained by always placing it in a nitrogen gas stream at -178°C. A total of 300 X-ray diffraction images were collected while rotating the crystal by 0.6° each time using a CCD detector MX-225HE (RAYONIX) equipped on the beamline. For the determination of the lattice constant, indexing of diffraction spots, and processing of diffraction data from the obtained diffraction images, the programs Xia2 (J. Appl. Cryst. 2010, 43, 186-190), XDS Package (Acta Cryst. 2010, D66, 125-132), and Scala (Acta Cryst. 2006, D62, 72-82) were used, and finally, diffraction intensity data up to a resolution of 2.81 Å were obtained. This crystal belonged to the space group C2221, with lattice constants a = 156.69 Å, b = 260.17 Å, c = 56.85 Å, α = 90°, β = 90°, and γ = 90°.

[0196] [X-ray Crystal Structure Analysis of Fc(P208) / FcγRIIb Extracellular Domain Complex] Structure determination was carried out by the molecular replacement method using the program Phaser (J. Appl. Cryst. 2007, 40, 658-674). From the size of the obtained crystal lattice and the molecular weight of the Fc(P208) / FcγRIIb extracellular domain complex, the number of complexes in the asymmetric unit was expected to be one. From the structural coordinates of PDB code: 3SGJ, which is the crystal structure of the Fc(WT) / FcγRIIIa extracellular domain complex, the amino acid residue portions of chain A positions 239-340 and chain B positions 239-340 were taken out as separate coordinates and used as search models for the CH2 domain of Fc, respectively. Similarly, from the structural coordinates of PDB code: 3SGJ, the amino acid residue portions of chain A positions 341-444 and chain B positions 341-443 were taken out as one coordinate and used as a search model for the Fc CH3 domain. Finally, from the structural coordinates of PDB code: 2FCB, which is the crystal structure of the FcγRIIb extracellular domain, the amino acid residue portion of chain A positions 6-178 was taken out and used as a search model for Fc(P208). When attempting to determine the orientation and position within the crystal lattice of each search model for the Fc CH3 domain, the FcγRIIb extracellular domain, and the Fc CH2 domain using the rotation function and the translation function, the positioning of one of the CH2 domains was unsuccessful. Therefore, referring to the crystal structure of the Fc(WT) / FcγRIIIa extracellular domain complex, the position of the last CH2 domain was determined for the electron density map calculated based on the phases calculated from the remaining three parts, and an initial model of the Fc(P208) / FcγRIIb extracellular domain complex crystal structure was obtained. Rigid body refinement was performed on the obtained initial model by moving the two Fc CH2 domains, the two Fc CH3 domains, and the FcγRIIb extracellular domain. At this point, for the diffraction intensity data at 25-3.0 Å, the crystallographic reliability factor R value was 42.6% and the Free R value was 43.7%.Furthermore, structure refinement was carried out using the program REFMAC5 (Acta Cryst. 2011, D67, 355 - 367), and model refinement was performed with the program Coot (Acta Cryst. 2010, D66, 486 - 501) while looking at the electron density maps with coefficients of 2Fo - Fc and Fo - Fc calculated from the experimentally determined structure factors Fo and the structure factors Fc calculated from the model as well as the phases calculated from the model. The model was refined by repeating these processes. Finally, water molecules were incorporated into the model based on the electron density maps with coefficients of 2Fo - Fc and Fo - Fc, and refinement was carried out. Ultimately, using 27,259 diffraction intensity data with a resolution of 25 - 2.81 Å, for a model containing 4,786 non - hydrogen atoms, the crystallographic reliability factor R value was 24.5% and the Free R value was 28.2%.

[0197] 3 - 2. X - ray crystallographic analysis of the complex of Fc(P208) and the extracellular region of FcγRIIaR As a result of structural analysis, the crystal structure of the Fc(P208) / FcγRIIaR extracellular domain complex was determined at a resolution of 2.87 Å. When the crystal structure of the Fc(P208) / FcγRIIaR extracellular domain complex was compared with the crystal structure of the Fc(P208) / FcγRIIb extracellular domain complex shown in Example 3-1, reflecting the very high amino acid homology of both receptors, almost no difference was found in the overall structure (Figure 11). However, when looking at the structure in detail at the electron density level, differences that might be useful for improving selectivity were found. In the FcγRIIaR type, the 160th residue is Phe instead of Tyr, and as shown in Figure 12, a hydrogen bond cannot be formed between the main chain of the 237th amino acid residue of Fc CH2 domain A that existed when binding Fc containing the P238D modification to FcgRIIb. This is considered to be the main factor for improving the selectivity with the FcγRIIaR type due to the introduction of the P238D modification. Furthermore, when comparing at the electron density level, in the complex with FcγRIIb, the electron density of the side chains of Leu at EU numbering 235 and Leu at EU numbering 234 in Fc CH2 domain A can be confirmed, while in the complex with the FcγRIIaR type, the electron density of these side chains is not clear, and the loop in the vicinity of EU numbering 237 is considered to be fluctuating due to the decrease in the interaction with the FcgRIIaR type in this vicinity. On the other hand, when comparing the structure of the same region for CH2 domain B (Figure 13), the electron density up to Asp at EU numbering 237 can be confirmed in the complex structure with FcγRIIb, while in the complex with the FcγRIIaR type, the electron density can be confirmed up to about 3 residues before Asp at EU numbering 237, and it is considered that the interaction is formed using a wider region compared to when binding to FcgRIIb. From the above, it was suggested that in the region from the 234th to the 238th of the EU numbering of Fc(P208), the contribution of the CH2 domain A side is larger when binding to FcγRIIb, and the contribution of the CH2 domain B side is larger when binding to FcγRIIaR.

[0198] [Expression and Purification of FcγRIIaR Extracellular Domain] It was prepared according to the method of Reference Example 2.

[0199] [Purification of Fc (P208) / FcγRIIaR extracellular domain complex] To 1.5 mg of the purified FcγRIIa R extracellular domain sample, 0.15 mg of Endo F1 (Protein Science 1996, 5, 2617 - 2622) expressed and purified by Escherichia coli as a fusion protein with glutathione S-transferase, 20 μl of 5 U / ml Endo F2 (QA-bio), and 20 μl of 5 U / ml Endo F3 (QA-bio) were added. After standing at room temperature for 9 days under the buffer condition of 0.1 M Na Acetate pH 4.5, further, 0.07 mg of Endo F1 (Protein Science 1996, 5, 2617 - 2622) expressed and purified by Escherichia coli as a fusion protein with glutathione S-transferase, 7.5 μl of 5 U / ml Endo F2 (QA-bio), and 7.5 μl of 5 U / ml Endo F3 (QA-bio) were added and left standing for another 3 days to cleave, leaving N-acetylglucosamine directly bound to Asn at the N-type sugar chain. Next, the FcγRIIaR extracellular domain sample subjected to this sugar chain cleavage treatment was concentrated by an ultrafiltration membrane with a molecular weight cut-off of 10000 MWCO and purified by gel filtration column chromatography (Superdex200 10 / 300) equilibrated with 25 mM HEPES pH 7, 0.1 M NaCl. Further, Fc (P208) was added to the obtained sugar chain-cleaved FcγRIIaR extracellular domain fraction so that the molar ratio was slightly in excess of the FcγRIIaR extracellular domain. After concentration by an ultrafiltration membrane with a molecular weight cut-off of 10000 MWCO, it was purified by gel filtration column chromatography (Superdex200 10 / 300) equilibrated with 25 mM HEPES pH 7, 0.1 M NaCl to obtain a sample of the Fc (P208) / FcγRIIaR extracellular domain complex.

[0200] [Crystallization of Fc(P208) / FcγRIIaR extracellular domain complex] A sample of the Fc(P208) / FcγRIIa R extracellular domain complex was concentrated to approximately 10 mg / ml using a 10,000 MWCO ultrafiltration membrane, and crystallization was carried out by the sitting drop vapor diffusion method. For the reservoir solution of 0.1 M Bis-Tris pH 7.5, 26% (w / v) PEG3350, 0.2 M Ammonium Sulfate, the crystallization drop was prepared by mixing the reservoir solution:crystallization sample = 0.8 μl:1.0 μl. After sealing with a seal and allowing it to stand at 20 °C, plate-like crystals were successfully obtained.

[0201] [Measurement of X-ray diffraction data from crystals of the Fc(P208) / FcγRIIaR extracellular domain complex] One single crystal of the obtained Fc(P208) / FcγRIIaR extracellular domain complex was immersed in a solution of 0.1 M Bis-Tris pH 7.5, 27.5% (w / v) PEG3350, 0.2 M Ammonium Sulfate, 20% (v / v) Glycerol. Then, it was scooped up with a pin with a tiny nylon loop together with the solution and frozen in liquid nitrogen. X-ray diffraction data were measured at the Photon Factory BL-17A, a synchrotron radiation facility of the High Energy Accelerator Research Organization. During the measurement, the frozen state was maintained by always placing it in a nitrogen gas stream at -178 °C. A total of 225 X-ray diffraction images were collected while rotating the crystal by 0.6° each using a CCD detector Quantum 315r (ADSC) equipped with the beamline. For the determination of the lattice constant, indexing of the diffraction spots, and processing of the diffraction data from the obtained diffraction images, the programs Xia2 (J. Appl. Cryst. 2010, 43, 186 - 190), XDS Package (Acta Cryst. 2010, D66, 125 - 132), and Scala (Acta Cryst. 2006, D62, 72 - 82) were used, and finally diffraction intensity data up to a resolution of 2.87 Å were obtained. This crystal belonged to the space group C2221, with lattice constants a = 154.31 Å, b = 257.61 Å, c = 56.19 Å, α = 90°, β = 90°, γ = 90°.

[0202] [X-ray Crystallographic Analysis of the Fc(P208) / FcγRIIaR Extracellular Domain Complex] Structure determination was performed by the molecular replacement method using the program Phaser (J. Appl. Cryst. 2007, 40, 658-674). From the size of the obtained crystal lattice and the molecular weight of the Fc(P208) / FcγRIIaR extracellular domain complex, the number of complexes in the asymmetric unit was expected to be one. Using the crystal structure of the Fc(P208) / FcγRIIb extracellular domain complex obtained in Example 3-1 as a search model, the orientation and position within the crystal lattice were determined from the rotation function and translation function. Rigid body refinement was then performed on the obtained initial model by moving the CH2 domains of the two Fcs, the CH3 domains of the two Fcs, and the FcγRIIaR extracellular domain. At this point, for the diffraction intensity data of 25-3.0 Å, the crystallographic reliability factor R value was 38.4% and the Free R value was 38.0%. Further structure refinement using the program REFMAC5 (Acta Cryst. 2011, D67, 355-367), and model modification while observing the electron density map calculated with 2Fo-Fc and Fo-Fc, which are based on the experimentally determined structure factor Fo, the structure factor Fc calculated from the model, and the phase calculated from the model, were performed using the program Coot (Acta Cryst. 2010, D66, 486-501), and the model was refined by repeating these steps. Finally, water molecules were incorporated into the model based on the electron density map with 2Fo-Fc and Fo-Fc as coefficients, and refinement was performed. Ultimately, using 24,838 diffraction intensity data with a resolution of 25-2.87 Å, for the model containing 4,758 non-hydrogen atoms, the crystallographic reliability factor R value was 26.3% and the Free R value was 29.8%.

[0203] [Example 4] Fc Variant with Modification Sites Determined Based on the Crystal Structure As shown in Example 3, in the CH2 domain B of the FcγRIIb-binding enhanced variant Fc(P208), it was suggested that Asp at position 268 in the EU numbering forms an electrostatic interaction with Arg at position 292 in the EU numbering as a result of the surrounding structural changes accompanying the introduction of the P271G modification (Figure 9). It is conceivable that the formation of this interaction contributed to the stabilization of the loop structure at positions 266 - 271 in the EU numbering, and as a result, contributed to the enhanced binding to FcγRIIb. Therefore, by modifying Asp at position 268 in the EU numbering to Glu, the electrostatic interaction with Arg at position 292 in FcγRIIb was strengthened, and by further stabilizing this loop structure, it was examined whether it would lead to enhanced interaction with FcγRIIb. Also, as shown in Figure 8, Tyr at position 160 in the EU numbering of FcγRIIb forms a hydrogen bond with the main chain of Asp at position 237 in the EU numbering of the CH2 domain A of Fc(P208) and plays an important role in the binding to FcγRIIb. On the other hand, although the side chain portion of Asp at position 237 in the EU numbering does not form a specific interaction, Ile at position 332, Glu at position 333, and Lys at position 334 in the EU numbering are located in the vicinity within the molecule. By substituting these sites with hydrophilic residues to strengthen the interaction with Asp at position 237 in the EU numbering and stabilizing the loop structure in the vicinity of this residue, it was also verified whether the interaction with Tyr at position 160 in FcγRIIb would be enhanced.

[0204] Variants were prepared by introducing H268E, I332T, I332S, I332E, I332K, E333K, E333R, E333S, E333T, K334S, K334T, and K334E, respectively, into IL6R-BP230 / IL6R-L (SEQ ID NO: 27 / SEQ ID NO: 21) prepared in Example 2. IL6R-L (SEQ ID NO: 21) was commonly used as the antibody light chain. Antibodies were expressed and purified from these variants according to the method of Reference Example 1, and the binding to each FcγR (FcγRIa, FcγRIIa H-type, FcγRIIa R-type, FcγRIIb, FcγRIIIa V-type) was evaluated by the method of Reference Example 2.

[0205] The KD values for each FcgR of each variant are shown in Table 2. The modifications in the table indicate the modifications introduced to IL6R-B3 (SEQ ID NO: 23). However, for IL6R-B3 / IL6R-L used as a template when preparing IL6R-BP230, it is indicated as *. The KD (IIb) of the parental polypeptide / KD (IIb) of the modified polypeptide in the table refers to the value obtained by dividing the KD value for FcgRIIb of IL6R-B3 / IL6R-L by the KD value for FcgRIIb of each variant. Also, the KD (IIaR) of the parental polypeptide / KD (IIaR) of the modified polypeptide refers to the value obtained by dividing the KD value for FcgR IIaR of IL6R-B3 / IL6R-L by the KD value for FcgR IIaR of each variant. KD (IIaR) / KD (IIb) is the value obtained by dividing the KD for FcgRIIaR of each variant by the KD for FcgRIIb of each variant, and the larger this value, the higher the selectivity for FcgRIIb. Note that the cells shaded in gray in Table 2 had weak binding of FcgR to IgG and were judged to be unable to be correctly analyzed by kinetic analysis. Therefore, the value calculated using the formula of [Formula 2] TIFF0007698004000003.tif9156 is used.

[0206]

Table 2

[0207] For IL6R-BP264 / IL6R-L with H268E introduced, IL6R-BP465 / IL6R-L with E333K introduced, IL6R-BP466 / IL6R-L with E333R introduced, and IL6R-BP470 with E333T introduced, compared with IL6R-BP230 / IL6R-L, both the binding to FcgRIIb and the selectivity were improved. Also, for IL6R-BP391 / IL6R-L with I332T introduced, although the selectivity for FcgRIIb decreased compared with IL6R-BP230 / IL6R-L, the binding to FcgRIIb was improved.

[0208] [Example 5] Introduction of Comprehensive Modifications around EU Number 271 When comparing the X-ray crystal structure of the Fc(P238D) and FcγRIIb extracellular region complex with the P238D modification and the X-ray crystal structure of the Fc(P208) and FcγRIIb extracellular region complex, the most structurally significant changes are in the structure near EU number 271 (Figure 9). As shown in Reference Example 8, in Fc (P238D), when the 270th Asp of the EU numbering forms a strong electrostatic interaction with the 131st Arg of FcγRIIb, it is suggested that there may be steric stress on the 271st Pro part of the EU numbering. In the structure of Fc (P208) / FcγRIIb, due to the introduction of the P271G modification, a positional change at the main chain level occurs to relieve this structural strain. As a result, it is considered that the structure near EU number 271 has changed significantly. If modifications can be successfully introduced to further stabilize this changed structure, the entropic energy loss associated with the formation of the electrostatic interaction with the 131st Arg of FcγRIIb can be further reduced, which may lead to an improvement in binding activity. Therefore, comprehensive modifications were introduced around EU number 271 to search for modifications that show an effect of enhancing binding or improving selectivity for FcgRIIb. As a template for introducing comprehensive modifications, IL6R-BP267 (SEQ ID NO: 29) with E233D, G237D, P238D, H268E, and P271G introduced with respect to IL6R-B3 (SEQ ID NO: 23) was prepared and used. For IL6R-BP267, the amino acids at positions 264, 265, 266, 267, 269, and 272 in EU numbering were each substituted with the original amino acid and 18 amino acids excluding Cys. IL6R-L (SEQ ID NO: 21) was commonly used as the antibody L chain. According to the method of Reference Example 1, antibodies were expressed and purified from these variants, and the binding to each FcgR (FcgRIa, FcgRIIa H-type, FcgRIIa R-type, FcgRIIb, FcgRIIIa V-type) was evaluated by the method of Reference Example 2. Among the obtained variants, those with enhanced binding to FcgRIIb or improved selectivity for FcgRIIb compared to IL6R-BP267 / IL6R-L before introducing the modifications were summarized in Table 3.

[0209] [Table 3]

[0210] The KD values for each FcgR of each variant are shown in Table 3. The "modifications added to IL6R-BP267" in the table indicate the modifications introduced to IL6R-BP267 (SEQ ID NO: 29) used as a template. However, for IL6R-B3 / IL6R-L which was the original when preparing IL6R-B3, it is indicated with *. The KD(IIb) of the parental polypeptide / KD(IIb) of the modified polypeptide in the table refers to the value obtained by dividing the KD value for FcgRIIb of IL6R-B3 / IL6R-L by the KD value for FcgRIIb of each variant. Also, the KD(IIaR) of the parental polypeptide / KD(IIaR) of the modified polypeptide refers to the value obtained by dividing the KD value for FcgR IIaR of IL6R-B3 / IL6R-L by the KD value for FcgR IIaR of each variant. KD(IIaR) / KD(IIb) is the value obtained by dividing the KD for FcgRIIaR of each variant by the KD for FcgRIIb of each variant, and the larger this value, the higher the selectivity for FcgRIIb. Note that the cells shaded in gray in Table 3 have weak binding of FcgR to IgG and were judged to be unable to be correctly analyzed by kinetic analysis. Therefore, the [Formula 2] It is the value calculated using the formula of TIFF0007698004000006.tif9156.

[0211] All of the variants shown in Table 3 maintained or decreased their binding to FcgRIa, FcgRIIaH, and FcgRIIIaV compared to IL6R-B3 / IL6R-L. Also, the variants with S267A, V264I, E269D, S267E, V266F, S267G, and V266M added to IL6R-BP267 / IL6R-L respectively had enhanced binding to FcgRIIb compared to IL6R-BP267 / IL6R-L before the modification. In addition, the variants with S267A, S267G, E272M, E272Q, D265E, E272D, E272N, V266L, E272I, and E272F added to IL6R-BP267 / IL6R-L respectively had an increased value of KD(IIaR) / KD(IIb) compared to IL6R-BP267 / IL6R-L before the modification, indicating an effect of improving the selectivity for FcgRIIb.

[0212] 〔Example 6〕Enhanced binding to FcgRIIb by introducing modifications into the CH3 region It has been reported that the modification of substituting Pro at the 396th position of EU numbering with Leu enhances the binding to FcgRIIb (Cancer Res., 2007, 67, 8882 - 8890). The 396th position of EU numbering is a site that is not directly involved in the interaction with FcgR, but it is considered to affect the interaction with FcgR by changing the antibody structure. Therefore, by introducing comprehensive modifications at the 396th position of EU numbering, it was verified whether the binding or selectivity to FcgRIIb was improved. IL6R - BP423 (SEQ ID NO: 33) with E233D, G237D, P238D, S267A, H268E, P271G, A330R introduced into IL6R - B3 (SEQ ID NO: 23) was prepared and used as a template. For IL6R - BP423, variants were prepared in which the 396th position of EU numbering was substituted with 18 types of amino acids excluding the original amino acid and cysteine. IL6R - L (SEQ ID NO: 21) was commonly used as the antibody L chain. According to the method of Reference Example 1, antibodies were expressed and purified from these variants, and the binding to each FcgR (FcgRIa, FcgRIIaH type, FcgRIIaR type, FcgRIIb, FcgRIIIaV type) was evaluated by the method of Reference Example 2. The binding of the obtained variants to each FcgR is summarized in Table 4.

[0213]

Table 4

[0214] Note that the "modifications added to IL6R-BP423" in the table indicate the modifications introduced to IL6R-BP423, while the IL6R-B3 / IL6R-L used as the template when preparing IL6R-BP423 is indicated as *. The KD(IIb) of the parent polypeptide in the table divided by the KD(IIb) of the modified polypeptide refers to the value obtained by dividing the KD value of IL6R-B3 / IL6R-L for FcgRIIb by the KD value of each variant for FcgRIIb. Also, the KD(IIaR) of the parent polypeptide divided by the KD(IIaR) of the modified polypeptide refers to the value obtained by dividing the KD value of IL6R-B3 / IL6R-L for FcgR IIaR by the KD value of each variant for FcgR IIaR. KD(IIaR) / KD(IIb) is the value obtained by dividing the KD of each variant for FcgRIIaR by the KD of each variant for FcgRIIb, and the larger this value, the higher the selectivity for FcgRIIb. Note that the cells shaded in gray in Table 4 have weak binding of FcgR to IgG and were judged to be unable to be correctly analyzed by kinetic analysis. Therefore, the value calculated using the formula of [Formula 2] TIFF0007698004000008.tif9156 is the value calculated using the formula.

[0215] From the results in Table 4, for IL6R-BP456 / IL6R-L with P396M introduced into IL6R-BP423 / IL6R-L, IL6R-BP455 / IL6R-L with P396L introduced, IL6R-BP464 / IL6R-L with P396Y introduced, IL6R-BP450 / IL6R-L with P396F introduced, IL6R-BP448 / IL6R-L with P396D introduced, IL6R-BP458 / IL6R-L with P396Q introduced, IL6R-BP453 / IL6R-L with P396I introduced, IL6R-BP449 / IL6R-L with P396E introduced, IL6R-BP454 / IL6R-L with P396K introduced, and IL6R-BP459 / IL6R-L with P396R introduced, the binding to FcgRIIb was improved compared to IL6R-BP423 / IL6R-L before the introduction of the modification. Also, from the value of KD(IIaR) / KD(IIb), IL6R-BP456 / IL6R-L with P396M introduced into IL6R-BP423 / IL6R-L had a larger value of KD(IIaR) / KD(IIb) and an improved selectivity for FcgRIIb compared to IL6R-BP423 / IL6R-L before the introduction of the modification. All the variants prepared in Table 4 had a lower affinity for FcgRIa, FcgRIIaH, and FcgRIIIaV than the parental polypeptide IL6R-B3 / IL6R-L.

[0216] [Example 7] Preparation of FcgRIIb-enhanced variants by using subclass sequences Human IgG has subclasses with different binding profiles to FcgRs. Here, it was verified whether the difference in the affinity of IgG1 and IgG4 for each FcgR could be utilized to improve the binding and selectivity for FcgRIIb. First, the affinities of IgG1 and IgG4 to each FcγR were analyzed. As the antibody heavy chain, IL6R-G4d (SEQ ID NO: 30) having G4d in which Ser at the 228th position of the EU numbering of human IgG4 was replaced with Pro and the C-terminal Gly and Lys were removed was prepared. As the antibody light chain, IL6R-L (SEQ ID NO: 21) was commonly used. According to the method of Reference Example 1, IL6R-G1d / IL6R-L and IL6R-G4d / IL6R-L were expressed and purified, and the binding to each FcγR (FcγRIa, FcγRIIa H-type, FcγRIIa R-type, FcγRIIb, FcγRIIIa V-type) was evaluated by the method of Reference Example 2. The binding of the obtained variants to each FcγR is summarized in Table 5.

[0217]

Table 5

[0218] It was found that IL6R-G4d / IL6R-L had 1.5 times stronger binding to FcγRIIb and 2.2 times weaker binding to FcγRIIaR compared with IL6R-G1d / IL6R-L. Also, regarding the affinities to FcγRIa, FcγRIIa H, and FcγRIIIa V, IL6R-G4d / IL6R-L was weaker than IL6R-G1d / IL6R-L. From the above results, it became clear that IL6R-G4d was superior in both binding and selectivity to FcγRIIb compared with IL6R-G1d.

[0219] Figure 14 compares the sequences from CH1 to the C-terminus (positions 118 to 445 of the EU numbering) of G1d and G4d. The amino acids enclosed by the frames in Figure 14 indicate residues that are different between G1d and G4d. From among these different amino acids, several sites predicted to be involved in the interaction with FcγR were selected, and it was verified whether further improvement in binding and selectivity was possible by transplanting the sequence of G4d, which is excellent in both binding and selectivity to FcγRIIb, into the FcγRIIb-enhanced variant.

[0220] Specifically, for IL6R-BP230, IL6R-BP473 with A327G introduced, IL6R-BP472 with A330S introduced, IL6R-BP471 with P331S introduced, IL6R-BP474 with A330S and P331S introduced, IL6R-BP475 with A327G and A330S introduced, IL6R-BP476 with A327G, A330S, and P331S introduced, and IL6R-BP477 with A327G and P331S introduced were prepared. Also, IL6R-BP478 (SEQ ID NO: 31) was prepared by replacing the region from Ala at position 118 to Thr at position 225 of IL6R-BP230 with the sequence of G4d (from Ala at position 118 to Pro at position 222) according to EU numbering. As the antibody light chain, IL6R-L (SEQ ID NO: 21) was commonly used. According to the method of Reference Example 1, antibodies were expressed and purified from these variants, and the binding to each FcgR (FcgRIa, FcgRIIa H-type, FcgRIIa R-type, FcgRIIb, FcgRIIIa V-type) was evaluated by the method of Reference Example 2.

[0221] The KD values for each FcgR of each variant are shown in Table 6. In the table, "KD(IIb) of the parental polypeptide / KD(IIb) of the modified polypeptide" refers to the value obtained by dividing the KD value for FcgRIIb of IL6R-B3 / IL6R-L by the KD value for FcgRIIb of each variant. "Modification added to IL6R-BP230" indicates the modification introduced to IL6R-BP230. For IL6R-B3 / IL6R-L used as the template when preparing IL6R-BP230, it is indicated as *1. For IL6R-BP478 (SEQ ID NO: 31) in which the region from Ala at position 118 to Thr at position 225 in the EU numbering of IL6R-BP230 was replaced with the sequence of G4d (from Ala at position 118 to Pro at position 222 in the EU numbering), it is indicated as *2. "KD(IIaR) of the parental polypeptide / KD(IIaR) of the modified polypeptide" refers to the value obtained by dividing the KD value for FcgR IIaR of IL6R-B3 / IL6R-L by the KD value for FcgR IIaR of each variant. KD(IIaR) / KD(IIb) is the value obtained by dividing the KD for FcgRIIaR of each variant by the KD for FcgRIIb of each variant, and a larger value indicates higher selectivity for FcgRIIb. Note that the cells shaded in gray in Table 6 had weak binding of FcgR to IgG and were judged to be unable to be correctly analyzed by kinetic analysis. Therefore, the value calculated using the formula of 〔Formula 2〕 TIFF0007698004000010.tif9156 is shown.

[0222]

Table 6

[0223] Among the variants listed in Table 6, IL6R-BP473 / IL6R-L with the introduction of A327G had a 1.2-fold enhanced binding to FcgRIIb compared to IL6R-BP230 / IL6R-L. Also, IL6R-BP478 / IL6R-L, in which the sequence from Ala at position 118 to Thr at position 225 of IL6R-BP230 was replaced with the sequence of G4d (from Ala at position 118 to Pro at position 222 according to EU numbering), had a 1.1-fold enhanced binding to both FcgRIIb and FcgRIIaR compared to IL6R-BP230 / IL6R-L. The affinity of all variants to FcgRIa, FcgRIIaH, and FcgRIIIaV was lower than that of the parental polypeptide IL6R-B3 / IL6R-L.

[0224] Also, as shown in Figure 14, sites with different amino acids other than G1d and G4d include positions 268, 274, 296, 355, 356, 358, 409, 419, and 445 according to EU numbering. Therefore, it is conceivable that substituting these sites with amino acids derived from IgG4 may improve the binding and selectivity to FcgRIIb. In the studies so far, it has been shown that transplanting A327G, which is the sequence of human IgG4, into the variant IL6R-BP230 / IL6R-L enhances the binding activity to FcγRIIb. Therefore, further studies were conducted on the sites where the sequences of IgG4 and IgG1 differ. Specifically, as the antibody heavy chain, IL6R-BP541 with K274Q introduced, IL6R-BP542 with Y296F introduced, IL6R-BP543 with H268Q introduced, IL6R-BP544 with R355Q introduced, IL6R-BP545 with D356E introduced, IL6R-BP546 with L358M introduced, IL6R-BP547 with K409R introduced, and IL6R-BP548 with Q419E introduced were prepared against IL6R-BP230. As the antibody light chain, IL6R-L was commonly used. According to the method of Reference Example 1, antibodies containing the modified heavy chain and the light chain of IL6R-L were purified. The binding of the purified antibodies to each FcγR (FcγRIa, FcγIIaH, FcγIIaR, FcγIIb, FcγIIIaV) was evaluated by the method of Reference Example 2. The KD values of each variant for each FcγR are shown in Table 7. In the table, "KD (IIb) of the parental polypeptide / KD (IIb) of the modified polypeptide" refers to the value obtained by dividing the KD value of IL6R-B3 / IL6R-L for FcγRIIb by the KD value of each variant for FcγRIIb. "Modification added to IL6R-BP230" in the table indicates the modification introduced to IL6R-BP230. However, IL6R-B3 / IL6R-L used as the template when preparing IL6R-BP230 is indicated as *1. "KD (IIaR) of the parental polypeptide / KD (IIaR) of the modified polypeptide" refers to the value obtained by dividing the KD value of IL6R-B3 / IL6R-L for FcγR IIaR by the KD value of the said variant for FcγR IIaR. KD (IIaR) / KD (IIb) is the value obtained by dividing the KD of each variant for FcγRIIaR by the KD of the said variant for FcγRIIb, and the larger this value is, the higher the selectivity for FcγRIIb. Note that the numerical values in the cells shaded in gray in Table 7 have weak binding of the FcγR to IgG and were determined to be unable to be correctly analyzed by kinetic analysis, so they are the numerical values calculated using the formula of [Formula 2] TIFF0007698004000012.tif9156. [Table 7] As shown in Table 7, for IL6R-BP230 / IL6R-L, IL6R-BP541 / IL6R-L with K274Q introduced, IL6R-BP544 / IL6R-L with R355Q introduced, IL6R-BP545 / IL6R-L with D356E introduced, and IL6R-BP546 / IL6R-L with L358M introduced, the binding to FcγRIIb was enhanced compared to IL6R-BP230 / IL6R-L before the modification. Among these, for IL6R-BP230 / IL6R-L, IL6R-BP544 / IL6R-L with R355Q introduced, IL6R-BP545 / IL6R-L with D356E introduced, and IL6R-BP546 / IL6R-L with L358M introduced, the value of KD(IIaR) / KD(IIb) increased compared to IL6R-BP230 / IL6R-L before the modification, indicating that the modification is also an improvement in the selectivity for FcγRIIb.

[0225] 〔Example 8〕Examination of combinations of modifications that enhance binding to FcgRIIb and improve selectivity Combinations of modifications that improve the binding activity or selectivity to FcγRIIb found in previous studies were examined, and further optimization was attempted. Combinations of modifications that enhanced the binding to FcγRIIb and / or improved the selectivity in previous studies were introduced into IL6R-B3. Also, as a comparative control, IL6R-BP253 was prepared by introducing the modifications of S267E and L328F, which are modifications that enhance the binding to existing FcγRIIb (Seung et al. (Mol. Immunol. (2008) 45, 3926 - 3933)). IL6R-L was used as the antibody L chain. Antibodies containing the modified heavy chain and the light chain of IL6R-L expressed according to the method of Reference Example 1 were purified. The binding of the purified antibodies to each FcγR (FcγRIa, FcγRIIaH, FcγRIIaR, FcγRIIb, FcγRIIIaV) was evaluated by the method of Reference Example 2. The KD values for each FcγR of each variant are shown in Table 8. The modifications in the table indicate the modifications introduced to IL6R-B3. However, IL6R-B3 / IL6R-L used as the template when preparing each variant was indicated as *. The KD (IIb) of the parental polypeptide / the KD (IIb) of the modified polypeptide refers to the value obtained by dividing the KD value for FcγRIIb of IL6R-B3 / IL6R-L by the KD value for FcγRIIb of each variant. Also, the KD (IIaR) of the parental polypeptide / the KD (IIaR) of the modified polypeptide refers to the value obtained by dividing the KD value for FcγR IIaR of IL6R-B3 / IL6R-L by the KD for FcγR IIaR of the said variant. KD (IIaR) / KD (IIb) is the value obtained by dividing the KD for FcγRIIaR of each variant by the KD for FcγRIIb of the said variant, and the larger this value is, the higher the selectivity for FcγRIIb compared to FcγRIIaR. Also, KD (IIaH) / KD (IIb) is the value obtained by dividing the KD for FcγRIIaH of each variant by the KD for FcγRIIb of the said variant, and the larger this value is, the higher the selectivity for FcγRIIb compared to FcγRIIaH. In Table 8, the numerical values in the cells filled with gray were determined to have weak binding of FcγR to IgG and could not be correctly analyzed by kinetic analysis. Therefore, the [Formula 2] The numerical value calculated using the formula of TIFF0007698004000014.tif9156.

[0226] [Table 8]

[0227] Among the variants described in Table 8, the binding activities of IL6R-BP253 / IL6R-L with existing modifications that enhance binding to FcγRIIb to FcγRIIb and FcγRIIaR were enhanced 277-fold and 529-fold, respectively, compared to those of IL6R-B3 / IL6R-L before the introduction of the modifications. In addition, the binding activity of IL6R-BP253 / IL6R-L to FcγRIa was also enhanced compared to that of IL6R-B3 / IL6R-L. On the other hand, the binding of IL6R-BP253 / IL6R-L to FcγRIIaH and FcγRIIIaV was attenuated compared to that of IL6R-B3 / IL6R-L. Among the other variants, the binding of IL6R-BP436 / IL6R-L, IL6R-BP438 / IL6R-L, IL6R-BP567 / IL6R-L, and IL6R-BP568 / IL6R-L to FcγRIa was slightly enhanced compared to that of IL6R-B3 / IL6R-L before the introduction of the modifications, but the binding of the other variants to FcγRIa was attenuated. In addition, the binding of any of the variants to FcγRIIaH and FcγRIIIaV was attenuated compared to that of IL6R-B3 / IL6R-L. When comparing the variants prepared in this study with the IL6R-BP253 / IL6R-L, which is a variant with enhanced binding to existing FcγRIIb, the values of KD(IIaH) / KD(IIb) were 107.7 for the lowest IL6R-BP480 / IL6R-L and 8362 for the highest IL6R-BP426 / IL6R-L, and all variants were higher compared to 107.1 of IL6R-BP253 / IL6R-L. Also, the values of KD(IIaR) / KD(IIb) were 16.1 for the lowest IL6R-BP479 / IL6R-L and 64.4 for the highest IL6R-BP567 / IL6R-L, and all variants were higher compared to 0.2 of IL6R-BP253 / IL6R-L. From these results, it was shown that all the variants described in Table 8 are variants with improved selectivity for FcγRIIb compared to the variants with enhanced binding to existing FcγRIIb. In particular, IL6R-BP559 / IL6R-L, IL6R-BP493 / IL6R-L, IL6R-BP557 / IL6R-L, IL6R-BP492 / IL6R-L, IL6R-BP500 / IL6R-L, and IL6R-BP567 / IL6R-L all maintained the binding to FcγRIIaR at 1.5-fold or less compared to IL6R-B3 / IL6R-L while enhancing the binding activity to FcγRIIb by 100-fold or more. Therefore, it is expected to show the effect of enhanced binding to FcγRIIb while avoiding the side effects caused by enhancing the binding to FcγRIIaR. In addition, the binding of IL6R-BP489 / IL6R-L, IL6R-BP487 / IL6R-L, IL6R-BP499 / IL6R-L, IL6R-BP498 / IL6R-L, IL6R-BP503 / IL6R-L, IL6R-BP488 / IL6R-L, IL6R-BP490 / IL6R-L, IL6R-BP445 / IL6R-L, IL6R-BP552 / IL6R-L, IL6R-BP507 / IL6R-L, IL6R-BP536 / IL6R-L, IL6R-BP534 / IL6R-L, IL6R-BP491 / IL6R-L, IL6R-BP553 / IL6R-L, IL6R-BP532 / IL6R-L, IL6R-BP506 / IL6R-L, IL6R-BP511 / IL6R-L, IL6R-BP502 / IL6R-L, IL6R-BP531 / IL6R-L, IL6R-BP510 / IL6R-L, IL6R-BP535 / IL6R-L, IL6R-BP497 / IL6R-L, IL6R-BP533 / IL6R-L, IL6R-BP555 / IL6R-L, IL6R-BP554 / IL6R-L, IL6R-BP436 / IL6R-L, IL6R-BP423 / IL6R-L, IL6R-BP440 / IL6R-L, IL6R-BP538 / IL6R-L, IL6R-BP429 / IL6R-L, IL6R-BP438 / IL6R-L, IL6R-BP565 / IL6R-L, IL6R-BP540 / IL6R-L, IL6R-BP426 / IL6R-L, IL6R-BP437 / IL6R-L, IL6R-BP439 / IL6R-L, IL6R-BP551 / IL6R-L, IL6R-BP494 / IL6R-L, IL6R-BP537 / IL6R-L, IL6R-BP550 / IL6R-L, IL6R-BP556 / IL6R-L, IL6R-BP539 / IL6R-L, IL6R-BP558 / IL6R-L, IL6R-BP425 / IL6R-L, IL6R-BP495 / IL6R-L to FcγRIIb is higher than that of IL6R-BP253 / IL6R-L to which an existing modification that enhances the binding to FcγRIIb has been added. The range of enhancement from the lowest IL6R-BP495 / IL6R-L to the highest IL6R-BP489 / IL6R-L was from 321-fold to 3100-fold when the binding of IL6R-B3 / IL6R-L was set to 1.Therefore, it can be said that these variants are superior to the prior art in terms of both binding to FcγRIIb and selectivity. Here, the variants related to IL6R-BP567 / IL6R-L, which are considered to be the most excellent from the perspective of selectivity for FcγRIIb, were examined from the perspective of immunogenicity. The most selective IL6R-BP567 / IL6R-L and IL6R-BP493 / IL6R-L, in which the binding to FcγRIIaR is completely equivalent to that of the native type and the binding to FcγRIIb is enhanced 147-fold, have the Y296D modification introduced. Y296 has been reported to be included in the Tregitope sequence (De Groot et al. (Blood (2008) 112, 3303-3311)), and the introduction of a modification at this site may impair the immunosuppressive function originally possessed by native IgG1. Therefore, from the perspective of immunogenicity, variants not containing the Y296D modification are more preferable. IL6R-BP568 / IL6R-L and IL6R-BP492 / IL6R-L are obtained by removing the Y296D modification from IL6R-BP567 / IL6R-L and IL6R-BP493 / IL6R-L, respectively. Regarding the binding activity and selectivity for FcγRIIb, IL6R-BP492 / IL6R-L and IL6R-BP568 / IL6R-L showed a decrease in both selectivity and binding activity compared to the case containing Y296D due to the removal of the Y296D modification. However, IL6R-BP568 / IL6R-L has a 1.6-fold binding to FcγRIIaR and a 211-fold binding to FcγRIIb compared to the native type, and IL6R-BP492 / IL6R-L still maintains a high selectivity and binding activity with a 1.2-fold binding to FcγRIIaR and a 131-fold binding to FcγRIIb. From these results, it can be said that IL6R-BP568 / IL6R-L and IL6R-BP492 / IL6R-L are excellent variants not only in terms of binding activity and selectivity for FcγRIIb but also in terms of immunogenicity.

[0228] [Example 9] Enhancement of Binding to FcgRIIb by Heterodimeric Antibodies [9-1] Examination of Introducing P238D Only into a Single Chain As shown in Fig. 26 of Reference Example 7, the reason why Fc(P238D) acquired high binding to FcgRIIb is that, by introducing the P238D modification, what formed the peripheral residues and the hydrophobic core in Pro changed to Asp, resulting in the inability to exist in the hydrophobic core and facing the solvent side, which caused a large change in the loop structure of domain A. However, there is room for consideration as to whether it is necessary to introduce the P238D modification to both chains, and whether it is acceptable to introduce P238D to one chain and other modifications to the other chain. Therefore, these points were verified using heterodimeric antibodies in which different modifications were introduced into each H chain of the antibody. For the heavy chain of the antibody, the variable region (SEQ ID NO: 15) of a glypican 3 antibody containing the CDRs of GpH7, which is an anti-glypican 3 antibody with improved pharmacokinetics disclosed in WO2009 / 041062, was used. For GpH7-G1d (SEQ ID NO: 34) obtained by removing the C-terminal Gly and Lys of IgG1 having GpH7 in the variable region, GpH7-A5 (SEQ ID NO: 35) with D356K and H435R modifications introduced, and GpH7-B3 (SEQ ID NO: 17) with K439E modification introduced into GpH7-G1d were utilized. The D356K and K439E modifications introduced into each heavy chain were introduced to efficiently form heterodimers of each heavy chain when producing a heterodimeric antibody consisting of two heavy chains (WO2006 / 106905). H435R is a modification that hinders binding to Protein A and was introduced to efficiently separate a homodimer consisting of two heavy chains with the same modification as a heterodimer consisting of two heavy chains with different modifications introduced. As one heavy chain, a variant in which the amino acids at positions 236th, 237th, and 238th in EU numbering were replaced with the original amino acids and Cys in GpH7-B3 (SEQ ID NO: 17) prepared in Reference Example 1 was used. As the other chain, GpH7-AP001 with P238D introduced into GpH7-A5 (SEQ ID NO: 35) was prepared. For the light chain of the antibody, GpL16-k0 (SEQ ID NO: 16) of a glypican 3 antibody with improved pharmacokinetics disclosed in WO2009 / 041062 was commonly used. These variants were expressed and purified by the method of Reference Example 1, and the binding to each FcgRIIaR type and FcgRIIb was evaluated by the method of Reference Example 2. The binding amounts of each variant to FcgR are shown in Fig. 15.

[0229] The modified G237W, G237F, G236N, P238G, P238N, P238E, P238D shown in Fig. 15 refer to the modifications introduced into GpH7-B3. Also, A5 / B3 indicates GpH7-A5 / GpH7-B3 / GpL16-k0 where no modification was introduced into either chain, and the variant containing P238D only in one chain is shown as GpH7-A5 / GpH7-BF648 / GpL16-k0. These results are also shown in Table 9.

[0230]

Table 9

[0231] The "Binding amount to FcgRIIb / Binding amount to FcgRIIaR" in Table 9 is the value obtained by dividing the binding amount of each variant to FcgRIIb by the binding amount of each variant to FcgRIIaR. The larger this value is, the higher the selectivity for FcgRIIb. Also, the "Modifications introduced into GpH7-A5" and "Modifications introduced into GpH7-B3" indicate the modifications introduced into GpH7-A5 and GpH7-B3, respectively. For GpH7-G1d, which was used as the template when preparing GpH7-A5 and GpH7-B3, it is shown as *. From the results in Table 9, GpH7-AP001 / GpH7-BF648 / GpL16-k0, which has the P238D modification on both chains, had the highest selectivity for FcgRIIb. Also, GpH7-AP001 / GpH7-BP061 / GpL16-k0, GpH7-AP001 / GpH7-BP069 / GpL16-k0, and GpH7-AP001 / GpH7-BP063 / GpL16-k0, which have P238E, P238N, and P238G on the other chain, had a binding amount to FcgRIIb / binding amount to FcgRIIaR of 2.9, 2.2, and 1.7, respectively, and maintained a high selectivity for FcgRIIb even when compared with GpH7-AP001 / GpH7-BF648 / GpL16-k0, which has the P238D modification on both chains. Also, regarding the affinity for FcgRIIb, since it maintained more than 69% of GpH7-AP001 / GpH7-BF648 / GpL16-k0, which has the P238D modification on both chains, it can be said that if the P238D modification exists on one chain, the other chain can be replaced with P238E, P238N, or P238G.Furthermore, focusing on the binding to FcgRIIb, compared with GpH7-AP001 / GpH7-BF648 / GpL16-k0 containing P238D in both chains, GpH7-A5 / GpH7-BF648 / GpL16-k0 containing P238D only in one chain and no modification in the other chain binds more strongly to FcgRIIb. It was also revealed that GpH7-AP001 / GpH7-BP032 / GpL16-k0, GpH7-AP001 / GpH7-BP044 / GpL16-k0, and GpH7-AP001 / GpH7-BP057 / GpL16-k0, which contain P238D in one chain and G236N, G237F, and G237W, respectively, in the other chain, bind more strongly to FcgRIIb.

[0232] 9-2. Verification of Modifications Based on the Structural Information of Fc(P208) / FcgRIIb As shown in Figure 10, in the crystal structure of Fc(P208) / FcgRIIb, the electron density of Lys at position 117 of FcgRIIb was not observed, and this residue is considered not to be significantly involved in the binding to Fc(P208). By substituting Ser at position 239 (EU numbering) of CH2 domain B, which is located in the vicinity, with Asp or Glu, it is possible to form an electrostatic interaction with Lys at position 117 of this FcgRIIb. On the other hand, as shown in Figure 7, in CH2 domain A, Ser at position 239 (EU numbering) forms a hydrogen bond with Gly at position 236 (EU numbering) and contributes to strengthening the binding to Tyr at position 160 (EU numbering) of FcgRIIb by stabilizing the loop structure from position 233 to 239. Substitution at this site is expected to cause destabilization of the loop structure and a consequent decrease in binding activity in CH2 domain A, and it was predicted that these effects would cancel each other out in homomutations. Therefore, in this study, modifications of S239D or S239E were introduced only into one chain by heterodimerization to verify the effect of enhancing the binding to FcgRIIb.

[0233] As the antibody H chain of the film side, IL6R-BP256 with S239D introduced into IL6R-BP208 (SEQ ID NO: 24) and IL6R-BP257 with S239E introduced were prepared. Similarly, IL6R-BP259 with S239D introduced into IL6R-BP230 (SEQ ID NO: 27) and IL6R-BP260 with S239E introduced were prepared. As the other antibody H chain, IL6R-AP002 with E233D, G237D, P238D, H268D, P271G, A330R introduced, which are the same modifications as those contained in CH2 of IL6R-BP208, into IL6R-A5 (SEQ ID NO: 69), and IL6R-AP009 with E233D, G237D, P238D, H268D, P271G, Y296D, A330R introduced, which are the same modifications as those contained in CH2 of IL6R-BP230, were prepared and used. Also, as a comparison target, IL6R-BP253 (SEQ ID NO: 32) with S267E and L328F introduced into IL6R-B3, which is an existing FcgRIIb enhancement technology (Non-Patent Document 28), was prepared. As the antibody L chain, IL6R-L (SEQ ID NO: 21) was commonly used. According to the method of Reference Example 1, antibodies were expressed and purified from these variants, and the binding to each FcgR (FcgRIa, FcgRIIaH type, FcgRIIaR type, FcgRIIb, FcgRIIIaV type) was evaluated by the method of Reference Example 2.

[0234] The KD values for each FcgR of each variant are shown in Table 10. In the table, "KD of the parental polypeptide (IIb) / KD of the modified polypeptide (IIb)" refers to the value obtained by dividing the KD value of IL6R-B3 / IL6R-L for FcgRIIb by the KD value of each variant for FcgRIIb. Also, "KD of the parental polypeptide (IIaR) / KD of the modified polypeptide (IIaR)" refers to the value obtained by dividing the KD value of IL6R-B3 / IL6R-L for FcgR IIaR by the KD value of each variant for FcgR IIaR. "KD(IIaR) / KD(IIb)" is the value obtained by dividing the KD of each variant for FcgRIIaR by the KD of each variant for FcgRIIb, and the larger this value, the higher the selectivity for FcgRIIb. Note that the cells shaded in gray in Table 10 have weak binding of FcgR to IgG and were judged to be unable to be correctly analyzed by kinetic analysis. Therefore, they are the values calculated using the formula of 〔Formula 2〕 TIFF0007698004000017.tif9156 as shown in the formula.

[0235]

Table 10

[0236] From the results in Table 10, for IL6R-AP002 / IL6R-BP256 / IL6R-L with S239D introduced into one strand and IL6R-AP002 / IL6R-BP257 / IL6R-L with S239E introduced into one strand against IL6R-BP208 / IL6R-L, the binding to FcgRIIb was enhanced in both cases compared to IL6R-BP208 / IL6R-L. Also, the value of KD(IIaR) / KD(IIb) exceeded that of IL6R-BP256 / IL6R-L, indicating an improvement in the selectivity for FcgRIIb. On the other hand, for IL6R-BP256 / IL6R-L with S239D introduced into both strands and IL6R-BP257 / IL6R-L with S239E introduced into both strands of IL6R-BP208 / IL6R-L, both the binding to FcgRIIb and the selectivity were significantly decreased compared to IL6R-BP208 / IL6R-L. Thus, when S239D or S239E was introduced into only one strand, an effect of enhancing the binding to FcgRIIb was observed, while when introduced into both strands, the binding to FcgRIIb was significantly decreased. As described above, this is considered to be due to the destabilization of the loop structure in CH2 domain A. Similar results were also obtained when S239D and S239E were introduced using IL6R-BP230 / IL6R-L as a template. For IL6R-AP009 / IL6R-BP259 / IL6R-L and IL6R-AP009 / IL6R-BP260 / IL6R-L with S239D and S239E introduced into one strand of IL6R-BP230 / IL6R-L respectively, both the binding to FcgRIIb and the selectivity exceeded those of IL6R-BP230 / IL6R-L. However, for IL6R-BP259 / IL6R-L and IL6R-BP260 / IL6R-L with S239D and S239E introduced into both strands respectively, both the binding to FcgRIIb and the selectivity were significantly decreased compared to IL6R-BP230 / IL6R-L. In addition, the variants with S239D or S239E introduced into one strand of IL6R-BP208 / IL6R-L and IL6R-BP230 / IL6R-L exceeded both the binding to FcgRIIb and the selectivity of IL6R-BP253 / IL6R-L that utilized existing FcgRIIb enhancement technology.

[0237] Verification of Modifications Based on the Structural Information of Fc(P208) / FcgRIIaR In Example 3, when the crystal structure of Fc(P208) with FcgRIIb was compared with the crystal structure with FcgRIIaR, a difference in electron density was observed near position 237 in the EU numbering, which forms a hydrogen bond with Tyr at position 160 of FcgRIIb. It was suggested that the contribution from the A side of the CH2 domain is large for the binding to FcgRIIb, while the contribution from the B side of the CH2 domain is large for the binding to FcgRIIaR (Figs. 12 and 13). For example, from the appearance of the potential density, in the binding with the FcgRIIaR type, Leu at position 234 and Leu at position 235 in the CH2 domain B of the EU numbering are considered to be involved in the binding to the receptor. On the other hand, in the binding with FcgRIIb, the involvement of these residues is considered to be small. Therefore, by substituting these two residues with residues other than hydrophobic residues, it is conceivable that the interaction with the FcgRIIaR type can be more greatly reduced. However, on the A side of the CH2 domain, the residues of Leu at position 234 and Leu at position 235 in the EU numbering are considered to contribute to the stabilization of the loop structure near position 237, and are particularly likely to be highly involved in the binding with FcgRIIb. Therefore, substituting these residues with residues other than hydrophobic residues may reduce the interaction with FcgRIIb in the CH2 domain A. In particular, Leu at position 235 in the EU numbering forms a good hydrophobic interaction in the CH2 domain A of the complex structure with FcgRIIb and is considered to make a large contribution to the stabilization of the loop structure near position 237 in the EU numbering. Therefore, for this residue, a study was conducted to substitute only one strand with a residue other than a hydrophobic residue. Also, by substituting Leu at position 235 in the EU numbering of both strands with other hydrophobic amino acids, if the hydrophobic interaction in the CH2 domain A can be particularly strengthened and the loop structure near position 237 in the EU numbering can be more stabilized, it may lead to a reduction in the entropic energy loss associated with the formation of a hydrogen bond with Tyr at position 160 of FcgRIIb, and the binding and selectivity to FcgRIIb may be improved. Therefore, this was also studied together.

[0238] As the antibody heavy chain, IL6R-BP264 (SEQ ID NO: 28) with E233D, G237D, P238D, H268E, P271G, Y296D, and A330R introduced with respect to IL6R-B3 (SEQ ID NO: 23) was prepared and used as a template. Variants were prepared by substituting the 234th Leu of IL6R-BP264 in EU numbering with Asn, Ser, Asp, Gln, Glu, Thr, Arg, His, Gly, Lys, and Tyr, respectively. Also, variants were prepared by substituting the 235th amino acid of IL6R-BP264 in EU numbering with 18 types of amino acids excluding the original amino acid and Cys. As the other antibody heavy chain, IL6R-AP029 (SEQ ID NO: 42) with E233D, G237D, P238D, H268E, P271G, Y296D, and A330R introduced with respect to IL6R-A5 (SEQ ID NO: 69) was prepared. As the antibody light chain, IL6R-L (SEQ ID NO: 21) was commonly used. For variants with L234N, L234S, L234D, L234Q, L234E, L234T, L234R, L234H, L234G, L234K, and L234Y introduced into IL6R-BP264, and variants with L235W, L235M, L235P, L235F, L235A, L235V, and L235I introduced, homodimers containing the same modification in both chains were considered. For variants with L235N, L235S, L235D, L235Q, L235E, L235T, L235R, L235H, L235G, L235K, and L235Y introduced, they were examined as heterodimeric antibodies combined with IL6R-AP029. According to the method of Reference Example 1, antibodies were expressed and purified from these variants, and the binding to each FcgR (FcgRIa, FcgIIaH type, FcgIIaR type, FcgIIb, FcgIIIaV type) was evaluated by the method of Reference Example 2. A graph showing the KD for FcgIIb of each variant on the horizontal axis and the KD for FcgIIaR on the vertical axis is shown in Figure 16.

[0239] As shown in Fig. 16, IL6R-BP404 / IL6R-L with L234Y introduced into both strands with respect to IL6R-BP264 / IL6R-L showed a slightly enhanced binding to FcgRIIb compared to IL6R-BP264 / IL6R-L before the introduction of the modification.

[0240] Among these variants, IL6R-BP404 / IL6R-L with enhanced binding to FcgRIIb and the variants with improved selectivity for FcgRIIb were summarized in Table 11. The KD(IIb) of the parental polypeptide / KD(IIb) of the modified polypeptide in the table refers to the value obtained by dividing the KD value of IL6R-B3 / IL6R-L for FcgRIIb by the KD value of each variant for FcgRIIb. Also, the KD(IIaR) of the parental polypeptide / KD(IIaR) of the modified polypeptide refers to the value obtained by dividing the KD value of IL6R-B3 / IL6R-L for FcgR IIaR by the KD value of each variant for FcgR IIaR. KD(IIaR) / KD(IIb) is the value obtained by dividing the KD of each variant for FcgRIIaR by the KD of each variant for FcgRIIb, and the larger this value, the higher the selectivity for FcgRIIb. Note that the cells shaded in gray in Table 11 had weak binding of FcgR to IgG and were judged to be unable to be correctly analyzed by kinetic analysis. Therefore, the value calculated using the formula of 〔Equation 2〕 TIFF0007698004000019.tif9156 is shown.

[0241]

Table 11

[0242] As shown in Table 11, IL6R-BP404 / IL6R-L with L234Y introduced into both strands with respect to IL6R-BP264 / IL6R-L had a 1.1-fold increase in binding to FcgRIIb compared to IL6R-BP264 / IL6R-L before the introduction of the modification. IL6R-BP408 / IL6R-L with L235Q introduced into both strands of IL6R-BP264 / IL6R-L, IL6R-BP419 / IL6R-L with L235F introduced into both strands, IL6R-AP029 / IL6R-BP407 / IL6R-L with L235D introduced into one strand, IL6R-AP029 / IL6R-BP408 / IL6R-L with L235Q introduced into one strand, IL6R-AP029 / IL6R-BP409 / IL6R-L with L235E introduced into one strand, and IL6R-AP029 / IL6R-BP410 / IL6R-L with L235T introduced into one strand all had a larger KD(IIaR) / KD(IIb) value compared to IL6R-BP264 / IL6R-L before the introduction of the modification and were variants with improved selectivity for FcgRIIb.

[0243] Example 10: Evaluation of Immunogenicity of FcgRIIb-Binding Enhanced Fc Modification Variants by an In Silico Immunogenicity Prediction Tool When the Fc variant described in this example is used as an antibody pharmaceutical, it is preferably not to induce the production of anti-drug antibodies that attenuate its pharmacological action. Since antibodies with high immunogenicity are likely to induce the production of anti-drug antibodies, it is preferable that the immunogenicity of the antibody pharmaceutical is as low as possible. In order not to increase the immunogenicity of the variant as much as possible, in silico immunogenicity prediction tools for predicting T-cell epitopes such as Epibase and EpiMatrix can be used. Epibase Light (Lonza) is an in silico immunogenicity prediction tool that calculates the binding ability of 9-mer peptides to MHC class II containing the major DRB1 allele using the FASTER algorism (Expert Opin Biol Ther. 2007 Mar;7(3):405-18.). This tool can identify T-cell epitopes with strong binding to MHC class II (Strong epitopes) and T-cell epitopes with moderate binding (Medium epitopes). The calculation reflects the abundance of DRB1 allotypes, and the abundances in Caucasians shown in Table 12 below can be used for this.

Table 12

Table 13

[0244] [Example 11] Evaluation of the Pharmacokinetics of an Fc Variant with Enhanced Binding to Human FcgRIIb Using Human FcgRIIb Transgenic Mice (11-1) Outline of the Test As shown in WO2013 / 047752, by administering an antigen-binding molecule having a binding activity to human FcRn under conditions of an acidic pH range, having an antigen-binding domain whose binding activity to an antigen changes depending on ionic concentration conditions, and having a higher binding activity to FcgR than the binding domain to FcgR of the Fc region of natural human IgG in which the sugar chain bound to position 297 of EU numbering is a fucose-containing sugar chain, it is possible to significantly reduce the plasma concentration of its target soluble antigen in the living body as compared with natural human IgG. Among FcgRs, it has also been reported that when an antigen-binding molecule with enhanced binding activity to FcgRIIb in particular is administered in vivo, it is possible to accelerate the disappearance of soluble antigen in plasma and effectively reduce the concentration of soluble antigen in plasma. In this example, by administering an Fc variant with enhanced binding to human FcgRIIb to a transgenic mouse into which human FcgRIIb has been introduced by genetic modification, it was verified whether the disappearance rate of its target soluble antigen can be accelerated with the Fc variant that actually has enhanced binding to human FcgRIIb described herein. (11-2) Preparation of an Antibody with Enhanced Binding to FcgRIIb As the Fc variants with enhanced binding to human FcγRIIb, the following antibodies were used. IL6R-P587 was prepared by introducing the mutations E233D, P238D, S264I, S267A, H268E, and P271G into IL6R-G1d (SEQ ID NO: 19), which consists of the variable region of an antibody against human interleukin 6 receptor (human IL-6R) disclosed in WO2009 / 125825 and the constant region of G1d with the C-terminal Gly and Lys of human IgG1 removed, in the same manner as BP568. Fv4-P587 having IL-6R-P587 as the antibody heavy chain and IL6R-L2 (SEQ ID NO: 74), which is the light chain of the antibody against human IL-6R disclosed in WO2009 / 125825, as the antibody light chain was prepared according to the method of Reference Example 1. Further, as a comparison target, Fv4-IgG1 having IL6R-G1d (SEQ ID NO: 19) and IL6R-L2 (SEQ ID NO: 74) as the antibody heavy chain and light chain, respectively, was prepared in the same manner according to the method of Reference Example 1. Fv4-G1d and Fv4-P587 prepared herein have an antigen-binding domain in which the binding activity of the antigen-binding molecule to the antigen changes depending on the proton ion concentration condition, that is, they bind weakly to the antigen human IL-6R under acidic pH conditions compared to neutral pH conditions, as described in WO2009 / 125825. (11-3) Generation of human FcγRIIb transgenic mice Human FcγRIIb transgenic mice were generated by the following method. Transgenic mice were generated by introducing the human FcgRIIb gene into C57BL / 6 (B6) mice. The generation of transgenic mice was carried out according to the procedures described in "Nagy et al. (Manipulating the mouse embryo, CSHL press. (2003) 399-506)" and "Ueda et al. (The latest techniques of gene targeting, Yodosha. (2000) 190-207)". That is, a bacterial artificial chromosome in which the genomic region of the human FcgRIIb gene (GeneBank # NW_004077999:18,307,411-18,381,603) was cloned was microinjected into the pronucleus of fertilized eggs of B6 mice. Among the obtained mice, mice into which the human FcgRIIb gene was introduced were selected by Southern blotting and PCR using a probe that specifically hybridizes to the human FcgRIIb gene. Blood and liver were collected from the human FcgRIIb transgenic mice, and the expression of the human FcgRIIb gene was confirmed by RT-PCR (Reverse Transcription Polymerase Chain Reaction) using primers that specifically amplify the human FcgRIIb gene. As a result, the expression of the human FcgRIIb gene was detected. In addition, mouse PBMC (Peripheral Blood Mononuclear Cell) was isolated from the blood of the human FcgRIIb transgenic mice, and the expression of human FcgRIIb in PBMC was confirmed by FACS (Fluorescence Activated Cell Sorting) analysis. As a result, the expression of human FcgRIIb was detected. From the above, it was confirmed that human FcgRIIb transgenic mice expressing human FcgRIIb could be established. (11-4) In vivo test of simultaneous administration of antigen and antibody using human FcgRIIb transgenic mice Using the human FcgRIIb transgenic mice generated in (11-3), soluble human IL-6R as an antigen and the anti-human IL-6R antibody prepared in (11-2) were simultaneously administered, and the soluble human IL-6R concentration and anti-human IL-6R antibody concentration in plasma after the administration were evaluated. A mixed solution of soluble human IL-6R and anti-human IL-6R antibody (5 μg / mL and 0.1 mg / mL respectively) was administered once via the tail vein at 10 mL / kg. At this time, since the anti-human IL-6R antibody is present in a sufficient excess amount relative to soluble human IL-6R, it is considered that almost all of the soluble human IL-6R is bound to the antibody. Blood was collected 5 minutes, 1 hour, 4 hours, 7 hours, 1 day, 3 days, 7 days, 14 days, 21 days, and 28 days after the administration. The collected blood was immediately centrifuged at 4°C and 15,000 rpm for 15 minutes to obtain plasma. The separated plasma was stored in a freezer set at -20°C or lower until the measurement was carried out. As the anti-human IL-6R antibody, the above-mentioned Fv4-P587 and Fv4-IgG1 were used. (11-5) Measurement of anti-human IL-6R antibody concentration in plasma by ELISA The anti-human IL-6R antibody concentration in mouse plasma was measured by ELISA. First, anti-human IgG (γ-chain specific) F(ab')2 antibody fragment (Sigma) was dispensed into Nunc-ImmunoPlate, MaxiSorp (Nalge Nunc International) and left standing overnight at 4°C to prepare an anti-human IgG immobilized plate. Calibration curve samples with concentrations of 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125 μg / mL as plasma concentrations and mouse plasma measurement samples diluted 100-fold or more were prepared. 200 μL of soluble human IL-6R at 20 ng / mL was added to 100 μL of these calibration curve samples and plasma measurement samples, and the mixture was stirred at room temperature for 1 hour. Then, it was dispensed into the anti-human IgG immobilized plate and further stirred at room temperature for 1 hour. Thereafter, a biotinylated anti-human IL-6R antibody (R&D) was reacted at room temperature for 1 hour, and further Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was reacted at room temperature for 1 hour. A color reaction was performed using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as a substrate. After stopping the reaction with 1N sulfuric acid (Showa Chemical), the absorbance at 450 nm was measured using a microplate reader. The concentration in mouse plasma was calculated using the analysis software SOFTmax PRO (Molecular Devices) from the absorbance of the calibration curve. The change in plasma antibody concentration over time in human FcgRIIb transgenic mice after intravenous administration measured by this method is shown in Fig. 33. (11-5) Measurement of human IL-6R concentration in plasma by electrochemiluminescence method The concentration of human IL-6R in mouse plasma was measured by electrochemiluminescence method. Human IL-6R calibration curve samples adjusted to 12.5, 6.25, 3.13, 1.56, 0.781, 0.391, 0.195 ng / mL as plasma concentrations and mouse plasma measurement samples diluted 50-fold or more were prepared. Monoclonal Anti-human IL-6R Antibody (R&D) ruthenated with ruthenium by SULFO-TAG NHS Ester (Meso Scale Discovery), Biotinylated Anti-human IL-6 R Antibody (R&D), and tocilizumab solution were mixed and reacted at 37°C overnight. Then, it was dispensed into a Streptavidin Gold Multi-ARRAY Plate (Meso Scale Discovery) blocked overnight at 5°C using a PBS-Tween solution containing 0.5% BSA (w / v). After further reacting at room temperature for 2 hours, washing, and dispensing Read Buffer T (×2) (Meso Scale Discovery), measurement was immediately performed using a SECTOR Imager 2400 (Meso Scale Discovery). The hSIL-6R concentration was calculated using analysis software SOFTmax PRO (Molecular Devices) from the response of the calibration curve. The change in the plasma soluble human IL-6R concentration in human FcgRIIb transgenic mice after intravenous administration measured by this method is shown in Fig. 34. (11-6) Effect of enhancing human FcgRIIb binding The results of in vivo tests of Fv4-IgG1 and Fv4-P587, whose binding to human FcgRIIb was enhanced, were compared. As shown in Fig. 33, the retention of both antibodies in plasma was almost the same. However, as shown in Fig. 34, it was confirmed that human IL-6R co-administered with Fv4-P587, whose binding to human FcgRIIb was enhanced, disappeared faster than human IL-6R co-administered with Fv4-IgG1. That is, it was found that an antibody that binds to human IL-6R in a pH-dependent manner can reduce the soluble human IL-6R concentration by enhancing the human FcgRIIb binding ability. Although not bound by a specific theory, from these results, it is also possible to consider that the soluble antigen in plasma that binds to the antibody disappears by being taken up into cells expressing FcγRIIb via human FcγRIIb according to the mechanism shown in FIG. 35. Soluble human IL-6R bound to an antibody that binds to soluble human IL-6R is recycled into plasma by FcRn together with the antibody, whereas Fv4-IgG1, an antibody that binds to soluble human IL-6R in a pH-dependent manner, dissociates the soluble human IL-6R bound to the antibody under acidic conditions within the endosome. Since the dissociated soluble human IL-6R is degraded by lysosomes, it becomes possible to significantly accelerate the disappearance of soluble human IL-6R. Furthermore, Fv4-IgG1, an antibody that binds to soluble human IL-6R in a pH-dependent manner, is recycled into plasma after binding to FcRn within the endosome. Since the recycled antibody can bind to soluble human IL-6R again, the binding to the antigen (soluble human IL-6R) and the recycling in plasma by FcRn are repeated. As a result, it is considered that one antibody molecule can bind to soluble human IL-6R repeatedly multiple times. Furthermore, by enhancing the FcgRIIb binding activity of Fv4-IgG1 that binds to the antigen in a pH-dependent manner, the complex of the antibody that binds to soluble human IL-6R and soluble human IL-6R is rapidly taken into cells via FcgRIIb, and it is considered that the soluble human IL-6R concentration can be reduced more efficiently (FIG. 35).

[0245] [Example 12] Evaluation of the blood pharmacokinetics of an Fc variant with enhanced human FcgRIIb binding using human FcgRIIb- and human FcRn-transgenic mice (12-1) Outline of the test As shown in WO2013 / 047752, by using an antigen-binding molecule having a binding activity to FcγR higher than that of the Fc region of natural IgG and an enhanced human FcRn binding activity under acidic pH conditions, an improvement in plasma retention was confirmed as compared with an antigen-binding molecule having no enhanced human FcRn binding activity under acidic pH conditions. On the other hand, it has also been reported that the target antigen concentration in plasma was lower for an antigen-binding molecule having a binding activity to FcγR higher than that of the Fc region of natural human IgG and an enhanced human FcRn binding activity under acidic pH conditions, as compared with an antigen-binding molecule having a binding activity to FcγR higher than that of the Fc region of natural human IgG and no enhanced human FcRn binding activity under acidic pH conditions. Therefore, it was verified whether an antigen-binding molecule having an Fc region variant with enhanced binding to human FcgRIIb described in this example has the same properties. (12-2) Preparation of an antigen-binding molecule having a binding activity to FcγR higher than that of the Fc region of natural human IgG and an enhanced human FcRn binding activity under acidic pH conditions In addition to Fv4-IgG1 and Fv4-P587 described in Example 11-2, Fv4-P587-LS (SEQ ID NO: 73) having a modification consisting of substitution of Met at position 428 with Leu and substitution of Asn at position 434 with Ser represented by EU numbering, which has been reported to improve the pharmacokinetics of an antibody in the past (Nat. Biotechnol. 2010. 28; 157-159) as the H chain of the antibody, and IL6R-L2 as the L chain of the antibody were prepared according to the method of Reference Example 1. (12-3) Interaction analysis with human FcRn Interaction analysis of the prepared antibody with human FcRn was performed using a Biacore T200. Protein L (BioVision) was immobilized in an appropriate amount on a sensor chip CM4 (GE Healthcare) described below by an amine coupling method, and the antibody of interest was captured thereon. Next, an FcRn dilution solution and a running buffer (as a reference control solution) were injected to allow human FcRn to interact with the antibody captured on the sensor chip. The running buffer used was 50 mmol / L sodium phosphate, 150 mmol / L NaCl, 0.05% (w / v) Tween 20, pH 6.0, and the running buffer was also used for diluting FcRn. 10 mmol / L glycine-HCl, pH 1.5 was used for chip regeneration. All measurements were carried out at 25°C. From the sensorgrams obtained in the measurements, the kinetic parameters, the association rate constant ka (1 / Ms) and the dissociation rate constant kd (1 / s), were calculated, and based on these values, the KD (M) of each antibody for human FcRn was calculated. Biacore T200 Evaluation Software (GE Healthcare) was used for calculating each parameter. The KD values of the prepared antibody for human FcRn when measured by this method are shown in Table 14. As shown in Table 14, it was confirmed that the binding of Fv4-P587-LS to FcRn was enhanced under acidic conditions compared to Fv4-P587.

Table 14

Claims

A method for producing an Fc region variant, comprising a step of introducing an amino acid modification into the Fc region, wherein the amino acid modification includes the modifications described in the following (1) and (2), (1) Modification of the amino acid at position 238 in EU numbering to Asp, and (2) At least one modification selected from the group consisting of modification of the amino acid at position 264 in EU numbering to Ile, modification of the amino acid at position 268 to Asp, Gln or Glu, modification of the amino acid at position 271 to Gly, and modification of the amino acid at position 330 to Lys, Arg or Ser, the Fc region variant is an Fc region variant with enhanced binding selectivity for FcγRIIb compared to FcγRIIa (R type), or an Fc region variant with enhanced binding activity for FcγRIIb compared to an Fc region without an introduced amino acid modification and enhanced binding selectivity for FcγRIIb compared to FcγRIIa (R type), a method wherein the value of [KD value of the polypeptide containing the Fc region variant for FcγRIIa (R type)] / [KD value of the polypeptide containing the Fc region variant for FcγRIIb] is 20.0 or more. A method for producing an Fc region variant, comprising a step of introducing an amino acid modification into the Fc region, wherein the amino acid modification includes the modifications described in the following (1) to (3), (1) Modification of the amino acid at position 238 in EU numbering to Asp, (2) Modification of the amino acid at position 271 in EU numbering to Gly, and (3) At least one modification selected from the group consisting of modification of the amino acid at position 233 in EU numbering to Asp, modification of the amino acid at position 237 to Asp, modification of the amino acid at position 268 to Asp, and modification of the amino acid at position 330 to Arg, the Fc region variant is an Fc region variant with enhanced binding activity for FcγRIIb compared to an Fc region without an introduced amino acid modification and enhanced binding selectivity for FcγRIIb compared to FcγRIIa (R type). A method wherein the value of [KD value of a polypeptide containing an Fc region without amino acid modification for FcγRIIb] / [KD value of a polypeptide containing an Fc region variant for FcγRIIb] is 15.0 or more, and the value of [KD value of a polypeptide containing an Fc region variant for FcγRIIa (R type)] / [KD value of a polypeptide containing an Fc region variant for FcγRIIb] is 10.0 or more.

3. The method according to claim 1 or 2, wherein the amino acid modification includes the modifications described in the following (1) to (4): (1) Modification of the amino acid at position 238 according to EU numbering to Asp; (2) Modification of the amino acid at position 268 according to EU numbering to Asp or Glu; (3) Modification of the amino acid at position 271 according to EU numbering to Gly; and (4) At least one modification selected from the modification of the amino acid at position 233 according to EU numbering to Asp, the modification of the amino acid at position 237 to Asp, the modification of the amino acid at position 264 to Ile, the modification of the amino acid at position 267 to Ala or Gly, the modification of the amino acid at position 272 to Asp or Pro, the modification of the amino acid at position 296 to Asp, the modification of the amino acid at position 327 to Gly, the modification of the amino acid at position 330 to Arg, the modification of the amino acid at position 332 to Thr, and the modification of the amino acid at position 396 to Leu or Met.

4. The method according to claim 1 or 2, wherein the amino acid modification includes the modifications described in any of the following (a) to (x): (a) Modification of the amino acid at position 238 according to EU numbering in the Fc region to Asp, the modification of the amino acid at position 233 to Asp, the modification of the amino acid at position 237 to Asp, the modification of the amino acid at position 268 to Asp, the modification of the amino acid at position 271 to Gly, the modification of the amino acid at position 296 to Asp, and the modification of the amino acid at position 330 to Arg; (b) Modification of the amino acid at position 238 according to EU numbering in the Fc region to Asp, the modification of the amino acid at position 237 to Asp, the modification of the amino acid at position 268 to Asp or Glu, the modification of the amino acid at position 271 to Gly, the modification of the amino acid at position 296 to Asp, and the modification of the amino acid at position 330 to Arg; (c) Modification of the 238th amino acid in the Fc region to Asp, modification of the 233rd amino acid to Asp, modification of the 237th amino acid to Asp, modification of the 268th amino acid to Asp, modification of the 271st amino acid to Gly, modification of the 296th amino acid to Asp, modification of the 330th amino acid to Arg, and modification of the 332nd amino acid to Thr; (d) Modification of the 238th amino acid in the Fc region to Asp, modification of the 233rd amino acid to Asp, modification of the 237th amino acid to Asp, modification of the 264th amino acid to Ile, modification of the 267th amino acid to Gly or Ala, modification of the 268th amino acid to Glu, modification of the 271st amino acid to Gly, and modification of the 330th amino acid to Arg; (e) Modification of the 238th amino acid in the Fc region to Asp, modification of the 233rd amino acid to Asp, modification of the 237th amino acid to Asp, modification of the 267th amino acid to Ala, modification of the 268th amino acid to Glu, modification of the 271st amino acid to Gly, modification of the 296th amino acid to Asp, modification of the 330th amino acid to Arg, and modification of the 332nd amino acid to Thr; (f) Modification of the 238th amino acid in the Fc region to Asp, modification of the 237th amino acid to Asp, modification of the 267th amino acid to Ala, modification of the 268th amino acid to Glu, modification of the 271st amino acid to Gly, modification of the 296th amino acid to Asp, modification of the 330th amino acid to Arg, and modification of the 332nd amino acid to Thr; (g) Modification of the 238th amino acid in the Fc region to Asp, modification of the 233rd amino acid to Asp, modification of the 237th amino acid to Asp, modification of the 268th amino acid to Asp, modification of the 271st amino acid to Gly, modification of the 296th amino acid to Asp, modification of the 327th amino acid to Gly, and modification of the 330th amino acid to Arg; (h) Modification of the 238th amino acid in the Fc region to Asp, the 233rd amino acid to Asp, the 237th amino acid to Asp, the 264th amino acid to Ile, the 267th amino acid to Ala, the 268th amino acid to Glu, and the 271st amino acid to Gly; (i) Modification of the 238th amino acid in the Fc region to Asp, the 233rd amino acid to Asp, the 237th amino acid to Asp, the 264th amino acid to Ile, the 267th amino acid to Ala, the 268th amino acid to Glu, the 271st amino acid to Gly, the 296th amino acid to Asp, and the 330th amino acid to Arg; (j) Modification of the 238th amino acid in the Fc region to Asp, the 233rd amino acid to Asp, the 237th amino acid to Asp, the 264th amino acid to Ile, the 267th amino acid to Ala, the 268th amino acid to Glu, the 271st amino acid to Gly, the 296th amino acid to Asp, the 330th amino acid to Arg, and the 396th amino acid to Met or Leu; (k) Modification of the 238th amino acid in the Fc region to Asp, the 237th amino acid to Asp, the 264th amino acid to Ile, the 267th amino acid to Ala, the 268th amino acid to Glu, the 271st amino acid to Gly, and the 330th amino acid to Arg; (l) Modification of the 238th amino acid in the Fc region to Asp, the 237th amino acid to Asp, the 264th amino acid to Ile, the 267th amino acid to Ala, the 268th amino acid to Glu, the 271st amino acid to Gly, the 296th amino acid to Asp, and the 330th amino acid to Arg; (m) Modification of the 238th amino acid in the Fc region to Asp, the 264th amino acid to Ile, the 267th amino acid to Ala, the 268th amino acid to Glu, and the 271st amino acid to Gly; (n) Modification of the 238th amino acid of the EU numbering in the Fc region to Asp, modification of the 264th amino acid to Ile, modification of the 267th amino acid to Ala, modification of the 268th amino acid to Glu, modification of the 271st amino acid to Gly, and modification of the 296th amino acid to Asp; (o) Modification of the 238th amino acid of the EU numbering in the Fc region to Asp, modification of the 237th amino acid to Asp, modification of the 267th amino acid to Ala or Gly, modification of the 268th amino acid to Glu, modification of the 271st amino acid to Gly, modification of the 296th amino acid to Asp, and modification of the 330th amino acid to Arg; (p) Modification of the 238th amino acid of the EU numbering in the Fc region to Asp, modification of the 233rd amino acid to Asp, modification of the 237th amino acid to Asp, modification of the 264th amino acid to Ile, modification of the 267th amino acid to Ala, modification of the 268th amino acid to Glu, modification of the 271st amino acid to Gly, modification of the 330th amino acid to Arg, and modification of the 396th amino acid to Met or Leu; (q) Modification of the 238th amino acid of the EU numbering in the Fc region to Asp, modification of the 233rd amino acid to Asp, modification of the 237th amino acid to Asp, modification of the 264th amino acid to Ile, modification of the 267th amino acid to Ala, modification of the 268th amino acid to Glu, modification of the 271st amino acid to Gly, modification of the 296th amino acid to Asp, modification of the 327th amino acid to Gly, modification of the 330th amino acid to Arg, and modification of the 396th amino acid to Met; (r) Modification of the 238th amino acid of the EU numbering in the Fc region to Asp, modification of the 233rd amino acid to Asp, modification of the 237th amino acid to Asp, modification of the 264th amino acid to Ile, modification of the 267th amino acid to Ala, modification of the 268th amino acid to Glu, modification of the 271st amino acid to Gly, modification of the 272nd amino acid to Asp, and modification of the 296th amino acid to Asp; (s) Modification of the 238th amino acid in the Fc region to Asp, modification of the 237th amino acid to Asp, modification of the 264th amino acid to Ile, modification of the 267th amino acid to Ala, modification of the 268th amino acid to Glu, modification of the 271st amino acid to Gly, modification of the 272nd amino acid to Pro, and modification of the 330th amino acid to Arg; (t) Modification of the 238th amino acid in the Fc region to Asp, modification of the 237th amino acid to Asp, modification of the 264th amino acid to Ile, modification of the 267th amino acid to Ala, modification of the 268th amino acid to Glu, modification of the 271st amino acid to Gly, modification of the 272nd amino acid to Pro, modification of the 296th amino acid to Asp, and modification of the 330th amino acid to Arg; (u) Modification of the 238th amino acid in the Fc region to Asp, modification of the 233rd amino acid to Asp, modification of the 264th amino acid to Ile, modification of the 267th amino acid to Ala, modification of the 268th amino acid to Glu, and modification of the 271st amino acid to Gly; (v) Modification of the 238th amino acid in the Fc region to Asp, modification of the 237th amino acid to Asp, modification of the 267th amino acid to Gly, modification of the 268th amino acid to Asp, modification of the 271st amino acid to Gly, modification of the 296th amino acid to Asp, and modification of the 330th amino acid to Arg; (w) Modification of the 238th amino acid in the Fc region to Asp, modification of the 264th amino acid to Ile, modification of the 267th amino acid to Ala, modification of the 268th amino acid to Glu, modification of the 271st amino acid to Gly, modification of the 272nd amino acid to Asp, and modification of the 296th amino acid to Asp; and (x) Modification of the 238th amino acid in the Fc region to Asp, modification of the 233rd amino acid to Asp, modification of the 264th amino acid to Ile, modification of the 267th amino acid to Ala, modification of the 268th amino acid to Glu, modification of the 271st amino acid to Gly, and modification of the 296th amino acid to Asp. Claim 5 The method according to claim 1, 3, or 4, wherein the value of [KD value of the polypeptide containing the Fc region without amino acid modification for FcγRIIb] / [KD value of the polypeptide containing the Fc region variant for FcγRIIb] is 15.0 or more.

6. The method according to any one of claims 1 to 4, wherein the value of [KD value of the polypeptide containing the Fc region without amino acid modification for FcγRIIb] / [KD value of the polypeptide containing the Fc region variant for FcγRIIb] is 50.0 or more.

7. The method according to any one of claims 1 to 4, wherein the value of [KD value of the polypeptide containing the Fc region without amino acid modification for FcγRIIb] / [KD value of the polypeptide containing the Fc region variant for FcγRIIb] is 100.0 or more.

8. The method according to any one of claims 2 to 7, wherein the value of [KD value of the polypeptide containing the Fc region variant for FcγRIIa (R type)] / [KD value of the polypeptide containing the Fc region variant for FcγRIIb] is 20.0 or more.

9. A method for producing a polypeptide containing an Fc region variant, comprising the step of introducing an amino acid modification into the Fc region in a polypeptide containing the Fc region, wherein the amino acid modification includes the modifications described in the following (1) and (2), (1) Modification of the amino acid at position 238 in EU numbering to Asp, and (2) At least one modification selected from the modification of the amino acid at position 264 in EU numbering to Ile, the modification of the amino acid at position 268 to Asp, Gln, or Glu, the modification of the amino acid at position 271 to Gly, and the modification of the amino acid at position 330 to Lys, Arg, or Ser, the Fc region variant is an Fc region variant with enhanced binding selectivity for FcγRIIb compared to FcγRIIa (R type), or an Fc region variant with enhanced binding activity for FcγRIIb compared to the Fc region without amino acid modification and enhanced binding selectivity for FcγRIIb compared to FcγRIIa (R type), The method, wherein the value of [KD value of the polypeptide containing the Fc region variant for FcγRIIa (R type)] / [KD value of the polypeptide containing the Fc region variant for FcγRIIb] is 20.0 or more. **Claim 10**: A method for producing a polypeptide comprising an Fc region variant, the method comprising the step of introducing an amino acid modification into the Fc region in a polypeptide comprising the Fc region, wherein the amino acid modification comprises the modifications described in (1) to (3) below, (1) Modification of the amino acid at position 238 according to EU numbering to Asp, (2) Modification of the amino acid at position 271 according to EU numbering to Gly, and (3) At least one modification selected from the group consisting of modification of the amino acid at position 233 according to EU numbering to Asp, modification of the amino acid at position 237 according to EU numbering to Asp, modification of the amino acid at position 268 according to EU numbering to Asp, and modification of the amino acid at position 330 according to EU numbering to Arg, wherein the Fc region variant is an Fc region variant in which the binding activity to FcγRIIb is enhanced as compared to an Fc region into which no amino acid modification has been introduced, and the binding selectivity to FcγRIIb is enhanced as compared to FcγRIIa (R type), and the value of [KD value of the polypeptide containing the Fc region without amino acid modification for FcγRIIb] / [KD value of the polypeptide containing the Fc region variant for FcγRIIb] is 15.0 or more, and the value of [KD value of the polypeptide containing the Fc region variant for FcγRIIa (R type)] / [KD value of the polypeptide containing the Fc region variant for FcγRIIb] is 10.0 or more. **Claim 11** A method for producing a pharmaceutical composition containing a polypeptide comprising an Fc region variant, the method comprising the step of introducing an amino acid modification into the Fc region in a polypeptide comprising the Fc region and the step of formulating by adding a pharmaceutically acceptable carrier to the polypeptide comprising the Fc region variant, wherein the amino acid modification comprises the modifications described in (1) and (2) below, (1) Modification of the amino acid at position 238 according to EU numbering to Asp, and (2) At least one modification selected from the group consisting of modification of the amino acid at position 264 according to EU numbering to Ile, modification of the amino acid at position 268 according to EU numbering to Asp, Gln or Glu, modification of the amino acid at position 271 according to EU numbering to Gly, and modification of the amino acid at position 330 according to EU numbering to Lys, Arg or Ser, An Fc region variant, which has enhanced binding selectivity for FcγRIIb as compared to FcγRIIa (R type), or an Fc region variant, which has enhanced binding activity for FcγRIIb as compared to an Fc region without amino acid modification and has enhanced binding selectivity for FcγRIIb as compared to FcγRIIa (R type). A method wherein the value of [KD value of the polypeptide containing the Fc region variant for FcγRIIa (R type)] / [KD value of the polypeptide containing the Fc region variant for FcγRIIb] is 20.0 or more. The method for producing a pharmaceutical composition containing a polypeptide containing an Fc region variant according to claim 12, which comprises the step of introducing an amino acid modification into the Fc region in a polypeptide containing the Fc region, and the step of formulating by adding a pharmaceutically acceptable carrier to the polypeptide containing the Fc region variant. The amino acid modification includes the modifications described in the following (1) to (3). (1) Modification of the amino acid at position 238 according to EU numbering to Asp. (2) Modification of the amino acid at position 271 according to EU numbering to Gly, and (3) At least one modification selected from the group consisting of modification of the amino acid at position 233 according to EU numbering to Asp, modification of the amino acid at position 237 to Asp, modification of the amino acid at position 268 to Asp, and modification of the amino acid at position 330 to Arg. An Fc region variant, which has enhanced binding activity for FcγRIIb as compared to an Fc region without amino acid modification and has enhanced binding selectivity for FcγRIIb as compared to FcγRIIa (R type). A method wherein the value of [KD value of the polypeptide containing an Fc region without amino acid modification for FcγRIIb] / [KD value of the polypeptide containing the Fc region variant for FcγRIIb] is 15.0 or more, and the value of [KD value of the polypeptide containing the Fc region variant for FcγRIIa (R type)] / [KD value of the polypeptide containing the Fc region variant for FcγRIIb] is 10.0 or more.

13. The method according to any one of claims 9 to 12, wherein the polypeptide containing the Fc region variant is a polypeptide containing at least two Fc region variants, and the two Fc region variants are associated with each other.

14. The method according to claim 13, wherein the amino acid sequences of the two associated Fc region variants are identical. **Claim 15** The method according to claim 13, wherein the amino acid sequences of the two associated Fc region variants are different. **Claim 16** The method according to claim 15, wherein in the amino acid sequences of the two associated Fc region variants, at least one amino acid selected from the 235th, 236th, 237th, and 239th amino acids of the EU numbering of the Fc region variant is different. **Claim 17** The method according to claim 16, wherein the amino acid sequence of either one of the two associated Fc region variants has at least one amino acid selected from the 235th amino acid being Asp, Gln, Glu or Thr, the 236th amino acid being Asn, the 237th amino acid being Phe or Trp, and the 239th amino acid being Asp or Glu. **Claim 18** The method according to any one of claims 9 to 17, wherein the polypeptide containing the Fc region variant is an IgG antibody. **Claim 19** The method according to any one of claims 9 to 17, wherein the polypeptide containing the Fc region variant is an Fc fusion protein molecule.

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