Fcγriib-specific fc antibody

Abstract

To provide a polypeptide comprising a Fc region with a maintained or decreased binding activity to H-type or R-type, which is a genetic polymorphism of FcγRIIa, and an increased binding activity to FcγRIIb; a pharmaceutical composition comprising the polypeptide; a therapeutic or prophylactic agent for immune inflammatory diseases comprising the pharmaceutical composition; manufacturing methods therefor; and a method for maintaining or decreasing binding activity to the genetic polymorphism of FcγRIIa and increasing binding activity to FcγRIIb.SOLUTION: The present invention provides a polypeptide comprising an antibody Fc region including a modification that substitutes Asp for Pro at position 238 (EU numbering) and Glu for Leu at position 328 (EU numbering) or a peptide comprising a modification that substitutes Asp for Pro at position 238 (EU numbering) and any of other modifications.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a polypeptide comprising an Fc region in which amino acid substitutions have been introduced into the Fc region of IgG, thereby maintaining or decreasing binding activity to both H type (H type) FcγRIIa genetic polymorphisms in which the amino acid at EU numbering position 131 of FcγRIIa is His and R type (R type) in which the amino acid at EU numbering position 131 of FcγRIIa is Arg, and increasing binding activity to FcγRIIb, as compared to a parent polypeptide, a pharmaceutical composition containing the polypeptide, a therapeutic or preventive agent for immunoinflammatory disease containing the polypeptide, and methods for producing these. The present invention also relates to a method for maintaining or decreasing binding activity to both H type (H type) FcγRIIa genetic polymorphisms in which the amino acid at EU numbering position 131 of FcγRIIa is His and R type (R type) in which the amino acid at EU numbering position 131 of FcγRIIa is Arg, as compared to a parent polypeptide, and increasing binding activity to FcγRIIb, as compared to a parent polypeptide, and a method for suppressing antibody production when administered to a living body, as compared to the parent polypeptide. Furthermore, the present invention relates to a method for producing a polypeptide that has maintained or reduced binding activity to FcγRIIa of both H type (H type) in which the 131st amino acid of FcγRIIa is His) and R type (R type) in which the 131st amino acid of FcγRIIa is Arg, as compared to the parent polypeptide, and has enhanced binding activity to FcγRIIb, as well as a method for producing a polypeptide that suppresses antibody production when administered to a living body, as compared to the parent polypeptide. [Background technology]

[0002] Antibodies are attracting attention as pharmaceuticals because they are highly stable in the blood and have few side effects (Non-Patent Documents 1 and 2). Most of the antibody drugs currently on the market are human IgG1 subclass antibodies. Antibody-dependent cellular cytotoxicity (hereinafter referred to as ADCC activity) is known as one of the functions of IgG class antibodies (Non-Patent Document 3). In order for an antibody to exert ADCC activity, it is necessary for the Fc region of the antibody to bind to Fcγ receptors (hereinafter referred to as FcγR), which are antibody-binding receptors present on the surfaces of effector cells such as killer cells, natural killer cells, and activated macrophages.

[0003] In humans, the FcγR protein family includes the isoforms FcγRIa (CD64A), FcγRIIa (CD32A), FcγRIIb (CD32B), FcγRIIIa (CD16A), and FcγRIIIb (CD16B), and their allotypes have also been reported (Non-Patent Document 7). FcγRIa, FcγRIIa, and FcγRIIIa have immune-activating functions and are therefore called activating FcγR, while FcγRIIb has immune-suppressing functions and is called inhibitory FcγR (Non-Patent Document 8).

[0004] It has been shown that several amino acid residues in the hinge region and CH2 domain of an antibody and a sugar chain attached to Asn at EU numbering position 297 attached to the CH2 domain are important for the binding between the Fc region and FcγR (Non-Patent Documents 4, 5, and 6). Mutants with various FcγR binding properties have been studied, mainly focusing on antibodies in which mutations have been introduced into these sites, and Fc region mutants with higher binding activity to activating FcγR have been obtained (Patent Documents 1, 2, 3, and 4).

[0005] When activated FcγR is cross-linked by immune complexes, it induces phosphorylation of immunoreceptor tyrosine-based activating motifs (ITAMs) contained in the intracellular domain or its interacting partner, the FcR common γ-chain, which activates the signal transduction substance SYK and initiates an activation signal cascade, thereby causing 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 interaction of the Fc region of an antibody with FcγRIIb suppresses the primary immunity of B cells (Non-Patent Document 11). It has also been reported that when FcγRIIb on B cells and the B cell receptor (BCR) are crosslinked via immune complexes in the blood, B cell activation is suppressed and antibody production by B cells is suppressed (Non-Patent Document 12). The immunoreceptor tyrosine-based inhibitory motif (ITIM) contained in the intracellular domain of FcγRIIb is required for the transmission of immunosuppressive signals via this BCR and FcγRIIb (Non-Patent Document 13, Non-Patent Document 14). When a signal is received and ITIM is phosphorylated, SH2-containing inositol polyphosphate 5-phosphatase (SHIP) is recruited, which inhibits the transmission of signal cascades of other activating FcγRs and suppresses inflammatory immune responses (Non-Patent Document 15). It has also been reported that association of only FcγRIIb transiently suppresses B cell proliferation and calcium influx induced by BCR cross-linking without inducing apoptosis of IgM-producing B cells in a BCR-independent manner (Non-Patent Document 16).

[0007] FcγRIIb is also expressed in dendritic cells, macrophages, activated neutrophils, mast cells, and basophils. In these cells, FcγRIIb inhibits the functions of activating FcγR, such as phagocytosis and release of inflammatory cytokines, and suppresses inflammatory immune responses (Non-Patent Document 8).

[0008] The importance of the immunosuppressive function of FcγRIIb has been elucidated by studies using FcγRIIb knockout mice. It has been reported that FcγRIIb knockout mice have inappropriate humoral immunity (Non-Patent Document 17), have increased susceptibility to collagen-induced arthritis (CIA) (Non-Patent Document 18), and exhibit lupus-like symptoms and Goodpasture syndrome-like symptoms (Non-Patent Document 19).

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

[0010] Thus, based on mouse models and clinical findings, FcγRIIb is thought to play a role in controlling autoimmune and inflammatory diseases, primarily through its involvement with B cells, and is a promising target molecule for controlling autoimmune and inflammatory diseases.

[0011] IgG1, which is mainly used as a commercially available antibody drug, is known to bind strongly not only to FcγRIIb but also to activating FcγR (Non-Patent Document 27). By using an Fc region with enhanced binding to FcγRIIb or improved selectivity of binding to FcγRIIb compared to activating FcγR, it is possible to develop an antibody drug with 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 with enhanced binding to FcγRIIb (Non-Patent Document 28). It has been reported that the differentiation of B cells into plasma cells and the resulting IgE production are suppressed by crosslinking FcγRIIb on B cells with IgE bound to the B-cell receptor, and that in mice transplanted with human PBMC, the human IgG and IgM concentrations are maintained, but the human IgE concentration is reduced (Non-Patent Document 29). It has been reported that not only IgE but also CD79b and FcγRIIb, which form the B-cell receptor complex, were cross-linked with an antibody to suppress B cell proliferation in vitro and alleviate symptoms in a collagen-induced arthritis model (Non-patent Document 30). In addition to B cells, it has been reported that a molecule formed by fusing the Fc portion of IgE, which binds to the IgE receptor FcεRI, with the Fc portion of IgG with enhanced binding to FcγRIIb can be used to cross-link FcεRI and FcγRIIb on mast cells, causing phosphorylation of FcγRIIb and suppressing FcεRI-dependent calcium influx, suggesting that enhancing binding to FcγRIIb makes it possible to inhibit degranulation mediated by stimulation of FcγRIIb (Non-Patent Document 31). These findings suggest that antibodies having Fc with improved binding activity to FcγRIIb may be promising therapeutic agents for inflammatory diseases such as autoimmune diseases.

[0012] It has also been suggested that mutants with enhanced binding to FcγRIIb are promising as therapeutic agents for cancer, in addition to therapeutic agents for inflammatory diseases such as autoimmune diseases. It has been revealed that FcγRIIb also plays an important role in the agonistic activity of agonistic antibodies against the anti-TNF receptor family. Specifically, it has been suggested that the agonistic activity of antibodies against CD40, DR4, DR5, CD30, and CD137, which are included in the TNF receptor family, requires interaction with FcγRIIb (Non-Patent Documents 32, 33, 34, 35, 36, 37). Non-Patent Document 32 shows that the antitumor effect of anti-CD40 antibodies is enhanced by using antibodies with enhanced binding to FcγRIIb. From this, it is expected that antibodies with enhanced binding to FcγRIIb have the effect of enhancing the agonistic action of agonistic antibodies, including antibodies against the anti-TNF receptor family.

[0013] There have been reports on antibodies with Fc having improved binding activity to FcγRIIb (Non-Patent Document 28). In this document, the binding activity to FcγRIIb was improved by adding modifications such as S267E / L328F, G236D / S267E, and S239D / S267E to the Fc region of the antibody. Among these, an antibody into which the S267E / L328F mutation was introduced bound most strongly to FcγRIIb, and the binding to FcγRIa and H type of FcγRIIa was maintained at the same level as that of natural IgG1. However, according to another report, this modification enhanced the binding to R type of FcγRIIa by several hundred times to the same level as that to FcγRIIb, and the selectivity of binding to FcγRIIb was not improved compared to FcγRIIa R type (Patent Document 5). Even if the binding to FcγRIIb is enhanced compared to IgG1, it is considered that only the effect of enhanced binding to FcγRIIa, not enhanced binding to FcγRIIb, is affected in cells such as platelets that express FcγRIIa but not FcγRIIb (Non-Patent Document 8). For example, it is known that the risk of thromboembolism increases in patients administered bevacizumab, an antibody against VEGF (Non-Patent Document 38). Similarly, thromboembolism was observed in a clinical development test of an antibody against CD40 ligand, and the clinical trial was discontinued (Non-Patent Document 39). In the case of both of these antibodies, subsequent studies using animal models have suggested that the administered antibody aggregates platelets through binding to FcγRIIa on platelets to form thrombi (Non-Patent Document 40, Non-Patent Document 41). It has been reported that in systemic lupus erythematosus, an autoimmune disease, platelets are activated by an FcγRIIa-dependent mechanism, and platelet activation correlates with the severity (Non-Patent Document 42). Administering an antibody with enhanced binding to FcγRIIa to patients who are already at high risk of developing such thromboembolism would be extremely dangerous, as it would further increase the risk of developing thromboembolism, even if binding to FcγRIIb was also enhanced. It has also been reported that antibodies with enhanced binding to FcγRIIa have enhanced antibody-dependent phagocytic activity (ADCP) mediated by macrophages (Non-Patent Document 43). When the antigen of an antibody is phagocytosed by a macrophage, the antibody itself is also phagocytosed. In this case, a peptide fragment derived from the antibody is also presented as an antigen, which is thought to increase antigenicity and increase the risk of production of antibodies against the antibody (anti-antibody). In other words, enhancing the binding to FcγRIIa increases the risk of production of antibodies against the antibody, significantly reducing its value as a pharmaceutical. In other words, enhancing binding to FcγRIIa increases the risk of thrombus formation via platelet aggregation, and also increases antigenicity and the risk of anti-antibody production, significantly reducing its value as a pharmaceutical.

[0014] From this perspective, when considering the Fc with enhanced binding to FcγRIIb, the binding to FcγRIIa R type is significantly enhanced compared to natural IgG1, so that its value as a medicine for patients with FcγRIIa R type is significantly reduced. FcγRIIa H type and R type are observed at approximately the same frequency in Caucasians and African-Americans (Non-Patent Documents 44 and 45). For this reason, when using this Fc to treat autoimmune diseases, the number of patients who can safely use it while enjoying the effects of the medicine is limited. It has also been reported that dendritic cell maturation occurs spontaneously in dendritic cells lacking FcγRIIb or in dendritic cells in which the interaction between FcγRIIb and the Fc portion of the antibody is inhibited by an anti-FcγRIIb antibody (Non-Patent Document 46, Non-Patent Document 47). This report suggests that FcγRIIb actively suppresses dendritic cell maturation in a steady state in which inflammation is not occurring. 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 and the like is also enhanced, it is considered that this will ultimately promote the maturation of dendritic cells. In other words, it is considered important to improve not only the binding activity to FcγRIIb but also the ratio of the binding activity to FcγRIIb to the binding activity to FcγRIIa in order to bring about an immunosuppressive effect in an antibody. For this reason, when considering the creation of pharmaceuticals that utilize the immunosuppressive effect mediated by FcγRIIb binding, there is a demand for an Fc that not only has enhanced binding activity to FcγRIIb, but also maintains binding to both FcγRIIa H and R genetic polymorphisms at the same level as that of native IgG1 or has even weaker binding than that. In response to this, there have been reports of increasing the selectivity of binding to FcγRIIb by introducing amino acid modifications into the Fc region (Non-Patent Document 48). However, all of the mutants reported in this document that are said to have improved selectivity for FcγRIIb showed reduced binding to FcγRIIb compared to native IgG1. Therefore, it is considered difficult for these mutants to actually induce an immunosuppressive reaction mediated by FcγRIIb more than that of IgG1. In addition, since FcγRIIb plays an important role in the agonist antibody described above, enhancing its binding activity is expected to enhance the agonist activity. However, if the binding to FcγRIIa is also enhanced in the same way, unintended ADCC activity or ADCP activity may be exhibited, which may cause side effects. From this perspective, it is preferable to selectively enhance the binding activity to FcγRIIb.

[0015] From these results, when creating antibody drugs using FcγRIIb for the purpose of treating autoimmune diseases or cancer, it is important that the binding activity to any genetic polymorphism of FcγRIIa is maintained or decreased and the binding activity to FcγRIIb is enhanced, as compared to natural IgG. However, FcγRIIb and FcγRIIa, which is one of the activating FcγRs, share 93% sequence identity in the extracellular domain and are extremely similar in structure, and furthermore, FcγRIIa has genetic polymorphisms of H type (H type) in which the 131st amino acid is His and R type (R type) in which the 131st amino acid is Arg, and the interactions with antibodies differ between these types (Non-Patent Document 49). Therefore, in order to create an Fc region that selectively binds to FcγRIIb, it is considered to be the most difficult task to distinguish between these similar sequences and to impart to the Fc region of an antibody a property that selectively improves the binding activity to FcγRIIb, that is, to increase the binding activity to FcγRIIb while not increasing or decreasing the binding activity to each genetic polymorphism of FcγRIIa, and thus no mutants with sufficient selectivity to FcγRIIb have been obtained so far. Patent Document 5 reports mutants with enhanced binding activity to FcγRIIb, but the degree of the enhancement is weak, and there has been a demand for the development of mutants with the above-mentioned properties. [Prior art documents] [Patent documents]

[0016] [Patent Document 1] WO 2000 / 42072 [Patent Document 2] WO 2006 / 019447 [Patent Document 3] WO 2004 / 99249 [Patent Document 4] WO 2004 / 29207 [Patent Document 5] US2009 / 0136485 [Non-patent literature]

[0017] [Non-licensed document 1] Nat Biotechnol, 23(9), 1073-1078, 2005 [Non-licensed document 2] Eur J Pharm Biopharm, 59(3), 389-96, 2005 [Non-licensed document 3] Chem Immunol, 65, 88-110, 1997

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[0018] The present invention has been made in view of such circumstances, and its purpose is to provide a polypeptide comprising an Fc region in which the binding activity to both genetic polymorphisms of FcγRIIa, H type (H type) in which the amino acid at EU numbering 131 of FcγRIIa is His and R type (R type) in which the amino acid at EU numbering 131 of FcγRIIa is Arg, is maintained or decreased, and the binding activity to FcγRIIb is enhanced, as compared to a parent polypeptide, a pharmaceutical composition containing the polypeptide, a therapeutic or preventive agent for immune inflammatory diseases containing the polypeptide, and a method for producing these. In addition, the present invention is to provide a method for maintaining or decreasing the binding activity to both genetic polymorphisms of FcγRIIa, H type (H type) in which the amino acid at EU numbering 131 of FcγRIIa is His and R type (R type) in which the amino acid at EU numbering 131 of FcγRIIa is Arg, as compared to a parent polypeptide, and enhancing the binding activity to FcγRIIb, as well as a method for suppressing antibody production, as compared to a parent polypeptide, when administered to a living body. Furthermore, the present invention provides a method for producing a polypeptide that maintains or reduces binding activity to FcγRIIa of both H type (H type) in which the 131st amino acid of FcγRIIa is His) and R type (R type) in which the 131st amino acid of FcγRIIa is Arg, as compared to the parent polypeptide, and has enhanced binding activity to FcγRIIb, as well as a method for producing a polypeptide that suppresses antibody production when administered to a living body, as compared to the parent polypeptide. [Means for solving the problem]

[0019] The present inventors have conducted extensive research into polypeptides containing an Fc region that exhibits reduced Fc-mediated binding to FcγRIIa and enhanced binding to FcγRIIb compared to the parent polypeptide. As a result, the present inventors have found that a polypeptide containing an antibody Fc region that includes a modification in which Pro at position 238 (EU numbering) is replaced with Asp or Leu at position 328 (EU numbering) is replaced with Glu has enhanced binding activity to FcγRIIb and reduced binding activity to both H-type and R-type genetic polymorphisms of FcγRIIa via the Fc region. Furthermore, the present inventors have found that a polypeptide containing an antibody Fc region that includes a modification in which Pro at position 238 (EU numbering) is replaced with Asp and several other modifications has enhanced binding activity to FcγRIIb and maintained or reduced binding activity to both H-type and R-type genetic polymorphisms of FcγRIIa via the Fc region.

[0020] That is, the present invention relates to the following. [1] A polypeptide variant comprising an antibody Fc region in which at least one amino acid has been altered, which, compared to the parent polypeptide, maintains or decreases its binding activity to FcγRIIa (R type) and FcγRIIa (H type) and enhances its binding activity to FcγRIIb, and the value of [KD value of the polypeptide variant for FcγRIIa (R type)] / [KD value of the polypeptide variant for FcγRIIb] is 1.2 or greater. [2] The polypeptide according to [1], wherein the value of [KD value of the polypeptide mutant for FcγRIIa (H type)] / [KD value of the polypeptide mutant for FcγRIIb] is 4.2 or more. [3] The polypeptide according to [1] or [2], wherein the value of [KD value of parent polypeptide for FcγRIIb] / [KD value of polypeptide mutant for FcγRIIb] is 1.6 or more. [4] The polypeptide according to any one of [1] to [3], wherein the value of [the stronger KD value of the binding activity of the polypeptide mutant to FcγRIIa (R type) and FcγRIIa (H type)] / [the stronger KD value of the binding activity of the parent polypeptide to FcγRIIa (R type) and FcγRIIa (H type)] is 0.7 or more. [5] The polypeptide according to any one of [1] to [4], which has a binding activity to FcγRIIIa that is maintained or reduced compared to the parent polypeptide. [6] The polypeptide according to any one of [1] to [5], which has a binding activity to FcγRIa that is maintained or reduced compared to the parent polypeptide. [7] the polypeptide according to any one of [1] to [6], wherein the amino acid modification is a substitution of Pro at position 238 (EU numbering) with Asp, or a substitution of Leu at position 328 (EU numbering) with Glu; [8] The amino acid modifications are: substitution of Pro at position 238 (EU numbering) with Asp, substitution of Gly at position 237 (EU numbering) with Trp, substitution of Gly at position 237 (EU numbering) with Phe, substitution of Ser at position 267 (EU numbering) with Val, substitution of Ser at position 267 (EU numbering) with Gln, substitution of His at position 268 (EU numbering) with Asn, substitution of Pro at position 271 (EU numbering) with Gly, substitution of Lys at position 326 (EU numbering) with Leu, substitution of Lys at position 326 (EU numbering) with Gl ... substitution of Lys at position 326 (EU numbering) by Glu, substitution of Lys at position 326 (EU numbering) by Met, substitution of Ser at position 239 (EU numbering) by Asp, substitution of Ser at position 267 (EU numbering) by Ala, substitution of Leu at position 234 (EU numbering) by Trp, substitution of Leu at position 234 (EU numbering) by Tyr, substitution of Gly at position 237 (EU numbering) by Ala, substitution of Gly at position 237 (EU numbering) by Asp, substitution of Gly at position 237 (EU numbering) by Glu, substitution of Gly at position 237 (EU numbering) by substitution of Leu, substitution of Gly at position 237 (EU numbering) by Met, substitution of Gly at position 237 (EU numbering) by Tyr, substitution of Ala at position 330 (EU numbering) by Lys, substitution of Ala at position 330 (EU numbering) by Arg, substitution of Glu at position 233 (EU numbering) by Asp, substitution of His at position 268 (EU numbering) by Asp, substitution of His at position 268 (EU numbering) by Glu, substitution of Lys at position 326 (EU numbering) by Asp, substitution of Lys at position 326 (EU numbering) by Ser, EU numbering The polypeptide of any one of [1] to [7], wherein the substitution is at least one selected from the group consisting of substitution of Lys at position 326 with Thr, substitution of Val at position 323 (EU numbering) with Ile, substitution of Val at position 323 (EU numbering) with Leu, substitution of Val at position 323 (EU numbering) with Met, substitution of Tyr at position 296 (EU numbering) with Asp, substitution of Lys at position 326 (EU numbering) with Ala, substitution of Lys at position 326 (EU numbering) with Asn, and substitution of Ala at position 330 (EU numbering) with Met. [9] The polypeptide according to any one of [1] to [8], wherein the polypeptide comprising an antibody Fc region is an IgG antibody.

[10] The polypeptide according to any one of [1] to [8], wherein the polypeptide comprising an antibody Fc region is an Fc fusion protein molecule.

[11] A method for maintaining or decreasing the binding activity to FcγRIIa (R type) and FcγRIIa (H type) and enhancing the binding activity to FcγRIIb, compared to a parent polypeptide, comprising making at least one amino acid modification to the Fc region, wherein the amino acid modification is a substitution of Pro at position 238 (EU numbering) with Asp, or a substitution of Leu at position 328 (EU numbering) with Glu.

[12] A method for suppressing antibody production against a polypeptide comprising an antibody Fc region, as compared to a parent polypeptide, when administered to a living body, comprising making at least one amino acid modification to the Fc region, wherein the amino acid modification is a substitution of Pro at position 238 (EU numbering) with Asp, or a substitution of Leu at position 328 (EU numbering) with Glu.

[13] The amino acid modifications are: substitution of Pro at position 238 (EU numbering) with Asp; substitution of Gly at position 237 (EU numbering) with Trp; substitution of Gly at position 237 (EU numbering) with Phe; substitution of Ser at position 267 (EU numbering) with Val; substitution of Ser at position 267 (EU numbering) with Gln; substitution of His at position 268 (EU numbering) with Asn; substitution of Pro at position 271 (EU numbering) with Gly; substitution of Lys at position 326 (EU numbering) with Leu; substitution of Lys at position 326 (EU numbering) with Gln; Lys at position 326 (EU numbering) replaced by Glu, Lys at position 326 (EU numbering) replaced by Met, Ser at position 239 (EU numbering) replaced by Asp, Ser at position 267 (EU numbering) replaced by Ala, Leu at position 234 (EU numbering) replaced by Trp, Leu at position 234 (EU numbering) replaced by Tyr, Gly at position 237 (EU numbering) replaced by Ala, Gly at position 237 (EU numbering) replaced by Asp, Gly at position 237 (EU numbering) replaced by Glu, Gl at position 237 (EU numbering) replaced by substitution of y by Leu, substitution of Gly by Met at position 237 (EU numbering), substitution of Gly by Tyr at position 237 (EU numbering), substitution of Ala by Lys at position 330 (EU numbering), substitution of Ala by Arg at position 330 (EU numbering), substitution of Glu by Asp at position 233 (EU numbering), substitution of His by Asp at position 268 (EU numbering), substitution of His by Glu at position 268 (EU numbering), substitution of Lys by Asp at position 326 (EU numbering), substitution of Lys by Ser at position 326 (EU numbering), The method according to

[11] or

[12] , wherein the substitution is at least one selected from the group consisting of substitution of Lys at position 326 (EU numbering) with Thr, substitution of Val at position 323 (EU numbering) with Ile, substitution of Val at position 323 (EU numbering) with Leu, substitution of Val at position 323 (EU numbering) with Met, substitution of Tyr at position 296 (EU numbering) with Asp, substitution of Lys at position 326 (EU numbering) with Ala, substitution of Lys at position 326 (EU numbering) with Asn, and substitution of Ala at position 330 (EU numbering) with Met.

[14] The method according to any one of

[11] to

[13] , wherein the polypeptide comprising an antibody Fc region is an IgG antibody.

[15] The method according to any one of

[11] to

[13] , wherein the polypeptide comprising an antibody Fc region is an Fc fusion protein molecule.

[16] A method for producing a polypeptide comprising an antibody Fc region, which has maintained or decreased binding activity to FcγRIIa (R type) and FcγRIIa (H type) and enhanced binding activity to FcγRIIb, as compared to a parent polypeptide, comprising making at least one amino acid modification to the Fc region, wherein the amino acid modification is a substitution of Pro at position 238 (EU numbering) with Asp, or a substitution of Leu at position 328 (EU numbering) with Glu.

[17] A method for producing a polypeptide that contains an antibody Fc region and suppresses production of antibodies against the polypeptide when administered to a living body, as compared to a parent polypeptide, comprising making at least one amino acid modification to the Fc region, wherein the amino acid modification is a substitution of Pro at position 238 (EU numbering) with Asp, or a substitution of Leu at position 328 (EU numbering) with Glu.

[18] The amino acid modifications are: substitution of Pro at position 238 (EU numbering) with Asp, substitution of Gly at position 237 (EU numbering) with Trp, substitution of Gly at position 237 (EU numbering) with Phe, substitution of Ser at position 267 (EU numbering) with Val, substitution of Ser at position 267 (EU numbering) with Gln, substitution of His at position 268 (EU numbering) with Asn, substitution of Pro at position 271 (EU numbering) with Gly, substitution of Lys at position 326 (EU numbering) with Leu, substitution of Lys at position 326 (EU numbering) with Gl ... Lys at position 326 (EU numbering) replaced by Glu, Lys at position 326 (EU numbering) replaced by Met, Ser at position 239 (EU numbering) replaced by Asp, Ser at position 267 (EU numbering) replaced by Ala, Leu at position 234 (EU numbering) replaced by Trp, Leu at position 234 (EU numbering) replaced by Tyr, Gly at position 237 (EU numbering) replaced by Ala, Gly at position 237 (EU numbering) replaced by Asp, Gly at position 237 (EU numbering) replaced by Glu, Gl at position 237 (EU numbering) replaced by substitution of y by Leu, substitution of Gly by Met at position 237 (EU numbering), substitution of Gly by Tyr at position 237 (EU numbering), substitution of Ala by Lys at position 330 (EU numbering), substitution of Ala by Arg at position 330 (EU numbering), substitution of Glu by Asp at position 233 (EU numbering), substitution of His by Asp at position 268 (EU numbering), substitution of His by Glu at position 268 (EU numbering), substitution of Lys by Asp at position 326 (EU numbering), substitution of Lys by Ser at position 326 (EU numbering), The method according to

[16] or

[17] , wherein the substitution is at least one selected from the group consisting of substitution of Lys at position 326 (EU numbering) with Thr, substitution of Val at position 323 (EU numbering) with Ile, substitution of Val at position 323 (EU numbering) with Leu, substitution of Val at position 323 (EU numbering) with Met, substitution of Tyr at position 296 (EU numbering) with Asp, substitution of Lys at position 326 (EU numbering) with Ala, substitution of Lys at position 326 (EU numbering) with Asn, and substitution of Ala at position 330 (EU numbering) with Met.

[19] The method according to any one of

[16] to

[18] , wherein the polypeptide comprising an antibody Fc region is an IgG antibody.

[20] The method according to any one of

[16] to

[18] , wherein the polypeptide comprising an antibody Fc region is an Fc fusion protein molecule.

[21] A polypeptide produced by the method according to any one of

[16] to

[20] .

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

[10] and

[21] .

[23] An agent for suppressing the activation of B cells, mast cells, dendritic cells, and / or basophils, comprising the polypeptide according to any one of [1] to

[10] and

[21] .

[24] A therapeutic or preventive agent for an immunoinflammatory disease, comprising the polypeptide according to any one of [1] to

[10] and

[21] .

[25] The therapeutic or prophylactic agent according to

[24] , wherein the immunoinflammatory disease is an autoimmune disease believed to be caused by production of an antibody against an autoantigen.

[26] A therapeutic agent for a disease comprising the polypeptide according to any one of [1] to

[10] and

[21] , wherein the disease is caused by a deficiency of a protein necessary for the living body.

[27] An antiviral agent comprising the polypeptide according to any one of [1] to

[10] and

[21] . The present invention also relates to a method for treating or preventing an immunoinflammatory disease, comprising the step of administering to a subject a polypeptide of the present invention or a polypeptide produced by the production method of the present invention. The present invention also relates to a kit for use in the therapeutic or preventive method of the present invention, comprising the polypeptide of the present invention or a polypeptide produced by the production method of the present invention, or a pharmaceutical composition of the present invention. The present invention also relates to the use of the polypeptide of the present invention or a polypeptide produced by the production method of the present invention in the production of a therapeutic or preventive agent for an immunoinflammatory disease. The present invention also relates to the polypeptide of the present invention or a polypeptide produced by the production method of the present invention for use in the therapeutic or preventive method 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, comprising the step of administering to a subject a polypeptide of the present invention or a polypeptide produced by the production method of the present invention. The present invention also relates to a kit for use in the suppression method of the present invention, comprising the polypeptide of the present invention or a polypeptide produced by the production method of the present invention, or a pharmaceutical composition of the present invention. The present invention also relates to the use of the polypeptide of the present invention or a polypeptide produced by the production method of the present invention in the production of an inhibitor of the activation of B cells, mast cells, dendritic cells and / or basophils. The present invention also relates to the polypeptide of the present invention or a polypeptide produced by the production method of the present invention for use in the suppression method of the present invention. The present invention also relates to a method for treating a disease in which a protein necessary for the living body is lacking, comprising the step of administering to a subject the polypeptide of the present invention or a polypeptide produced by the production method of the present invention. The present invention also relates to a kit for use in the treatment method of the present invention, comprising the polypeptide of the present invention or a polypeptide produced by the production method of the present invention, or the pharmaceutical composition of the present invention. The present invention also relates to the use of the polypeptide of the present invention or a polypeptide produced by the production method of the present invention in the production of a therapeutic agent for a disease in which a protein necessary for the living body is lacking. The present invention also relates to the polypeptide of the present invention or a polypeptide produced by the production method of the present invention for use in the treatment method of the present invention.The present invention also relates to a method for inhibiting viruses, comprising the step of administering to a subject a polypeptide of the present invention or a polypeptide produced by the production method of the present invention. The present invention also relates to a kit for use in the inhibition method of the present invention, comprising the polypeptide of the present invention or a polypeptide produced by the production method of the present invention, or the pharmaceutical composition of the present invention. The present invention also relates to use of the polypeptide of the present invention or a polypeptide produced by the production method of the present invention in the production of an antiviral agent. The present invention also relates to the polypeptide of the present invention or a polypeptide produced by the production method of the present invention for use in the inhibition method of the present invention. Effect of the Invention

[0021] The present invention provides a polypeptide comprising an Fc region in which the binding activity to both H-type and R-type FcγRIIa gene polymorphisms is maintained or decreased, and the binding activity to FcγRIIb is enhanced, as compared with the parent polypeptide. By using the polypeptide in which the binding selectivity to FcγRIIb is enhanced for both FcγRIIa gene polymorphisms (H-type, R-type), it becomes possible to transmit an inhibitory signal of an inflammatory immune response via phosphorylation of ITIM of FcγRIIb to patients with both R-type and H-type gene polymorphisms. In addition, by imparting the property of selectively binding to FcγRIIb to the Fc of an antibody, it is possible to suppress the production of anti-antibodies through the immunosuppressive action mediated by FcγRIIb. [Brief description of the drawings]

[0022] [Figure 1] This figure shows a comparison of binding to FcγRIa and FcγRIIb, and labels the binding of an antibody in which Pro at position 238 (EU numbering) is substituted with Asp and an antibody in which Leu at position 328 (EU numbering) is substituted with Glu. "Mutation A" refers to the modification in which Pro at position 238 (EU numbering) is substituted with Asp, and "mutation B" refers to the modification in which Leu at position 328 (EU numbering) is substituted with Glu. [Diagram 2]This figure shows a comparison of binding to FcγRIIa H type and FcγRIIb, and labels the binding of an antibody in which Pro at position 238 (EU numbering) is substituted with Asp and an antibody in which Leu at position 328 (EU numbering) is substituted with Glu. "Mutation A" refers to the modification in which Pro at position 238 (EU numbering) is substituted with Asp, and "mutation B" refers to the modification in which Leu at position 328 (EU numbering) is substituted with Glu. [Diagram 3] This figure shows a comparison of binding to FcγRIIa R type and FcγRIIb, and labels the binding of an antibody in which Pro at position 238 (EU numbering) is substituted with Asp and an antibody in which Leu at position 328 (EU numbering) is substituted with Glu. "Mutation A" refers to the modification in which Pro at position 238 (EU numbering) is substituted with Asp, and "mutation B" refers to the modification in which Leu at position 328 (EU numbering) is substituted with Glu. [Figure 4] This figure shows a comparison of binding to FcγRIIIa and FcγRIIb, and labels the binding of an antibody in which Pro at position 238 (EU numbering) is substituted with Asp and an antibody in which Leu at position 328 (EU numbering) is substituted with Glu. "Mutation A" refers to the modification in which Pro at position 238 (EU numbering) is substituted with Asp, and "mutation B" refers to the modification in which Leu at position 328 (EU numbering) is substituted with Glu. [Diagram 5] The relationship between the amino acid residues constituting the Fc domain of IgG1, IgG2, IgG3, and IgG4 and EU numbering (also referred to as EU INDEX herein) is shown. [Figure 6]The horizontal axis shows the relative binding activity of each PD variant to FcγRIIb, and the vertical axis shows the relative binding activity of each PD variant to FcγRIIa R. The binding amount of each PD variant to each FcγR was divided by the binding amount of the control antibody before modification, IL6R-F652 (modified Fc in which Pro at position 238 (EU numbering) is replaced with Asp), and the resulting value was multiplied by 100 to obtain the relative binding activity of each PD variant to each FcγR. The plot labeled F652 in the figure shows the value for IL6R-F652. [Figure 7] The vertical axis shows the relative binding activity to FcγRIIb of variants obtained by introducing each alteration into GpH7-B3 that does not have the P238D alteration, and the horizontal axis shows the relative binding activity to FcγRIIb of variants obtained by introducing each alteration into IL6R-F652 that has the P238D alteration. The value of the FcγRIIb binding amount of each variant is divided by the value of the FcγRIIb binding amount of the antibody before the alteration is introduced, and then multiplied by 100 to obtain the relative binding activity value. Here, modifications that exert an FcγRIIb binding-enhancing effect when introduced into GpH7-B3 that does not have P238D and when introduced into IL6R-F652 that has P238D are included in region A, and modifications that exert an FcγRIIb binding-enhancing effect when introduced into GpH7-B3 that does not have P238D but do not exert an FcγRIIb binding-enhancing effect when introduced into IL6R-F652 that has P238D are included in region B. [Figure 8] 1 shows the crystal structure of the Fc(P238D) / FcγRIIb extracellular region complex. [Figure 9] FIG. 1 shows a diagram of a superposition of the crystal structure of the Fc(P238D) / FcγRIIb extracellular region complex and the model structure of the Fc(WT) / FcγRIIb extracellular region complex, performed by a least-squares method based on the Cα atomic distances for the FcγRIIb extracellular region and Fc CH2 domain A. [Figure 10]The crystal structure of the Fc(P238D) / FcγRIIb extracellular region complex and the model structure of the Fc(WT) / FcγRIIb extracellular region complex were superimposed by the least-squares method based on the Cα atomic distances for the Fc CH2 domain A and the Fc CH2 domain B alone, and the detailed structures around P238D were compared. [Figure 11] FIG. 13 shows that in the crystal structure of the Fc (P238D) / FcγRIIb extracellular region complex, a hydrogen bond is observed between the main chain of Gly at position 237 (EU numbering) in Fc CH2 domain A and Tyr at position 160 in FcγRIIb. [Figure 12] FIG. 13 shows that electrostatic interaction is observed between Asp at position 270 (EU numbering) of Fc CH2 domain B and Arg at position 131 (EU numbering) of FcγRIIb in the crystal structure of the Fc (P238D) / FcγRIIb extracellular region complex. [Figure 13] The horizontal axis shows the relative binding activity of each 2B variant to FcγRIIb, and the vertical axis shows the relative binding activity of each 2B variant to FcγRIIa R. The binding amount of each 2B variant to each FcγR was divided by the binding amount of the control antibody before the introduction of the modification (modified Fc in which Pro at position 238 (EU numbering) is replaced with Asp) to each FcγR, and the resulting value was multiplied by 100 to obtain the relative binding activity of each 2B variant to each FcγR. [Figure 14] FIG. 13 shows the Glu at position 233 (EU numbering) of Fc Chain A and its surrounding residues in the extracellular region of FcγRIIb in the crystal structure of the Fc (P238D) / FcγRIIb extracellular region complex. [Figure 15] FIG. 13 shows Ala at position 330 (EU numbering) of Fc Chain A and its surrounding residues in the extracellular region of FcγRIIb in the crystal structure of the Fc (P238D) / FcγRIIb extracellular region complex. [Figure 16]FIG. 1 shows the structure of Pro at position 271 (EU numbering) of Fc Chain B, obtained by superimposing the crystal structures of the Fc(P238D) / FcγRIIb extracellular region complex and the Fc(WT) / FcγRIIIa extracellular region complex using the least-squares method based on the Cα atomic distances with respect to Fc Chain B. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present invention provides polypeptides comprising an IgG Fc region, in which amino acid substitutions have been introduced into the Fc region, thereby maintaining or decreasing binding activity to FcγRIIa and enhancing binding activity to FcγRIIb, as compared to a parent polypeptide. More specifically, the present invention provides a polypeptide comprising an antibody Fc region comprising a substitution of Pro at position 238 (EU numbering) with Asp or a substitution of Leu at position 328 (EU numbering) with Glu, and a polypeptide comprising an antibody Fc region comprising a combination of a substitution of Pro at position 238 (EU numbering) with Asp and several specific amino acid substitutions. Furthermore, the present invention provides a method for maintaining or decreasing the binding activity of any gene polymorphism to FcγRIIa and enhancing the binding activity to FcγRIIb, as compared to the parent polypeptide. The present invention also provides a method for suppressing antibody production, as compared to the parent polypeptide, when administered to a living body.

[0024] The term "polypeptide" as used herein generally refers to a peptide or protein having a length of about 10 amino acids or more. Generally, the polypeptide is derived from an organism, but is not particularly limited thereto, and may be, for example, a polypeptide having an artificially designed sequence. The polypeptide may be any of a natural polypeptide, a synthetic polypeptide, a recombinant polypeptide, and the like.

[0025] An Fcγ receptor (sometimes referred to as Fcγ receptor or FcγR herein) refers to a receptor capable of binding to the Fc region of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody, and means any member of a family of proteins substantially encoded by the Fcγ receptor gene. In humans, this family includes, but is not limited to, FcγRI (CD64) including isoforms FcγRIa, FcγRIb and FcγRIc; FcγRII (CD32) including isoforms FcγRIIa (including allotypes H131 (H type) and R131 (R type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) and FcγRIIc; and FcγRIII (CD16) including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any undiscovered human FcγRs or FcγR isoforms or allotypes. FcγRs may be derived from any organism, including, but not limited to, humans, mice, rats, rabbits and monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16) and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRs or FcγR isoforms or allotypes. Suitable examples of such Fcγ receptors include human FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16) and / or FcγRIIIB (CD16).

[0026] The polynucleotide and amino acid sequences of FcγRI are set forth in SEQ ID NOs: 1 (NM_000566.3) and 2 (NP_000557.1), respectively. The polynucleotide and amino acid sequences of FcγRIIA are set forth in SEQ ID NOs: 3 (BC020823.1) and 4 (AAH20823.1), respectively. The polynucleotide and amino acid sequences of FcγRIIB are set forth in SEQ ID NOs: 5 (BC146678.1) and 6 (AAI46679.1), respectively. The polynucleotide and amino acid sequences of FcγRIIIA are set forth in SEQ ID NOs: 7 (BC033678.1) and 8 (AAH33678.1), respectively. The polynucleotide sequence and amino acid sequence of FcγRIIIB are set forth in SEQ ID NOs: 9 (BC128562.1) and 10 (AAI28563.1), respectively (RefSeq accession numbers are indicated in parentheses).

[0027] Furthermore, there are two types of genetic polymorphisms in FcγRIIa, in which the 131st amino acid of FcγRIIa is substituted with histidine (H type) or arginine (R type) (J. Exp. Med, 172, 19-25, 1990).

[0028] As used herein, the term "parent polypeptide" refers to a polypeptide that serves as the basis for the preparation of a polypeptide comprising an antibody Fc region of the present invention. That is, it can be a polypeptide comprising an antibody Fc region, prior to modification of at least one amino acid in the Fc region. The parent polypeptide of the present invention may be, for example, a polypeptide comprising the Fc region of a native IgG, or a polypeptide comprising the Fc region of an IgG in which a modification other than the amino acid modification of the present invention has been added to a native IgG.

[0029] Native IgG refers to a polypeptide that includes the same amino acid sequence as IgG found in nature and belongs to the class of antibodies substantially encoded by immunoglobulin gamma genes. For example, native human IgG refers to native human IgG1, native human IgG2, native human IgG3, native human IgG4, etc. Native IgG also includes naturally occurring mutants thereof.

[0030] The Fc region of native IgG refers to an Fc region that includes the same amino acid sequence as an Fc region originating from naturally occurring IgG. The Fc region of native IgG is shown in FIG. 5 (SEQ ID NOs: 11 to 14), and refers to, for example, an Fc region originating from native human IgG1, an Fc region originating from native human IgG2, an Fc region originating from native human IgG3, or an Fc region originating from native human IgG4. The Fc region of native IgG also includes naturally occurring mutants thereof.

[0031] In the present invention, whether the binding activity of the polypeptide or Fc region of the present invention to various FcγRs has been enhanced, or whether the binding activity has been maintained or decreased can be determined, for example, as shown in this Example, by using BIACORE, an interaction analysis device that utilizes the surface plasmon resonance (SPR) phenomenon, and observing whether the dissociation constant (KD) value obtained from the analysis of sensorgrams in which various FcγRs are allowed to interact as an analyte with a sensor chip on which an antibody has been immobilized or captured with Protein A, Protein L, Protein A / G, Protein G, anti-lamda chain antibody, anti-kappa chain antibody, antigen peptide, antigen protein, etc. has decreased or increased. Alternatively, it can be judged whether the value obtained by dividing the change in resonance unit (RU) value on the sensorgram before and after interaction of various FcγRs as an analyte with an antibody on a sensor chip immobilized on a sensor chip or captured with Protein A, Protein L, Protein A / G, Protein G, an anti-lamda chain antibody, an anti-kappa chain antibody, an antigen peptide, an antigen protein, or the like by the change in resonance unit (RU) value before and after immobilization or capture of the antibody on the sensor chip has decreased or increased. It can also be judged whether the value of the dissociation constant (KD) obtained from the analysis of a sensorgram in which a sample such as an antibody to be evaluated is interacted as an analyte using a sensor chip on which FcγR is directly immobilized on a sensor chip or immobilized via an anti-tag antibody, or the like, has decreased or increased. Alternatively, it can be judged whether the change in the sensorgram value has decreased or increased before and after interaction of a sample such as an antibody to be evaluated as an analyte with a sensor chip on which FcγR is directly immobilized on a sensor chip or immobilized via an anti-tag antibody, or the like.

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

[0033] The ALPHA screen is carried out by ALPHA technology using two beads, donor and acceptor, based on the following principle: A luminescence signal is detected only when a molecule bound to a donor bead biologically interacts with a molecule bound to an acceptor bead and the two beads are in close proximity. A photosensitizer in the donor bead excited by a laser converts surrounding oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and, when it reaches a nearby acceptor bead, triggers a chemiluminescence reaction in the bead, which ultimately emits light. If there is no interaction between the molecules bound to the donor bead and the acceptor bead, the singlet oxygen produced by the donor bead does not reach the acceptor bead, and no chemiluminescence reaction occurs.

[0034] For example, a biotin-labeled polypeptide complex 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 competing polypeptide complex containing a mutant Fc region, a polypeptide complex containing a wild-type Fc region interacts with the Fcγ receptor and generates a signal at 520-620 nm. A polypeptide complex containing an untagged mutant Fc region competes with the interaction between a polypeptide complex containing a wild-type Fc region and the Fcγ receptor. Relative binding activity can be determined by quantifying the decrease in fluorescence that occurs as a result of competition. It is known that polypeptide complexes such as antibodies can be biotinylated using sulfo-NHS-biotin or the like. An example of a method for tagging an Fcγ receptor with GST is to express a fusion gene in which a polynucleotide encoding an Fcγ receptor and a polynucleotide encoding GST are fused in frame in a cell or the like that harbors the fusion gene, and purify the fusion gene using a glutathione column in an expression vector, or the like. The resulting signals are suitably analyzed by fitting to a one-site competition model utilizing non-linear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).

[0035] One of the substances (ligand) for which interaction is to be observed is fixed on the gold thin film of the sensor chip, and light is applied from the back of the sensor chip so that it is totally reflected at the interface between the gold thin film and the glass. When the other substance (analyte) for which interaction is to be observed is poured onto the surface of the sensor chip and the ligand and analyte bind, the mass of the immobilized ligand molecule increases, and the refractive index of the solvent on the sensor chip surface changes. This change in refractive index shifts the position of the SPR signal (conversely, when the bond is dissociated, the position of the signal returns). The Biacore system takes the amount of shift mentioned above, that is, the change in mass on the sensor chip surface, as the vertical axis, and displays the change in mass over time as measurement data (sensorgram). The amount of binding of the analyte to the ligand captured on the sensor chip surface can be obtained from the sensorgram. In addition, the kinetics: binding rate constant (ka) and dissociation rate constant (kd) can be obtained from the curve of the sensorgram, and the dissociation constant (KD) can be obtained from the ratio of these constants. Inhibition measurement methods are also preferably used in the BIACORE method. An example of an inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

[0036] A polypeptide with reduced binding activity to FcγR refers to one that binds to FcγR with essentially weaker binding activity than the parent polypeptide when assayed using essentially the same amounts of the parent polypeptide and a polypeptide (also called a polypeptide variant) that contains at least one amino acid modification in the Fc region of the parent polypeptide.

[0037] For example, in the KD values ​​measured by the above-mentioned measurement method, the KD value ratio (KD value of the polypeptide mutant / KD value of the parent polypeptide) is preferably 1.25 or more, 2 or more, or 3 or more, more preferably 5 or more, 10 or more, 100 or more, 1000 or more, or 10000 or more. In addition, in the KD value measured by the above-mentioned measuring method, the KD value is preferably increased by 1 μM or more, more preferably increased by 2 μM or more, 3 μM or more, 5 μM or more, 10 μM or more, 20 μM or more, 50 μM or more, or 100 μM or more. In addition, in the KD value measured by the above-mentioned measuring method, the KD value is preferably increased by 0.0001 μM or more, more preferably increased by 0.001 μM or more, 0.01 μM or more, 0.1 μM or more, 0.5 μM or more, 1 μM or more, 2 μM or more, 3 μM or more, 5 μM or more, 10 μM or more, 100 μM or more, or 1000 μM or more.

[0038] A polypeptide with enhanced binding activity to FcγR refers to one that binds to FcγR with substantially stronger binding activity than the parent polypeptide when assayed using essentially the same amount of the parent polypeptide and the polypeptide mutant.

[0039] For example, in the KD values ​​measured by the above-mentioned measurement method, the KD value ratio (KD value of parent polypeptide / KD value of polypeptide mutant) is preferably 1.25 or more, 2 or more, or 3 or more, more preferably 5 or more, 10 or more, 100 or more, 1000 or more, or 10000 or more. Furthermore, in the KD value measured by the above-mentioned measurement method, it is preferable that the KD value is decreased by 0.001 μM or more, and it is even more preferable that the KD value is decreased by 0.01 μM, 0.1 μM, 1 μM or more, 2 μM or more, 3 μM or more, 5 μM or more, 10 μM or more, 20 μM or more, 50 μM or more, or 100 μM or more. Furthermore, in the KD value measured by the above-mentioned measurement method, the KD value is preferably 5 μM or less, and more preferably 3 μM or less, 1 μM or less, 0.5 μM or less, 0.1 μM or less, 0.01 μM or less, 0.001 μM or less, or 0.0001 μM or less.

[0040] A polypeptide that maintains (retains) binding activity to FcγR refers to one that binds to FcγR with essentially the same binding activity as the parent polypeptide when assayed using essentially the same amounts of the parent polypeptide and a polypeptide (also called a polypeptide variant) that contains at least one amino acid modification in the Fc region of the parent polypeptide.

[0041] Whether or not a polypeptide has a maintained or decreased binding activity to FcγRIIa and an enhanced binding activity to FcγRIIb can be determined by using the KD value of the polypeptide for FcγRIIa and the KD value of the polypeptide for FcγRIIb, which are determined according to the above example. For example, the KD value of the polypeptide of the present invention for FcγRIIb is decreased compared to the KD value of the parent polypeptide for FcγRIIb, and the KD value of the polypeptide of the present invention for FcγRIIa (R type, H type) is increased or maintained compared to the KD value of the parent polypeptide for FcγRIIa (R type, H type). It is also possible to determine by appropriately combining the KD value of the polypeptide for FcγRIa and the KD value of the polypeptide for FcγRIIIa, which are determined according to the above example. In the present invention, enhanced binding activity to FcγRIIb means, for example, that the KD value ratio (KD value of parent polypeptide) / (KD value of polypeptide mutant) measured by the above-mentioned measurement method is preferably 1.6 or more, 2 or more, or 3 or more, and more preferably 5 or more, 10 or more, 20 or more, 30 or more, or 50 or more. Furthermore, "the binding activity to FcγRIIa (R type) and FcγRIIa (H type) is maintained or decreased" means, for example, that in the KD values ​​measured by the above-mentioned measurement method, the KD value ratio of [the stronger KD value of the binding activity of the polypeptide mutant to FcγRIIa (R type) and FcγRIIa (H type)] / [the stronger KD value of the binding activity of the parent polypeptide to FcγRIIa (R type) and FcγRIIa (H type)] is preferably 0.7 or more, 1 or more, 2 or more, or 3 or more, and more preferably 5 or more, 10 or more, 20 or more, 30 or more, or 50 or more.

[0042] The polypeptide of the present invention preferably has a binding activity maintained or decreased to FcγRIIa R and FcγRIIa H. It is more preferable that the binding activity to FcγRIIa R and FcγRIIa H is maintained or decreased, and that the binding activity to FcγRIIIa is maintained or decreased. Furthermore, it is preferable that the binding activity to FcγRIa is also maintained or decreased. "The binding activity to FcγRIIIa or FcγRIa is maintained or decreased" means, for example, that the KD value ratio (KD value of the polypeptide mutant) / (KD value of the parent polypeptide) measured by the above-mentioned measurement method is preferably 1 or more, 2 or more, or 3 or more, and more preferably 5 or more, 10 or more, 20 or more, 30 or more, or 50 or more.

[0043] Furthermore, whether or not the polypeptide of the present invention is a polypeptide with improved binding selectivity for FcγRIIb over FcγRIIa can be determined by comparing the ratio of the KD value of the polypeptide of the present invention for FcγRIIa to the KD value of FcγRIIb (KD value for FcγRIIa / KD value for FcγRIIb) determined according to the above example with the ratio of the KD value of the parent polypeptide for FcγRIIa to the KD value of FcγRIIb (KD value for FcγRIIa / KD value for FcγRIIb). Specifically, when the above KD value ratio is larger for the polypeptide of the present invention than for the parent polypeptide, it can be determined that the polypeptide of the present invention has improved binding selectivity for FcγRIIb over FcγRIIa compared to the parent polypeptide. Regarding the binding selectivity between FcγRIIa (R type) and FcγRIIb, for example, in the KD values ​​measured by the above-mentioned measurement method, the KD value ratio (KD value of the polypeptide mutant for FcγRIIa (R type)) / (KD value of the polypeptide mutant for FcγRIIb) is preferably 1.2 or more, 2 or more, or 3 or more. More preferably, it is 5 or more, 10 or more, 20 or more, or 30 or more. Regarding the binding selectivity between FcγRIIa (H type) and FcγRIIb, for example, in the KD values ​​measured by the above-mentioned measurement method, the KD value ratio (KD value of the polypeptide mutant for FcγRIIa (H type)) / (KD value of the polypeptide mutant for FcγRIIb) is preferably 4.2 or more, 5 or more, or 10 or more. More preferably, it is 20 or more, 30 or more, 50 or more, 100 or more, or 200 or more.

[0044] Furthermore, whether the binding activity of the polypeptide of the present invention to various FcγRs has been maintained, enhanced, or decreased can also be determined from the increase or decrease in the amount of binding of various FcγRs to the polypeptide of the present invention determined according to the above example. Here, the amount of binding of various FcγRs to a polypeptide means the value obtained by dividing the difference in RU values ​​in sensorgrams showing changes before and after interaction of each polypeptide with various FcγRs as analytes by the difference in RU values ​​in sensorgrams showing changes before and after capture of the polypeptide on a sensor chip.

[0045] Whether a polypeptide of the present invention has maintained or decreased binding activity to FcγRIIa (R type, H type) and enhanced binding activity to FcγRIIb can be determined by using the amount of binding of the polypeptide to FcγRIIa and the amount of binding of the polypeptide to FcγRIIb, determined according to the above example.

[0046] For example, this is the case when the binding amount of the polypeptide of the present invention to FcγRIIb is increased compared to the binding amount of the parent polypeptide to FcγRIIb, and the binding amount of the polypeptide of the present invention to FcγRIIa (R type, H type) is equivalent (maintained) to, or preferably decreased, the binding amount of the parent polypeptide to FcγRIIa (R type, H type). It is also possible to judge by appropriately combining the binding amount of the polypeptide to FcγRIa and the binding amount of the polypeptide to FcγRIIIa, which are determined according to the above example.

[0047] The term "Fc region" refers to a region in an antibody molecule that includes a fragment consisting of the hinge region or a part thereof, and the CH2 and CH3 domains. The Fc region of an IgG class refers to, for example, but is not limited to, from cysteine ​​at position 226 to the C-terminus, or from proline at position 230 to the C-terminus, in the EU numbering (also referred to as EU INDEX herein) (see FIG. 5).

[0048] The Fc region can be suitably obtained by partially digesting IgG1, IgG2, IgG3, IgG4 monoclonal antibodies, etc. with a protease such as pepsin, and then re-eluting the fraction adsorbed to a Protein A column. Such a protease is not particularly limited as long as it can digest full-length antibodies so as to produce Fab or F(ab')2 in a limited manner by appropriately setting the enzyme reaction conditions, such as pH, and examples of such an enzyme include pepsin and papain.

[0049] The present invention provides antibody constant regions comprising an Fc region comprising an alteration in which Pro at position 238 (EU numbering) is replaced with Asp or Leu at position 328 (EU numbering) is replaced with Glu in human IgG (IgG1, IgG2, IgG3, IgG4). By introducing an alteration in which Pro at position 238 (EU numbering) is replaced with Asp or an alteration in which Leu at position 328 (EU numbering) is replaced with Glu into human IgG, it is possible to provide a polypeptide that maintains or decreases binding activity to any of the R-type and H-type genetic polymorphisms of FcγRIa, FcγRIIIa, and FcγRIIa, and has enhanced binding activity to FcγRIIb, compared to the parent polypeptide.

[0050] The present invention allows for the addition of at least one other modification to the Fc region of human IgG containing the modification of replacing Pro at position 238 (EU numbering) with Asp or Leu at position 328 (EU numbering) with Glu. The modification means any of substitution, deletion, addition, and insertion, or a combination thereof. In addition to these modifications, an additional modification may be included. The additional modification may be selected from, for example, any of amino acid substitution, deletion, and modification, or a combination thereof. For example, it is possible to add a modification that enhances the binding activity to FcγRIIb and maintains or reduces the binding activity to FcγRIIa (H type) and FcγRIIa (R type). By adding such a modification, the binding selectivity to FcγRIIb over FcγRIIa is improved.

[0051] Among these, preferred are modifications that improve the binding selectivity for FcγRIIb over FcγRIIa (R type), and further preferred are modifications that improve the binding selectivity for FcγRIIb over FcγRIIa (H type). Preferred amino acid substitutions as such modifications include, for example, substitution of Gly at position 237 (EU numbering) with Trp, substitution of Gly at position 237 (EU numbering) with Phe, substitution of Pro at position 238 (EU numbering) with Phe, substitution of Asn at position 325 (EU numbering) with Met, substitution of Ser at position 267 (EU numbering) with Ile, substitution of Leu at position 328 (EU numbering) with Asp, and substitution of Ser at position 267 (EU numbering) with Val. at position 327 (EU numbering) to Asn; at position 325 (EU numbering) to Ser; at position 235 (EU numbering) to Leu; at position 267 (EU numbering) to Trp; at position 267 (EU numbering) to Ser; at position 267 (EU numbering) to Met; at position 236 (EU numbering) to Gly; at position 236 (EU numbering) to Asp; at position 327 (EU numbering) to Asn; at position 325 (EU numbering) to Ser; at position 235 (EU numbering) to Leu; at position 266 (EU numbering) to Va I is substituted with Met, Leu at position 328 (EU numbering) is substituted with Tyr, Leu at position 235 (EU numbering) is substituted with Trp, Leu at position 235 (EU numbering) is substituted with Phe, Ser at position 239 (EU numbering) is substituted with Gly, Ala at position 327 (EU numbering) is substituted with Glu, Ala at position 327 (EU numbering) is substituted with Gly, Pro at position 238 (EU numbering) is substituted with Leu, Ser at position 239 is replaced with Leu, Leu at position 328 (EU numbering) is replaced with Thr, Leu at position 328 (EU numbering) is replaced with Ser, Leu at position 328 (EU numbering) is replaced with Met, Pro at position 331 (EU numbering) is replaced with Trp, Pro at position 331 (EU numbering) is replaced with Tyr, Pro at position 331 (EU numbering) is replaced with Phe, Ala at position 327 (EU numbering) is replaced with Asp,Leu at position 328 (EU numbering) is replaced with Phe, Pro at position 271 (EU numbering) is replaced with Leu, Ser at position 267 (EU numbering) is replaced with Glu, Leu at position 328 (EU numbering) is replaced with Ala, Leu at position 328 (EU numbering) is replaced with Ile, Leu at position 328 (EU numbering) is replaced with Gln, Leu at position 328 (EU numbering) is replaced with Val, Lys at position 326 (EU numbering) is replaced with Trp, Lys is replaced with Arg, His at position 268 (EU numbering) is replaced with Gly, His at position 268 (EU numbering) is replaced with Asn, Ser at position 324 (EU numbering) is replaced with Val, Val at position 266 (EU numbering) is replaced with Leu, Pro at position 271 (EU numbering) is replaced with Gly, Ile at position 332 (EU numbering) is replaced with Phe, Ser at position 324 (EU numbering) is replaced with Ile, Glu at position 333 (EU numbering) is replaced with Pro, Tyr at position 300 (EU numbering) is replaced with Asp, Ser at position 337 (EU numbering) is replaced with Asp, Tyr at position 300 (EU numbering) is replaced with Gln, Thr at position 335 (EU numbering) is replaced with Asp, Ser at position 239 (EU numbering) is replaced with Asn, Lys at position 326 (EU numbering) is replaced with Leu, Lys at position 326 (EU numbering) is replaced with Ile, Ser at position 239 (EU numbering) is replaced with Glu, Lys is replaced with Phe, Lys at position 326 (EU numbering) is replaced with Val, Lys at position 326 (EU numbering) is replaced with Tyr, Ser at position 267 (EU numbering) is replaced with Asp, Lys at position 326 (EU numbering) is replaced with Pro, Lys at position 326 (EU numbering) is replaced with His, Lys at position 334 (EU numbering) is replaced with Ala, Lys at position 334 (EU numbering) is replaced with Trp, His at position 268 (EU numbering) is replaced with Gln,Lys at position 326 (EU numbering) is replaced with Gln, Lys at position 326 (EU numbering) is replaced with Glu, Lys at position 326 (EU numbering) is replaced with Met, Val at position 266 (EU numbering) is replaced with Ile, Lys at position 334 (EU numbering) is replaced with Glu, Tyr at position 300 (EU numbering) is replaced with Glu, Lys at position 334 (EU numbering) is replaced with Met, Lys at position 334 (EU numbering) is replaced with Val, Lys is replaced with Thr, Lys at position 334 (EU numbering) is replaced with Ser, Lys at position 334 (EU numbering) is replaced with His, Lys at position 334 (EU numbering) is replaced with Phe, Lys at position 334 (EU numbering) is replaced with Gln, Lys at position 334 (EU numbering) is replaced with Pro, Lys at position 334 (EU numbering) is replaced with Tyr, Lys at position 334 (EU numbering) is replaced with Ile, Gln at position 295 (EU numbering) is replaced with Leu, Lys at position 334 (EU numbering) is replaced with Leu, Lys at position 334 (EU numbering) is replaced with Asn, His at position 268 (EU numbering) is replaced with Ala, Ser at position 239 (EU numbering) is replaced with Asp, Ser at position 267 (EU numbering) is replaced with Ala, Leu at position 234 (EU numbering) is replaced with Trp, Leu at position 234 (EU numbering) is replaced with Tyr, Gly at position 237 (EU numbering) is replaced with Ala, Gly is replaced with Asp, Gly at position 237 (EU numbering) is replaced with Glu, Gly at position 237 (EU numbering) is replaced with Leu, Gly at position 237 (EU numbering) is replaced with Met, Gly at position 237 (EU numbering) is replaced with Tyr, Ala at position 330 (EU numbering) is replaced with Lys, Ala at position 330 (EU numbering) is replaced with Arg, Glu at position 233 (EU numbering) is replaced with Asp, His at position 268 (EU numbering) is replaced with Asp,Examples of such an alteration include a substitution of His at position 268 (EU numbering) with Glu, a substitution of Lys at position 326 (EU numbering) with Asp, a substitution of Lys at position 326 (EU numbering) with Ser, a substitution of Lys at position 326 (EU numbering) with Thr, a substitution of Val at position 323 (EU numbering) with Ile, a substitution of Val at position 323 (EU numbering) with Leu, a substitution of Val at position 323 (EU numbering) with Met, a substitution of Tyr at position 296 (EU numbering) with Asp, a substitution of Lys at position 326 (EU numbering) with Ala, a substitution of Lys at position 326 (EU numbering) with Asn, and a substitution of Ala at position 330 (EU numbering) with Met.

[0052] Further, among these modifications, preferred amino acid substitutions include, for example, a modification in which Gly at position 237 (EU numbering) is replaced with Trp, a modification in which Gly at position 237 (EU numbering) is replaced with Phe, a modification in which Ser at position 267 (EU numbering) is replaced with Val, a modification in which Ser at position 267 (EU numbering) is replaced with Gln, a modification in which His at position 268 (EU numbering) is replaced with Asn, a modification in which Pro at position 271 (EU numbering) is replaced with Gly, a modification in which Lys at position 326 (EU numbering) is replaced with Leu, and a modification in which L at position 326 (EU numbering) is replaced with L. a substitution of Lys with Gln, a substitution of Lys at position 326 (EU numbering) with Glu, a substitution of Lys at position 326 (EU numbering) with Met, a substitution of Ser at position 239 (EU numbering) with Asp, a substitution of Ser at position 267 (EU numbering) with Ala, a substitution of Leu at position 234 (EU numbering) with Trp, a substitution of Leu at position 234 (EU numbering) with Tyr, a substitution of Gly at position 237 (EU numbering) with Ala, a substitution of Gly at position 237 (EU numbering) with Asp, Gly at position 237 (EU numbering) is replaced with Glu, Gly at position 237 (EU numbering) is replaced with Leu, Gly at position 237 (EU numbering) is replaced with Met, Gly at position 237 (EU numbering) is replaced with Tyr, Ala at position 330 (EU numbering) is replaced with Lys, Ala at position 330 (EU numbering) is replaced with Arg, Glu at position 233 (EU numbering) is replaced with Asp, His at position 268 (EU numbering) is replaced with Asp, His is replaced with Glu, Lys at position 326 (EU numbering) is replaced with Asp, Lys at position 326 (EU numbering) is replaced with Ser, Lys at position 326 (EU numbering) is replaced with Thr, Val at position 323 (EU numbering) is replaced with Ile, Val at position 323 (EU numbering) is replaced with Leu, Val at position 323 (EU numbering) is replaced with Met, Tyr at position 296 (EU numbering) is replaced with Asp, Lys at position 326 (EU numbering) is replaced with Ala,Examples of such modifications include substitution of Lys at position 326 (EU numbering) with Asn, and substitution of Ala at position 330 (EU numbering) with Met. The above modifications may be made at one site or in combination of two or more sites. Preferred examples of such modifications include the modifications shown in Tables 6 to 7 and Tables 9 to 12.

[0053] Furthermore, for example, amino acid substitutions that improve binding activity to FcRn (J Immunol. 2006 Jan 1;176(1):346-56, J Biol Chem. 2006 Aug 18;281(33):23514-24, Int Immunol. 2006 Dec;18(12):1759-69, Nat Biotechnol. 2010 Feb;28(2):157-9, WO / 2006 / 019447, WO / 2006 / 053301, WO / 2009 / 086320), or amino acid substitutions to improve antibody heterogeneity or stability (WO / 2009 / 041613) may be added to the antibody constant region. Alternatively, the present invention also includes polypeptides to which the properties of promoting antigen elimination as described in WO2011 / 122011 and PCT / JP2011 / 072550 have been imparted to the polypeptides of the present invention, and polypeptides to which the properties of repeatedly binding to multiple antigen molecules as described in WO2009 / 125825 and PCT / JP2011 / 077619 have been imparted to the polypeptides of the present invention.

[0054] A preferred example of the polypeptide of the present invention is an IgG antibody. When an IgG antibody is used as the antibody, the type of its constant region is not limited, and IgG isotypes (subclasses) such as IgG1, IgG2, IgG3, and IgG4 can be used. The IgG antibody of the present invention is preferably human IgG, more preferably human IgG1 or human IgG4, and the amino acid sequences of the heavy chain constant regions of human IgG1 and human IgG4 are known. As for the human IgG1 constant region, a number of allotype sequences due to genetic polymorphism are described in Sequences of proteins of immunological interest, NIH Publication No. 91-3242, and any of these may be used in the present invention.

[0055] <replacement> When an amino acid residue is substituted, the purpose is to effect modification in, for example, the following points (a) to (c) by substituting another amino acid residue. (a) the backbone structure of the polypeptide in the form of sheet or helical regions; (b) the charge or hydrophobicity at the target site, or (c) Side chain size.

[0056] Amino acid residues are classified into the following groups based on common side chain properties: (1) Hydrophobic: norleucine, met, ala, val, leu, ile; (2) Neutral hydrophilic: cys, ser, thr, asn, gln; (3) Acidic: asp, glu; (4) Basic: his, lys, arg; (5) Residues that affect chain orientation: gly, pro; and (6) Aromaticity: trp, tyr, phe.

[0057] Substitutions of amino acid residues within each of these groups are called conservative substitutions, while substitutions of amino acid residues between other groups are called non-conservative substitutions. Substitutions in the present invention may be conservative substitutions, non-conservative substitutions, or a combination of conservative and non-conservative substitutions.

[0058] Amino acid sequence modifications are prepared by a variety of methods known in the art. These methods include, but are not limited to, site-directed mutagenesis (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis. Gene 152, 271-275; Zoller, MJ, and Smith, M.(1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors.Methods Enzymol. 100, 468-500; Kramer, W, Drutsa, V, Jansen, HW, Kramer, B, Pflugfelder, M, and Fritz, HJ(1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456; Kramer W, and Fritz Mutations can be performed by methods such as PCR mutagenesis and cassette mutagenesis, etc. (HJ (1987) Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154, 350-367; Kunkel, TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci US A. 82, 488-492), PCR mutagenesis, and cassette mutagenesis.

[0059] The amino acid modification of the present invention includes post-translational modification. Specific examples of post-translational modification include the addition or deletion of a sugar chain. For example, in the IgG1 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. In general, antibodies expressed in eukaryotic cells include sugar chain modifications in the constant region. Therefore, antibodies expressed in the following cells are usually modified with some kind of sugar chain. Mammalian antibody-producing cells A eukaryotic cell transformed with an expression vector containing DNA encoding the antibody.

[0060] The eukaryotic cells shown here include yeast and animal cells. For example, CHO cells and HEK293H cells are typical animal cells for transformation with an expression vector containing DNA encoding an antibody. On the other hand, those without glycosylation at the relevant position are also included in the constant region of the present invention. Antibodies whose constant region is not modified with a glycosylation can be obtained by expressing a gene encoding the antibody in a prokaryotic cell such as E. coli.

[0061] More specifically, for example, sialic acid may be added to the sugar chain of the Fc region (MAbs. 2010 Sep-Oct;2(5):519-27.).

[0062] <Antibody> Furthermore, the present invention provides antibodies comprising an Fc region in which any of the above-mentioned amino acid sequences have been altered.

[0063] The term "antibody" in the present invention is used in the broadest sense and includes any antibody, such as a monoclonal antibody (including a full-length monoclonal antibody), a polyclonal antibody, an antibody mutant, an antibody fragment, a multispecific antibody (e.g., a bispecific antibody), a chimeric antibody, or a humanized antibody, so long as it exhibits the desired biological activity.

[0064] The antibody of the present invention is not limited by the type of antigen, the origin of the antibody, etc., and may be any antibody. The origin of the antibody is not particularly limited, but examples thereof include human antibody, mouse antibody, rat antibody, rabbit antibody, etc.

[0065] Methods for producing antibodies are well known to those skilled in the art, and for example, monoclonal antibodies may be produced by hybridoma techniques (Kohler and Milstein, Nature 256:495 (1975)), recombinant techniques (U.S. Patent No. 4,816,567), or isolated from phage antibody libraries (Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1991)).

[0066] A humanized antibody is also called a reshaped human antibody. Specifically, humanized antibodies in which the CDRs of an antibody from a non-human animal, such as a mouse antibody, are grafted onto a human antibody are known. General gene recombination techniques for obtaining humanized antibodies are also known. Specifically, overlap extension PCR is known as a method for grafting the CDRs of a mouse antibody onto human FRs.

[0067] A vector for expressing a humanized antibody can be prepared by inserting a DNA encoding an antibody variable region in which three CDRs and four FRs are linked with a DNA encoding a human antibody constant region into an expression vector so that they are fused in frame. After introducing the integration vector into a host to establish a recombinant cell, the recombinant cell is cultured to express the DNA encoding the humanized antibody, and the humanized antibody is produced in the culture of the cultured cell (see European Patent Publication EP 239400 and International Publication WO1996 / 002576).

[0068] If necessary, amino acid residues in the FR can be substituted so that the CDRs of the reshaped human antibody form an appropriate antigen-binding site. For example, amino acid sequence mutations can be introduced into the FR by applying the PCR method used for grafting mouse CDRs to human FRs.

[0069] Transgenic animals carrying the entire repertoire of human antibody genes (see International Publications WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, WO1996 / 033735) are used as immunized animals, and desired human antibodies can be obtained by DNA immunization.

[0070] Furthermore, a technique for obtaining a human antibody by panning using a human antibody library is also known. For example, the V region of a human antibody is expressed on the surface of a phage as a single chain antibody (scFv) by phage display. A phage expressing an scFv that binds to an antigen can be selected. By analyzing the gene of the selected phage, the DNA sequence encoding the V region of a human antibody that binds to an antigen can be determined. After determining the DNA sequence of the scFv that binds to an antigen, the V region sequence can be fused in frame with the sequence of the C region of a desired human antibody and then inserted into an appropriate expression vector to prepare an expression vector. The expression vector is introduced into a suitable expression cell as listed above, and the gene encoding the human antibody is expressed to obtain the human antibody. These methods are already known (see International Publications WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, and WO1995 / 015388).

[0071] The variable region constituting the antibody of the present invention can be a variable region that recognizes any antigen.

[0072] In the present specification, the antigen is not particularly limited and may be any antigen. Examples of the antigen include ligands (cytokines, chemokines, etc.), receptors, cancer antigens, MHC antigens, differentiation antigens, immunoglobulins, and immune complexes that contain immunoglobulins as a part.

[0073] Examples of cytokines include interleukins 1 to 18, colony-stimulating factors (G-CSF, M-CSF, GM-CSF, etc.), interferons (IFN-α, IFN-β, IFN-γ, etc.), growth factors (EGF, FGF, IGF, NGF, PDGF, TGF, HGF, etc.), tumor necrosis factors (TNF-α, TNF-β), lymphotoxin, erythropoietin, leptin, SCF, TPO, MCAF, and BMP. Examples of chemokines include CC chemokines such as CCL1 to CCL28, CXC chemokines such as CXCL1 to CXCL17, C chemokines such as XCL1 to XCL2, and CX3C chemokines such as CX3CL1.

[0074] Examples of receptors include receptors belonging to receptor families such as the hematopoietic factor receptor family, cytokine receptor family, tyrosine kinase receptor family, serine / threonine kinase receptor family, TNF receptor family, G protein-coupled receptor family, GPI-anchored receptor family, tyrosine phosphatase receptor family, adhesion factor family, and hormone receptor family. The receptors belonging to these receptor families and their characteristics are described in many publications, for example, Cooke BA., King RJB., van der Molen HJ. ed. New Comprehesive Biochemistry Vol.18B "Hormones and their Actions Part II" pp.1-46 (1988) Elsevier Science Publishers BV., Patthy (Cell (1990) 61 (1), 13-14), Ullrich et al. (Cell (1990) 61 (2), 203-212), Massague (e has an acute accent mark) (Cell (1992) 69 (6), 1067-1070), Miyajima et al. (Annu. Rev. Immunol. (1992) 10, 295-331), Taga et al. (FASEB J. (1992) 6, (Annu. Rev. Biochem. (1993), 62, 453-481), Smith et al. (Cell (1994) 76 (6) 959-962), Flower DR. (Biochim. Biophys. Acta (1999) 1422 (3) 207-234), etc.

[0075] Specific examples of receptors belonging to the above receptor families include human or mouse erythropoietin (EPO) receptors (Blood (1990) 76 (1), 31-35; Cell (1989) 57 (2), 277-285), human or mouse granulocyte colony-stimulating factor (G-CSF) receptors (Proc. Natl. Acad. Sci. USA. (1990) 87 (22), 8702-8706; mG-CSFR, Cell (1990) 61 (2), 341-350), human or mouse thrombopoietin (TPO) receptors (Proc Natl Acad Sci USA. (1992) 89 (12), 5640-5644; EMBO J. (1993) 12(7), 2645-53), human or mouse insulin receptors (Nature (1985) 313 (6005), 756-761), human or mouse Flt-3 ligand receptor (Proc. Natl. Acad. Sci. USA. (1994) 91 (2), 459-463), human or mouse platelet-derived growth factor (PDGF) receptor (Proc. Natl. Acad. Sci. USA. (1988) 85 (10) 3435-3439), human or mouse interferon (IFN)-α, β receptor (Cell (1990) 60 (2), 225-234. and Cell (1994) 77 (3), Suitable examples of such an antibody include human or mouse leptin receptor, human or mouse growth hormone (GH) receptor, human or mouse interleukin (IL)-10 receptor, human or mouse insulin-like growth factor (IGF)-I receptor, human or mouse leukemia inhibitory factor (LIF) receptor, and human or mouse ciliary neurotrophic factor (CNTF) receptor.

[0076] Cancer antigens are antigens that are expressed in association with the malignant transformation of cells, and are also called tumor-specific antigens. In addition, abnormal sugar chains that appear on the cell surface or protein molecules when cells become cancerous are also cancer antigens, and are also called cancer sugar chain antigens. Examples of cancer antigens include, for example, GPC3 (Int J Cancer. (2003) 103 (4), 455-65), which belongs to the GPI-anchored receptor family as the above-mentioned receptor and is expressed in several cancers including liver cancer, EpCAM (Proc Natl Acad Sci US A. (1989) 86 (1), 27-31), CA19-9, CA15-3, cereal SSEA-1 (SLX), and the like, which are expressed in several cancers including lung cancer.

[0077] MHC antigens are mainly classified into MHC class I antigens and MHC class II antigens. MHC class I antigens include HLA-A, -B, -C, -E, -F, -G, and -H, while MHC class II antigens include HLA-DR, -DQ, and -DP.

[0078] Differentiation antigens include CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15s, CD16, CD18, CD19, CD20, CD21, CD23, CD25, CD28, CD29, CD30, CD32, CD33, CD34, CD35, CD38, CD40, CD41a, CD41b, CD42a, CD42b, These may include CD43, CD44, CD45, CD45RO, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD51, CD54, CD55, CD56, CD57, CD58, CD61, CD62E, CD62L, CD62P, CD64, CD69, CD71, CD73, CD95, CD102, CD106, CD122, CD126, and CDw130.

[0079] Immunoglobulins include IgA, IgM, IgD, IgG, and IgE, and immune complexes contain at least one of the immunoglobulin components. Other antigens include the following molecules: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, and activin RIB. ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, A RC, ART, Artemin, Anti-Id, ASPARTIC, Atrial Natriuretic Factor, av / b3 Integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD 8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD3 3 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV,UL, 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, サイトケラチン tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Decay accelerating factorfactor), des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2 / E phB4, EPO, ERCC, E-selectin, ET-1, Factor IIa, Factor VII, Factor VIIIc, Factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalka In, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GD F-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut4, glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GRO, growth hormone releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFGPEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, insulin Tegrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta 1, integrin alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta 1, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta 1, integrin beta 2, interferon gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1bp1, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES , MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, Mucin (Muc1), MUC18, Müllerian inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, NCADHERIN, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDK-1, P ECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PlGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV)F, RSVFgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, Thrombin, Thymic Ck-1, Thyroid Stimulating Hormone, Tie, TIMP, TIQ, Tissue Factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B(OPG OCIF, TR1), TNFRSF12(TWEAK R FN14), TNFRSF13B(TACI), TNFRSF13C(BAFF R), TNFRSF14(HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16(NGFR p75NTR), TNFRSF17(BCMA), TNFRSF18(GITR AITR), TNFRSF19(TROY TAJ, TRADE), TNFRSF19L(RELT), TNFRSF1A(TNF RI CD120a, p55-60), TNFRSF1B(TNF RIICD120b, p75-80), TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1 R), TNFRSF5(CD40 p50), TNFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B(DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB CD137, ILA), TNFRSF21(DR6), TNFRSF22(DcTRAIL R2 TNFRH2), TNFRST23(DcTRAIL R1 TNFRH1), TNFRSF25(DR3) Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-a Connectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand, Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand, CD70), TNFSF8 (CD30 ligand, CD153), TNFSF9 (4-1BB ligand, CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAILR, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expressed Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, Virus rus antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, Aβ, CD81, CD97, CD98, DDR1, DKK1, EREG, Hsp90, IL-17 / IL-17R, IL-20 / IL-20R, oxidized LDL, PCSK9, prekallikrein, RON, TMEM16F, SOD1, Chromogranin A, Chromogranin B, tau, VAP1, polymeric kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, factor B, factor D, factor H, properdin, sclerostin, fibrinogen, fibrin, prothrombin, thrombin, tissue factor, factor V, factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X, factor Xa,Examples include factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, Syndecan-1, Syndecan-2, Syndecan-3, Syndecan-4, LPA, S1P and receptors for hormones and growth factors.

[0080] The amino acid sequence constituting the variable region is allowed to be modified in one or more amino acid residues as long as the antigen-binding activity is maintained. When modifying the amino acid sequence of the variable region, the site to be modified and the number of amino acids to be modified are not particularly limited. For example, amino acids present in CDR and / or FR can be modified as appropriate. When modifying the amino acids of the variable region, although there are no particular limitations, it is preferable that the binding activity is maintained, for example, it is preferable that the binding activity is 50% or more, preferably 80% or more, and more preferably 100% or more compared to before modification. Furthermore, the binding activity may be increased by the amino acid modification, for example, the binding activity may be 2-fold, 5-fold, 10-fold, etc. compared to before 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.

[0081] For example, modification of the N-terminus of a variable region from glutamine to pyroglutamic acid by pyroglutamylation is a modification well known to those skilled in the art. Thus, when the N-terminus of the heavy chain of the antibody of the present invention is glutamine, it comprises a variable region in which it has been modified to pyroglutamic acid.

[0082] The variable region of the antibody of the present invention may have any sequence, and may be the variable region of an antibody of any origin, such as a mouse antibody, a rat antibody, a rabbit antibody, a goat antibody, a camel antibody, a humanized antibody obtained by humanizing these non-human antibodies, and a human antibody. A "humanized antibody", also called a reshaped human antibody, is an antibody derived from a mammal other than a human, for example, an antibody in which the complementarity determining region (CDR) of a mouse antibody is grafted onto the CDR of a human antibody. Methods for identifying CDRs are known (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al., Nature (1989) 342: 877). General gene recombination techniques are also known (see European Patent Application Publication No. EP 125023 and WO 96 / 02576). Furthermore, various amino acid substitutions may be introduced into the variable regions of these antibodies in order to improve antigen binding, pharmacokinetics, stability, and antigenicity. The variable regions of the antibodies of the present invention may have pH-dependency in antigen binding, and thus may be capable of repeatedly binding to antigens (WO2009 / 125825).

[0083] The light chain constant region of an antibody may be either of the κ-chain and λ-chain types. Furthermore, the light chain constant region of the present invention may be a light chain constant region that has been modified by amino acid substitution, deletion, addition, and / or insertion.

[0084] The heavy chain constant region of the antibody of the present invention can be, for example, the heavy chain constant region of a human IgG antibody, preferably the heavy chain constant region of a human IgG1 antibody or a human IgG4 antibody.

[0085] Furthermore, the polypeptides of the present invention can be bound to other proteins, physiologically active peptides, etc. to form Fc fusion protein molecules. Examples of other proteins and biologically active peptides include, but are not limited to, receptors, adhesion molecules, ligands, and enzymes.

[0086] Preferred examples of the Fc fusion protein molecule of the present invention include proteins in which an Fc domain is fused to a receptor protein that binds to a target, such as TNFR-Fc fusion protein, IL1R-Fc fusion protein, VEGFR-Fc fusion protein, and CTLA4-Fc fusion protein (Nat Med. 2003 Jan;9(1):47-52, BioDrugs. 2006;20(3):151-60). Furthermore, the protein fused to the polypeptide of the present invention may be any molecule as long as it binds to a target molecule, and examples of such molecules include scFv molecules (WO2005 / 037989), single domain antibody molecules (WO2004 / 058821, WO2003 / 002609), antibody-like molecules (Current Opinion in Biotechnology 2006, 17:653-658, Current Opinion in Biotechnology 2007, 18:1-10, Current Opinion in Structural Biology 1997, 7:463-469, Protein Science 2006, 15:14-27), DARPins (WO2002 / 020565), Affibodies (WO1995 / 001937), Avimers (WO2004 / 044011, (WO2005 / 040229), Adnectin (WO2002 / 032925), etc. Furthermore, the antibody and Fc fusion protein molecule may be a multispecific antibody that binds to multiple types of target molecules or epitopes.

[0087] The antibody of the present invention also includes modified antibodies. Examples of modified antibodies include antibodies bound to various molecules such as polyethylene glycol (PEG) and cytotoxic substances. Such modified antibodies can be obtained by chemically modifying the antibody of the present invention. Methods for modifying antibodies have already been established in this field.

[0088] Furthermore, the antibody of the present invention may be a bispecific antibody. A bispecific antibody is an antibody having variable regions that recognize different epitopes in the same antibody molecule, and the epitopes may be present in different molecules or may be present in the same molecule.

[0089] The polypeptide of the present invention can be produced by a method known to those skilled in the art. For example, the antibody can be produced by the following method, but is not limited thereto.

[0090] A DNA encoding an antibody heavy chain in which one or more amino acid residues in the Fc region have been substituted with other amino acids of interest, and a DNA encoding an antibody light chain are expressed. A DNA encoding a heavy chain in which one or more amino acid residues in the Fc region have been substituted with other amino acids of interest can be obtained, for example, by obtaining the Fc region portion of DNA encoding a natural heavy chain and introducing appropriate substitutions so that codons encoding specific amino acids in the Fc region code for other amino acids of interest.

[0091] It is also possible to obtain DNA encoding a heavy chain in which one or more amino acid residues in the Fc region of a native heavy chain have been substituted with other amino acids of interest by designing a DNA encoding a protein in which one or more amino acid residues in the Fc region have been substituted with other amino acids of interest in advance and chemically synthesizing the DNA. The amino acid substitution site and type of substitution are not particularly limited. Furthermore, the substitution is not limited to substitution, and may be any of deletion, addition, insertion, or a combination of these.

[0092] Furthermore, a DNA encoding a heavy chain in which one or more amino acid residues in the Fc region have been substituted with other amino acids of interest can be produced by dividing it into partial DNAs. 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 by dividing it into partial DNAs in the same manner.

[0093] Methods for expressing the above DNA include the following methods. 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. These heavy and light chain genes can also be incorporated into a single vector.

[0094] When DNA encoding the antibody of interest is incorporated into an expression vector, it is incorporated into the expression vector so that it is expressed under the control of an expression control region, for example, an enhancer or a promoter. Next, a host cell is transformed with this expression vector to express the antibody. In this case, a suitable combination of a host and an expression vector can be used.

[0095] Examples of vectors include M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script, etc. Furthermore, for the purpose of subcloning or excision of cDNA, in addition to the above vectors, for example, pGEM-T, pDIRECT, pT7, etc. can be used.

[0096] When a vector is used for the purpose of producing the polypeptide of the present invention, an expression vector is particularly useful. For example, when the host is Escherichia coli such as JM109, DH5α, HB101, or XL1-Blue, it is essential that the expression vector has a promoter capable of efficient expression in Escherichia coli, such as the lacZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427, the entirety of which is incorporated herein by reference), the araB promoter (Better et al., Science (1988) 240, 1041-1043, the entirety of which is incorporated herein by reference), or the T7 promoter. In addition to the above vectors, such vectors include pGEX-5X-1 (Pharmacia), "QIAexpress system" (QIAGEN), pEGFP, or pET (in this case, the host is preferably BL21, which expresses T7 RNA polymerase).

[0097] The vector may also contain a signal sequence for polypeptide secretion. When the polypeptide is produced in the periplasm of E. coli, the signal sequence for polypeptide secretion may be the pelB signal sequence (Lei, SP et al. J. Bacteriol. (1987) 169, 4397, the entirety of which is incorporated herein by reference). The vector can be introduced into the host cell by, for example, the lipofectin method, the calcium phosphate method, or the DEAE-Dextran method.

[0098] In addition to E. coli expression vectors, examples of vectors for producing the polypeptides of the present invention include mammalian expression vectors (e.g., pcDNA3 (Invitrogen), pEGF-BOS (Nucleic Acids. Res. 1990, 18(17), p5322, the entirety of which is incorporated herein by reference), pEF, and pCDM8), insect cell expression vectors (e.g., "Bac-to-BAC baculovairus expression system" (GIBCO BRL), and pBacPAK8), plant expression vectors (e.g., pMH1 and pMH2), animal virus expression vectors (e.g., pHSV, pMV, and pAdexLcw), retrovirus expression vectors (e.g., pZIPneo), yeast expression vectors (e.g., "Pichia Expression Kit" (Invitrogen), pNV11, and SP-Q01), and Bacillus subtilis expression vectors (e.g., pPL608 and pKTH50).

[0099] When the purpose is to express in animal cells such as CHO cells, COS cells, and NIH3T3 cells, it is essential to have a promoter necessary for expression in the cells, such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108, the entirety of which is incorporated herein by reference), the MMTV-LTR promoter, the EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322, the entirety of which is incorporated herein by reference), the CAG promoter (Gene. (1991) 108, 193, the entirety of which is incorporated herein by reference), the CMV promoter, etc., and it is even more preferable if it has a gene for selecting transformed cells (for example, a drug resistance gene that can be distinguished by a drug (neomycin, G418, etc.)). Examples of vectors having such characteristics include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0100] Furthermore, when the aim is to stably express a gene and to amplify the copy number of the gene in the cell, a method can be used in which a vector (e.g., pCHOI, etc.) having a DHFR gene that complements the nucleic acid synthesis pathway is introduced into a CHO cell lacking the nucleic acid synthesis pathway and the vector is amplified with methotrexate (MTX). When the aim is to transiently express a gene, a method can be used in which a COS cell having a gene expressing SV40 T antigen on its chromosome is transformed with a vector (pcD, etc.) having an SV40 replication origin. Replication origins derived from polyoma virus, adenovirus, bovine papilloma virus (BPV), etc. can also be used. Furthermore, in order to amplify the copy number of the gene in the host cell system, the expression vector can contain, as a selection marker, an aminoglycoside transferase (APH) gene, a thymidine kinase (TK) gene, an Escherichia coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, a dihydrofolate reductase (dhfr) gene, etc.

[0101] The antibody can be recovered, for example, by culturing the transformed cells and then isolating the antibody from the cells or culture medium of the molecularly transformed cells. The antibody can be separated and purified by an appropriate combination of methods such as centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, 1q, FcRn, protein A, protein G column, affinity chromatography, ion exchange chromatography, and gel filtration chromatography.

[0102] Furthermore, the present invention provides a method for producing a polypeptide comprising an antibody Fc region, which comprises adding at least one amino acid modification to the Fc region, and which has maintained or decreased binding activity to FcγRIIa and enhanced binding activity to FcγRIIb compared to a parent polypeptide. For example, the production method may include the following steps: (a) modifying at least one amino acid in an antibody Fc region of a polypeptide comprising the Fc region; (b) measuring the binding activity to FcγRIIa and the binding activity to FcγRIIb of the polypeptide altered in step (a); and (c) selecting a polypeptide that has maintained or decreased binding activity to FcγRIIa and enhanced binding activity to FcγRIIb, compared to the parent polypeptide.

[0103] A preferred embodiment of the present invention is a method for producing a polypeptide comprising an antibody Fc region, comprising the steps of: (a) modifying a nucleic acid encoding the polypeptide so that the binding activity to FcγRIIa is maintained or decreased and the binding activity to FcγRIIb is enhanced compared to the parent polypeptide; (b) introducing the nucleic acid into a host cell and culturing it so as to express the nucleic acid; (c) recovering the polypeptide from the host cell culture. Furthermore, antibodies and Fc fusion protein molecules produced by this production method are also included in the present invention. The present invention also provides a method for producing a polypeptide comprising an antibody Fc region, which comprises making at least one amino acid modification in the Fc region, and which, when administered to a living body, suppresses the production of antibodies against the polypeptide compared to a parent polypeptide. For example, the production method may include the following steps: (a) modifying at least one amino acid in an antibody Fc region of a polypeptide comprising the Fc region; and (b) confirming that the polypeptide modified in step (a) suppresses antibody production when administered to a living body, as compared to the parent polypeptide. Whether or not the production of antibodies against the polypeptide is suppressed can be confirmed by actually administering the polypeptide to an animal. Alternatively, the binding activity to FcγRIIa and the binding activity to FcγRIIb can be measured, and the suppression of antibody production can be judged by an increase in the value obtained by dividing the KD value for FcγRIIa by the KD value for FcγRIIb. Such a polypeptide can suppress the production of antibodies without activating activating FcγR, and is therefore considered to be useful as a pharmaceutical.

[0104] In the above-mentioned manufacturing method, it is preferable to enhance the binding activity to FcγRIIb and maintain or decrease the binding activity to FcγRIIa (R type) and FcγRIIa (H type), and it is further preferable to decrease the binding activity to FcγRIa and / or FcγRIIIa. In a preferred embodiment of the above-mentioned production method, for example, a polypeptide comprising the Fc region of human IgG is modified so that Pro at position 238 (EU numbering) is replaced with Asp, or Leu at position 328 (EU numbering) is replaced with Glu.In another preferred embodiment, in addition to the substitution of Pro at position 238 (EU numbering) with Asp, further substitutions include substitution of Gly at position 237 (EU numbering) with Trp, substitution of Gly at position 237 (EU numbering) with Phe, substitution of Ser at position 267 (EU numbering) with Val, substitution of Ser at position 267 (EU numbering) with Gln, substitution of His at position 268 (EU numbering) with Asn, substitution of Pro at position 271 (EU numbering) with Gly, substitution of Lys at position 326 (EU numbering) with Leu, substitution of Lys at position 326 (EU numbering) with Gln, or substitution of any of the following: substitution of Lys at position 326 (EU numbering) by Glu; substitution of Lys at position 326 (EU numbering) by Met; substitution of Ser at position 239 (EU numbering) by Asp; substitution of Ser at position 267 (EU numbering) by Ala; substitution of Leu at position 234 (EU numbering) by Trp; substitution of Leu at position 234 (EU numbering) by Tyr; substitution of Gly at position 237 (EU numbering) by Ala; substitution of Gly at position 237 (EU numbering) by Asp; substitution of Gly at position 237 (EU numbering) by Glu; Gly at position 237 (EU numbering) replaced by Leu, Gly at position 237 (EU numbering) replaced by Met, Gly at position 237 (EU numbering) replaced by Tyr, Ala at position 330 (EU numbering) replaced by Lys, Ala at position 330 (EU numbering) replaced by Arg, Glu at position 233 (EU numbering) replaced by Asp, His at position 268 (EU numbering) replaced by Asp, His at position 268 (EU numbering) replaced by Glu, Lys at position 326 (EU numbering) replaced by Asp, Lys at position 326 (EU numbering) replaced by Ser, E The polypeptide is modified so that at least one substitution selected from the group consisting of substitution of Lys at position 326 (EU numbering) with Thr, substitution of Val at position 323 (EU numbering) with Ile, substitution of Val at position 323 (EU numbering) with Leu, substitution of Val at position 323 (EU numbering) with Met, substitution of Tyr at position 296 (EU numbering) with Asp, substitution of Lys at position 326 (EU numbering) with Ala, substitution of Lys at position 326 (EU numbering) with Asn, and substitution of Ala at position 330 (EU numbering) with Met.

[0105] Furthermore, the present invention provides methods for modifying polypeptides to prepare polypeptides that have maintained or decreased FcγRIIa-binding activity and enhanced FcγRIIb-binding activity, as compared to the parent polypeptide. The present invention also provides methods for modifying polypeptides to produce polypeptides that, when administered to a living body, induce reduced antibody production compared to the parent polypeptide. In a preferred embodiment, for example, in a polypeptide comprising the Fc region of human IgG, the polypeptide is modified so that Pro at position 238 (EU numbering) is replaced with Asp, or Leu at position 328 (EU numbering) is replaced with Glu.In another preferred embodiment, in addition to the substitution of Pro at position 238 (EU numbering) with Asp, further substitutions include substitution of Gly at position 237 (EU numbering) with Trp, substitution of Gly at position 237 (EU numbering) with Phe, substitution of Ser at position 267 (EU numbering) with Val, substitution of Ser at position 267 (EU numbering) with Gln, substitution of His at position 268 (EU numbering) with Asn, substitution of Pro at position 271 (EU numbering) with Gly, substitution of Lys at position 326 (EU numbering) with Leu, substitution of Lys at position 326 (EU numbering) with Gln, or substitution of any of the following: substitution of Lys at position 326 (EU numbering) by Glu; substitution of Lys at position 326 (EU numbering) by Met; substitution of Ser at position 239 (EU numbering) by Asp; substitution of Ser at position 267 (EU numbering) by Ala; substitution of Leu at position 234 (EU numbering) by Trp; substitution of Leu at position 234 (EU numbering) by Tyr; substitution of Gly at position 237 (EU numbering) by Ala; substitution of Gly at position 237 (EU numbering) by Asp; substitution of Gly at position 237 (EU numbering) by Glu; Gly at position 237 (EU numbering) replaced by Leu, Gly at position 237 (EU numbering) replaced by Met, Gly at position 237 (EU numbering) replaced by Tyr, Ala at position 330 (EU numbering) replaced by Lys, Ala at position 330 (EU numbering) replaced by Arg, Glu at position 233 (EU numbering) replaced by Asp, His at position 268 (EU numbering) replaced by Asp, His at position 268 (EU numbering) replaced by Glu, Lys at position 326 (EU numbering) replaced by Asp, Lys at position 326 (EU numbering) replaced by Ser, E The polypeptide is modified so that at least one substitution selected from the group consisting of substitution of Lys at position 326 (EU numbering) with Thr, substitution of Val at position 323 (EU numbering) with Ile, substitution of Val at position 323 (EU numbering) with Leu, substitution of Val at position 323 (EU numbering) with Met, substitution of Tyr at position 296 (EU numbering) with Asp, substitution of Lys at position 326 (EU numbering) with Ala, substitution of Lys at position 326 (EU numbering) with Asn, and substitution of Ala at position 330 (EU numbering) with Met.

[0106] Furthermore, the present invention provides a nucleic acid encoding a polypeptide comprising an antibody Fc region, which has at least one amino acid alteration and has maintained or decreased binding activity to FcγRIIa and enhanced binding activity to FcγRIIb compared to a parent polypeptide. The nucleic acid of the present invention may be in any form, such as DNA or RNA.

[0107] Furthermore, the present invention provides a vector comprising the nucleic acid of the present invention. The type of vector can be appropriately selected by those skilled in the art depending on the host cell into which the vector is to be introduced, and for example, the vectors described above can be used.

[0108] Furthermore, the present invention relates to a host cell transformed with the vector of the present invention. The host cell can be appropriately selected by those skilled in the art, and for example, the host cells described above can be used.

[0109] Furthermore, the present invention provides a method for maintaining or decreasing the binding activity to FcγRIIa and enhancing the binding activity to FcγRIIb in a polypeptide comprising an antibody Fc region, compared to a parent polypeptide, which comprises making at least one amino acid modification to the Fc region. The present invention also provides a method for suppressing the production of antibodies against a polypeptide comprising an antibody Fc region, when administered to a living body, compared to a parent polypeptide, comprising making at least one amino acid modification in the Fc region.

[0110] In a preferred embodiment, for example, in a polypeptide comprising the Fc region of human IgG, the polypeptide is modified so that Pro at position 238 (EU numbering) is replaced with Asp, or Leu at position 328 (EU numbering) is replaced with Glu.In another preferred embodiment, in addition to the substitution of Pro at position 238 (EU numbering) with Asp, further substitutions include substitution of Gly at position 237 (EU numbering) with Trp, substitution of Gly at position 237 (EU numbering) with Phe, substitution of Ser at position 267 (EU numbering) with Val, substitution of Ser at position 267 (EU numbering) with Gln, substitution of His at position 268 (EU numbering) with Asn, substitution of Pro at position 271 (EU numbering) with Gly, substitution of Lys at position 326 (EU numbering) with Leu, substitution of Lys at position 326 (EU numbering) with Gln, or substitution of any of the following: substitution of Lys at position 326 (EU numbering) by Glu; substitution of Lys at position 326 (EU numbering) by Met; substitution of Ser at position 239 (EU numbering) by Asp; substitution of Ser at position 267 (EU numbering) by Ala; substitution of Leu at position 234 (EU numbering) by Trp; substitution of Leu at position 234 (EU numbering) by Tyr; substitution of Gly at position 237 (EU numbering) by Ala; substitution of Gly at position 237 (EU numbering) by Asp; substitution of Gly at position 237 (EU numbering) by Glu; Gly at position 237 (EU numbering) replaced by Leu, Gly at position 237 (EU numbering) replaced by Met, Gly at position 237 (EU numbering) replaced by Tyr, Ala at position 330 (EU numbering) replaced by Lys, Ala at position 330 (EU numbering) replaced by Arg, Glu at position 233 (EU numbering) replaced by Asp, His at position 268 (EU numbering) replaced by Asp, His at position 268 (EU numbering) replaced by Glu, Lys at position 326 (EU numbering) replaced by Asp, Lys at position 326 (EU numbering) replaced by Ser, E The polypeptide is modified so that at least one substitution selected from the group consisting of substitution of Lys at position 326 (EU numbering) with Thr, substitution of Val at position 323 (EU numbering) with Ile, substitution of Val at position 323 (EU numbering) with Leu, substitution of Val at position 323 (EU numbering) with Met, substitution of Tyr at position 296 (EU numbering) with Asp, substitution of Lys at position 326 (EU numbering) with Ala, substitution of Lys at position 326 (EU numbering) with Asn, and substitution of Ala at position 330 (EU numbering) with Met.

[0111] In the above-mentioned method, it is preferable to enhance the binding activity to FcγRIIb and maintain or decrease the binding activity to FcγRIIa (R type) and FcγRIIa (H type), and it is further preferable to maintain or decrease the binding activity to FcγRIa and / or FcγRIIIa. The present invention also includes polypeptides produced by any of the above methods.

[0112] <Pharmaceutical Composition> The present invention provides a pharmaceutical composition comprising a polypeptide of the present invention. The pharmaceutical composition of the present invention can be formulated by a known method by introducing a pharma- ceutical acceptable carrier in addition to the antibody or Fc fusion protein molecule of the present invention. For example, it can be used parenterally in the form of a sterile solution or suspension injection in water or other pharma- ceutical acceptable liquid. For example, it can be formulated by appropriately combining with a pharmacologically acceptable carrier or vehicle, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc., and mixing in a unit dose form required for generally accepted pharmaceutical practice. Specific examples of carriers include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl acetal diethyl amino acetate, polyvinyl pyrrolidone, gelatin, medium chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts, etc. The amount of active ingredient in these preparations is such that an appropriate dose within the indicated range can be obtained. Sterile compositions for injection can be formulated according to standard pharmaceutical practice using vehicles such as distilled water for injection.

[0113] Examples of aqueous solutions for injection include isotonic solutions containing physiological saline, glucose or other auxiliary drugs, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, which may be used in combination with an appropriate solubilizing agent, such as alcohol, specifically ethanol, polyalcohols, such as propylene glycol and polyethylene glycol, and nonionic surfactants, such as polysorbate 80(TM) and HCO-50.

[0114] The oily liquid may be sesame oil or soybean oil, and may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. Buffers such as phosphate buffer, sodium acetate buffer, soothing agents such as procaine hydrochloride, stabilizers such as benzyl alcohol, phenol, and antioxidants may also be used. The prepared injection solution is usually filled into a suitable ampule.

[0115] The administration is preferably parenteral administration, and specific examples include injection dosage forms, intranasal administration dosage forms, pulmonary administration dosage forms, transdermal administration dosage forms, etc. The injection dosage forms can be administered systemically or locally, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.

[0116] Furthermore, the method of administration of the pharmaceutical composition of the present invention can be appropriately selected depending on the age and symptoms of the patient. The dosage of the pharmaceutical composition containing an antibody or a polynucleotide encoding the antibody can be selected, for example, from the range of 0.0001 mg to 1000 mg per kg of body weight per administration. Alternatively, the dosage can be selected, for example, from the range of 0.001 to 100,000 mg / body per patient, but is not necessarily limited to these values. The dosage and method of administration vary depending on the patient's body weight, age, symptoms, etc., but can be appropriately selected by those skilled in the art.

[0117] The above-mentioned polypeptide 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 of the present invention can selectively act on FcγRIIb without activating activating FcγR to 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, and the like. The above-mentioned polypeptide of the present invention suppresses IgE production by B cells by crosslinking FcγRIIb with IgE, suppresses IgM production by B cells by crosslinking with IgM, and suppresses IgA production by crosslinking with IgA. In addition, the same inhibitory effect as above can be exerted by directly or indirectly crosslinking FcγRIIb with a molecule expressed on B cells, such as BCR, CD19, CD79b, which contains an ITAM domain intracellularly or interacts with the ITAM domain. The activation of mast cells also includes proliferation, activation by IgE, etc., degranulation, and the like. The polypeptide of the present invention can inhibit proliferation, activation by IgE, etc., and degranulation in mast cells by directly or indirectly crosslinking FcγRIIb with a molecule that contains an ITAM domain or interacts with an ITAM domain expressed on mast cells, such as IgE receptors FcεRI, DAP12, and CD200R3. Activation of basophils includes proliferation, degranulation, and the like. The polypeptide of the present invention can inhibit activation, degranulation, and proliferation in basophils by directly or indirectly crosslinking FcγRIIb with a molecule on the cell membrane that contains an ITAM domain intracellularly or interacts with an ITAM domain. Activation of dendritic cells includes proliferation, degranulation, and the like. The polypeptide of the present invention can inhibit activation, degranulation, and proliferation in dendritic cells by directly or indirectly crosslinking FcγRIIb with a molecule on the cell membrane that contains an ITAM domain intracellularly or interacts with an ITAM domain.

[0118] In the present invention, the above-mentioned polypeptide of the present invention is useful as an active ingredient of a therapeutic or preventive agent for immunoinflammatory diseases. As described above, the polypeptide of the present invention can suppress the activation of B cells, mast cells, dendritic cells and / or basophils, and as a result, it is possible to treat or prevent immunoinflammatory diseases by administering the polypeptide of the present invention. "Immunoinflammatory diseases" include, but are not limited to, the following: rheumatoid arthritis, autoimmune hepatitis, autoimmune thyroiditis, autoimmune bullous disease, autoimmune adrenocorticitis, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, megalocytic anemia, autoimmune atrophic gastritis, autoimmune neutropenia, autoimmune orchitis, autoimmune encephalomyelitis, autoimmune receptor disease, autoimmune infertility, chronic active hepatitis, glomerulonephritis, interstitial pulmonary fibrosis, multiple sclerosis, Paget's disease, osteoporosis, multiple myeloma, uveitis, acute and chronic spondylitis, gouty arthritis, inflammatory bowel disease, adult respiratory distress syndrome (ARDS), psoriasis, Crohn's disease, Graves' disease, juvenile diabetes mellitus, Addison's disease, myasthenia gravis, phaco 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, I Diabetes mellitus type II and II, bone resorption diseases, graft-versus-host reaction, ischemia-reperfusion injury, atherosclerosis, brain trauma, cerebral malaria, sepsis, septic shock, toxic shock syndrome, fever, malgias due to staining, aplastic anemia, hemolytic anemia, idiopathic thrombocytopenia, Goodpasture's syndrome, Guillain-Barre syndrome, Hashimoto's disease, pemphigus, IgA nephropathy, hay fever, antiphospholipid syndrome, polymyositis, Wegener's granulomatosis, arteritis nodosa, mixed tuberculosis Synthetic tissue disease, fibromyalgia, asthma, atopic dermatitis, chronic atrophic gastritis, primary biliary cirrhosis, primary sclerosing cholangitis, autoimmune pancreatitis, aortitis syndrome, rapidly progressive glomerulonephritis, megaloblastic anemia, idiopathic thrombocytopenic purpura, primary hypothyroidism, idiopathic Addison's disease, insulin-dependent diabetes mellitus, chronic discoid lupus erythematosus, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, epidermolysis bullosa acquisita, alopecia areata, vitiligo vulgaris, Sutton's acquired centrifugal vitiligo, Harada's disease,Autoimmune optic neuropathy, idiopathic azoospermia, habitual abortion, hypoglycemia, chronic urticaria, ankylosing spondylitis, psoriatic arthritis, enteropathic arthritis, reactive arthritis, spondyloarthropathy, enthesitis, irritable bowel syndrome, chronic fatigue syndrome, dermatomyositis, inclusion body myositis, Schmidt's syndrome, Graves' disease, pernicious anemia, lupoid hepatitis, presenile dementia, Alzheimer's disease, demyelinating diseases, amyotrophic lateral sclerosis, hypoparathyroidism, Dressler's syndrome, Eaton-Lambert syndrome, dermatitis herpetiformis, alopecia, progressive systemic sclerosis, CREST syndrome (calcification , Raynaud's phenomenon, esophageal motility disorder, sclerodactyly and telangiectasia), sarcoidosis, rheumatic fever, erythema multiforme, Cushing's syndrome, transfusion reactions, leprosy, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, giant cell arteritis, eczema, lymphomatoid granulomatosis, Kawasaki disease, endocarditis, endomyocardial fibrosis, endophthalmitis, erythroblastosis fetalis, eosinophilic fasciitis, Felty's syndrome, Henoch-Schönlein purpura, transplant rejection, mumps, cardiomyopathy, septic arthritis, familial Mediterranean fever, Muckle-Wells syndrome, hyper-IgD syndrome.

[0119] In addition, the polypeptide of the present invention is useful as an active ingredient of a drug for treating or preventing an autoimmune disease in which the production of an antibody (autoantibody) against an autoantigen is considered to be the cause of the disease, by suppressing the production of the autoantibody. It has been reported that the use of a molecule in which AchR, an autoantigen of myasthenia gravis, is fused with the Fc portion of an antibody can suppress the proliferation of B cells expressing a BCR that recognizes AchR and induce apoptosis (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 the BCR of a B cell expressing a BCR against the autoantigen with FcγRIIb, suppress the proliferation of B cells expressing a BCR against the autoantigen, and induce apoptosis. These autoimmune diseases include Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, autoimmune pancreatitis, aortitis syndrome, Goodpasture's syndrome, rapidly progressive glomerulonephritis, megaloblastic anemia, autoimmune hemolytic anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Graves' disease, Hashimoto's disease, and primary hypothyroidism. These conditions include, but are not limited to, laxatives, idiopathic Addison's disease, insulin-dependent diabetes mellitus, chronic discoid lupus erythematosus, localized scleroderma, pemphigus, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, epidermolysis bullosa acquisita, alopecia areata, vitiligo vulgaris, Sutton's acquired centrifugal vitiligo, Harada's disease, autoimmune optic neuropathy, idiopathic azoospermia, recurrent abortions, type 2 diabetes mellitus, hypoglycemia, and chronic urticaria.

[0120] In addition, the polypeptide of the present invention is useful as an active ingredient of a therapeutic agent for diseases in which a protein necessary for the living body is missing. For diseases in which a protein necessary for the living body is missing, a treatment method is used in which the protein is administered as a drug to supplement the disease, but since the patient is originally missing the protein, the protein supplemented from the outside is recognized as a foreign body, and antibodies against the protein are produced. As a result, the protein becomes more likely to be removed, and the effect of the drug is weakened. 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 the production of antibodies against the protein. Proteins 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, and glucocerebrosidase. Diseases to be supplemented with these proteins include hemophilia, idiopathic thrombocytopenic purpura, renal anemia, lysosomal diseases (mucopolysaccharidosis, Fabry disease, Pompe disease, and Gaucher disease), etc., but are not limited to these.

[0121] The polypeptide of the present invention is also useful as an active ingredient of an antiviral agent. An antibody against a virus that includes the Fc region described in the present invention can suppress antibody-dependent enhancement observed in antibodies against viruses. Antibody-dependent enhancement is a phenomenon in which a virus is phagocytosed via active FcγR using a neutralizing antibody against the virus, and infects an FcγR-expressing cell, thereby spreading the infection. It has been reported that the binding of a neutralizing antibody against the dengue virus to FcγRIIb plays an important role in suppressing antibody-dependent enhancement (Proc Natl Acad Sci USA, 108, 12479-12484, 2011). An immune complex formed by a neutralizing antibody against the dengue virus crosslinks FcγRIIb, thereby inhibiting phagocytosis via FcγR, and as a result suppressing antibody-dependent enhancement. Viruses include dengue viruses (DENV1, DENV2, DENV4) and HIV. However, they are not limited to these.

[0122] Moreover, the above-mentioned polypeptide 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 FcγRIIa-dependent adhesion of inflammatory cells. Anti-oxidized LDL antibodies inhibit the interaction between oxidized LDL and CD36, but it has been reported that anti-oxidized LDL antibodies bind to endothelial cells, and monocytes recognize and adhere to the Fc portion in an FcγRIIa or FcγRI-dependent manner (Immunol Lett, 108, 52-61, 2007). By using an antibody containing the Fc region described in the present invention as such an antibody, it is considered that FcγRIIa-dependent binding is inhibited and adhesion of monocytes is suppressed by an inhibitory signal mediated by FcγRIIb.

[0123] In the present invention, the polypeptide of the present invention is useful as an active ingredient of a therapeutic or preventive agent for cancer. As described above, it is known that enhancing the binding to FcγRIIb enhances the agonistic activity of an agonistic antibody and also enhances the antitumor effect of the antibody, so that an agonistic antibody using the Fc region described in the present invention is useful for treating or preventing cancer. The Fc region described in the present invention enhances the agonistic activity of agonistic antibodies against TNF receptor family members 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, and Ectodysplasin A2 receptor. It also enhances the agonistic activity of agonistic antibodies other than those mentioned above.Cancers include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), colorectal cancer, rectal cancer, colon cancer, breast cancer, liver cancer, stomach cancer, pancreatic cancer, kidney cancer, prostate cancer, ovarian cancer, thyroid cancer, bile duct cancer, peritoneal cancer, mesothelioma, squamous cell carcinoma, cervical cancer, uterine cancer, bladder cancer, esophageal cancer, head and neck cancer, nasopharyngeal cancer, salivary gland tumors, thymoma, skin cancer, basal cell carcinoma, malignant melanoma, anal cancer, penile cancer, testicular cancer, Wilms' tumor, acute myeloid leukemia (including acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, and acute monocytic leukemia), chronic myeloid leukemia, chronic myeloblastic leukemia, acute myelomonocytic leukemia, and acute monocytic leukemia, chronic myeloma ... Myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma (Burkitt's lymphoma, chronic lymphocytic leukemia, mycosis fungoides, mantle cell lymphoma, follicular lymphoma, diffuse large 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 tumors (including glioma, astrocytoma, glioblastoma, meningioma and ependymoma), neuroblastoma, retinoblastoma, osteosarcoma, Kaposi's sarcoma, Ewing's sarcoma, angiosarcoma, hemangiopericytoma.

[0124] The present invention also relates to a method for treating or preventing an immunoinflammatory disease, which comprises the step of administering to a subject (patient) the polypeptide of the present invention or a polypeptide produced by the production method of the present invention.

[0125] The present invention also provides a kit for use in the therapeutic or preventive method of the present invention, comprising at least the polypeptide of the present invention or a polypeptide produced by the production method of the present invention, or the pharmaceutical composition of the present invention. The kit may also be packaged with other pharma- ceutically acceptable carriers, vehicles, instructions describing the method of use, and the like. The present invention also relates to the use of the polypeptide of the present invention or a polypeptide produced by the production method of the present invention in the production of a therapeutic or preventive agent for immunoinflammatory diseases. The present invention also relates to the polypeptide of the present invention or a polypeptide produced by the production method of the present invention for use in the therapeutic or preventive method of the present invention.

[0126] The correspondence between the three-letter and one-letter amino acid codes used in this specification is as follows: Alanine: Ala: A Arginine:Arg:R Asparagine:Asn:N Aspartic acid:Asp:D Cysteine:Cys:C Glutamine:Gln:Q Glutamic acid: Glu:E Glycine:Gly:G Histidine:His:H Isoleucine:Ile:I Leucine:Leu:L Lysine:Lys:K Methionine:Met:M Phenylalanine:Phe:F Proline: Pro:P Serine:Ser:S Threonine: Thr:T Tryptophan:Trp:W Tyrosine:Tyr:Y Valin:Val:V

[0127] All prior art documents cited in this specification are hereby incorporated by reference. EXAMPLES

[0128] The present invention is further illustrated by, but not limited to, the following examples.

[0129] [Example 1] Comprehensive analysis of Fc variant binding to FcγR In order to generate an antibody that, compared to IgG1, has reduced Fc-mediated binding to both activating FcγRs, particularly the H and R genetic polymorphisms of FcγRIIa, and enhanced binding to FcγRIIb, mutations were introduced into the IgG1 antibody and comprehensive analysis of binding to each FcγR was performed.

[0130] The antibody H chain used was the variable region of a glypican 3 antibody containing the CDR of GpH7 (SEQ ID NO: 15), an anti-glypican 3 antibody with improved plasma kinetics disclosed in WO2009 / 041062. Similarly, the antibody L chain used was GpL16-k0 (SEQ ID NO: 16), an anti-glypican 3 antibody with improved plasma kinetics disclosed in WO2009 / 041062. In addition, the antibody H chain constant region used was B3 (SEQ ID NO: 17), which is G1d with the C-terminus of IgG1 removed and the mutation K439E introduced. This H chain is called GpH7-B3 (SEQ ID NO: 18), and the L chain is called GpL16-k0 (SEQ ID NO: 16).

[0131] In GpH7-B3, amino acids thought to be involved in FcγR binding and the amino acids around them (234th to 239th, 265th to 271st, 295th, 296th, 298th, 300th, and 324th to 337th in EU numbering) were replaced with the original amino acids and 18 types of amino acids excluding Cys. These Fc mutants are called B3 variants. The B3 variants were expressed and purified by the method of Reference Example 1, and binding to each FcγR (FcγRIa, FcγRIIa H type, FcγRIIa R type, FcγRIIb, FcγRIIIa) was comprehensively evaluated by the method of Reference Example 2. The results of the interaction analysis with each FcγR were plotted according to the following method. The value of the binding amount of the antibody derived from each B3 variant to FcγR was divided by the value of the binding amount of the antibody to FcγR of the comparative antibody without mutations introduced into B3 (antibody having the sequence of human native IgG1 at positions 234 to 239, 265 to 271, 295, 296, 298, 300, and 324 to 337 in EU numbering). The value was further multiplied by 100 to obtain an index of the relative binding activity of each variant to FcγR. The horizontal axis shows the relative binding activity of each variant to FcγRIIb, and the vertical axis shows the relative binding activity of each variant to the activating FcγRs FcγRIa, FcγRIIa H type, FcγRIIa R type, and FcγRIIIa, respectively (Figures 1, 2, 3, and 4).

[0132] As a result, as labeled in Figures 1 to 4, it was found that when only mutation A (replacing Pro at position 238, EU numbering, with Asp) and mutation B (replacing Leu at position 328, EU numbering, with Glu) were introduced among all the modifications, the binding to FcγRIIb was significantly enhanced and the binding to both types of FcγRIIa was significantly suppressed compared to before the introduction.

[0133] [Example 2] SPR analysis of FcγRIIb selective binding variants The binding to each FcγR of the alteration discovered in Example 1 in which Pro at position 238 (EU numbering) was replaced with Asp was analyzed in more detail.

[0134] The antibody H-chain variable region was the variable region of IL6R-H (SEQ ID NO: 19), which is the variable region of an antibody against human interleukin 6 receptor disclosed in WO2009 / 125825, and the antibody H-chain constant region was IL6R-G1d (SEQ ID NO: 20), which has G1d with Gly and Lys removed from the C-terminus of human IgG1, as the IgG1 H-chain. IL6R-G1d-v1 (SEQ ID NO: 21) was prepared by replacing Pro at position 238 (EU numbering) of IL6R-G1d with Asp. Next, IL6R-G1d-v2 (SEQ ID NO: 23) was prepared by replacing Leu at position 328 (EU numbering) of IL6R-G1d with Glu. For comparison, IL6R-G1d-v3 (SEQ ID NO: 24) was prepared by substituting Ser at position 267 (EU numbering) of IL6R-G1d described in Non-Patent Document 27 with Glu and Leu at position 328 (EU numbering) with Phe. IL6R-L (SEQ ID NO: 22), which is the L chain of tocilizumab, was used in common as the antibody L chain, and the antibody was expressed and purified together with each H chain according to the method of Reference Example 1. The antibodies having amino acid sequences derived from IL6R-G1d, IL6R-G1d-v1, IL6R-G1d-v2, and IL6R-G1d-v3 as the antibody H chain obtained here are referred to as IgG1, IgG1-v1, IgG1-v2, and IgG1-v3, respectively.

[0135] Next, kinetic analysis of the interaction between these antibodies and FcγR was carried out using Biacore T100 (GE Healthcare). HBS-EP+ (GE Healthcare) was used as the running buffer, and the measurement temperature was 25°C. A chip with Protein A immobilized by the amine coupling method was used for Series S Sensor Chip CM5 (GE Healthcare). The target antibody was captured on this chip, and each FcγR diluted with the running buffer was allowed to interact with it, and the binding to the antibody was measured. After the measurement, the antibody captured on the chip was washed by reacting with 10 mM glycine-HCl, pH 1.5, and the chip was regenerated and used repeatedly. The sensorgram obtained as the measurement result was analyzed using the Biacore Evaluation Software with a 1:1 Langmuir binding model to calculate the binding rate constant ka (L / mol / s) and the dissociation rate constant kd (1 / s), and the dissociation constant KD (mol / L) was calculated from the values.

[0136] In this study, the binding of IgG1-v1 and IgG1-v2 to FcγRIIa H and FcγRIIIa was weak, so kinetic parameters such as KD could not be calculated using the above analysis method. For these interactions, KD was calculated using the following 1:1 binding model equation described in the Biacore T100 Software Handbook BR1006-48 Edition AE. The behavior of molecules interacting via a 1:1 binding model on a Biacore can be represented by the following Equation 1.

[0137] [Formula 1] TIFF2025010300000001.tif13144

[0138] R eq :a plot of steady state binding levels against analyte concentration C: concentration RI:bulk refractive index contribution in the sample R max :analyte binding capacity of the surface

[0139] By rearranging this equation, KD can be expressed as the following Equation 2. [Formula 2] TIFF2025010300000002.tif10136

[0140] In this equation, R max By substituting the values ​​of , RI, and C, it is possible to calculate KD. From the measurement conditions in this study, RI = 0, C = 2 μmol / L. max The R values ​​obtained by global fitting the sensorgrams obtained as the result of the interaction analysis of IgG1 with each FcγR using a 1:1 Langmuir binding model are shown in max The value was divided by the capture amount of IgG1 and multiplied by the capture amount of IgG1-v1 and IgG1-v2 to obtain the value. This is because, in all of the variants in which mutations were introduced into IgG1, there was no change in the limit amount that each FcγR could bind compared to IgG1, and the R max This calculation is based on the assumption that R is proportional to the amount of antibody bound to the chip at the time of measurement. eq was defined as the amount of binding of each FcγR to each variant on the sensor chip observed during measurement.

[0141] Under the measurement conditions used here, the binding amounts of IgG1-v1 and IgG1-v2 to FcγRIIa H type (R eq ) were approximately 2.5 and 10 RU, respectively, and the binding amounts of IgG1-v1 and IgG1-v2 to FcγRIIIa (R eq) were approximately 2.5 and 5 RU, respectively. The capture amounts of IgG1, IgG1-v1, and IgG1-v2 in the interaction analysis with FcγRIIa H type were 452, 469.2, and 444.2 RU, and the capture amounts of IgG1, IgG1-v1, and IgG1-v2 in the interaction analysis with FcγRIIIa were 454.5, 470.8, and 447.1 RU. The R max The R values ​​for IgG1-v1 and IgG1-v2 against FcγRIIa H type were 69.8 and 63.8 RU, respectively. max The R of IgG1-v1 and IgG1-v2 against FcγRIIIa was 72.5 and 68.6 RU, respectively. max were calculated to be 66.0 and 62.7 RU, respectively. [Formula 2] The KD of IgG1-v1 and IgG1-v2 for FcγRIIa H type and FcγRIIIa was calculated by substituting the results into the equation in TIFF2025010300000003.tif10136.

[0142] The KD values ​​of IgG1, IgG1-v1, IgG1-v2, and IgG1-v3 for each FcγR are shown in Table 1 (KD values ​​of each antibody for each FcγR), and the relative KD values ​​of IgG1-v1, IgG1-v2, and IgG1-v3, calculated by dividing the KD value of IgG1 for each FcγR by the KD value of IgG1-v1, IgG1-v2, and IgG1-v3 for each FcγR, are shown in Table 2 (relative KD values ​​of each antibody for each FcγR).

[0143] [Table 1]

[0144] In Table 1 above, * indicates that FcγR binding to IgG was not sufficiently observed. [Formula 2] This means the KD calculated using the formula in TIFF2025010300000005.tif10136.

[0145] [Table 2]

[0146] As shown in Table 2, compared with IgG1, IgG1-v1 had a 0.047-fold decreased binding activity to FcγRIa, a 0.10-fold decreased binding activity to the R type of FcγRIIa, a 0.014-fold decreased binding activity to the H type of FcγRIIa, and a 0.061-fold decreased binding activity to FcγRIIIa. On the other hand, the binding activity to FcγRIIb was improved by 4.8-fold.

[0147] Furthermore, as shown in Table 2, compared with IgG1, IgG1-v2 had a 0.74-fold reduced binding activity to FcγRIa, a 0.41-fold reduced binding activity to FcγRIIa R type, a 0.064-fold reduced binding activity to FcγRIIa H type, and a 0.14-fold reduced binding activity to FcγRIIIa. On the other hand, the binding activity to FcγRIIb was improved by 2.3-fold.

[0148] In other words, these results demonstrated that IgG1-v1, which has an alteration in which Pro at position 238 (EU numbering) is replaced with Asp, and IgG1-v2, which has an alteration in which Leu at position 328 (EU numbering) is replaced with Glu, have the property of weakening binding to all activating FcγR, including both FcγRIIa genetic polymorphisms, while increasing binding to FcγRIIb, an inhibitory FcγR.

[0149] Next, the selectivity of the obtained variants for FcγRIIb was evaluated using the ratio of the binding activity to FcγRIIb and the binding activity to FcγRIIa R or H as an index. Specifically, I / A (R) or I / A (H), which is the value obtained by dividing the KD value for FcγRIIa R or H by the KD value for FcγRIIb, was used as an index of the selectivity of FcγRIIb for each FcγRIIa. This index shows a larger value as the KD value for FcγRIIb becomes smaller or the KD value for FcγRIIa becomes larger. In other words, a variant showing a larger value indicates that the relative binding activity to FcγRIIb compared to FcγRIIa is improved. This index is summarized in Table 3 for each variant.

[0150] [Table 3]

[0151] The results in Table 3 show that, compared to IgG1, IgG1-v3 to which existing technology was applied showed a higher I / A (H) value than IgG1 and was more selective for FcγRIIb, but showed a lower I / A (R) value than IgG1, indicating improved selectivity for FcγRIIb. On the other hand, IgG1-v1 and IgG1-v2 discovered in this example showed higher I / A (R) and I / A (H) values ​​than IgG1, indicating improved selectivity for FcγRIIb for all FcγRIIa gene polymorphisms. Modifications with such properties have not been reported so far, and are extremely rare, as shown in Figures 1, 2, 3, and 4. The substitution of Pro at position 238 (EU numbering) with Asp and the substitution of Leu at position 328 (EU numbering) with Glu are extremely useful for the development of therapeutic agents for immune inflammatory diseases.

[0152] Furthermore, IgG1-v3 described in Table 2 and Non-Patent Document 27 certainly enhances the binding to FcγRIIb by 408 times, and the binding to FcγRIIa H type is decreased to 0.51 times, but on the other hand, enhances the binding to FcγRIIa R type by 522 times. From these results, IgG1-v1 and IgG1-v2 suppress the binding to both FcγRIIa R type and H type, and enhance the binding to FcγRIIb, and therefore are considered to be variants that bind more selectively to FcγRIIb than IgG1-v3. That is, the modification in which Pro at position 238 (EU numbering) is replaced with Asp and the modification in which Leu at position 328 (EU numbering) is replaced with Glu are extremely useful for the development of therapeutic agents for immune inflammatory diseases and the like.

[0153] [Example 3] Effect of combining FcγRIIb selective binding alteration with amino acid substitutions in other Fc regions Based on the variants in which Pro at position 238 (EU numbering) was substituted with Asp, which had improved selectivity for FcγRIIb as found in Examples 1 and 2, an attempt was made to further enhance selectivity for FcγRIIb. First, IL6R-G1d_v1 (SEQ ID NO: 21) was prepared by replacing Pro at position 238 (EU numbering) with Asp to prepare IL6R-G1d-v4 (SEQ ID NO: 25) by replacing Leu at position 328 (EU numbering) with Glu to enhance selectivity to FcγRIIb as described in Example 2. This was combined with IL6R-L (SEQ ID NO: 22) and prepared according to the method of Reference Example 1. The antibody having an amino acid sequence derived from IL6R-G1d-v4 as the antibody H chain obtained here is referred to as IgG1-v4. The binding activity of IgG1, IgG1-v1, IgG1-v2, and IgG1-v4 to FcγRIIb was evaluated according to the method of Reference Example 2, and the results are shown in Table 4.

[0154] [Table 4]

[0155] As the results in Table 4 show, because L328E improves FcγRIIb-binding activity by 2.3-fold compared to IgG1, it was expected that combining it with P238D, which similarly improves FcγRIIb-binding activity by 4.8-fold compared to IgG1, would further increase the degree of improvement in FcγRIIb-binding activity, but it was revealed that the FcγRIIb-binding activity of the variant combining these modifications was actually reduced to 0.47-fold compared to IgG1. This result is an effect that could not be predicted from the effect of each modification.

[0156] Similarly, IL6R-G1d-v1 (SEQ ID NO: 21), which is an IL6R-G1d modified by substituting Pro at position 238 (EU numbering) with Asp, was used to prepare a variant IL6R-G1d-v5 (SEQ ID NO: 26) in the manner described in Reference Example 1, in which Ser at position 267 (EU numbering) was substituted with Glu and Leu at position 328 (EU numbering) was substituted with Phe to improve the binding activity to FcγRIIb as described in Example 2. The antibody having an amino acid sequence derived from IL6R-G1d-v5 as the antibody H chain obtained here is referred to as IgG1-v5. The binding activity of IgG1, IgG1-v1, IgG1-v3, and IgG1-v5 to FcγRIIb was evaluated according to the method of Reference Example 2, and the results are shown in Table 5. S267E / L328F, which had an enhancing effect on FcγRIIb binding in Example 2, was introduced into the P238D variant, and the FcγRIIb-binding activity was evaluated before and after the introduction of the alterations. The results are shown in Table 5.

[0157] [Table 5]

[0158] As the results in Table 5 show, because S267E / L328F has a 408-fold improved FcγRIIb-binding activity compared to IgG1, it was expected that when combined with P238D, which similarly has a 4.8-fold improved FcγRIIb-binding activity compared to IgG1, the degree of improvement in FcγRIIb-binding activity would be further increased, but in reality, as in the previous example, it was revealed that the FcγRIIb-binding activity of the variant combining these modifications was only about 12-fold improved compared to IgG1. This result, too, is an effect that could not be predicted from the effect of each modification.

[0159] From these results, it was revealed that the substitution of Pro at position 238 (EU numbering) with Asp alone improves the binding activity to FcγRIIb, but when combined with other modifications that improve the binding activity to FcγRIIb, the effect is not exerted. One factor for this is that the structure of the interaction interface between Fc and FcγR is changed by the introduction of the substitution of Pro at position 238 (EU numbering) with Asp, and the effect of the modification observed in the natural antibody is no longer reflected. For this reason, it was considered extremely difficult to create an Fc with excellent selectivity for FcγRIIb using an Fc containing a substitution of Pro at position 238 (EU numbering) with Asp as a template, since the information on the effect of the modification obtained in the natural antibody cannot be utilized.

[0160] [Example 4] Comprehensive analysis of FcγRIIb binding of variants containing modifications in the hinge region in addition to the P238D modification As shown in Example 3, when an Fc in which Pro at position 238 (EU numbering) of human native IgG1 was substituted with Asp was combined with other modifications predicted to further enhance binding to FcγRIIb, the expected combination effect was not obtained. Therefore, based on a modified Fc in which Pro at position 238 (EU numbering) was substituted with Asp, a study was carried out to find a modified Fc that further enhances binding to FcγRIIb by comprehensively introducing modifications to the Fc. As the antibody H chain, IL6R-F11 (SEQ ID NO: 27) was prepared by introducing modifications to IL6R-G1d (SEQ ID NO: 20) in which Met at position 252 (EU numbering) was substituted with Tyr and Asn at position 434 (EU numbering) was substituted with Tyr, and IL6R-F652 (SEQ ID NO: 28) was prepared by further introducing a modification to replace Pro at position 238 (EU numbering) with Asp. For IL6R-F652, expression plasmids were prepared containing antibody H-chain sequences in which the regions near residue 238 (EU numbering positions 234 to 237 and 239) were substituted with the original amino acid and 18 types of amino acids excluding cysteine. IL6R-L (SEQ ID NO: 22) was commonly used for the antibody L-chain. These variants were expressed, purified, and expressed by the method of Reference Example 1. These Fc mutants are referred to as PD variants. The interactions of each PD variant with FcγRIIa R type and FcγRIIb were comprehensively evaluated by the method of Reference Example 2.

[0161] A graph was created using the following method to plot the results of the analysis of the interaction with each FcγR. The binding amount of each PD variant to each FcγR was divided by the binding amount of the control antibody before the introduction of the alteration (IL6R-F652 / IL6R-L, an alteration in which Pro at position 238 (EU numbering) was replaced with Asp) to each FcγR, and the resulting value was multiplied by 100 to obtain the relative binding activity of each PD variant to each FcγR. The horizontal axis shows the relative binding activity of each PD variant to FcγRIIb, and the vertical axis shows the relative binding activity of each PD variant to FcγRIIa R type (FIG. 6).

[0162] As a result, it was found that 11 types of alterations have the effect of enhancing binding to FcγRIIb and maintaining or enhancing binding to FcγRIIa R, compared to the antibody before the introduction of the alterations. The results of the binding activity of these 11 types of alterations to FcγRIIb and FcγRIIa R are summarized in Table 6. Note that the sequence numbers in the table represent the sequence numbers of the H chains of the evaluated alterations, and the alterations represent the alterations introduced into IL6R-F11 (SEQ ID NO: 27).

[0163] [Table 6]

[0164] The relative FcγRIIb binding activity values ​​when these 11 types of alterations were further introduced into the variants into which P238D had been introduced, and the relative FcγRIIb binding activity values ​​when these alterations were introduced into Fc not containing P238D in Example 1 are shown in FIG. 7. When these 11 types of alterations were further introduced into the P238D alteration, the amount of binding to FcγRIIb was enhanced compared to before introduction, but on the contrary, when eight types of alterations other than G237F, G237W, and S239D were introduced into the P238D-free variant (GpH7-B3 / GpL16-k0) used in Example 1, they showed the effect of decreasing the binding to FcγRIIb. From the results of Example 3 and this result, it became clear that it is difficult to predict the effect of introducing the P238D alteration into a variant containing the same alteration in Fc from the effect of the alteration introduced into the native IgG1. In other words, the eight types of modifications discovered this time would not have been possible without this study. The KD values ​​of the variants shown in Table 6 for FcγRIa, FcγRIIaR, FcγRIIaH, FcγRIIb, and FcγRIIIaV were measured by the method of Reference Example 2, and the results are summarized in Table 7. In the table, the sequence number indicates the sequence number of the H chain of the variant evaluated, and the modification indicates the modification introduced into IL6R-F11 (SEQ ID NO: 27). However, IL6R-G1d / IL6R-L used as the template for producing IL6R-F11 is indicated with *. In addition, KD(IIaR) / KD(IIb) and KD(IIaH) / KD(IIb) in the table respectively indicate the value obtained by dividing the KD value of each variant for FcγRIIaR by the KD value of each variant for FcγRIIb, and the value obtained by dividing the KD value of each variant for FcγRIIaH by the KD value of each variant for FcγRIIb. KD(IIb) of parent polypeptide / KD(IIb) of modified polypeptide refers to the value obtained by dividing the KD value of the parent polypeptide for FcγRIIb by the KD value of each variant for FcγRIIb. In addition, the stronger KD value of each variant's binding activity to FcγRIIaR and FcγRIIaH / the stronger KD value of the parent polypeptide's binding activity to FcγRIIaR and FcγRIIaH is shown in Table 7. The parent polypeptide here refers to a variant having IL6R-F11 (SEQ ID NO: 27) in its H chain. Note that the grey-filled cells in Table 7 indicate that the binding of FcγR to IgG is weak and cannot be properly analyzed by kinetic analysis, and therefore the binding activity of each variant is determined to be weak and cannot be properly analyzed by kinetic analysis, and therefore the binding activity of each variant is determined to be weak and cannot be properly analyzed by kinetic analysis, as described in Reference Example 2. [Formula 2] This value was calculated using the formula TIFF2025010300000011.tif10136. As shown in Table 7, all variants had improved affinity for FcγRIIb compared to IL6R-F11, with the improvement ranging from 1.9-fold to 5.0-fold. The ratio of the KD value of each variant for FcγRIIaR / KD value of each variant for FcγRIIb, and the ratio of the KD value of each variant for FcγRIIaH / KD value of each variant for FcγRIIb represent the binding activity to FcγRIIb relative to the binding activity to FcγRIIaR and FcγRIIaH. In other words, this value indicates the high binding selectivity of each variant for FcγRIIb, and the higher this value, the higher the binding selectivity to FcγRIIb. The ratio of the KD value for FcγRIIaR / KD value for FcγRIIb and the ratio of the KD value for FcγRIIaH / KD value for FcγRIIb of the parent polypeptide IL6R-F11 / IL6R-L were both 0.7, so all of the variants in Table 7 had improved binding selectivity to FcγRIIb compared to the parent polypeptide. The stronger KD value of the variant's binding activity to FcγRIIaR and FcγRIIaH / the stronger KD value of the parent polypeptide's binding activity to FcγRIIaR and FcγRIIaH being 1 or more means that the stronger binding activity of the variant to FcγRIIaR and FcγRIIaH is equivalent to or reduced to the stronger binding activity of the parent polypeptide to FcγRIIaR and FcγRIIaH. In the variants obtained this time, this value was 0.7 to 5.0, so it can be said that the stronger binding activity of the variants obtained this time to FcγRIIaR and FcγRIIaH was almost the same as or lower than that of the parent polypeptide. These results revealed that the variants obtained this time have enhanced binding activity to FcγRIIb while maintaining or reducing binding activity to FcγRIIa R and H types compared to the parent polypeptide, thereby improving selectivity for FcγRIIb. In addition, all variants had lower affinity for FcγRIa and FcγRIIIaV compared to IL6R-F11. [Table 7]

[0165] [Example 5] X-ray crystal structure analysis of the complex between Fc containing P238D and the extracellular region of FcγRIIb As shown in Example 3 above, it was revealed that even when modifications were introduced into an Fc containing P238D to improve the binding activity to FcγRIIb or to improve selectivity to FcγRIIb, the binding activity to FcγRIIb was weakened, and this was thought to be due to the change in the structure of the interaction interface between Fc and FcγRIIb caused by the introduction of P238D. Therefore, in order to investigate the cause of this phenomenon, the three-dimensional structure of a complex between an IgG1 Fc having a P238D mutation (hereinafter, Fc(P238D)) and the extracellular region of FcγRIIb was clarified by X-ray crystal structure analysis, and the three-dimensional structure and binding mode were compared with those of a complex between a natural IgG1 Fc (hereinafter, Fc(WT)) and the extracellular region of FcγRIIb. There have already been several reports on the three-dimensional structure of the complex between Fc and the extracellular region of FcγR, and the three-dimensional structures of the Fc(WT) / FcγRIIIb extracellular region complex (Nature, 2000, 400, 267-273; J.Biol.Chem. 2011, 276, 16469-16477), Fc(WT) / FcγRIIIa extracellular region complex (Proc.Natl.Acad.Sci.USA, 2011, 108, 12669-126674), and Fc(WT) / FcγRIIa extracellular region complex (J.Imunol. 2011, 187, 3208-3217) have been analyzed. Although the three-dimensional structure of the Fc(WT) / FcγRIIb extracellular region complex has not been analyzed to date, the three-dimensional structure of the complex with Fc(WT) is known between FcγRIIa and FcγRIIb, which share 93% of the amino acid sequence in their extracellular regions, demonstrating extremely high homology; therefore, the three-dimensional structure of the Fc(WT) / FcγRIIb extracellular region complex was predicted by modeling from the crystal structure of the Fc(WT) / FcγRIIa extracellular region complex.

[0166] The three-dimensional structure of the Fc(P238D) / FcγRIIb extracellular region complex was determined by X-ray crystal structure analysis at a resolution of 2.6 Å. The structure resulting from this analysis is shown in Figure 8. The FcγRIIb extracellular region is bound so as to be sandwiched between the two Fc CH2 domains, and is similar to the three-dimensional structures of the complexes between Fc(WT) and the extracellular regions of FcγRIIIa, FcγRIIIb, and FcγRIIa that have been analyzed so far. Next, for detailed comparison, the crystal structure of the Fc(P238D) / FcγRIIb extracellular region complex and the model structure of the Fc(WT) / FcγRIIb extracellular region complex were superimposed on the FcγRIIb extracellular region and Fc CH2 domain A by the least squares method based on the Cα atomic distance (Figure 9). At that time, the degree of superimposition between the Fc CH2 domain Bs was not good, revealing that there is a three-dimensional structural difference in this area. Furthermore, using the crystal structure of the Fc(P238D) / FcγRIIb extracellular region complex and the model structure of the Fc(WT) / FcγRIIb extracellular region complex, atom pairs with a distance of 3.7 Å or less between the FcγRIIb extracellular region and Fc CH2 domain B were extracted and compared, and the differences in the atomic interactions between FcγRIIb and Fc CH2 domain B were compared between Fc(WT) and Fc(P238D). As shown in Table 8, the atomic interactions between Fc CH2 domain B and FcγRIIb are not consistent between Fc(P238D) and Fc(WT).

[0167] [Table 8] TIFF2025010300000014.tif47154

[0168] Furthermore, the X-ray crystal structure of the Fc(P238D) / FcγRIIb extracellular domain complex and the model structure of the Fc(WT) / FcγRIIb extracellular domain complex were superimposed by the least squares method based on the Cα atomic distance between the Fc CH2 domain A and the Fc CH2 domain B alone, and the detailed structure around P238D was compared. The position of the amino acid residue at EU numbering 238, which is the position where the mutation is introduced, changes between Fc(P238D) and Fc(WT), and the loop structure in this vicinity continuing from the hinge region changes between Fc(P238D) and Fc(WT) (Figure 10). Originally, Pro at EU numbering 238 is located inside the protein in Fc(WT) and forms a hydrophobic core with the surrounding residues, but if it is changed to Asp, which has a charge and is very hydrophilic, remaining in the hydrophobic core becomes energetically unfavorable in terms of desolvation. In order to eliminate this energetic disadvantage, the amino acid residue at position 238 (EU numbering) in Fc(P238D) was changed to face the solvent, which is thought to have caused a change in the loop structure in the vicinity. Furthermore, since this loop continues from the hinge region bridged by SS bonds, it is presumed that the structural change was not limited to a local change but also affected the relative arrangement of Fc CH2 domain A and domain B, resulting in a difference in the atomic interaction between FcγRIIb and Fc CH2 domain B. For this reason, it is thought that the expected effect was not obtained even when an Fc already having the P238D modification was combined with a modification that improves the selectivity and binding activity for FcγRIIb in native IgG.

[0169] As a result of the structural change caused by the introduction of P238D, a hydrogen bond is observed between the main chain of the adjacent Gly at position 237 (EU numbering) and Tyr at position 160 of FcγRIIb in Fc CH2 domain A (Figure 11). The residue corresponding to this Tyr160 is Phe in FcγRIIa, and this hydrogen bond is not formed in the case of binding to FcγRIIa. Considering that the amino acid at position 160 is one of the few differences between FcγRIIa and FcγRIIb at the interaction interface with Fc, it is speculated that the presence or absence of this hydrogen bond, which is specific to FcγRIIb, leads to the improvement of the binding activity of Fc(P238D) to FcγRIIb and the decrease of the binding activity to FcγRIIa, which is the cause of the improvement of selectivity. In addition, electrostatic interaction is observed between Asp at position 270 (EU numbering) and Arg at position 131 of FcγRIIb in Fc CH2 domain B (Figure 12). In FcγRIIa H type, one of the FcγRIIa allotypes, the corresponding residue is His, and this electrostatic interaction cannot be formed. This explains why FcγRIIa H type has reduced binding activity to Fc(P238D) compared to FcγRIIa R type. Considerations based on the results of such X-ray crystal structure analysis revealed that the change in the loop structure near P238D and the associated relative change in domain arrangement form a new interaction not found in native IgG, leading to the selective binding profile of the P238D variant to FcγRIIb.

[0170] [Expression and purification of Fc(P238D)] Fc containing the P238D modification was prepared as follows. First, Cys at position 220 (EU numbering) of hIL6R-IgG1-v1 (SEQ ID NO: 21) was replaced with Ser, and the gene sequence Fc (P238D) was cloned by PCR from Glu at position 236 (EU numbering) to its C-terminus, and an expression vector was prepared, expressed, and purified according to the method described in Reference Example 1. Note that, in normal IgG1, Cys at position 220 (EU numbering) forms a disulfide bond with Cys in the L chain, but when preparing only Fc, in order to prevent co-expression of the L chain, it was replaced with Ser to avoid unnecessary disulfide bond formation.

[0171] [Expression and purification of the extracellular domain of FcγRIIb] Prepared according to the method of Reference Example 2.

[0172] [Purification of Fc(P238D) / FcγRIIb extracellular domain complex] To 2 mg of the FcγRIIb extracellular domain sample obtained for crystallization, 0.29 mg of Endo F1 (Protein Science 1996, 5, 2617-2622), which was expressed and purified in E. coli as a fusion protein with glutathione S-transferase, was added, and the mixture was left at room temperature for 3 days in 0.1 M Bis-Tris pH 6.5 buffer conditions to cleave the N-glycosylation leaving behind N-acetylglucosamine directly bound to Asn. Next, the FcγRIIb extracellular domain sample subjected to this glycan cleavage treatment was concentrated using a 5000 MWCO ultrafiltration membrane and purified by gel filtration column chromatography (Superdex200 10 / 300) equilibrated with 20 mM HEPS pH 7.5, 0.05 M NaCl. Furthermore, Fc(P238D) was added to the obtained glycosylated FcγRIIb extracellular region fraction so that the FcγRIIb extracellular region was in slight excess in terms of molar ratio, and the mixture was concentrated using a 10,000 MWCO ultrafiltration membrane. After that, the mixture was purified by gel filtration column chromatography (Superdex200 10 / 300) equilibrated with 20 mM HEPS pH 7.5, 0.05 M NaCl to obtain a sample of the Fc(P238D) / FcγRIIb extracellular region complex.

[0173] [Crystallization of Fc (P238D) / FcγRIIb extracellular domain complex] A sample of Fc(P238D) / FcγRIIb extracellular domain complex was concentrated to approximately 10mg / ml using a 10000MWCO ultrafiltration membrane, and crystallized by sitting drop vapor diffusion. A Hydra II Plus One (MATRIX) was used for crystallization, and a reservoir solution of 100mM Bis-Tris pH6.5, 17% PEG3350, 0.2M ammonium acetate, 2.7%(w / v) D-galactose was mixed with the reservoir solution:crystallization sample = 0.2μl:0.2μl to create a crystallization drop, which was then sealed and left to stand at 20℃, resulting in thin plate-like crystals.

[0174] [Measurement of X-ray diffraction data from crystals of the Fc(P238D) / FcγRIIb extracellular domain complex] A single crystal of the Fc(P238D) / FcγRIIb extracellular domain complex was immersed in a solution of 100 mM Bis-Tris pH 6.5, 20% PEG3350, ammonium acetate, 2.7% (w / v) D-galactose, and 22.5% (v / v) Ethylene glycol, then scooped up with a pin with a small nylon loop and frozen in liquid nitrogen. X-ray diffraction data was measured at the Photon Factory BL-1A, a synchrotron radiation facility of the High Energy Accelerator Research Organization. During the measurement, the crystal was kept frozen by placing it in a nitrogen gas flow at -178°C, and a total of 225 X-ray diffraction images were collected by rotating the crystal by 0.8° at a time using a CCD detector Quantum 270 (ADSC) installed on the beamline. The programs Xia2 (CCP4 Software Suite), XDS Package (Walfgang Kabsch), and Scala (CCP4 Software Suite) were used to determine the lattice constants from the obtained diffraction images, index the diffraction spots, and process the diffraction data, and finally, diffraction intensity data up to a resolution of 2.46 Å were obtained. The crystal belongs to the space group P21, with the lattice constants a = 48.85 Å, b = 76.01 Å, c = 115.09 Å, α = 90°, β = 100.70°, and γ = 90°.

[0175] [X-ray crystal structure analysis of Fc(P238D) / FcγRIIb extracellular domain complex] The crystal structure of the Fc(P238D) / FcγRIIb extracellular domain complex was determined by molecular replacement using the program Phaser (CCP4 Software Suite). Based on the size of the obtained crystal lattice and the molecular weight of the Fc(P238D) / FcγRIIb extracellular domain complex, the number of complexes in the asymmetric unit was predicted to be one. From the structural coordinates of the crystal structure of the Fc(WT) / FcγRIIIa extracellular domain complex (PDB code:3SGJ), the amino acid residues at positions 239-340 of the A chain and 239-340 of the B chain were taken as separate coordinates and used as models for searching the Fc CH2 domain. Similarly, from the structural coordinates of PDB code:3SGJ, the amino acid residues at positions 341-444 of the A chain and 341-443 of the B chain were taken as one coordinate and used as a model for searching the Fc CH3 domain. Finally, the amino acid residues 6-178 of the A chain were extracted from the structural coordinates of the crystal structure of the FcγRIIb extracellular domain (PDB code: 2FCB) and used as a search model for the FcγRIIb extracellular domain. The orientation and position of each search model in the crystal lattice were determined in the order of the Fc CH3 domain, the FcγRIIb extracellular domain, and the Fc CH2 domain using rotation and translation functions to obtain an initial model of the Fc(P238D) / FcγRIIb extracellular domain complex crystal structure. Rigid-body refinement was performed on the initial model obtained by moving the two Fc CH2 domains, the two Fc CH3 domains, and the FcγRIIb extracellular domain. At this point, the crystallographic reliability factor R value for the diffraction intensity data from 25-3.0 Å was 40.4% and the free R value was 41.9%. Furthermore, the structure was refined using the program Refmac5 (CCP4 Software Suite), and the model was modified using the program Coot (Paul Emsley) while looking at the electron density map with coefficients 2Fo-Fc and Fo-Fc calculated based on the experimentally determined structure factor Fo, the structure factor Fc calculated from the model, and the phase calculated from the model.The model was refined by repeating these steps.Finally, water molecules were incorporated into the model based on the electron density map with coefficients of 2Fo-Fc and Fo-Fc, and refinement was performed. Finally, using 24,291 diffraction intensity data with a resolution of 25-2.6 Å, the crystallographic reliability factor R value was 23.7% and the free R value was 27.6% for the model containing 4,846 non-hydrogen atoms.

[0176] [Modeling the structure of the Fc(WT) / FcγRIIb extracellular domain complex] Based on the structure coordinates of the crystal structure of the Fc(WT) / FcγRIIa extracellular domain complex (PDB code: 3RY6), the Build Mutants function of the program Discovery Studio 3.1 (Accelrys) was used to introduce mutations into FcγRIIa in the structure coordinates so that it matches the amino acid sequence of FcγRIIb. At that time, the optimization level was set to High and the cut radius was set to 4.5, and five models were generated. The one with the best energy score was adopted as the model structure of the Fc(WT) / FcγRIIb extracellular domain complex.

[0177] [Example 6] Analysis of FcγR binding of Fc variants in which modification sites were determined based on crystal structure Based on the results of the X-ray crystal structure analysis of the complex between Fc (P238D) and the extracellular region of FcγRIIb obtained in Example 5, comprehensive modifications were introduced into the modified Fc in which Pro at position 238 (EU numbering) was replaced with Asp at sites predicted to affect the interaction with FcγRIIb (residues at positions 233, 240, 241, 263, 265, 266, 267, 268, 271, 273, 295, 296, 298, 300, 323, 325, 326, 327, 328, 330, 332, and 334 (EU numbering)), and combination modified forms that further enhance binding to FcγRIIb were examined.

[0178] IL6R-B3 (SEQ ID NO: 40) was prepared by introducing an alteration into IL6R-G1d (SEQ ID NO: 20) prepared in Example 2, in which Lys at position 439 (EU numbering) was replaced with Glu. Next, IL6R-BF648 (SEQ ID NO: 41) was prepared by introducing an alteration into IL6R-B3, in which Pro at position 238 (EU numbering) was replaced with Asp. IL6R-L (SEQ ID NO: 22) was used in common as the antibody L chain. These alterations were expressed and purified according to the method of Reference Example 1, and binding to each FcγR (FcγRIa, FcγRIIa H type, FcγRIIa R type, FcγRIIb, FcγRIIIa V type) was comprehensively evaluated according to the method of Reference Example 2.

[0179] A graph was created using the following method to plot the results of the analysis of the interaction with each FcγR. The value of the binding amount of each variant to each FcγR was divided by the value of the binding amount of the control antibody (IL6R-BF648 / IL6R-L in which Pro at position 238 (EU numbering) was replaced with Asp) to each FcγR, and the result was multiplied by 100 to obtain the relative binding activity value of each variant to each FcγR. The horizontal axis shows the relative binding activity value of each variant to FcγRIIb, and the vertical axis shows the relative binding activity value of each variant to FcγRIIa R type (FIG. 13). As a result, as shown in Figure 13, 24 of all the modifications were found to have the effect of maintaining or enhancing the binding to FcγRIIb compared to the antibody before the modification was introduced. The binding of these modifications to FcγR for each of them is shown in Table 9. In the table, the sequence number indicates the sequence number of the H chain of the evaluated modified antibody, and the modification indicates the modification introduced into IL6R-B3 (SEQ ID NO: 40). However, IL6R-G1d / IL6R-L used as the template for producing IL6R-B3 is indicated with *.

[0180] [Table 9] The KD values ​​of the variants shown in Table 9 against FcγRIa, FcγRIIaR, FcγRIIaH, FcγRIIb, and FcγRIIIa V type were measured by the method of Reference Example 2, and the results are summarized in Table 10. In the table, the sequence number indicates the sequence number of the H chain of the variant evaluated, and the modification indicates the modification introduced into IL6R-B3 (SEQ ID NO: 40). However, IL6R-G1d / IL6R-L used as the template for producing IL6R-B3 is indicated with *. In addition, KD(IIaR) / KD(IIb) and KD(IIaH) / KD(IIb) in the table respectively indicate the value obtained by dividing the KD value of each variant against FcγRIIaR by the KD value of each variant against FcγRIIb, and the value obtained by dividing the KD value of each variant against FcγRIIaH by the KD value of each variant against FcγRIIb. KD(IIb) of parent polypeptide / KD(IIb) of modified polypeptide refers to the value obtained by dividing the KD value of the parent polypeptide for FcγRIIb by the KD value of each variant for FcγRIIb. In addition, the stronger KD value of each variant's binding activity to FcγRIIaR and FcγRIIaH / the stronger KD value of the parent polypeptide's binding activity to FcγRIIaR and FcγRIIaH is shown in Table 10. The parent polypeptide here refers to a variant having IL6R-B3 (SEQ ID NO: 40) in its H chain. Note that the grey-filled cells in Table 10 indicate that the binding of FcγR to IgG is weak and cannot be properly analyzed by kinetic analysis, and therefore the binding activity of each variant is determined to be weak and cannot be properly analyzed by kinetic analysis, and therefore the binding activity of each variant is determined to be weak and cannot be properly analyzed by kinetic analysis, as described in Reference Example 2. [Formula 2] This value was calculated using the formula TIFF2025010300000016.tif10136. As shown in Table 10, all variants had improved affinity for FcγRIIb compared to IL6R-B3, with the improvement ranging from 2.1-fold to 9.7-fold. The ratio of the KD value of each variant for FcγRIIaR / KD value of each variant for FcγRIIb, and the ratio of the KD value of each variant for FcγRIIaH / KD value of each variant for FcγRIIb represent the binding activity to FcγRIIb relative to the binding activity to FcγRIIaR and FcγRIIaH. In other words, this value indicates the high binding selectivity of each variant for FcγRIIb, and the higher this value, the higher the binding selectivity to FcγRIIb. The ratios of the KD value for FcγRIIaR / KD value for FcγRIIb and the KD value for FcγRIIaH / KD value for FcγRIIb of the parent polypeptide IL6R-B3 / IL6R-L were 0.3 and 0.2, respectively, and therefore all variants in Table 10 had improved binding selectivity to FcγRIIb compared to the parent polypeptide. The stronger KD value of the variant's binding activity to FcγRIIaR and FcγRIIaH / the stronger KD value of the parent polypeptide's binding activity to FcγRIIaR and FcγRIIaH being 1 or more means that the stronger binding activity of the variant to FcγRIIaR and FcγRIIaH is equivalent to or reduced to the stronger binding activity of the parent polypeptide to FcγRIIaR and FcγRIIaH. In the variants obtained this time, this value was 4.6 to 34.0, so it can be said that the stronger binding activity of the variants obtained this time to FcγRIIaR and FcγRIIaH was reduced compared to that of the parent polypeptide. These results revealed that the variants obtained this time have enhanced binding activity to FcγRIIb while maintaining or reducing binding activity to FcγRIIa R and H types compared to the parent polypeptide, thereby improving selectivity for FcγRIIb. In addition, all variants had reduced affinity for FcγRIa and FcγRIIIaV compared to IL6R-B3. [Table 10]

[0181] Among the obtained combination variants, the factors of the effect of promising ones were considered from the crystal structure. Figure 14 shows the crystal structure of the Fc (P238D) / FcγRIIb extracellular domain complex. In this figure, the H chain located on the left side is Fc Chain A, and the H chain located on the right side is Fc Chain B. Here, it can be seen that the site at EU numbering 233 in Fc Chain A is located near Lys at EU numbering 113 of FcγRIIb. However, in this crystal structure, the electron density of the side chain of E233 is not well observed, and it is in a state of considerable mobility. Therefore, the modification of replacing Glu at EU numbering 233 with Asp shortens the side chain by one carbon, thereby reducing the degree of freedom of the side chain, and as a result, it is presumed that the entropy loss during interaction formation with Lys at EU numbering 113 of FcγRIIb is reduced, which contributes to the improvement of the binding free energy.

[0182] FIG. 15 also shows the environment near position 330 (EU numbering) in the structure of the Fc (P238D) / FcγRIIb extracellular region complex. This figure shows that the area around position 330 (EU numbering) of Fc Chain A of Fc (P238D) is a hydrophilic environment composed of Ser at position 85 (EU numbering), Glu at position 86, and Lys at position 163 (EU numbering) of FcγRIIb. Therefore, it is speculated that the modification of substituting Ala at position 330 (EU numbering) with Lys or Arg contributes to strengthening the interaction with Ser at position 85 (EU numbering) or Glu at position 86 (EU numbering) of FcγRIIb.

[0183] FIG. 16 shows the structure of Pro at position 271 (EU numbering), superimposed on the crystal structures of the Fc(P238D) / FcγRIIb extracellular region complex and the Fc(WT) / FcγRIIIa extracellular region complex by the least squares method based on the Cα atomic distance with respect to Fc Chain B. These two structures match well, but have different three-dimensional structures at Pro at position 271 (EU numbering). In addition, considering that the electron density around this position is weak in the crystal structure of the Fc(P238D) / FcγRIIb extracellular region complex, it was suggested that the fact that Pro is at position 271 (EU numbering) places a large strain on the structure in Fc(P238D) / FcγRIIb, which may prevent this loop structure from taking an optimal structure. Therefore, it is presumed that the modification of replacing Pro at position 271 (EU numbering) with Gly confers flexibility to this loop structure and reduces the energetic hindrance to the optimal structure for interacting with FcγRIIb, thereby contributing to enhanced binding.

[0184] [Example 7] Verification of the combined effect of alterations that enhance FcγRIIb binding when combined with P238D Among the alterations obtained in Examples 4 and 6, alterations that showed the effect of enhancing binding to FcγRIIb or the effect of maintaining binding to FcγRIIb and suppressing binding to other FcγRs were combined, and the effects were examined. As in the method of Example 6, particularly excellent alterations were selected from Tables 6 and 9 and introduced in combination into the antibody H chain IL6R-BF648. IL6R-L was used for all the antibody L chains, and the antibodies were expressed and purified according to the method of Reference Example 1, and binding to each FcγR (FcγRIa, FcγRIIa H type, FcγRIIa R type, FcγRIIb, FcγRIIIa V type) was comprehensively evaluated by the method of Reference Example 2. The relative binding activity of each variant to each FcγR was calculated from the analysis results of the interaction with each FcγR according to the following method. The value of the binding amount of each variant to each FcγR was divided by the value of the binding amount of the control antibody before the introduction of the modification (IL6R-BF648 / IL6R-L in which Pro at position 238 (EU numbering) was replaced with Asp) to each FcγR, and the value was multiplied by 100 to obtain the value of the relative binding activity of each variant to each FcγR. The horizontal axis shows the relative binding activity of each variant to FcγRIIb, and the vertical axis shows the relative binding activity of each variant to FcγRIIa R type (Table 11). In the table, the sequence numbers indicate the sequence numbers of the H chains of the variants evaluated, and the modifications indicate the modifications introduced into IL6R-B3 (SEQ ID NO: 40). However, IL6R-G1d / IL6R-L, which was used as a template to prepare IL6R-B3, is indicated with an *.

[0185] [Table 11]

[0186] The KD values ​​of the variants shown in Table 11 against FcγRIa, FcγRIIaR, FcγRIIaH, FcγRIIb, and FcγRIIIa V type were measured by the method of Reference Example 2, and the results are summarized in Table 12. In the table, the sequence number indicates the sequence number of the H chain of the variant evaluated, and the modification indicates the modification introduced into IL6R-B3 (SEQ ID NO: 40). However, IL6R-G1d / IL6R-L used as the template for producing IL6R-B3 is indicated with *. In addition, KD(IIaR) / KD(IIb) and KD(IIaH) / KD(IIb) in the table respectively indicate the value obtained by dividing the KD value of each variant against FcγRIIaR by the KD value of each variant against FcγRIIb, and the value obtained by dividing the KD value of each variant against FcγRIIaH by the KD value of each variant against FcγRIIb. KD(IIb) of parent polypeptide / KD(IIb) of modified polypeptide refers to the value obtained by dividing the KD value of the parent polypeptide for FcγRIIb by the KD value of each variant for FcγRIIb. In addition, the stronger KD value of each variant's binding activity to FcγRIIaR and FcγRIIaH / the stronger KD value of the parent polypeptide's binding activity to FcγRIIaR and FcγRIIaH is shown in Table 12. The parent polypeptide here refers to a variant having IL6R-B3 (SEQ ID NO: 40) in its H chain. Note that the grey-filled cells in Table 12 are those in which the binding of FcγR to IgG is weak and it was determined that the binding cannot be properly analyzed by kinetic analysis, and therefore the binding activity of each variant is determined by the binding activity of each variant to FcγRIIaR and FcγRIIaH, as described in Reference Example 2. [Formula 2] This value was calculated using the formula in TIFF2025010300000019.tif10136. As shown in Table 12, all variants had improved affinity for FcγRIIb compared to IL6R-B3, with the improvement ranging from 3.0-fold to 99.0-fold. The ratio of the KD value of each variant for FcγRIIaR / KD value of each variant for FcγRIIb, and the ratio of the KD value of each variant for FcγRIIaH / KD value of each variant for FcγRIIb represent the binding activity to FcγRIIb relative to the binding activity to FcγRIIaR and FcγRIIaH. In other words, this value indicates the high binding selectivity of each variant for FcγRIIb, and the higher this value, the higher the binding selectivity to FcγRIIb. The ratios of the KD value for FcγRIIaR / KD value for FcγRIIb and the KD value for FcγRIIaH / KD value for FcγRIIb of the parent polypeptide IL6R-B3 / IL6R-L were 0.3 and 0.2, respectively, and therefore all variants in Table 12 had improved binding selectivity to FcγRIIb compared to the parent polypeptide. The stronger KD value of the variant's binding activity to FcγRIIaR and FcγRIIaH / the stronger KD value of the parent polypeptide's binding activity to FcγRIIaR and FcγRIIaH being 1 or more means that the stronger binding activity of the variant to FcγRIIaR and FcγRIIaH is equivalent to or lower than the stronger binding activity of the parent polypeptide to FcγRIIaR and FcγRIIaH. In the variants obtained this time, this value was 0.7 to 29.9, so it can be said that the stronger binding activity of the variants obtained this time to FcγRIIaR and FcγRIIaH was almost the same as or lower than that of the parent polypeptide. These results revealed that the variants obtained this time have enhanced binding activity to FcγRIIb while maintaining or reducing binding activity to FcγRIIa R and H types compared to the parent polypeptide, thereby improving selectivity for FcγRIIb. In addition, all variants had lower affinity for FcγRIa and FcγRIIIaV compared to IL6R-B3.

[0187] [Table 12] TIFF2025010300000021.tif234102

[0188] [Reference Example 1] Construction of an expression vector for an antibody and expression and purification of the antibody The synthesis of the full-length gene encoding the base sequence of the H chain and L chain of the variable region of the antibody was performed by a method known to those skilled in the art using Assemble PCR or the like. The introduction of amino acid substitution was performed by a method known to those skilled in the art using PCR or the like. The obtained plasmid fragment was inserted into an animal cell expression vector to prepare an H chain expression vector and an L chain expression vector. The base sequence of the obtained expression vector was determined by a method known to those skilled in the art. The prepared plasmid was transiently introduced into human fetal kidney cancer cell-derived HEK293H strain (Invitrogen) or FreeStyle293 cells (Invitrogen) to express the antibody. The obtained culture supernatant was collected and then passed through a 0.22 μm filter MILLEX(R)-GV (Millipore) or a 0.45 μm filter MILLEX(R)-GV (Millipore) to obtain the culture supernatant. Antibodies were purified from the obtained culture supernatant using rProtein A Sepharose Fast Flow (GE Healthcare) or Protein G Sepharose 4 Fast Flow (GE Healthcare) by methods known to those skilled in the art. The purified antibody concentration was calculated from the absorbance at 280 nm measured using a spectrophotometer and the antibody concentration was calculated from the obtained value using the extinction coefficient calculated by a method such as PACE (Protein Science 1995; 4: 2411-2423).

[0189] [Reference Example 2] Method for preparing FcγR and method for analyzing the interaction between modified antibodies and FcγR The extracellular domain of FcγR was prepared by the following method. First, the gene for the extracellular domain of FcγR was synthesized by a method known to those skilled in the art. At that time, the sequence of each FcγR was prepared based on the information registered in NCBI. Specifically, FcγRI was prepared based on the sequence of NCBI accession number NM_000566.3, FcγRIIa was prepared based on the sequence of NCBI accession number NM_001136219.1, FcγRIIb was prepared based on the sequence of NCBI accession number NM_004001.3, FcγRIIIa was prepared based on the sequence of NCBI accession number NM_001127593.1, and FcγRIIIb was prepared based on the sequence of NCBI accession number NM_000570.3, and a His tag was added to the C-terminus. Polymorphisms are known for FcγRIIa, FcγRIIIa, and FcγRIIIb, and the polymorphic sites were prepared with reference to J. Exp. Med., 1990, 172: 19-25 for FcγRIIa, J. Clin. Invest., 1997, 100 (5): 1059-1070 for FcγRIIIa, and J. Clin. Invest., 1989, 84, 1688-1691 for FcγRIIIb.

[0190] The obtained gene fragment was inserted into an animal cell expression vector to prepare an expression vector. The prepared expression vector was transiently introduced into human fetal renal carcinoma cell-derived FreeStyle293 cells (Invitrogen) to express the target protein. For FcγRIIb used for crystal structure analysis, the target protein was expressed in the presence of Kifunesine at a final concentration of 10 μg / mL, so that the sugar chain added to FcγRIIb was of the high mannose type. After culturing and collecting the resulting culture supernatant, the culture supernatant was obtained through a 0.22 μm filter. In principle, the culture supernatant was purified in the following four steps. The first step was cation exchange column chromatography (SP Sepharose FF), the second step was affinity column chromatography for the His tag (HisTrap HP), the third step was gel filtration column chromatography (Superdex200), and the fourth step was sterile filtration. However, for FcγRI, anion exchange column chromatography using Q Sepharose FF was performed in the first step. The absorbance of the purified protein was measured at 280 nm using a spectrophotometer, and the concentration of the purified protein was calculated from the obtained value using the extinction coefficient calculated by a method such as PACE (Protein Science 1995; 4: 2411-2423).

[0191] Using Biacore T100 (GE Healthcare), Biacore T200 (GE Healthcare), Biacore A100, and Biacore 4000, the interaction analysis between each modified antibody and the Fcγ receptor prepared above was performed. HBS-EP+ (GE Healthcare) was used as the running buffer, and the measurement temperature was 25°C. A chip on which antigen peptide, Protein A (Thermo Scientific), Protein A / G (Thermo Scientific), Protein L (ACTIGEN or BioVision) was immobilized by the amine coupling method on Series S Sensor Chip CM5 (GE Healthcare) or Series S sensor Chip CM4 (GE Healthcare), or a chip on which an antigen peptide that had been biotinylated in advance was interacted with and immobilized on Series S Sensor Chip SA (certified) (GE Healthcare) was used.

[0192] The target antibodies were captured on these sensor chips, and the Fcγ receptor diluted in running buffer was allowed to interact with them, and the amount of binding to the antibody was measured and compared between the antibodies. However, since the amount of Fcγ receptor binding depends on the amount of captured antibody, the amount of Fcγ receptor binding was divided by the amount of each antibody captured to obtain a corrected value for comparison. In addition, the antibody captured on the sensor chip was washed by reacting with 10 mM glycine-HCl, pH 1.5, and the sensor chip was regenerated and used repeatedly.

[0193] Kinetic analysis to calculate the KD value of each modified antibody against FcγR was performed according to the following method: First, the antibody of interest was captured on the sensor chip described above, and allowed to interact with Fcγ receptor diluted with running buffer. The measurement results of the obtained sensorgram were globally fitted with a 1:1 Langmuir binding model using Biacore Evaluation Software to calculate the binding rate constant ka (L / mol / s) and dissociation rate constant kd (1 / s), and the dissociation constant KD (mol / L) was calculated from these values.

[0194] Furthermore, when it was determined that the interaction between each modified antibody and FcγR was too weak to be correctly analyzed by the above-mentioned kinetic analysis, the KD for that interaction was calculated using the following 1:1 binding model equation described in the Biacore T100 Software Handbook BR1006-48 Edition AE. The behavior of molecules interacting in a 1:1 binding model on Biacore can be expressed by the following equation 1. [Formula 1] TIFF2025010300000022.tif13144R eq :a plot of steady state binding levels against analyte concentration C: concentration RI:bulk refractive index contribution in the sample R max :analyte binding capacity of the surface By rearranging this equation, KD can be expressed as the following Equation 2. [Formula 2] TIFF2025010300000023.tif10136This formula is R maxIt is possible to calculate KD by substituting the values ​​of R, RI, and C. The values ​​of RI and C can be calculated from the sensorgram of the measurement results and the measurement conditions. max The calculation of R was performed according to the following method. For antibodies with sufficiently strong interactions that were evaluated simultaneously during the measurement, the R obtained by global fitting with the 1:1 Langmuir binding model was used. max The value of is divided by the amount of the antibody captured on the sensor chip to be compared, and multiplied by the amount of the modified antibody to be evaluated to obtain R max It was decided. [Industrial Applicability]

[0195] The present invention provides a polypeptide comprising an Fc region in which the binding activity to both H-type and R-type FcγRIIa gene polymorphisms is maintained or decreased, and the binding activity to FcγRIIb is enhanced, as compared with the parent polypeptide. By using the polypeptide in which the binding selectivity to FcγRIIb is enhanced for both FcγRIIa gene polymorphisms (H-type, R-type), it becomes possible to transmit an inhibitory signal of an inflammatory immune response via phosphorylation of ITIM of FcγRIIb to patients with both R-type and H-type gene polymorphisms. In addition, by imparting the property of selectively binding to FcγRIIb to the Fc of an antibody, it is possible to suppress the production of anti-antibodies through the immunosuppressive action mediated by FcγRIIb.

Claims

[Claim 1] The invention described herein.

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