Anti-myostatin antibody, polypeptide containing a mutated Fc region, and method of use

Abstract

An object of the present invention is to provide anti-myostatin antibodies, polypeptides comprising mutated Fc regions, and methods of using the same. [Solution] The present disclosure provides anti-myostatin antibodies and methods for making and using them. Nucleic acids encoding the anti-myostatin antibodies and host cells containing the nucleic acids are also provided. The present disclosure also provides polypeptides comprising mutant Fc regions and methods for making and using them. Nucleic acids encoding the polypeptides and host cells containing the nucleic acids are also provided.

Description

[Technical Field]

[0001] The present invention relates to anti-myostatin antibodies and methods for using them. The present invention also relates to polypeptides comprising a mutated Fc region and methods for using them. [Background technology]

[0002] Myostatin, also known as growth and differentiation factor 8 (GDF8), is a secreted protein and a member of the transforming growth factor β (TGF-β) superfamily of proteins. Members of this superfamily possess growth-regulating and morphogenic properties (see, e.g., Non-Patent Documents 1, 2, and 1). Myostatin is primarily expressed in developing and adult skeletal muscle and functions as a negative regulator of muscle growth. Systemic overexpression of myostatin in adult mice causes muscle wasting (see, e.g., Non-Patent Document 3), while myostatin knockout mice are characterized by skeletal muscle hypertrophy and hyperplasia, resulting in 2-3 times more muscle mass than wild-type littermates (see, e.g., Non-Patent Document 4).

[0003] Like other members of the TGF-β family, myostatin is synthesized as a large precursor protein containing an N-terminal propeptide domain and a C-terminal domain considered to be the active molecule (see, e.g., Non-Patent Document 5; Patent Document 2). The two molecules of the myostatin precursor are covalently linked via a single disulfide bond present in the C-terminal growth factor domain. Active mature myostatin (a disulfide-bonded homodimer consisting of the C-terminal growth factor domain) is released from the myostatin precursor through several steps of proteolytic processing. In the first step of the myostatin activation pathway, the peptide bond Arg266-Asp267 between the N-terminal propeptide domain and the C-terminal growth factor domain is cleaved by a furin-type precursor protein convertase in both chains of the homodimer precursor. However, the resulting three peptides (two propeptides and one mature myostatin (i.e., a disulfide-bonded homodimer consisting of a growth factor domain)) remain associated, forming a non-covalent, inactive complex called "latent myostatin." Mature myostatin can then be released from latent myostatin via the degradation of the propeptides. Members of the bone morphogenetic protein 1 (BMP1) family of metalloproteinases cleave one peptide bond Arg98-Asp99 within the propeptide simultaneously with the release of the active mature myostatin homodimer (see, for example, Non-Patent Document 6). Furthermore, latent myostatin can also be activated in vitro by dissociating the complex with either acid treatment or heat treatment (see, for example, Non-Patent Document 7).

[0004] Myostatin exerts its effects via the transmembrane serine / threonine kinase heterotetrameric receptor family; its activation enhances receptor transphosphorylation, thereby stimulating serine / threonine kinase activity. The myostatin pathway involves active myostatin dimers binding to the activin IIB receptor (ActRIIB) with high affinity, which has been shown to then recruit and activate the transphosphorylation of lower-affinity receptors such as activin-like kinase 4 (ALK4) or activin-like kinase 5 (ALK5). Proteins Smad 2 and Smad 3 are subsequently activated and form a complex with Smad 4, which has also been shown to translocate to the nucleus for transcriptional activation of target genes. In mice, expression of dominant-negative morphology ActRIIB mimics myostatin gene knockout, demonstrating that ActRIIB can mediate the effects of myostatin in vivo (see, for example, Non-Patent Document 8).

[0005] Many disorders or conditions are associated with muscle wasting (i.e., loss or dysfunction of muscle tissue), such as muscular dystrophy (MD; including Duchenne muscular dystrophy), amyotrophic lateral sclerosis (ALS), muscle atrophy, organ atrophy, frailty, congestive obstructive pulmonary disease (COPD), sarcopenia, and cachexia resulting from cancer or other diseases, as well as renal disease, heart failure or heart disease, and hepatic disease. Patients would benefit from increased muscle mass and / or muscle strength, but the treatments available for these disorders are currently limited. Therefore, due to its role as a negative regulator of skeletal muscle growth, myostatin is a desirable target for therapeutic or prophylactic interventions for such disorders or conditions, or for monitoring the progression of such disorders or conditions. In particular, agents that inhibit the activity of myostatin may be therapeutically beneficial.

[0006] Inhibition of myostatin expression results in both muscle hypertrophy and hyperplasia (Non-Patent Literature 4). Myostatin negatively regulates muscle regeneration after injury, and myostatin deficiency in myostatin-null mice accelerates muscle regeneration (see, for example, Non-Patent Literature 9). Anti-myostatin (GDF8) antibodies described in, for example, Patent Literature 3, 4, 5, 6, and 7, and Patent Literature 8, 9, and 10 have been shown to bind to myostatin and inhibit myostatin activity (including myostatin activity associated with the negative regulation of skeletal muscle mass) in vitro and in vivo. Myostatin-neutralizing antibodies increase body weight, skeletal muscle mass, and muscle size and strength in skeletal muscle in wild-type mice (see, for example, Non-Patent Literature 10) and mdx mice, a model of muscular dystrophy (see, for example, Non-Patent Literature 11; Non-Patent Literature 12). However, all of these prior art antibodies are specific to mature myostatin but not to latent myostatin, and the strategies described for inhibiting myostatin activity utilize antibodies that can bind to and neutralize mature myostatin.

[0007] Antibodies are attracting attention as drugs because they are very stable in the blood and have few side effects (see, for example, Non-Patent Documents 13 and 14). Almost all therapeutic antibodies currently on the market are antibodies of the human IgG1 subclass. One of the known functions of IgG class antibodies is antibody-dependent cell-mediated cytotoxicity (hereinafter referred to as ADCC activity) (see, for example, Non-Patent Document 15). For an antibody to exhibit ADCC activity, the antibody Fc region must bind to the Fcγ receptor (hereinafter referred to as FcγR), which is an antibody-binding receptor present on the surface of effector cells such as killer cells, natural killer cells, and activated macrophages.

[0008] In humans, the FcγRIa(CD64A), FcγRIIa(CD32A), FcγRIIb(CD32B), FcγRIIIa(CD16A), and FcγRIIIb(CD16B) isoforms have been reported as the FcγR protein family, and their allotypes have also been reported (see, for example, Non-Patent Document 16). FcγRIa, FcγRIIa, and FcγRIIIa are called activating FcγRs because they have immunologically active functions, while FcγRIIb is called inhibitory FcγRs because it has immunosuppressive functions (see, for example, Non-Patent Document 17).

[0009] In the binding between the Fc region and FcγR, it has been shown that several amino acid residues in the antibody hinge region and CH2 domain, as well as the glycan attached to Asn at position 297 (EU numbering) attached to the CH2 domain, are important (see, for example, Non-Patent Documents 18, 19, and 20). Various mutants with FcγR binding properties, mainly antibodies in which mutations have been introduced at these sites, have been studied, and Fc region mutants with higher binding activity to activated FcγR have been obtained (see, for example, Patent Documents 11, 12, 13, and 14).

[0010] When activated FcγR is cross-linked with an immune complex, it phosphorylates the immune receptor tyrosine activation motif (ITAM) contained in the intracellular domain or the FcR common gamma chain (interaction partner), activating the signaling molecule SYK and initiating an activation signal cascade, thereby inducing an inflammatory immune response (see, for example, Non-Patent Document 21).

[0011] FcγRIIb is the only FcγR expressed on B cells (see, for example, Non-Patent Document 22). Interaction between the antibody Fc region and FcγRIIb has been reported to suppress the initial immune response of B cells (see, for example, Non-Patent Document 23). Furthermore, it has been reported that when FcγRIIb on B cells and the B cell receptor (BCR) are cross-linked via immune complexes in the blood, B cell activation and antibody production by B cells are suppressed (see, for example, Non-Patent Document 24). In this immunosuppressive signaling mediated by BCR and FcγRIIb, the immune receptor tyrosine inhibitory motif (ITIM) contained in the intracellular domain of FcγRIIb is essential (see, for example, Non-Patent Documents 25 and 26). During signaling, ITIM is phosphorylated, recruiting SH2-containing inositol polyphosphate 5-phosphatase (SHIP), inhibiting the transmission of other activated FcγR signaling cascades, and suppressing the inflammatory immune response (see, for example, Non-Patent Document 27). Furthermore, it has been reported that only the aggregation of FcγRIIb transiently suppresses calcium influx caused by BCR crosslinking and transiently inhibits BCR-dependent B cell proliferation without inducing apoptosis of IgM-producing B cells (see, for example, Non-Patent Document 28).

[0012] FcγRIIb is also expressed in dendritic cells, macrophages, activated neutrophils, mast cells, and basophils. FcγRIIb inhibits the function of activated FcγR, such as phagocytosis, and the release of inflammatory cytokines in these cells, thereby suppressing the inflammatory immune response (see, for example, Non-Patent Document 17).

[0013] The importance of the immunosuppressive function of FcγRIIb has been revealed through studies using FcγRIIb knockout mice. Reports indicate that FcγRIIb knockout mice exhibit improperly regulated humoral immunity (see, for example, Non-Patent Document 29), increased susceptibility to collagen-induced arthritis (CIA) (see, for example, Non-Patent Document 30), lupus-like symptoms, and Goodpasture syndrome-like symptoms (see, for example, Non-Patent Document 31).

[0014] Furthermore, dysregulation of FcγRIIb has been reported to be associated with human autoimmune diseases. For example, a relationship has been reported between genetic polymorphisms in the transmembrane and promoter regions of FcγRIIb and the incidence of systemic lupus erythematosus (SLE) (see, for example, Non-Patent Documents 32, 33, 34, 35, and 36), as well as decreased FcγRIIb expression on the surface of B cells in SLE patients (see, for example, Non-Patent Documents 37 and 38).

[0015] Based on mouse models and such clinical findings, FcγRIIb is thought to play a role in controlling autoimmune and inflammatory diseases, particularly through its interaction with B cells, making it a promising target molecule for controlling autoimmune and inflammatory diseases.

[0016] IgG1, which is primarily used as a commercially available therapeutic antibody, is known to bind strongly not only to FcγRIIb but also to activated FcγR (see, for example, Non-Patent Document 39). By using an Fc region in which FcγRIIb binding is enhanced or FcγRIIb binding selectivity is improved compared to activated FcγR, it may be possible to develop therapeutic antibodies with higher immunosuppressive properties compared to the properties of IgG1. For example, it has been suggested that the use of an antibody having a variable region that binds to BCR and an Fc region with enhanced FcγRIIb binding may inhibit B cell activation (see, for example, Non-Patent Document 40). Crosslinking FcγRIIb on B cells with IgE bound to the B cell receptor suppresses the differentiation of B cells into plasma cells, which in turn leads to suppression of IgE production; and it has also been reported that in mice transplanted with human PBMCs, human IgG and IgM concentrations are maintained while human IgE concentrations decrease (see, for example, Non-Patent Document 41). It has been reported that when IgE, along with FcγRIIb and CD79b (a component of the B cell receptor complex), are crosslinked by an antibody, B cell proliferation is suppressed in vitro, and arthritis symptoms in a collagen arthritis model are alleviated (see, for example, Non-Patent Document 42).

[0017] In addition to B cells, it has been reported that cross-linking of FcεRI and FcγRIIb on mast cells using a molecule in which the Fc portion of IgG with enhanced FcγRIIb binding is fused to the Fc portion of IgE that binds to the IgE receptor FcεRI induces phosphorylation of FcγRIIb, thereby suppressing FcεRI-dependent calcium influx. This suggests that enhancing FcγRIIb binding can inhibit degranulation mediated by FcγRIIb stimulation (see, for example, Non-Patent Document 43).

[0018] Therefore, antibodies containing Fc with improved FcγRIIb binding activity are suggested to be promising therapeutic agents for inflammatory diseases such as autoimmune diseases.

[0019] Furthermore, it has been reported that the activation of macrophages and dendritic cells via Toll-like receptor 4, induced by LPS stimulation, is suppressed in the presence of antibody-antigen immune complexes, and this effect is also suggested to be due to the action of immune complexes mediated by FcγRIIb (see, for example, Non-Patent Documents 44 and 45). Therefore, the use of antibodies with enhanced FcγRIIb binding is expected to enable enhanced suppression of TLR-mediated activation signaling, and for this reason, such antibodies are suggested to be promising therapeutic agents for inflammatory diseases such as autoimmune diseases.

[0020] Furthermore, mutations that enhance FcγRIIb binding suggest promising therapeutic agents for cancer, as well as for inflammatory diseases such as autoimmune diseases. To date, FcγRIIb has been found to play a crucial role in the agonist activity of agonist antibodies against the anti-TNF receptor superfamily. Specifically, interaction with FcγRIIb is suggested to be necessary for the agonist activity of antibodies against CD40, DR4, DR5, CD30, and CD137, which are included in the TNF receptor family (see, for example, Non-Patent Documents 46, 47, 48, 49, 50, 51, and 52). Non-Patent Document 46 shows that the use of antibodies with enhanced FcγRIIb binding enhances the antitumor effect of anti-CD40 antibodies. Therefore, it is expected that antibodies with enhanced FcγRIIb binding will enhance the agonist activity of agonist antibodies, including antibodies against the anti-TNF receptor superfamily.

[0021] Furthermore, it has been shown that when Kit is crosslinked with FcγRIIb on Kit-expressing cells using an antibody that recognizes Kit, a type of receptor tyrosine kinase (RTK), cell proliferation is suppressed. Similar effects have been reported even when Kit is constitutively activated and has mutations that cause tumorigenesis (see, for example, Non-Patent Document 53). Therefore, it is expected that the use of an antibody with enhanced FcγRIIb binding may enhance the inhibitory effect against cells expressing RTKs with constitutively activated mutations.

[0022] Antibodies with improved Fc binding activity have been reported (see, for example, Non-Patent Document 40). In this document, FcγRIIb binding activity was improved by adding modifications such as S267E / L328F, G236D / S267E, and S239D / S267E to the antibody Fc region. Among these, the antibody with the S267E / L328F mutation bound to FcγRIIb most strongly, and its binding to FcγRIa and H-type FcγRIIa (where the residue at position 131 of FcγRIIa is His) remained at the same level as natural IgG1. However, other reports have shown that this modification enhances binding to R-type FcγRIIa (where the residue at position 131 of FcγRIIa is Arg) to the same level as FcγRIIb binding, by several hundred times, which means that the FcγRIIb binding selectivity is not improved compared to R-type FcγRIIa (see, for example, Patent Document 15).

[0023] The effect of enhancing only FcγRIIa binding while not enhancing FcγRIIb binding is thought to affect cells such as platelets that express FcγRIIa but not FcγRIIb (see, for example, Non-Patent Document 17). For example, it is known that patients administered bevacizumab, an antibody against VEGF, have an increased risk of thromboembolism (see, for example, Non-Patent Document 54). Furthermore, thromboembolism was observed in a similar manner in a clinical development trial of an antibody against CD40 ligand, and this clinical trial was discontinued (see, for example, Non-Patent Document 55). In both cases of these antibodies, subsequent studies using animal models suggested that the administered antibody caused platelet aggregation via FcγRIIa binding on platelets, leading to thrombus formation (see, for example, Non-Patent Documents 56 and 57). In systemic lupus erythematosus, an autoimmune disease, platelets are activated via an FcγRIIa-dependent mechanism, and platelet activation has been reported to correlate with the severity of symptoms (see, for example, Non-Patent Document 58). Administering antibodies with enhanced FcγRIIa binding to such patients already at high risk of developing thromboembolism increases the risk of developing thromboembolism and is therefore extremely dangerous.

[0024] Furthermore, antibodies with enhanced FcγRIIa binding have been reported to enhance macrophage-mediated antibody-dependent phagocytosis (ADCP) (see, for example, Non-Patent Document 59). When the antigen to which an antibody binds is phagocytosed by macrophages, it is thought that the antibody itself is also phagocytosed simultaneously. When an antibody is administered as a drug, peptide fragments derived from the administered antibody may also be presented as antigens, and therefore the risk of producing antibodies against the therapeutic antibody (anti-therapeutic antibodies) is expected to increase. More specifically, enhanced FcγRIIa binding increases the risk of producing antibodies against the therapeutic antibody, which significantly reduces its value as a drug. In addition, FcγRIIb on dendritic cells is suggested to contribute to peripheral tolerance by inhibiting dendritic cell activation caused by immune complexes formed between antigens and antibodies, or by suppressing antigen presentation to T cells via Fcγ receptor activation (see, for example, Non-Patent Document 60). Since FcγRIIa is also expressed on dendritic cells, when using an antibody containing Fc that selectively binds to FcγRIIb as a drug, the antigen is not immediately presented by dendritic cells, etc., because the selective binding to FcγRIIb is enhanced, and the risk of producing anti-drug antibodies can be relatively reduced. Such antibodies may be useful in this respect as well.

[0025] More specifically, if FcγRIIa binding is enhanced, it leads to an increased risk of thrombus formation via platelet aggregation and an increased risk of anti-therapeutic antibody production due to increased immunogenicity, significantly reducing the drug's value.

[0026] From this perspective, the aforementioned Fc variant with enhanced FcγRIIb binding exhibits significantly enhanced R-type FcγRIIa binding compared to natural IgG1. Therefore, its value as a drug for patients with R-type FcγRIIa is significantly reduced. H-type and R-type FcγRIIa are observed with almost equal frequency in Caucasians and African Americans (see, for example, Non-Patent Documents 61 and 62). Consequently, even if this Fc variant is used to treat autoimmune diseases, the number of patients who can safely use it while receiving drug efficacy is limited.

[0027] Furthermore, it has been reported that dendritic cells lacking FcγRIIb, or dendritic cells in which the interaction between FcγRIIb and the antibody Fc portion is inhibited by an anti-FcγRIIb antibody, mature (see, for example, Non-Patent Documents 63 and 64). This report suggests that in a steady state where there is no inflammation or other factors and no activation occurs, FcγRIIb actively suppresses dendritic cell maturation. Even if FcγRIIa is expressed on the surface of dendritic cells in addition to FcγRIIb, and therefore binding to inhibitory FcγRIIb is enhanced, if binding to activated FcγR such as FcγRIIa is also enhanced, dendritic cell maturation may be promoted as a result. More specifically, it is considered important to improve not only FcγRIIb binding activity but also the ratio of FcγRIIb binding activity to FcγRIIa binding activity when providing antibodies with immunosuppressive effects.

[0028] Therefore, when considering the manufacture of drugs that utilize immunosuppressive effects mediated by FcγRIIb binding, Fc variants are needed in which not only is FcγRIIb binding activity enhanced, but the binding of FcγRIIa to both the H and R allotypes is maintained at levels equivalent to or at lower than those of natural IgG1.

[0029] On the other hand, there have been reports of cases in which amino acid modifications have been introduced into the Fc region to increase FcγRIIb binding selectivity (see, for example, Non-Patent Document 65). However, all the mutants reported in this document that were said to have improved FcγRIIb selectivity showed decreased FcγRIIb binding compared to natural IgG1. Therefore, it is unlikely that these mutants can actually induce a stronger FcγRIIb-mediated immunosuppressive response than IgG1.

[0030] Furthermore, since FcγRIIb plays an important role in the aforementioned agonist antibodies, it is expected that enhancing their binding activity will enhance agonist activity. However, if FcγRIIa binding is also enhanced, unintended activities such as ADCC activity and ADCP activity may occur, which could cause side effects. From this perspective, it is preferable to selectively enhance FcγRIIb binding activity.

[0031] These results suggest that, in the production of therapeutic antibodies for use in the treatment of autoimmune diseases and cancer using FcγRIIb, it is important that the binding activity to both allotypes of FcγRIIa is maintained or reduced compared to natural IgG, and that FcγRIIb binding is enhanced. However, FcγRIIb shares 93% sequence identity with FcγRIIa, one of the activated FcγRs, in its extracellular domain, and they are also structurally very similar. FcγRIIa has H-type and R-type allotypes, with the amino acid at position 131 being His (H-type) or Arg (R-type), each reacting with antibodies in different ways (see, for example, Non-Patent Literature 66). Therefore, producing Fc-region variants with enhanced selective FcγRIIb binding compared to each allotype of FcγRIIa may be a challenging task, involving distinguishing highly homologous sequences between FcγRIIa and FcγRIIb. Despite these challenges, several Fc region variants that exhibit selective binding activity to FcγRIIb compared to FcγRIIa have been identified by conducting comprehensive amino acid modification analyses of the Fc region (see, for example, Patent Documents 16, 17, 18, 19, and 20).

[0032] While there have been reports of Fc region variants in human FcγR that exhibit binding selectivity to FcγRIIb, there have been no reports of such variants in monkey FcγR. Because such Fc variants do not exist, the effects of Fc variants that selectively bind to FcγRIIb have not yet been sufficiently studied in monkeys.

[0033] Separately from the above, it has been reported that the half-life of an antibody in the blood can be controlled by modifying the charge of amino acid residues that can be exposed on the antibody surface in order to increase or decrease the isoelectric point (pI) of the antibody (see, for example, Patent Documents 21 and 22). These documents show that it is possible to extend the plasma half-life of an antibody by decreasing the pI of the antibody, and vice versa.

[0034] Furthermore, it has been reported that the uptake of antigens into cells can be promoted by modifying the charge of specific amino acid residues, particularly in the CH3 domain, in order to increase the probative I (see, for example, Patent Document 23). Similarly, it has been reported that the half-life of antibodies in plasma can be extended by modifying the charge of amino acid residues in the constant region of the antibody (mainly the CH1 domain) in order to decrease the probative I (see, for example, Patent Document 24). [Prior art documents] [Patent Documents]

[0035] [Patent Document 1] US Patent No. 5,827,733 [Patent Document 2] WO 1994 / 021681 [Patent Document 3] US Patent No. 6,096,506 [Patent Document 4] US Patent No. 7,261,893 [Patent Document 5] US Patent No. 7,320,789 [Patent Document 6] US Patent No. 7,807,159 [Patent Document 7] US Patent No. 7,888,486 [Patent Document 8] WO 2005 / 094446 [Patent Document 9] WO 2007 / 047112 [Patent Document 10] WO 2010 / 070094 [Patent Document 11] WO 2000 / 042072 [Patent Document 12] WO 2006 / 019447 [Patent Document 13] WO 2004 / 099249 [Patent Document 14] WO 2004 / 029207 [Patent Document 15] US Appl. Publ. No. US2009 / 0136485 [Patent Document 16] WO 2012 / 115241 [Patent Document 17] WO 2013 / 047752 [Patent Document 18] WO 2013 / 125667 [Patent Document 19] WO 2014 / 030728 [Patent Document 20] WO 2014 / 163101 [Patent Document 21] WO 2007 / 114319 [Patent Document 22] WO 2009 / 041643 [Patent Document 23] WO 2014 / 145159 [Patent Document 24] WO 2012 / 016227 [Non-patent literature]

[0036] [Non-Patent Document 1] Kingsley et al., Genes Dev. 8(2):133-146 (1994) [Non-Patent Document 2] Hoodless et al., Curr. Top. Microbiol. Immunol. 228:235-272 (1998) [Non-licensed document 3] Zimmers et al., Science 296(5572):1486-1488 (2002)

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[0037] The object of the present invention is to provide an anti-myostatin antibody, a polypeptide containing a mutated Fc region, and a method for using the same. [Means for solving the problem]

[0038] This invention provides an anti-myostatin antibody and a method for using the same. The invention also provides a protein containing a mutated Fc region and a method for using the same.

[0039] In some embodiments, the isolated anti-myostatin antibody of the present invention binds to latent myostatin. In a further embodiment, the antibody binds to an epitope within a fragment of myostatin propeptide (SEQ ID NO: 78) consisting of amino acids 21-100. In some embodiments, the isolated anti-myostatin antibody of the present invention inhibits myostatin activation. In a further embodiment, the antibody prevents the release of mature myostatin from latent myostatin. In a further embodiment, the antibody prevents the proteolytic release of mature myostatin. In a further embodiment, the antibody prevents the spontaneous release of mature myostatin. In a further embodiment, the antibody does not bind to mature myostatin. In a further embodiment, the antibody binds to the same epitope as the antibodies listed in Table 13. In a further embodiment, the antibody binds to the same epitope as the antibodies containing the VH and VL pairs listed in Table 13. In a further embodiment, the antibody binds to the same epitope as the antibodies listed in Table 2a. In a further embodiment, the antibody binds to the same epitope as the antibody containing the VH and VL pair described in Table 2a. In a further embodiment, the antibody binds to the same epitope as the antibody described in Table 11a. In a further embodiment, the antibody binds to the same epitope as the antibody containing the VH and VL pair described in Table 11a. In a further embodiment, the antibody binds to the same epitope as the antibody described in Table 2a, 11a, or 13. In a further embodiment, the antibody binds to the same epitope as the antibody containing the VH and VL pair described in Table 2a, 11a, or 13.

[0040] In some embodiments, the isolated anti-myostatin antibody of the present invention binds to latent myostatin with higher affinity at neutral pH than at acidic pH. In some embodiments, the anti-myostatin antibody binds to latent myostatin with higher affinity at pH 7.4 than at pH 5.8. In some embodiments, the isolated anti-myostatin antibody of the present invention binds to a polypeptide fragment consisting of amino acids 21-100 of myostatin propeptide (SEQ ID NO: 78) with higher affinity at pH 7.4 than at pH 5.8. In some embodiments, the antibody binds to the same myostatin epitope as the antibody listed in Table 13 with higher affinity at neutral pH than at acidic pH. In additional embodiments, the anti-myostatin antibody binds to the same epitope as the antibody listed in Table 13 with higher affinity at pH 7.4 than at pH 5.8. In a further embodiment, the antibody binds to the same epitope as the antibody containing the VH and VL pair listed in Table 13, with higher affinity at pH 7.4 than at pH 5.8. In some embodiments, the antibody binds to the same myostatin epitope as the antibody listed in Table 2a, with higher affinity at neutral pH than at acidic pH. In some embodiments, the antibody binds to the same myostatin epitope as the antibody listed in Table 2a, with higher affinity at pH 7.4 than at pH 5.8. In a further embodiment, the antibody binds to the same epitope as the antibody containing the VH and VL pair listed in Table 2a, with higher affinity at pH 7.4 than at pH 5.8. In an additional embodiment, the anti-myostatin antibody binds to the same epitope as the antibody listed in Table 11a, with higher affinity at neutral pH than at acidic pH. In a further embodiment, the antibody binds to the same myostatin epitopes as the antibodies listed in Table 11a, with higher affinity at pH 7.4 than at pH 5.8. In a further embodiment, the antibody binds to the same epitopes as the antibodies containing the VH and VL pairs listed in Table 11a, with higher affinity at pH 7.4 than at pH 5.8.In an additional embodiment, the anti-myostatin antibody binds to the same epitopes as the antibodies listed in Table 2a, 11a, or 13 with higher affinity at neutral pH than at acidic pH. In a further embodiment, the antibody binds to the same myostatin epitopes as the antibodies listed in Table 2a, 11a, or 13 with higher affinity at pH 7.4 than at pH 5.8. In a further embodiment, the antibody binds to the same epitopes as the antibodies containing the VH and VL pairs listed in Table 2a, 11a, or 13 with higher affinity at pH 7.4 than at pH 5.8.

[0041] In some embodiments, the isolated anti-myostatin antibody of the present invention competes with the antibodies described herein in terms of binding to latent myostatin. In some embodiments, the isolated anti-myostatin antibody of the present invention competes with the antibodies listed in Table 13 in terms of binding to latent myostatin. In some embodiments, the isolated anti-myostatin antibody of the present invention competes with the antibodies containing the VH and VL pairs listed in Table 13 in terms of binding to latent myostatin. In some embodiments, the antibody competes with the antibodies listed in Table 2a in terms of binding to latent myostatin. In some embodiments, the isolated anti-myostatin antibody of the present invention competes with the antibodies containing the VH and VL pairs listed in Table 2a in terms of binding to latent myostatin. In some embodiments, the antibody competes with the antibodies listed in Table 11a in terms of binding to latent myostatin. In some embodiments, the isolated anti-myostatin antibody of the present invention competes with antibodies containing VH and VL pairs as described in Table 11a for binding to latent myostatin. In further embodiments, the anti-myostatin antibody competes with antibodies described in Tables 2a, 11a, or 13 for binding to latent myostatin. In further embodiments, the anti-myostatin antibody competes with antibodies containing VH and VL pairs as described in Tables 2a, 11a, or 13 for binding to latent myostatin. In further embodiments, the anti-myostatin antibody binds to latent myostatin with higher affinity at neutral pH than at acidic pH. In further embodiments, the anti-myostatin antibody binds to latent myostatin with higher affinity at pH 7.4 than at pH 5.8. In a further embodiment, the anti-myostatin antibody binds to a polypeptide fragment consisting of amino acids 21-100 of myostatin propeptide (SEQ ID NO: 78) with higher affinity at pH 7.4 than at pH 5.8. Methods for evaluating the ability of an antibody to compete with a reference antibody for binding to latent myostatin are described herein and are known in the art.

[0042] In some embodiments, the isolated anti-myostatin antibody of the present invention is a monoclonal antibody. In some embodiments, the isolated anti-myostatin antibody of the present invention is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the isolated anti-myostatin antibody of the present invention is an antibody fragment that binds to myostatin. In some embodiments, the isolated anti-myostatin antibody of the present invention is an antibody fragment that binds to latent myostatin. In some embodiments, the isolated anti-myostatin antibody of the present invention is an antibody fragment that binds to a polypeptide fragment consisting of amino acids 21-100 of myostatin propeptide (SEQ ID NO: 78). In some embodiments, the isolated anti-myostatin antibody of the present invention is a full-length IgG antibody.

[0043] In some embodiments, the anti-myostatin antibody of the present invention is (a)(i) HVR-H3 comprising the amino acid sequence GVPAX1SX2GGDX3 (where X1 is Y or H, X2 is T or H, and X3 is L or K) (Sequence ID: 128), (ii) HVR-L3 comprising the amino acid sequence AGGYGGGX1YA (where X1 is L or R) (Sequence ID: 131), and (iii) HVR-H2 comprising the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7 (where X1 is Y or H, X2 is S or K, X3 is T, M or K, X4 is Y or K, X5 is A, M or E, X6 is S or E, and X7 is G or K) (Sequence ID: 127); (b) HVR-H1 comprising (i) amino acid sequence X1X2DIS (where X1 is S or H, X2 is Y, T, D, or E) (Sequence ID: 126), (ii) HVR-H2 comprising amino acid sequence IISX1AGX2X3YX4X5X6WAKX7 (where X1 is Y or H, X2 is S or K, X3 is T, M, or K, X4 is Y or K, X5 is A, M, or E, X6 is S or E, X7 is G or K) (Sequence ID: 127), and (iii) HVR-H3 comprising amino acid sequence GVPAX1SX2GGDX3 (where X1 is Y or H, X2 is T or H, X3 is L or K) (Sequence ID: 128); (c)(i) HVR-H1 containing the amino acid sequence X1X2DIS (where X1 is S or H, X2 is Y, T, D or E) (Sequence ID: 126), (ii) HVR-H2 containing the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7 (where X1 is Y or H, X2 is S or K, X3 is T, M or K, X4 is Y or K, X5 is A, M or E, X6 is S or E, X7 is G or K) (Sequence ID: 127), (iii) amino acid sequence GVPAX1SX2GGDX3 (where X1 is Y or H, X2 is T or H, X3 is L or K) (sequence ID: 127) HVR-H3 containing (number: 128), HVR-L1 containing (iv) amino acid sequence X1X2SQX3VX4X5X6NWLS (where X1 is Q or T, X2 is S or T, X3 is S or E, X4 is Y or F, X5 is D or H, X6 is N, D, A or E) (Sequence ID: 129), HVR-L2 containing (v) amino acid sequence WAX1TLAX2 (where X1 is S or E, X2 is S, Y, F or W) (Sequence ID: 130), and HVR-L3 containing (vi) amino acid sequence AGGYGGGX1YA (where X1 is L or R) (Sequence ID: 131); (d)(i) HVR-L1 containing the amino acid sequence X1X2SQX3VX4X5X6NWLS (where X1 is Q or T, X2 is S or T, X3 is S or E, X4 is Y or F, X5 is D or H, X6 is N, D, A or E) (Sequence ID: 129), (ii) HVR-L2 containing the amino acid sequence WAX1TLAX2 (where X1 is S or E, X2 is S, Y, F or W) (Sequence ID: 130), and (iii) HVR-L3 containing the amino acid sequence AGGYGGGX1YA (where X1 is L or R) (Sequence ID: 131) This includes. In some embodiments, the antibody of (b) further comprises: a heavy chain variable domain framework FR1 containing any one amino acid sequence of SEQ ID NO: 132-134; FR2 containing any one amino acid sequence of SEQ ID NO: 135-136; FR3 containing the amino acid sequence of SEQ ID NO: 137; and FR4 containing the amino acid sequence of SEQ ID NO: 138. In some embodiments, the antibody of (d) further comprises: a light chain variable domain framework FR1 containing the amino acid sequence of SEQ ID NO: 139; FR2 containing any one amino acid sequence of SEQ ID NO: 140-141; FR3 containing any one amino acid sequence of SEQ ID NO: 142-143; and FR4 containing the amino acid sequence of SEQ ID NO: 144.

[0044] In some embodiments, the isolated anti-myostatin antibody of the present invention comprises (a) HVR-H3 comprising the amino acid sequence GVPAX1SX2GGDX3 (where X1 is Y or H, X2 is T or H, and X3 is L or K) (SEQ ID NO: 128), (b) HVR-L3 comprising the amino acid sequence AGGYGGGX1YA (where X1 is L or R) (SEQ ID NO: 131), and (c) HVR-H2 comprising the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7 (where X1 is Y or H, X2 is S or K, X3 is T, M or K, X4 is Y or K, X5 is A, M or E, X6 is S or E, and X7 is G or K) (SEQ ID NO: 127).

[0045] In some embodiments, the isolated anti-myostatin antibody of the present invention comprises (a) HVR-H1 comprising the amino acid sequence X1X2DIS (where X1 is S or H, X2 is Y, T, D or E) (SEQ ID NO: 126), (b) HVR-H2 comprising the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7 (where X1 is Y or H, X2 is S or K, X3 is T, M or K, X4 is Y or K, X5 is A, M or E, X6 is S or E, X7 is G or K) (SEQ ID NO: 127), and (c) HVR-H3 comprising the amino acid sequence GVPAX1SX2GGDX3 (where X1 is Y or H, X2 is T or H, X3 is L or K) (SEQ ID NO: 128). In a further embodiment, the antibody comprises a heavy chain variable domain framework FR1 containing any one amino acid sequence of SEQ ID NOs: 132-134; FR2 containing any one amino acid sequence of SEQ ID NOs: 135-136; FR3 containing the amino acid sequence of SEQ ID NO: 137; and FR4 containing the amino acid sequence of SEQ ID NO: 138. In a further embodiment, the antibody further comprises (a) HVR-L1 comprising the amino acid sequence X1X2SQX3VX4X5X6NWLS (where X1 is Q or T, X2 is S or T, X3 is S or E, X4 is Y or F, X5 is D or H, and X6 is N, D, A or E) (Sequence ID: 129), (b) HVR-L2 comprising the amino acid sequence WAX1TLAX2 (where X1 is S or E, X2 is S, Y, F or W) (Sequence ID: 130), and (c) HVR-L3 comprising the amino acid sequence AGGYGGGX1YA (where X1 is L or R) (Sequence ID: 131).

[0046] In some embodiments, the isolated anti-myostatin antibody of the present invention comprises (a) HVR-L1 comprising the amino acid sequence X1X2SQX3VX4X5X6NWLS (wherein X1 is Q or T, X2 is S or T, X3 is S or E, X4 is Y or F, X5 is D or H, and X6 is N, D, A or E) (Sequence ID: 129), (b) HVR-L2 comprising the amino acid sequence WAX1TLAX2 (wherein X1 is S or E, X2 is S, Y, F or W) (Sequence ID: 130), and (c) HVR-L3 comprising the amino acid sequence AGGYGGGX1YA (wherein X1 is L or R) (Sequence ID: 131). In some embodiments, the antibody further comprises a light chain variable domain framework FR1 comprising the amino acid sequence of SEQ ID NO: 139; FR2 comprising any one amino acid sequence of SEQ ID NO: 140-141; FR3 comprising any one amino acid sequence of SEQ ID NO: 142-143; and FR4 comprising the amino acid sequence of SEQ ID NO: 144.

[0047] In some embodiments, the isolated anti-myostatin antibody of the present invention comprises a heavy chain variable domain framework FR1 containing any one amino acid sequence of SEQ ID NO: 132-134; FR2 containing any one amino acid sequence of SEQ ID NO: 135-136; FR3 containing the amino acid sequence of SEQ ID NO: 137; and FR4 containing the amino acid sequence of SEQ ID NO: 138. In some embodiments, the isolated anti-myostatin antibody of the present invention comprises a light chain variable domain framework FR1 containing the amino acid sequence of SEQ ID NO: 139; FR2 containing any one amino acid sequence of SEQ ID NO: 140-141; FR3 containing any one amino acid sequence of SEQ ID NO: 142-143; and FR4 containing the amino acid sequence of SEQ ID NO: 144.

[0048] In some embodiments, the isolated anti-myostatin antibody of the present invention comprises (a) a VH sequence having at least 95% sequence identity to any one amino acid sequence of SEQ ID NOs: 13, 16-30, 32-34, and 86-95; (b) a VL sequence having at least 95% sequence identity to any one amino acid sequence of SEQ ID NOs: 15, 31, 35-38, and 96-99; or (c) the VH sequence of (a) and the VL sequence of (b). In a further embodiment, the antibody comprises the VH sequence of any one of SEQ ID NOs: 13, 16-30, 32-34, and 86-95. In a further embodiment, the antibody comprises the VL sequence of any one of SEQ ID NOs: 15, 31, 35-38, and 96-99. In some embodiments, the antibody comprises the VH sequence of any one of SEQ ID NOs: 13, 16-30, 32-34, and 86-95. In a further embodiment, the antibody comprises one VH sequence of sequence numbers 13, 16-30, 32-34, and 86-95; and one VL sequence of sequence numbers 15, 31, 35-38, and 96-99.

[0049] The present invention also provides isolated nucleic acids encoding the anti-myostatin antibody of the present invention. The present invention also provides host cells containing the nucleic acid of the present invention. The present invention also provides a method for producing an antibody, comprising the step of culturing the host cells of the present invention so that the antibody can be produced.

[0050] In some aspects, the present invention provides a method for producing an anti-myostatin antibody, comprising the steps of (a) culturing host cells of the present invention so as to produce the antibody; or (b) immunizing an animal with a polypeptide comprising a region corresponding to amino acids at positions 21-100 of a myostatin propeptide (SEQ ID NO: 78).

[0051] The present invention further provides a method for producing anti-myostatin antibodies. In some embodiments, the method comprises the step of immunizing an animal with a polypeptide comprising a region corresponding to amino acids at positions 21-100 of myostatin propeptide (SEQ ID NO: 78).

[0052] The present invention also provides a pharmaceutical formulation comprising the anti-myostatin antibody of the present invention and a pharmaceutically acceptable carrier.

[0053] The anti-myostatin antibody of the present invention may be for use as a pharmaceutical. In some embodiments, the antibody may be used in the manufacture of pharmaceuticals for (a) the treatment of muscle wasting diseases; (b) increasing the volume of muscle tissue; (c) increasing the strength of muscle tissue; or (d) reducing body fat accumulation. In some embodiments, the anti-myostatin antibody of the present invention may be for use in the treatment of muscle wasting diseases. The anti-myostatin antibody of the present invention may be for use in increasing the volume of muscle tissue. The anti-myostatin antibody of the present invention may be for use in increasing the strength of muscle tissue. The anti-myostatin antibody of the present invention may be for use in reducing body fat accumulation.

[0054] In some embodiments, the anti-myostatin antibodies provided herein have applications in (a) the treatment of muscle wasting diseases; (b) increasing the volume of muscle tissue; (c) increasing the strength of muscle tissue; or (d) reducing body fat accumulation.

[0055] The anti-myostatin antibody of the present invention may be used in the manufacture of pharmaceuticals. In some embodiments, the antibody is used in the manufacture of pharmaceuticals for (a) the treatment of muscle wasting diseases; (b) increasing the volume of muscle tissue; (c) increasing the strength of muscle tissue; or (d) reducing body fat accumulation. In some embodiments, the pharmaceutical is for the treatment of muscle wasting diseases. In some embodiments, the pharmaceutical is for increasing the volume of muscle tissue. In some embodiments, the pharmaceutical is for increasing the strength of muscle tissue. In some embodiments, the pharmaceutical is for reducing body fat accumulation.

[0056] The present invention also provides a method for treating an individual having muscle wasting disease. In some embodiments, the method comprises administering an effective amount of the anti-myostatin antibody of the present invention to the individual. The present invention also provides a method for increasing the amount of muscle tissue in an individual. In some embodiments, the method comprises administering an effective amount of the anti-myostatin antibody of the present invention to the individual in order to increase the amount of muscle tissue. The present invention also provides a method for increasing the strength of muscle tissue in an individual. In some embodiments, the method comprises administering an effective amount of the anti-myostatin antibody of the present invention to the individual in order to increase the strength of muscle tissue. The present invention also provides a method for reducing body fat accumulation in an individual. In some embodiments, the method comprises administering an effective amount of the anti-myostatin antibody of the present invention to the individual in order to reduce body fat accumulation.

[0057] This invention provides polypeptides containing a mutated Fc region, as well as methods for producing and using the same.

[0058] In one embodiment, the present invention provides an FcγRIIb-binding polypeptide comprising a mutant Fc region and a method of using the same. In some embodiments, the mutant Fc region of the present invention with enhanced FcγRIIb-binding activity comprises at least one amino acid modification in the parent Fc region. In a further embodiment, the ratio of [KD value of parent Fc region to monkey FcγRIIb] / [KD value of mutant Fc region to monkey FcγRIIb] is 2.0 or higher. In a further embodiment, the ratio of [KD value of parent Fc region to monkey FcγRIIIa] / [KD value of mutant Fc region to monkey FcγRIIIa] is 0.5 or lower. In a further embodiment, the ratio of [KD value of parent Fc region to human FcγRIIb] / [KD value of mutant Fc region to human FcγRIIb] is 2.0 or higher. In a further embodiment, the ratio of [KD value of parent Fc region to human FcγRIIIa] / [KD value of mutant Fc region to human FcγRIIIa] is 0.5 or lower. In a further embodiment, the ratio of [KD value of the parental Fc region for human FcγRIIa (type H)] / [KD value of the mutant Fc region for human FcγRIIa (type H)] is 5.0 or less. In another embodiment, the ratio of [KD value of the parental Fc region for human FcγRIIa (type R)] / [KD value of the mutant Fc region for human FcγRIIa (type R)] is 5.0 or less. In yet another embodiment, the KD value of the mutant Fc region for monkey FcγRIIb is 1.0 × 10⁻¹⁰ -6 It is less than or equal to M. In another embodiment, the KD value of the mutant Fc region for monkey FcγRIIIa is 5.0 × 10⁻⁶. -7 It is M or higher. In another embodiment, the KD value of the mutant Fc region for human FcγRIIb is 2.0 × 10⁻⁶. -6 It is less than or equal to M. In another embodiment, the KD value of the mutant Fc region for human FcγRIIIa is 1.0 × 10⁻⁶. -6 It is M or higher. In another embodiment, the KD value of the mutant Fc region for human FcγRIIa (type H) is 1.0 × 10⁻⁶. -7 It is M or higher. In another embodiment, the KD value of the mutant Fc region for human FcγRIIa (R type) is 2.0 × 10⁻⁶. -7 It is M or higher.

[0059] In some embodiments, the mutant Fc region of the present invention with enhanced FcγRIIb binding activity includes at least one amino acid modification at at least one position selected from the group consisting of positions 231, 232, 233, 234, 235, 236, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334, and 396, represented by EU numbering.

[0060] In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity comprises, as represented by EU numbering, (a) one amino acid modification at position 236, and (b) at least two amino acid modifications, including (i) at positions 231, 232, 233, 234, 235, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334, and 396; (ii) at least one amino acid modification at at least one position selected from the group consisting of positions 231, 232, 235, 239, 268, 295, 298, 326, 330, and 396; or (iii) at positions 268, 295, 326, and 330.

[0061] In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity comprises at least two amino acid modifications, represented by EU numbering, including (a) one amino acid modification at position 236 and (b) at least one amino acid modification at at least one position selected from the group consisting of positions 231, 232, 233, 234, 235, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334, and 396.

[0062] In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity comprises at least two amino acid modifications, represented by EU numbering, including (a) one amino acid modification at position 236 and (b) at least one amino acid modification at at least one position selected from the group consisting of positions 231, 232, 235, 239, 268, 295, 298, 326, 330, and 396.

[0063] In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity comprises at least two amino acid modifications, represented by EU numbering, including (a) one amino acid modification at position 236 and (b) at least one amino acid modification at at least one position selected from the group consisting of positions 268, 295, 326, and 330.

[0064] In some embodiments, the mutant Fc region with enhanced FcγRIIb binding activity of the present invention is represented by EU numbering as: (a) Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 231; (b) Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn at position 232. , Gln, Arg, Ser, Thr, Val, Trp, Tyr; (c) Asp at position 233; (d) Trp, Tyr at position 234; (e) Trp at position 235; (f) Ala, Asp, Glu, His, Ile, Leu, Met, Asn, Gln, Ser, Thr, Val at position 236; (g) Asp, Tyr at position 237; (h) Glu, Ile, Met, Gln, Tyr at position 238; (i) Ile, L at position 239 eu, Asn, Pro, Val; (j) Ile at position 264; (k) Phe at position 266; (l) Ala, His, Leu at position 267; (m) Asp, Glu at position 268; (n) Asp, Glu, Gly at position 271; (o) Leu at position 295; (p) Leu at position 298; (q) Glu, Phe, Ile, Leu at position 325; (r) Thr at position 326; (s) Ile, Asn at position 327; (t) position 32 It contains at least one amino acid selected from the group consisting of Thr at position 8; Lys and Arg at position 330 (u); Glu at position 331 (v); Asp at position 332 (w); Asp, Ile, Met, Val, and Tyr at position 334 (x); and Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at position 396 (y).

[0065] In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity contains at least one amino acid selected from the group consisting of (a) Gly, Thr at position 231; (b) Asp at position 232; (c) Trp at position 235; (d) Asn, Thr at position 236; (e) Val at position 239; (f) Asp, Glu at position 268; (g) Leu at position 295; (h) Leu at position 298; (i) Thr at position 326; (j) Lys, Arg at position 330; and (k) Lys, Met at position 396, as represented by EU numbering.

[0066] In another embodiment, the present invention provides a polypeptide comprising an isoelectric point (pI)-elevating mutant Fc region and a method of using the same. In some embodiments, the polypeptide comprising the pI-elevating mutant Fc region comprises at least two amino acid modifications in the parent Fc region. In a further embodiment, each of the amino acid modifications elevates the isoelectric point (pI) of the mutant Fc region compared to the parent Fc region. In a further embodiment, the amino acids may be exposed on the surface of the mutant Fc region. In a further embodiment, the polypeptide comprises the mutant Fc region and an antigen-binding domain. In a further embodiment, the antigen-binding activity of the antigen-binding domain is variable depending on the ion concentration conditions. In a further embodiment, the pI-elevated mutant Fc region of the present invention comprises at least two amino acid modifications at at least two positions selected from the group consisting of positions 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422, and 431, as represented by EU numbering. In a further embodiment, the pI-elevated mutant Fc region comprises Arg or Lys at each of the selected positions.

[0067] In some embodiments, the mutant Fc region of the present invention includes the amino acid modifications described in Tables 14-30.

[0068] In some embodiments, the polypeptide comprises a mutant Fc region of the present invention. In a further embodiment, the parental Fc region is derived from human IgG1. In a further embodiment, the polypeptide is an antibody. In a further embodiment, the polypeptide is an Fc fusion protein.

[0069] The present invention provides a polypeptide comprising any one amino acid sequence from SEQ ID NOs: 229 to 381.

[0070] The present invention also provides isolated nucleic acids encoding polypeptides containing the mutant Fc region of the present invention. The present invention also provides host cells containing the nucleic acids of the present invention. The present invention also provides a method for producing polypeptides containing the mutant Fc region, comprising the step of culturing the host of the present invention so that the polypeptides can be produced.

[0071] The present invention further provides a pharmaceutical formulation comprising a polypeptide containing the mutated Fc region of the present invention and a pharmaceutically acceptable carrier. [Brief explanation of the drawing]

[0072] [Figure 1] Figure 1 shows the inhibition of proteolytic activation of latent myostatin by an anti-latent myostatin antibody, as described in Example 3. The activity of active myostatin released from latent myostatin by BMP1 protease was measured using the HEK Blue assay in the presence of the anti-latent myostatin antibody. [Figure 2] Figure 2 shows the inhibition of spontaneous activation of latent myostatin by an anti-latent myostatin antibody, as described in Example 4. The activity of active myostatin released from latent myostatin by incubation at 37°C was measured using the HEK Blue assay in the presence of the anti-latent myostatin antibody. [Figure 3] Figure 3 shows the binding of an anti-latent myostatin antibody to the propeptide domain, as described in Example 5. [Figure 4]Figure 4 shows the Western blot analysis of myostatin propeptide, as described in Example 6. Proteolytic cleavage of myostatin propeptide by BMP1 was evaluated in the presence or absence of an anti-latent myostatin antibody. [Figure 5] Figures 5A to 5C show BIACORE® sensorgrams of the anti-latent myostatin antibody MST1032-G1m against human latent myostatin (A), cynomolgus monkey latent myostatin (B), and mouse latent myostatin (C), as described in Example 7. [Figure 6A] Figure 6A shows the in vivo effects of anti-latent myostatin antibodies and anti-mature myostatin antibodies on muscle mass and fat mass, as described in Example 8. Anti-latent myostatin antibody (MST1032-G1m; labeled as MST1032 in the figure) or anti-mature myostatin antibody (41C1E4) was administered to SCID mice, and whole-body lean body mass (or lean body mass: LBM) was measured. [Figure 6B] Figure 6B shows the in vivo effects of anti-latent myostatin antibodies and anti-mature myostatin antibodies on muscle mass and fat mass, as described in Example 8. Anti-latent myostatin antibody (MST1032-G1m; labeled as MST1032 in the figure) or anti-mature myostatin antibody (41C1E4) was administered to SCID mice, and the change in total body fat mass from day 0 to day 14 was measured. [Figure 6C] Figure 6C shows the in vivo effects of anti-latent myostatin antibodies and anti-mature myostatin antibodies on muscle mass and fat mass, as described in Example 8. Anti-latent myostatin antibody (MST1032-G1m; labeled as MST1032 in the figure) or anti-mature myostatin antibody (41C1E4) was administered to SCID mice, and the volume of gastrocnemius and quadriceps femoris muscles was measured. [Figure 7A]Figure 7A shows a comparison of the in vivo effects of several anti-myostatin antibodies, as described in Example 9. Anti-latent myostatin antibody (MST1032-G1m; labeled as MST1032 in the figure) or anti-mature myostatin antibody (41C1E4, REGN, OGD, or MYO-029) was administered to SCID mice, and the total body fat percentage was measured. [Figure 7B] Figure 7B shows a comparison of the in vivo effects of several anti-myostatin antibodies, as described in Example 9. Anti-latent myostatin antibody (MST1032-G1m; labeled as MST1032 in the figure) or anti-mature myostatin antibody (41C1E4, REGN, OGD, or MYO-029) was administered to SCID mice, and grip strength was measured. [Figure 7C] Figure 7C shows a comparison of the in vivo effects of several anti-myostatin antibodies, as described in Example 9. Anti-latent myostatin antibody (MST1032-G1m; labeled as MST1032 in the figure) or anti-mature myostatin antibody (41C1E4, REGN, OGD, or MYO-029) was administered to SCID mice, and the change in total body fat mass from day 0 to day 14 was measured. [Figure 8] Figure 8 shows the inhibition of proteolytic and spontaneous activation of latent myostatin by a humanized anti-latent myostatin antibody, as described in Example 10. The activity of active myostatin, released from latent myostatin by BMP1 protease (proteolytically) or by incubation at 37°C in the absence of BMP1 (spontaneously), was measured using the HEK Blue assay in the presence of the anti-latent myostatin antibody. [Figure 9]Figure 9 shows BIACORE® sensorgrams of histidine substitution variants of anti-latent myostatin antibodies, as described in Example 11. The antibody / antigen complex was dissociated at pH 7.4 and then further dissociated at pH 5.8 (indicated by arrows) to evaluate pH-dependent interactions. The antibodies tested in this experiment were Ab001 (solid black line), Ab002 (short black dashed line), Ab003 (dotted black line), Ab004 (short gray dashed line), Ab005 (solid gray line), Ab006 (long gray dashed line), and Ab007 (long black dashed line). [Figure 10] Figure 10 shows the inhibition of proteolytic activity and spontaneous activation of latent myostatin by a pH-dependent anti-latent myostatin antibody, as described in Example 13. The activity of active myostatin, released from latent myostatin by BMP1 protease (proteolytically) or by incubation at 37°C in the absence of BMP1 (spontaneously), was measured using the HEK Blue assay in the presence of the anti-latent myostatin antibody. Antibodies MS1032LO01-SG1, MS1032LO02-SG1, MS1032LO03-SG1, and MS1032LO04-SG1 are referred to as MSLO-01, MSLO-02, MSLO-03, and MSLO-04, respectively, in the figure. Similar levels of inhibition of latent myostatin proteolytic activity and spontaneous activation were achieved by MS1032LO01-SG1, MS1032LO02-SG1, MS1032LO03-SG1, and MS1032LO04-SG1 as by MS1032LO00-SG1. [Figure 11] Figures 11A to 11F show BIACORE® sensorgrams of pH-dependent anti-latent myostatin antibodies, as described in Example 14. The reaction rate parameters of MST1032-SG1(A), MS1032LO00-SG1(B), MS1032LO01-SG1(C), MS1032LO02-SG1(D), MS1032LO03-SG1(E), and MS1032LO04-SG1(F) were measured at neutral and acidic pH. [Figure 12]Figure 12 shows the time course of plasma myostatin concentration after intravenous administration of an anti-myostatin antibody in mice, as described in Example 15. The effect of FcγR-mediated cell uptake of antibody / antigen complexes on myostatin clearance in vivo was evaluated by comparing an anti-myostatin antibody with FcγR binding ability (MS1032LO00-SG1) with an anti-myostatin antibody that has lost FcγR binding ability (MS1032LO00-F760). [Figure 13] Figure 13 shows the time course of plasma myostatin concentration after intravenous administration of anti-myostatin antibodies in mice, as described in Example 16. The effect of pH-dependent binding of anti-myostatin antibodies on in vivo myostatin clearance was evaluated by comparing pH-dependent anti-myostatin antibodies (MS1032LO01-SG1 or MS1032LO01-F760) with pH-independent anti-myostatin antibodies (MS1032LO00-SG1 or MS1032LO00-F760). [Figure 14] Figures 14A-14E show the in vivo effects of pH-dependent and pH-independent anti-latent myostatin antibodies, as described in Example 17. A pH-dependent anti-latent myostatin antibody (MS1032LO01-SG1; labeled MSLO1 in the figure) or a pH-independent anti-latent myostatin antibody (MS1032LO00-SG1; labeled MSLO0 in the figure) was administered to SCID mice, and whole-body lean body mass (A), total body fat mass (B), quadriceps femoris muscle mass (C), gastrocnemius muscle mass (D), and grip strength (E) were measured. [Figure 15] Figure 15 shows the binding activity of the anti-latent myostatin antibody MST1032 to latent myostatin and GDF11, as described in Example 19. [Figure 16]Figure 16 shows the inhibitory activity of the anti-latent myostatin antibody MST1032 against the proteolytic activity and spontaneous activation of GDF11, as described in Example 20. The activity of active GDF11, released (spontaneously) by BMP1 protease (proteolytically) or by incubation at 37°C in the absence of BMP1, was measured using the HEK Blue assay in the presence of the anti-latent myostatin antibody. [Figure 17] Figure 17 shows the inhibition of proteolytic activation of latent myostatin by an anti-latent myostatin antibody, as described in Example 22. The activity of active myostatin released from latent myostatin by BMP1 protease was measured using the HEK Blue assay in the presence of the anti-latent myostatin antibody. [Figure 18] Figure 18 shows the time course of plasma myostatin concentration after intravenous administration of an anti-latent myostatin antibody in mice, as described in Example 23. The effect of pH dependence on myostatin clearance in vivo was evaluated by comparing a pH-independent anti-latent myostatin antibody (MS1032LO00-SG1) with various pH-dependent anti-latent myostatin antibodies (MS1032LO01-SG1, MS1032LO06-SG1, MS1032LO11-SG1, MS1032LO18-SG1, MS1032LO19-SG1, MS1032LO21-SG1, and MS1032LO25-SG1). [Figure 19]Figures 19A and 19B show the time course of plasma myostatin concentration after intravenous administration of anti-latent myostatin antibody in cynomolgus monkeys, as described in Example 24. (A) The effects of pH dependence and Fc modification on in vivo myostatin clearance were evaluated by comparing pH-independent anti-latent myostatin antibody (MS1032LO00-SG1) with pH-dependent anti-latent myostatin antibodies with Fc modification (MS1032LO06-SG1012, MS1032LO06-SG1016, MS1032LO06-SG1029, MS1032LO06-SG1031, MS1032LO06-SG1033, MS1032LO06-SG1034). (B) The effect of Fc modification on in vivo myostatin clearance was evaluated by comparing anti-latent myostatin antibodies (MS1032LO19-SG1079, MS1032LO19-SG1071, MS1032LO19-SG1080, MS1032LO19-SG1074, MS1032LO19-SG1081, and MS1032LO19-SG1077). [Figure 20A] Figure 20A, along with Figures 20B-20I, shows the in vivo effects of the anti-latent myostatin antibody (MS1032 variant) on lean body mass (LBM), grip strength, and body fat mass, as described in Example 25. MS1032LO06-SG1, MS1032LO11-SG1, and MS1032LO18-SG1 were administered to SCID mice, and lean body mass (A) was measured. [Figure 20B] Figure 20B, along with Figures 20A, 20C-20I, shows the in vivo effects of the anti-latent myostatin antibody (MS1032 variant) on lean body mass (LBM), grip strength, and body fat mass, as described in Example 25. MS1032LO06-SG1, MS1032LO19-SG1, and MS1032LO25-SG1 were administered to SCID mice, and lean body mass (B) was measured. [Figure 20C]Figure 20C, along with Figures 20A-B and 20D-20I, shows the in vivo effects of the anti-latent myostatin antibody (MS1032 variant) on lean body mass (LBM), grip strength, and body fat mass, as described in Example 25. MS1032LO01-SG, MS1032LO06-SG1, and MS1032LO11-SG1 were administered to SCID mice, and lean body mass (C) was measured. [Figure 20D] Figure 20D, along with Figures 20A-C and 20E-20I, shows the in vivo effects of the anti-latent myostatin antibody (MS1032 variant) on lean body mass (LBM), grip strength, and body fat mass, as described in Example 25. MS1032LO06-SG1, MS1032LO11-SG1, and MS1032LO18-SG1 were administered to SCID mice, and grip strength (D) was measured. [Figure 20E] Figure 20E, along with Figures 20A-D and 20F-20I, shows the in vivo effects of the anti-latent myostatin antibody (MS1032 variant) on lean body mass (LBM), grip strength, and body fat mass, as described in Example 25. MS1032LO06-SG1, MS1032LO19-SG1, and MS1032LO25-SG1 were administered to SCID mice, and grip strength (E) was measured. [Figure 20F] Figure 20F, along with Figures 20A-E and 20G-I, shows the in vivo effects of the anti-latent myostatin antibody (MS1032 variant) on lean body mass (LBM), grip strength, and body fat mass, as described in Example 25. MS1032LO01-SG, MS1032LO06-SG1, and MS1032LO11-SG1 were administered to SCID mice, and grip strength (F) was measured. [Figure 20G] Figure 20G, along with Figures 20A-F and 20H-I, shows the in vivo effects of the anti-latent myostatin antibody (MS1032 variant) on lean body mass (LBM), grip strength, and body fat mass, as described in Example 25. MS1032LO06-SG1, MS1032LO11-SG1, and MS1032LO18-SG1 were administered to SCID mice, and body fat mass (G) was measured. [Figure 20H]Figure 20H, along with Figures 20A-G and 20I, shows the in vivo effects of the anti-latent myostatin antibody (MS1032 variant) on lean body mass (LBM), grip strength, and body fat mass, as described in Example 25. MS1032LO06-SG1, MS1032LO19-SG1, and MS1032LO25-SG1 were administered to SCID mice, and body fat mass (H) was measured. [Figure 20I] Figure 20I, along with Figures 20A-H, shows the in vivo effects of the anti-latent myostatin antibody (MS1032 variant) on lean body mass (LBM), grip strength, and body fat mass, as described in Example 25. MS1032LO01-SG, MS1032LO06-SG1, and MS1032LO11-SG1 were administered to SCID mice, and body fat mass (I) was measured. [Figure 21] Figure 21 shows the inhibitory activity of anti-latent myostatin antibodies against the activation of latent myostatin, as described in Example 26. The amount of mature myostatin released from latent myostatin by BMP1 protease was measured in the presence of anti-latent myostatin antibodies (MST1032, MST1504, MST1538, MST1551, MST1558, MST1572, and MST1573). [Figure 22A] Figure 22A shows schematic diagrams of 100-amino acid latent myostatin fragments designed for epitope mapping of anti-latent myostatin antibodies, as described in Example 26. [Figure 22B] Figure 22B shows a Western blot analysis of GST-tagged human latent myostatin fragments (GST-hMSTN) using an anti-GST antibody, as described in Example 26. Each lane represents: 1, GST-hMSTN 1-100aa; 2, GST-hMSTN 21-120aa; 3, GST-hMSTN 41-140aa; 4, GST-hMSTN 61-160aa; 5, GST-hMSTN 81-180aa; 6, GST-hMSTN 101-200aa; 7, GST-hMSTN 121-220aa; 8, GST-hMSTN 141-241aa; 9, GST control. [Figure 22C]Figure 22C shows Western blot analysis of GST-tagged human latent myostatin fragments (GST-hMSTN) using anti-latent myostatin antibodies (MST1032, MST1538, MST1572, and MST1573) as described in Example 26. Each lane represents the following: 1. GST-hMSTN 1~100aa; 2. GST-hMSTN 21~120aa; 3. GST-hMSTN 41~140aa; 4. GST-hMSTN 61~160aa; 5. GST-hMSTN 81~180aa; 6. GST-hMSTN 101~200aa; 7. GST-hMSTN 121~220aa; 8. GST-hMSTN 141~241aa; 9. GST control; 10. Human latent myostatin (100ng). [Figure 22D] Figure 22D shows a summary of the Western blot analysis results and estimated epitope locations for the anti-latent myostatin antibodies (MST1032, MST1538, MST1572, and MST1573) as described in Example 26. [Figure 23] Figure 23 shows the amino acid sequence alignment of cynomolgus monkey (cyno) FcγRIIa1, FcγRIIa2, FcγRIIa3, FcγRIIb, human FcγRIIaH, FcγRIIaR, and FcγRIIb. The regions enclosed in squares indicate residues that are presumed to interact with the Fc domain. [Figure 24] Figure 24 shows the time course of total plasma myostatin concentration in whole-human FcγR transgenic mice after intravenous administration of an anti-myostatin antibody containing an FcγRIIb-enhanced Fc variant, as described in Example 28. The effect of the FcγRIIb-enhanced Fc variant on antigen elimination mediated by human FcγRIIb was evaluated. [Figure 25] Figure 25 shows the time course of plasma antibody concentration in whole-human FcγR transgenic mice after intravenous administration of an anti-myostatin antibody containing an FcγRIIb-enhanced Fc variant, as described in Example 28. The effect of the FcγRIIb-enhanced Fc variant on the pharmacokinetics of the antibody was evaluated. [Figure 26]Figures 26A and 26B show the time course of plasma myostatin concentration after intravenous administration of anti-latent myostatin antibody in cynomolgus monkeys, as described in Example 29. (A) The effects of pH dependence and Fc modification on in vivo myostatin clearance were evaluated by comparing pH-independent anti-latent myostatin antibody (MS1032LO00-SG1) with pH-dependent anti-latent myostatin antibodies with Fc modification (MS1032LO06-SG1012, MS1032LO06-SG1016, MS1032LO06-SG1029, MS1032LO06-SG1031, MS1032LO06-SG1033, MS1032LO06-SG1034). (B) The effect of Fc modification on in vivo myostatin clearance was evaluated by comparing anti-latent myostatin antibodies (MS1032LO19-SG1079, MS1032LO19-SG1071, MS1032LO19-SG1080, MS1032LO19-SG1074, MS1032LO19-SG1081, and MS1032LO19-SG1077). [Figure 27A] Figure 27A shows the time course of total plasma myostatin concentration in human FcRn transgenic mice after intravenous administration of an anti-myostatin antibody with a pI-elevated Fc variant, as described in Example 30. The effect of the pI-elevated Fc variant on antigen elimination was evaluated. [Figure 27B] Figure 27B shows the time course of plasma antibody concentration in human FcRn transgenic mice after intravenous administration of an anti-myostatin antibody with a pI-elevating Fc variant, as described in Example 30. The effect of the pI-elevating Fc variant on the pharmacokinetics of the antibody was evaluated. [Figure 28A] Figure 28A shows the time course of total plasma myostatin concentration in human FcRn transgenic mice after intravenous administration of an anti-myostatin antibody with a pI-elevated Fc variant, as described in Example 30. The effect of the pI-elevated Fc variant on antigen elimination was evaluated. In this assay, an excess amount of human normal immunoglobulin was administered co-administered with the anti-myostatin antibody to mimic the conditions of human plasma. [Figure 28B] Figure 28B shows the time course of plasma antibody concentration in human FcRn transgenic mice after intravenous administration of an anti-myostatin antibody with a pI-elevated Fc variant, as described in Example 30. The effect of the pI-elevated Fc variant on the pharmacokinetics of the antibody was evaluated. In this assay, an excess amount of human normal immunoglobulin was administered co-administered with the anti-myostatin antibody to mimic the conditions of human plasma. [Figure 29] Figure 29 shows the time course of total plasma myostatin concentration in human FcγRIIb transgenic mice after intravenous administration of an anti-myostatin antibody containing an FcγRIIb-enhanced Fc variant, as described in Example 31. The effect of the FcγRIIb-enhanced Fc variant on antigen elimination mediated by human FcγRIIb was evaluated. [Figure 30] Figure 30 shows the time course of plasma antibody concentration in human FcγRIIb transgenic mice after intravenous administration of an anti-myostatin antibody containing an FcγRIIb-enhanced Fc variant, as described in Example 31. The effect of the FcγRIIb-enhanced Fc variant on the pharmacokinetics of the antibody was evaluated. [Figure 31] Figure 31 shows the results of cell imaging analysis of anti-myostatin antibodies with FcγRIIb-enhanced Fc variants, as described in Example 33. Each antibody was complexed with fluorescently labeled myostatin, and the intracellular uptake of the antigen-antibody complex into cells expressing human FcγRIIb was measured. [Modes for carrying out the invention]

[0073] Description of the manner The methods and procedures described or cited herein are generally well understood, and refer to, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FM Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney), ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DM Weir and C.C.Blackwell, eds.);Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987);PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994);Current Protocols in Immunology (JE Coligan et al., eds., 1991);Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (CA Janeway and P. Travers, 1997);Antibodies (P. Finch, 1997);Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989);Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using Antibodies: A Laboratory Manual (E. Conventional techniques, such as those widely used by those skilled in the art, are commonly employed by those skilled in the art, as described in Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993).

[0074] I. Definition Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which this invention pertains. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, NY 1992) provide general guidance for many of the terms used herein. All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety.

[0075] For the purpose of interpreting this Spec., the following definitions apply, and wherever applicable, a term used in the singular also includes the plural, and vice versa. It should be understood that the terms used herein are intended solely to describe a particular aspect and not to limit it. In the event of any conflict between the following definitions and any document incorporated herein by reference, the following definitions shall prevail.

[0076] In the spirit of this specification, “acceptor human framework” is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from the human immunoglobulin framework or human consensus framework as defined below. An acceptor human framework “derived” from the human immunoglobulin framework or human consensus framework may contain the same amino acid sequence or may contain a modification of the amino acid sequence. In some embodiments, the number of amino acid modifications is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is sequence-identical to the VL human immunoglobulin framework sequence or human consensus framework sequence.

[0077] "Affinity" refers to the strength of the combined non-covalent interactions between one binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X to its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific examples and exemplary embodiments for measuring binding affinity are described below.

[0078] An "affinity-matured" antibody is an antibody that, compared to a parent antibody without modifications, has one or more modifications in one or more hypervariable regions (HVRs) that result in improved affinity of the antibody to the antigen.

[0079] The term "anti - myostatin antibody" or "antibody that binds to myostatin" refers to an antibody that can bind to myostatin with sufficient affinity such that the antibody is useful as a diagnostic agent and / or therapeutic agent when it targets myostatin. In one embodiment, the degree of binding of an anti - myostatin antibody to an unrelated non - myostatin protein is less than about 10% of the binding of the antibody to myostatin when measured (e.g., by radioimmunoassay (RIA)). In certain embodiments, an antibody that binds to myostatin has a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M). In certain embodiments, the anti - myostatin antibody binds to an epitope of myostatin that is conserved among myostatins from different species.

[0080] As used herein, the term "antibody" is used in the broadest sense and encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen - binding activity.

[0081] "Antibody fragment" refers to a molecule other than a full - length antibody that includes a portion of the full - length antibody that binds to an antigen to which the full - length antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single - chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0082] An antibody that "binds to the same epitope as the reference antibody" is an antibody that, in a competitive assay, prevents the reference antibody from binding to its own antigen, and / or conversely, the reference antibody prevents the aforementioned antibody from binding to its own antigen in a competitive assay. An exemplary competitive assay is provided herein.

[0083] The term "chimeric" refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remaining heavy chain and / or light chain originates from a different source or species.

[0084] The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0085] As used herein, the term “cytotoxic agent” means a substance that inhibits or interferes with the function of a cell and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At). 211 , I 131 , I 125 , Y 90 Re 186 Re 188 Sm 153 , Bi 212 , P 32 Pb 212, and radioisotopes of Lu); chemotrexate or chemotherapeutic agents (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; enzymes such as nucleases and their fragments; antibiotics; toxins such as low molecular weight toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including their fragments and / or variants); and various antitumor or anticancer agents as disclosed below.

[0086] "Effector function" refers to the biological activity that varies depending on the antibody isotype, stemming from the Fc region of the antibody. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0087] The “effective dose” of a drug (for example, a pharmaceutical formulation) refers to the amount in the required dosage and over the required period that is effective in achieving the desired therapeutic or prophylactic outcome.

[0088] The term "epitope" includes any determinant that can be bound by an antibody. An epitope is a region of an antigen that is bound by an antibody targeting that antigen and contains specific amino acids that are in direct contact with the antibody. Epitope determinants can include a group of chemically active surface molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and can possess specific three-dimensional structural properties and / or specific charge properties. Generally, antibodies specific to a particular target antigen preferentially recognize epitopes on that target antigen in a complex mixture of proteins and / or macromolecules.

[0089] An "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR is one that binds to an IgG antibody (gamma receptor) and includes the FcγRI, FcγRII, and FcγRIII subclass receptors, including allelic variants and alternative splicing forms of these receptors. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, for example, Daeron, Annu. Rev. Immunol. 15:203-234 (1997).) FcRs have been reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med 126:330-41 (1995). Other FcRs, including those to be identified in the future, are also included in the term “FcR” as used herein.

[0090] The term “Fc receptor” or “FcR” also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are publicly known (see, e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO2004 / 92219 (Hinton et al.)). The in vivo binding to human FcRn and the serum half-life of human FcRn high-affinity binding polypeptides can be measured, for example, in transgenic mice expressing human FcRn or transfected human cell lines, or in primates administered polypeptides with mutant Fc regions. WO2000 / 42072 (Presta) describes antibody variants with improved or reduced binding to FcR. See also, for example, Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

[0091] In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes both the native sequence Fc region and mutant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain, provided that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region is present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0092] The term "Fc region-containing antibody" refers to an antibody that contains an Fc region. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) or the C-terminal glycine-lysine of the Fc region (residues 446-447) can be removed, for example, during antibody purification or by recombination operations of the nucleic acid encoding the antibody. Therefore, a composition containing an antibody having an Fc region according to the present invention may include an antibody with G446-K447, an antibody with G446 but without K447, an antibody from which G446-K447 has been completely removed, or a mixture of the above three types of antibodies.

[0093] The "framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the sequences of HVR and FR usually appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0094] The terms "full-length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a native antibody, or having a heavy chain containing an Fc region as defined herein.

[0095] A "functional Fc region" possesses "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptors: BCRs). Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody-variable domain) and can be evaluated using various measurement methods disclosed, for example, within the definitions herein.

[0096] The terms “host cell,” “host cell line,” and “host cell culture” refer to cells (including their offspring) that are interchangeably used and into which foreign nucleic acids have been introduced. Host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and their offspring, regardless of passage number. Offspring do not have to be completely identical to the parent cells in terms of nucleic acid content and may contain mutations. Mutant offspring that have the same function or biological activity as those used when the original transformed cells were screened or selected are also included herein.

[0097] A "human antibody" is an antibody that possesses an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or an antibody derived from a non-human source that uses the human antibody repertoire or other human antibody coding sequences. This definition of a human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues.

[0098] The "Human Consensus Framework" is a framework that shows the most commonly occurring amino acid residues in selected human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from subgroups of variable domain sequences. Typically, the sequence subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup κI by Kabat et al. As described above. In another embodiment, for VH, the subgroup is subgroup III by Kabat et al. As described above.

[0099] A “humanized” antibody is a chimeric antibody that contains amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments, a humanized antibody contains substantially all of at least one, typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The “humanized form” of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0100] As used herein, the term “hypervariable region” or “HVR” refers to each region of the variable domain of an antibody that is hypervariable in sequence (a “complementarity determining region” or “CDR”), and / or forms a structurally defined loop (a “hypervariable loop”), and / or contains an antigen contact residue (a “antigen contact”). Typically, an antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Illustrative HVRs as used herein include: (a) Hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contact occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and, (d) A combination of (a), (b), and / or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

[0101] Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein in accordance with Kabat et al.

[0102] An "immunoconjugate" is an antibody that has been conjugated to one or more heterologous molecules (the heterologous molecules may include, but are not limited to, cytotoxic agents).

[0103] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, horses), primates (e.g., humans, and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0104] "Isolated" antibodies are those separated from the components of their original environment. In some embodiments, antibodies are purified to a purity of over 95% or 99% by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0105] "Isolated" nucleic acids are nucleic acid molecules that have been separated from the components of their original environment. Isolated nucleic acids include nucleic acid molecules that would normally be found in the cell containing them, but these nucleic acid molecules are located outside the chromosome or in a chromosomal location different from their original chromosomal location.

[0106] "Isolated nucleic acids encoding an anti-myostatin antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, and includes nucleic acid molecules borne on one or more vectors, and nucleic acid molecules present at one or more locations within a host cell.

[0107] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies constituting that population are identical and / or bind to the same epitope, except for any possible mutant antibodies (e.g., mutant antibodies containing naturally occurring mutations, or mutant antibodies that arise during the production of a monoclonal antibody preparation; such variants are usually present in small amounts). In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen. Therefore, the modifier “monoclonal” indicates a characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be prepared by a variety of methods, including, but are not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0108] A "naked antibody" is an antibody that is not conjugated with a different part (e.g., a cytotoxic part) or a radioactive label. Naked antibodies may be present in pharmaceutical preparations.

[0109] "Natural antibodies" refer to immunoglobulin molecules with various structures that occur naturally. For example, a natural IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. Based on the amino acid sequence of its constant domain, the light chains of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ).

[0110] The "natural-type Fc region" contains amino acid sequences identical to those of Fc regions found in nature. The natural-type human Fc region includes the natural-type human IgG1 Fc region (non-A and A allotypes); the natural-type human IgG2 Fc region; the natural-type human IgG3 Fc region; and the natural-type human IgG4 Fc region, as well as naturally occurring variants thereof.

[0111] The term “package insert” is used to refer to instructions for use that are typically included in the commercial packaging of therapeutic products and contain information about indications, usage, dosage, method of administration, combination therapies, contraindications, and / or warnings regarding the use of such therapeutic products.

[0112] Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage ratio of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after the sequences have been aligned to obtain the greatest possible percentage sequence identity and gaps have been introduced where necessary, and no conservative substitutions are considered part of the sequence identity. Alignment for the purpose of determining percentage amino acid sequence identity can be achieved by using various methods within the scope of the art, such as publicly available computer software, including BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetics Co., Ltd.). A person skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm necessary to achieve the greatest possible alignment over the entire length of the sequences being compared. The ALIGN-2 sequence comparison computer program is copyrighted by Genentech, Inc., and its source code has been filed with the U.S. Copyright Office, Washington DC, 20559, along with user documentation, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, and can also be compiled from source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

[0113] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, or with, or relative to, a given amino acid sequence B (or, a given amino acid sequence A having or containing a certain % amino acid sequence identity to, or with, or relative to, a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical in the alignment of A and B by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0114] The term "pharmaceutical preparation" refers to a preparation in which the biological activity of the active ingredient contained therein can exert its effect, and which does not contain additional elements that are toxic to an extent unacceptable to the subject to which the preparation is administered.

[0115] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical preparation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0116] As used herein, the term "myostatin" may refer to any native myostatin from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). Unless otherwise specified, the term "myostatin" refers to the human myostatin protein having the amino acid sequence shown in SEQ ID NO: 1 and containing the terminal propeptide domain of human myostatin shown in SEQ ID NO: 75 or 78. This term encompasses both "full-length," i.e., unprocessed myostatin, and any form of myostatin resulting from processing in cells. This term also encompasses naturally occurring myostatin variants, such as splice variants and allele variants. The amino acid sequence of an exemplary human myostatin (promyostatin) is shown in SEQ ID NO: 1. The amino acid sequence of an exemplary C-terminal growth factor domain of human myostatin is shown in SEQ ID NO: 2. The amino acid sequence of an exemplary N-terminal propeptide domain of human myostatin is shown in SEQ ID NO: 75 or 78. Active mature myostatin is a disulfide-bonded homodimer consisting of two C-terminal growth factor domains. Inactive latent myostatin is a complex in which two propeptides and mature myostatin are non-covalently associated. As disclosed herein, the antibodies of the present invention bind to inactive latent myostatin but not to mature active myostatin homodimer. In some embodiments, the antibodies of the present invention bind to an epitope in a fragment of amino acids 21-100 of myostatin propeptide (SEQ ID NO: 78) but not to mature active myostatin homodimer. The amino acid sequences of exemplary cynomolgus monkey and mouse myostatin (promyostatin) are shown in SEQ ID NOs: 3 and 5, respectively. The amino acid sequences of exemplary C-terminal growth factor domains of cynomolgus monkey and mouse myostatin are shown in SEQ ID NOs: 4 and 6, respectively. Exemplary amino acid sequences of the N-terminal propeptide domains of cynomolgus monkey and mouse myostatin are shown in SEQ ID NOs: 76 or 79 and 77 or 80, respectively. GDF-11 (BMP-11) is a molecule closely related to myostatin, and both are members of the TGF-β superfamily.Similar to myostatin, GDF11 is first synthesized as a precursor polypeptide and then cleaved into an N-terminal prodomain and a C-terminal mature GDF11. The amino acid sequence of human GDF11 (precursor) is shown in SEQ ID NO: 81. The amino acid sequence of C-terminal mature human GDF11 is shown in SEQ ID NO: 82. The amino acid sequence of the N-terminal prodomain of human GDF11 is shown in SEQ ID NO: 83 or 84. The amino acid sequences of SEQ ID NOs: 1, 3, 5, 78, 79, 80, 81, and 84 contain signal sequences. Of these, amino acids 1-24 correspond to the signal sequence and are removed during intracellular processing.

[0117] As used herein, “treatment” (and its grammatical derivatives, e.g., “to treat,” “to treat,” etc.) means a clinical intervention intended to modify the natural course of the individual being treated, and may be carried out for preventive purposes or during the course of a clinical condition. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, reduction of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction of the rate of disease progression, recovery or mitigation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of disease or to slow the progression of disease.

[0118] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) typically have a similar structure, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated by screening complementary libraries of VL or VH domains, respectively, using the VH or VL domains from antibodies that bind to that antigen. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0119] A "mutant Fc region" includes an amino acid sequence that differs from that of the native sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the mutant Fc region has at least one amino acid substitution, for example about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, compared to the native sequence Fc region or the parent polypeptide Fc region. The mutant Fc regions described herein preferably have at least about 80% homology to the native sequence Fc region and / or the parent polypeptide Fc region, most preferably at least about 90% homology, and more preferably at least about 95% homology.

[0120] As used herein, the term "vector" refers to a nucleic acid molecule that can amplify another nucleic acid molecule to which it is ligated. This term includes vectors as self-replicating nucleic acid structures and vectors that are incorporated into the genome of a host cell into which they are introduced. A certain vector can effect the expression of a nucleic acid operably linked thereto. Such vectors are also referred to herein as "expression vectors".

[0121] II. Compositions and Methods In one aspect, the present invention is based in part on anti-myostatin antibodies and their use. In certain embodiments, antibodies that bind to myostatin are provided. The antibodies of the present invention are useful, for example, for the diagnosis or treatment of muscle wasting diseases.

[0122] In another aspect, the present invention is based in part on polypeptides comprising a mutated Fc region and their use. In one embodiment, polypeptides comprising a mutated Fc region with enhanced FcγRIIb binding activity are provided. In another embodiment, polypeptides comprising a mutated Fc region with an increased pI are provided. In certain embodiments, the polypeptides of the present invention are antibodies. The polypeptides comprising a mutated Fc region of the present invention are useful, for example, for the diagnosis or treatment of diseases.

[0123] A. Exemplary Anti-Myostatin Antibodies and Polypeptides Comprising a Mutated Fc Region In one aspect, the present invention provides an isolated antibody that binds to myostatin. In a particular aspect, the anti-myostatin antibody of the present invention binds to latent myostatin. In a further aspect, the anti-myostatin antibody of the present invention binds to myostatin propeptide (human: SEQ ID NO: 75 or 78; cynomolgus monkey: SEQ ID NO: 76 or 79; mouse: SEQ ID NO: 77 or 80). In a further aspect, the antibody binds to an epitope in a fragment of myostatin propeptide (SEQ ID NO: 78) consisting of amino acids 21 to 100. The propeptide is contained within latent myostatin as one of its components, as described above. In a particular aspect, the anti-myostatin antibody of the present invention inhibits myostatin activation. In a particular aspect, the anti-myostatin antibody prevents the release of mature myostatin from latent myostatin. Mature myostatin has been reported to be released from latent myostatin via proteolytic and non-proteolytic processes. The anti-myostatin antibodies of the present invention can inhibit the proteolytic and / or non-proteolytic release of mature myostatin from latent myostatin. In certain embodiments, the anti-myostatin antibody inhibits the proteolytic cleavage of latent myostatin. In certain embodiments, the anti-myostatin antibody inhibits the access of a protease to latent myostatin (particularly the proteolytic cleavage site (Arg98-Asp99) of latent myostatin). In further embodiments, the protease may be a BMP1 / TLD family metalloprotease, e.g., BMP1, TED, tolloid-like protein 1 (TLL-1), or tolloid-like protein 2 (TLL-2). In another embodiment, the anti-myostatin antibody inhibits the non-proteolytic release of mature myostatin from latent myostatin. As used herein, non-proteolytic release refers to the spontaneous release of mature myostatin from latent myostatin, which does not involve proteolytic cleavage of latent myostatin. Non-proteolytic release includes, for example, the release of mature myostatin by incubating latent myostatin at, for example, 37°C, in the absence of a protease that cleaves latent myostatin.In certain embodiments, the anti-myostatin antibody of the present invention does not bind to mature myostatin. In some embodiments, the anti-myostatin antibody binds to the same epitopes as the antibodies listed in Table 2a. In some embodiments, the anti-myostatin antibody competes with the antibodies listed in Table 2a for binding to latent myostatin. In additional embodiments, the anti-myostatin antibody competes with the antibodies containing the VH and VL pairs listed in Table 2a for binding to latent myostatin. In some embodiments, the anti-myostatin antibody competes with the antibodies listed in Table 2a for binding to the fragment of myostatin propeptide (SEQ ID NO: 78) consisting of amino acids 21-100. In further embodiments, the anti-myostatin antibody binds to the same epitopes as the antibodies listed in Table 11a or 13. In some embodiments, the anti-myostatin antibody competes with the antibodies listed in Table 11a or 13 for binding to latent myostatin. In some embodiments, anti-myostatin antibodies compete with the antibodies listed in Table 11a or 13 for binding to the 21-100 amino acid fragment of myostatin propeptide (SEQ ID NO: 78). In some embodiments, the anti-myostatin antibody of the present invention binds to latent myostatin and inhibits myostatin activation. In further embodiments, the antibody (a) prevents the release of mature myostatin from latent myostatin; (b) prevents the proteolytic release of mature myostatin; (c) prevents the spontaneous release of mature myostatin; or (d) does not bind to mature myostatin, or binds to an epitope in a fragment of amino acids 21-100 of myostatin propeptide (SEQ ID NO: 78). In further embodiments, the antibody competes for binding to latent myostatin with antibodies containing VH and VL pairs as described in Table 2a, 11a, or 13, or binds to the same epitope. In further embodiments, the antibody binds to latent myostatin with higher affinity at a neutral pH (e.g., pH 7.4) than at an acidic pH (e.g., pH 5.8). In a further embodiment, the antibody is (a) a monoclonal antibody; (b) a human, humanized, or chimeric antibody; (c) a full-length IgG antibody; or (d) an antibody fragment that binds to latent myostatin or myostatin propeptide.

[0124] In another embodiment, the anti-myostatin antibody of the present invention does not bind to GDF11. In a particular embodiment, the anti-myostatin antibody of the present invention does not inhibit the activation of GDF11. In a particular embodiment, the anti-myostatin antibody does not prevent the release of mature GDF11 from latent GDF11. The anti-myostatin antibody of the present invention does not prevent either the proteolytic or non-proteolytic release of mature GDF11 from latent GDF11. In a particular embodiment, the anti-myostatin antibody does not prevent the proteolytic cleavage of latent GDF11. In a particular embodiment, the anti-myostatin antibody does not prevent the protease from approaching latent GDF11 (particularly the proteolytic cleavage site of latent GDF11). In a further embodiment, the protease may be a BMP1 / TLD family metalloprotease, e.g., BMP1, TED, tolloid-like protein 1 (TLL-1), or tolloid-like protein 2 (TLL-2). As used herein, non-proteolytic release refers to the spontaneous release of mature GDF11 from latent GDF11, without proteolytic cleavage of latent GDF11. Non-proteolytic release includes, for example, the release of mature GDF11 by incubating latent GDF11, for example at 37°C, in the absence of a protease that cleaves latent GDF11. Most anti-myostatin antibodies known to date have not been myostatin specific. These antibodies have high affinity for other members of the TGF-β superfamily, such as GDF11, and neutralize their biological activity. GDF11 plays a crucial role in embryonic development and is responsible for homeotic transformation of the axial skeleton. Homozygous GDF11 knockout mice are perinatal lethal, while mice with one wild-type copy of the GDF11 gene are viable but have skeletal defects. Because GDF11 plays a crucial role in embryonic development, antagonists that inhibit GDF11 present a theoretical safety risk, either as toxicity in treated patients or as reproductive toxicity in women of childbearing age.Therefore, in the treatment of myostatin-related diseases, where it is desirable to increase muscle mass, size, and strength, especially in women of childbearing age, there is a need to specifically inhibit myostatin activity.

[0125] In another aspect, the present invention provides anti-myostatin antibodies exhibiting pH-dependent binding properties. As used herein, “pH-dependent binding” means that the antibody exhibits “a decrease in binding to myostatin at acidic pH compared to its binding at neutral pH” (both expressions may be used interchangeably for the purposes of this disclosure). For example, antibodies “having pH-dependent binding properties” include antibodies that bind to myostatin with higher affinity at neutral pH than at acidic pH. In certain embodiments, antibodies of the present invention bind to myostatin with at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times, or more, at neutral pH than at acidic pH. In some embodiments, these antibodies bind to myostatin (e.g., latent myostatin or propeptide myostatin) with higher affinity at pH 7.4 than at pH 5.8. In further embodiments, these antibodies bind to myostatin with higher affinity at pH 7.4 than at pH 5.8, by at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times, or more.

[0126] When the antigen is a soluble protein, the antibody can have a longer half-life in plasma than the antigen itself and can act as a carrier for the antigen. Therefore, antibody binding to the antigen can lead to an extension of the antigen's half-life in plasma (i.e., reduced clearance of the antigen from plasma). This is due to the reuse of the antigen-antibody complex by FcRn via the endosomal pathway in cells (Roopenian, Nat. Rev. Immunol. 7(9): 715-725 (2007)). However, antibodies with pH-dependent binding properties, which bind to the antigen in a neutral extracellular environment while releasing the antigen into the acidic endosomal compartment after entering the cell, are expected to have superior properties in terms of antigen neutralization and clearance compared to their pH-independent binding counterparts (Igawa et al., Nature Biotechnol. 28(11):1203-1207 (2010); Devanaboyina et al., mAbs 5(6):851-859 (2013); WO 2009 / 125825).

[0127] In the spirit of this disclosure, the “affinity” of an antibody against myostatin is expressed as the antibody’s KD. The KD of an antibody is the equilibrium dissociation constant of the antibody-antigen interaction. The larger the KD value of an antibody for binding to its antigen, the weaker its binding affinity to that particular antigen. Therefore, as used herein, the expression “higher affinity at neutral pH than at acidic pH” (or the equivalent expression “pH-dependent binding”) means that the KD of antibody binding to myostatin at acidic pH is greater than the KD of antibody binding to myostatin at neutral pH. For example, in the context of the present invention, if the KD of antibody binding to myostatin at acidic pH is at least twice as large as the KD of antibody binding to myostatin at neutral pH, the antibody is considered to bind to myostatin with higher affinity at neutral pH than at acidic pH. Accordingly, the present invention includes an antibody that binds to myostatin at an acidic pH with a KD of at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times greater than the KD of the antibody binding to myostatin at a neutral pH. In another embodiment, the KD value of the antibody at a neutral pH is 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 It may be M or less. In another embodiment, the KD value of the antibody at an acidic pH is 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 It can be M or a larger value.

[0128] In a further embodiment, if the KD of antibody binding to myostatin at pH 5.8 is at least twice as large as the KD of antibody binding to myostatin at pH 7.4, the antibody is considered to bind to myostatin (e.g., latent myostatin or propeptide myostatin) with higher affinity at neutral pH than at acidic pH. In some embodiments, the provided antibody binds to myostatin at pH 5.8 with a KD of at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times, or greater than the KD of antibody binding to myostatin at pH 7.4. In another embodiment, the KD value of the antibody at pH 7.4 is 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 It may be M or less. In another embodiment, the KD value of the antibody at pH 5.8 is 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 It can be M or a larger value.

[0129] The binding characteristics of an antibody to a particular antigen may also be expressed as the antibody's kd. The antibody's kd refers to the dissociation rate constant of the antibody with respect to a particular antigen, and is the reciprocal of seconds (i.e., sec). -1 The kd value is expressed in units of 10. A larger kd value means that the antibody binds to the antigen more weakly. The present invention therefore includes an antibody that binds to myostatin at an acidic pH with a higher kd value than at a neutral pH. The present invention includes an antibody that binds to myostatin at an acidic pH with a kd value at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times greater than the kd of antibody binding to myostatin at a neutral pH. In another embodiment, the kd value of the antibody at a neutral pH is 10 -2 1 / s, 10-3 1 / s, 10 -4 1 / s, 10 -5 1 / s, 10 -6 It may be 1 / s or less. In another embodiment, the kd value of the antibody at an acidic pH is 10 -3 1 / s, 10 -2 1 / s, 10 -1 The value may be 1 / s or greater. The present invention also includes antibodies that bind to myostatin (e.g., latent myostatin or propeptide myostatin) at a higher kd value at pH 5.8 than at pH 7.4. The present invention includes antibodies that bind to myostatin at pH 5.8 at a kd value at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times, or greater than the kd of antibody binding to myostatin at pH 7.4. In another embodiment, the kd value of the antibody at pH 7.4 is 10 -2 1 / s, 10 -3 1 / s, 10 -4 1 / s, 10 -5 1 / s, 10 -6 It may be 1 / s or less. In another embodiment, the kd value of the antibody at pH 5.8 is 10 -3 1 / s, 10 -2 1 / s, 10 -1 It can be 1 / s or a value greater than that.

[0130] In certain embodiments, "decreased binding to myostatin at acidic pH compared to binding at neutral pH" is expressed as the ratio of the KD value of antibody binding to myostatin at acidic pH to the KD value of antibody binding to myostatin at neutral pH (or vice versa). For example, in the spirit of the present invention, if an antibody exhibits an acidic / neutral KD ratio of 2 or higher, the antibody may be considered to exhibit "decreased binding to myostatin at acidic pH compared to binding at neutral pH." In certain embodiments, the KD ratio of the anti-myostatin antibody of the present invention at pH 5.8 / pH 7.4 is 2 or higher. In certain exemplary embodiments, the acidic / neutral KD ratio for the antibodies of the present invention may be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or greater. In another embodiment, the KD value of the antibody at neutral pH is 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 It may be M or less. In another embodiment, the KD value of the antibody at an acidic pH is 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 The value may be M or greater. In a further example, if an antibody exhibits a KD ratio of 2 or greater at pH 5.8 / pH 7.4, the antibody may be considered to exhibit "decreased binding to myostatin (e.g., latent myostatin) at acidic pH compared to binding at neutral pH." In certain exemplary embodiments, the KD ratio at pH 5.8 / pH 7.4 for an antibody may be 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or greater. In another embodiment, the KD value of an antibody at pH 7.4 may be 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12It may be M or less. In another embodiment, the KD value of the antibody at pH 5.8 is 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 It can be M or a larger value.

[0131] In certain examples, "decreased binding to myostatin at acidic pH compared to binding at neutral pH" is expressed as the ratio of the kd value of antibody binding to myostatin at acidic pH to the kd value of antibody binding to myostatin at neutral pH (or vice versa). For example, in the spirit of the present invention, if an antibody exhibits an acidic / neutral kd ratio of 2 or higher, the antibody may be considered to exhibit "decreased binding to myostatin at acidic pH compared to binding at neutral pH." In certain exemplary embodiments, the pH 5.8 / pH 7.4 kd ratio for the antibody of the present invention is 2 or higher. In certain exemplary embodiments, the acid / neutral kd ratio for the antibodies of the present invention may be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or greater. In another embodiment, the kd value of the antibody at neutral pH is 10 -2 1 / s, 10 -3 1 / s, 10 -4 1 / s, 10 -5 1 / s, 10 -6 It may be 1 / s or less. In another embodiment, the kd value of the antibody at an acidic pH is 10 -3 1 / s, 10 -2 1 / s, 10 -1 The value may be 1 / s or greater. In certain exemplary embodiments, the kd ratio at pH 5.8 / pH 7.4 for the antibody of the present invention may be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or greater. In another embodiment, the kd value of the antibody at pH 7.4 may be 10 -2 1 / s, 10 -3 1 / s, 10 -4 1 / s, 10-5 1 / s, 10 -6 1 / s, or may be less than that. In another embodiment, the kd value of the antibody at pH 5.8 is 10 -3 1 / s, 10 -2 1 / s, 10 -1 1 / s, or may be a value greater than that.

[0132] As used herein, the expression "acidic pH" means pH 4.0 to 6.5. This expression "acidic pH" includes any one pH value of 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5. In a particular aspect, "acidic pH" is 5.8.

[0133] As used herein, the expression "neutral pH" means pH 6.7 to about 10.0. This expression "neutral pH" includes any one pH value of 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and 10.0. In a particular aspect, "neutral pH" is 7.4.

[0134] The KD value and kd value represented herein can be determined using a biosensor based on surface plasmon resonance to characterize the antibody-antigen interaction. (See, for example, Example 7 of this specification). The KD value and kd value can be determined at 25°C or 37°C.

[0135] In certain embodiments, the anti-myostatin antibody of the present invention binds to myostatin derived from multiple species. In further embodiments, the anti-myostatin antibody binds to myostatin derived from humans and non-human animals. In further embodiments, the anti-myostatin antibody binds to myostatin derived from humans, mice, and monkeys (e.g., cynomolgus monkeys, rhesus monkeys, marmosets, chimpanzees, or baboons).

[0136] In certain embodiments, the anti-myostatin antibody of the present invention binds to latent myostatin derived from multiple species. In further embodiments, the anti-myostatin antibody binds to latent myostatin derived from humans and non-human animals. In further embodiments, the anti-myostatin antibody binds to latent myostatin derived from humans, mice, and monkeys.

[0137] In certain embodiments, the anti-myostatin antibody of the present invention binds to propeptide myostatin derived from multiple species. In further embodiments, the anti-myostatin antibody binds to propeptide myostatin derived from humans and non-human animals. In further embodiments, the anti-myostatin antibody binds to propeptide myostatin derived from humans, mice, and monkeys.

[0138] In a further aspect, the present invention provides anti-myostatin antibodies that form immune complexes (i.e., antigen-antibody complexes) with myostatin. In certain embodiments, two or more anti-myostatin antibodies bind to two or more myostatin molecules to form immune complexes. This is possible because myostatin exists as a homodimer containing two myostatin molecules, while the antibody has two antigen-binding sites. The anti-myostatin antibodies may bind to the same epitope on the myostatin molecule, or, as with bispecific antibodies, they may bind to different epitopes on the myostatin molecule. Generally speaking, when two or more antibodies form immune complexes with two or more antigens, the resulting immune complex can strongly bind to Fc receptors present on the cell surface due to the avidity effect mediated by the Fc region of the antibodies in the complex, and can then be taken up into the cell with high efficiency. Therefore, the aforementioned anti-myostatin antibodies, which can form an immune complex containing two or more anti-myostatin antibodies and two or more myostatin molecules, can lead to rapid clearance of myostatin from plasma in the body through strong binding to Fc receptors due to the avidity effect.

[0139] Furthermore, antibodies with pH-dependent binding properties are thought to have superior characteristics in terms of antigen neutralization and clearance compared to their counterparts that bind in a pH-independent manner (Igawa et al., Nature Biotech. 28(11):1203-1207 (2010); Devanaboyina et al. mAbs 5(6):851-859 (2013); WO 2009 / 125825). Therefore, antibodies possessing both of the above characteristics, i.e., antibodies that have pH-dependent binding properties and form immune complexes containing two or more antigens and two or more antibodies, are expected to have superior characteristics in terms of very rapid antigen elimination from plasma (WO 2013 / 081143).

[0140] In one aspect, the present invention provides an anti-myostatin antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising any one amino acid sequence of SEQ ID NOs: 55-57, 114-115, and 126; (b) HVR-H2 comprising any one amino acid sequence of SEQ ID NOs: 58-60, 116-120, and 127; (c) HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 61-64, 121, and 128; (d) HVR-L1 comprising any one amino acid sequence of SEQ ID NOs: 65-69, 122-124, and 129; (e) HVR-L2 comprising any one amino acid sequence of SEQ ID NOs: 70-72, 125, and 130; and (f) HVR-L3 comprising any one amino acid sequence of SEQ ID NOs: 73-74 and 131.

[0141] In one aspect, the present invention provides an anti-myostatin antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising any one amino acid sequence of SEQ ID NOs: 55 to 57; (b) HVR-H2 comprising any one amino acid sequence of SEQ ID NOs: 58 to 60; (c) HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 61 to 64; (d) HVR-L1 comprising any one amino acid sequence of SEQ ID NOs: 65 to 69; (e) HVR-L2 comprising any one amino acid sequence of SEQ ID NOs: 70 to 72; and (f) HVR-L3 comprising any one amino acid sequence of SEQ ID NOs: 73 to 74.

[0142] In one aspect, the present invention provides an anti-myostatin antibody comprising at least one, two, three, four, five, or six hypervariable regions (HVRs) selected from: (a) HVR-H1 comprising any one of the amino acid sequences of SEQ ID NO: 114 to 115; (b) HVR-H2 comprising any one of the amino acid sequences of SEQ ID NO: 116 to 120; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) HVR-L1 comprising any one of the amino acid sequences of SEQ ID NO: 122 to 124; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 125; and (f) HVR-L3 comprising any one of the amino acid sequences of SEQ ID NO: 73 to 74.

[0143] In another aspect, the present invention provides an anti-myostatin antibody comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 114; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74. In another aspect, the present invention provides an anti-myostatin antibody comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 114; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 123; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74.

[0144] In another aspect, the present invention provides an anti-myostatin antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 126; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 127; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 128; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 130; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 131.

[0145] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising any one amino acid sequence of SEQ ID NOs: 55-57, 114-115, and 126; (b) HVR-H2 comprising any one amino acid sequence of SEQ ID NOs: 58-60, 116-120, and 127; and (c) HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 61-64, 121, and 128. In one embodiment, the antibody comprises HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 61-64, 121, and 128. In another embodiment, the antibody comprises HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 61-64, 121, and 128, and HVR-L3 comprising any one amino acid sequence of SEQ ID NOs: 73-74 and 131. In a further embodiment, the antibody comprises HVR-H3 containing one amino acid sequence of any one of SEQ ID NOs: 61-64, 121, or 128; HVR-L3 containing one amino acid sequence of any one of SEQ ID NOs: 73-74 or 131; and HVR-H2 containing one amino acid sequence of any one of SEQ ID NOs: 58-60, 116-120, or 127. In a further embodiment, the antibody comprises (a) HVR-H1 containing one amino acid sequence of any one of SEQ ID NOs: 55-57, 114-115, or 126; (b) HVR-H2 containing one amino acid sequence of any one of SEQ ID NOs: 58-60, 116-120, or 127; and (c) HVR-H3 containing one amino acid sequence of any one of SEQ ID NOs: 61-64, 121, or 128.

[0146] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising any one amino acid sequence of SEQ ID NOs: 55 to 57; (b) HVR-H2 comprising any one amino acid sequence of SEQ ID NOs: 58 to 60; and (c) HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 61 to 64. In one aspect, the antibody comprises HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 61 to 64. In another aspect, the antibody comprises HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 61 to 64 and HVR-L3 comprising any one amino acid sequence of SEQ ID NOs: 73 to 74. In a further aspect, the antibody comprises HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 61 to 64, HVR-L3 comprising any one amino acid sequence of SEQ ID NOs: 73 to 74, and HVR-H2 comprising any one amino acid sequence of SEQ ID NOs: 58 to 60. In a further embodiment, the antibody comprises (a) HVR-H1 containing any one amino acid sequence of SEQ ID NOs: 55-57, (b) HVR-H2 containing any one amino acid sequence of SEQ ID NOs: 58-60, and (c) HVR-H3 containing any one amino acid sequence of SEQ ID NOs: 61-64.

[0147] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising any one amino acid sequence of SEQ ID NO: 114 to 115; (b) HVR-H2 comprising any one amino acid sequence of SEQ ID NO: 116 to 120; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121 and HVR-L3 comprising any one amino acid sequence of SEQ ID NO: 73 to 74. In a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121, HVR-L3 comprising any one amino acid sequence of SEQ ID NO: 73 to 74, and HVR-H2 comprising any one amino acid sequence of SEQ ID NO: 116 to 120. In a further embodiment, the antibody comprises (a) HVR-H1 comprising any one amino acid sequence of SEQ ID NO: 114-115, (b) HVR-H2 comprising any one amino acid sequence of SEQ ID NO: 116-120, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121. In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 114; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74. In a further embodiment, the antibody comprises HVR-H3 having the amino acid sequence of SEQ ID NO: 63, HVR-L3 having the amino acid sequence of SEQ ID NO: 74, and HVR-H2 having the amino acid sequence of SEQ ID NO: 58. In a further embodiment, the antibody comprises (a) HVR-H1 having the amino acid sequence of SEQ ID NO: 114, (b) HVR-H2 having the amino acid sequence of SEQ ID NO: 58, and (c) HVR-H3 having the amino acid sequence of SEQ ID NO: 63.

[0148] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 126; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 127; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 128. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 128. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 128 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 131. In a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 128, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 131, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 127. In a further embodiment, the antibody comprises (a) HVR-H1 having the amino acid sequence of SEQ ID NO: 126, (b) HVR-H2 having the amino acid sequence of SEQ ID NO: 127, and (c) HVR-H3 having the amino acid sequence of SEQ ID NO: 128.

[0149] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising any one amino acid sequence of SEQ ID NOs: 65-69, 122-124, and 129; (b) HVR-L2 comprising any one amino acid sequence of SEQ ID NOs: 70-72, 125, and 130; and (c) HVR-L3 comprising any one amino acid sequence of SEQ ID NOs: 73-74 and 131. In one embodiment, the antibody comprises (a) HVR-L1 comprising any one amino acid sequence of SEQ ID NOs: 65-69, 122-124, and 129; (b) HVR-L2 comprising any one amino acid sequence of SEQ ID NOs: 70-72, 125, and 130; and (c) HVR-L3 comprising any one amino acid sequence of SEQ ID NOs: 73-74 and 131.

[0150] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising any one amino acid sequence of SEQ ID NOs: 65-69; (b) HVR-L2 comprising any one amino acid sequence of SEQ ID NOs: 70-72; and (c) HVR-L3 comprising any one amino acid sequence of SEQ ID NOs: 73-74. In one embodiment, the antibody comprises (a) HVR-L1 comprising any one amino acid sequence of SEQ ID NOs: 65-69; (b) HVR-L2 comprising any one amino acid sequence of SEQ ID NOs: 70-72; and (c) HVR-L3 comprising any one amino acid sequence of SEQ ID NOs: 73-74.

[0151] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising any one amino acid sequence of SEQ ID NO: 122 to 124; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 125; and (c) HVR-L3 comprising any one amino acid sequence of SEQ ID NO: 73 to 74. In one embodiment, the antibody comprises (a) HVR-L1 comprising any one amino acid sequence of SEQ ID NO: 122 to 124; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 125; and (c) HVR-L3 comprising any one amino acid sequence of SEQ ID NO: 73 to 74. In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74. In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 123; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74. In one embodiment, the antibody comprises (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 123, (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 71, and (c) HVR-L3 containing the amino acid sequence of SEQ ID NO: 74.

[0152] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 130; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 131. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 130, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 131.

[0153] In another aspect, the antibody of the present invention is a VH domain comprising at least one, at least two, or all three VH domains selected from (i) HVR-H1 comprising one amino acid sequence of any one of SEQ ID NOs: 55-57, 114, 115, or 126; (ii) HVR-H2 comprising one amino acid sequence of any one of SEQ ID NOs: 58-60, 116-120, or 127; and (iii) HVR-H3 comprising one amino acid sequence of any one of SEQ ID NOs: 61-64, 121, or 128. (b) a VH domain containing an HVR sequence; and a VL domain containing at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 containing one amino acid sequence of sequence numbers 65-69, 122-124, and 129; (ii) HVR-L2 containing one amino acid sequence of sequence numbers 70-72, 125, and 130; and (iii) HVR-L3 containing one amino acid sequence of sequence numbers 73-74 and 131.

[0154] In another aspect, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising any one amino acid sequence of SEQ ID NOs: 55 to 57, (ii) HVR-H2 comprising any one amino acid sequence of SEQ ID NOs: 58 to 60, and (iii) HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 61 to 64; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising any one amino acid sequence of SEQ ID NOs: 65 to 69, (ii) HVR-L2 comprising any one amino acid sequence of SEQ ID NOs: 70 to 72, and (iii) HVR-L3 comprising any one amino acid sequence of SEQ ID NOs: 73 to 74.

[0155] In another aspect, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising any one amino acid sequence of SEQ ID NO: 114 to 115, (ii) HVR-H2 comprising any one amino acid sequence of SEQ ID NO: 116 to 120, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising any one amino acid sequence of SEQ ID NO: 122 to 124, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 125, and (iii) HVR-L3 comprising any one amino acid sequence of SEQ ID NO: 73 to 74. In another aspect, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 114, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74. In another aspect, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 114, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 123, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74.

[0156] In another aspect, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 126, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 127, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 128; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 130, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 131.

[0157] In another aspect, the present invention provides antibodies comprising (a) HVR-H1 comprising any one amino acid sequence of SEQ ID NOs: 55-57, 114-115, and 126; (b) HVR-H2 comprising any one amino acid sequence of SEQ ID NOs: 58-60, 116-120, and 127; (c) HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 61-64, 121, and 128; (d) HVR-L1 comprising any one amino acid sequence of SEQ ID NOs: 65-69, 122-124, and 129; (e) HVR-L2 comprising any one amino acid sequence of SEQ ID NOs: 70-72, 125, and 130; and (f) HVR-L3 comprising any one amino acid sequence of SEQ ID NOs: 73-74 and 131.

[0158] In another aspect, the present invention provides antibodies comprising (a) HVR-H1 comprising any one amino acid sequence of SEQ ID NOs: 55-57 and 114-115; (b) HVR-H2 comprising any one amino acid sequence of SEQ ID NOs: 58-60 and 116-120; (c) HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 61-64 and 121; (d) HVR-L1 comprising any one amino acid sequence of SEQ ID NOs: 65-69 and 122-124; (e) HVR-L2 comprising any one amino acid sequence of SEQ ID NOs: 70-72 and 125; and (f) HVR-L3 comprising any one amino acid sequence of SEQ ID NOs: 73-74.

[0159] In another aspect, the present invention provides antibodies comprising (a) HVR-H1 comprising any one amino acid sequence of SEQ ID NOs: 114 to 115; (b) HVR-H2 comprising any one amino acid sequence of SEQ ID NOs: 116 to 120; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NOs: 121; (d) HVR-L1 comprising any one amino acid sequence of SEQ ID NOs: 122 to 124; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NOs: 125; and (f) HVR-L3 comprising any one amino acid sequence of SEQ ID NOs: 73 to 74. In another aspect, the present invention provides antibodies comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 114; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74. In another aspect, the present invention provides antibodies comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 114; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 123; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74.

[0160] In another aspect, the present invention provides antibodies comprising (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 126; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 127; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 128; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 129; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 130; and (f) HVR-L3 containing the amino acid sequence of SEQ ID NO: 131.

[0161] In a particular embodiment, one or more amino acids of the above-mentioned anti-myostatin antibody are substituted at the following HVR positions: (a) in HVR-H1 (SEQ ID NO: 55): positions 1 and 2; (b) in HVR-H2 (SEQ ID NO: 58): positions 4, 7, 8, 10, 11, 12, and 16; (c) in HVR-H3 (SEQ ID NO: 61): positions 5, 7, and 11; (d) in HVR-L1 (SEQ ID NO: 65): positions 1, 2, 5, 7, 8, and 9; (e) in HVR-L2 (SEQ ID NO: 70): positions 3 and 7; and (f) in HVR-L3 (SEQ ID NO: 73): position 8.

[0162] In certain embodiments, any one or more amino acid substitutions in the anti-myostatin antibodies provided herein are conservative substitutions. In certain embodiments, one or more of the following substitutions may be made in any combination: (a) in HVR-H1 (sequence number: 55): S1H;Y2T, D, or E; (b) in HVR-H2 (sequence number: 58): Y4H;S7K;T8M or K;Y10K;A11M or E;S12E;G16K; (c) in HVR-H3 (sequence number: 61): Y5H;T7H;L11K; (d) in HVR-L1 (sequence number: 65): Q1T;S2T;S5E;Y7F;D8H;N9D or A or E; (e) in HVR-L2 (sequence number: 70): S3E;S7Y, F, or W; and (f) in HVR-L3 (sequence number: 73): L8R.

[0163] All possible substitution combinations described above are included in the consensus sequences of sequence numbers 126, 127, 128, 129, 130, and 131 for HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, respectively.

[0164] In any of the embodiments described above, the anti-myostatin antibody can be humanized. In one embodiment, the anti-myostatin antibody comprises HVR in any of the embodiments described above and further comprises an acceptor human framework (e.g., a human immunoglobulin framework or a human consensus framework). In another embodiment, the anti-myostatin antibody comprises HVR in any of the embodiments described above and further comprises VH or VL containing an FR sequence. In a further embodiment, the anti-myostatin antibody comprises the following heavy chain and / or light chain variable domain FR sequences: for the heavy chain variable domain, FR1 comprises one amino acid sequence of SEQ ID NOs: 132-134, FR2 comprises one amino acid sequence of SEQ ID NOs: 135-136, FR3 comprises the amino acid sequence of SEQ ID NO: 137, and FR4 comprises the amino acid sequence of SEQ ID NO: 138. Regarding the light chain variable domains, FR1 contains the amino acid sequence of SEQ ID NO: 139, FR2 contains one amino acid sequence from SEQ ID NO: 140 to 141, FR3 contains one amino acid sequence from SEQ ID NO: 142 to 143, and FR4 contains the amino acid sequence of SEQ ID NO: 144.

[0165] In one aspect, the present invention provides an anti-myostatin antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising any one amino acid sequence of SEQ ID NOs: 157 to 162; (b) HVR-H2 comprising any one amino acid sequence of SEQ ID NOs: 163 to 168; (c) HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 169 to 174; (d) HVR-L1 comprising any one amino acid sequence of SEQ ID NOs: 175 to 180; (e) HVR-L2 comprising any one amino acid sequence of SEQ ID NOs: 181 to 186; and (f) HVR-L3 comprising any one amino acid sequence of SEQ ID NOs: 187 to 192.

[0166] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising any one amino acid sequence of SEQ ID NOs: 157 to 162; (b) HVR-H2 comprising any one amino acid sequence of SEQ ID NOs: 163 to 168; and (c) HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 169 to 174. In one embodiment, the antibody comprises HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 169 to 174. In another embodiment, the antibody comprises HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 169 to 174 and HVR-L3 comprising any one amino acid sequence of SEQ ID NOs: 187 to 192. In a further embodiment, the antibody comprises HVR-H3 containing any one amino acid sequence of SEQ ID NOs: 169-174, HVR-L3 containing any one amino acid sequence of SEQ ID NOs: 187-192, and HVR-H2 containing any one amino acid sequence of SEQ ID NOs: 163-168. In a further embodiment, the antibody comprises (a) HVR-H1 containing any one amino acid sequence of SEQ ID NOs: 157-162, (b) HVR-H2 containing any one amino acid sequence of SEQ ID NOs: 163-168, and (c) HVR-H3 containing any one amino acid sequence of SEQ ID NOs: 169-174.

[0167] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising any one amino acid sequence of SEQ ID NOs: 175-180; (b) HVR-L2 comprising any one amino acid sequence of SEQ ID NOs: 181-186; and (c) HVR-L3 comprising any one amino acid sequence of SEQ ID NOs: 187-192. In one embodiment, the antibody comprises (a) HVR-L1 comprising any one amino acid sequence of SEQ ID NOs: 175-180; (b) HVR-L2 comprising any one amino acid sequence of SEQ ID NOs: 181-186; and (c) HVR-L3 comprising any one amino acid sequence of SEQ ID NOs: 187-192.

[0168] In another aspect, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising any one amino acid sequence of SEQ ID NO: 157 to 162, (ii) HVR-H2 comprising any one amino acid sequence of SEQ ID NO: 163 to 168, and (iii) HVR-H3 comprising any one amino acid sequence of SEQ ID NO: 169 to 174; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising any one amino acid sequence of SEQ ID NO: 175 to 180, (ii) HVR-L2 comprising any one amino acid sequence of SEQ ID NO: 181 to 186, and (iii) HVR-L3 comprising any one amino acid sequence of SEQ ID NO: 187 to 192.

[0169] In another aspect, the present invention provides antibodies comprising (a) HVR-H1 comprising any one amino acid sequence of SEQ ID NOs: 157 to 162; (b) HVR-H2 comprising any one amino acid sequence of SEQ ID NOs: 163 to 168; (c) HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 169 to 174; (d) HVR-L1 comprising any one amino acid sequence of SEQ ID NOs: 175 to 180; (e) HVR-L2 comprising any one amino acid sequence of SEQ ID NOs: 181 to 186; and (f) HVR-L3 comprising any one amino acid sequence of SEQ ID NOs: 187 to 192.

[0170] In another aspect, an anti-myostatin antibody contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one amino acid sequence of SEQ ID NOs: 13, 16-30, 32-34, and 86-95. In a particular embodiment, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the reference sequence includes substitutions (e.g., conservative substitutions), insertions, or deletions, but the anti-myostatin antibody containing such sequence retains the ability to bind to myostatin. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in one of the sequence numbers: 13, 16-30, 32-34, and 86-95. In certain embodiments, the substitution, insertion, or deletion occurs in the region outside the HVR (i.e., within the FR). Optionally, the anti-myostatin antibody contains the VH sequence in one of the sequence numbers: 13, 16-30, 32-34, and 86-95, including post-translational modifications of said sequence. In certain embodiments, VH comprises one, two, or three HVRs selected from (a) HVR-H1 containing one amino acid sequence of any one of SEQ ID NOs: 55-57, 114-115, or 126; (b) HVR-H2 containing one amino acid sequence of any one of SEQ ID NOs: 58-60, 116-120, or 127; and (c) HVR-H3 containing one amino acid sequence of any one of SEQ ID NOs: 61-64, 121, or 128. Post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamate at the N-terminus of the heavy or light chain.

[0171] In another aspect, an anti-myostatin antibody contains a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to one of the amino acid sequences of SEQ ID NOs: 13, 16-30, 32, 33, and 34. In a particular embodiment, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the reference sequence includes substitutions (e.g., conservative substitutions), insertions, or deletions, but the anti-myostatin antibody containing such sequence retains the ability to bind to myostatin. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in one of the SEQ ID NOs: 13, 16-30, 32, 33, and 34. In certain embodiments, substitutions, insertions, or deletions occur in the outer region of the HVR (i.e., within the FR). Optionally, the anti-myostatin antibody includes the VH sequence in any one of SEQ ID NOs: 13, 16-30, 32, 33, and 34, including post-translational modifications of said sequence. In certain embodiments, the VH includes one, two, or three HVRs selected from (a) HVR-H1 containing any one amino acid sequence of SEQ ID NOs: 55-57, (b) HVR-H2 containing any one amino acid sequence of SEQ ID NOs: 58-60, and (c) HVR-H3 containing any one amino acid sequence of SEQ ID NOs: 61-64. Post-translational modifications include, but are not limited to, modifications to pyroglutamic acid by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0172] In another aspect, the anti-myostatin antibody contains a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one amino acid sequence of SEQ ID NOs: 86-95. In a particular embodiment, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions with respect to the reference sequence, but the anti-myostatin antibody containing such sequence retains the ability to bind to myostatin. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in any one of SEQ ID NOs: 86-95. In a particular embodiment, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., within the FR). Optionally, the anti-myostatin antibody includes the VH sequence in any one of SEQ ID NOs: 86-95, including post-translational modifications of said sequence. In certain embodiments, the VH includes one, two, or three HVRs selected from (a) HVR-H1 containing any one amino acid sequence of SEQ ID NOs: 57, 114-115, or 126; (b) HVR-H2 containing any one amino acid sequence of SEQ ID NOs: 58, 116-120, or 127; and (c) HVR-H3 containing any one amino acid sequence of SEQ ID NOs: 63, 121, or 128. Post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain. In another aspect, the anti-myostatin antibody contains a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 86. In a particular embodiment, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the reference sequence includes substitutions (e.g., conservative substitutions), insertions, or deletions, but the anti-myostatin antibody containing such sequence retains the ability to bind to myostatin.In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 86. In certain embodiments, the substitution, insertion, or deletion occurs in the outer region of the HVR (i.e., within the FR). Optionally, the anti-myostatin antibody includes the VH sequence in SEQ ID NO: 86, including those with post-translational modifications of said sequence. In certain embodiments, the VH includes one, two, or three HVRs selected from (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 114, (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 58, and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 63. Post-translational modifications include, but are not limited to, modifications to pyroglutamic acid by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain. In another aspect, the anti-myostatin antibody contains a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 92. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the reference sequence includes substitutions (e.g., conservative substitutions), insertions, or deletions, but the anti-myostatin antibody containing such sequence retains the ability to bind to myostatin. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 92. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., within the FR). Optionally, the anti-myostatin antibody includes the VH sequence in SEQ ID NO: 92, including the sequence with post-translational modifications. In certain embodiments, VH includes one, two, or three HVRs selected from (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 114, (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 58, and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 63. Post-translational modifications include, but are not limited to, modifications to pyroglutamic acid by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0173] In another aspect, an anti-myostatin antibody is provided, comprising a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to one of the amino acid sequences of SEQ ID NOs: 15, 31, 35-38, and 96-99. In a particular embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the reference sequence includes substitutions (e.g., conservative substitutions), insertions, or deletions, but the anti-myostatin antibody comprising such sequence retains the ability to bind to myostatin. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in one of the SEQ ID NOs: 15, 31, 35-38, and 96-99. In certain embodiments, substitutions, insertions, or deletions occur in the outer region of the HVR (i.e., within the FR). Optionally, the anti-myostatin antibody includes the VL sequence in any one of SEQ ID NOs: 15, 31, 35-38, and 96-99, including post-translational modifications of said sequence. In certain embodiments, the VL includes one, two, or three HVRs selected from (a) HVR-L1 containing any one amino acid sequence of SEQ ID NOs: 65-69, 122-124, and 129; (b) HVR-L2 containing any one amino acid sequence of SEQ ID NOs: 70-72, 125, and 130; and (c) HVR-L3 containing any one amino acid sequence of SEQ ID NOs: 73-74 and 131. Post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamate at the N-terminus of the heavy or light chain.

[0174] In another aspect, an anti-myostatin antibody is provided, comprising a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to any one amino acid sequence of SEQ ID NOs: 15, 31, 35, 36, 37, and 38. In a particular embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the reference sequence includes substitutions (e.g., conservative substitutions), insertions, or deletions, but the anti-myostatin antibody comprising such sequence retains the ability to bind to myostatin. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in any one of SEQ ID NOs: 15, 31, 35, 36, 37, and 38. In certain embodiments, substitutions, insertions, or deletions occur in the outer region of the HVR (i.e., within the FR). Optionally, the anti-myostatin antibody includes the VL sequence in any one of SEQ ID NOs: 15, 31, 35, 36, 37, and 38, including post-translational modifications of said sequence. In certain embodiments, the VL includes one, two, or three HVRs selected from (a) HVR-L1 containing any one amino acid sequence of SEQ ID NOs: 65-69, (b) HVR-L2 containing any one amino acid sequence of SEQ ID NOs: 70-72, and (c) HVR-L3 containing any one amino acid sequence of SEQ ID NOs: 73-74. Post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0175] In another aspect, an anti-myostatin antibody is provided, comprising a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one amino acid sequence of SEQ ID NOs: 96-99. In a particular embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions with respect to the reference sequence, but the anti-myostatin antibody comprising such sequence retains the ability to bind to myostatin. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in any one of SEQ ID NOs: 96-99. In a particular embodiment, the substitutions, insertions, or deletions occur in the region outside the HVR. Optionally, the anti-myostatin antibody includes the VL sequence in any one of SEQ ID NOs: 96-99, including post-translational modifications of said sequence. In certain embodiments, the VL includes one, two, or three HVRs selected from (a) HVR-L1 containing the amino acid sequence in any one of SEQ ID NOs: 122-124, 129; (b) HVR-L2 containing the amino acid sequence in any one of SEQ ID NOs: 71, 125, 130; and (c) HVR-L3 containing the amino acid sequences in SEQ ID NOs: 74, 131. Post-translational modifications include, but are not limited to, modifications to pyroglutamic acid by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain. In another aspect, an anti-myostatin antibody is provided, comprising a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 96. In a particular embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the reference sequence includes substitutions (e.g., conservative substitutions), insertions, or deletions, but the anti-myostatin antibody comprising such sequence retains the ability to bind to myostatin. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 96.In certain embodiments, substitutions, insertions, or deletions occur in the outer region of the HVR. Optionally, the anti-myostatin antibody includes the VL sequence in SEQ ID NO: 96, including those with post-translational modifications of said sequence. In certain embodiments, the VL includes one, two, or three HVRs selected from (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 122, (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 71, and (c) HVR-L3 containing the amino acid sequence of SEQ ID NO: 74. Post-translational modifications include, but are not limited to, modifications to pyroglutamic acid by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain. In another aspect, an anti-myostatin antibody is provided, comprising a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 97. In a particular embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the reference sequence includes substitutions (e.g., conservative substitutions), insertions, or deletions, but the anti-myostatin antibody comprising such sequence retains the ability to bind to myostatin. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 97. In a particular embodiment, the substitutions, insertions, or deletions occur in the region outside the HVR. Optionally, the anti-myostatin antibody includes the VL sequence in SEQ ID NO: 97, including the sequence with post-translational modifications. In certain embodiments, the VL includes one, two, or three HVRs selected from (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 123, (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 71, and (c) HVR-L3 containing the amino acid sequence of SEQ ID NO: 74. Post-translational modifications include, but are not limited to, modifications to pyroglutamic acid by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0176] In another aspect, an anti-myostatin antibody is provided comprising VH in any of the embodiments described above, and VL in any of the embodiments described above. In one embodiment, the antibody comprises the VH and VL sequences in any one of SEQ ID NOs: 13, 16-30, 32-34, and 86-95, and any one of SEQ ID NOs: 15, 31, 35-38, and 96-99, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in any one of SEQ ID NOs: 13, 16-30, and 32-34, and any one of SEQ ID NOs: 15, 31, and 35-38, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in any one of SEQ ID NOs: 86-95 and any one of SEQ ID NOs: 96-99, including post-translational modifications of said sequences. Post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamate at the N-terminus of the heavy or light chain.

[0177] In another aspect, an anti-myostatin antibody is provided comprising VH in any of the above embodiments and VL in any of the above embodiments. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 86 and SEQ ID NO: 96, respectively, including those with post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 92 and SEQ ID NO: 97, respectively, including those with post-translational modifications of said sequences. Post-translational modifications include, but are not limited to, modification to pyroglutamic acid by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0178] In another aspect, an anti-myostatin antibody contains a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one amino acid sequence of SEQ ID NOs: 12, 145-150. In a particular embodiment, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions with respect to the reference sequence, but the anti-myostatin antibody containing such sequence retains the ability to bind to myostatin. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in any one of SEQ ID NOs: 12, 145-150. In certain embodiments, substitutions, insertions, or deletions occur in the outer region of the HVR (i.e., within the FR). Optionally, the anti-myostatin antibody includes the VH sequence in any one of SEQ ID NOs: 12, 145–150, including post-translational modifications of said sequence. In certain embodiments, the VH includes one, two, or three HVRs selected from (a) HVR-H1 containing any one amino acid sequence of SEQ ID NOs: 55, 157–162, (b) HVR-H2 containing any one amino acid sequence of SEQ ID NOs: 58, 163–168, and (c) HVR-H3 containing any one amino acid sequence of SEQ ID NOs: 61, 169–174. Post-translational modifications include, but are not limited to, modifications to pyroglutamic acid by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0179] In another aspect, an anti-myostatin antibody is provided comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one amino acid sequence of SEQ ID NOs: 14, 151-156. In a particular embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions with respect to the reference sequence, but the anti-myostatin antibody comprising such sequence retains the ability to bind to myostatin. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in any one of SEQ ID NOs: 14, 151-156. In certain embodiments, substitutions, insertions, or deletions occur in the outer region of the HVR (i.e., within the FR). Optionally, the anti-myostatin antibody includes the VL sequence in any one of SEQ ID NOs: 14, 151-156, including post-translational modifications of said sequence. In certain embodiments, the VL includes one, two, or three HVRs selected from (a) HVR-L1 containing any one amino acid sequence of SEQ ID NOs: 65, 175-180, (b) HVR-L2 containing any one amino acid sequence of SEQ ID NOs: 70, 181-186, and (c) HVR-L3 containing any one amino acid sequence of SEQ ID NOs: 73, 187-192. Post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0180] In another aspect, an anti-myostatin antibody is provided comprising VH in any of the above embodiments and VL in any of the above embodiments. In one embodiment, the antibody comprises the VH and VL sequences in any one of SEQ ID NOs: 12, 145-150 and any one of SEQ ID NOs: 14, 151-156, including post-translational modifications of said sequences. Post-translational modifications include, but are not limited to, modifications to pyroglutamic acid by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0181] In a particular embodiment, the anti-myostatin antibody of the present invention comprises a heavy chain constant region comprising VH in any of the embodiments described above, and one amino acid sequence of any one of SEQ ID NOs: 7, 9, 11, 193, 195-198, 227, 228, 229-381. In a particular embodiment, the anti-myostatin antibody of the present invention comprises a light chain constant region comprising VL in any of the embodiments described above, and one amino acid sequence of any one of SEQ ID NOs: 8 and 10.

[0182] In a further aspect, the present invention provides antibodies that bind to the same epitopes as the anti-myostatin antibodies provided herein. In a further aspect, the present invention provides antibodies that bind to the same epitopes as the antibodies listed in Table 2a. In a further aspect, the present invention provides antibodies that bind to the same epitopes as the antibodies listed in Table 11a or 13. In a particular embodiment, an antibody is provided that binds to an epitope in a fragment of myostatin propeptide consisting of amino acids 21-100 of SEQ ID NO: 78. Alternatively, the antibody binds to a myostatin propeptide fragment consisting of amino acids 21-80, 41-100, 21-60, 41-80, 61-100, 21-40, 41-60, 61-80, or 81-100 of SEQ ID NO: 78.

[0183] In a further aspect of the present invention, the anti-myostatin antibody according to any of the above embodiments is a monoclonal antibody comprising a chimeric, humanized, or human antibody. In one embodiment, the anti-myostatin antibody is an antibody fragment, such as Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length IgG antibody, such as a complete IgG1 or IgG4 antibody, or any other antibody class or isotype as defined herein.

[0184] In further contexts, anti-myostatin antibodies in any of the above embodiments may, alone or in combination, incorporate any of the characteristics described in items 1 to 7 below.

[0185] 1. Affinity of the antibody In certain embodiments, the antibodies provided herein have a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M).

[0186] In one embodiment, the Kd is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using the Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is measured by equilibrating the Fab with a minimum concentration of ( 125 I) radiolabeled antigen in the presence of a serial increasing amount of unlabeled antigen and then capturing the bound antigen with a plate coated with an anti-Fab antibody. (See, e.g., Chen et al., J. Mol. Biol. 293:865 - 881 (1999)). To construct the measurement conditions, a MICROTITER® multiwell plate (Thermo Scientific) is coated overnight with 5 μg / ml of the capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then blocked with 2% (w / v) bovine serum albumin in PBS for 2 - 5 hours at room temperature (approx. 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM of 125Mix the [I]-antigen with serial dilutions of the Fab of interest (e.g., as in the evaluation of anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). Then incubate the Fab of interest overnight, although this incubation may be extended for a longer period (e.g., about 65 hours) to ensure equilibrium is achieved. Subsequently, transfer the mixture to a capture plate for incubation at room temperature (e.g., 1 hour). Then remove the solution and wash the plate eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate is dry, add 150 μl / well of scintillant (MICROSCINT-20®, Packard) and count the plate for 10 minutes on a TOPCOUNT® gamma counter (Packard). Select concentrations of each Fab that give less than 20% of maximum binding for use in competitive binding assays.

[0187] In another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, the assay using BIACORE®-2000 or BIACORE®-3000 (BIACORE®, Inc., Piscataway, NJ) is performed at 25°C using a CM5 chip immobilized with approximately 10 response units (RUs) of antigen. In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE®, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8 before being injected at a flow rate of 5 μl / min to achieve binding of approximately 10 response units (RUs) of protein. After antigen injection, 1M ethanolamine is injected to block unreacted groups. For reaction rate measurement, two-fold serial dilutions (0.78 nM to 500 nM) of Fab in PBS (PBST) containing 0.05% polysorbate 20 (TWEEN-20®) surfactant are injected at 25°C and a flow rate of approximately 25 μl / min. Binding rate (k on ) and dissociation rate (k off ) is calculated by simultaneously fitting the coupling and dissociation sensorgrams using a simple one-to-one Langmuir coupling model (BIACORE® evaluation software version 3.2). The equilibrium dissociation constant (Kd) is given by k off / k on It is calculated as a ratio. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). The on velocity is 10 by the surface plasmon resonance assay described above. 6 M -1 s -1If it exceeds this, the ON rate can be determined using fluorescence quenching techniques, which measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, bandpass 16 nm) at 25°C in the presence of gradually increasing concentrations of antigen, using a spectrometer (e.g., a stop-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO® spectrophotometer (ThermoSpectronic) using a stirred cuvette).

[0188] 2. Antibody fragment In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described below. For a review of specific antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp.269-315 (1994); in addition, see WO1993 / 16185; and U.S. Patents 5,571,894 and 5,587,458. For a discussion on the Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and exhibiting extended in vivo half-lives, see U.S. Patent No. 5,869,046.

[0189] A diabody is an antibody fragment containing two antigen-binding sites, which may be bivalent or bispecific. See, for example, EP404,097; WO1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0190] A single-domain antibody is an antibody fragment containing all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (see, for example, Domantis, Inc., Waltham, MA; U.S. Patent No. 6,248,516B1).

[0191] Antibody fragments can be produced by various methods, including, but are not limited to, the proteolytic digestion of complete antibodies and production by recombinant host cells (e.g., Escherichia coli or phages) as described herein.

[0192] 3. Chimeric and humanized antibodies In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and in Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody includes a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In further examples, a chimeric antibody is a “class-switched” antibody in which the class or subclass of the parent antibody has been changed. A chimeric antibody also includes its antigen-binding fragment.

[0193] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity to humans while maintaining the specificity and affinity of the parent non-human antibody. A humanized antibody usually contains one or more variable domains, in which the HVR (e.g., CDR (or a portion thereof)) is derived from the non-human antibody and the FR (or a portion thereof) is derived from the human antibody sequence. The humanized antibody optionally contains at least a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from the non-human antibody (e.g., the antibody from which the HVR residues originated) to restore or improve the specificity or affinity of the antibody, for example.

[0194] Humanized antibodies and their preparation methods have been reviewed in Almagro, Front. Biosci. 13:1619-1633 (2008), and also in, for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patents No. 5,821,337, No. 7,527,791, No. 6,982,321, and No. 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describes specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describes resurfacing); Further details can be found in Dall'Acqua et al., Methods 36:43-60 (2005) (which describes FR shuffling), as well as in Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (which describes a "guided selection" approach for FR shuffling).

[0195] The human framework regions that can be used for humanization are not limited to these, but include: framework regions selected using the "best fit" method (see Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subgroups of light chain or heavy chain variable regions (see Carter et al. Proc. Natl. Acad. Sci. USA 89:4285 (1992) and Presta et al. J. Immunol. 151:2623 (1993)); human maturation (somatic mutation) framework regions or human germline framework regions (see, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and (See Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0196] 4. Human antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced by various methods known in the art. Human antibodies are outlined in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-374 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

[0197] Human antibodies may be prepared by administering immunogens to transgenic animals modified to produce fully human antibodies or fully human antibodies with human variable regions in response to antigen challenge (loading). Such animals typically contain all or part of a human immunoglobulin locus, which either replaces an endogenous immunoglobulin locus or is randomly incorporated extrachromosomally or within the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin locus is usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE® technology; U.S. Patent No. 5,770,429 describing HUMAB® technology; U.S. Patent No. 7,041,870 describing KM MOUSE® technology; and U.S. Patent Application Publication 2007 / 0061900 describing VELOCIMOUSE® technology. Human variable regions from complete antibodies produced by such animals may be further modified, for example, by combining them with different human constant regions.

[0198] Human antibodies can also be produced using hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have already been described. (See, for example, Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol. 147: 86 (1991).) Human antibodies produced via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006). Additional methods include, for example, those described in U.S. Patent No. 7,189,826 (describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describes human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-191 (2005).

[0199] Human antibodies can also be generated by isolating selected Fv clone variable domain sequences from a human-derived phage display library. Such variable domain sequences can then be combined with a desired human constant domain. A method for selecting human antibodies from an antibody library is described below.

[0200] 5. Library-derived antibodies The antibodies of the present invention may be isolated by screening a combinatorial library for antibodies exhibiting one or more desired activities. For example, various methods are known in the art for generating phage display libraries and for screening such libraries for antibodies possessing desired binding properties. Such methods have been reviewed in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (2000); O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further, for example, McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. This is described in Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

[0201] In certain phage display methods, the VH and VL gene repertoires are cloned separately by polymerase chain reaction (PCR), randomly recombined in a phage library, and screened for antigen-binding phages as described in Winter et al., Ann. Rev. Immunol. 12: 433-455 (1994). The phages typically present antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies against the immunosource without requiring the construction of hybridomas. Alternatively, as described in Griffiths et al., EMBO J, 12: 725-734 (1993), naive repertoires (e.g., from humans) can be cloned to provide a single source of antibodies against a wide range of non-self and self-antigens without immunization. Finally, naive libraries can also be synthesized synthetically, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992), by cloning the pre-reorganization V-gene segment from stem cells and using PCR primers containing random sequences that encode the hypervariable CDR3 region and achieve in vitro rearrangement. Patent documents describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, as well as U.S. Patent Application Publications 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0202] Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments in this specification.

[0203] 6. Multispecific antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies (e.g., bispecific antibodies). A multispecific antibody is a monoclonal antibody having binding specificity to at least two different sites. In certain embodiments, one binding specificity is to myostatin and the other is to any other antigen. In certain embodiments, a bispecific antibody may bind to two different epitopes of myostatin. A bispecific antibody may be used to localize a cytotoxic agent to cells expressing myostatin. A bispecific antibody may be prepared as a full-length antibody or as an antibody fragment.

[0204] Methods for producing multispecific antibodies are not limited to these, but include, the recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO 1993 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and knob-in-hole techniques (see, for example, U.S. Patent No. 5,731,168). Multispecific antibodies can be produced by manipulating electrostatic steering effects to create Fc heterodimer molecules (WO 2009 / 089004A1); crosslinking two or more antibodies or fragments (see U.S. Patent No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); creating antibodies with two specificities using a leucine zipper (see Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)); producing bispecific antibody fragments using "diabody" technology (see Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (Gruber et al., J. Immunol. 152:5368). (See 1994); and may also be prepared by preparing a trispecific antibody, for example, as described in Tutt et al., J. Immunol. 147: 60 (1991).

[0205] Modified antibodies containing three or more functional antigen-binding sites, including "octopus antibodies," are also included herein (see, for example, U.S. Patent Application Publication 2006 / 0025576A1).

[0206] In this specification, an antibody or fragment also includes a “dual-acting Fab” or “DAF” comprising one antigen-binding site that binds to myostatin and another different antigen (see, for example, U.S. Patent Application Publication No. 2008 / 0069820).

[0207] 7. Antibody variants In certain embodiments, amino acid sequence variants of antibodies provided herein are also considered. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies may be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from the amino acid sequence of the antibody, and / or insertions into the amino acid sequence of the antibody, and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be performed to arrive at the final construct, provided that the final construct possesses the desired characteristics (e.g., antigen-binding ability).

[0208] a. Substitution, insertion, and deletion variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. The target sites for substitutional mutagenesis include HVR and FR. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions." More substantial modifications are provided in Table 1 under the heading "Exemplary Substitutions" and are described in detail below, with reference to the classes of amino acid side chains. Amino acid substitutions may be introduced into the antibody of interest, and the product may be screened for desired activity, such as retained / improved antigen-binding, reduced immunogenicity, or improved ADCC or CDC. [Table 1] TIFF0007829387000001.tif131148

[0209] Amino acids can be grouped according to their common side-chain characteristics: (1) Hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile); (2) Neutral hydrophilic: cysteine ​​(Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln); (3) Acidic: Aspartic acid (Asp), glutamic acid (Glu); (4) Basic: histidine (His), lysine (Lys), arginine (Arg); (5) Residues that affect chain orientation: glycine (Gly), proline (Pro); and (6) Aromatic: Tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe). A non-conservative permutation is the exchange of one member of one group for a member of another group.

[0210] One type of substitution mutant involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting mutants, and those selected for further study, will have modifications (e.g., improvements) in specific biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody, and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitution mutant is an affinity-matured antibody, which can be appropriately produced using, for example, a phage display-based affinity-mature technique (e.g., one described herein). Briefly, one or more HVR residues are mutated, and the mutant antibody is displayed on a phage and screened for specific biological activity (e.g., binding affinity).

[0211] Modifications (e.g., substitutions) may be made in HVRs, for example, to improve antibody affinity. Such modifications may be made in HVR "hot spots," i.e., residues encoded by codons that frequently mutate during the somatic cell maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or residues that come into contact with the antigen, and the resulting mutant VH or VL may be tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into variable genes selected for maturation by any variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). Next, a secondary library is prepared. This library is then screened to identify any antibody variant with the desired affinity. Another method for introducing diversity involves an HVR-directed approach that randomizes several HVR residues (e.g., 4-6 residues at a time). HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0212] In certain embodiments, substitutions, insertions, or deletions may be made within one or more HVRs, provided that such modifications do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative modifications that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made within an HVR. Such modifications may be, for example, outside the antigen-contact residue of the HVR. In certain embodiments of the mutant VH and VL sequences described above, each HVR is either unmodified or contains only one, two, or three amino acid substitutions.

[0213] A useful method for identifying antibody residues or regions that can be targeted for mutational introduction is called "alanine scanning mutagenesis," described by Cunningham and Wells, (1989) Science, 244:1081-1085. In this method, one or a group of target residues (e.g., charged residues, e.g., arginine, aspartic acid, histidine, lysine, and glutamic acid) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to this initial substitution. Alternatively, the crystal structure of the antigen-antibody complex may be analyzed to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted as substitution candidates or excluded from the list of substitution candidates. Mutants may be screened to determine whether they possess the desired properties.

[0214] Amino acid sequence insertions include not only the insertion of single or multiple amino acid residues within a sequence, but also the fusion of polypeptides ranging in length from one to over 100 residues at the amino and / or carboxyl terminals. An example of terminal insertion is an antibody with a methionyl residue at the N-terminus. Other insertion variants of antibody molecules include those in which a polypeptide that increases the blood half-life of an enzyme (e.g., for ADEPT) or an antibody is fused to the N- or C-terminus of the antibody.

[0215] b. Glycosylated mutants In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. Adding or removing glycosylation sites to an antibody can be easily achieved by modifying the amino acid sequence to create or remove one or more glycosylation sites.

[0216] If the antibody contains an Fc region, the carbohydrate to which it is attached may be modified. Native antibodies produced by mammalian cells typically contain branched oligosaccharides, which are usually attached to Asn297 of the CH2 domain of the Fc region by N-linkage. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the branched oligosaccharide structure. In some embodiments, the modification of the oligosaccharide in the antibody of the present invention may be carried out to produce antibody variants with specific improved properties.

[0217] In one embodiment, antibody variants are provided having a carbohydrate structure lacking fucose (directly or indirectly) attached to the Fc region. For example, the amount of fucose in such an antibody may be 1%–80%, 1%–65%, 5%–65%, or 20%–40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all sugar structures (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, measured by MALDI-TOF mass spectrometry, as described, for example, in WO 2008 / 077546. Asn297 represents an asparagine residue located around position 297 of the Fc region (EU numbering of Fc region residues). However, due to slight sequence variability among multiple antibodies, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, for example, U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L.) and No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications concerning "defucosylated" or "fucose-deficient" antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; WO 2002 / 031140; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication US 2003 / 0157108A1, Presta, L; and WO 2004 / 056312, Adams et al., particularly Example 11) and knockout cell lines, such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda et al., Biotechnol. Bioeng. 94(4):680-688 (2006); and WO 2003 / 085107).

[0218] Further antibody variants are provided having a bifid oligosaccharide, for example, in which a bifid branched oligosaccharide attached to the Fc region of the antibody is bifid by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and U.S.2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.); WO1998 / 58964 (Raju, S.); and WO1999 / 22764 (Raju, S.).

[0219] c.Fc region variant In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibodies provided herein to generate Fc region variants. The Fc region variants may include a human Fc region sequence (e.g., the Fc region of human IgG1, IgG2, IgG3, or IgG4) that includes amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0220] In certain embodiments, antibody variants possessing some, but not all, effector functions are also within consideration of the present invention, such effector functions making the antibody a desirable candidate for application when its in vivo half-life is important, but certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity measurements can be performed to confirm reduced / deficient CDC and / or ADCC activity. For example, Fc receptor (FcR) binding measurements may be performed to confirm that the antibody lacks FcγR binding (and therefore is likely to lack ADCC activity) while maintaining FcRn binding ability. NK cells, the primary cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of the target molecule are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); and U.S. Patent No. 5,821,337 (see Bruggemann, et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive measurement methods may be used (see, for example, ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assays (Promega, Madison, WI)).Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, the ADCC activity of the molecule of interest may be evaluated in vivo in animal models, such as those described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. Furthermore, CDC measurements may be performed to evaluate complement activation (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, et al., Blood 101:1045-1052 (2003); and Cragg, Blood 103:2738-2743 (2004)). In addition, FcRn binding and in vivo clearance / half-life can be determined using methods known in the art (see, for example, Petkova, et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0221] Antibodies with reduced effector function include those with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include the so-called "DANA" Fc variant with alanine substitutions at residues 265 and 297 (U.S. Patent No. 7,332,581), and Fc variants with two or more substitutions at amino acid positions 265, 269, 270, 297, and 327.

[0222] Certain antibody variants exhibiting improved or reduced binding affinity to FcRs have been described. (See U.S. Patent No. 6,737,056; WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

[0223] In certain embodiments, the antibody variant includes an Fc region with one or more amino acid substitutions that improve ADCC (e.g., substitutions at positions 298, 333, and / or 334 (residue in EU numbering) of the Fc region).

[0224] In some embodiments, modifications are made in the Fc region that result in altered (i.e., either improved or attenuated) C1q binding and / or complement-dependent cell injury (CDC), as described, for example, in U.S. Patent No. 6,194,551, WO 1999 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0225] Antibodies with increased half-life and improved binding affinity to the neonatal Fc receptor (FcRn: which plays a role in transferring maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994))) are described in U.S. Patent Application Publication No. 2005 / 0014934A1 (Hinton et al.). These antibodies contain an Fc region with one or more substitutions that improve its binding affinity to FcRn. Such Fc variants include those with substitutions at one or more of the Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (e.g., substitution of Fc region residue 434 (U.S. Patent No. 7,371,826)). For other examples of Fc region variants, see also Duncan, Nature 322:738-40 (1988); U.S. Patents No. 5,648,260 and 5,624,821; and WO 1994 / 29351.

[0226] d. Cysteine-modified antibody variants In certain embodiments, it would be desirable to produce cysteine-modified antibodies (e.g., "thioMAbs") in which one or more residues of the antibody are substituted with cysteine ​​residues. In certain embodiments, the residues to be substituted occur in accessible sites of the antibody. By substituting these residues with cysteine, a reactive thiol group is located in an accessible site of the antibody, and this reactive thiol group may be used to conjugate the antibody to other parts (such as a drug part or a linker-drug part) to create an immunoconjugate as further detailed herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine-modified antibodies may be produced, for example, as described in U.S. Patent No. 7,521,541.

[0227] e. Antibody derivative In certain embodiments, the antibodies provided herein may be further modified to include additional non-protein moieties known and readily available in the art. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde would be advantageous in production due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if one or more polymers are attached, they may be the same molecule or different molecules. Generally, the number and / or type of polymers used in derivatization can be determined based on considerations such as the specific properties or functions of the antibody to be improved, and whether the antibody derivative will be used for therapy under specified conditions, although these are not limited to these.

[0228] In another embodiment, a conjugate is provided of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation may be of any wavelength, but is not limited thereto, and includes wavelengths that heat the non-protein moiety to a temperature that does not harm normal cells but kills cells adjacent to the antibody-non-protein moiety.

[0229] 8. Mutant Fc region In one aspect, the present invention provides an isolated polypeptide comprising a mutant Fc region with enhanced FcγRIIb binding activity. In several aspects, the polypeptide is an antibody. In several aspects, the polypeptide is an Fc fusion protein. In certain embodiments, the mutant Fc region comprises at least one amino acid residue modification (e.g., substitution) compared to the corresponding sequence in the Fc region of the native sequence or a reference mutant sequence (which may be collectively referred to herein as the “parent” Fc region). In certain embodiments, the mutant Fc region of the present invention has enhanced binding activity to monkey FcγRIIb compared to the parent Fc region. In certain embodiments, the monkey FcγRIIb is cynomolgus monkey FcγRIIb (SEQ ID NO: 223).

[0230] In certain embodiments, the ratio of [KD value of the parental Fc region to monkey FcγRIIb] / [KD value of the mutant Fc region to monkey FcγRIIb] may be 2.0 or greater, 3.0 or greater, 4.0 or greater, 5.0 or greater, 6.0 or greater, 7.0 or greater, 8.0 or greater, 9.0 or greater, 10 or greater, 15 or greater, 20 or greater, 25 or greater, 30 or greater, 40 or greater, or 50 or greater. In further embodiments, the mutant Fc region has reduced binding activity to monkey FcγRIIIa. In certain embodiments, the ratio of [KD value of the parental Fc region relative to monkey FcγRIIIa] / [KD value of the mutant Fc region relative to monkey FcγRIIIa] may be ≤0.50, ≤0.40, ≤0.30, ≤0.20, ≤0.10, ≤0.09, ≤0.08, ≤0.07, ≤0.06, ≤0.05, ≤0.04, ≤0.03, ≤0.02, or ≤0.01. In certain embodiments, monkey FcγRIIb has the sequence of sequence number: 223 (cynomolgus monkey). In certain embodiments, monkey FcγRIIIa has the sequence of sequence number: 224 (cynomolgus monkey).

[0231] In a further embodiment, the mutant Fc region has enhanced binding activity to human FcγRIIb. In a particular embodiment, the ratio of [KD value of the parental Fc region to human FcγRIIb] / [KD value of the mutant Fc region to human FcγRIIb] may be ≥2.0, ≥3.0, ≥4.0, ≥5.0, ≥6.0, ≥7.0, ≥8.0, ≥9.0, ≥10, ≥15, ≥20, ≥25, ≥30, ≥40, or ≥50. In a further embodiment, the mutant Fc region has reduced binding activity to human FcγRIIIa. In certain embodiments, the ratio of [KD value of the parental Fc region relative to human FcγRIIIa] / [KD value of the mutant Fc region relative to human FcγRIIIa] may be ≤0.50, ≤0.40, ≤0.30, ≤0.20, ≤0.10, ≤0.09, ≤0.08, ≤0.07, ≤0.06, ≤0.05, ≤0.04, ≤0.03, ≤0.02, or ≤0.01. In certain embodiments, human FcγRIIb has sequence number 212, 213, or 214. In certain embodiments, human FcγRIIIa has sequence number 215, 216, 217, or 218.

[0232] In a further embodiment, the mutant Fc region has lower binding activity to human FcγRIIa(H type) than to human FcγRIIb. In a particular embodiment, the ratio of [KD value of the parent Fc region to human FcγRIIa(H type)] / [KD value of the mutant Fc region to human FcγRIIa(H type)] may be ≤5.0, ≤4.0, ≤3.0, ≤2.0, ≤1.0, ≤0.9, ≤0.8, ≤0.7, ≤0.6, ≤0.5, ≤0.4, ≤0.3, ≤0.2, or ≤0.1. In a further embodiment, the mutant Fc region has lower binding activity to human FcγRIIa(R type) than to human FcγRIIb. In certain embodiments, the ratio of [KD value of the parental Fc region relative to human FcγRIIa(R type)] / [KD value of the mutant Fc region relative to human FcγRIIa(R type)] may be 5.0 or less, 4.0 or less, 3.0 or less, 2.0 or less, 1.0 or less, 0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, or 0.1 or less. In certain embodiments, human FcγRIIa(H type) has the sequence of sequence number: 211. In certain embodiments, human FcγRIIa(R type) has the sequence of sequence number: 210.

[0233] In a particular embodiment, the ratio of [KD value of the parental Fc region relative to monkey FcγRIIa] / [KD value of the mutant Fc region relative to monkey FcγRIIa] may be 2.0 or greater, 3.0 or greater, 4.0 or greater, 5.0 or greater, 6.0 or greater, 7.0 or greater, 8.0 or greater, 9.0 or greater, 10 or greater, 15 or greater, 20 or greater, 25 or greater, 30 or greater, 40 or greater, or 50 or greater. In a particular embodiment, monkey FcγRIIa is selected from monkey FcγRIIa1 (e.g., cynomolgus monkey FcγRIIa1 (sequence number: 220)), monkey FcγRIIa2 (e.g., cynomolgus monkey FcγRIIa2 (sequence number: 221)), and monkey FcγRIIa3 (e.g., cynomolgus monkey FcγRIIa3 (sequence number: 222)).

[0234] In another embodiment, the KD value of the mutant Fc region for monkey FcγRIIb is 1.0 × 10⁻⁶. -6 M or less, 9.0×10 -7M or less, 8.0×10 -7 M or less, 7.0×10 -7 M or less, 6.0×10 -7 M or less, 5.0×10 -7 M or less, 4.0×10 -7 M or less, 3.0×10 -7 M or less, 2.0×10 -7 M or less, or 1.0 × 10 -7 It may be less than M. In another embodiment, the KD value of the mutant Fc region for monkey FcγRIIIa is 5.0 × 10⁻⁶. -7 M or more, 6.0×10 -7 M or higher, 7.0×10 -7 M or higher, 8.0×10 -7 M or higher, 9.0×10 -7 M or more, 1.0×10 -6 M or more, 2.0×10 -6 M or higher, 3.0×10 -6 M or higher, 4.0×10 -6 M or more, 5.0×10 -6 M or more, 6.0×10 -6 M or higher, 7.0×10 -6 M or higher, 8.0×10 -6 M or higher, 9.0×10 -6 M or larger, or 1.0 × 10 -5 It may be M or greater. In another embodiment, the KD value of the mutant Fc region for human FcγRIIb is 2.0 × 10⁻⁶. -6 M or less, 1.0×10 -6 M or less, 9.0×10 -7 M or less, 8.0×10 -7 M or less, 7.0×10 -7 M or less, 6.0×10 -7 M or less, 5.0×10 -7 M or less, 4.0×10 -7 M or less, 3.0×10 -7 M or less, 2.0×10 -7 M or less, or 1.0 × 10 -7 It may be less than M. In another embodiment, the KD value of the mutant Fc region for human FcγRIIIa is 1.0 × 10⁻⁶. -6 M or more, 2.0×10 -6 M or higher, 3.0×10 -6 M or higher, 4.0×10 -6M or more, 5.0×10 -6 M or more, 6.0×10 -6 M or higher, 7.0×10 -6 M or higher, 8.0×10 -6 M or higher, 9.0×10 -6 M or more, 1.0×10 -5 M or more, 2.0×10 -5 M or higher, 3.0×10 -5 M or higher, 4.0×10 -5 M or larger, or 5.0 × 10 -5 It may be M or higher. In another embodiment, the KD value of the mutant Fc region for human FcγRIIa (type H) is 1.0 × 10⁻⁶. -7 M or more, 2.0×10 -7 M or higher, 3.0×10 -7 M or higher, 4.0×10 -7 M or more, 5.0×10 -7 M or more, 6.0×10 -7 M or higher, 7.0×10 -7 M or higher, 8.0×10 -7 M or higher, 9.0×10 -7 M or more, 1.0×10 -6 M or more, 2.0×10 -6 M or higher, 3.0×10 -6 M or higher, 4.0×10 -6 M or larger, or 5.0 × 10 -6 It may be M or higher. In another embodiment, the KD value of the mutant Fc region for human FcγRIIa (R type) is 2.0 × 10⁻⁶. -7 M or higher, 3.0×10 -7 M or higher, 4.0×10 -7 M or more, 5.0×10 -7 M or more, 6.0×10 -7 M or higher, 7.0×10 -7 M or higher, 8.0×10 -7 M or higher, 9.0×10 -7 M or more, 1.0×10 -6 M or more, 2.0×10 -6 M or higher, 3.0×10 -6 M or higher, 4.0×10 -6 M or larger or 5.0 × 10 -6 It could be M or higher.

[0235] In another embodiment, the KD value of the mutant Fc region in monkey FcγRIIa is 1.0 × 10⁻⁶. -6 M or less, 9.0×10 -7 M or less, 8.0×10 -7 M or less, 7.0×10 -7 M or less, 6.0×10 -7 M or less, 5.0×10 -7 M or less, 4.0×10 -7 M or less, 3.0×10 -7 M or less, 2.0×10 -7 M or less, or 1.0 × 10 -7 It may be less than or equal to M. In certain embodiments, monkey FcγRIIa may be selected from one of monkey FcγRIIa1, monkey FcγRIIa2, and monkey FcγRIIa3.

[0236] When developing pharmaceutical products for the treatment of human diseases, evaluating their efficacy and safety in monkeys is important because monkeys are biologically similar to humans. From this perspective, it is preferable that the developed pharmaceutical products exhibit cross-reactivity with both humans and monkeys in terms of target-binding activity.

[0237] "Fcγ receptor" (hereinafter referred to as Fcγ receptor, FcγR, or FcgR) refers to a receptor capable of binding to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies, and effectively means any member of the protein family encoded by the Fcγ receptor gene. In humans, this family includes, but is not limited to, FcγRI(CD64), which contains isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII(CD32), which contains isoforms FcγRIIa (including allotypes H131 (H type) and R131 (R type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII(CD16), which contains isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any undiscovered human FcγR, FcγR isoform, or allotype. FcγRIIb1 and FcγRIIb2 have been reported as splicing variants of human FcγRIIb. Furthermore, a splicing variant called FcγRIIb3 has been reported (J Exp Med, 1989, 170: 1369-1385). In addition to these splicing variants, human FcγRIIb includes all splicing variants registered in NCBI: NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1, and NP_003992.3. Furthermore, human FcγRIIb includes, in addition to FcγRIIb, all previously reported genetic polymorphisms (Arthritis Rheum. 48:3242-3252(2003); Kono et al., Hum. Mol. Genet. 14:2881-2892(2005); and Kyogoju et al., Arthritis Rheum. 46:1242-1254(2002)) and all genetic polymorphisms that may be reported in the future.

[0238] FcγRIIa has two allotypes: one in which the amino acid at position 131 of FcγRIIa is histidine (H type), and the other in which the amino acid at position 131 is substituted with arginine (R type) (Warrmerdam, J. Exp. Med. 172:19-25 (1990)).

[0239] FcγR includes, but is not limited to, FcγR derived from humans, mice, rats, rabbits, and monkeys, and may originate from any organism. Mouse FcγR includes, but is not limited to, FcγRI(CD64), FcγRII(CD32), FcγRIII(CD16), and FcγRIII-2(CD16-2), as well as any mouse FcγR or FcγR isoform. Unless otherwise specified, the term "monkey FcγR" or its derivatives refers to cynomolgus macaque FcγRIIa1(SEQ ID NO: 220), FcγRIIa2(SEQ ID NO: 221), FcγRIIa3(SEQ ID NO: 222), FcγRIIb(SEQ ID NO: 223), or FcγRIIIaS(SEQ ID NO: 224).

[0240] The polynucleotide sequence of human FcγRI is described in SEQ ID NO: 199 (NM_000566.3); the polynucleotide sequence of human FcγRIIa is described in SEQ ID NO: 200 (BC020823.1) or SEQ ID NO: 201 (NM_001136219.1); the polynucleotide sequence of human FcγRIIb is described in SEQ ID NO: 202 (BC146678.1) or SEQ ID NO: 203 (NM_004001.3); the polynucleotide sequence of human FcγRIIIa is described in SEQ ID NO: 204 (BC033678.1) or SEQ ID NO: 205 (NM_001127593.1); and the polynucleotide sequence of human FcγRIIIb is described in SEQ ID NO: 206 (BC128562.1).

[0241] The amino acid sequence of human FcγRI is described in SEQ ID NO: 207 (NP_000557.1); the amino acid sequence of human FcγRIIa is described in SEQ ID NO: 208 (AAH20823.1), SEQ ID NO: 209, SEQ ID NO: 210, or SEQ ID NO: 211; the amino acid sequence of human FcγRIIb is described in SEQ ID NO: 212 (AAI46679.1), SEQ ID NO: 213, or SEQ ID NO: 214; the amino acid sequence of human FcγRIIIa is described in SEQ ID NO: 215 (AAH33678.1), SEQ ID NO: 216, SEQ ID NO: 217, or SEQ ID NO: 218; and the amino acid sequence of human FcγRIIIb is described in SEQ ID NO: 219 (AAI28563.1).

[0242] The amino acid sequence of cynomolgus monkey FcγRIIa is described in SEQ ID NO: 220 (FcγRIIa1), SEQ ID NO: 221 (FcγRIIa2), or SEQ ID NO: 222 (FcγRIIa3); the amino acid sequence of cynomolgus monkey FcγRIIb is described in SEQ ID NO: 223; and the amino acid sequence of cynomolgus monkey FcγRIIIa is described in SEQ ID NO: 224.

[0243] In one aspect, the present invention provides a polypeptide comprising a mutant Fc region having enhanced FcγRIIb binding activity compared to a corresponding reference FcγRIIb-binding polypeptide. In a further aspect, the polypeptide of the present invention comprises at least one amino acid modification at at least one position selected from the group consisting of positions 231, 232, 233, 234, 235, 236, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334, and 396, as represented by EU numbering. In a particular embodiment, the FcγRIIb has the sequence of cynomolgus monkey FcγRIIb (SEQ ID NO: 223). In a particular embodiment, the FcγRIIb has the sequence of human FcγRIIb (e.g., SEQ ID NO: 212, 213, or 214).

[0244] In one aspect, the present invention provides a polypeptide comprising a mutant Fc region with enhanced FcγRIIb binding activity, comprising at least two amino acid modifications, represented by EU numbering: (a) one amino acid modification at position 236 and (b) at least one amino acid modification at at least one position selected from the group consisting of positions 231, 232, 233, 234, 235, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334, and 396. In certain embodiments, FcγRIIb has the sequence of cynomolgus monkey FcγRIIb (SEQ ID NO: 223). In certain embodiments, FcγRIIb has the sequence of human FcγRIIb (e.g., SEQ ID NO: 212, 213, or 214).

[0245] In one aspect, the present invention provides a polypeptide comprising a mutant Fc region with enhanced FcγRIIb binding activity, which includes an amino acid modification at position 236, as represented by EU numbering.

[0246] In one aspect, the present invention provides a polypeptide comprising a mutant Fc region with enhanced FcγRIIb binding activity, comprising at least two amino acid modifications, represented by EU numbering: (a) one amino acid modification at position 236 and (b) at least one amino acid modification at at least one position selected from the group consisting of positions 231, 232, 235, 239, 268, 295, 298, 326, 330, and 396. In a further embodiment, the mutant Fc region comprises an amino acid modification at at least one position selected from the group consisting of positions 231, 232, 235, 239, 268, 295, 298, 326, 330, and 396, represented by EU numbering. In a further embodiment, the mutant Fc region comprises an amino acid modification at at least one position selected from the group consisting of positions 268, 295, 326, and 330, represented by EU numbering. In certain embodiments, FcγRIIb has the sequence of cynomolgus monkey FcγRIIb (sequence number: 223). In certain embodiments, FcγRIIb has the sequence of human FcγRIIb (e.g., sequence numbers: 212, 213, or 214).

[0247] In another aspect, the present invention provides polypeptides comprising mutant Fc regions with enhanced FcγRIIb binding activity, comprising any one of the following amino acid modifications: (1) positions 231, 236, 239, 268, and 330, represented by EU numbering; (2) positions 231, 236, 239, 268, 295, and 330; (3) positions 231, 236, 268, and 330; (4) positions 231, 236, 268, 295, and 330; (5) positions 232, 236, 239, 268, 295, and 330; (6) positions 232, 2 36, 268, 295, and 330; (7) Positions 232, 236, 268, and 330; (8) Positions 235, 236, 268, 295, 326, and 330; (9) Positions 235, 236, 268, 295, and 330; (10) Positions 235, 236, 268, and 330; (11) Positions 235, 236, 268, 330, and 396; (12) Positions 235, 236, 268, and 396; (13) Positions 236, 239, 268, 295, 298, and 330; (14) Positions 236, 239, 268, 295, 326, and 330; ( 15) Positions 236, 239, 268, 295, and 330; (16) Positions 236, 239, 268, 298, and 330; (17) Positions 236, 239, 268, 326, and 330; (18) Positions 236, 239, 268, and 330; (19) Positions 236, 239, 268, 330, and 396; (20) Positions 236, 239, 268, and 396; (21) Positions 236 and 268; (22) Positions 236, 268, and 295; (23) Positions 236, 268, 295, 298, and 330; (24) Positions 236, 268, 295 , 326, and 330; (25) Positions 236, 268, 295, 326, 330, and 396; (26) Positions 236, 268, 295, and 330; (27) Positions 236, 268, 295, 330, and 396; (28) Positions 236, 268, 298, and 330; (29) Positions 236, 268, 298, and 396; (30) Positions 236, 268, 326, and 330; (31) Positions 236, 268, 326, 330, and 396; (32) Positions 236, 268, and 330; (33) Positions 236, 268, 330, and 396;(34) positions 236, 268, and 396; (35) positions 236 and 295; (36) positions 236, 330, and 396; and (37) positions 236 and 396. In certain embodiments, FcγRIIb has the sequence of cynomolgus monkey FcγRIIb (sequence number: 223). In certain embodiments, FcγRIIb has the sequence of human FcγRIIb (e.g., sequence numbers: 212, 213, or 214).

[0248] In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity contains at least one amino acid selected from the group consisting of: represented by EU numbering, (a) Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 231; (b) Ala, Asp, Glu, Phe, Gl at position 232 y, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr; (c) Asp at position 233; (d) Trp, Tyr at position 234; (e) Trp at position 235; (f) Ala, Asp, Glu, His, Ile, Leu, Met, Asn, Gln, Ser, Thr, Val at position 236; (g) Asp, Tyr at position 237; (h) Glu, Ile at position 238, Met, Gln, Tyr; (i) Ile, Leu, Asn, Pro, Val at position 239; (j) Ile at position 264; (k) Phe at position 266; (l) Ala, His, Leu at position 267; (m) Asp, Glu at position 268; (n) Asp, Glu, Gly at position 271; (o) Leu at position 295; (p) Leu at position 298; (q) Glu, Phe, Ile, Leu at position 325; (r) Th at position 326 r;(s) Ile, Asn at position 327;(t) Thr at position 328;(u) Lys, Arg at position 330;(v) Glu at position 331;(w) Asp at position 332;(x) Asp, Ile, Met, Val, Tyr at position 334; and(y) Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 396. In certain embodiments, FcγRIIb has the sequence of cynomolgus monkey FcγRIIb (sequence number: 223). In certain embodiments, FcγRIIb has the sequence of human FcγRIIb (e.g., sequence numbers: 212, 213, or 214).

[0249] In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity comprises an amino acid modification (e.g., substitution) represented by EU numbering, which is at least one selected from the group consisting of (a) Gly, Thr at position 231; (b) Asp at position 232; (c) Trp at position 235; (d) Asn, Thr at position 236; (e) Val at position 239; (f) Asp, Glu at position 268; (g) Leu at position 295; (h) Leu at position 298; (i) Thr at position 326; (j) Lys, Arg at position 330; and (k) Lys, Met at position 396. In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity comprises an amino acid modification (e.g., substitution) represented by EU numbering, which is Asn at position 236, Glu at position 268, Lys at position 330, and Met at position 396. In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity comprises an amino acid modification (e.g., substitution) represented by EU numbering as Asn at position 236, Asp at position 268, and Lys at position 330. In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity comprises an amino acid modification (e.g., substitution) represented by EU numbering as Asn at position 236, Asp at position 268, Leu at position 295, and Lys at position 330. In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity comprises an amino acid modification (e.g., substitution) represented by EU numbering as Thr at position 236, Asp at position 268, and Lys at position 330. In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity comprises amino acid modifications (e.g., substitutions) represented by EU numbering as Asn at position 236, Asp at position 268, Leu at position 295, Thr at position 326, and Lys at position 330. In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity comprises amino acid modifications (e.g., substitutions) represented by EU numbering as Trp at position 235, Asn at position 236, Asp at position 268, Leu at position 295, Thr at position 326, and Lys at position 330.

[0250] In one aspect, the present invention provides a polypeptide comprising a mutant Fc region with enhanced FcγRIIb binding activity, which includes an amino acid modification at position 238, as represented by EU numbering.

[0251] In one aspect, the present invention provides a polypeptide comprising a mutant Fc region with enhanced FcγRIIb binding activity, comprising at least one amino acid modification at at least one position selected from the group consisting of positions 234, 238, 250, 264, 267, 307, and 330, as represented by EU numbering. In a further aspect, the polypeptide comprises at least one amino acid modification at at least one position selected from the group consisting of positions 234, 250, 264, 267, 307, and 330, as represented by EU numbering. In a particular aspect, the FcγRIIb has the sequence of cynomolgus monkey FcγRIIb (SEQ ID NO: 223). In a particular aspect, the FcγRIIb has the sequence of human FcγRIIb (e.g., SEQ ID NO: 212, 213, or 214).

[0252] In another aspect, the present invention provides polypeptides comprising mutant Fc regions with enhanced FcγRIIb binding activity, comprising any one of the following amino acid modifications: (1) positions 234, 238, 250, 307, and 330, represented by EU numbering; (2) positions 234, 238, 250, 264, 307, and 330; (3) positions 234, 238, 250, 264, 267, (4) positions 234, 238, 250, 267, 307, and 330; (5) positions 238, 250, 264, 307, and 330; (6) positions 238, 250, 264, 267, 307, and 330; (7) positions 238, 250, 267, 307, and 330; (8) positions 238, 250, and 307; and (9) positions 238, 250, 307, and 330. In certain embodiments, FcγRIIb has the sequence of cynomolgus monkey FcγRIIb (sequence number: 223). In certain embodiments, FcγRIIb has the sequence of human FcγRIIb (e.g., sequence numbers: 212, 213, or 214).

[0253] In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity includes an amino acid modification (e.g., substitution) selected from the group consisting of (a) Tyr at position 234; (b) Asp at position 238; (c) Val at position 250; (d) Ile at position 264; (e) Ala at position 267; (f) Pro at position 307; and (g) Lys at position 330, as represented by EU numbering. In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity includes an amino acid modification (e.g., substitution) of Asp at position 238, as represented by EU numbering. In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity includes amino acid modifications (e.g., substitution) of Asp at position 238, Val at position 250, and Pro at position 307, as represented by EU numbering. In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity comprises an amino acid modification (e.g., substitution) represented by EU numbering as Asp at position 238, Val at position 250, Pro at position 307, and Lys at position 330. In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity comprises an amino acid modification (e.g., substitution) represented by EU numbering as Asp at position 238, Val at position 250, Ile at position 264, Pro at position 307, and Lys at position 330. In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity comprises an amino acid modification (e.g., substitution) represented by EU numbering as Asp at position 238, Val at position 250, Ala at position 267, Pro at position 307, and Lys at position 330. In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity comprises amino acid modifications (e.g., substitutions) represented by EU numbering as Tyr at position 234, Asp at position 238, Val at position 250, Pro at position 307, and Lys at position 330. In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity comprises amino acid modifications (e.g., substitutions) represented by EU numbering as Tyr at position 234, Asp at position 238, Val at position 250, Ala at position 267, Pro at position 307, and Lys at position 330.In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity comprises amino acid modifications (e.g., substitutions) represented by EU numbering as Asp at position 238, Val at position 250, Ile at position 264, Ala at position 267, Pro at position 307, and Lys at position 330. In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity comprises amino acid modifications (e.g., substitutions) represented by EU numbering as Tyr at position 234, Asp at position 238, Val at position 250, Ile at position 264, Pro at position 307, and Lys at position 330. In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity comprises amino acid modifications (e.g., substitutions) represented by EU numbering as Tyr at position 234, Asp at position 238, Val at position 250, Ile at position 264, Ala at position 267, Pro at position 307, and Lys at position 330. In a particular embodiment, FcγRIIb has the sequence of cynomolgus monkey FcγRIIb (SEQ ID NO: 223). In a particular embodiment, FcγRIIb has the sequence of human FcγRIIb (e.g., SEQ ID NOs: 212, 213, or 214).

[0254] In another aspect, the present invention provides isolated polypeptides comprising a mutant Fc region with an elevated isoelectric point (pI). In certain embodiments, the mutant Fc region described herein comprises at least two amino acid modifications in the parent Fc region. In certain embodiments, each of the amino acid modifications increases the isoelectric point (pI) of the mutant Fc region compared to the parent Fc region. This is based on the finding that using an antibody having an elevated pI due to modifications of at least two amino acid residues can accelerate the elimination of an antigen from plasma, for example, when the antibody is administered in vivo.

[0255] In this invention, pI may be either theoretical or experimental. The value of pI can be measured, for example, by isoelectric focusing methods known to those skilled in the art. The theoretical value of pI can be calculated, for example, using gene and amino acid sequence analysis software (such as Genetyx).

[0256] In one embodiment, the pI value may increase compared to before modification by, for example, at least 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 or more, at least 0.6, 0.7, 0.8, 0.9 or more, at least 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 or more, or at least 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0 or more.

[0257] In certain embodiments, amino acids related to pI elevation may be exposed on the surface of the mutant Fc region. In this invention, an amino acid that can be exposed on the surface usually refers to an amino acid residue located on the surface of the polypeptide constituting the mutant Fc region. An amino acid residue located on the surface of a polypeptide is an amino acid residue whose side chain can come into contact with solvent molecules (usually mostly water molecules). However, the side chain does not necessarily have to be in contact with the solvent molecule as a whole; even if only a part of the side chain is in contact with the solvent molecule, the amino acid is still defined as a "surface-located amino acid residue." Furthermore, surface-located amino acid residues also include amino acid residues located near the surface that can be affected by the charge from other amino acid residues whose side chains are in contact with solvent molecules, even if only partially. Those skilled in the art can create homology models of polypeptides using, for example, commercially available software. Alternatively, methods known to those skilled in the art, such as X-ray crystallography, can be used. Amino acid residues that can be exposed on the surface are determined using coordinates derived from a three-dimensional model using, for example, the Insight II program (Accelrys). The areas that can be exposed on the surface may be determined using algorithms known in the art (e.g., Lee and Richards (J. Mol. Biol. 55:379-400 (1971)); Connolly (J. Appl. Cryst. 16:548-558 (1983))). The areas that can be exposed on the surface can be determined using software suitable for protein modeling and three-dimensional structural information. Software available for such purposes includes, for example, the SYBYL Biopolymer Module software (Tripos Associates). If the algorithm requires a user input size parameter, the "size" of the probe used in the calculation may be set to a radius of approximately 1.4 angstroms (Å) or less.Furthermore, a method for determining the area that can be exposed on the surface using personal computer software has been described by Pacios (Comput. Chem. 18(4):377-386(1994); J. Mol. Model. 1:46-53(1995)). Based on such information as described above, appropriate amino acid residues located on the surface of the polypeptide constituting the mutant Fc region can be selected.

[0258] In certain embodiments, the polypeptide comprises both a variant Fc region and an antigen-binding domain. In further embodiments, the antigen is a soluble antigen. In one embodiment, the antigen is present in the biological fluid of the subject (e.g., plasma, interstitial fluid, lymph, ascites, and pleural fluid). The antigen may also be a membrane antigen.

[0259] In a further embodiment, the antigen-binding activity of the antigen-binding domain varies with ion concentration conditions. In one embodiment, the ion concentration is not particularly limited and refers to hydrogen ion concentration (pH) or metal ion concentration. In this specification, metal ions refer to ions of Group I elements such as alkali metals and copper group elements, Group II elements such as alkaline earth metals and zinc group elements, Group III elements except boron, Group IV elements except carbon and silicon, Group VIII elements such as iron group elements and platinum group elements, elements belonging to subgroups A of Groups V, VI, and VII, and metal elements such as antimony, bismuth, and polonium. In the present invention, metal ions include, for example, calcium ions, as described in WO2012 / 073992 and WO2013 / 125667. In one embodiment, “ion concentration conditions” may be conditions that focus on the difference in the biological behavior of the antigen-binding domain between low and high ion concentrations. Furthermore, the statement that "the antigen-binding activity of the antigen-binding domain changes with ion concentration conditions" means that the antigen-binding activity of the antigen-binding domain changes between low and high ion concentrations (in this specification, such an antigen-binding domain is referred to as an "ion-concentration-dependent antigen-binding domain"). The antigen-binding activity of an antigen-binding domain under high ion concentration conditions may be higher (stronger) or lower (weaker) than that under low ion concentration conditions. In one embodiment, an ion-concentration-dependent antigen-binding domain (e.g., a pH-dependent antigen-binding domain or a calcium ion concentration-dependent antigen-binding domain) can be obtained by known methods described, for example, WO2009 / 125825, WO2012 / 073992, and WO2013 / 046722.

[0260] In the present invention, the antigen-binding activity of the antigen-binding domain under high calcium ion concentration conditions may be higher than that under low calcium ion concentration conditions. The high calcium ion concentration may be selected from among 100 μM to 10 mM, 200 μM to 5 mM, 400 μM to 3 mM, 200 μM to 2 mM, 400 μM to 1 mM, or 500 μM to 2.5 mM, and is preferably close to the in vivo plasma (blood) calcium ion concentration. On the other hand, the low calcium ion concentration may be selected from among 0.1 μM to 30 μM, 0.2 μM to 20 μM, 0.5 μM to 10 μM, 1 μM to 5 μM, or 2 μM to 4 μM, and is preferably close to the in vivo calcium ion concentration in early endosomes.

[0261] In one embodiment, the ratio of antigen-binding activity under low calcium ion concentration conditions to antigen-binding activity under high calcium ion concentration conditions is not limited, but the ratio of the dissociation constant (KD) under low calcium ion concentration conditions to the dissociation constant (KD) under high calcium ion concentration conditions, i.e., KD(low calcium ion concentration conditions) / KD(high calcium ion concentration conditions), is 2 or greater, 10 or greater, or 40 or greater. The upper limit of this ratio may be 400, 1000, or 10000, provided that such an antigen-binding domain can be prepared by techniques known to those skilled in the art. Alternatively, instead of KD, for example, the dissociation rate constant (kd) can be used. In this case, the ratio of the kd under low calcium ion concentration conditions to the kd under high calcium ion concentration conditions, i.e., kd(low calcium ion concentration conditions) / kd(high calcium ion concentration conditions), is 2 or greater, 5 or greater, 10 or greater, or 30 or greater. The upper limit of this ratio may be 50, 100, or 200, provided that the antigen-binding domain can be prepared based on the common technical knowledge of those skilled in the art.

[0262] In the present invention, the antigen-binding activity of the antigen-binding domain under low hydrogen ion concentration (neutral pH) may be higher than that under high hydrogen ion concentration (acidic pH). The acidic pH may be, for example, selected from pH 4.0 to pH 6.5, selected from pH 4.5 to pH 6.5, selected from pH 5.0 to pH 6.5, or selected from pH 5.5 to pH 6.5, and is preferably close to the pH in early endosomes in vivo. The acidic pH may also be, for example, pH 5.8 or pH 6.0. In certain embodiments, the acidic pH is pH 5.8. On the other hand, the neutral pH may be, for example, selected from pH 6.7 to pH 10.0, selected from pH 6.7 to pH 9.5, selected from pH 7.0 to pH 9.0, or selected from pH 7.0 to pH 8.0, and is preferably close to the pH in plasma (blood) in vivo. A neutral pH may also be, for example, pH 7.4 or pH 7.0. In a particular embodiment, the neutral pH is pH 7.4.

[0263] In one embodiment, the ratio of antigen-binding activity under acidic pH conditions to antigen-binding activity under neutral pH conditions is not limited, but the ratio of the dissociation constant (KD) under acidic pH conditions to the dissociation constant (KD) under neutral pH conditions, i.e., KD(acidic pH conditions) / KD(neutral pH conditions), is 2 or greater, 10 or greater, or 40 or greater. The upper limit of this ratio may be 400, 1000, or 10000, provided that such an antigen-binding domain can be prepared by techniques known to those skilled in the art. Alternatively, instead of KD, for example, the dissociation rate constant (kd) can be used. In this case, the ratio of the kd under acidic pH conditions to the kd under neutral pH conditions, i.e., kd(acidic pH conditions) / kd(neutral pH conditions), is 2 or greater, 5 or greater, 10 or greater, or 30 or greater. The upper limit of this ratio may be 50, 100, or 200, provided that the antigen-binding domain can be prepared based on the common technical knowledge of those skilled in the art.

[0264] In one embodiment, as described in WO2009 / 125825, at least one amino acid residue is substituted in the antigen-binding domain, for example, with an amino acid residue having a side-chain pKa of 4.0 to 8.0, and / or at least one amino acid having a side-chain pKa of 4.0 to 8.0 is inserted. The amino acids may be substituted and / or inserted at any site, as long as the antigen-binding activity of the antigen-binding domain is weaker under acidic pH conditions than under neutral pH conditions compared to before substitution or insertion. If the antigen-binding domain has a variable region or CDR, the site may be located within the variable region or CDR. The number of amino acids to be substituted or inserted can be appropriately determined by those skilled in the art and may be one or more. Amino acids having a side-chain pKa of 4.0 to 8.0 can be used to alter the antigen-binding activity of the antigen-binding domain depending on the hydrogen ion concentration conditions. Such amino acids include, for example, natural amino acids such as His(H) and Glu(E), as well as non-natural amino acids such as histidine analogs (US2009 / 0035836), m-NO2-Tyr (pKa 7.45), 3,5-Br2-Tyr (pKa 7.21), and 3,5-I2-Tyr (pKa 7.38) (Heyl et al., Bioorg. Med. Chem. 11(17):3761-3768(2003)). Amino acids with side-chain pKas of 6.0-7.0 can also be used, including, for example, His(H).

[0265] In another embodiment, an antigen-binding domain suitable for a mutated Fc region with elevated pI has been described, which can be obtained by the method described in Japanese Patent Application Nos. 2015-021371 and 2015-185254.

[0266] In a particular embodiment, the pI-elevated mutant Fc region includes at least two amino acid modifications at at least two positions selected from the group consisting of positions 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422, and 431, as represented by EU numbering.

[0267] In a further embodiment, the pI-elevated mutant Fc region includes at least two amino acid modifications at at least two positions selected from the group consisting of positions 311, 341, 343, 384, 399, 400, 401, 402, and 413, as represented by EU numbering.

[0268] In another aspect, the present invention provides polypeptides comprising a pI-enhanced mutant Fc region comprising any one of the following amino acid modifications: (1) positions 311 and 341; (2) positions 311 and 343; (3) positions 311, 343 and 413; (4) positions 311, 384 and 413; (5) positions 311 and 399; (6) positions 311 and 401; (7) positions 311 and 413; (8) positions 400 and 413; (9) positions 401 and 413; and (10) positions 402 and 413.

[0269] Methods for increasing the pI of a protein include, for example, reducing the number of amino acids with negatively charged side chains (e.g., aspartic acid and glutamic acid) and / or increasing the number of amino acids with positively charged side chains (e.g., arginine, lysine, and histidine) under neutral pH conditions. Amino acids with negatively charged side chains have a negative charge represented as -1 under pH conditions well above their side chain pKa, a theory well known to those skilled in the art. For example, the theoretical pKa of the side chain of aspartic acid is 3.9, and this side chain has a negative charge represented as -1 under neutral pH conditions (e.g., in a solution at pH 7.0). Conversely, amino acids with positively charged side chains have a positive charge represented as +1 under pH conditions well below their side chain pKa. For example, the theoretical pKa of the side chain of arginine is 12.5, and this side chain has a positive charge represented as +1 under neutral pH conditions (e.g., in a solution at pH 7.0). In contrast, it is known that amino acids whose side chains do not carry a charge under neutral pH conditions (for example, in a solution with pH 7.0) include 15 natural amino acids: alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan, and tyrosine. Naturally, it is understood that non-natural amino acids can also be used to increase pI.

[0270] As described above, a method for increasing the pI of a protein under neutral pH conditions (for example, in a pH 7.0 solution) is to substitute aspartic acid or glutamic acid (whose side chains have a negative charge of -1) in the amino acid sequence of the protein with an amino acid whose side chain has no charge, thereby conferring a +1 charge modification to the protein of interest. Furthermore, a +1 charge modification can be conferred to the protein by substituting an amino acid whose side chain has no charge with arginine or lysine (whose side chains have a positive charge of +1). In addition, a +2 charge modification can be conferred to the protein at once by substituting aspartic acid or glutamic acid (whose side chains have a negative charge of -1) with arginine or lysine (whose side chains have a positive charge of +1). Alternatively, in order to increase the pI of a protein, amino acids with uncharged side chains and / or preferably amino acids with positively charged side chains can be added to or inserted into the amino acid sequence of the protein, or amino acids with uncharged side chains and / or preferably amino acids with negatively charged side chains that are present in the amino acid sequence of the protein can be deleted. For example, the N-terminal and C-terminal amino acid residues of a protein have charges derived from the main chain in addition to the charges derived from their side chains (NH3 of the amino group at the N-terminus). + and COO of the carbonyl group at the C-terminus - It is understood that the protein has the following properties. Therefore, the protein pI can also be increased by adding, deleting, substituting, or inserting functional groups derived from the main chain.

[0271] Amino acid substitutions to increase pI include, for example, substituting an amino acid with a negatively charged side chain with an amino acid with an uncharged side chain in the amino acid sequence of the parent Fc region, substituting an amino acid with an uncharged side chain with an amino acid with a positively charged side chain, and substituting an amino acid with a negatively charged side chain with an amino acid with a positively charged side chain. These substitutions can be performed individually or in appropriate combinations.

[0272] Insertions or additions of amino acids to increase pI include, for example, the insertion or addition of amino acids with uncharged side chains in the amino acid sequence of the parent Fc region, and / or the insertion or addition of amino acids with positively charged side chains, which are carried out individually or in appropriate combinations.

[0273] Deletions of amino acids to increase pI include, for example, deletions of amino acids in the amino acid sequence of the parent Fc region whose side chains are uncharged, and / or deletions of amino acids whose side chains are negatively charged, and these are carried out individually or in appropriate combinations.

[0274] In one embodiment, the native amino acids used to increase pI can be classified as follows: (a) amino acids with negatively charged side chains may be Glu(E) or Asp(D); (b) amino acids with uncharged side chains may be Ala(A), Asn(N), Cys(C), Gln(Q), Gly(G), His(H), Ile(I), Leu(L), Met(M), Phe(F), Pro(P), Ser(S), Thr(T), Trp(W), Tyr(Y), or Val(V); and (c) amino acids with positively charged side chains may be His(H), Lys(K), or Arg(R). In one embodiment, the insertion or substitution of the modified amino acid is Lys(K) or Arg(R).

[0275] In another aspect, the present invention provides isolated polypeptides comprising a mutant Fc region with enhanced FcγRIIb binding activity and increased pI. In a particular embodiment, the mutant Fc region described herein comprises at least two amino acid modifications in the parent Fc region.

[0276] In one aspect, the present invention provides (a) at least one amino acid modification at at least one position selected from the group consisting of positions 231, 232, 233, 234, 235, 236, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334, and 396, represented by EU numbering, and (b) position 28 The present invention provides a polypeptide comprising a mutant Fc region with enhanced FcγRIIb binding activity and increased pI, comprising at least three amino acid modifications, including at least two amino acid modifications at at least two positions selected from the group consisting of 5, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422, and 431.

[0277] In one aspect, the present invention provides a polypeptide comprising a mutant Fc region with enhanced FcγRIIb binding activity and increased pI, comprising at least three amino acid modifications, including (a) at least one amino acid modification selected from the group consisting of positions 231, 232, 235, 236, 239, 268, 295, 298, 326, 330, and 396 represented by EU numbering, and (b) at least two amino acid modifications selected from the group consisting of positions 311, 341, 343, 384, 399, 400, 401, 402, and 413 represented by EU numbering.

[0278] In another aspect, the present invention provides polypeptides comprising a mutant Fc region with enhanced FcγRIIb binding activity and increased pI, comprising any one of the following amino acid modifications: (1) positions 235, 236, 268, 295, 311, 326, 330 and 343, represented by EU numbering; (2) positions 236, 268, 295, 311, 326, 330 and 343; (3) positions 236, 268, 295, 311 , 330 and 413; (4) positions 236, 268, 311, 330, 396 and 399; (5) positions 236, 268, 311, 330 and 343; (6) positions 236, 268, 311, 330, 343 and 413; (7) positions 236, 268, 311, 330, 384 and 413; (8) positions 236, 268, 311, 330 and 413; and (9) positions 236, 268, 330, 396, 400 and 413. In certain embodiments, FcγRIIb has the sequence of cynomolgus monkey FcγRIIb (sequence number: 223). In certain embodiments, FcγRIIb has the sequence of human FcγRIIb (e.g., sequence numbers: 212, 213, or 214).

[0279] In one aspect, the present invention provides a polypeptide comprising a mutant Fc region with enhanced FcγRIIb binding activity and increased pI, comprising at least three amino acid modifications, including (a) at least one amino acid modification at at least one position selected from the group consisting of positions 234, 238, 250, 264, 267, 307, and 330, as represented by EU numbering, and (b) at least two amino acid modifications at at least two positions selected from the group consisting of positions 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422, and 431. In a further embodiment, the polypeptide comprises at least two amino acid modifications at at least two positions selected from the group consisting of 311, 341, 343, 384, 399, 400, 401, 402, and 413, as represented by EU numbering. In a particular embodiment, FcγRIIb has the sequence of cynomolgus monkey FcγRIIb (SEQ ID NO: 223). In a particular embodiment, FcγRIIb has the sequence of human FcγRIIb (e.g., SEQ ID NOs: 212, 213, or 214).

[0280] In another aspect, the present invention provides polypeptides comprising a mutant Fc region with enhanced FcγRIIb binding activity and increased pI, comprising any one of the following amino acid modifications: (1) positions 234, 238, 250, 264, 307, 311, 330 and 343, represented by EU numbering; (2) positions 234, 238, 250, 264, 307, 311, 330 and 4 13; (3) Positions 234, 238, 250, 264, 267, 307, 311, 330 and 343; (4) Positions 234, 238, 250, 264, 267, 307, 311, 330 and 413; (5) Positions 234, 238, 250, 267, 307, 311, 330 and 343; (6) Positions 234, 238, 250, 267, 307, 311, 330 and 413; (7) Position 234, 238, 250, 307, 311, 330 and 343; (8) Positions 234, 238, 250, 307, 311, 330 and 413; (9) Positions 238, 250, 264, 267, 307, 311, 330 and 343; (10) Positions 238, 250, 264, 267, 307, 311, 330 and 413; (11) Positions 238, 250, 264, 307, 311, 330 and 343; (12) positions 238, 250, 264, 307, 311, 330 and 413; (13) positions 238, 250, 267, 307, 311, 330 and 343; (14) positions 238, 250, 267, 307, 311, 330 and 413; (15) positions 238, 250, 307, 311, 330 and 343; and (16) positions 238, 250, 307, 311, 330 and 413.

[0281] In a further embodiment, the mutant Fc region includes an amino acid modification selected from any single modification, a combination of single modifications, or a combination of modifications listed in Tables 14-30.

[0282] In some embodiments, the polypeptide comprises a mutated Fc region of the present invention. In a further embodiment, the polypeptide is a constant region of an antibody heavy chain. In a further embodiment, the polypeptide is an antibody heavy chain. In a further embodiment, the polypeptide is an antibody. In a further embodiment, the polypeptide is an Fc fusion protein.

[0283] In a further embodiment, the present invention provides a polypeptide comprising any one amino acid sequence of SEQ ID NOs: 229 to 381.

[0284] As used herein, “parental Fc region” refers to the Fc region before the introduction of the amino acid modifications described herein. Preferred examples of parental Fc regions include Fc regions derived from native antibodies. Antibodies include, for example, IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), and IgM. Antibodies may be derived from humans or monkeys (e.g., cynomolgus macaques, rhesus macaques, marmosets, chimpanzees, or baboons). Native antibodies may contain naturally occurring mutations. Several allotype sequences of IgG due to genetic polymorphisms are described in “Sequences of proteins of immunological interest,” NIH Publication No. 91-3242, and any of them may be used in the present invention. In particular, for human IgG1, the amino acid sequence at positions 356-358 (EU numbering) may be either DEL or EEM. Preferred examples of parent Fc regions include Fc regions derived from the heavy chain constant regions of human IgG1 (SEQ ID NO: 195), human IgG2 (SEQ ID NO: 196), human IgG3 (SEQ ID NO: 197), and human IgG4 (SEQ ID NO: 198). Another preferred example of a parent Fc region is an Fc region derived from the heavy chain constant region SG1 (SEQ ID NO: 9). Furthermore, the parent Fc region may be an Fc region created by adding amino acid modifications other than those described herein to an Fc region derived from a native antibody.

[0285] Furthermore, amino acid modifications performed for other purposes can be combined in the mutant Fc region described herein. For example, amino acid substitutions that enhance FcRn binding activity (Hinton et al., J. Immunol. 176(1):346-356(2006); Dall'Acqua et al., J. Biol. Chem. 281(33):23514-23524(2006); Petkova et al., Intl. Immunol. 18(12):1759-1769(2006); Zalevsky et al., Nat. Biotechnol. 28(2):157-159(2010); WO2006 / 019447; WO2006 / 053301; and WO2009 / 086320), and amino acid substitutions to improve the heterogeneity or stability of the antibody (WO2009 / 041613) may be added. Alternatively, polypeptides having properties that promote antigen clearance as described in WO2011 / 122011, WO2012 / 132067, WO2013 / 046704, or WO2013 / 180201, polypeptides having properties that specifically bind to target tissues as described in WO2013 / 180200, or polypeptides having properties that repeatedly bind to multiple antigen molecules as described in WO2009 / 125825, WO2012 / 073992, or WO2013 / 047752 may be combined with the mutant Fc region described herein. Alternatively, amino acid modifications disclosed in EP1752471 and EP1772465 may be combined at CH3 of the mutant Fc region described herein for the purpose of conferring binding ability to other antigens. Alternatively, an amino acid modification that lowers the constant region pI (WO2012 / 016227) may be combined with the mutant Fc region described herein for the purpose of increasing plasma retention. Alternatively, an amino acid modification that increases the constant region pI (WO2014 / 145159) may be combined with the mutant Fc region described herein for the purpose of promoting uptake into cells.Alternatively, amino acid modifications that increase the constant region pI (Japanese Patent Application Nos. 2015-021371 and 2015-185254) may be combined in the mutant Fc region described herein for the purpose of promoting the elimination of the target molecule from plasma. In one embodiment, such modifications may include substitutions at at least one position selected from the group consisting of positions 311, 343, 384, 399, 400, and 413, as represented by EU numbering. In a further embodiment, such substitutions may be Lys or Arg substitutions of the amino acid at each position.

[0286] Amino acid modifications that enhance human FcRn binding activity under acidic pH conditions can also be combined in the mutant Fc region described herein. Specifically, such modifications include, for example, the substitution of Met at position 428 with Leu and the substitution of Asn at position 434 with Ser, as represented by EU numbering (Zalevsky et al., Nat. Biotechnol. 28:157-159(2010)); the substitution of Asn at position 434 with Ala (Deng et al., Metab. Dispos. 38(4):600-605(2010)); the substitution of Met at position 252 with Tyr, the substitution of Ser at position 254 with Thr and the substitution of Thr at position 256 with Glu (Dall'Acqua et al., J. Biol. Chem. 281:23514-23524(2006)); the substitution of Thr at position 250 with Gln and the substitution of Met at position 428 with Leu (Hinton et al., J. Immunol. 176(1):346-356 (2006)); Substitution of Asn to His at position 434 (Zheng et al., Clin. Pharmacol. Ther. This may include modifications as described in 89(2):283-290(2011)) and WO2010 / 106180, WO2010 / 045193, WO2009 / 058492, WO2008 / 022152, WO2006 / 050166, WO2006 / 053301, WO2006 / 031370, WO2005 / 123780, WO2005 / 047327, WO2005 / 037867, WO2004 / 035752, or WO2002 / 060919. Such modifications may include, for example, at least one modification selected from the group consisting of the substitution of Met at position 428 with Leu, the substitution of Asn at position 434 with Ala, and the substitution of Tyr at position 436 with Thr. These modifications may further include the substitution of Gln at position 438 with Arg and / or the substitution of Ser at position 440 with Glu (Japanese Patent Application Nos. 2015-021371 and 2015-185254).

[0287] Two or more polypeptides containing the mutated Fc region described herein can be included in a single molecule, where two polypeptides containing the mutated Fc region are conjugated, much like in a single antibody. The type of antibody is not limited, and IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), and IgM can be used.

[0288] The two bound polypeptides containing mutant Fc regions may be polypeptides containing mutant Fc regions with identical amino acid modifications (hereinafter referred to as homologous mutant Fc regions), polypeptides containing mutant Fc regions with different amino acid modifications, or polypeptides containing mutant Fc regions with amino acid modifications in only one of the Fc regions (hereinafter referred to as non-homologous polypeptides containing mutant Fc regions). One preferred amino acid modification is a modification of the loop structure at positions 233-239 (EU numbering) within the CH2 domain of the Fc region, which is involved in binding to FcγRIIb and FcγRIIa. Preferably, a modification is introduced to the loop structure of the CH2 domain of one Fc region to improve FcγRIIb binding activity and / or selectivity, and another modification is introduced to the loop structure of the CH2 domain of the other Fc region to destabilize it. An example of an amino acid modification that can destabilize the loop structure of the CH2 domain is the substitution of at least one amino acid selected from the amino acids at positions 235, 236, 237, 238, and 239 with another amino acid. In particular, this can be destabilized, for example, by changing the amino acid at position 235, represented by EU numbering, to Asp, Gln, Glu, or Thr; changing the amino acid at position 236 to Asn; changing the amino acid at position 237 to Phe or Trp; changing the amino acid at position 238 to Glu, Gly, or Asn; and changing the amino acid at position 239 to Asp or Glu.

[0289] Regarding the binding of non-homologous polypeptides containing mutated Fc regions, techniques can be applied to suppress unintended binding of homologous polypeptides containing mutated Fc regions by introducing electrostatic repulsion at the interface of the CH2 or CH3 domain of the Fc region, as described in WO2006 / 106905.

[0290] Examples of amino acid residues in contact with the interface of the CH2 or CH3 domain in the Fc region include the residues at positions 356 (EU numbering), 439 (EU numbering), 357 (EU numbering), 370 (EU numbering), 399 (EU numbering), and 409 (EU numbering) within the CH3 domain.

[0291] More specifically, for example, 1 to 3 pairs of amino acid residues selected from (1) to (3) below can be used to create an Fc region having the same charge: (1) amino acid residues at positions 356 and 439 (EU numbering) in the CH3 domain; (2) amino acid residues at positions 357 and 370 (EU numbering) in the CH3 domain; and (3) amino acid residues at positions 399 and 409 (EU numbering) in the CH3 domain.

[0292] Furthermore, it is possible to create non-homologous polypeptides containing mutant Fc regions in which 1 to 3 pairs of amino acid residues selected from (1) to (3) above have the same charge in the CH3 domain of the first Fc region, and the same pair of amino acid residues selected in the first Fc region also has the same charge in the CH3 domain of the second Fc region, but the charges of the first and second Fc regions are diametrically opposed.

[0293] In the Fc region described above, for example, the negatively charged amino acid residue is preferably selected from glutamic acid (E) and aspartic acid (D), and the positively charged amino acid residue is preferably selected from lysine (K), arginine (R), and histidine (H).

[0294] Further known techniques can be used for the binding of non-homologous polypeptides containing mutant Fc regions. Specifically, such techniques are carried out by substituting an amino acid side chain present in one Fc region with a larger side chain (knob) and substituting an amino acid side chain present in the Fc region with a smaller side chain (hole), thereby placing the knob within the hole. This promotes efficient binding between Fc region-containing polypeptides having different amino acid sequences (WO1996 / 027011; Ridgway et al., Prot. Eng. 9:617-621(1996); Merchant et al., Nat. Biotech. 16, 677-681(1998)).

[0295] Furthermore, other known techniques can be used for non-homologous binding of polypeptides containing mutant Fc regions. Binding of polypeptides containing Fc regions can be efficiently induced using a chain-exchange recombination domain CH3 heterodimer (Davis et al., Prot. Eng. Des. & Sel., 23:195-202 (2010)). This technique can also be used to efficiently induce binding between Fc region-containing polypeptides having different amino acid sequences.

[0296] In addition, heterodimerized antibody production techniques using the conjugation of antibody CH1 and CL, and VH and VL, as described in WO2011 / 028952, can also be used.

[0297] It is also possible to use a heterodimerization antibody production technique, similar to the methods described in WO2008 / 119353 and WO2011 / 131746, which involves preparing two types of homodimerized antibodies in advance, incubating these antibodies under reducing conditions to dissociate them, and then rejoining them.

[0298] Similar to the method described in Strop (J. Mol. Biol. 420:204-219 (2012)), it is also possible to use heterodimerized antibody production techniques by introducing charged residues such as Lys, Arg, Glu, and Asp to introduce electrostatic repulsion into the CH3 domain.

[0299] Furthermore, it is also possible to use heterodimerized antibody production techniques by modifying the CH2 and CH3 domains, similar to the method described in WO2012 / 058768.

[0300] When two polypeptides containing mutant Fc regions with different amino acid sequences are simultaneously expressed to produce polypeptides containing non-homologous mutant Fc regions, polypeptides containing homologous mutant Fc regions are usually also produced as impurities. In such cases, polypeptides containing non-homologous mutant Fc regions can be efficiently obtained by separating and purifying them from polypeptides containing homologous mutant Fc regions using known techniques. A method has been reported for efficiently separating and purifying heterodimerized antibodies from homodimerized antibodies using ion-exchange chromatography by introducing amino acid modifications into the variable regions of the two types of antibody heavy chains that create a difference in isoelectric points between homodimerized and heterodimerized antibodies (WO2007 / 114325). Another method has been reported for purifying heterodimerized antibodies using protein A chromatography by constructing heterodimerized antibodies containing two types of heavy chains derived from mouse IgG2a, which binds to protein A, and rat IgG2b, which does not bind to protein A (WO1998 / 050431 and WO1995 / 033844).

[0301] Furthermore, by substituting amino acid residues at positions 435 and 436 (EU numbering) located at the protein A binding site of the antibody heavy chain with amino acids such as Tyr or His, heterodimerized antibodies can be efficiently purified using protein A chromatography to obtain different protein A binding affinities.

[0302] In this invention, amino acid modification means any substitution, deletion, addition, insertion, and modification, or a combination thereof. In this invention, amino acid modification can be rephrased as amino acid mutation.

[0303] When substituting amino acid residues, substitutions with other amino acid residues may be carried out with the aim of modifying any of the following: (a) the polypeptide backbone structure in the sheet or helical region; (b) the charge or hydrophobicity of the target site; or (c) the size of the side chain.

[0304] Amino acid residues are classified into the following groups based on the general characteristics of their side chains: (a) hydrophobic: norleucine, Met, Ala, Val, Leu, and Ile; (b) neutral hydrophilic: Cys, Ser, Thr, Asn, and Gln; (c) acidic: Asp and Glu; (d) basic: His, Lys, and Arg; (e) residues that affect chain orientation: Gly and Pro; and (f) aromatic: Trp, Tyr, and Phe.

[0305] Amino acid modification can be achieved by a wide variety of methods known to those skilled in the art. Such methods include, but are not limited to, site-directed mutagenesis (Hashimoto-Gotoh et al., Gene 152:271-275(1995); Zoller, Meth. Enzymol. 100:468-500(1983); Kramer et al., Nucleic Acids Res. 12: 9441-9456(1984)); Kramer and Fritz, Methods Enzymol. 154: 350-367(1987); and Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492(1985)), PCR mutagenesis, and cassette mutagenesis.

[0306] The number of amino acid modifications introduced into the Fc region is not limited. In certain embodiments, it may be 1, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 8 or less, 10 or less, 12 or less, 14 or less, 16 or less, 18 or less, or 20 or less.

[0307] Amino acid modifications include post-translational modifications. Certain post-translational modifications may involve the addition or deletion of glycans. For example, the amino acid residue at position 297 (EU numbering) within the IgG1 constant region may be glycan-modified. The glycan structure for modification is not limited. For example, sialic acid can be added to the glycan in the Fc region (MAbs 2010 Sep-Oct, 2(5): 519-527). Generally, antibodies expressed in eukaryotic cells contain glycosylation in their constant region. For example, antibodies expressed in cells such as naturally occurring mammalian antibody-producing cells or eukaryotic cells transformed with expression vectors containing antibody-coding DNA are known to typically have some type of glycan attached.

[0308] The eukaryotic cells described herein include yeast and animal cells. For example, CHO cells and HEK293 cells are typical animal cells used for transformation with expression vectors containing antibody-encoding DNA. On the other hand, the present invention also includes unglycosylated constant regions. Antibodies with an unglycosylated constant region can be obtained by expressing the antibody-encoding gene in prokaryotic cells such as Escherichia coli.

[0309] Furthermore, the polypeptide containing the mutated Fc region of the present invention may be chemically modified with various molecules such as polyethylene glycol (PEG) and cytotoxic substances. Such chemical modification methods for polypeptides are well established in the art.

[0310] In one aspect, the present invention provides an isolated polypeptide comprising a mutant Fc region with enhanced FcγRIIb binding activity. In several aspects, the polypeptide is an antibody. In several aspects, the polypeptide is an Fc fusion protein. In certain embodiments, the antibody is a chimeric antibody or a humanized antibody. The origin of the antibody is not particularly limited, but examples include human antibodies, mouse antibodies, rat antibodies, and rabbit antibodies. In several aspects, the polypeptide is an Fc fusion protein.

[0311] The variable regions of antibodies containing mutated Fc regions and the protein-binding motifs of Fc fusion proteins containing mutated Fc regions provided herein can recognize any antigen. Examples of antigens that can be bound by such antibodies and fusion proteins include, but are not limited to, ligands (such as cytokines and chemokines), receptors, cancer antigens, MHC antigens, differentiation antigens, immunoglobulins, and immunocomplexes partially containing immunoglobulins.

[0312] Examples of cytokines that can be conjugated by antibodies or fusion proteins containing the mutated Fc region of the present invention and / or recombinantly fused with polypeptides containing the disclosed mutated Fc region include, but are not limited to, interleukins 1-18, 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 and TNF-β), lymphotoxins, erythropoietin, leptin, SCF, TPO, MCAF, and BMP.

[0313] Examples of chemokines that can be conjugated by an antibody or fusion protein containing the mutated Fc region of the present invention and / or recombinantly fused with a polypeptide containing the disclosed mutated Fc region include, but are not limited to, 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.

[0314] Examples of receptors that can be conjugated by an antibody or fusion protein containing the mutated Fc region of the present invention and / or recombinantly fused with a polypeptide containing the disclosed mutated Fc region include, but are not limited to, receptors belonging to receptor families such as the hematopoietic growth factor receptor family, cytokine receptor family, tyrosine kinase receptor family, serine / threonine kinase receptor family, TNF receptor family, G protein-linked receptor family, GPI-anchored receptor family, tyrosine phosphatase receptor family, adhesion factor family, and hormone receptor family. Receptors belonging to these receptor families and their characteristics are described in Cooke, ed. New Comprehensive Biochemistry Vol.18B "Hormones and their Actions Part II" pp.1-46 (1988) Elsevier Science Publishers BV; Patthy (Cell 61(1):13-14 (1990)); Ullrich (Cell 61(2):203-212 (1990)); Massague (Cell 69(6):1067-1070 (1992)); Miyajima et al. (Annu. Rev. Immunol. 10:295-331 (1992)); Taga et al. (FASEB J. 6:3387-3396 (1992)); Fantl et al. (Annu. Rev. Biochem. 62:453-481 (1993)); Smith et al. It is described in numerous publications, including al. (Cell 76(6):959-962(1994)) and Flower (Biochim. Biophys. Acta 1422(3): 207-234(1999)).

[0315] Examples of specific receptors belonging to the above receptor families include the human or mouse erythropoietin (EPO) receptor (Jones et al., Blood 76(1):31-35(1990); D'Andrea et al., Cell 57(2):277-285(1989)), the human or mouse granulocyte colony-stimulating factor (G-CSF) receptor (Fukunaga et al., Proc. Natl. Acad. Sci. USA 87(22):8702-8706(1990), mG-CSFR; Fukunaga et al., Cell 61(2): 341-350(1990)), and the human or mouse thrombopoietin (TPO) receptor (Vigon et al., Proc. Natl. Acad. Sci. USA. 89(12):5640-5644(1992); Skoda et al., EMBO J. 12(7):2645-2653(1993)), human or mouse insulin receptor (Ullrich et al., Nature 313(6005):756-761(1985)), human or mouse Flt-3 ligand receptor (Small et al., Proc. Natl. Acad. Sci. USA. 91(2):459-463(1994)), human or mouse platelet-derived growth factor (PDGF) receptor (Gronwald et al., Proc. Natl. Acad. Sci. USA. 85(10):3435-3439(1988)), human or mouse interferon (IFN)-α and β receptors (Uze et al., Cell 60(2): 225-234(1990); Novick et al., Cell These include human or mouse leptin receptors, human or mouse growth hormone (GH) receptors, human or mouse interleukin (IL)-10 receptors, human or mouse insulin-like growth factor (IGF)-I receptors, human or mouse leukemia suppressor (LIF) receptors, and human or mouse ciliary neurotrophic factor (CNTF) receptors.

[0316] Cancer antigens are antigens that are expressed as cells become malignant, and are also called tumor-specific antigens. Abnormal glycans that appear on the cell surface or protein molecules when cells become cancerous are also cancer antigens, and these are also called glycan cancer antigens. Examples of cancer antigens that can be bound by antibodies or fusion proteins containing the mutated Fc region of the present invention include, but are not limited to, GPC3 (Midorikawa et al., Int. J. Cancer 103(4):455-465(2003)), a receptor belonging to the GPI-anchored receptor family mentioned above and expressed in several cancers including liver cancer, and EpCAM (Linnenbach et al., Proc. Natl. Acad. Sci. USA 86(1):27-31(1989)), CA19-9, CA15-3, and sialyl SSEA-1 (SLX), which are expressed in several cancers including lung cancer.

[0317] MHC antigens are broadly 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.

[0318] Examples of differentiation antigens that can be conjugated by an antibody or fusion protein containing the mutant Fc region of the present invention and / or recombinantly fused with a polypeptide containing the disclosed mutant Fc region include CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15s, CD16, CD18, CD19, CD20, CD21, CD23, CD25, CD28, CD29, CD30, CD32, CD33, CD34, CD35 CD38, CD40, CD41a, CD41b, CD42a, CD42b, CD43, CD44, CD45, CD45RO, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD51, CD54, CD55, CD56, CD57, CD58, CD61, CD62E, CD62L, CD62P, CD64, CD69, CD71, CD73, CD95, CD102, CD106, CD122, CD126, and CDw130 are included without limitation.

[0319] Immunoglobulins include IgA, IgM, IgD, IgG, and IgE. Immune complexes contain at least one component of immunoglobulin.

[0320] Other examples of antigens that can be conjugated by antibodies or fusion proteins containing the mutant Fc region of the present invention and / or recombinantly fused with polypeptides containing the disclosed mutant Fc region include 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, and activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressi aFGF, ALCAM, ALK, ALK-1, ALK-7, α-1-antitrypsin, α-V / β-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, Al Artemin, Anti-Id, Aspartic, Atrial Natriuretic Peptide, av / b3 Integrin, Axl, b2M, B7-1, B7-2, B7-H, B Lymphocyte-Stimulating 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-6 Vgr-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, β-NGF, BOK, Bombecin, 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, CCR11, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Botulinum toxin, Clostridium perfringens toxin, Ckb8-1, CLC, CMV, 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, Cytokeratin tumor-related antigen, DAN, DCC, DcR3, DC-SIGN, Complement regulatory factor (decay accelerating factor), des(1-3)-IGF-I (Brain IGF-1), Dhh, Digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAREGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, factor IIa, factor VII, factor VIIIc, factor IX, fibroblast-activating protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF8 (Myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GFAP, GFRa-1, GFR-α1, GFR-α2, GFR-α3, GITR, Glucagon, Glut4, Glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GRO, Growth hormone-releasing hormone, 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, HMFG PEM, 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 (IFN)-α, IFN-β, IFN-γ, Inhibin, iNOS, Insulin A chain, Insulin B chain, Insulin-like growth factor 1, Integrin α2, Integrin α3, Integrin α4, Integrin α4 / β1, Integrin α4 / β7, In Tegrin α5 (αV), Integrin α5 / β1, Integrin α5 / β3, Integrin α6, Integrin β1, Integrin β2, Interferon γ, 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-1 bp1, 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 β receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinase, MGDF receptor Phytoplasm, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-α, 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 duct inhibitor, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA 90, NCAM, Neprilysin, Neurotrophin-3, -4, or -6, Neuroturin, Nerve growth factor (NGF), NGFR, NGF-β,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, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSV Fgp, 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 α / β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, TGF-β Pan-specific, TGF-βRI (ALK-5), TGF-βRII, TGF-βRIIb, TGF-βRIII, TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, thrombin, thymic Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-α, TNF-αβ, TNF-β2, 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(WHEM ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR). p75NTR), TNFRSF17(BCMA), TNFRSF18(GITR AITR), TNFRSF19(TROY CROWN), TNFRSF19L(RELT), TNFRSF1A(TNF RI CD120a, p55-60), TNFRSF1B(TNF RII). CD120b, 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 polymer TL2) TNFSF11(TRANCE / RANK polymer ODF) TNFSF12(TWEAK). Apo-3リンド, DR3リンド, TNFSF13(APRIL TALL2), TNFSF13B(BAFF LIGHT, TALL1, THANK, TNFSF20), TNFSF14(LIGHT HVEM LTg, TNFSF15(TL1A / VEGI), TNFSF18 (GITR AITR ligand TL6), TNFSF1A(TNF-a ligand DIF, TNFSF2), TNFSF1B(TNF-b LTa, TNFSF1), TNFSF3(LTb). TNFC, p33, TNFSF4(OX40functional gp34, TXGP1), TNFSF5(CD40 functional 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, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expression Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM Viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, Aβ CD81, CD97, CD98, DDR1, DKK1, EREG, Hsp90, IL-17 / IL-17R, IL-20 / IL-20R, Oxidized LDL, PCSK9, Prekallikrein, RON, TMEM16F, SOD1, Chromogranin A, Chromogranin B, Tau, VAP1, High Molecular Weight Kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, E PCR, 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, fibrino Gen, 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, 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, and S1P; as well as hormone and growth factor receptors, are not limited to these.

[0321] As described herein, in the amino acid sequence constituting the variable region, one or more amino acid residues can be modified as long as their antigen-binding activity is maintained. When modifying the amino acid sequence of the variable region, there are no particular limitations on the modification site or the number of amino acids modified. For example, amino acids present in the CDR and / or FR can be modified as appropriate. When modifying amino acids within the variable region, it is preferable that the binding activity is maintained without particular limitations; for example, the binding activity may be 50% or more, 80% or more, and 100% or more compared to the original binding activity. Furthermore, the binding activity may be enhanced by the amino acid modification. For example, the binding activity may be 2 times, 5 times, or 10 times higher than the binding activity before modification. Modification of the amino acid sequence may consist of at least one of amino acid residue substitution, addition, deletion, and modification.

[0322] For example, the modification of the N-terminus of the variable region to pyroglutamic acid by pyroglutamylation is a modification well known to those skilled in the art. Therefore, when the N-terminus of the heavy chain is glutamine, the antibody described herein may include a variable region in which glutamine has been modified to pyroglutamic acid.

[0323] The antibody variable regions described herein may have any sequence and may be antibody variable regions of any origin, such as mouse antibodies, rat antibodies, rabbit antibodies, goat antibo...

Claims

1. An isolated antibody that binds to latent myostatin, The antibody contains a mutant Fc region with amino acid modifications in the parent Fc region, which is the Fc region derived from human IgG1. The ratio of [KD value of the parental Fc region for monkey FcγRIIb] / [KD value of the mutant Fc region for monkey FcγRIIb] is 2.0 or greater, and the ratio of [KD value of the parental Fc region for human FcγRIIIa] / [KD value of the mutant Fc region for human FcγRIIIa] is 0.5 or less, and, This antibody has an Fc region that exhibits enhanced binding affinity to both human FcγRIIb and cynomolgus monkey FcγRIIb, and reduced binding affinity to both human FcγRIIIa and cynomolgus monkey FcγRIIIa. The mutated Fc region is It includes at least one amino acid modification at at least one position selected from the group consisting of positions 231, 232, 234, 235, 236, 237, 239, 267, 268, 295, 298, 326, 330, 331, and 396 according to EU numbering, and Includes at least two amino acid modifications at positions 236, 268, and 396. antibody.

2. The mutated Fc region, according to EU numbering, (a) Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr, (b) Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr, (d) Trp, Tyr, at position 234 (e) Trp at position 235, (f) Ala, Asp, Glu, His, Ile, Leu, Met, Asn, Gln, Ser, Thr, Val, at position 236 (g) Asp, Tyr, at position 237 (i) Ile, Leu, Asn, Pro, Val at position 239 (l) Ala, His, Leu at position 267, (m) Asp, Glu at position 268 (o) Leu at position 295, (p) Leu at position 298, (r) Thr at position 326, (u) Lys, Arg, at position 330 (v) Glu at position 331, and (y) Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 396 The antibody according to claim 1, comprising at least one amino acid selected from the group consisting of the following.

3. The mutated Fc region, according to EU numbering, (a) Gly, Thr at position 231 (b) Asp at position 232, (c) Trp at position 235, (d) Asn, Thr, at position 236 (e) Val at position 239, (f) Asp, Glu, at position 268 (g) Leu at position 295, (h) Leu at position 298, (i) Thr at position 326, (j) Lys, Arg, and at position 330 (k) Lys, Met at position 396 The antibody according to claim 2, comprising at least one amino acid selected from the group consisting of the following.

4. The antibody according to any one of claims 1 to 3, comprising the following: (i) HVR-H1 containing the amino acid sequence of SEQ ID NO: 114, HVR-H2 containing the amino acid sequence of SEQ ID NO: 58, HVR-H3 containing the amino acid sequence of SEQ ID NO: 63, HVR-L1 containing the amino acid sequence of sequence number 122, HVR-L2 containing the amino acid sequence of SEQ ID NO: 71, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 74; or, (ii) HVR-H1 containing the amino acid sequence of sequence number 114, HVR-H2 containing the amino acid sequence of sequence number 116, HVR-H3 containing the amino acid sequence of SEQ ID NO: 121, HVR-L1 containing the amino acid sequence of sequence number 122, HVR-L2 containing the amino acid sequence of SEQ ID NO: 71, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 74; or, (iii) HVR-H1 containing the amino acid sequence of sequence number 57, HVR-H2 containing the amino acid sequence of sequence number 117, HVR-H3 containing the amino acid sequence of SEQ ID NO: 63, HVR-L1 containing the amino acid sequence of sequence number 122, HVR-L2 containing the amino acid sequence of SEQ ID NO: 71, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 74; or, (iv) HVR-H1 containing the amino acid sequence of sequence number 57, HVR-H2 containing the amino acid sequence of sequence number 118, HVR-H3 containing the amino acid sequence of SEQ ID NO: 63, HVR-L1 containing the amino acid sequence of sequence number 122, HVR-L2 containing the amino acid sequence of SEQ ID NO: 71, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 74; or, (v) HVR-H1 containing the amino acid sequence of Sequence ID: 57, HVR-H2 containing the amino acid sequence of sequence number: 119, HVR-H3 containing the amino acid sequence of SEQ ID NO: 63, HVR-L1 containing the amino acid sequence of sequence number 122, HVR-L2 containing the amino acid sequence of SEQ ID NO: 71, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 74; or, (vi) HVR-H1 containing the amino acid sequence of sequence number:114, HVR-H2 containing the amino acid sequence of sequence number 118, HVR-H3 containing the amino acid sequence of SEQ ID NO: 63, HVR-L1 containing the amino acid sequence of sequence number 122, HVR-L2 containing the amino acid sequence of SEQ ID NO: 71, and Sequence ID: HVR-L3 containing amino acid sequence 74; or, (vii) HVR-H1 containing the amino acid sequence of sequence number 114, HVR-H2 containing the amino acid sequence of SEQ ID NO: 58, HVR-H3 containing the amino acid sequence of SEQ ID NO: 63, HVR-L1 containing the amino acid sequence of sequence number: 123, HVR-L2 containing the amino acid sequence of SEQ ID NO: 71, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 74; or, (viiii) HVR-H1 containing the amino acid sequence of sequence number:115, HVR-H2 containing the amino acid sequence of SEQ ID NO: 58, HVR-H3 containing the amino acid sequence of SEQ ID NO: 63, HVR-L1 containing the amino acid sequence of sequence number: 123, HVR-L2 containing the amino acid sequence of SEQ ID NO: 71, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 74; or, (ix) HVR-H1 containing the amino acid sequence of sequence number 114, HVR-H2 containing the amino acid sequence of SEQ ID NO: 58, HVR-H3 containing the amino acid sequence of SEQ ID NO: 63, HVR-L1 containing the amino acid sequence of sequence number 124, HVR-L2 containing the amino acid sequence of SEQ ID NO: 125, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 74; or, (x) HVR-H1 containing the amino acid sequence of sequence number: 115, HVR-H2 containing the amino acid sequence of SEQ ID NO: 120, HVR-H3 containing the amino acid sequence of SEQ ID NO: 63, HVR-L1 containing the amino acid sequence of sequence number: 123, HVR-L2 containing the amino acid sequence of SEQ ID NO: 71, and HVR-L3 containing the amino acid sequence of SEQ ID NO:

74.

5. An isolated nucleic acid encoding the antibody according to claim 4.

6. A host cell comprising the nucleic acid described in claim 5.

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