Anti-myostatin antibodies, polypeptides comprising variant fc regions, and methods of use

Abstract

It is an object of the invention to provide anti-myostatin antibodies, polypeptides comprising a variant Fc region, and methods of use thereof.SOLUTION: The present disclosure provides anti-myostatin antibodies and methods of making and using the same. Nucleic acids encoding the anti-myostatin antibodies and host cells comprising the nucleic acids are also provided. The present disclosure also provides polypeptides comprising variant Fc regions and methods of making and using the same. Nucleic acids encoding the polypeptides and host cells comprising the nucleic acids are also provided.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] Myostatin, also known as growth 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 regulatory and morphogenetic properties (see, for example, Non-Patent Document 1, Non-Patent Document 2, and Patent Document 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, for example, Non-Patent Document 3). Conversely, myostatin knockout mice are characterized by skeletal muscle hypertrophy and hyperplasia resulting in muscle mass two to three times greater than that of wild-type littermates (see, for example, 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 that is thought to be the active molecule (see, for example, Non-Patent Document 5; Patent Document 2). 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-linked homodimer consisting of the C-terminal growth factor domain) is released from the myostatin precursor through multiple 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 in both chains of the homodimeric precursor by a furin-type proprotein convertase. However, the resulting three peptides (two propeptides and one mature myostatin (i.e., a disulfide-linked homodimer consisting of the growth factor domain)) remain associated, forming a noncovalent, inactive complex called "latent myostatin." Mature myostatin can then be released from latent myostatin via degradation of the propeptide. Members of the bone morphogenetic protein 1 (BMP1) family of metalloproteinases cleave a single peptide bond, Arg98-Asp99, in the propeptide, concomitantly releasing the active, mature myostatin homodimer (see, e.g., Non-Patent Document 6). Furthermore, latent myostatin can also be activated in vitro by dissociating the complex with either acid or heat treatment (see, e.g., Non-Patent Document 7).

[0004] Myostatin exerts its effects through the transmembrane serine / threonine kinase heterotetrameric receptor family, activation of which enhances receptor transphosphorylation, thereby stimulating serine / threonine kinase activity. The myostatin pathway involves the high-affinity binding of active myostatin dimers to the activin type IIB receptor (ActRIIB), which then recruits and activates transphosphorylation of lower-affinity receptors, such as activin-like kinase 4 (ALK4) or activin-like kinase 5 (ALK5). The proteins Smad2 and Smad3 are subsequently activated, forming a complex with Smad4 that then translocates to the nucleus for transcriptional activation of target genes. Expression of a dominant-negative form of ActRIIB in mice 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., muscle tissue loss or dysfunction), 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 kidney disease, heart failure or heart disease, and liver disease. Patients benefit from increased muscle mass and / or strength, but available treatments 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 preventive intervention in 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 Document 4). Myostatin negatively regulates muscle regeneration after injury, and its absence in myostatin null mice accelerates muscle regeneration (see, e.g., Non-Patent Document 9). Anti-myostatin (GDF8) antibodies, described in, for example, Patent Documents 3, 4, 5, 6, and 7, and Patent Documents 8, 9, and 10, have been shown to bind to myostatin and inhibit myostatin activity (including myostatin activity related to 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 wild-type mice (see, e.g., Non-Patent Document 10) and mdx mice, a model of muscular dystrophy (see, e.g., Non-Patent Document 11; Non-Patent Document 12). However, all of these prior art antibodies are specific for mature myostatin but not for 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 pharmaceutical agents because they are highly stable in the blood and have few side effects (see, for example, Non-Patent Document 13 and Non-Patent Document 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 an Fcγ receptor (hereinafter referred to as FcγR), an antibody-binding receptor present on the surface of effector cells such as killer cells, natural killer cells, and activated macrophages.

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

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

[0010] When activated FcγR is crosslinked with immune complexes, it phosphorylates immunoreceptor tyrosine-based activation motifs (ITAMs) contained in the intracellular domain or the FcR common γ chain (interacting partner), activating the signal transducer 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). It has been reported that the interaction between the antibody Fc region and FcγRIIb suppresses the early 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 crosslinked in the blood via immune complexes, B cell activation and antibody production by B cells are suppressed (see, for example, Non-Patent Document 24). The immunoreceptor tyrosine-based inhibitory motif (ITIM) contained in the intracellular domain of FcγRIIb is essential for this immunosuppressive signaling mediated by BCR and FcγRIIb (see, for example, Non-Patent Document 25 and Non-Patent Document 26). During signal transduction, the ITIM is phosphorylated, recruiting SH2-containing inositol polyphosphate 5-phosphatase (SHIP), inhibiting the transmission of other activating FcγR signaling cascades and suppressing inflammatory immune responses (see, for example, Non-Patent Document 27). Furthermore, it has been reported that FcγRIIb aggregation alone transiently suppresses calcium influx caused by BCR cross-linking and transiently suppresses 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 activated FcγR functions, such as phagocytosis, and the release of inflammatory cytokines in these cells, thereby suppressing inflammatory immune responses (see, for example, Non-Patent Document 17).

[0013] The importance of the immunosuppressive function of FcγRIIb has been elucidated by studies using FcγRIIb knockout mice. It has been reported that FcγRIIb knockout mice have inadequately regulated humoral immunity (see, for example, Non-Patent Document 29), increased susceptibility to collagen-induced arthritis (CIA) (see, for example, Non-Patent Document 30), and the presence of lupus-like symptoms and Goodpasture's 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 between genetic polymorphisms in the transmembrane and promoter regions of FcγRIIb and the incidence of systemic lupus erythematosus (SLE) has been reported (see, for example, Non-Patent Documents 32, 33, 34, 35, and 36), and reduced expression of FcγRIIb on the B cell surface 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 engagement with B cells, and is 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 strongly bind not only to FcγRIIb but also to activating FcγRs (see, for example, Non-Patent Document 39). By using an Fc region with enhanced FcγRIIb binding or improved FcγRIIb-binding selectivity relative to activating FcγRs, it may be possible to develop therapeutic antibodies with more immunosuppressive properties than those of IgG1. For example, it has been suggested that the use of an antibody with a variable region that binds to the BCR and an Fc with enhanced FcγRIIb binding can inhibit B cell activation (see, for example, Non-Patent Document 40). It has been reported that cross-linking FcγRIIb on B cells with IgE bound to the B cell receptor inhibits B cell differentiation into plasma cells, resulting in suppression of IgE production; and that in mice transplanted with human PBMCs, human IgG and IgM concentrations are maintained, while human IgE concentrations are reduced (see, for example, Non-Patent Document 41). It has been reported that when IgE, as well as FcγRIIb, and CD79b, a component of the B cell receptor complex, are cross-linked by an antibody, B cell proliferation is suppressed in vitro and arthritis symptoms are alleviated in a collagen-induced arthritis model (see, for example, non-patent document 42).

[0017] In addition to B cells, it has been reported that cross-linking of FcγRIIb 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 causes phosphorylation of FcγRIIb, thereby suppressing FcεRI-dependent calcium influx. This suggests that enhancing FcγRIIb binding can inhibit FcγRIIb-mediated degranulation (see, for example, Non-Patent Document 43).

[0018] Therefore, it is suggested that antibodies having an Fc with improved FcγRIIb binding activity are promising as therapeutic agents for inflammatory diseases such as autoimmune diseases.

[0019] Furthermore, it has been reported that the activation of macrophages and dendritic cells mediated by Toll-like receptor 4 due to LPS stimulation is suppressed in the presence of antibody-antigen immune complexes, suggesting that this effect is also 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 enhance the inhibitory effect of TLR-mediated activation signals, suggesting that such antibodies are promising therapeutic agents for inflammatory diseases such as autoimmune diseases.

[0020] Furthermore, mutations with enhanced FcγRIIb binding have been suggested to be promising therapeutic agents for cancer, as well as for inflammatory diseases such as autoimmune diseases. FcγRIIb has been found to play an important role in the agonistic activity of agonistic antibodies against the anti-TNF receptor superfamily. Specifically, it has been suggested that interaction with FcγRIIb is necessary for the agonistic activity of antibodies against CD40, DR4, DR5, CD30, and CD137, which are members of the TNF receptor family (see, for example, Non-Patent Document 46, Non-Patent Document 47, Non-Patent Document 48, Non-Patent Document 49, Non-Patent Document 50, Non-Patent Document 51, and Non-Patent Document 52). Non-Patent Document 46 demonstrates that the use of antibodies with enhanced FcγRIIb binding enhances the anti-tumor activity of anti-CD40 antibodies. Therefore, antibodies with enhanced FcγRIIb binding are expected to have the effect of enhancing the agonistic activity of agonistic antibodies, including antibodies against the anti-TNF receptor superfamily.

[0021] Furthermore, it has been shown that cross-linking Kit and FcγRIIb in Kit-expressing cells using an antibody that recognizes Kit, a type of receptor tyrosine kinase (RTK), inhibits cell proliferation. A similar effect has been reported even when Kit is constitutively activated and harbors a mutation that causes tumorigenesis (see, for example, Non-Patent Document 53). Therefore, it is expected that the use of antibodies with enhanced FcγRIIb binding will enhance the inhibitory effect on cells expressing RTKs with constitutively activated mutations.

[0022] Antibodies having Fc with improved FcγRIIb-binding activity have been reported (see, for example, Non-Patent Document 40). In this document, FcγRIIb-binding activity was improved by adding alterations such as S267E / L328F, G236D / S267E, and S239D / S267E to the antibody Fc region. Among these, an antibody into which the S267E / L328F mutation has been introduced exhibits the strongest FcγRIIb binding, while its binding to FcγRIa and H-type FcγRIIa (residue 131 of FcγRIIa is His) remains at the same level as that of native IgG1. However, another report showed that this alteration enhances the binding to R-type FcγRIIa (in which the residue at position 131 of FcγRIIa is Arg) several hundred times, which is the same level as the FcγRIIb binding, meaning 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, but not FcγRIIb binding, is thought to affect cells such as platelets that express FcγRIIa but not FcγRIIb (see, e.g., Non-Patent Document 17). For example, patients administered bevacizumab, an antibody against VEGF, are known to have an increased risk of thromboembolism (see, e.g., 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, e.g., Non-Patent Document 55). In the case of both of these antibodies, subsequent studies using animal models suggested that the administered antibody aggregates platelets via FcγRIIa binding to platelets, resulting in thrombus formation (see, e.g., Non-Patent Document 56 and Non-Patent Document 57). It has been reported that in systemic lupus erythematosus, an autoimmune disease, platelets are activated via an FcγRIIa-dependent mechanism, and platelet activation correlates with the severity of symptoms (see, for example, Non-Patent Document 58). Administration of an antibody with enhanced FcγRIIa binding to such patients, who are 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 cellular phagocytosis (ADCP) (see, for example, Non-Patent Document 59). When an antigen bound by an antibody is phagocytosed by macrophages, the antibody itself is also thought to be phagocytosed at the same time. When an antibody is administered as a drug, peptide fragments derived from the administered antibody may also be presented as antigens, thus increasing the risk of the production of antibodies against the therapeutic antibody (anti-therapeutic antibodies). More specifically, enhanced FcγRIIa binding increases the risk of the production of antibodies against the therapeutic antibody, significantly reducing its value as a drug. Furthermore, FcγRIIb on dendritic cells has been suggested to contribute to peripheral tolerance by inhibiting dendritic cell activation triggered 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 an antibody having an Fc with enhanced selective binding to FcγRIIb is used as a drug, the antigen is not immediately presented by dendritic cells or the like due to the enhanced selective binding to FcγRIIb, and the risk of anti-drug antibody production can be relatively reduced. Such an antibody can be useful in this regard as well.

[0025] More specifically, enhanced FcγRIIa binding 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 above-mentioned Fc variants with enhanced FcγRIIb binding exhibit significantly enhanced R-type FcγRIIa binding compared to native IgG1. Therefore, their value as a drug for patients with R-type FcγRIIa is significantly reduced. H-type and R-type FcγRIIa are observed with approximately the same frequency in Caucasians and African Americans (see, for example, Non-Patent Documents 61 and 62). Therefore, even if this Fc variant were used to treat autoimmune diseases, the number of patients who could safely use it while still receiving its therapeutic benefits would be limited.

[0027] Furthermore, it has been reported that dendritic cells lacking FcγRIIb or in which the interaction between FcγRIIb and the Fc portion of antibodies has been inhibited by anti-FcγRIIb antibodies undergo dendritic cell maturation (see, for example, Non-Patent Documents 63 and 64). This report suggests that FcγRIIb actively suppresses dendritic cell maturation under steady-state conditions in the absence of inflammation or activation. In addition to FcγRIIb, FcγRIIa is expressed on the surface of dendritic cells. Therefore, even if binding to inhibitory FcγRIIb is enhanced, dendritic cell maturation can be promoted if binding to activating FcγRs such as FcγRIIa is also enhanced. More specifically, improving not only FcγRIIb-binding activity but also the ratio of FcγRIIb-binding activity to FcγRIIa-binding activity is considered important when providing antibodies with immunosuppressive activity.

[0028] Therefore, when considering the production of drugs that utilize immunosuppressive effects mediated by FcγRIIb binding, an Fc variant is required that not only has enhanced FcγRIIb binding activity, but also maintains binding to both H and R FcγRIIa allotypes at a level equivalent to that of native IgG1 or is weakened to a lower level.

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

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

[0031] These results suggest that, in the production of therapeutic antibodies using FcγRIIb for the treatment of autoimmune diseases and cancer, it is important to maintain or reduce binding activity to both FcγRIIa allotypes and enhance FcγRIIb binding compared to native IgG. However, FcγRIIb shares 93% sequence identity in its extracellular region with FcγRIIa, one of the activating FcγRs, and the two are structurally very similar. FcγRIIa exists in H-type and R-type allotypes, in which the amino acid at position 131 is His (H-type) or Arg (R-type), and each reacts with antibodies in a different manner (see, for example, Non-Patent Document 66). Therefore, generating Fc region variants with enhanced selective FcγRIIb binding compared to each FcγRIIa allotype may be a difficult task, which involves distinguishing between highly homologous sequences between FcγRIIa and FcγRIIb. Despite these challenges, several Fc region variants that have selective binding activity to FcγRIIb compared to FcγRIIa have been identified by comprehensive amino acid modification analysis in the Fc region (see, for example, Patent Documents 16, 17, 18, 19, and 20).

[0032] Although there have been reports of Fc region mutants of human FcγR that have binding selectivity for FcγRIIb, there have been no reports of Fc region mutants of monkey FcγR that have binding selectivity for FcγRIIb. Because no such Fc mutants exist, the effects of Fc mutants that selectively bind to FcγRIIb have not yet been thoroughly studied in monkeys.

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

[0034] Furthermore, it has been reported that modifying the charge of specific amino acid residues, particularly in the CH3 domain, to increase the pI of an antibody can promote the uptake of antigens into cells (see, for example, Patent Document 23). Similarly, it has been reported that modifying the charge of amino acid residues in the constant region (mainly the CH1 domain) of an antibody to decrease its pI can extend the half-life of the antibody in plasma (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] An object of the present invention is to provide anti-myostatin antibodies, polypeptides comprising mutated Fc regions, and methods of using the same. [Means for solving the problem]

[0038] The present invention provides anti-myostatin antibodies and methods of using them. The present invention also provides proteins comprising mutant Fc regions and methods of using them.

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

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

[0041] In some embodiments, an isolated anti-myostatin antibody of the invention competes with an antibody described herein for binding to latent myostatin. In some embodiments, an isolated anti-myostatin antibody of the invention competes with an antibody described in Table 13 for binding to latent myostatin. In some embodiments, an isolated anti-myostatin antibody of the invention competes with an antibody comprising a VH and VL pair described in Table 13 for binding to latent myostatin. In some embodiments, the antibody competes with an antibody described in Table 2a for binding to latent myostatin. In some embodiments, an isolated anti-myostatin antibody of the invention competes with an antibody comprising a VH and VL pair described in Table 2a for binding to latent myostatin. In some embodiments, the antibody competes with an antibody described in Table 11a for binding to latent myostatin. In some embodiments, an isolated anti-myostatin antibody of the present invention competes for binding to latent myostatin with an antibody comprising a VH and VL pair set forth in Table 11a. In further embodiments, an anti-myostatin antibody competes for binding to latent myostatin with an antibody set forth in Table 2a, 11a, or 13. In further embodiments, an anti-myostatin antibody competes for binding to latent myostatin with an antibody comprising a VH and VL pair set forth in Table 2a, 11a, or 13. In further embodiments, the anti-myostatin antibody binds to latent myostatin with greater affinity at neutral pH than at acidic pH. In further embodiments, the anti-myostatin antibody binds to latent myostatin with greater affinity at pH 7.4 than at pH 5.8. In a further aspect, the anti-myostatin antibody binds to a polypeptide fragment consisting of amino acids 21 to 100 of the myostatin propeptide (SEQ ID NO: 78) with greater affinity at pH 7.4 than at pH 5.8. Methods for assessing the ability of an antibody to compete with a reference antibody for binding to latent myostatin are described herein and 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 to 100 of the 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 antibodies of the invention (a) (i) an HVR-H3 comprising the amino acid sequence GVPAX1SX2GGDX3 (wherein Xi is Y or H, X2 is T or H, and X3 is L or K) (SEQ ID NO: 128), (ii) an HVR-L3 comprising the amino acid sequence AGGYGGGX1YA (wherein Xi is L or R) (SEQ ID NO: 131), and (iii) an HVR-H2 comprising the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7 (wherein Xi 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); (b) (i) HVR-H1 comprising the amino acid sequence X1X2DIS (wherein X1 is S or H and X2 is Y, T, D, or E) (SEQ ID NO: 126), (ii) HVR-H2 comprising the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7 (wherein 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), and (iii) HVR-H3 comprising the amino acid sequence GVPAX1SX2GGDX3 (wherein X1 is Y or H, X2 is T or H, and X3 is L or K) (SEQ ID NO: 128); (c) (i) HVR-H1 comprising the amino acid sequence X1X2DIS (wherein X1 is S or H, and X2 is Y, T, D, or E) (SEQ ID NO: 126), (ii) HVR-H2 comprising the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7 (wherein 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), (iii) HVR-H2 comprising the amino acid sequence GVPAX1SX2GGDX3 (wherein X1 is Y or H, X2 is T or H, and X3 is L or K) (SEQ ID NO: 128), (iv) 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) (SEQ ID NO: 129), (v) HVR-L2 comprising the amino acid sequence WAX1TLAX2 (wherein X1 is S or E and X2 is S, Y, F, or W) (SEQ ID NO: 130), and (vi) HVR-L3 comprising the amino acid sequence AGGYGGGX1YA (wherein X1 is L or R) (SEQ ID NO: 131); (d) (i) 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) (SEQ ID NO: 129), (ii) HVR-L2 comprising the amino acid sequence WAX1TLAX2 (wherein X1 is S or E and X2 is S, Y, F, or W) (SEQ ID NO: 130), and (iii) HVR-L3 comprising the amino acid sequence AGGYGGGX1YA (wherein X1 is L or R) (SEQ ID NO: 131). In some embodiments, the antibody of (b) further comprises a heavy chain variable domain framework FR1 comprising the amino acid sequence of any one of SEQ ID NOs: 132 to 134; FR2 comprising the amino acid sequence of any one of SEQ ID NOs: 135 to 136; FR3 comprising the amino acid sequence of SEQ ID NO: 137; and FR4 comprising 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 comprising the amino acid sequence of SEQ ID NO: 139; FR2 comprising the amino acid sequence of any one of SEQ ID NOs: 140 to 141; FR3 comprising the amino acid sequence of any one of SEQ ID NOs: 142 to 143; and FR4 comprising the amino acid sequence of SEQ ID NO: 144.

[0044] In some embodiments, an isolated anti-myostatin antibody of the present invention comprises (a) an HVR-H3 having the amino acid sequence GVPAX1SX2GGDX3 (wherein X1 is Y or H, X2 is T or H, and X3 is L or K) (SEQ ID NO: 128), (b) an HVR-L3 having the amino acid sequence AGGYGGGX1YA (wherein X1 is L or R) (SEQ ID NO: 131), and (c) an HVR-H2 having the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7 (wherein 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) an HVR-H1 comprising the amino acid sequence X1X2DIS (wherein X1 is S or H and X2 is Y, T, D or E) (SEQ ID NO: 126), (b) an HVR-H2 comprising the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7 (wherein 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), and (c) an HVR-H3 comprising the amino acid sequence GVPAX1SX2GGDX3 (wherein X1 is Y or H, X2 is T or H, and X3 is L or K) (SEQ ID NO: 128). In a further aspect, the antibody comprises a heavy chain variable domain framework FR1 comprising the amino acid sequence of any one of SEQ ID NOs: 132 to 134; FR2 comprising the amino acid sequence of any one of SEQ ID NOs: 135 to 136; FR3 comprising the amino acid sequence of SEQ ID NO: 137; and FR4 comprising the amino acid sequence of SEQ ID NO: 138. In a further embodiment, the antibody additionally comprises (a) an 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) (SEQ ID NO: 129), (b) an HVR-L2 comprising the amino acid sequence WAX1TLAX2 (wherein X1 is S or E and X2 is S, Y, F, or W) (SEQ ID NO: 130), and (c) an HVR-L3 comprising the amino acid sequence AGGYGGGX1YA (wherein X1 is L or R) (SEQ ID NO: 131).

[0046] In some embodiments, an isolated anti-myostatin antibody of the present invention comprises (a) an 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) (SEQ ID NO: 129), (b) an HVR-L2 comprising the amino acid sequence WAX1TLAX2 (wherein X1 is S or E and X2 is S, Y, F, or W) (SEQ ID NO: 130), and (c) an HVR-L3 comprising the amino acid sequence AGGYGGGX1YA (wherein X1 is L or R) (SEQ ID NO: 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 the amino acid sequence of any one of SEQ ID NOs: 140-141; FR3 comprising the amino acid sequence of any one of SEQ ID NOs: 142-143; and FR4 comprising the amino acid sequence of SEQ ID NO: 144.

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

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

[0049] The invention also provides isolated nucleic acids encoding the anti-myostatin antibodies of the invention. The invention also provides host cells comprising nucleic acids of the invention. The invention also provides methods of producing antibodies, comprising culturing host cells of the invention so that the antibodies are produced.

[0050] In some aspects, the present invention provides a method for producing an anti-myostatin antibody, the method comprising: (a) culturing a host cell of the present invention so that the antibody is produced; or (b) immunizing an animal against a polypeptide comprising a region corresponding to amino acids 21 to 100 of the myostatin propeptide (SEQ ID NO: 78).

[0051] The present invention further provides methods for producing anti-myostatin antibodies, which in some embodiments comprise immunizing an animal against a polypeptide comprising a region corresponding to amino acids 21-100 of the myostatin propeptide (SEQ ID NO: 78).

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

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

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

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

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

[0057] The present invention provides polypeptides comprising variant Fc regions and methods for making and using the same.

[0058] In one embodiment, the present invention provides FcγRIIB-binding polypeptides comprising mutant Fc regions and methods for using the same. In some embodiments, the mutant Fc regions of the present invention with enhanced FcγRIIb-binding activity comprise at least one amino acid modification in the parent Fc region. In a further embodiment, the ratio of [KD value of the parent Fc region for monkey FcγRIIb] / [KD value of the mutant Fc region for monkey FcγRIIb] is 2.0 or higher. In a further embodiment, the ratio of [KD value of the parent Fc region for monkey FcγRIIIa] / [KD value of the mutant Fc region for monkey FcγRIIIa] is 0.5 or lower. In a further embodiment, the ratio of [KD value of the parent Fc region for human FcγRIIb] / [KD value of the mutant Fc region for human FcγRIIb] is 2.0 or higher. In a further embodiment, the ratio of [KD value of the parent Fc region for human FcγRIIIa] / [KD value of the mutant Fc region for human FcγRIIIa] is 0.5 or lower. In a further embodiment, the ratio of [KD value of parent Fc region for human FcγRIIa (H type)] / [KD value of mutant Fc region for human FcγRIIa (H type)] is 5.0 or less. In a further embodiment, the ratio of [KD value of parent Fc region for human FcγRIIa (R type)] / [KD value of mutant Fc region for human FcγRIIa (R type)] is 5.0 or less. In another embodiment, the KD value of mutant Fc region for monkey FcγRIIb is 1.0 × 10 -6 In another embodiment, the KD value of the mutant Fc region for monkey FcγRIIIa is 5.0×10 -7 In another embodiment, the KD value of the mutant Fc region for human FcγRIIb is 2.0 × 10 -6 In another embodiment, the KD value of the mutant Fc region for human FcγRIIIa is 1.0 × 10 -6 In another embodiment, the KD value of the mutant Fc region for human FcγRIIa (H type) is 1.0 × 10 -7 In another embodiment, the KD value of the mutant Fc region for human FcγRIIa (R type) is 2.0 × 10 -7 M or above.

[0059] In some embodiments, the mutant Fc region of the present invention with enhanced FcγRIIb binding activity 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 expressed by EU numbering.

[0060] In a further aspect, the mutant Fc region with enhanced FcγRIIb binding activity comprises at least two amino acid modifications, 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: (i) 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) positions 231, 232, 235, 239, 268, 295, 298, 326, 330, and 396; or (iii) positions 268, 295, 326, and 330, as represented by EU numbering.

[0061] In a further aspect, the mutant Fc region with enhanced FcγRIIb binding activity comprises at least two amino acid modifications, 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, as represented by EU numbering.

[0062] In a further aspect, the mutant Fc region with enhanced FcγRIIb binding activity comprises at least two amino acid modifications, 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, as represented by EU numbering.

[0063] In a further aspect, the mutant Fc region with enhanced FcγRIIb binding activity comprises at least two amino acid modifications, 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, as represented by EU numbering.

[0064] In some embodiments, the mutant Fc regions of the present invention with enhanced FcγRIIb binding activity comprise, as 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, Gly, His, Ile, Lys, Leu, Met, Asn at position 232; (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 (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) Ala, His, Leu at position 267; (u) Thr 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.

[0065] In a further aspect, the mutant Fc region with enhanced FcγRIIb binding activity comprises 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 aspect, the present invention provides polypeptides comprising a mutant Fc region with an increased isoelectric point (pI), and methods of using the same. In some embodiments, the polypeptide comprising the mutant Fc region with an increased pI comprises at least two amino acid modifications in the parent Fc region. In further embodiments, each of the amino acid modifications increases the isoelectric point (pI) of the mutant Fc region compared to the parent Fc region. In further embodiments, the amino acids may be exposed on the surface of the mutant Fc region. In further embodiments, the polypeptide comprises a mutant Fc region and an antigen-binding domain. In further embodiments, the antigen-binding activity of the antigen-binding domain is altered by ionic concentration conditions. In a further embodiment, the pI-increased 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 (EU numbering). In a further embodiment, the pI-increased mutant Fc region comprises Arg or Lys at each of the selected positions.

[0067] In some embodiments, the mutant Fc regions of the present invention comprise the amino acid modifications set forth in Tables 14-30.

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

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

[0070] The present invention also provides isolated nucleic acids encoding polypeptides comprising mutant Fc regions of the present invention. The present invention also provides host cells comprising nucleic acids of the present invention. The present invention also provides methods for producing polypeptides comprising mutant Fc regions, the methods comprising culturing a host of the present invention so that the polypeptide is produced.

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

[0072] [Figure 1] Figure 1 shows the inhibition of proteolytic activation of latent myostatin by anti-latent myostatin antibodies, as described in Example 3. The activity of active myostatin released from latent myostatin by BMP1 protease was measured in the presence of anti-latent myostatin antibodies using the HEK Blue assay. [Figure 2] Figure 2 shows the inhibition of spontaneous activation of latent myostatin by anti-latent myostatin antibodies, as described in Example 4. The activity of active myostatin released from latent myostatin by incubation at 37°C was measured in the presence of anti-latent myostatin antibodies using the HEK Blue assay. [Figure 3] Figure 3 shows binding of anti-latent myostatin antibodies to the propeptide domain, as described in Example 5. [Figure 4]Figure 4 shows Western blot analysis for myostatin propeptide, as described in Example 6. Proteolytic cleavage of myostatin propeptide by BMP1 was assessed in the presence or absence of anti-latent myostatin antibody. [Figure 5] Figures 5A-5C show BIACORE (registered trademark) 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 antibody and anti-mature myostatin antibody on muscle mass and fat mass, as described in Example 8. Anti-latent myostatin antibody (MST1032-G1m; referred to as MST1032 in the figure) or anti-mature myostatin antibody (41C1E4) was administered to SCID mice, and total body lean mass (or lean body mass: LBM) was measured. [Figure 6B] Figure 6B shows the in vivo effects of anti-latent myostatin antibody and anti-mature myostatin antibody 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 antibody and anti-mature myostatin antibody on muscle mass and fat mass, as described in Example 8. Anti-latent myostatin antibody (MST1032-G1m; referred to as MST1032 in the figure) or anti-mature myostatin antibody (41C1E4) was administered to SCID mice, and the mass of the gastrocnemius and quadriceps muscles was measured. [Figure 7A]Figure 7A shows a comparison of the in vivo effects among several anti-myostatin antibodies, as described in Example 9. An anti-latent myostatin antibody (MST1032-G1m; referred to as MST1032 in the figure) or an anti-mature myostatin antibody (41C1E4, REGN, OGD, or MYO-029) was administered to SCID mice, and whole body lean mass was measured. [Figure 7B] Figure 7B shows a comparison of the in vivo effects of several anti-myostatin antibodies, as described in Example 9. An anti-latent myostatin antibody (MST1032-G1m; referred to as MST1032 in the figure) or an 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. An anti-latent myostatin antibody (MST1032-G1m; referred to as MST1032 in the figure) or an 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 humanized anti-latent myostatin antibodies, as described in Example 10. The activity of active myostatin released from latent myostatin by BMP1 protease (proteolytic) or by incubation at 37°C in the absence of BMP1 (spontaneous) was measured in the presence of anti-latent myostatin antibodies using the HEK Blue assay. [Figure 9]Figure 9 shows BIACORE® sensorgrams of histidine-substituted mutants of anti-latent myostatin antibodies, as described in Example 11. The antibody / antigen complexes were dissociated at pH 7.4, followed by further dissociation at pH 5.8 (indicated by arrows) to assess pH-dependent interactions. The antibodies tested in this experiment are Ab001 (solid black line), Ab002 (short-dashed black line), Ab003 (dotted black line), Ab004 (short-dashed gray line), Ab005 (solid gray line), Ab006 (long-dashed gray line), and Ab007 (long-dashed black line). [Figure 10] Figure 10 shows the inhibition of proteolytic and spontaneous activation of latent myostatin by pH-dependent anti-latent myostatin antibodies, as described in Example 13. The activity of active myostatin released from latent myostatin by BMP1 protease (proteolytic) or by incubation at 37°C in the absence of BMP1 (spontaneous) was measured in the presence of anti-latent myostatin antibodies using the HEK Blue assay. Antibodies MS1032LO01-SG1, MS1032LO02-SG1, MS1032LO03-SG1, and MS1032LO04-SG1 are referred to in the figure as MSLO-01, MSLO-02, MSLO-03, and MSLO-04, respectively. Inhibition of proteolytic and spontaneous activation of latent myostatin to the same extent as MS1032LO00-SG1 was achieved by MS1032LO01-SG1, MS1032LO02-SG1, MS1032LO03-SG1, and MS1032LO04-SG1. [Figure 11] Figures 11A-11F show BIACORE® sensorgrams of pH-dependent anti-latent myostatin antibodies, as described in Example 14. Kinetic 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 concentrations after intravenous administration of anti-myostatin antibodies in mice, as described in Example 15. The effect of FcγR-mediated cellular uptake of antibody / antigen complexes on myostatin clearance in vivo was assessed by comparing an anti-myostatin antibody with FcγR binding ability (MS1032LO00-SG1) with an anti-myostatin antibody with lost FcγR binding ability (MS1032LO00-F760). [Figure 13] Figure 13 shows the time course of plasma myostatin concentrations 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 myostatin clearance in vivo was assessed 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 total body lean mass (A), total body fat mass (B), quadriceps mass (C), gastrocnemius mass (D), and grip strength (E) were measured. [Figure 15] Figure 15 shows the binding activity of anti-latent myostatin antibody MST1032 to latent myostatin and GDF11, as described in Example 19. [Figure 16]Figure 16 shows the inhibitory activity of anti-latent myostatin antibody MST1032 on the proteolytic and spontaneous activation of GDF11, as described in Example 20. The activity of active GDF11 released by BMP1 protease (proteolytic) or by incubation at 37°C in the absence of BMP1 (spontaneous) was measured in the presence of anti-latent myostatin antibody using the HEK Blue assay. [Figure 17] Figure 17 shows the inhibition of proteolytic activation of latent myostatin by anti-latent myostatin antibodies, as described in Example 22. The activity of active myostatin released from latent myostatin by BMP1 protease was measured in the presence of anti-latent myostatin antibodies using the HEK Blue assay. [Figure 18] Figure 18 shows the time course of plasma myostatin concentrations after intravenous administration of anti-latent myostatin antibodies in mice, as described in Example 23. The effect of pH dependence on myostatin clearance in vivo was assessed 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 concentrations following intravenous administration of anti-latent myostatin antibodies in cynomolgus monkeys, as described in Example 24. (A) The effect of pH dependence and Fc modifications on myostatin clearance in vivo was assessed by comparing a pH-independent anti-latent myostatin antibody (MS1032LO00-SG1) with pH-dependent anti-latent myostatin antibodies with Fc modifications (MS1032LO06-SG1012, MS1032LO06-SG1016, MS1032LO06-SG1029, MS1032LO06-SG1031, MS1032LO06-SG1033, MS1032LO06-SG1034). (B) The effect of Fc modifications on myostatin clearance in vivo was assessed by comparing anti-latent myostatin antibodies (MS1032LO19-SG1079, MS1032LO19-SG1071, MS1032LO19-SG1080, MS1032LO19-SG1074, MS1032LO19-SG1081, and MS1032LO19-SG1077). [Figure 20A] Figure 20A, together with Figures 20B-20I, shows the in vivo effects of anti-latent myostatin antibodies (MS1032 variants) on lean body mass (LBM), grip strength, and 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, together with Figures 20A, 20C-20I, shows the in vivo effects of anti-latent myostatin antibodies (MS1032 variants) on lean body mass (LBM), grip strength, and 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, together with Figures 20A-B and 20D-20I, shows the in vivo effects of anti-latent myostatin antibodies (MS1032 variants) on lean body mass (LBM), grip strength, and fat mass, as described in Example 25. MS1032LO01-SG, MS1032LO06-SG1, and MS1032LO11-SG1 were administered to SCID mice, and lean body mass (LBM) was measured. [Figure 20D] Figure 20D, together with Figures 20A-C, 20E-20I, shows the in vivo effects of anti-latent myostatin antibodies (MS1032 variants) on lean body mass (LBM), grip strength, and 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, together with Figures 20A-D and 20F-20I, shows the in vivo effects of anti-latent myostatin antibodies (MS1032 variants) on lean body mass (LBM), grip strength, and 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, together with Figures 20A-E and 20G-I, shows the in vivo effects of anti-latent myostatin antibodies (MS1032 variants) on lean body mass (LBM), grip strength, and 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, together with Figures 20A-F and 20H-I, shows the in vivo effects of anti-latent myostatin antibodies (MS1032 variants) on lean body mass (LBM), grip strength, and fat mass, as described in Example 25. MS1032LO06-SG1, MS1032LO11-SG1, and MS1032LO18-SG1 were administered to SCID mice, and fat mass (G) was measured. [Figure 20H]Figure 20H, together with Figures 20A-G and 20I, shows the in vivo effects of anti-latent myostatin antibodies (MS1032 variants) on lean body mass (LBM), grip strength, and fat mass, as described in Example 25. MS1032LO06-SG1, MS1032LO19-SG1, and MS1032LO25-SG1 were administered to SCID mice, and fat mass (H) was measured. [Figure 20I] Figure 20I, together with Figures 20A-H, shows the in vivo effects of anti-latent myostatin antibodies (MS1032 variants) on lean body mass (LBM), grip strength, and fat mass, as described in Example 25. MS1032LO01-SG, MS1032LO06-SG1, and MS1032LO11-SG1 were administered to SCID mice, and 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 a schematic diagram 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 Western blot analysis of GST-tagged human latent myostatin fragment (GST-hMSTN) using anti-GST antibody, as described in Example 26. Lanes represent: 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; and 9, GST control. [Figure 22C]Figure 22C shows Western blot analysis of GST-tagged human latent myostatin fragment (GST-hMSTN) using anti-latent myostatin antibodies (MST1032, MST1538, MST1572, and MST1573), as described in Example 26. Lanes represent: 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; and 10, human latent myostatin (100 ng). [Figure 22D] Figure 22D shows a summary of the Western blot analysis results and predicted epitope locations for anti-latent myostatin antibodies (MST1032, MST1538, MST1572, and MST1573), as described in Example 26. [Figure 23] Figure 23 shows an alignment of the amino acid sequences of cynomolgus monkey (cyno) FcγRIIa1, FcγRIIa2, FcγRIIa3, FcγRIIb, and human FcγRIIaH, FcγRIIaR, and FcγRIIb. The boxed regions indicate residues predicted to interact with the Fc domain. [Figure 24] Figure 24 shows the time course of plasma total myostatin concentrations after intravenous administration of anti-myostatin antibodies with FcγRIIb-enhancing Fc variants in fully human FcγR transgenic mice, as described in Example 28. The effect of FcγRIIb-enhancing Fc variants on human FcγRIIb-mediated antigen elimination was assessed. [Figure 25] Figure 25 shows the time course of plasma antibody concentrations following intravenous administration of an anti-myostatin antibody with an FcγRIIb-enhancing Fc variant in fully human FcγR transgenic mice, as described in Example 28. The effect of the FcγRIIb-enhancing Fc variant on the pharmacokinetics of the antibody was assessed. [Figure 26]Figures 26A and 26B show the time course of plasma myostatin concentrations following intravenous administration of anti-latent myostatin antibodies in cynomolgus monkeys, as described in Example 29. (A) The effect of pH dependence and Fc modifications on myostatin clearance in vivo was assessed by comparing a pH-independent anti-latent myostatin antibody (MS1032LO00-SG1) with pH-dependent anti-latent myostatin antibodies with Fc modifications (MS1032LO06-SG1012, MS1032LO06-SG1016, MS1032LO06-SG1029, MS1032LO06-SG1031, MS1032LO06-SG1033, MS1032LO06-SG1034). (B) The effect of Fc modifications on myostatin clearance in vivo was assessed 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 plasma total myostatin concentrations in human FcRn transgenic mice after intravenous administration of anti-myostatin antibodies with pI-increased Fc variants, as described in Example 30. The effect of pI-increased Fc variants on antigen clearance was assessed. [Figure 27B] Figure 27B shows the time course of plasma antibody concentrations after intravenous administration of anti-myostatin antibodies with pI-increased Fc variants in human FcRn transgenic mice, as described in Example 30. The effect of the pI-increased Fc variants on the pharmacokinetics of the antibody was assessed. [Figure 28A] Figure 28A shows the time course of plasma total myostatin concentrations in human FcRn transgenic mice after intravenous administration of anti-myostatin antibodies with pI-increased Fc variants, as described in Example 30. The effect of pI-increased Fc variants on antigen clearance was assessed. In this assay, an excess of human normal immunoglobulin was co-administered with the anti-myostatin antibody to mimic the situation in human plasma. [Figure 28B] Figure 28B shows the time course of plasma antibody concentrations after intravenous administration of an anti-myostatin antibody with a pI-increased Fc variant in human FcRn transgenic mice, as described in Example 30. The effect of the pI-increased Fc variant on the pharmacokinetics of the antibody was assessed. In this assay, an excess of human normal immunoglobulin was co-administered with the anti-myostatin antibody to mimic the situation in human plasma. [Figure 29] Figure 29 shows the time course of plasma total myostatin concentrations after intravenous administration of anti-myostatin antibodies with FcγRIIb-enhancing Fc variants in human FcγRIIb transgenic mice, as described in Example 31. The effect of FcγRIIb-enhancing Fc variants on human FcγRIIb-mediated antigen elimination was evaluated. [Figure 30] Figure 30 shows the time course of plasma antibody concentrations following intravenous administration of an anti-myostatin antibody with an FcγRIIb-enhancing Fc variant in human FcγRIIb transgenic mice, as described in Example 31. The effect of the FcγRIIb-enhancing Fc variant on the pharmacokinetics of the antibody was assessed. [Figure 31] Figure 31 shows the results of cellular imaging analysis of anti-myostatin antibodies with FcγRIIb-enhancing 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. DETAILED DESCRIPTION OF THE INVENTION

[0073] Description of Aspects The techniques and procedures described or cited herein are generally well understood and can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. 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. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993), using conventional techniques commonly used by those skilled in the art.

[0074] I. Definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. 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 those skilled in the art with general guidance for many of the terms used in this application. All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety.

[0075] For purposes of interpreting this specification, the following definitions will apply, and wherever applicable, terms used in the singular will also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. In the event that any of the definitions below conflict with any document incorporated herein by reference, the definition below shall control.

[0076] An "acceptor human framework," for purposes of this specification, is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise those same amino acid sequences or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes 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 identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

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

[0078] An "affinity matured" antibody refers to an antibody with one or more modifications in one or more hypervariable regions (HVRs) that result in improved affinity of the antibody for antigen, compared to a parent antibody that does not possess such modifications.

[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 and / or therapeutic agent when targeted to myostatin. In one embodiment, the extent of binding of the anti-myostatin antibody to an unrelated, non-myostatin protein is less than about 10% of the binding of the antibody to myostatin, as measured (e.g., by radioimmunoassay (RIA)). In certain embodiments, an antibody that binds to myostatin has an affinity 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~10 -13 M, e.g., 10 -9 M~10 -13 In certain embodiments, the anti-myostatin antibody binds to an epitope of myostatin that is conserved among myostatin from different species.

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

[0081] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact 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 a reference antibody refers to an antibody that blocks the binding of the reference antibody to its own antigen in a competition assay, and / or conversely, the reference antibody blocks the binding of the antibody to its own antigen in a competition assay. Exemplary competition assays are provided herein.

[0083] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived 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 major 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 the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0085] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes the death or destruction of cells. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 Pb 212, and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; enzymes and fragments thereof, such as nucleases; antibiotics; toxins, such as, for example, small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including fragments and / or variants thereof); and various anti-tumor or anti-cancer agents, as disclosed below.

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

[0087] An "effective amount" of an agent (eg, a pharmaceutical formulation) refers to an amount, at dosages and for periods of time necessary, effective to achieve a desired therapeutic or prophylactic result.

[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 that targets that antigen and includes specific amino acids that directly contact the antibody. Epitopic determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics and / or specific charge characteristics. Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on that target antigen in a complex mixture of proteins and / or macromolecules.

[0089] "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 binds to IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997).) FcRs are reviewed, e.g., 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 identified in the future, are also encompassed by the term "FcR" herein.

[0090] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for regulating maternal IgG transfer to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and immunoglobulin homeostasis. Methods for measuring binding to FcRn are 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 serum half-life of human FcRn high-affinity binding polypeptides can be measured, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which polypeptides with mutant Fc regions are administered. WO2000 / 42072 (Presta) describes antibody mutants with improved or reduced binding to FcR. See, for example, Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

[0091] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain, except that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) residues of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to as the EU index) as 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 (residue 447 according to the EU numbering system) or the C-terminal glycine-lysine (residues 446-447) of the Fc region can be removed, for example, during antibody purification or by recombinant engineering of a nucleic acid encoding the antibody. Thus, a composition containing an antibody with an Fc region according to the present invention can contain an antibody with G446-K447, an antibody with G446 but without K447, an antibody with G446-K447 completely removed, or a mixture of the above three types of antibodies.

[0093] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences typically 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 an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

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

[0096] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived from that cell regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as that for which the original transformed cell was screened or selected are also included herein.

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

[0098] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup 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 according to Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III according to Kabat et al., supra.

[0099] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and 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 comprise at least a portion of an antibody constant region derived from a human antibody. A "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 an antibody variable domain that is hypervariable in sequence (the "complementarity determining region" or "CDR") and / or forms structurally defined loops (the "hypervariable loops") and / or contains antigen-contacting residues (the "antigen contacts"). Typically, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein include the following: (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) antigenic contacts 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), comprising 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 according to Kabat et al., supra.

[0102] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to, cytotoxic agents.

[0103] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, 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 human.

[0104] An "isolated" antibody is one that has been separated from a component of its original environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as measured by 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 assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0105] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its original environment. Isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0106] An "isolated nucleic acid encoding an anti-myostatin antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of the antibody, including nucleic acid molecules carried on a single vector or separate vectors, and nucleic acid molecules present in one or more locations within a host cell.

[0107] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variants (e.g., variants containing naturally occurring mutations or variants 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 include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0108] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. Naked antibodies may be present in a pharmaceutical formulation.

[0109] "Native antibodies" refer to immunoglobulin molecules with various naturally occurring structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy 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 domain or light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa (κ) or lambda (λ), based on the amino acid sequence of its constant domain.

[0110] A "native-sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. Native-sequence human Fc regions include native-sequence human IgG1 Fc regions (non-A and A allotypes); native-sequence human IgG2 Fc regions; native-sequence human IgG3 Fc regions; and native-sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

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

[0112] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps, if necessary, and excluding any conservative substitutions from the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetyx Corporation). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the entire length of the sequences being compared. The ALIGN-2 sequence comparison computer program is the property of Genentech, Inc., and its source code, along with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the 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 vary.

[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, with, or against a given amino acid sequence B (alternatively, one can say that a given amino acid sequence A has or contains a certain % amino acid sequence identity to, with, or against 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 matches by the sequence alignment program ALIGN-2 in its alignment of A and B, 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 will not equal 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 immediately preceding paragraph.

[0114] The term "pharmaceutical formulation" refers to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

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

[0116] As used herein, the term "myostatin" can refer to any naturally occurring myostatin from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). Unless otherwise indicated, the term "myostatin" refers to a human myostatin protein having the amino acid sequence set forth in SEQ ID NO:1 and containing the terminal propeptide domain of human myostatin set forth in SEQ ID NO:75 or 78. The term encompasses "full-length," i.e., unprocessed, myostatin, as well as any form of myostatin resulting from processing in cells. The term also encompasses naturally occurring variants of myostatin, such as splice variants and allelic variants. An exemplary amino acid sequence of human myostatin (promyostatin) is set forth in SEQ ID NO:1. An exemplary amino acid sequence of the C-terminal growth factor domain of human myostatin is set forth in SEQ ID NO:2. An exemplary amino acid sequence of the N-terminal propeptide domain of human myostatin is set forth 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 formed by non-covalent association of two propeptides with mature myostatin. As disclosed herein, the antibodies of the present invention bind to inactive latent myostatin but do not bind to mature active myostatin homodimers. In some embodiments, the antibodies of the present invention bind to an epitope within a fragment consisting of amino acids 21-100 of the myostatin propeptide (SEQ ID NO: 78) but do not bind to mature active myostatin homodimers. The amino acid sequences of exemplary cynomolgus monkey and mouse myostatin (promyostatin) are set forth 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 set forth in SEQ ID NOs: 4 and 6, respectively. The amino acid sequences of exemplary N-terminal propeptide domains of cynomolgus monkey and mouse myostatin are shown in SEQ ID NO: 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.Like 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 to 24 correspond to the signal sequence, and these are removed during intracellular processing.

[0117] As used herein, "treatment" (and its grammatical derivatives, such as "treat," "treating," etc.) refers to a clinical intervention intended to alter the natural course of the individual being treated and can be performed prophylactically or during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or slow the progression of disease.

[0118] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. 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 specific antigen may be isolated by screening a complementary library of VL or VH domains, respectively, using a VH or VL domain from an antibody that binds to that antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0119] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by at least one amino acid modification (alteration), preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution, for example, about one to about ten amino acid substitutions, preferably about one to about five amino acid substitutions, in the native-sequence Fc region or in the Fc region of the parent polypeptide, compared to the native-sequence Fc region or the Fc region of the parent polypeptide. The variant Fc region herein preferably has at least about 80% homology with the native-sequence Fc region and / or the Fc region of the parent polypeptide, most preferably at least about 90% homology thereto, and more preferably at least about 95% homology thereto.

[0120] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of effecting the expression of nucleic acids to which they are operatively linked. Such vectors are also referred to herein as "expression vectors."

[0121] II. Compositions and Methods In one aspect, the invention is based in part on anti-myostatin antibodies and their uses. In certain embodiments, antibodies that bind to myostatin are provided. The antibodies of the 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 mutant Fc regions and their uses. In one embodiment, a polypeptide comprising a mutant Fc region with enhanced FcγRIIb binding activity is provided. In another embodiment, a polypeptide comprising a mutant Fc region with an increased pI is provided. In a specific embodiment, the polypeptide of the present invention is an antibody. Polypeptides comprising mutant Fc regions of the present invention are useful, for example, for the diagnosis or treatment of diseases.

[0123] A. Exemplary Anti-myostatin Antibodies and Polypeptides Comprising Mutant Fc Regions In one aspect, the present invention provides isolated antibodies that bind to myostatin. In certain embodiments, the anti-myostatin antibodies of the present invention bind to latent myostatin. In a further embodiment, the anti-myostatin antibodies of the present invention bind to the 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 embodiment, the antibody binds to an epitope in a fragment consisting of amino acids 21 to 100 of the myostatin propeptide (SEQ ID NO: 78). As described above, the propeptide is contained within latent myostatin as one of its components. In certain embodiments, the anti-myostatin antibodies of the present invention inhibit the activation of myostatin. In certain embodiments, the anti-myostatin antibodies prevent the release of mature myostatin from latent myostatin. Mature myostatin has been reported to be released from latent myostatin through proteolytic and non-proteolytic processes. The anti-myostatin antibodies of the present invention can prevent the proteolytic and / or non-proteolytic release of mature myostatin from latent myostatin. In certain embodiments, the anti-myostatin antibodies prevent the proteolytic cleavage of latent myostatin. In certain embodiments, the anti-myostatin antibodies prevent proteases from accessing latent myostatin, particularly the proteolytic cleavage site (Arg98-Asp99) of latent myostatin. In further embodiments, the protease may be a BMP1 / TLD family metalloprotease, such as BMP1, TED, tolloid-like protein 1 (TLL-1), or tolloid-like protein 2 (TLL-2). In another embodiment, the anti-myostatin antibody prevents 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, for example, at 37° C., in the absence of proteases that cleave latent myostatin.In certain embodiments, the anti-myostatin antibodies of the present invention do not bind to mature myostatin. In some embodiments, the anti-myostatin antibodies bind to the same epitope as the antibodies listed in Table 2a. In some embodiments, the anti-myostatin antibodies compete with the antibodies listed in Table 2a for binding to latent myostatin. In additional embodiments, the anti-myostatin antibodies compete with antibodies comprising a VH and VL pair listed in Table 2a for binding to latent myostatin. In some embodiments, the anti-myostatin antibodies compete with the antibodies listed in Table 2a for binding to a fragment consisting of amino acids 21-100 of the myostatin propeptide (SEQ ID NO: 78). In further embodiments, the anti-myostatin antibodies bind to the same epitope as the antibodies listed in Table 11a or 13. In some embodiments, the anti-myostatin antibodies compete with the antibodies listed in Table 11a or 13 for binding to latent myostatin. In some embodiments, the anti-myostatin antibody competes with an antibody listed in Table 11a or 13 for binding to a fragment consisting of amino acids 21 to 100 of the myostatin propeptide (SEQ ID NO: 78). In some embodiments, the anti-myostatin antibodies of the present invention bind to latent myostatin and inhibit myostatin activation. In further embodiments, the antibodies (a) prevent the release of mature myostatin from latent myostatin; (b) prevent the proteolytic release of mature myostatin; (c) prevent the spontaneous release of mature myostatin; or (d) do not bind to mature myostatin or bind to an epitope in a fragment consisting of amino acids 21-100 of the myostatin propeptide (SEQ ID NO: 78). In further embodiments, the antibodies compete for binding to latent myostatin with, or bind to the same epitope as, an antibody comprising a VH and VL pair set forth in Tables 2a, 11a, or 13. In further embodiments, the antibodies bind to latent myostatin with greater affinity at neutral pH (e.g., pH 7.4) than at acidic pH (e.g., pH 5.8). In further embodiments, 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 antibodies of the present invention do not bind to GDF11. In certain embodiments, the anti-myostatin antibodies of the present invention do not inhibit the activation of GDF11. In certain embodiments, the anti-myostatin antibodies do not prevent the release of mature GDF11 from latent GDF11. The anti-myostatin antibodies of the present invention do not prevent either the proteolytic or non-proteolytic release of mature GDF11 from latent GDF11. In certain embodiments, the anti-myostatin antibodies do not prevent the proteolytic cleavage of latent GDF11. In certain embodiments, the anti-myostatin antibodies do not prevent proteases from accessing latent GDF11 (particularly the proteolytic cleavage site of latent GDF11). In further embodiments, the protease may be a BMP1 / TLD family metalloprotease, such as 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, which does not involve proteolytic cleavage of latent GDF11. Non-proteolytic release includes, for example, the release of mature GDF11 by incubating latent GDF11 at, for example, 37°C in the absence of a protease that cleaves latent GDF11. Most anti-myostatin antibodies known to date are not specific to myostatin. These antibodies have high affinity for other members of the TGF-β superfamily, such as GDF11, and neutralize their biological activity. GDF11 plays an important role in embryonic development and is responsible for the 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 an important role during embryonic development, antagonists that inhibit GDF11 pose a theoretical safety risk that could exist either as toxicity in the treated patient or as reproductive toxicity in, for example, women of childbearing potential.Thus, there is a need to specifically inhibit myostatin activity in the treatment of myostatin-related disorders where it is desirable to increase muscle mass, size, strength, etc., particularly in women of childbearing age.

[0125] In another aspect, the present invention provides anti-myostatin antibodies that exhibit pH-dependent binding properties. As used herein, the phrase "pH-dependent binding" means that an antibody exhibits "reduced binding to myostatin at acidic pH compared to its binding at neutral pH" (for purposes of this disclosure, both phrases can be used interchangeably). For example, antibodies with "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, 10,000, or more times higher affinity at neutral pH than at acidic pH. In some embodiments, these antibodies bind to myostatin (e.g., latent myostatin or propeptide myostatin) with greater affinity at pH 7.4 than at pH 5.8. In further embodiments, these antibodies 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, 10,000, or more times greater affinity at pH 7.4 than at pH 5.8.

[0126] When the antigen is a soluble protein, the antibody may have a longer half-life in plasma than the antigen itself and may act as a carrier of the antigen, so that binding of the antibody to the antigen may result in an increase in the half-life of the antigen in plasma (i.e., a decrease in clearance of the antigen from plasma). This is due to recycling 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 their antigens in a neutral extracellular environment but release them into acidic endosomal compartments after intracellular entry, are expected to have superior antigen neutralization and clearance properties compared to their counterparts that bind in a pH-independent manner (Igawa et al., Nature Biotechnol. 28(11):1203-1207 (2010); Devanaboyina et al., mAbs 5(6):851-859 (2013); WO 2009 / 125825).

[0127] For purposes of this disclosure, the "affinity" of an antibody for myostatin is expressed as the KD of the antibody. The KD of an antibody refers to the equilibrium dissociation constant of the antibody-antigen interaction. The higher the KD value for binding of an antibody to its antigen, the weaker its binding affinity to that particular antigen. Thus, as used herein, the phrase "higher affinity at neutral pH than at acidic pH" (or the equivalent phrase "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, an antibody is considered to bind to myostatin with higher affinity at neutral pH than at acidic pH if the KD of antibody binding to myostatin at acidic pH is at least two-fold greater than the KD of antibody binding to myostatin at neutral pH. Thus, the present invention includes antibodies that bind to myostatin at acidic pH with a KD that is 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, or more times greater than the KD of the antibody binding to myostatin at neutral pH. -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 In another embodiment, the KD value of the antibody at acidic pH is 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M, or a larger value.

[0128] In further embodiments, an antibody is considered to bind myostatin (e.g., latent myostatin or propeptide myostatin) with greater affinity at neutral pH than at acidic pH if the KD of the antibody binding to myostatin at pH 5.8 is at least 2-fold greater than the KD of the antibody binding to myostatin at pH 7.4. In some embodiments, the provided antibodies bind to myostatin at pH 5.8 with a KD that is 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, 10,000, or more times greater than the KD of the 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 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 M, or a larger value.

[0129] The binding properties of an antibody for 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 expressed in reciprocal seconds (i.e., sec -1 ) units. A higher kd value indicates weaker binding of the antibody to the antigen. The present invention therefore includes antibodies that bind to myostatin with a higher kd value at acidic pH than at neutral pH. The present invention includes antibodies that bind to myostatin at acidic pH with a kd that is 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, 10,000, or more times greater than the kd of the antibody binding to myostatin at neutral pH. 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 In another embodiment, the kd value of the antibody at acidic pH is 10 -3 1 / s, 10 -2 1 / s, 10 -1 1 / s, or greater. The present invention also includes antibodies that bind to myostatin (e.g., latent myostatin or propeptide myostatin) with a higher kd at pH 5.8 than at pH 7.4. The present invention includes antibodies that bind to myostatin at pH 5.8 with a kd that is 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, 10,000, or greater than the kd of the antibody binding to myostatin at pH 7.4. In another embodiment, the kd 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 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 larger.

[0130] In certain embodiments, "reduced 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, for the purposes of the present invention, an antibody can be considered to exhibit "reduced binding to myostatin at acidic pH compared to binding at neutral pH" if it exhibits an acidic / neutral KD ratio of 2 or greater. In certain embodiments, the pH 5.8 / pH 7.4 KD ratio for an anti-myostatin antibody of the present invention is 2 or greater. In certain exemplary embodiments, the acidic / neutral KD ratio for an antibody of the invention can 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 more. In another embodiment, the KD value of an antibody at neutral pH is 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 In another embodiment, the KD value of the antibody at acidic pH is 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M, or greater. In a further example, if an antibody exhibits a pH 5.8 / pH 7.4 KD ratio of 2 or greater, the antibody can be considered to exhibit "reduced binding to myostatin (e.g., latent myostatin) at acidic pH compared to binding at neutral pH." In certain exemplary embodiments, the pH 5.8 / pH 7.4 KD ratio for an antibody can 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 is 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12In another embodiment, the KD value of the antibody at pH 5.8 is 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M, or a larger value.

[0131] In certain instances, "reduced 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, for purposes of the present invention, an antibody can be considered to exhibit "reduced binding to myostatin at acidic pH compared to binding at neutral pH" if the antibody exhibits an acidic / neutral kd ratio of 2 or greater. In certain exemplary embodiments, the pH5.8 / pH7.4 kd ratio for an antibody of the present invention is 2 or greater. In certain exemplary embodiments, the acidic / neutral kd ratio for the antibodies of the invention can 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 more. 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 In another embodiment, the kd value of the antibody at acidic pH is 10 -3 1 / s, 10 -2 1 / s, 10 -1 1 / s, or greater. In certain exemplary embodiments, the pH 5.8 / pH 7.4 kd ratio for antibodies of the invention can 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 an antibody at pH 7.4 is 10 -2 1 / s, 10 -3 1 / s, 10 -4 1 / s, 10-5 1 / s, 10 -6 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 larger.

[0132] As used herein, the term "acidic pH" refers to a pH range of 4.0 to 6.5. This term "acidic pH" includes any one of the following pH values: 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 certain aspects, the "acidic pH" is 5.8.

[0133] As used herein, the term "neutral pH" refers to a pH of 6.7 to about 10.0. This term "neutral pH" includes any one of the following pH values: 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 certain aspects, "neutral pH" is 7.4.

[0134] The KD and kd values ​​presented herein can be determined using a surface plasmon resonance-based biosensor to characterize antibody-antigen interactions (see, e.g., Example 7 herein). The KD and kd values ​​can be determined at 25°C or 37°C.

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

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

[0137] In certain embodiments, the anti-myostatin antibodies of the present invention bind to propeptide myostatin from multiple species. In further embodiments, the anti-myostatin antibodies bind to propeptide myostatin from humans and non-human animals. In still further embodiments, the anti-myostatin antibodies bind to propeptide myostatin 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, 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 complexes can strongly bind to Fc receptors present on the cell surface due to the avidity effect mediated by the Fc regions of the antibodies in the complex, and can then be internalized into cells with high efficiency. Therefore, the above-mentioned anti-myostatin antibodies, which can form immune complexes containing two or more anti-myostatin antibodies and two or more myostatin molecules, can bring about 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 properties 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 with both of the above properties, 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 properties 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 the amino acid sequence of any one of SEQ ID NOs: 55-57, 114-115, and 126; (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 58-60, 116-120, and 127; (c) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 61-64, 121, and 128; (d) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 65-69, 122-124, and 129; (e) HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 70-72, 125, and 130; and (f) HVR-L3 comprising the amino acid sequence of any one 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 the amino acid sequence of any one of SEQ ID NOs: 55 to 57; (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 58 to 60; (c) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 61 to 64; (d) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 65 to 69; (e) HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 70 to 72; and (f) HVR-L3 comprising the amino acid sequence of any one 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 the amino acid sequence of any one of SEQ ID NOs: 114 to 115; (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 116 to 120; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 122 to 124; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 125; and (f) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 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 the amino acid sequence of any one of SEQ ID NOs: 55 to 57, 114 to 115, and 126; (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 58 to 60, 116 to 120, and 127; and (c) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 61 to 64, 121, and 128. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 61 to 64, 121, and 128. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 61 to 64, 121, and 128, and HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 73 to 74, and 131. In a further embodiment, the antibody comprises an HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 61 to 64, 121, and 128, an HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 73 to 74, and 131, and an HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 58 to 60, 116 to 120, and 127. In a further embodiment, the antibody comprises (a) an HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 55 to 57, 114 to 115, and 126; (b) an HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 58 to 60, 116 to 120, and 127; and (c) an HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 61 to 64, 121, and 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 the amino acid sequence of any one of SEQ ID NOs: 55-57; (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 58-60; and (c) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 61-64. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 61-64. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 61-64 and HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 73-74. In a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 61-64, HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 73-74, and HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 58-60. In a further aspect, the antibody comprises (a) HVR-H1 having the amino acid sequence of any one of SEQ ID NOs: 55 to 57, (b) HVR-H2 having the amino acid sequence of any one of SEQ ID NOs: 58 to 60, and (c) HVR-H3 having the amino acid sequence of any one of SEQ ID NOs: 61 to 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 the amino acid sequence of any one of SEQ ID NOs: 114 to 115; (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 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 and HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 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 the amino acid sequence of any one of SEQ ID NOs: 73 to 74, and HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 116 to 120. In a further embodiment, the antibody comprises (a) an HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 114-115, (b) an HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 116-120, and (c) an 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) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 114; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58; and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63. In one embodiment, the antibody comprises an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63. In another embodiment, the antibody comprises an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63 and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74. In a further embodiment, the antibody comprises an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74, and an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58. In a further embodiment, the antibody comprises (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 114, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, and (c) an HVR-H3 comprising 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 aspect, the antibody comprises (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.

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

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

[0151] In another aspect, the present invention provides antibodies comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 122 to 124; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 125; and (c) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 73 to 74. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 122 to 124; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 125; and (c) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 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) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 123, (b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and (c) an HVR-L3 comprising 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 comprises: (a) a VH domain comprising at least one, at least two, or all three VH domains selected from (i) HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 55 to 57, 114, 115, and 126; (ii) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 58 to 60, 116 to 120, and 127; and (iii) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 61 to 64, 121, and 128. (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 any one of SEQ ID NOs: 65 to 69, 122 to 124, and 129, (ii) HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 70 to 72, 125, and 130, and (iii) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 73 to 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 the amino acid sequence of any one of SEQ ID NOs: 55 to 57, (ii) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 58 to 60, and (iii) HVR-H3 comprising the amino acid sequence of any one 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 the amino acid sequence of any one of SEQ ID NOs: 65 to 69, (ii) HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 70 to 72, and (iii) HVR-L3 comprising the amino acid sequence of any one 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 the amino acid sequence of any one of SEQ ID NOs: 114 to 115, (ii) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 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 the amino acid sequence of any one of SEQ ID NOs: 122 to 124, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 125, and (iii) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 73 to 74. In another aspect, an 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, an 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, an 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 of the amino acid sequences of SEQ ID NOs: 55 to 57, 114 to 115, and 126; (b) HVR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 58 to 60, 116 to 120, and 127; (c) HVR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 61 to 64, 121, and 128; (d) HVR-L1 comprising any one of the amino acid sequences of SEQ ID NOs: 65 to 69, 122 to 124, and 129; (e) HVR-L2 comprising any one of the amino acid sequences of SEQ ID NOs: 70 to 72, 125, and 130; and (f) HVR-L3 comprising any one of the amino acid sequences of SEQ ID NOs: 73 to 74 and 131.

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

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

[0161] In certain embodiments, any one or more amino acids of the above-mentioned anti-myostatin antibodies 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 of the anti-myostatin antibodies provided herein are conservative substitutions. In certain embodiments, any one or more of the following substitutions may be made in any combination: (a) in HVR-H1 (SEQ ID NO: 55): S1H; Y2T, D, or E; (b) in HVR-H2 (SEQ ID NO: 58): Y4H; S7K; T8M or K; Y10K; A11M or E; S12E; G16K; (c) in HVR-H3 (SEQ ID NO: 61): Y5H; T7H; L11K; (d) in HVR-L1 (SEQ ID NO: 65): Q1T; S2T; S5E; Y7F; D8H; N9D or A or E; (e) in HVR-L2 (SEQ ID NO: 70): S3E; S7Y, F or W; and (f) in HVR-L3 (SEQ ID NO: 73): L8R.

[0163] All possible combinations of the above substitutions are encompassed by the consensus sequences of SEQ ID NOs: 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 above-described embodiments, the anti-myostatin antibody can be humanized. In one embodiment, the anti-myostatin antibody comprises the HVR of any of the above-described embodiments 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 the HVR of any of the above-described embodiments and further comprises a VH or VL comprising FR sequences. In a further embodiment, the anti-myostatin antibody comprises the following heavy and / or light chain variable domain FR sequences: for the heavy chain variable domain, FR1 comprises the amino acid sequence of any one of SEQ ID NOs: 132-134, FR2 comprises the amino acid sequence of any one 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. For the light chain variable domain, FR1 comprises the amino acid sequence of SEQ ID NO: 139, FR2 comprises the amino acid sequence of any one of SEQ ID NOs: 140 to 141, FR3 comprises the amino acid sequence of any one of SEQ ID NOs: 142 to 143, and FR4 comprises 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 the amino acid sequence of any one of SEQ ID NOs: 157 to 162; (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 163 to 168; (c) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 169 to 174; (d) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 175 to 180; (e) HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 181 to 186; and (f) HVR-L3 comprising the amino acid sequence of any one 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 the amino acid sequence of any one of SEQ ID NOs: 157-162; (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 163-168; and (c) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 169-174. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 169-174. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 169-174 and HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 187-192. In a further aspect, the antibody comprises an HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 169 to 174, an HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 187 to 192, and an HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 163 to 168. In a further aspect, the antibody comprises (a) an HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 157 to 162, (b) an HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 163 to 168, and (c) an HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 169 to 174.

[0167] In another aspect, the present invention provides antibodies comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 175 to 180; (b) HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 181 to 186; and (c) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 187 to 192. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 175 to 180; (b) HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 181 to 186; and (c) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 187 to 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 the amino acid sequence of any one of SEQ ID NOs: 157 to 162, (ii) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 163 to 168, and (iii) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 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 the amino acid sequence of any one of SEQ ID NOs: 175 to 180, (ii) HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 181 to 186, and (iii) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 187 to 192.

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

[0170] In another aspect, an anti-myostatin antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 13, 16-30, 32-34, and 86-95. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-myostatin antibody comprising such a 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 any one of SEQ ID NOs: 13, 16-30, 32-34, and 86-95. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the HVR (i.e., within the FR). Optionally, the anti-myostatin antibody comprises a VH sequence in any one of SEQ ID NOs: 13, 16-30, 32-34, and 86-95, including post-translational modifications of that sequence. In a specific embodiment, the VH comprises one, two, or three HVRs selected from (a) HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 55 to 57, 114 to 115, and 126, (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 58 to 60, 116 to 120, and 127, and (c) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 61 to 64, 121, and 128. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

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

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

[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 to the amino acid sequence of any one of SEQ ID NOs: 15, 31, 35-38, and 96-99. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, 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 have been substituted, inserted, and / or deleted in any one of SEQ ID NOs: 15, 31, 35-38, and 96-99. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., within the FRs). Optionally, the anti-myostatin antibody comprises a VL sequence in any one of SEQ ID NOs: 15, 31, 35-38, and 96-99, including post-translational modifications of such sequences. In certain embodiments, the VL comprises one, two, or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 65-69, 122-124, and 129, (b) HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 70-72, 125, and 130, and (c) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 73-74, and 131. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

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

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

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

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

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

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

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

[0181] In certain embodiments, an anti-myostatin antibody of the invention comprises a VH of any of the above-described embodiments and a heavy chain constant region comprising the amino acid sequence of any one of SEQ ID NOs: 7, 9, 11, 193, 195-198, 227, 228, and 229-381. In certain embodiments, an anti-myostatin antibody of the invention comprises a VL of any of the above-described embodiments and a light chain constant region comprising the 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 epitope as the anti-myostatin antibodies provided herein. In a further aspect, the present invention provides antibodies that bind to the same epitope as the antibodies listed in Table 2a. In a further aspect, the present invention provides antibodies that bind to the same epitope as the antibodies listed in Tables 11a or 13. In certain embodiments, antibodies are provided that bind to an epitope in a fragment of the 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 further aspects of the invention, the anti-myostatin antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized, or human antibody. In one embodiment, the anti-myostatin antibody is an antibody fragment, such as, for example, an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length IgG antibody, such as, for example, a complete IgG1 or IgG4 antibody, or other antibody classes or isotypes defined herein.

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

[0185] 1. Antibody affinity In certain embodiments, the antibodies provided herein have a cytotoxicity 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~10 -13 M, e.g. 10 -9 M~10 -13 It has a dissociation constant (Kd) of 1 M.

[0186] In one embodiment, Kd is measured by radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is measured using the lowest concentration ( 125 I) Measurement is performed by equilibrating Fab with labeled antigen and then capturing the bound antigen using a plate coated with anti-Fab antibody. (See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish measurement conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125[I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., as in the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight, although this incubation can be continued for longer periods (e.g., approximately 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μl / well of scintillant (MICROSCINT-20™, Packard) is added, and the plate is counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that gives 20% or less of maximum binding is selected for use in the competitive binding assay.

[0187] In another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, measurements using a BIACORE®-2000 or BIACORE®-3000 (BIACORE®, Inc., Piscataway, NJ) are performed at 25°C using a CM5 chip with approximately 10 response units (RU) of antigen immobilized. 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 injection at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of protein binding. After injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25°C and a flow rate of approximately 25 μl / min. The binding rate (k on ) and dissociation rate (k off ) is calculated by simultaneously fitting the association and dissociation sensorgrams with a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2). The equilibrium dissociation constant (Kd) is given by k off / k on The on-rate is calculated as a ratio of 10 to 10. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). 6 M -1 s -1If the on-rate exceeds , the on-rate can be determined using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, bandpass 16 nm) of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 at 25°C in the presence of increasing concentrations of antigen, as measured in a spectrometer (e.g., a stopped-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); see also WO1993 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F(ab')2 fragments containing salvage receptor binding epitope residues and having increased half-lives in vivo.

[0189] Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 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 that contains all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0191] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of whole antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0192] 3. Chimeric and humanized antibodies In certain embodiments, the antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises 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 another example, a chimeric antibody is a "class-switched" antibody whose class or subclass is changed from that of the parent antibody. Chimeric antibodies also include antigen-binding fragments thereof.

[0193] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans while maintaining the specificity and affinity of the parent non-human antibody. A humanized antibody usually comprises one or more variable domains, in which the HVRs (e.g., CDRs (or portions thereof)) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues were derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0194] Humanized antibodies and methods for their production are reviewed in Almagro, Front. Biosci. 13:1619-1633 (2008), and also see, e.g., Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing resurfacing); Further described in Dall'Acqua et al., Methods 36:43-60 (2005) (describing FR shuffling); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guide selection" approach for FR shuffling).

[0195] Human framework regions that can be used for humanization include, but are not limited to, 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 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 mature (somatically mutated) framework regions or human germline framework regions (see, e.g., 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 techniques known in the art. Human antibodies are reviewed 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 an immunogen to transgenic animals that have been engineered to produce fully human antibodies or complete antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or a portion of human immunoglobulin loci, which either replace endogenous immunoglobulin loci or are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are 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, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HUMAB® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes VELOCIMOUSE® technology. The human variable regions from whole antibodies produced by such animals may be further modified, for example, by combining 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 been described. (See, e.g., 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 generated 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 those described, for example, in U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (which 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 Fv clone variable domain sequences selected from human-derived phage display libraries. These variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0200] 5. Library-derived Antibodies Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. Such methods are 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 described, for example, in 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. 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 a specific phage display method, VH and VL gene repertoires are separately cloned by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol. 12: 433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies to a wide range of non-self and self antigens without immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can be generated synthetically by cloning unrearranged V-gene segments from stem cells and using PCR primers encoding the hypervariable CDR3 regions and containing random sequences to achieve rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent literature describing human antibody phage libraries includes, for example: U.S. Pat. No. 5,750,373, and U.S. Patent Application Publication Nos. 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 herein to be human antibodies or human antibody fragments.

[0203] 6. Multispecific antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies (e.g., bispecific antibodies). Multispecific antibodies are monoclonal antibodies that have binding specificities at at least two different sites. In certain embodiments, one of the binding specificities is for myostatin and the other is for any other antigen. In certain embodiments, bispecific antibodies may bind to two different epitopes of myostatin. Bispecific antibodies may also be used to localize cytotoxic agents to cells that express myostatin. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0204] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-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 technology (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can be made by manipulating electrostatic steering effects to create Fc heterodimeric molecules (WO 2009 / 089004A1); cross-linking two or more antibodies or fragments (see U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to generate antibodies with two specificities (see Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)); using "diabody" technology to create bispecific antibody fragments (see Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see Gruber et al., J. Immunol. 152:5368 (1993)). (1994)); and by preparing trispecific antibodies as described, for example, in Tutt et al., J. Immunol. 147: 60 (1991).

[0205] Engineered antibodies with three or more functional antigen binding sites, including "octopus antibodies," are also included herein (see, eg, US Patent Application Publication No. 2006 / 0025576 A1).

[0206] As used herein, antibodies or fragments also include "dual-acting Fabs" or "DAFs" that contain one antigen-binding site that binds to myostatin and another distinct antigen (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820).

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

[0208] Substitution, insertion, and deletion mutants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading of "Preferred Substitutions." More substantial changes are provided in Table 1 under the heading of "Exemplary Substitutions" and are detailed below with reference to classes of amino acid side chains. Amino acid substitutions may be introduced into the antibody of interest, and the products may be screened for a desired activity, such as, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. [Table 1] TIFF2026001164000001.tif131148

[0209] Amino acids can be divided into groups according to common side chain properties: (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). Non-conservative substitutions refer to the exchange of a member of one of these groups for a member of another group.

[0210] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variant selected for further study will have a modified (e.g., improved) specific biological property compared to the parent antibody (e.g., increased affinity, decreased immunogenicity) and / or will substantially retain the specific biological property of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage-display-based affinity maturation techniques (e.g., those described herein). Briefly, one or more HVR residues are mutated, and the mutated antibodies are displayed on phage and screened for a specific biological activity (e.g., binding affinity).

[0211] Modifications (e.g., substitutions) can be made in HVRs, for example, to improve antibody affinity. Such modifications can be made in HVR "hot spots," i.e., residues encoded by codons that frequently mutate during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or residues that contact antigen, and the resulting mutant VH or VL can 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 the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. 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, as long as such modifications do not substantially reduce the antibody's ability to bind to antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such modifications may, for example, be outside the antigen-contacting residues of the HVRs. In certain embodiments of the above-described mutant VH and VL sequences, each HVR is unaltered 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 mutagenesis is called "alanine scanning mutagenesis," described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues, such as arginine, aspartic acid, histidine, lysine, and glutamic acid) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and it is determined whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to this initial substitution. Alternatively or additionally, a crystal structure of the antigen-antibody complex can be analyzed to identify contact points between the antibody and the antigen. Such contact residues and neighboring residues can be targeted as substitution candidates or excluded from the list. Mutants can be screened to determine whether they contain desired properties.

[0214] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as internal insertions of single or multiple amino acid residues. An example of a terminal insertion includes an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody of an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0215] B glycosylation mutants In certain embodiments, the antibodies provided herein have been modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0216] If the antibody contains an Fc region, the carbohydrate attached thereto may be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, which are usually attached to Asn297 in the CH2 domain of the Fc region via an N-linkage. See, e.g., 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 biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the present invention may be performed to create antibody variants with specific improved properties.

[0217] In one embodiment, antibody variants are provided that have carbohydrate structures lacking fucose added (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 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 glycostructures (e.g., complex, hybrid, and high-mannose structures) added to Asn297, as measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 represents an asparagine residue located approximately at position 297 in the Fc region (EU numbering of Fc region residues). However, due to slight sequence variability between multiple antibodies, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Application Publication Nos. 2003 / 0157108 (Presta, L.); 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "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; 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, which lack protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312, Adams et al., especially Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., 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 provided are antibody variants having bisected oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody, bisected 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 US2005 / 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 mutants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that includes an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0220] In certain embodiments, antibody variants that retain some, but not all, effector functions are also contemplated by the present invention, making them desirable candidates for applications where in vivo half-life is important but certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / lack of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that an antibody lacks FcγR binding (and thus likely lacks ADCC activity) while retaining FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcR 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 to assess ADCC activity of a molecule of interest are described in U.S. Pat. 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); U.S. Pat. No. 5,821,337 (see, Bruggemann, et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, e.g., ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assays (Promega, Madison, WI)).Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of a molecule of interest may be assessed in vivo in an animal model, e.g., as 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 is unable to bind C1q and thus lacks CDC activity. See, e.g., the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. CDC measurements may also be performed to assess complement activation (see, e.g., 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)). Furthermore, determination of FcRn binding and in vivo clearance / half-life may also be performed using methods known in the art (see, e.g., 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 Fc variants with substitutions at two or more amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0222] Certain antibody variants with improved or diminished binding to FcRs have been described (see U.S. Pat. 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 comprises an Fc region with one or more amino acid substitutions that improve ADCC (e.g., substitutions at positions 298, 333, and / or 334 (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 cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 1999 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0225] Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn, which is responsible for the transfer of 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 / 0014934 A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of 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. Patent Nos. 5,648,260 and 5,624,821; and WO 1994 / 29351.

[0226] d. Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to generate cysteine-engineered antibodies (e.g., "thioMAbs") in which one or more residues of an antibody have been substituted with a cysteine ​​residue. In certain embodiments, the substituted residues occur at accessible sites of the antibody. By substituting these residues with cysteine, reactive thiol groups are placed at accessible sites of the antibody, which may be used to conjugate the antibody to other moieties (such as drug moieties or linker-drug moieties) to generate immunoconjugates, as further detailed herein. In certain embodiments, any one or more of the following residues may be substituted with a 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-engineered antibodies may be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0227] e. Antibody derivative In certain embodiments, the antibodies provided herein may be further modified to contain additional nonproteinaceous moieties known in the art and readily available. Suitable moieties for derivatization of antibodies 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 homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. Polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and if more than one polymer is attached, they can be the same molecule or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in therapy under defined conditions, etc.

[0228] In another embodiment, a conjugate of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation is provided. 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 can be of any wavelength, including but not limited to, wavelengths that heat the non-protein moiety to temperatures that are not harmful to normal cells but that kill cells in close proximity to the antibody-non-protein moiety.

[0229] 8. Mutant Fc Regions In one aspect, the present invention provides an isolated polypeptide comprising a mutant Fc region with enhanced FcγRIIb binding activity. In some aspects, the polypeptide is an antibody. In some 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 a native sequence or a reference mutant sequence (sometimes collectively referred to herein as a "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 parent Fc region for monkey FcγRIIb] / [KD value of mutant Fc region for monkey FcγRIIb] can be 2.0 or more, 3.0 or more, 4.0 or more, 5.0 or more, 6.0 or more, 7.0 or more, 8.0 or more, 9.0 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, or 50 or more. In a further embodiment, the mutant Fc region has reduced binding activity to monkey FcγRIIIa. In certain embodiments, the ratio of [KD value of parent Fc region for monkey FcγRIIIa] / [KD value of mutant Fc region for monkey FcγRIIIa] can be 0.50 or less, 0.40 or less, 0.30 or less, 0.20 or less, 0.10 or less, 0.09 or less, 0.08 or less, 0.07 or less, 0.06 or less, 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, or 0.01 or less. In certain embodiments, monkey FcγRIIb has the sequence of SEQ ID NO: 223 (cynomolgus monkey). In certain embodiments, monkey FcγRIIIa has the sequence of SEQ ID NO: 224 (cynomolgus monkey).

[0231] In a further embodiment, the mutant Fc region has enhanced binding activity to human FcγRIIb. In a specific embodiment, the ratio of [KD value of parent Fc region for human FcγRIIb] / [KD value of mutant Fc region for human FcγRIIb] can be 2.0 or more, 3.0 or more, 4.0 or more, 5.0 or more, 6.0 or more, 7.0 or more, 8.0 or more, 9.0 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, or 50 or more. In a further embodiment, the mutant Fc region has reduced binding activity to human FcγRIIIa. In certain embodiments, the ratio of [KD value of parent Fc region for human FcγRIIIa] / [KD value of mutant Fc region for human FcγRIIIa] can be 0.50 or less, 0.40 or less, 0.30 or less, 0.20 or less, 0.10 or less, 0.09 or less, 0.08 or less, 0.07 or less, 0.06 or less, 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, or 0.01 or less. In certain embodiments, human FcγRIIb has the sequence of SEQ ID NO: 212, 213, or 214. In certain embodiments, human FcγRIIIa has the sequence of SEQ ID NO: 215, 216, 217, or 218.

[0232] In a further embodiment, the mutant Fc region has reduced binding activity to human FcγRIIa (H type) compared to human FcγRIIb. In a specific embodiment, the ratio of [KD value of parent Fc region for human FcγRIIa (H type)] / [KD value of mutant Fc region for human FcγRIIa (H type)] can 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 a further embodiment, the mutant Fc region has reduced binding activity to human FcγRIIa (R type) compared to human FcγRIIb. In certain embodiments, the ratio of [KD value of parent Fc region for human FcγRIIa (R type)] / [KD value of mutant Fc region for human FcγRIIa (R type)] can 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 SEQ ID NO: 211. In certain embodiments, human FcγRIIa (R type) has the sequence of SEQ ID NO: 210.

[0233] In certain embodiments, the ratio of [KD value of parent Fc region for monkey FcγRIIa] / [KD value of mutant Fc region for monkey FcγRIIa] can be 2.0 or more, 3.0 or more, 4.0 or more, 5.0 or more, 6.0 or more, 7.0 or more, 8.0 or more, 9.0 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, or 50 or more. In certain embodiments, monkey FcγRIIa is selected from monkey FcγRIIa1 (e.g., cynomolgus monkey FcγRIIa1 (SEQ ID NO: 220)), monkey FcγRIIa2 (e.g., cynomolgus monkey FcγRIIa2 (SEQ ID NO: 221)), and monkey FcγRIIa3 (e.g., cynomolgus monkey FcγRIIa3 (SEQ ID NO: 222)).

[0234] In another embodiment, the KD value of the mutant Fc region for monkey FcγRIIb is 1.0 × 10 -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 x 10 -7 In another embodiment, the KD value of the mutant Fc region for monkey FcγRIIIa can be 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 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, or 1.0 x 10 -5 In another embodiment, the KD value of the mutant Fc region for human FcγRIIb can be 2.0 × 10 -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 x 10 -7 In another embodiment, the KD value of the mutant Fc region for human FcγRIIIa can be 1.0 × 10 -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 more, or 5.0 x 10 -5 In another embodiment, the KD value of the mutant Fc region for human FcγRIIa (H type) can be 1.0 × 10 -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 more, or 5.0 x 10 -6 In another embodiment, the KD value of the mutant Fc region for human FcγRIIa (R type) can be 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 more or 5.0 x 10 -6 It can be M or more.

[0235] In another embodiment, the KD value of the mutant Fc region for monkey FcγRIIa is 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 x 10 -7 In a specific embodiment, the monkey FcγRIIa may be selected from any one of monkey FcγRIIa1, monkey FcγRIIa2, and monkey FcγRIIa3.

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

[0237] The term "Fcγ receptor" (herein referred to as Fcγ receptor, FcγR, or FcgR) refers to a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies, and effectively refers to any member of a family of proteins encoded by Fcγ receptor genes. In humans, this family includes FcγRI (CD64), which includes the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 (H type) and R131 (R type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which includes the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any unidentified human FcγR, FcγR isoform, or allotype. FcγRIIb1 and FcγRIIb2 have been reported as splice variants of human FcγRIIb. Furthermore, a splice variant designated FcγRIIb3 has been reported (J Exp Med, 1989, 170: 1369-1385). In addition to these splice variants, human FcγRIIb includes all splice variants registered with 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 genetic polymorphisms that have been reported in the past (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)), as well as all genetic polymorphisms that will be reported in the future.

[0238] There are two allotypes of FcγRIIa: 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] FcγRs may be derived from any organism, including, but not limited to, FcγRs from humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any mouse FcγR or FcγR isoform. Unless otherwise specified, the term "monkey FcγR" or derivatives thereof refers to cynomolgus monkey 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 set forth in SEQ ID NO: 199 (NM_000566.3); the polynucleotide sequence of human FcγRIIa is set forth in SEQ ID NO: 200 (BC020823.1) or SEQ ID NO: 201 (NM_001136219.1); the polynucleotide sequence of human FcγRIIb is set forth in SEQ ID NO: 202 (BC146678.1) or SEQ ID NO: 203 (NM_004001.3); the polynucleotide sequence of human FcγRIIIa is set forth in SEQ ID NO: 204 (BC033678.1) or SEQ ID NO: 205 (NM_001127593.1); and the polynucleotide sequence of human FcγRIIIb is set forth in SEQ ID NO: 206 (BC128562.1).

[0241] The amino acid sequence of human FcγRI is set forth in SEQ ID NO: 207 (NP_000557.1); the amino acid sequence of human FcγRIIa is set forth 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 set forth in SEQ ID NO: 212 (AAI46679.1), SEQ ID NO: 213, or SEQ ID NO: 214; the amino acid sequence of human FcγRIIIa is set forth 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 set forth in SEQ ID NO: 219 (AAI28563.1).

[0242] The amino acid sequence of cynomolgus monkey FcγRIIa is set forth 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 set forth in SEQ ID NO: 223; and the amino acid sequence of cynomolgus monkey FcγRIIIa is set forth in SEQ ID NO: 224.

[0243] In one aspect, the present invention provides polypeptides comprising a mutant Fc region having enhanced FcγRIIb-binding activity compared to a corresponding reference FcγRIIb-binding polypeptide. In a further aspect, the polypeptides of the present invention comprise 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 (EU numbering). In a specific embodiment, the FcγRIIb has the sequence of cynomolgus monkey FcγRIIb (SEQ ID NO: 223). In a specific 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 polypeptides comprising a mutant Fc region with enhanced FcγRIIb-binding activity, the mutant Fc region comprising at least two amino acid modifications, 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, as indicated by EU numbering. In a specific embodiment, the FcγRIIb has the sequence of cynomolgus monkey FcγRIIb (SEQ ID NO: 223). In a specific embodiment, the 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 comprises an amino acid modification at position 236 (EU numbering).

[0246] In one aspect, the present invention provides polypeptides comprising a mutant Fc region with enhanced FcγRIIb binding activity, which comprises at least two amino acid modifications, 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, as expressed 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 231, 232, 235, 239, 268, 295, 298, 326, 330, and 396, as expressed 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, as expressed by EU numbering. 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).

[0247] In another aspect, the present invention provides a polypeptide comprising a mutant Fc region with enhanced FcγRIIb binding activity, which comprises any one of the following amino acid modifications (1) to (37): (1) positions 231, 236, 239, 268, and 330; (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, 236, 239, 268, 295, and 330, as indicated by EU numbering. 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, and 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 (SEQ ID NO: 223). In certain embodiments, FcγRIIb has the sequence of human FcγRIIb (e.g., SEQ ID NO: 212, 213, or 214).

[0248] In a further aspect, the mutant Fc region with enhanced FcγRIIb binding activity comprises at least one amino acid selected from the group consisting of: (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; (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) 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 a specific embodiment, FcγRIIb has the sequence of cynomolgus monkey FcγRIIb (SEQ ID NO: 223). In a specific embodiment, FcγRIIb has the sequence of human FcγRIIb (e.g., SEQ ID NO: 212, 213, or 214).

[0249] In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity comprises at least one amino acid modification (e.g., substitution) 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 the following amino acid modifications (e.g., substitutions) as represented by EU numbering: 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 the following amino acid modifications (e.g., substitutions) as indicated by EU numbering: 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 the following amino acid modifications (e.g., substitutions) as indicated by EU numbering: 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 the following amino acid modifications (e.g., substitutions) as indicated by EU numbering: 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 the following amino acid modifications (e.g., substitutions) as represented by EU numbering: 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 the following amino acid modifications (e.g., substitutions) as represented by EU numbering: 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 comprises an amino acid modification at position 238 (EU numbering).

[0251] In one aspect, the present invention provides a polypeptide comprising a mutant Fc region with enhanced FcγRIIb binding activity, which comprises 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 indicated by EU numbering. In a further embodiment, 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 indicated by EU numbering. In a specific embodiment, the FcγRIIb has the sequence of cynomolgus monkey FcγRIIb (SEQ ID NO: 223). In a specific embodiment, 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 a mutant Fc region with enhanced FcγRIIb binding activity, which comprises any one of the following amino acid alterations (1) to (9): (1) positions 234, 238, 250, 307, and 330; (2) positions 234, 238, 250, 264, 307, and 330, as indicated by EU numbering; (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 (SEQ ID NO: 223). In certain embodiments, FcγRIIb has the sequence of human FcγRIIb (e.g., SEQ ID NO: 212, 213, or 214).

[0253] In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity comprises at least one 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 indicated by EU numbering. In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity comprises the amino acid modification (e.g., substitution) of Asp at position 238, as indicated by EU numbering. In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity comprises the amino acid modifications (e.g., substitutions) of Asp at position 238, Val at position 250, and Pro at position 307, as indicated by EU numbering. In a further embodiment, the mutant Fc region with enhanced FcγRIIb binding activity comprises the following amino acid modifications (e.g., substitutions) as indicated by EU numbering: 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 the following amino acid modifications (e.g., substitutions) as indicated by EU numbering: 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 the following amino acid modifications (e.g., substitutions) as indicated by EU numbering: 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 the following amino acid modifications (e.g., substitutions) as indicated by EU numbering: 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 the following amino acid modifications (e.g., substitutions) as indicated by EU numbering: 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 the following amino acid modifications (e.g., substitutions) as indicated by EU numbering: 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 the following amino acid modifications (e.g., substitutions) as indicated by EU numbering: 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 the following amino acid modifications (e.g., substitutions), as represented by EU numbering: 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 specific embodiment, the FcγRIIb has the sequence of cynomolgus monkey FcγRIIb (SEQ ID NO: 223). In a specific embodiment, the FcγRIIb has the sequence of human FcγRIIb (e.g., SEQ ID NO: 212, 213, or 214).

[0254] In another aspect, the present invention provides isolated polypeptides comprising a mutant Fc region with an increased isoelectric point (pI). In certain embodiments, the mutant Fc region described herein comprises at least two amino acid modifications in a 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 with an increased pI due to modifications of at least two amino acid residues can facilitate antigen elimination from plasma, for example, when the antibody is administered in vivo.

[0255] In the present invention, the pI may be a theoretical pI or an actually measured pI. The pI value can be measured, for example, by isoelectric focusing, which is known to those skilled in the art. The theoretical pI value can be calculated, for example, using gene and amino acid sequence analysis software (such as Genetyx).

[0256] In one embodiment, the pI value may be increased 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, compared to before modification.

[0257] In certain embodiments, amino acids related to the increased pI may be exposed on the surface of the mutant Fc region. In the present invention, surface-exposed amino acids generally refer to amino acid residues located on the surface of the polypeptide constituting the mutant Fc region. An amino acid residue located on the surface of a polypeptide refers to an amino acid residue whose side chain can contact solvent molecules (usually mostly water molecules). However, the entire side chain does not necessarily have to be in contact with solvent molecules; even if only a portion of the side chain is in contact with solvent molecules, the amino acid is still defined as a "surface-exposed amino acid residue." Furthermore, amino acid residues located on the surface of a polypeptide also include amino acid residues located near the surface and thus potentially influenced by the charge of another amino acid residue whose side chain is in contact, even partially, with solvent molecules. Those skilled in the art can create a homology model of a polypeptide using, for example, commercially available software. Alternatively, methods known to those skilled in the art, such as X-ray crystallography, can be used. Surface-exposed amino acid residues can be determined using coordinates derived from a three-dimensional model using a computer program such as the Insight II program (Accelrys). Surface-exposable sites 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)). Surface-exposable sites can be determined using suitable software for protein modeling and three-dimensional structural information. Software that can be used for such purposes includes, for example, SYBYL Biopolymer Module software (Tripos Associates). Where the algorithm requires a user-input size parameter, the "size" of the probe used in the calculation can be set to a radius of about 1.4 angstroms (Å) or less.Furthermore, a method for determining surface-exposable regions using software for personal computers has been described by Pacios (Comput. Chem. 18(4):377-386(1994); J. Mol. Model. 1:46-53(1995)). Based on such information, appropriate amino acid residues to be 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 a subject's biological fluid (e.g., plasma, interstitial fluid, lymph, ascites, and pleural effusion). The antigen may also be a membrane antigen.

[0259] In a further embodiment, the antigen-binding activity of an antigen-binding domain varies depending on ion concentration conditions. In one embodiment, the ion concentration is not particularly limited and refers to hydrogen ion concentration (pH) or metal ion concentration. As used herein, metal ions refer to ions of Group I elements excluding hydrogen, such as alkali metals and copper group elements; Group II elements, such as alkaline earth metals and zinc group elements; Group III elements excluding boron; Group IV elements excluding 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 differences in the biological behavior of antigen-binding domains between low and high ion concentrations. Furthermore, the phrase "the antigen-binding activity of an antigen-binding domain changes depending on ion concentration conditions" means that the antigen-binding activity of the antigen-binding domain changes between low and high ion concentrations (herein, such antigen-binding domains are referred to as "ion concentration-dependent antigen-binding domains"). 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, ion concentration-dependent antigen-binding domains (e.g., pH-dependent antigen-binding domains or calcium ion concentration-dependent antigen-binding domains) can be obtained by known methods, for example, as described in WO2009 / 125825, WO2012 / 073992, and WO2013 / 046722.

[0260] In the present invention, the antigen-binding activity of an antigen-binding domain under high calcium ion concentrations may be higher than that under low calcium ion concentrations. A high calcium ion concentration may be, but is not limited to, a concentration selected from the range of 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, preferably close to the calcium ion concentration in plasma (blood) in vivo. A low calcium ion concentration may be, but is not limited to, a concentration selected from the range of 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, preferably close to the calcium ion concentration in early endosomes in vivo.

[0261] In one embodiment, the ratio of antigen-binding activity under low calcium ion concentration conditions to that 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 under high calcium ion concentration conditions, i.e., KD (low calcium ion concentration conditions) / KD (high calcium ion concentration conditions), is 2 or more, 10 or more, or 40 or more. As long as such antigen-binding domains can be produced using techniques known to those skilled in the art, the upper limit of this ratio can be 400, 1,000, or 10,000. Alternatively, for example, the dissociation rate constant (kD) can be used instead of KD. In this case, the ratio of kD under low calcium ion concentration conditions to kD under high calcium ion concentration conditions, i.e., kD (low calcium ion concentration conditions) / kD (high calcium ion concentration conditions), is 2 or more, 5 or more, 10 or more, or 30 or more. As long as the antigen-binding domains can be produced using common technical knowledge of those skilled in the art, the upper limit of this ratio can be 50, 100, or 200.

[0262] In the present invention, the antigen-binding activity of an antigen-binding domain at a low hydrogen ion concentration (neutral pH) may be higher than that at a high hydrogen ion concentration (acidic pH). The acidic pH may be, for example, selected from pH 4.0 to pH 6.5, pH 4.5 to pH 6.5, pH 5.0 to pH 6.5, or pH 5.5 to pH 6.5, which 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 a specific embodiment, 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, pH 6.7 to pH 9.5, pH 7.0 to pH 9.0, or pH 7.0 to pH 8.0, which is preferably close to the pH in plasma (blood) in vivo. The neutral pH may also be, for example, pH 7.4 or pH 7.0. In certain embodiments, the neutral pH is pH 7.4.

[0263] In one embodiment, the ratio of the antigen-binding activity under acidic pH conditions to the antigen-binding activity under neutral pH conditions is not limited, but the ratio of the dissociation constant (KD) under acidic pH conditions to the neutral pH condition, i.e., KD(acidic pH conditions) / KD(neutral pH conditions), is 2 or more, 10 or more, or 40 or more. As long as such an antigen-binding domain can be prepared by techniques known to those skilled in the art, the upper limit of this ratio can be 400, 1,000, or 10,000. Alternatively, for example, the dissociation rate constant (kD) can be used instead of KD. 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 more, 5 or more, 10 or more, or 30 or more. As long as the antigen-binding domain can be prepared based on the common technical knowledge of those skilled in the art, the upper limit of this ratio can be 50, 100, or 200.

[0264] In one embodiment, as described in WO2009 / 125825, for example, at least one amino acid residue in the antigen-binding domain is substituted with an amino acid residue having a side chain pKa of 4.0 to 8.0, and / or at least one amino acid residue having a side chain pKa of 4.0 to 8.0 is inserted. 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 the substitution or insertion. When the antigen-binding domain has a variable region or CDR, the site may be within the variable region or CDR. The number of amino acids to be substituted or inserted can be determined appropriately by those skilled in the art and may be one or more. To change the antigen-binding activity of the antigen-binding domain depending on the hydrogen ion concentration conditions, amino acids with a side chain pKa of 4.0 to 8.0 can be used. Such amino acids include, for example, natural amino acids such as His (H) and Glu (E), as well as unnatural 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 pKa values ​​of 6.0 to 7.0 can also be used, including, for example, His (H).

[0265] In another aspect, antigen-binding domains suitable for mutant Fc regions with increased pI are described, which are obtainable by the methods described in Japanese Patent Application Nos. 2015-021371 and 2015-185254.

[0266] In certain embodiments, the mutant Fc region with increased pI 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 expressed in EU numbering.

[0267] In a further embodiment, the mutant Fc region with increased pI comprises 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 expressed in EU numbering.

[0268] In another aspect, the present invention provides a polypeptide comprising a mutant Fc region with an increased pI, which comprises any one of the following amino acid modifications (1) to (10): (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, as indicated by EU numbering.

[0269] For example, the method for increasing the pI of protein is to reduce the number of amino acids whose side chains have negative charges (e.g., aspartic acid and glutamic acid) under neutral pH conditions, and / or increase the number of amino acids whose side chains have positive charges (e.g., arginine, lysine, and histidine).Amino acids whose side chains have negative charges have a negative charge represented as -1 under pH conditions that are sufficiently higher than the pKa of their side chains, which is 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 the side chain has a negative charge represented as -1 under neutral pH conditions (e.g., in a solution of pH 7.0).Conversely, amino acids whose side chains have positive charges have a positive charge represented as +1 under pH conditions that are sufficiently lower than the pKa of their side chains.For example, the theoretical pKa of the side chain of arginine is 12.5, and the side chain has a positive charge represented as +1 under neutral pH conditions (e.g., in a solution of pH 7.0). In contrast, amino acids whose side chains do not have a charge under neutral pH conditions (e.g., in a solution of pH 7.0) are known to include the 15 naturally occurring amino acids, i.e., alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan, and tyrosine. Of course, it is understood that amino acids for increasing pI may also be unnatural amino acids.

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

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

[0272] Insertion or addition of amino acids to increase the pI includes, for example, insertion or addition of amino acids with uncharged side chains and / or insertion or addition of amino acids with positively charged side chains in the amino acid sequence of the parent Fc region, which may be performed alone or in appropriate combination.

[0273] Amino acid deletions to increase the pI include, for example, deletion of amino acids with uncharged side chains and / or deletion of amino acids with negatively charged side chains in the amino acid sequence of the parent Fc region, either alone or in appropriate combination.

[0274] In one embodiment, the natural amino acids used to increase the pI can be classified as follows: (a) amino acids with a negatively charged side chain can be Glu (E) or Asp (D); (b) amino acids with an uncharged side chain can 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 a positively charged side chain can be His (H), Lys (K), or Arg (R). In one embodiment, the amino acid inserted or substituted after modification 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 certain embodiments, the mutant Fc region described herein comprises at least two amino acid modifications in a parent Fc region.

[0276] In one aspect, the present invention provides a method for the preparation of a human ovarian tumor suppressor comprising: (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, as indicated by EU numbering; and (b) at least one amino acid modification at position 28, as indicated by EU numbering. and 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 polypeptides comprising a mutant Fc region with enhanced FcγRIIb binding activity and increased pI, the mutant Fc region 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 231, 232, 235, 236, 239, 268, 295, 298, 326, 330, and 396, as expressed in EU numbering; and (b) 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 expressed in EU numbering.

[0278] In another aspect, the present invention provides polypeptides comprising mutant Fc regions with enhanced FcγRIIb binding activity and increased pI, comprising any one of the following amino acid alterations (1) to (9): (1) positions 235, 236, 268, 295, 311, 326, 330, and 343; (2) positions 236, 268, 295, 311, 326, 330, and 343; (3) positions 236, 268, 295, 311, 326, 330, and 343, as indicated by EU numbering; , 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 (SEQ ID NO: 223). In certain embodiments, FcγRIIb has the sequence of human FcγRIIb (e.g., SEQ ID NO: 212, 213, or 214).

[0279] In one aspect, the present invention provides polypeptides 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, 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, as expressed in EU numbering. 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 specific embodiment, FcγRIIb has the sequence of cynomolgus monkey FcγRIIb (SEQ ID NO: 223). In a specific embodiment, FcγRIIb has the sequence of human FcγRIIb (e.g., SEQ ID NO: 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 alterations (1) to (16): (1) positions 234, 238, 250, 264, 307, 311, 330, and 343, as indicated 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) positions 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 variant Fc region comprises an amino acid modification selected from any single modification, combination of single modifications, or combination modifications listed in Tables 14-30.

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

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

[0284] As used herein, the term "parent Fc region" refers to an Fc region prior to the introduction of the amino acid modifications described herein. Preferred examples of parent Fc regions include Fc regions derived from natural antibodies. Examples of antibodies include IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), and IgM. Antibodies may be derived from humans or monkeys (e.g., cynomolgus monkeys, rhesus monkeys, marmosets, chimpanzees, or baboons). Natural antibodies may contain naturally occurring mutations. Multiple 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 these may be used in the present invention. In particular, for human IgG1, the amino acid sequence at positions 356 to 358 (EU numbering) may be either DEL or EEM. Preferred examples of the parent Fc region 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 the 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 prepared by adding amino acid modifications other than those described herein to an Fc region derived from a native antibody.

[0285] Additionally, amino acid modifications made for other purposes can be combined in the variant Fc regions 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 that improve antibody heterogeneity or stability (WO2009 / 041613) may be made. Alternatively, the mutant Fc regions described herein can be combined with polypeptides having the property of promoting antigen clearance as described in WO2011 / 122011, WO2012 / 132067, WO2013 / 046704, or WO2013 / 180201, polypeptides having the property of specifically binding to target tissues as described in WO2013 / 180200, or polypeptides having the property of repeatedly binding to multiple antigen molecules as described in WO2009 / 125825, WO2012 / 073992, or WO2013 / 047752. Alternatively, the amino acid modifications disclosed in EP1752471 and EP1772465 can be combined in the CH3 of the mutant Fc regions described herein to confer binding ability to other antigens. Alternatively, the mutant Fc region described herein may be combined with amino acid modifications that decrease the pI of the constant region (WO2012 / 016227) to increase plasma retention, or with amino acid modifications that increase the pI of the constant region (WO2014 / 145159) to promote cellular uptake.Alternatively, amino acid modifications that increase the pI of the constant region (Japanese Patent Application Nos. 2015-021371 and 2015-185254) may be combined in the mutant Fc region described herein to promote plasma clearance of the target molecule. In one embodiment, such modifications may include, for example, substitutions at at least one position selected from the group consisting of positions 311, 343, 384, 399, 400, and 413 (EU numbering). In a further embodiment, such substitutions may be substitutions of the amino acid at each position with Lys or Arg.

[0286] Amino acid modifications that enhance human FcRn-binding activity at acidic pH can also be combined in the mutant Fc regions described herein. Specifically, such modifications include, for example, substitution of Met at position 428 with Leu and substitution of Asn at position 434 with Ser, as represented by EU numbering (Zalevsky et al., Nat. Biotechnol. 28:157-159(2010)); substitution of Asn at position 434 with Ala (Deng et al., Metab. Dispos. 38(4):600-605(2010)); substitution of Met at position 252 with Tyr, substitution of Ser at position 254 with Thr, and substitution of Thr at position 256 with Glu (Dall'Acqua et al., J. Biol. Chem. 281:23514-23524(2006)); substitution of Thr at position 250 with Gln and substitution of Met at position 428 with Leu (Hinton et al., J. Immunol. 176(1):346-356(2006)); substitution of Asn at position 434 with His (Zheng et al., Clin. Pharmacol. Ther. 89(2):283-290(2011)), as well as modifications described in 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 a substitution of Met with Leu at position 428, a substitution of Asn with Ala at position 434, and a substitution of Tyr with Thr at position 436. These modifications may further include a substitution of Gln with Arg at position 438 and / or a substitution of Ser with Glu at position 440 (Patent Application Nos. 2015-021371 and 2015-185254).

[0287] Two or more polypeptides containing the mutant Fc regions described herein can be contained in a single molecule, where the two polypeptides containing the mutant Fc regions are linked together in much the same manner as in a single antibody. The type of antibody is not limited, and can include IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), and IgM.

[0288] The two linked polypeptides containing mutant Fc regions may be polypeptides containing mutant Fc regions with the same amino acid modification introduced (hereinafter referred to as "homologous mutant Fc regions"), or polypeptides containing mutant Fc regions with different amino acid modifications introduced, or polypeptides containing mutant Fc regions in which an amino acid modification has been introduced in only one of the Fc regions (hereinafter referred to as "heterologous polypeptides containing mutant Fc regions"). One preferred amino acid modification is a modification in the loop structure at positions 233 to 239 (EU numbering) in the CH2 domain of the Fc region, which is involved in binding to FcγRIIb and FcγRIIa. Preferably, a modification that improves FcγRIIb-binding activity and / or selectivity is introduced into the loop structure of the CH2 domain of one Fc region, and a different modification that destabilizes the loop structure of the CH2 domain of the other Fc region is introduced. An example of an amino acid modification that can destabilize the loop structure of the CH2 domain is 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, destabilization can be achieved by, for example, changing the amino acid at position 235 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, as represented by EU numbering.

[0289] With regard to the binding of heterologous polypeptides containing mutant Fc regions, techniques can be applied that suppress unintended binding of homologous polypeptides containing mutant 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 of the Fc region include residues at positions 356 (EU numbering), 439 (EU numbering), 357 (EU numbering), 370 (EU numbering), 399 (EU numbering), and 409 (EU numbering) in the CH3 domain.

[0291] More specifically, for example, an Fc region can be produced in which one to three pairs of amino acid residues selected from the following (1) to (3) have 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, a heterologous polypeptide can be produced comprising a mutant Fc region in which one to three pairs of amino acid residues selected from (1) to (3) above have the same charge in the CH3 domain of a first Fc region, and the pairs of amino acid residues selected in the first Fc region above also have the same charge in the CH3 domain of a second Fc region, but the charges of the first and second Fc regions are opposite.

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

[0294] Other known techniques can also be used to bind heterologous polypeptides containing mutant Fc regions. Specifically, such techniques involve replacing the amino acid side chains present in one Fc region with larger side chains (knobs) and smaller side chains (holes) in the Fc region, thereby locating the knobs within the holes. This promotes efficient binding between Fc region-containing polypeptides with 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 heterologous binding of polypeptides containing mutant Fc regions. Binding of polypeptides containing Fc regions can be efficiently induced using chain-swapped recombinant CH3 domain heterodimers (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 with different amino acid sequences.

[0296] In addition, the heterodimerized antibody production technique described in WO2011 / 028952, which utilizes the combination of antibody CH1 and CL and VH and VL, can also be used.

[0297] Similar to the methods described in WO2008 / 119353 and WO2011 / 131746, it is also possible to use a technique for producing heterodimerized antibodies by first preparing two types of homodimerized antibodies, incubating the antibodies under reducing conditions to dissociate them, and then rebinding them.

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

[0299] Furthermore, it is also possible to use techniques for producing heterodimerized antibodies 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 a polypeptide containing a heterologous mutant Fc region, a polypeptide containing a homologous mutant Fc region is usually also produced as an impurity. In such cases, the polypeptide containing a heterologous mutant Fc region can be efficiently obtained by separating and purifying it from the polypeptide containing the homologous mutant Fc region using known techniques. A method for efficiently separating and purifying a heterodimerized antibody from a homodimerized antibody using ion exchange chromatography has been reported (WO2007 / 114325), by introducing amino acid modifications into the variable regions of the two antibody heavy chains that cause a difference in the isoelectric point between the homodimerized antibody and the heterodimerized antibody. Another method for purifying a heterodimerized antibody using protein A chromatography has been reported (WO1998 / 050431 and WO1995 / 033844), by constructing a heterodimerized antibody containing two heavy chains derived from mouse IgG2a, which binds to protein A, and rat IgG2b, which does not bind to protein A.

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

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

[0303] When substituting an amino acid residue, the substitution with another amino acid residue can be performed for the purpose of modifying the following aspects (a) to (c): (a) the polypeptide backbone structure in the sheet or helix region; (b) the charge or hydrophobicity at the target site; or (c) the size of the side chain.

[0304] Amino acid residues are classified into the following groups based on their general side chain properties: (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 modifications can be achieved by a variety of methods known to those skilled in the art, including, but 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, and in certain embodiments, 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. A specific post-translational modification can be the addition or deletion of a glycosylation site. For example, the amino acid residue at position 297 (EU numbering) in the IgG1 constant region may be glycosylated. The glycosylation site for modification is not limited. For example, sialic acid can be added to the glycosylation site of the Fc region (MAbs 2010 Sep-Oct, 2(5): 519-527). Generally, antibodies expressed in eukaryotic cells contain glycosylation in the constant region. For example, antibodies expressed in cells such as naturally occurring mammalian antibody-producing cells or eukaryotic cells transformed with an expression vector containing antibody-encoding DNA are known to usually have some type of glycosylation site.

[0308] The eukaryotic cells referred to herein include yeast and animal cells. For example, CHO cells and HEK293 cells are typical animal cells used for transformation with an expression vector containing an antibody-encoding DNA. On the other hand, the present invention also includes constant regions that are not glycosylated. Antibodies with non-glycosylated constant regions can be obtained by expressing a gene encoding the antibody in prokaryotic cells such as Escherichia coli.

[0309] Furthermore, polypeptides comprising the mutant Fc regions of the present invention may be chemically modified with various molecules, such as polyethylene glycol (PEG) and cytotoxic substances. Methods for such chemical modification of polypeptides are 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 some aspects, the polypeptide is an antibody. In some 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 some aspects, the polypeptide is an Fc fusion protein.

[0311] The variable regions of antibodies containing the mutant Fc regions provided herein and the protein-binding motifs of Fc fusion proteins containing the mutant Fc regions 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 immune complexes that partially comprise immunoglobulins.

[0312] Examples of cytokines that can be bound by antibodies or fusion proteins comprising the mutant Fc regions of the present invention and / or can be recombinantly fused to polypeptides comprising the disclosed mutant Fc regions include, but are not limited to, interleukins 1 to 18, colony-stimulating factors (such as G-CSF, M-CSF, and GM-CSF), interferons (such as IFN-α, IFN-β, and IFN-γ), growth factors (such as EGF, FGF, IGF, NGF, PDGF, TGF, and HGF), tumor necrosis factors (TNF-α and TNF-β), lymphotoxin, erythropoietin, leptin, SCF, TPO, MCAF, and BMP.

[0313] Examples of chemokines that can be bound by antibodies or fusion proteins comprising the mutant Fc regions of the present invention and / or can be recombinantly fused to polypeptides comprising the disclosed mutant Fc regions include, but are not limited to, CC chemokines such as CCL1-CCL28, CXC chemokines such as CXCL1-CXCL17, C chemokines such as XCL1-XCL2, and CX3C chemokines such as CX3CL1.

[0314] Examples of receptors that can be bound by antibodies or fusion proteins comprising the mutant Fc regions of the present invention and / or that can be recombinantly fused to polypeptides comprising the disclosed mutant Fc regions 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. The receptors belonging to these receptor families and their characteristics are described in Cooke, ed., New Comprehesive 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. 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 human or mouse erythropoietin (EPO) receptors (Jones et al., Blood 76(1):31-35(1990); D'Andrea et al., Cell 57(2):277-285(1989)), human or mouse granulocyte colony-stimulating factor (G-CSF) receptors (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 human or mouse thrombopoietin (TPO) receptors (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 77(3):391-400(1994)), human or mouse leptin receptor, human or mouse growth hormone (GH) receptor, human or mouse interleukin (IL)-10 receptor, human or mouse insulin-like growth factor (IGF)-I receptor, human or mouse leukemia inhibitory factor (LIF) receptor, and human or mouse ciliary neurotrophic factor (CNTF) receptor.

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

[0317] MHC antigens are broadly classified into MHC class I antigens and MHC class II antigens, which include HLA-A, -B, -C, -E, -F, -G, and -H, and MHC class II antigens which include HLA-DR, -DQ, and -DP.

[0318] Examples of differentiation antigens that can be bound by antibodies or fusion proteins comprising the variant Fc regions of the invention and / or that can be recombinantly fused to polypeptides comprising the disclosed variant Fc regions 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.

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

[0320] Other examples of antigens that can be bound by antibodies or fusion proteins comprising the variant Fc regions of the invention and / or that can be recombinantly fused to polypeptides comprising the disclosed variant Fc regions 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, activin RIB, activin B, activin C, activin D, activin E, activin F ... 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 stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2, BMP-2a, BMP-3, osteogenin, BMP-4, BMP-2b, BMP-5, BMP-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, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P,CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, 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 activation protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9 FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GFAP, GFR-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, 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 receptor, 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 inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA 90, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, 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, prolactin, 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 expressed Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM , viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, 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,Non-limiting examples include 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; and receptors for hormones and growth factors.

[0321] As described herein, one or more amino acid residues in the amino acid sequence constituting the variable region can be modified as long as the antigen-binding activity is maintained. When modifying the variable region amino acid sequence, the site of modification and the number of amino acids to be modified are not particularly limited. For example, amino acids present in the CDR and / or FR can be modified as appropriate. When modifying amino acids in the variable region, 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, or 100% or more compared to the binding activity before modification. Furthermore, the binding activity may be enhanced by the amino acid modification. For example, the binding activity may be 2-fold, 5-fold, or 10-fold higher than the binding activity before modification. The modification of the amino acid sequence may be at least one of substitution, addition, deletion, and modification of amino acid residues.

[0322] For example, modification of N-terminal glutamine of a variable region to pyroglutamic acid by pyroglutamylation is well known to those skilled in the art. Thus, if the N-terminus of a heavy chain is glutamine, the antibody described herein may comprise 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 of any origin, such as mouse, rat, rabbit, goat, or camel antibodies, humanized antibodies produced by humanizing these non-human antibodies, and human antibodies. Furthermore, these antibodies may have a wide variety of amino acid substitutions introduced into their variable regions to improve antigen binding, pharmacokinetics, stability, and immunogenicity. The variable regions may be capable of repeatedly binding to antigens due to their pH-dependent antigen binding ( WO2009 / 125825 ).

[0324] Both kappa and lambda chains are present in the light chain constant region of an antibody and are permissible. Furthermore, they may have several amino acid modifications such as substitutions, del...

Claims

1. 1. An isolated antibody that binds to latent myostatin, The antibody comprises a mutant Fc region comprising at least one amino acid modification in a parent Fc region that is an Fc region derived from human IgG1; the ratio of [KD value of the parent Fc region for monkey FcγRIIb] / [KD value of the mutant Fc region for monkey FcγRIIb] is 2.0 or more, and the ratio of [KD value of the parent Fc region for human FcγRIIIa] / [KD value of the mutant Fc region for human FcγRIIIa] is 0.5 or less; the variant Fc region comprises an amino acid modification at position 330 according to EU numbering; The antibody wherein the amino acid at position 330 according to EU numbering is Lys.

2. The antibody of claim 1, comprising: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 114; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58; HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63; HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 114; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 116; HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (iii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 57; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 117; HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63; HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 57; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118; HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63; HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (v) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 57; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 119; HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63; HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (vi) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 114; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118; HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63; HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (vii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 114; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58; HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63; HVR-L1 comprising the amino acid sequence of SEQ ID NO: 123; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (viii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 115; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58; HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63; HVR-L1 comprising the amino acid sequence of SEQ ID NO: 123; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (ix) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 114; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58; HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63; HVR-L1 comprising the amino acid sequence of SEQ ID NO: 124; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 125, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (x) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 115; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 120; HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63; HVR-L1 comprising the amino acid sequence of SEQ ID NO: 123; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74.

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