Anti-muscle growth suppressor antibodies, polypeptides comprising variant FC regions and methods of use

Abstract

The present disclosure provides anti-muscle growth inhibitor antibodies and methods of making and using the same. Also provided are nucleic acids encoding the anti-MGF antibodies and host cells comprising the nucleic acids. The disclosure also provides polypeptides containing 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.

Description

[0001] This application is a divisional application of the patent application with international application number PCT / JP2015 / 006323, international filing date December 18, 2015, Chinese application number 201580068857.X, and invention title "Anti-Myostatin Antibodies, Polypeptides Comprising Variant Fc Regions, and Methods of Use". Technical Field

[0002] The present invention relates to anti-myostatin antibodies and methods of using the same. The present invention also relates to polypeptides comprising variant Fc regions and methods of using the same. Background Art

[0003] Myostatin, also known as growth differentiation factor-8 (GDF8), is a secreted protein and a member of the transforming growth factor-β (TGF-β) protein superfamily. Members of this superfamily have growth-regulatory and morphogenetic properties (see, e.g., NPL1, NPL2, and PTL1). Myostatin is mainly expressed in developing and adult skeletal muscle and functions as a negative regulator of muscle growth. Systemic overexpression of myostatin in adult mice results in muscle wasting (see, e.g., NPL3), while, conversely, myostatin knockout mice are characterized by hypertrophy and hyperplasia of skeletal muscle, which results in a two- to three-fold higher muscle mass compared to their wild-type littermates (see, e.g., NPL4).

[0004] Similar to 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, e.g., NPL5; PTL2). Two myostatin precursor molecules are covalently linked via a single disulfide bond in the C-terminal growth factor domain. Through multi-step proteolytic processing, the active mature myostatin (a disulfide-bonded homodimer consisting of the C-terminal growth factor domain) is released from the myostatin precursor. In the first step of the myostatin activation pathway, in both chains of the homodimeric precursor, the peptide bond between the N-terminal propeptide domain and the C-terminal growth factor domain, Arg266-Asp267, is cleaved by a furin-type proprotein convertase. However, the three resulting peptides (two propeptides and one mature myostatin (i.e., a disulfide-bonded homodimer consisting of the growth factor domain)) remain linked, forming a non-covalently inactive complex called "latent myostatin". Then, via degradation of the propeptide, mature myostatin can be released from latent myostatin. A member of the bone morphogenetic protein 1 (BMP1) family of metalloproteinases cleaves a single peptide bond, Arg98-Asp99, within the propeptide, which is accompanied by the release of mature active myostatin as a homodimer (see, e.g., NPL6). In addition, latent myostatin can be activated in vitro by dissociating the complex using acid or heat treatment (see, e.g., NPL7).

[0005] Myostatin exerts its effects through a family of transmembrane serine / threonine kinase heterotetrameric receptors, the activation of which enhances receptor phosphotransfer, leading to stimulation of serine / threonine kinase activity. It has been demonstrated that the myostatin pathway involves the active myostatin dimer binding with high affinity to the activin receptor type IIB (ActRIIB), which then recruits and activates the phosphotransfer of the low-affinity receptor activin-like kinase 4 (ALK4) or activin-like kinase 5 (ALK5). It has also been demonstrated that the proteins Smad 2 and Smad 3 are subsequently activated and form a complex with Smad 4, which is then translocated to the nucleus for activation of target gene transcription. It has been demonstrated that ActRIIB is able to mediate the effects of myostatin in vivo, since the expression of a dominant-negative form of ActRIIB in mice is similar to myostatin gene knockout (see, e.g., NPL8).

[0006] Several diseases or conditions are associated with muscle wasting (i.e., loss of muscle tissue or impaired function), 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 would benefit from an increase in muscle mass and / or muscle strength; however, the treatments currently available for these conditions are limited. Accordingly, myostatin has emerged as a target for therapeutic or prophylactic intervention in such diseases or conditions or for monitoring the progression of such diseases or conditions due to its role as a negative regulator of skeletal muscle growth. In particular, agents that inhibit myostatin activity can be therapeutically beneficial.

[0007] Inhibition of myostatin expression results in muscle hypertrophy and hyperplasia (NPL9). After injury, myostatin negatively regulates muscle regeneration and the lack of myostatin in myostatin - null mice results in accelerated muscle regeneration (see, e.g., NPL10). Anti - myostatin (GDF8) antibodies described, for example, in PTL3, PTL4, PTL5, PTL6, PTL7, PTL8, PTL9, and PTL10 have been shown to bind myostatin and inhibit myostatin activity both in vitro and in vivo, including myostatin activity associated with the negative regulation of skeletal muscle mass. Myostatin - neutralizing antibodies increase body weight, skeletal muscle mass, and muscle size and strength in skeletal muscle of wild - type mice (see, e.g., NPL11) as well as mdx mice, a model of muscular dystrophy (see, e.g., NPL12; NPL13). However, these prior art antibodies are specific for mature myostatin and not for latent myostatin, and the strategies described for inhibiting myostatin activity have utilized antibodies that can bind and neutralize mature myostatin.

[0008] Antibodies have attracted attention as drugs because of their high stability in blood and few side effects (see, for example, NPL14 and NPL15). Almost all currently marketed therapeutic antibodies 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, NPL16). For antibodies showing ADCC activity, the antibody Fc region must bind to the Fcγ receptor (hereinafter referred to as FcγR), which is an antibody-binding receptor present on the surface of effector cells such as killer cells, natural killer cells, and activated macrophages.

[0009] In humans, the FcγRIa (CD64A), FcγRIIa (CD32A), FcγRIIb (CD32B), FcγRIIIa (CD16A), and FcγRIIIb (CD16B) isotypes have been reported as the FcγR protein family, and the corresponding allotypes have also been reported (see, for example, NPL17). FcγRIa, FcγRIIa, and FcγRIIIa are called activating FcγRs because they have immunologically active functions, while FcγRIIb is called an inhibitory FcγR because it has immunosuppressive functions (see, for example, NPL18).

[0010] In the binding between the Fc region and FcγR, several amino acid residues in the antibody hinge region and CH2 domain, as well as the sugar chain linked to Asn at position 297 (EU numbering) that binds to the CH2 domain, have been shown to be important (see, for example, NPL19, NPL20, and NPL21). Currently, various variants with FcγR-binding properties have been studied, mainly antibodies with mutations introduced at these sites; and Fc region variants with higher binding activity to activating FcγRs have been obtained (see, for example, PTL11, PTL12, PTL13, and PTL14).

[0011] When the activating FcγR is cross-linked with an immune complex, it phosphorylates the immunoreceptor tyrosine-based activation motif (ITAM) contained in its intracellular domain or the FcR common γ-chain (interaction partner), activates the signal transducer SYK, and triggers an inflammatory immune response by initiating an activation signal cascade (see, for example, NPL22).

[0012] FcγRIIb is the only FcγR expressed on B cells (see, e.g., NPL23). Interaction of the antibody Fc region with FcγRIIb has been reported to inhibit the primary immune response of B cells (see, e.g., NPL24). In addition, it has been reported that when FcγRIIb and the B cell receptor (BCR) on B cells are cross-linked via immune complexes in the blood, B cell activation and antibody production by B cells are inhibited (see, e.g., NPL25). In this immunosuppressive signal transduction mediated by BCR and FcγRIIb, the immunoreceptor tyrosine-based inhibitory motif (ITIM) contained in the intracellular domain of FcγRIIb is necessary (see, e.g., NPL26 and NPL27). When ITIM is phosphorylated after signal transduction, SH2-containing inositol polyphosphate 5-phosphatase (SHIP) is recruited, transduction of other activating FcγR signal cascades is inhibited, and the immune response is inhibited (see, e.g., NPL28). In addition, it has been reported that due to BCR cross-linking and B cell proliferation, aggregation of FcγRIIb alone transiently inhibits calcium influx in a BCR-independent manner without causing apoptosis of IgM-producing B cells (see, e.g., NPL29).

[0013] FcγRIIb is also expressed on dendritic cells, macrophages, activated neutrophils, columnar cells, and basophils. FcγRIIb inhibits the functions of activating FcγRs such as phagocytosis and release of inflammatory cytokines in these cells, and inhibits the inflammatory immune response (see, e.g., NPL30).

[0014] Current studies using FcγRIIb knockout mice have elucidated the importance of the immunosuppressive function of FcγRIIb. It has been reported that in FcγRIIb knockout mice, humoral immunity is not properly regulated (see, e.g., NPL31), sensitivity to collagen-induced arthritis (CIA) is increased (see, e.g., NPL32), lupus-like symptoms are presented, and Goodpasture's syndrome-like symptoms are presented (see, e.g., NPL33).

[0015] In addition, it has been reported that insufficient regulation of FcγRIIb is associated with human autoimmune diseases. For example, the relationship between genetic polymorphisms in the transmembrane region and promoter region of FcγRIIb and the incidence of systemic lupus erythematosus (SLE) has been reported (see, e.g., NPL34, NPL35, NPL36, NPL37, and NPL38), as well as a decrease in FcγRIIb expression on the surface of B cells in SLE patients (see, e.g., NPL39 and NPL40).

[0016] From mouse models and thus clinical findings, FcγRIIb is thought to play a role in controlling autoimmune and inflammatory diseases, particularly by involving B cells, and it is a promising target molecule for controlling autoimmune and inflammatory diseases.

[0017] It is known that IgG1, which is mainly used as a commercially available therapeutic antibody, not only binds to FcγRIIb but also strongly binds to activating FcγRs (see, for example, NPL41). It is possible to develop therapeutic antibodies with immunosuppressive properties greater than those of IgG1 by using an Fc region with enhanced FcγRIIb binding or increased FcγRIIb binding selectivity compared to activating FcγRs. For example, it has been proposed that antibodies with variable regions that bind to the BCR and an Fc with enhanced FcγRIIb binding can inhibit B cell activation (see, for example, NPL42). It has been reported that cross-linking FcγRIIb on B cells with IgE that binds to the B cell receptor inhibits the differentiation of B cells into plasma cells, which thus results in the inhibition of IgE production; and in mice transplanted with human PBMCs, the human IgG and IgM concentrations are maintained while the human IgE concentration decreases (see, for example, NPL43). In addition to IgE, it has been reported that when an antibody cross-links FcγRIIB and CD79b, which is a component molecule of the B-cell receptor complex, B cell proliferation in vitro is inhibited and arthritis symptoms are alleviated in a collagen arthritis model (see, for example, NPL44).

[0018] In addition to B cells, it has been reported that cross-linking of FcεRI and FcγRIIb on mast cells using a molecule in which the Fc portion of IgG with enhanced FcγRIIb binding is fused to the Fc portion of IgE that binds to the FcεRI receptor for IgE results in phosphorylation of FcγRIIb, thereby inhibiting FcεRI-dependent calcium influx. This suggests that it is possible to inhibit degranulation stimulated via FcγRIIb by enhancing FcγRIIb binding (see, for example, NPL45).

[0019] Therefore, it is shown that antibodies with an Fc having enhanced FcγRIIb-binding activity are promising as therapeutic agents for inflammatory diseases such as autoimmune diseases.

[0020] In addition, it has been reported that, in the presence of an antibody-antigen immune complex, the activation of macrophages and dendritic cells via Toll-like receptor 4 due to LPS stimulation is inhibited, and this effect is also thought to be the effect of the immune complex via FcγRIIb (see, for example, NPL46 and NPL47). Therefore, it is expected that an antibody with enhanced FcγRIIb binding can enhance the TLR-mediated activation signal-inhibiting effect; thus, it is suggested that such an antibody is promising as a therapeutic agent for inflammatory diseases such as autoimmune diseases.

[0021] In addition, mutants with enhanced FcγRIIb binding have been proposed as promising cancer therapeutic agents, as well as therapeutic agents for inflammatory diseases such as autoimmune diseases. Currently, it has been found that FcγRIIb plays an important role in the agonist activity of agonist antibodies against the TNF receptor superfamily. Specifically, it has been proposed that the agonist activity of antibodies against CD40, DR4, DR5, CD30, and CD137, which are included in the TNF receptor family, requires interaction with FcγRIIb (see, for example, NPL48, NPL49, NPL50, NPL51, NPL52, NPL53, and NPL54). NPL55 shows that the anti-tumor effect of an anti-CD40 antibody is enhanced by using an antibody with enhanced FcγRIIb binding. Therefore, it is expected that an antibody with enhanced FcγRIIb has the effect of enhancing the agonist activity of agonist antibodies including antibodies against the TNF receptor superfamily.

[0022] In addition, it has been demonstrated that when an antibody that recognizes Kit, a receptor tyrosine kinase (RTK), is used to crosslink FcγRIIb and Kit on cells expressing Kit, cell proliferation is inhibited. Even in cases where the Kit is constitutively activated and has a mutation that leads to tumor formation, a similar effect has been reported (see, for example, NPL56). Therefore, it is expected that the use of an antibody with enhanced FcγRIIb binding can enhance the inhibitory effect on cells expressing an RTK with a constitutively activating mutation.

[0023] Antibodies with an Fc having enhanced FcγRIIb-binding activity have been reported (see, for example, NPL57). In this literature, the FcγRIIb-binding activity was enhanced by adding alterations such as S267E / L328F, G236D / S267E, and S239D / S267E to the Fc region of the antibody. Among them, the antibody into which the S267E / L328F mutation was introduced bound most strongly to FcγRIIb and maintained the same level of binding to FcγRIa and FcγRIIa H type (where the residue at position 131 of FcγRIIa is His as in naturally occurring IgG1). However, another report showed that this alteration enhanced the binding to FcγRIIa R type (where the residue at position 131 of FcγRIIa is Arg) hundreds of times to the same level as the FcγRIIb binding, meaning that the FcγRIIb-binding selectivity was not improved compared to the R type FcγRIIa (see, for example, PTL15).

[0024] The effect of enhancing FcγRIIa binding without enhancing FcγRIIb binding has only been considered to have an impact on cells such as platelets that express FcγRIIa but not FcγRIIb (see, for example, NPL58). For example, it is known that the risk of thromboembolism increases in a patient group administered bevacizumab, an antibody against VEGF (see, for example, NPL59). In addition, thromboembolism was observed in a similar manner in the clinical development trial of an antibody against CD40 ligand, and the clinical study was interrupted (see, for example, NPL60). In the case of both of these antibodies, subsequent studies using animal models, etc. showed that the administered antibody caused platelet aggregation via binding to FcγRIIa on platelets and formed blood clots (see, for example, NPL61 and NPL62). In systemic lupus erythematosus, an autoimmune disease, platelets are activated via an FcγRIIa-dependent mechanism, and it has been reported that platelet activation is related to the severity of the symptoms (see, for example, NPL63). Administering an antibody with enhanced FcγRIIa binding to such patients who already have a high risk of developing thromboembolism will increase the risk of developing thromboembolism and is therefore extremely dangerous.

[0025] In addition, it has been reported that antibodies with enhanced FcγRIIa binding enhance macrophage-mediated antibody-dependent cell phagocytosis (ADCP) (see, e.g., NPL64). When the antigen to which the antibody is to bind is phagocytosed by a macrophage, the antibody itself is thought to be phagocytosed simultaneously. When the antibody is administered as a drug, it is speculated that peptide fragments derived from the administered antibody may also be presented as antigens, thereby increasing the risk of generating antibodies against the therapeutic antibody (anti-therapeutic antibodies). More specifically, enhanced FcγRIIa binding will increase the risk of generating antibodies against the therapeutic antibody, and this will significantly reduce its value as a drug. In addition, it has been proposed that FcγRIIb on dendritic cells contributes to peripheral tolerance by inhibiting dendritic cell activation caused by immune complexes formed between antigens and antibodies, or by inhibiting antigen presentation to T cells via activated Fcγ receptors (see, e.g., NPL65). Since FcγRIIa is also expressed on dendritic cells, when an antibody with an Fc that has enhanced selective binding to FcγRIIb is used as a drug, due to the enhanced selective binding to FcγRIIb, the antigen is not easily presented by dendritic cells, etc., and the risk of anti-drug antibody production can be relatively reduced. Such antibodies are also useful in this regard.

[0026] More specifically, when FcγRIIa binding is enhanced, the risk of thrombus formation via platelet aggregation increases and the risk of anti-therapeutic antibody production due to increased immunogenicity increases, and the value as a drug will significantly decline.

[0027] From this perspective, the aforementioned Fc variant with enhanced FcγRIIb binding shows significantly enhanced R-type FcγRIIa binding compared to naturally occurring IgG1. Therefore, its value as a drug for patients with R-type FcγRIIa is significantly reduced. H-type and R-type FcγRIIa are observed at approximately the same frequency in Caucasians and African Americans (see, e.g., NPL66 and NPL67). Therefore, when this Fc variant is used to treat autoimmune diseases, the number of patients who can safely use it while enjoying its drug effects will be limited.

[0028] In addition, in dendritic cells lacking FcγRIIb or in dendritic cells in which the interaction between FcγRIIb and the Fc portion of an antibody is inhibited by an anti-FcγRIIb antibody, the dendritic cells have been reported to be mature (see, for example, NPL68 and NPL69). This report suggests that FcγRIIb actively inhibits the maturation of dendritic cells in a steady state (no inflammation, etc. and no activation). In addition to FcγRIIb, FcγRIIa is also expressed on the surface of dendritic cells; thus, even if the binding to inhibitory FcγRIIb is enhanced and if the binding to activating FcγRs such as FcγRIIa is also enhanced, the maturation of dendritic cells can still be promoted thereby. More specifically, it is considered important in providing an antibody having an immunosuppressive effect not only to increase the FcγRIIb-binding activity but also to increase the ratio of the FcγRIIb-binding activity to the FcγRIIa-binding activity.

[0029] Therefore, when considering the production of a drug utilizing the immunosuppressive effect mediated by FcγRIIb binding, there is a need for such Fc variants that not only have enhanced FcγRIIb-binding activity but also can bind to both allotypes of FcγRIIa, the H type and the R type (maintained at a level similar to that of naturally occurring IgG1 or reduced to a lower level compared to naturally occurring IgG1).

[0030] Meanwhile, examples have been reported in which amino acid alterations are introduced into the Fc region to increase FcγRIIb-binding selectivity (see, for example, NPL70). However, all of the variants said to have improved FcγRIIb selectivity reported in this document show reduced FcγRIIb binding compared to naturally occurring IgG1. Therefore, it is considered that these variants are actually difficult to induce a stronger FcγRIIb-mediated immunosuppressive response than IgG1.

[0031] In addition, since FcγRIIb plays an important role in the agonist antibodies mentioned above, it is expected that enhancing its binding activity will enhance the agonist activity. However, when the FcγRIIa binding is similarly enhanced, unwanted activities such as ADCC activity and ADCP activity will be shown, and this may cause side effects. Also, from this perspective, it is preferable to be able to selectively enhance the FcγRIIb-binding activity.

[0032] From these results, in the preparation of therapeutic antibodies utilizing FcγRIIb for the treatment of autoimmune diseases and cancers, it is important that the activities of binding to both FcγRIIa allotypes are maintained or decreased compared to naturally occurring IgG, and that FcγRIIb binding is enhanced. However, the extracellular region of FcγRIIb has 93% sequence identity with the extracellular region of FcγRIIa, which is one of the activating FcγRs, and they are very similar in structure. There are allotypes of FcγRIIa, the H type and the R type, where the amino acid at position 131 is His (H type) or Arg (R type), and their reactions with antibodies are different (see, for example, NPL71). Therefore, a difficult problem may be to prepare Fc region variants with enhanced selective FcγRIIb binding compared to each allotype of FcγRIIa, which involves distinguishing sequences with high homology between FcγRIIa and FcγRIIb. Despite the said difficulties, several Fc region variants with selective binding activity to FcγRIIb compared to FcγRIIa have currently been identified through a comprehensive amino acid modification analysis in the Fc region (see, for example, PTL16, PTL17, PTL18, PTL19, and PTL20).

[0033] There have currently been reports on Fc region variants with binding selectivity for FcγRIIb related to human FcγRs, while there have been no reports on Fc region variants with binding selectivity for FcγRIIb related to monkey FcγRs. Due to the absence of such Fc variants, the effects of Fc variant selective binding to FcγRIIb have not been thoroughly tested in monkeys.

[0034] In addition to the above, it has been reported that by changing the charge of amino acid residues that may be exposed on the surface of the antibody to increase or decrease the isoelectric point (pI) of the antibody, it may be possible to regulate the half-life of the antibody in the blood (see, for example, PTL21 and PTL22). It has been shown that it may be possible to extend the plasma half-life of the antibody by decreasing the pI of the antibody, and vice versa.

[0035] Furthermore, it has been reported that by specifically changing the charge of designated amino acid residues in its CH3 domain to increase the pI of the antibody, antigen binding to cells can be promoted (see, for example, PTL23). In addition, it has been reported that by changing the charge of amino acid residues in the constant region of the antibody (mainly the CH1 domain) to decrease the pI, the half-life of the antibody in the plasma can be extended (see, for example, PTL24).

[0036] Citation List

[0037] Patent Documents

[0038] [PTL1] U.S. Patent No. 5,827,733

[0039] [PTL2] WO 1994 / 021681

[0040] [PTL3] U.S. Patent No. 6,096,506

[0041] [PTL4] U.S. Patent No. 7,261,893

[0042] [PTL5] U.S. Patent No. 7,320,789

[0043] [PTL6] U.S. Patent No. 7,807,159

[0044] [PTL7] U.S. Patent No. 7,888,486

[0045] [PTL8] WO 2005 / 094446

[0046] [PTL9] WO 2007 / 047112

[0047] [PTL10] WO 2010 / 070094

[0048] [PTL11] WO 2000 / 042072

[0049] [PTL12] WO 2006 / 019447

[0050] [PTL13] WO 2004 / 099249

[0051] [PTL14] WO 2004 / 029207

[0052] [PTL15] U.S. Application Publication No. US2009 / 0136485

[0053] [PTL16] WO 2012 / 115241

[0054] [PTL17] WO 2013 / 047752

[0055] [PTL18] WO 2013 / 125667

[0056] [PTL19] WO 2014 / 030728

[0057] [PTL20] WO 2014 / 163101

[0058] [PTL21] WO 2007 / 114319

[0059] [PTL22] WO 2009 / 041643

[0060] [PTL23] WO 2014 / 145159

[0061] [PTL24] WO 2012 / 016227

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[0130] [NPL68] Boruchov et al., J. Clin. Invest. 115:2914-2923 (2005)

[0131] [NPL69] Dhodapkar et al., Proc. Natl. Acad. Sci. USA 102:2910-2915 (2005)

[0132] [NPL70] Armour et al., Mol. Immunol. 40:585-593 (2003)

[0133] [NPL71] Warmerdam et al., J. Exp. Med. 172:19-25 (1990) Summary of the Invention

[0134] The present invention provides anti-myostatin antibodies and methods of using the same. The present invention also provides proteins comprising variant Fc regions and methods of using the same.

[0135] In some embodiments, the isolated anti - myostatin antibody of the invention binds latent myostatin. In additional embodiments, the antibody binds 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 invention inhibits the activation of myostatin. In additional embodiments, the antibody blocks the release of mature myostatin from latent myostatin. In additional embodiments, the antibody blocks the proteolytic release of mature myostatin. In additional embodiments, the antibody blocks the spontaneous release of mature myostatin. In additional embodiments, the antibody does not bind mature myostatin. In additional embodiments, the antibody binds the same epitope as the antibodies described in Table 13. In additional embodiments, the antibody binds the same epitope as an antibody comprising the VH and VL pair described in Table 13. In additional embodiments, the antibody binds the same epitope as the antibodies described in Table 2a. In additional embodiments, the antibody binds the same epitope as an antibody comprising the VH and VL pair described in Table 2a. In additional embodiments, the antibody binds the same epitope as the antibodies described in Table 11a. In additional embodiments, the antibody binds the same epitope as an antibody comprising the VH and VL pair described in Table 11a. In additional embodiments, the antibody binds the same epitope as the antibodies described in Table 2a, 11a, or 13. In additional embodiments, the antibody binds the same epitope as an antibody comprising the VH and VL pair described in Table 2a, 11a, or 13.

[0136] In some embodiments, the isolated anti - myostatin antibody of the invention binds latent myostatin with higher affinity at neutral pH than at acidic pH. In some embodiments, the anti - myostatin antibody binds latent myostatin with higher affinity at pH 7.4 than at pH 5.8. In some embodiments, the isolated anti - myostatin antibody of the invention binds a polypeptide fragment consisting of amino acids 21 - 100 of myostatin propeptide (SEQ ID NO: 78) with higher affinity at pH 7.4 than at pH 5.8. In some embodiments, the antibody binds the same myostatin epitope as the antibodies described in Table 13 with higher affinity at neutral pH than at acidic pH. In additional embodiments, the anti - myostatin antibody binds the same epitope as the antibodies described in Table 13 with higher affinity at pH 7.4 than at pH 5.8. In additional embodiments, the antibody binds the same epitope as the antibody comprising the VH and VL pair described in Table 13 with higher affinity at pH 7.4 than at pH 5.8. In some embodiments, the antibody binds the same myostatin epitope as the antibodies described in Table 2a with higher affinity at neutral pH than at acidic pH. In some embodiments, the antibody binds the same myostatin epitope as the antibodies described in Table 2a with higher affinity at pH 7.4 than at pH 5.8. In additional embodiments, the antibody binds the same epitope as the antibody comprising the VH and VL pair described in Table 2a with higher affinity at pH 7.4 than at pH 5.8. In additional embodiments, the anti - myostatin antibody binds the same epitope as the antibody described in Table 11a with higher affinity at neutral pH than at acidic pH. In additional embodiments, the antibody binds the same myostatin epitope as the antibody described in Table 11a with higher affinity at pH 7.4 than at pH 5.8. In additional embodiments, the antibody binds the same epitope as the antibody comprising the VH and VL pair described in Table 11a with higher affinity at pH 7.4 than at pH 5.8. In additional embodiments, the anti - myostatin antibody binds the same epitope as the antibodies described in Table 2a, 11a, or 13 with higher affinity at neutral pH than at acidic pH. In additional embodiments, the antibody binds the same myostatin epitope as the antibodies described in Table 2a, 11a, or 13 with higher affinity at pH 7.4 than at pH 5.8. In additional embodiments, the antibody binds the same epitope as the antibody comprising the VH and VL pair described in Table 2a, 11a, or 13 with higher affinity at pH 7.4 than at pH 5.8.

[0137] In some embodiments, the isolated anti - myostatin antibody of the invention competes with the antibodies provided herein for binding to latent myostatin. In some embodiments, the isolated anti - myostatin antibody of the invention competes with the antibodies described in Table 13 for binding to latent myostatin. In some embodiments, the isolated anti - myostatin antibody of the invention competes with an antibody comprising the VH and VL pair described in Table 13 for binding to latent myostatin. In some embodiments, the antibody competes with the antibodies described in Table 2a for binding to latent myostatin. In some embodiments, the isolated anti - myostatin antibody of the invention competes with an antibody comprising the VH and VL pair described in Table 2a for binding to latent myostatin. In some embodiments, the antibody competes with the antibodies described in Table 11a for binding to latent myostatin. In some embodiments, the isolated anti - myostatin antibody of the invention competes with an antibody comprising the VH and VL pair described in Table 11a for binding to latent myostatin. In additional embodiments, the anti - myostatin antibody competes with the antibodies described in Table 2a, 11a, or 13 for binding to latent myostatin. In additional embodiments, the anti - myostatin antibody competes with an antibody comprising the VH and VL pair described in Table 2a, 11a, or 13 for binding to latent myostatin. In additional embodiments, the anti - myostatin antibody has a higher affinity for binding to latent myostatin at neutral pH than at acidic pH. In additional embodiments, the anti - myostatin antibody has a higher affinity for binding to latent myostatin at pH 7.4 than at pH 5.8. In additional embodiments, the anti - myostatin antibody has a higher affinity for binding to a polypeptide fragment consisting of amino acids 21 - 100 of myostatin propeptide (SEQ ID NO: 78) 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 are known in the art.

[0138] In some embodiments, the isolated anti-myostatin antibody of the invention is a monoclonal antibody. In some embodiments, the isolated anti-myostatin antibody of the invention is a human, humanized or chimeric antibody. In some embodiments, the isolated anti-myostatin antibody of the invention is an antibody fragment that binds to myostatin. In some embodiments, the isolated anti-myostatin antibody of the invention is an antibody fragment that binds to latent myostatin. In some embodiments, the isolated anti-myostatin antibody of the invention is an antibody fragment that binds to a polypeptide 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 invention is a full-length IgG antibody.

[0139] In some embodiments, the anti-myostatin antibody of the invention comprises:

[0140] (a) (i) HVR-H3, which comprises the amino acid sequence GVPAX1SX2GGDX3, where X1 is Y or H, X2 is T or H, and X3 is L or K (SEQ ID NO: 128), (ii) HVR-L3, which comprises the amino acid sequence AGGYGGGX1YA, where X1 is L or R (SEQ ID NO: 131), and (iii) HVR-H2, which comprises the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7, where X1 is Y or H, X2 is S or K, X3 is T, M or K, X4 is Y or K, X5 is A, M or E, X6 is S or E, and X7 is G or K (SEQ ID NO: 127);

[0141] (b) (i) HVR-H1, which comprises the amino acid sequence X1X2DIS, where X1 is S or H, and X2 is Y, T, D or E (SEQ ID NO: 126), (ii) HVR-H2, which comprises the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7, where X1 is Y or H, X2 is S or K, X3 is T, M or K, X4 is Y or K, X5 is A, M or E, X6 is S or E, and X7 is G or K (SEQ ID NO: 127), and (iii) HVR-H3, which comprises the amino acid sequence GVPAX1SX2GGDX3, where X1 is Y or H, X2 is T or H, and X3 is L or K (SEQ ID NO: 128);

[0142] (c) (i) HVR-H1, wherein the HVR-H1 comprises the amino acid sequence X1X2DIS, where X1 is S or H and X2 is Y, T, D or E (SEQ ID NO: 126); (ii) HVR-H2, wherein the HVR-H2 comprises the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7, where X1 is Y or H, X2 is S or K, X3 is T, M or K, X4 is Y or K, X5 is A, M or E, X6 is S or E, and X7 is G or K (SEQ ID NO: 127); (iii) HVR-H3, wherein the HVR-H3 comprises the amino acid sequence GVPAX1SX2GGDX3, where X1 is Y or H, X2 is T or H, and X3 is L or K (SEQ ID NO: 128); (iv) HVR-L1, wherein the HVR-L1 comprises the amino acid sequence X1X2SQX3VX4X5X6NWLS, where X1 is Q or T, X2 is S or T, X3 is S or E, X4 is Y or F, X5 is D or H, and X6 is N, D, A or E (SEQ ID NO: 129); (v) HVR-L2, wherein the HVR-L2 comprises the amino acid sequence WAX1TLAX2, where X1 is S or E and X2 is S, Y, F or W (SEQ ID NO: 130); and (vi) HVR-L3, wherein the HVR-L3 comprises the amino acid sequence AGGYGGGX1YA, where X1 is L or R (SEQ ID NO: 131);

[0143] (d) (i) HVR-L1, wherein the HVR-L1 comprises the amino acid sequence X1X2SQX3VX4X5X6NWLS, where X1 is Q or T, X2 is S or T, X3 is S or E, X4 is Y or F, X5 is D or H, and X6 is N, D, A, or E (SEQ ID NO: 129); (ii) HVR-L2, wherein the HVR-L2 comprises the amino acid sequence WAX1TLAX2, where X1 is S or E and X2 is S, Y, F, or W (SEQ ID NO: 130); and (iii) HVR-L3, wherein the HVR-L3 comprises the amino acid sequence AGGYGGGX1YA, where 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, wherein the FR1 comprises the amino acid sequence of any one of SEQ ID NOs: 132 - 134; FR2, wherein the FR2 comprises the amino acid sequence of any one of SEQ ID NOs: 135 - 136; FR3, wherein the FR3 comprises the amino acid sequence of SEQ ID NO: 137; and FR4, wherein the FR4 comprises 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, wherein the FR1 comprises the amino acid sequence of SEQ ID NO: 139; FR2, wherein the FR2 comprises the amino acid sequence of any one of SEQ ID NOs: 140 - 141; FR3, wherein the FR3 comprises the amino acid sequence of any one of SEQ ID NOs: 142 - 143; and FR4, wherein the FR4 comprises the amino acid sequence of SEQ ID NO: 144.

[0144] In some embodiments, the isolated anti - myostatin antibody of the invention comprises (a) HVR-H3, wherein the HVR-H3 comprises the amino acid sequence GVPAX1SX2GGDX3, where X1 is Y or H, X2 is T or H, and X3 is L or K (SEQ ID NO: 128), (b) HVR-L3, wherein the HVR-L3 comprises the amino acid sequence AGGYGGGX1YA, where X1 is L or R (SEQ ID NO: 131), and (c) HVR-H2, wherein the HVR-H2 comprises the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7, where X1 is Y or H, X2 is S or K, X3 is T, M, or K, X4 is Y or K, X5 is A, M, or E, X6 is S or E, and X7 is G or K (SEQ ID NO: 127).

[0145] In some embodiments, the isolated anti - myostatin antibody of the invention comprises (a) HVR - H1, wherein HVR - H1 comprises the amino acid sequence X1X2DIS, where X1 is S or H, and X2 is Y, T, D or E (SEQ ID NO: 126); (b) HVR - H2, wherein HVR - H2 comprises the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7, where X1 is Y or H, X2 is S or K, X3 is T, M or K, X4 is Y or K, X5 is A, M or E, X6 is S or E, and X7 is G or K (SEQ ID NO: 127); and (c) HVR - H3, wherein HVR - H3 comprises the amino acid sequence GVPAX1SX2GGDX3, where X1 is Y or H, X2 is T or H, and X3 is L or K (SEQ ID NO: 128). In additional embodiments, the antibody comprises a heavy chain variable domain framework FR1, wherein FR1 comprises the amino acid sequence of any one of SEQ ID NOs: 132 - 134; FR2, wherein FR2 comprises the amino acid sequence of any one of SEQ ID NOs: 135 - 136; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 137; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: 138. In additional embodiments, the antibody further comprises (a) HVR - L1, wherein HVR - L1 comprises the amino acid sequence X1X2SQX3VX4X5X6NWLS, where X1 is Q or T, X2 is S or T, X3 is S or E, X4 is Y or F, X5 is D or H, and X6 is N, D, A or E (SEQ ID NO: 129); (b) HVR - L2, wherein HVR - L2 comprises the amino acid sequence WAX1TLAX2, where X1 is S or E, and X2 is S, Y, F or W (SEQ ID NO: 130); and (c) HVR - L3, wherein HVR - L3 comprises the amino acid sequence AGGYGGGX1YA, where X1 is L or R (SEQ ID NO: 131).

[0146] In some embodiments, the isolated anti - myostatin antibody of the invention comprises (a) HVR - L1, which comprises the amino acid sequence X1X2SQX3VX4X5X6NWLS, where X1 is Q or T, X2 is S or T, X3 is S or E, X4 is Y or F, X5 is D or H, and X6 is N, D, A or E (SEQ ID NO: 129); (b) HVR - L2, which comprises the amino acid sequence WAX1TLAX2, where X1 is S or E, and X2 is S, Y, F or W (SEQ ID NO: 130); and (c) HVR - L3, which comprises the amino acid sequence AGGYGGGX1YA, where X1 is L or R (SEQ ID NO: 131). In another embodiment, the antibody further comprises: a light chain variable domain framework FR1, which comprises the amino acid sequence of SEQ ID NO: 139; FR2, which comprises the amino acid sequence of any one of SEQ ID NOs: 140 - 141; FR3, which comprises the amino acid sequence of any one of SEQ ID NOs: 142 - 143; and FR4, which comprises the amino acid sequence of SEQ ID NO: 144.

[0147] In some embodiments, the isolated anti - myostatin antibody of the invention comprises a heavy chain variable domain framework FR1, which comprises the amino acid sequence of any one of SEQ ID NOs: 132 - 134; FR2, which comprises the amino acid sequence of any one of SEQ ID NOs: 135 - 136; FR3, which comprises the amino acid sequence of SEQ ID NO: 137; and FR4, which comprises the amino acid sequence of SEQ ID NO: 138. In some embodiments, the isolated anti - myostatin antibody of the invention comprises a light chain variable domain framework FR1, which comprises the amino acid sequence of SEQ ID NO: 139; FR2, which comprises the amino acid sequence of any one of SEQ ID NOs: 140 - 141; FR3, which comprises the amino acid sequence of any one of SEQ ID NOs: 142 - 143; and FR4, which comprises the amino acid sequence of SEQ ID NO: 144.

[0148] In some embodiments, the isolated anti - myostatin antibody of the invention comprises (a) a VH sequence having at least 95% sequence identity with 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 with the amino acid sequence of any one of SEQ ID NOs: 15, 31, 35 - 38, and 96 - 99; or (c) the VH sequence in (a) and the VL sequence in (b). In additional embodiments, the antibody comprises a VH sequence of any one of SEQ ID NOs: 13, 16 - 30, 32 - 34, and 86 - 95. In additional embodiments, the antibody comprises a VL sequence of any one of SEQ ID NOs: 15, 31, 35 - 38, and 96 - 99. In some embodiments, the antibody comprises a VH sequence of any one of SEQ ID NOs: 13, 16 - 30, 32 - 34, and 86 - 95. In additional embodiments, 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.

[0149] The invention also provides an isolated nucleic acid encoding the anti - myostatin antibody of the invention. The invention also provides a host cell comprising the nucleic acid of the invention. The invention also provides a method for preparing the antibody, the method comprising culturing the host cell of the invention to prepare the antibody.

[0150] In some aspects, the invention provides a method for preparing an anti - myostatin antibody, the method comprising: (a) culturing the host cell of the invention to prepare the antibody; or (b) immunizing an animal with a polypeptide, wherein the polypeptide comprises a region corresponding to the amino acids at positions 21 to 100 of the myostatin propeptide (SEQ ID NO: 78).

[0151] The invention also provides a method for preparing an anti - myostatin antibody. In some embodiments, the method comprises immunizing an animal with a polypeptide, wherein the polypeptide comprises a region corresponding to the amino acids at positions 21 - 100 of the myostatin propeptide (SEQ ID NO: 78).

[0152] The invention also provides a pharmaceutical formulation comprising the anti - myostatin antibody of the invention and a pharmaceutically acceptable carrier.

[0153] The invention provides a polypeptide comprising a variant Fc region, and methods for its preparation and use.

[0154] In one embodiment, the present invention provides an FcγRIIB-binding polypeptide comprising a variant Fc region and methods of using the same. In some embodiments, the variant Fc region having enhanced FcγRIIb-binding activity comprises at least one amino acid alteration in the parental Fc region. In additional embodiments, the ratio of [KD value of the parental Fc region for simian FcγRIIb] / [KD value of the variant Fc region for simian FcγRIIb] is 2.0 or greater. In additional embodiments, the ratio of [KD value of the parental Fc region for simian FcγRIIIa] / [KD value of the variant Fc region for simian FcγRIIIa] is 0.5 or less. In additional embodiments, the ratio of [KD value of the parental Fc region for human FcγRIIb] / [KD value of the variant Fc region for human FcγRIIb] is 2.0 or greater. In additional embodiments, the ratio of [KD value of the parental Fc region for human FcγRIIIa] / [KD value of the variant Fc region for human FcγRIIIa] is 0.5 or less. In additional embodiments, the ratio of [KD value of the parental Fc region for human FcγRIIa (H type)] / [KD value of the variant Fc region for human FcγRIIa (H type)] is 5.0 or less. In additional embodiments, the ratio of [KD value of the parental Fc region for human FcγRIIa (R type)] / [KD value of the variant Fc region for human FcγRIIa (R type)] is 5.0 or less. In another embodiment, the KD value of the variant Fc region for simian FcγRIIb is 1.0x10 -6 M or less. In another embodiment, the KD value of the variant Fc region for simian FcγRIIIa is 5.0x10 -7 M or greater. In another embodiment, the KD value of the variant Fc region for human FcγRIIb is 2.0x10 -6 M or less. In another embodiment, the KD value of the variant Fc region for human FcγRIIIa is 1.0x10 -6 M or greater. In another embodiment, the KD value of the variant Fc region for human FcγRIIa (H type) is 1.0x10 -7 M or greater. In another embodiment, the KD value of the variant Fc region for human FcγRIIa (R type) is 2.0x10 -7 M or greater.

[0155] In some embodiments, the variant Fc region of the present invention having enhanced FcγRIIb-binding activity comprises at least one amino acid alteration at at least one position selected from the group consisting of: 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 (according to EU numbering).

[0156] In further embodiments, the variant Fc region having enhanced FcγRIIb-binding activity comprises at least two amino acid alterations, said at least two amino acid alterations comprising: (a) one amino acid alteration at position 236, and (b) at least one amino acid alteration 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 (according to EU numbering).

[0157] In further embodiments, the variant Fc region having enhanced FcγRIIb-binding activity comprises at least two amino acid alterations, said at least two amino acid alterations comprising: (a) one amino acid alteration at position 236, and (b) at least one amino acid alteration at at least one position selected from the group consisting of: 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 (according to EU numbering).

[0158] In further embodiments, the variant Fc region having enhanced FcγRIIb-binding activity comprises at least two amino acid alterations, said at least two amino acid alterations comprising: (a) one amino acid alteration at position 236, and (b) at least one amino acid alteration at at least one position selected from the group consisting of: 231, 232, 235, 239, 268, 295, 298, 326, 330, and 396 (according to EU numbering).

[0159] In additional embodiments, a variant Fc region having enhanced FcγRIIb-binding activity comprises at least two amino acid alterations, said at least two amino acid alterations comprising: (a) one amino acid alteration at position 236, and (b) at least one amino acid alteration at at least one position selected from the group consisting of 268, 295, 326, and 330 (according to EU numbering).

[0160] In some embodiments, a variant Fc region of the invention having 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, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr 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, 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) Thr at position 326; (s) Ile, Asn at position 327; (t) Thr at position 328; (u) Lys, Arg at position 330; (v) Glu at position 331; (w) Asp at position 332; (x) Asp, Ile, Met, Val, Tyr at position 334; and (y) Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 396 (according to EU numbering).

[0161] In additional embodiments, a variant Fc region having 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 (according to EU numbering).

[0162] In another embodiment, the present invention provides polypeptides comprising a variant Fc region having an increased isoelectric point (pI) and methods of using the same. In some embodiments, a polypeptide comprising a variant Fc region having an increased pI comprises at least two amino acid alterations in the parental Fc region. In additional embodiments, each said amino acid alteration increases the isoelectric point (pI) of the variant Fc region as compared to the parental Fc region. In additional embodiments, the amino acids may be exposed on the surface of the variant Fc region. In additional embodiments, the polypeptide comprises a variant Fc region and an antigen-binding domain. In additional embodiments, the antigen-binding activity of the antigen-binding domain varies according to ionic concentration conditions. In additional embodiments, the variant Fc region having an increased pI of the present invention comprises at least two amino acid alterations at at least two positions selected from the group consisting of: 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422, and 431 (according to EU numbering). In additional embodiments, the variant Fc region having an increased pI comprises Arg or Lys at each selected position.

[0163] In some embodiments, the variant Fc region of the present invention comprises the amino acid alterations described in Table 14-30.

[0164] In some embodiments, a polypeptide comprises a variant Fc region of the present invention. In additional embodiments, the parental Fc region is derived from human IgG1. In additional embodiments, the polypeptide is an antibody. In additional embodiments, the polypeptide is an Fc fusion protein.

[0165] The present invention provides a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 229-381.

[0166] The present invention also provides an isolated nucleic acid encoding a polypeptide comprising a variant Fc region of the present invention. The present invention also provides a host cell comprising the nucleic acid of the present invention. The present invention also provides a method for preparing a polypeptide comprising a variant Fc region, the method comprising culturing the host of the present invention to produce the polypeptide.

[0167] The present invention also provides a pharmaceutical formulation comprising a polypeptide containing the variant Fc region of the present invention and a pharmaceutically acceptable carrier.

[0168] Specifically, the present invention relates to:

[0169] [1] An isolated antibody that binds latent myostatin, wherein the antibody inhibits the activation of myostatin.

[0170] [2] The antibody of [1], wherein the antibody:

[0171] (a) Blocks the release of mature myostatin from latent myostatin;

[0172] (b) Blocks the proteolytic release of mature myostatin;

[0173] (c) Blocks the spontaneous release of mature myostatin; or

[0174] (d) Does not bind mature myostatin; or binds to an epitope within a fragment consisting of amino acids 21 - 100 of the myostatin propeptide (SEQ ID NO: 78).

[0175] [3] The antibody of [1] or [2], wherein the antibody competes with an antibody comprising the VH and VL pairs described in Table 2a, 11a or 13 for binding to latent myostatin, or binds to the same epitope as an antibody comprising the VH and VL pairs described in Table 2a, 11a or 13.

[0176] [4] The antibody of any one of [1] to [3], which has a higher affinity for binding latent myostatin at neutral pH than at acidic pH, or has a higher affinity for binding latent myostatin at pH 7.4 than at pH 5.8.

[0177] [5] The antibody of any one of [1] to [4], which 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 myostatin.

[0178] [6] The antibody of any one of [1] to [5], wherein the antibody comprises:

[0179] (a) (i) HVR-H3, wherein the HVR-H3 comprises the amino acid sequence GVPAX1SX2GGDX3, where X1 is Y or H, X2 is T or H, and X3 is L or K (SEQ ID NO: 128); (ii) HVR-L3, wherein the HVR-L3 comprises the amino acid sequence AGGYGGGX1YA, where X1 is L or R (SEQ ID NO: 131); and (iii) HVR-H2, wherein the HVR-H2 comprises the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7, where X1 is Y or H, X2 is S or K, X3 is T, M or K, X4 is Y or K, X5 is A, M or E, X6 is S or E, and X7 is G or K (SEQ ID NO: 127);

[0180] (b) (i) HVR-H1, wherein the HVR-H1 comprises the amino acid sequence X1X2DIS, where X1 is S or H and X2 is Y, T, D or E (SEQ ID NO: 126); (ii) HVR-H2, wherein the HVR-H2 comprises the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7, where X1 is Y or H, X2 is S or K, X3 is T, M or K, X4 is Y or K, X5 is A, M or E, X6 is S or E, and X7 is G or K (SEQ ID NO: 127); and (iii) HVR-H3, wherein the HVR-H3 comprises the amino acid sequence GVPAX1SX2GGDX3, where X1 is Y or H, X2 is T or H, and X3 is L or K (SEQ ID NO: 128);

[0181] (c) (i) HVR-H1, wherein HVR-H1 comprises the amino acid sequence X1X2DIS, where X1 is S or H and X2 is Y, T, D or E (SEQ ID NO: 126); (ii) HVR-H2, wherein HVR-H2 comprises the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7, where X1 is Y or H, X2 is S or K, X3 is T, M or K, X4 is Y or K, X5 is A, M or E, X6 is S or E, and X7 is G or K (SEQ ID NO: 127); (iii) HVR-H3, wherein HVR-H3 comprises the amino acid sequence GVPAX1SX2GGDX3, where X1 is Y or H, X2 is T or H, and X3 is L or K (SEQ ID NO: 128); (iv) HVR-L1, wherein HVR-L1 comprises the amino acid sequence X1X2SQX3VX4X5X6NWLS, where X1 is Q or T, X2 is S or T, X3 is S or E, X4 is Y or F, X5 is D or H, and X6 is N, D, A or E (SEQ ID NO: 129); (v) HVR-L2, wherein HVR-L2 comprises the amino acid sequence WAX1TLAX2, where X1 is S or E and X2 is S, Y, F or W (SEQ ID NO: 130); and (vi) HVR-L3, wherein HVR-L3 comprises the amino acid sequence AGGYGGGX1YA, where X1 is L or R (SEQ ID NO: 131); or

[0182] (d) (i) HVR-L1, wherein HVR-L1 comprises the amino acid sequence X1X2SQX3VX4X5X6NWLS, where X1 is Q or T, X2 is S or T, X3 is S or E, X4 is Y or F, X5 is D or H, and X6 is N, D, A or E (SEQ ID NO: 129); (ii) HVR-L2, wherein HVR-L2 comprises the amino acid sequence WAX1TLAX2, where X1 is S or E and X2 is S, Y, F or W (SEQ ID NO:130); and (iii) HVR-L3, wherein HVR-L3 comprises the amino acid sequence AGGYGGGX1YA, where X1 is L or R (SEQ IDNO: 131).

[0183] [7] An antibody of [6](b), which further comprises a heavy chain variable domain framework FR1, said FR1 comprising the amino acid sequence of any one of SEQ ID NO: 132 - 134; FR2, said FR2 comprising the amino acid sequence of any one of SEQ ID NO: 135 - 136; FR3, said FR3 comprising the amino acid sequence of SEQ ID NO: 137; and FR4, said FR4 comprising the amino acid sequence of SEQ ID NO: 138.

[0184] [8] An antibody of [6](d), further comprising a light chain variable domain framework FR1, said FR1 comprising the amino acid sequence of SEQ ID NO: 139; FR2, said FR2 comprising the amino acid sequence of any one of SEQ ID NO: 140 - 141; FR3, said FR3 comprising the amino acid sequence of any one of SEQ ID NO: 142 - 143; and FR4, said FR4 comprising the amino acid sequence of SEQ ID NO: 144.

[0185] [9] An antibody of any one of [1] to [5], which comprises (a) a VH sequence having at least 95% sequence identity with the amino acid sequence of any one of SEQ ID NO: 13, 16 - 30, 32 - 34 and 86 - 95; (b) a VL sequence having at least 95% sequence identity with the amino acid sequence of any one of SEQ ID NO: 15, 31, 35 - 38 and 96 - 99; or (c) a VH sequence of any one of SEQ ID NO: 13, 16 - 30, 32 - 34 and 86 - 95, and a VL sequence of any one of SEQ ID NO: 15, 31, 35 - 38 and 96 - 99.

[0186]

[10] An isolated nucleic acid encoding an antibody of any one of [1] to [9].

[0187]

[11] A host cell comprising the nucleic acid of

[10] .

[0188]

[12] A method for preparing an anti - myostatin antibody, the method comprising:

[0189] (a) culturing the host cell of

[11] to prepare the antibody; or

[0190] (b) immunizing an animal with a polypeptide, wherein the polypeptide comprises a region corresponding to the amino acids at positions 21 to 100 of the myostatin propeptide (SEQ ID NO: 78).

[0191]

[13] A pharmaceutical preparation comprising an antibody of any one of [1] to [9] and a pharmaceutical carrier.

[0192]

[14] A polypeptide, the polypeptide comprising a variant Fc region, the variant Fc region comprising an amino acid alteration in at least one parental Fc region, wherein the ratio of [the KD value of the parental Fc region for simian FcγRIIb] / [the KD value of the variant Fc region for simian FcγRIIb] is 2.0 or greater, and the ratio of [the KD value of the parental Fc region for simian FcγRIIIa] / [the KD value of the variant Fc region for simian FcγRIIIa] is 0.5 or less.

[0193]

[15] The polypeptide of

[14] , wherein further, the ratio of [the KD value of the parental Fc region for human FcγRIIb] / [the KD value of the variant Fc region for human FcγRIIb] is 2.0 or greater, and the ratio of [the KD value of the parental Fc region for human FcγRIIIa] / [the KD value of the variant Fc region for human FcγRIIIa] is 0.5 or less.

[0194]

[16] The polypeptide of

[15] , wherein further, the ratio of [the KD value of the parental Fc region for human FcγRIIa (H type)] / [the KD value of the variant Fc region for human FcγRIIa (H type)] is 5.0 or less.

[0195]

[17] The polypeptide of

[16] , wherein further, the ratio of [the KD value of the parental Fc region for human FcγRIIa (R type)] / [the KD value of the variant Fc region for human FcγRIIa (R type)] is 5.0 or less.

[0196]

[18] The polypeptide of

[14] , wherein the KD value of the variant Fc region for simian FcγRIIb is 1.0x10 -6 M or less, and the KD value of the variant Fc region for simian FcγRIIIa is 5.0x10 -7 M or greater.

[0197]

[19] The polypeptide of

[15] , wherein the KD value of the variant Fc region for human FcγRIIb is 2.0x10 -6 M or less, and the KD value of the variant Fc region for human FcγRIIIa is 1.0x10 -6 M or greater.

[0198]

[20] The polypeptide of

[16] , wherein the KD value of the variant Fc region for human FcγRIIa (H type) is 1.0x10 -7 M or greater.

[0199]

[21] The polypeptide of

[17] , wherein the KD value of the variant Fc region for human FcγRIIa (R type) is 2.0x10 -7 M or greater.

[0200]

[22] A polypeptide according to any one of

[14] to

[21] , wherein the variant Fc region comprises at least one amino acid alteration at at least one position selected from the group consisting of: 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 (according to EU numbering).

[0201]

[23] The polypeptide of

[22] , wherein the variant Fc region comprises at least two amino acid alterations, said at least two amino acid alterations comprising:

[0202] (a) One amino acid alteration at position 236, and

[0203] (b) At least one amino acid alteration at at least one position selected from the group consisting of:

[0204] (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;

[0205] (ii) Positions 231, 232, 235, 239, 268, 295, 298, 326, 330, and 396; or

[0206] (iii) Positions 268, 295, 326, and 330; (according to EU numbering).

[0207]

[24] The polypeptide of

[22] or

[23] , wherein the variant Fc region comprises at least one amino acid selected from the group consisting of:

[0208] (a) Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 231,

[0209] (b) Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 232,

[0210] (c) Asp at position 233,

[0211] (d) Trp and Tyr at position 234,

[0212] (e) Trp at position 235,

[0213] (f) Ala, Asp, Glu, His, Ile, Leu, Met, Asn, Gln, Ser, Thr, Val at position 236,

[0214] (g) Asp and Tyr at position 237,

[0215] (h) Glu, Ile, Met, Gln, Tyr at position 238,

[0216] (i) Ile, Leu, Asn, Pro, Val at position 239,

[0217] (j) Ile at position 264,

[0218] (k) Phe at position 266,

[0219] (l) Ala, His, Leu at position 267,

[0220] (m) Asp and Glu at position 268,

[0221] (n) Asp, Glu, Gly at position 271,

[0222] (o) Leu at position 295,

[0223] (p) Leu at position 298,

[0224] (q) Glu, Phe, Ile, Leu at position 325,

[0225] (r) Thr at position 326,

[0226] (s) Ile and Asn at position 327,

[0227] (t) Thr at position 328,

[0228] (u) Lys and Arg at position 330,

[0229] (v) Glu at position 331,

[0230] (w) Asp at position 332,

[0231] (x) Asp, Ile, Met, Val, Tyr at position 334, and

[0232] (y) Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 396; (according to EU numbering).

[0233]

[25] A polypeptide of

[24] , wherein said variant Fc region comprises at least one amino acid selected from the group consisting of:

[0234] (a) Gly, Thr at position 231,

[0235] (b) Asp at position 232,

[0236] (c) Trp at position 235,

[0237] (d) Asn, Thr at position 236,

[0238] (e) Val at position 239,

[0239] (f) Asp, Glu at position 268,

[0240] (g) Leu at position 295,

[0241] (h) Leu at position 298,

[0242] (i) Thr at position 326,

[0243] (j) Lys, Arg at position 330, and

[0244] (k) Lys, Met at position 396; (according to EU numbering).

[0245]

[26] A polypeptide, said polypeptide comprising a variant Fc region, said variant Fc region comprising at least two amino acid alterations in the parental Fc region, wherein each said amino acid alteration increases the isoelectric point (pI) of the variant Fc region compared to the parental Fc region.

[0246]

[27] A polypeptide of

[26] , wherein said amino acid alterations are exposed on the surface of the variant Fc region.

[0247]

[28] A polypeptide of

[26] or

[27] , which further comprises an antigen-binding domain.

[0248]

[29] A polypeptide of

[28] , wherein the antigen-binding activity of said antigen-binding domain is altered according to ionic concentration conditions.

[0249]

[30] A polypeptide according to any one of

[26] to

[29] , wherein the variant Fc region comprises at least two amino acid alterations at at least two positions selected from the group consisting of: 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422 and 431 (according to EU numbering).

[0250]

[31] The polypeptide of

[30] , wherein the variant Fc region comprises Arg or Lys at each selected position.

[0251]

[32] A polypeptide comprising a variant Fc region, the variant Fc region comprising the amino acid alterations described in Table 14 - 30.

[0252]

[33] A polypeptide according to any one of

[14] to

[32] , wherein the parental Fc region is derived from human IgG1.

[0253]

[34] A polypeptide according to any one of

[14] to

[33] , wherein the polypeptide is an antibody or an Fc fusion protein.

[0254]

[35] A polypeptide comprising an amino acid sequence of any one of SEQ ID NO: 229 - 381.

[0255]

[36] An isolated nucleic acid encoding a polypeptide according to any one of

[14] to

[35] .

[0256]

[37] A host cell comprising the nucleic acid of

[36] .

[0257]

[38] A method for preparing a polypeptide comprising a variant Fc region, the method comprising culturing the host cell of

[37] to prepare the polypeptide.

[0258]

[39] A pharmaceutical formulation comprising a polypeptide according to any one of

[14] to

[35] and a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0259] Figure 1

[0260] Figure 1 The figure shows that an anti - latent myostatin antibody inhibits the proteolytic activation of latent myostatin, as described in Example 3. In the presence of the anti - latent myostatin antibody, the activity of active myostatin released from latent myostatin by BMP1 protease was measured using the HEKBlue assay.

[0261] Figure 2 ​​​​

[0262] Figure 2 The figure shows that the anti - latent myostatin antibody inhibits the spontaneous activation of latent myostatin, as described in Example 4. In the presence of the anti - latent myostatin antibody, the activity of the active myostatin released from latent myostatin by incubation at 37°C was measured using the HEK Blue assay.

[0263] Figure 3

[0264] Figure 3 The figure shows the binding of the anti - latent myostatin antibody to the propeptide domain, as described in Example 5.

[0265] Figure 4

[0266] Figure 4 The figure shows the Western blot analysis of myostatin propeptide, as described in Example 6. The proteolytic cleavage of myostatin propeptide by BMP1 was measured in the presence and absence of the anti - latent myostatin antibody.

[0267] Figure 5

[0268] Figure 5 A - 5C shows the BIACORE (registered trademark) sensorgrams of the anti - latent myostatin antibody MST1032 - G1m for human latent myostatin (A), cynomolgus monkey latent myostatin (B), and mouse latent myostatin (C), as described in Example 7.

[0269] Figure 6

[0270] Figure 6 The figure shows that the humanized anti - latent myostatin antibody inhibits the proteolysis and spontaneous activation of latent myostatin, as described in Example 8. In the presence of the anti - latent myostatin antibody, the activity of the active myostatin released from latent myostatin by BMP1 protease (proteolysis) or by incubation at 37°C in the absence of BMP1 (spontaneous) was measured using the HEK Blue assay.

[0271] Figure 7

[0272] Figure 7 ​​​​​​​​​​Graph showing the BIACORE (registered trademark) sensorgram of a histidine-substituted variant of an anti-latent myostatin antibody, as described in Example 9. The antibody / antigen complex was allowed to dissociate at pH 7.4 and then further dissociated at pH 5.8 (indicated by the arrow) to evaluate pH-dependent interactions. The antibodies tested in this experiment were: Ab001 (solid black curve), Ab002 (short dashed black curve), Ab003 (dotted black curve), Ab004 (short dashed gray curve), Ab005 (solid gray curve), Ab006 (long dashed gray curve), and Ab007 (long dashed black curve).

[0273] Figure 8

[0274] Figure 8 Graph showing pH-dependent inhibition of proteolysis and spontaneous activation of latent myostatin by anti-latent myostatin antibodies, as described in Example 11. In the presence of anti-latent myostatin antibodies, the activity of active myostatin released from latent myostatin was measured using the HEK Blue assay by BMP1 protease (proteolysis) or by incubation at 37 °C in the absence of BMP1 (spontaneous). Antibodies MS1032LO01-SG1, MS1032LO02-SG1, MS1032LO03-SG1, and MS1032LO04-SG1 are described as MSLO-01, MSLO-02, MSLO-03, and MSLO-04, respectively, in the figure. MS1032LO01-SG1, MS1032LO02-SG1, MS1032LO03-SG1, and MS1032LO04-SG1 achieved inhibition of proteolysis and spontaneous activation of latent myostatin comparable to that of MS1032LO00-SG1.

[0275] Figure 9

[0276] Figure 9 A-9F Graph showing the BIACORE (registered trademark) sensorgram of a pH-dependent anti-latent myostatin antibody, as described in Example 12. 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.

[0277] Figure 10 ​​​​​​

[0278] Figure 10 The time course of plasma myostatin concentration after intravenous administration of anti - myostatin antibody in mice is shown, as described in Example 13. By comparing the anti - myostatin antibody with FcγR binding (MS1032LO00 - SG1) and the anti - myostatin antibody with disrupted FcγR binding (MS1032LO00 - F760), the effect of FcγR - mediated cellular uptake of the antibody / antigen complex on myostatin clearance in vivo was evaluated.

[0279] Figure 11

[0280] Figure 11 The time course of plasma myostatin concentration after intravenous administration of anti - myostatin antibody in mice is shown, as described in Example 14. By comparing the pH - dependent anti - myostatin antibody (MS1032LO01 - SG1 or MS1032LO01 - F760) and the non - pH - dependent anti - myostatin antibody (MS1032LO00 - SG1 or MS1032LO00 - F760), the effect of pH - dependent binding of the anti - myostatin antibody on myostatin clearance in vivo was evaluated.

[0281] Figure 12

[0282] Figure 12 The binding activity of the anti - latent myostatin antibody MST1032 to latent myostatin and GDF11 is shown, as described in Example 16.

[0283] Figure 13

[0284] Figure 13 The inhibitory activity of the anti - latent myostatin antibody MST1032 against proteolysis and spontaneous activation of GDF11 is shown, as described in Example 17. In the presence of the anti - latent myostatin antibody, the activity of the released active GDF11 was measured using the HEKBlue assay by BMP1 protease (proteolysis) or by incubation at 37°C in the absence of BMP1 (spontaneous).

[0285] Figure 14

[0286] Figure 14 ​​​​​​​​The figure shows that the anti - latent myostatin antibody inhibits the proteolytic activation of latent myostatin, as described in Example 19. In the presence of the anti - latent myostatin antibody, the activity of the active myostatin released from latent myostatin by BMP1 protease was measured using the HEKBlue assay.

[0287] Figure 15

[0288] Figure 15 The figure shows the time course of plasma myostatin concentration after intravenous administration of an anti - latent myostatin antibody in mice, as described in Example 20. The effect of pH - dependence on in - vivo myostatin clearance was evaluated by comparing a non - pH - dependent anti - latent myostatin antibody (MS1032LO00 - SG1) with different pH - dependent anti - latent myostatin antibodies (MS1032LO01 - SG1, MS1032LO06 - SG1, MS1032LO11 - SG1, MS1032LO18 - SG1, MS1032LO19 - SG1, MS1032LO21 - SG1, and MS1032LO25 - SG1).

[0289] Figure 16

[0290] Figure 16 Figures 16A and 16B show the time course of plasma myostatin concentration after intravenous administration of an anti - latent myostatin antibody in cynomolgus monkeys, as described in Example 21. (A) The effect of pH - dependence and Fc engineering on in - vivo myostatin clearance was evaluated by comparing a non - pH - dependent anti - latent myostatin antibody (MS1032LO00 - SG1) with pH - dependent anti - latent myostatin antibodies with Fc engineering (MS1032LO06 - SG1012, MS1032LO06 - SG1016, MS1032LO06 - SG1029, MS1032LO06 - SG1031, MS1032LO06 - SG1033, MS1032LO06 - SG1034). (B) The effect of Fc engineering on in - vivo myostatin clearance was evaluated by comparing anti - latent myostatin antibodies (MS1032LO19 - SG1079, MS1032LO19 - SG1071, MS1032LO19 - SG1080, MS1032LO19 - SG1074, MS1032LO19 - SG1081, and MS1032LO19 - SG1077).

[0291] Figure 17 ​​​​​

[0292] Figure 17 The inhibitory activity of anti-latent myostatin antibodies against latent myostatin activation is shown, as described in Example 22. In the presence of anti-latent myostatin antibodies (MST1032, MST1504, MST1538, MST1551, MST1558, MST1572, and MST1573), the amount of mature myostatin released from latent myostatin by BMP1 protease was measured.

[0293] Figure 18A

[0294] Figure 18A Schematic diagrams of 100-amino acid latent myostatin fragments designed for epitope mapping of anti-latent myostatin antibodies are shown, as described in Example 22.

[0295] Figure 18B

[0296] Figure 18B Western blot analysis of GST-tagged human latent myostatin fragments (GST-hMSTN) with anti-GST antibody is shown, as described in Example 22. Each lane indicates: 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.

[0297] Figure 18C

[0298] Figure 18C ​​​​​​​Western blot analysis of GST-tagged human latent myostatin fragments (GST-hMSTN) with anti-latent myostatin antibodies (MST1032, MST1538, MST1572, and MST1573), as described in Example 22. Each lane indicates: 1, GST-hMSTN 1-100aa; 2, GST-hMSTN 21-120aa; 3, GST-hMSTN 41-140aa; 4, GST-hMSTN 61-160aa; 5, GST-hMSTN 81-180aa; 6, GST-hMSTN 101-200aa; 7, GST-hMSTN 121-220aa; 8, GST-hMSTN 141-241aa; 9, GST control; 10, human latent myostatin (100 ng).

[0299] Figure 18D

[0300] Figure 18D Schematic results of Western blot analysis of anti-latent myostatin antibodies (MST1032, MST1538, MST1572, and MST1573) and the predicted epitope locations, as described in Example 22.

[0301] Figure 19

[0302] Figure 19 Alignment of the amino acid sequences of cynomolgus monkey (cyno) FcγRIIa1, FcγRIIa2, FcγRIIa3, FcγRIIb, human FcγRIIaH, FcγRIIaR, and FcγRIIb. The boxed regions indicate the predicted residues that interact with the Fc domain.

[0303] Figure 20

[0304] Figure 20 Time course of total myostatin concentration in plasma after intravenous administration of an anti-myostatin antibody with an FcγRIIb-enhanced Fc variant in fully human FcγR transgenic mice, as described in Example 24. The effect of the FcγRIIb-enhanced Fc variant on antigen clearance via human FcγRIIb was evaluated.

[0305] Figure 21

[0306] Figure 21 ​​​​​​​​The time course of antibody concentration in plasma after intravenous administration of an anti - myostatin antibody with an Fc variant having enhanced FcγRIIb in fully human FcγR transgenic mice, as described in Example 24. The effect of the FcγRIIb - enhanced Fc variant on antibody pharmacokinetics was evaluated.

[0307] Figure 22

[0308] Figure 22 A and 22B show the time course of plasma myostatin concentration after intravenous administration of an anti - latent myostatin antibody in cynomolgus monkeys, as described in Example 25. (A) The effect of pH - dependence and Fc engineering on in - vivo myostatin clearance was evaluated by comparing a non - pH - dependent anti - latent myostatin antibody (MS1032LO00 - SG1) and pH - dependent anti - latent myostatin antibodies with Fc engineering (MS1032LO06 - SG1012, MS1032LO06 - SG1016, MS1032LO06 - SG1029, MS1032LO06 - SG1031, MS1032LO06 - SG1033, MS1032LO06 - SG1034). (B) The effect of Fc engineering on in - vivo myostatin clearance was evaluated by comparing anti - latent myostatin antibodies (MS1032LO19 - SG1079, MS1032LO19 - SG1071, MS1032LO19 - SG1080, MS1032LO19 - SG1074, MS1032LO19 - SG1081, and MS1032LO19 - SG1077).

[0309] Figure 23A

[0310] Figure 23A The time course of total myostatin concentration in plasma after intravenous administration of an anti - myostatin antibody with an Fc variant having an increased pI in human FcRn transgenic mice, as described in Example 26. The effect of the Fc variant with an increased pI on antigen elimination was evaluated.

[0311] Figure 23B

[0312] Figure 23B The time course of antibody concentration in plasma after intravenous administration of an anti - myostatin antibody with an Fc variant having an increased pI in human FcRn transgenic mice, as described in Example 26. The effect of the Fc variant with an increased pI on antibody pharmacokinetics was evaluated.

[0313] Figure 24A ​​​​​​​​

[0314] Figure 24A The time course of the total myostatin concentration in plasma after intravenous administration of an anti - myostatin antibody with an Fc variant having an increased pI in human FcRn transgenic mice, as described in Example 26, is shown. The effect of the Fc variant with increased pI on antigen clearance was evaluated. In this assay, an excess of human normal immunoglobulin was co - administered with the anti - myostatin antibody to mimic the situation of human plasma.

[0315] Figure 24B

[0316] Figure 24B The time course of the antibody concentration in plasma after intravenous administration of an anti - myostatin antibody with an Fc variant having an increased pI in human FcRn transgenic mice, as described in Example 26, is shown. The effect of the Fc variant with increased pI on antibody pharmacokinetics was evaluated. In this assay, an excess of human normal immunoglobulin was co - administered with the anti - myostatin antibody to mimic the situation of human plasma.

[0317] Figure 25

[0318] Figure 25 The time course of the total myostatin concentration in plasma after intravenous administration of an anti - myostatin antibody with an Fc variant having enhanced FcγRIIb in human FcγRIIb transgenic mice, as described in Example 27, is shown. The effect of the FcγRIIb - enhanced Fc variant on antigen clearance through human FcγRIIb was evaluated.

[0319] Figure 26

[0320] Figure 26 The time course of the antibody concentration in plasma after intravenous administration of an anti - myostatin antibody with an Fc variant having enhanced FcγRIIb in human FcγRIIb transgenic mice, as described in Example 27, is shown. The effect of the FcγRIIb - enhanced Fc variant on antibody pharmacokinetics was evaluated.

[0321] Figure 27

[0322] Figure 27 The results of the cell imaging analysis of an anti - myostatin antibody with an Fc variant having enhanced FcγRIIb, as described in Example 29, are shown. Each antibody was complexed with a fluorescently labeled myostatin and the intracellular uptake of the antigen - antibody complex into cells expressing human FcγRIIb was measured. Detailed Description

[0323] ​​​​​​​​The techniques and methods described or cited herein are generally well understood and routinely used by those of ordinary skill in the art using conventional methodologies, such as, for example, the widely used methods described in the following: Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd ed. (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; 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 (R.I. Freshney), ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley & Sons, 1999); Immunobiology (C.A. 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 (V.T. DeVita et al., eds., J.B. Lippincott Company, 1993).

[0324] I. Definitions

[0325] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994) and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992) provide one of ordinary skill in the art with a general guide to many of the terms used in this application. All documents cited herein (including patent applications and publications) are incorporated by reference in their entirety.

[0326] To explain this application, the following definitions will apply and, where appropriate, terms used in the singular will also include the plural and vice versa. It is to be understood that the techniques used herein are only for describing particular embodiments and are not intended to be limiting. If any definition given below conflicts with any document incorporated by reference herein, the definition given below shall control.

[0327] "Acceptor human framework" for purposes used herein is a framework comprising an 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 "derived from" human immunoglobulin framework or human consensus framework acceptor human framework may comprise the identical amino acid sequence thereof, or it may contain amino acid sequence variations. 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 sequence of the VL acceptor human framework is identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0328] "Affinity" refers to the sum of the forces of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects the 1:1 interaction between the 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 represented 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 of measuring binding affinity are described below.

[0329] An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more hypervariable regions (HVRs) compared to a parental antibody that does not have such alterations, and such alterations result in an increase in the affinity of the antibody for the antigen.

[0330] The terms "anti - myostatin antibody" and "antibody that binds myostatin" refer to an antibody that is capable of binding myostatin with sufficient affinity such that the antibody can be used as a diagnostic and / or therapeutic agent for targeting myostatin. In one embodiment, the anti - myostatin antibody binds to an unrelated, non - myostatin protein to an extent less than about 10% of the binding of the antibody to myostatin, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the dissociation constant (Kd) of the antibody that binds myostatin is 1 µM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M). In certain embodiments, the anti - myostatin antibody binds to an epitope of myostatin that is conserved among myostatins from different species.

[0331] The term "antibody" is used herein in the broadest sense and includes various 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.

[0332] An "antibody fragment" refers to a molecule that is different from a full - length antibody and that comprises a portion of the full - length antibody that binds the antigen to which the full - length antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single - chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0333] An "antibody that binds the same epitope as a reference antibody" refers to an antibody that blocks the binding of the reference antibody to its antigen in a competition assay, and / or conversely, the reference antibody blocks the binding of the antibody to its antigen in a competition assay. Exemplary competition assays are provided herein.

[0334] 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 remaining portion of the heavy and / or light chain is derived from a different source or species.

[0335] The "isotype" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (allotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different types of immunoglobulins are designated α, δ, ε, γ, and μ, respectively.

[0336] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., radioactive isotopes of At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloid (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitors; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as 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 disclosed hereinafter.

[0337] "Effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary with 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; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0338] An "effective amount" of a reagent (e.g., a pharmaceutical composition) is an amount effective to achieve the desired therapeutic or prophylactic result for a dosage and duration necessary therefor.

[0339] The term "epitope" includes any determinant capable of being bound by an antibody. An epitope is the region of an antigen that is bound by the antibody targeting the antigen and includes specific amino acids that directly contact the antibody. Epitope determinants can include chemical active surface clusters of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural features, and / or specific charge features. Generally, an antibody specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and / or macromolecules.

[0340] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR is an FcR that binds IgG antibodies (γ receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants of said receptors and alternatively spliced forms. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (See, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997).) FcRs are reviewed in, e.g., 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). The term "FcR" herein encompasses other FcRs, including those to be determined in the future.

[0341] The term "Fc receptor" or "FcR" also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are 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); WO 2004 / 92219 (Hinton et al.). In vivo binding to human FcRn and the serum half-life of polypeptides with variant Fc regions can be determined, for example, in transgenic mice expressing human FcRn or transfected human cell lines or in primates administered polypeptides with variant Fc regions. WO 2000 / 42072 (Presta) describes antibody variants with increased or decreased binding to FcR. See also, e.g., Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).

[0342] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated herein, the amino acid residue numbering in the Fc region or constant region is according to the EU numbering system, which is also known as the EU index, as defined in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0343] The term "antibody comprising an Fc region" refers to an antibody that comprises an Fc region. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, e.g., during antibody purification or by recombinant engineering of the nucleic acid encoding the antibody. Thus, a composition comprising an antibody having an Fc region according to the invention can comprise antibodies having K447, antibodies in which all K447s have been removed, or a mixture of antibodies having K447 residues and antibodies lacking K447 residues.

[0344] "Framework" or "FR" refers to variable domain residues that are different from the hypervariable region (HVR) residues. The FRs of a variable domain generally consist of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences in a VH (or VL) generally occur in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0345] The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein to refer to an antibody that has a structure substantially similar to a native antibody structure or has a heavy chain that contains an Fc region as defined herein.

[0346] A "functional Fc region" has the "effector functions" 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 receptor; BCR), etc. Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be evaluated using a variety of assays disclosed, e.g., in the Definitions section herein.

[0347] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably to refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells", which include the primary transformed cells and progeny therefrom (regardless of the number of passages). The nucleic acid content of the progeny may not be identical to that of the parental cell and may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected for in the original transformed cell are included herein.

[0348] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or a human cell or an amino acid sequence derived from a non-human source using a human antibody library or other human antibody-encoding sequences. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0349] "Human consensus framework" is a framework that represents the most common amino acid residues in the 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 such as Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In one embodiment, for VL, the subgroup is subgroup κ I as in Kabat et al. above. In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al. above.

[0350] A "humanized" antibody refers to a chimeric antibody that contains amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, a humanized antibody will comprise substantially all of at least one (and typically, two) variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of a non-human antibody, and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. The "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has been humanized.

[0351] As used herein, the term "hypervariable region" or "HVR" refers to each region in the variable domain of an antibody in which the sequences are highly variable ("complementary determining region" or "CDR") and / or form structurally defined loops ("hypervariable loops") and / or contain residues that contact an antigen ("antigen contact points"). Typically, an antibody comprises six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include: (a) hypervariable loops occurring at amino acid residues 26 - 32 (L1), 50 - 52 (L2), 91 - 96 (L3), 26 - 32 (H1), 53 - 55 (H2), and 96 - 101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901 - 917 (1987)); (b) CDRs occurring at amino acid residues 24 - 34 (L1), 50 - 56 (L2), 89 - 97 (L3), 31 - 35b (H1), 50 - 65 (H2), and 95 - 102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NIH, Bethesda, MD (1991)); (c) antigen contact points 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) combinations of (a), (b), and / or (c), including HVR amino acid residues 46 - 56 (L2), 47 - 56 (L2), 48 - 56 (L2), 49 - 56 (L2), 26 - 35 (H1), 26 - 35b (H1), 49 - 65 (H2), 93 - 102 (H3), and 94 - 102 (H3).

[0352] Unless otherwise indicated, herein, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered according to Kabat et al. as above.

[0353] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules including, but not limited to, a cytotoxic agent.

[0354] "Individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0355] An "isolated" antibody is an antibody that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0356] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from the components of its natural environment. Isolated nucleic acids include nucleic acid molecules that are contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0357] An "isolated nucleic acid encoding an anti - myostatin antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains of an antibody (or fragments thereof), including such nucleic acid molecules in a single vector or in separate vectors, and such nucleic acid molecules at one or more locations in a host cell.

[0358] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homologous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during the preparation of the monoclonal antibody preparation, which variants are usually present in minor amounts. In contrast to polyclonal antibody preparations (which typically include different antibodies directed against different determinants (epitopes)), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the adjective "monoclonal" indicates the nature of the antibody as being obtained from a substantially homologous population of antibodies and is not to be construed as requiring that the antibody be made by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be prepared by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage display methods, and methods using transgenic animals that contain all or part of the human immunoglobulin locus, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0359] "Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in pharmaceutical formulations.

[0360] "Native antibody" refers to immunoglobulin molecules that occur naturally and have a variety of structures. For example, a native IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains that are linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), which is also referred to as the variable heavy chain domain or the 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), which is also referred to as the variable light chain domain or the light chain variable domain, followed by a light chain constant (CL) domain. The light chains of an antibody can be assigned to one of two types based on the amino acid sequence of their constant domains, referred to as kappa (κ) and lambda (λ).

[0361] "Native sequence Fc region" contains an amino acid sequence that is the same as the amino acid sequence of the Fc region found in nature. Native sequence human Fc region includes native sequence human IgG1 Fc region (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region and its naturally occurring variants.

[0362] The term "package insert" is used to refer to the instructions for use that are typically included in the commercial packaging of a therapeutic product and that contain information about the indications, uses, dosage, administration, combination therapies, contraindications, and / or warnings for use of such therapeutic product.

[0363] "Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity, wherein no conservative substitutions are to be considered as part of the sequence identity. The alignment for determining percent amino acid sequence identity can be achieved in various ways within the skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. However, for the purposes herein, the % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The authors of the ALIGN-2 sequence comparison computer program are Genentech, Inc., and the source code has been filed with the U.S. Copyright Office, Washington D.C., 20559, which is registered with U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or the program can be compiled from the source code. The ALIGN-2 program should be compiled for the UNIX operating system, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and need not be changed.

[0364] In the case where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A with or relative to a given amino acid sequence B (which can alternatively be phrased as the given amino acid sequence A has or contains a particular % amino acid sequence identity with or relative to the 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 the program alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that when 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 be equal to the % amino acid sequence identity of B to A. Unless otherwise expressly stated, all % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph.

[0365] The term "pharmaceutical formulation" means a formulation that has a form in which the biological activity of the active ingredient contained therein is effective and that contains no additional components that are toxic to the subject to which the formulation is to be administered.

[0366] "Pharmaceutical carrier" refers to the components in a pharmaceutical preparation other than the active ingredient, which are non-toxic to the subject. Pharmaceutical carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0367] As used herein, the term "myostatin" can refer to any native myostatin from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). Unless otherwise indicated, the term "myostatin" refers to the human myostatin protein having the amino acid sequence shown in SEQ ID NO: 1 and containing the propeptide domain at the N-terminus of the human myostatin shown in SEQ ID NO: 75 or 78. The term encompasses "full-length" unprocessed myostatin as well as any form of myostatin produced by processing in cells. The term also encompasses naturally occurring variants of myostatin, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human myostatin (pro-myostatin) is shown in SEQ ID NO: 1. The amino acid sequence of an exemplary C-terminal growth factor domain of human myostatin is shown in SEQ ID NO: 2. The amino acid sequence of an exemplary N-terminal propeptide domain of human myostatin is shown in SEQ ID NO: 75 or 78. The active mature myostatin is a disulfide-bonded homodimer composed of two C-terminal growth factor domains. The inactive latent myostatin is a non-covalently associated complex of two propeptides and the mature myostatin. As disclosed herein, the antibodies of the present invention bind to the inactive latent myostatin but do not bind to the mature active myostatin homodimer. 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 the mature active myostatin homodimer. The amino acid sequences of exemplary cynomolgus monkey and murine myostatin (pro-myostatin) are shown in SEQ ID NO: 3 and 5, respectively. The amino acid sequences of exemplary C-terminal growth factor domains of cynomolgus monkey and murine myostatin are shown in SEQ ID NO: 4 and 6, respectively. The amino acid sequences of exemplary N-terminal propeptide domains of cynomolgus monkey and murine 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. Similar to myostatin, GDF11 is first synthesized as a precursor polypeptide and then cleaved into an N-terminal prodomain and a C-terminal mature GDF11. The amino acid sequence of human GDF11 (precursor) is shown in SEQ ID NO: 81. The amino acid sequence of C-terminal mature human GDF11 is shown in SEQ ID NO: 82.The amino acid sequence of the N-terminal prodomain of human GDF11 is shown in SEQ ID NO: 83 or 84. The amino acid sequences of SEQ ID NO: 1, 3, 5, 78, 79, 80, 81, and 84 include a signal sequence. Amino acids 1-24 thereof correspond to the signal sequence and are removed during intracellular processing.

[0368] As used herein, "treatment" (and its grammatical variants such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of an individual being treated and can be performed for prophylaxis or during the clinical course of a disease. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, eliminating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and eliminating or improving the prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or to slow the progression of a disease.

[0369] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in antigen binding by the antibody. The heavy and light chain variable domains (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al., Kuby Immunology, 6 th th ed., W.H. Freeman & Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. In addition, antibodies that bind a particular antigen can be isolated by screening libraries of complementary VL or VH domains using, respectively, the VH or VL domain from an antibody that binds the antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0370] A "variant Fc region" comprises an amino acid sequence that is different from the amino acid sequence of a native sequence Fc region due to at least one amino acid modification (change), preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or the Fc region of a parental polypeptide, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions, in the native sequence Fc region or in the Fc region of the parental polypeptide. The variant Fc region herein preferably has at least about 80% homology, and most preferably at least about 90% homology, more preferably at least about 95% homology, with the native sequence Fc region and / or the Fc region of the parental polypeptide.

[0371] As used herein, the term "vector" refers to a nucleic acid molecule capable of causing the proliferation of another nucleic acid linked thereto. 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 directing the expression of nucleic acids operably linked thereto. Such vectors are referred to herein as "expression vectors".

[0372] II. Compositions and Methods

[0373] In one aspect, the present invention is in part based on anti-myostatin antibodies and their uses. In certain embodiments, antibodies that bind myostatin are provided. The antibodies of the present invention can be used, for example, for the diagnosis or treatment of diseases.

[0374] In another aspect, the present invention is in part based on polypeptides comprising variant Fc regions and their uses. In one embodiment, polypeptides comprising variant Fc regions having enhanced FcγRIIb-binding activity are provided. In another embodiment, polypeptides comprising variant Fc regions having an increased pI are provided. In particular embodiments, the polypeptides of the present invention are antibodies. The polypeptides of the present invention comprising variant Fc regions can be used, for example, for the diagnosis or treatment of diseases.

[0375] A. Exemplary Anti-Myostatin Antibodies and Polypeptides Comprising Variant Fc Regions

[0376] In one aspect, the present invention provides an isolated antibody that binds myostatin. In certain embodiments, the anti-myostatin antibody of the present invention binds latent myostatin. In additional embodiments, the anti-myostatin antibody of the present invention binds 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 additional embodiments, the antibody binds to an epitope within a fragment consisting of amino acids 21 - 100 of the myostatin propeptide (SEQ ID NO: 78). As described above, the propeptide is included as one of the components in latent myostatin. In certain embodiments, the anti-myostatin antibody of the present invention inhibits the activation of myostatin. In certain embodiments, the anti-myostatin antibody blocks the release of mature myostatin from latent myostatin. It has been reported that mature myostatin is released via proteolytic and non-proteolytic processes from latent myostatin. The anti-myostatin antibody of the present invention can block the proteolytic and / or non-proteolytic release of mature myostatin from latent myostatin. In certain embodiments, the anti-myostatin antibody blocks the proteolytic cleavage of latent myostatin. In certain embodiments, the anti-myostatin antibody blocks the access of proteases to latent myostatin (specifically, to the proteolytic cleavage site (Arg98 - Asp99) of latent myostatin). In additional embodiments, the protease can 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 blocks 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 is not accompanied by proteolytic cleavage of latent myostatin. Non-proteolytic release includes, for example, releasing mature myostatin by incubating latent myostatin at 37°C in the absence of a protease that cleaves latent myostatin. In certain embodiments, the anti-myostatin antibody of the present invention does not bind mature myostatin. In some embodiments, the anti-myostatin antibody binds to the same epitope as the antibody described in Table 2a. In some embodiments, the anti-myostatin antibody competes with the antibody described in Table 2a for binding to latent myostatin.In additional embodiments, the anti - myostatin antibody competes with an antibody comprising the VH and VL pair described in Table 2a for binding to latent myostatin. In some embodiments, the anti - myostatin antibody competes with the antibody described in Table 2a for binding to a fragment consisting of amino acids 21 - 100 of the myostatin propeptide (SEQ ID NO: 78). In additional embodiments, the anti - myostatin antibody binds to the same epitope as the antibody described in Table 11a or 13. In some embodiments, the anti - myostatin antibody competes with the antibody described in Table 11a or 13 for binding to latent myostatin. In some embodiments, the anti - myostatin antibody competes with the antibody described in Table 11a or 13 for binding to a fragment consisting of amino acids 21 - 100 of the myostatin propeptide (SEQ ID NO: 78).

[0377] In some embodiments, the anti - myostatin antibody of the invention binds to latent myostatin and inhibits the activation of myostatin. In additional embodiments, the antibody: (a) blocks the release of mature myostatin from latent myostatin; (b) blocks the proteolytic release of mature myostatin; (c) blocks the spontaneous release of mature myostatin; or (d) does not bind to mature myostatin; or binds to an epitope within a fragment consisting of amino acids 21 - 100 of the myostatin propeptide (SEQ ID NO: 78). In additional embodiments, the antibody competes with an antibody comprising the VH and VL pair described in Table 2a, 11a or 13 for binding to latent myostatin, or binds to the same epitope as an antibody comprising the VH and VL pair described in Table 2a, 11a or 13. In additional embodiments, the antibody has a higher affinity for binding to latent myostatin at neutral pH (e.g., pH 7.4) than at acidic pH (e.g., pH 5.8). In additional 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 the myostatin propeptide.

[0378] In another embodiment, the anti - myostatin antibody of the present invention does not bind to GDF11. In certain embodiments, the anti - myostatin antibody of the present invention does not inhibit the activation of GDF11. In certain embodiments, the anti - myostatin antibody does not block the release of mature GDF11 from latent GDF11. The anti - myostatin antibody of the present invention does not block the hydrolysis or non - proteolytic release of mature GDF11 from latent GDF11. In certain embodiments, the anti - myostatin antibody does not block the proteolytic cleavage of latent GDF11. In certain embodiments, the anti - myostatin antibody does not block protease access to latent GDF11 (specifically, access to the proteolytic cleavage site of latent GDF11). In additional embodiments, the protease can be a BMP1 / TLD family metalloprotease such as BMP1, TED, tolloid - like protein - 1 (TLL - 1) or tolloid - like protein - 2 (TLL - 2). Non - proteolytic release as used herein refers to the spontaneous release of mature GDF11 from latent GDF11 that does not accompany proteolytic cleavage of latent GDF11. Non - proteolytic release includes, for example, releasing mature GDF11 by incubating latent GDF11 at 37°C in the absence of a protease that cleaves latent GDF11. Most currently known anti - myostatin antibodies are not specific for myostatin. These antibodies also have high affinity for other members of the TGF - β superfamily such as GDF11 and neutralize their biological activities. GDF11 plays an important role during embryogenesis and is responsible for the homeotic transformation of the axial skeleton. Homozygous GDF11 knockout mice are perinatally lethal, and mice with one wild - type GDF11 gene copy are viable but have skeletal defects. Because GDF11 plays an important role during embryogenesis, inhibitors of GDF11 pose theoretical safety risks that may manifest as toxicity in treated patients or reproductive toxicity in women, for example, who may be pregnant. Therefore, in the treatment of myostatin - related diseases, especially in women who may be pregnant, there is a need for specific inhibition of myostatin activity.

[0379] In another aspect, the present invention provides an anti - myostatin antibody that exhibits pH - dependent binding properties. As used herein, the phrase "pH - dependent binding" means that the antibody "shows reduced binding to myostatin at acidic pH compared to its binding at neutral pH" (for the present disclosure, the two expressions can be used interchangeably). For example, an antibody "having pH - dependent binding properties" includes an antibody that binds myostatin with a higher affinity at neutral pH than at acidic pH. In certain embodiments, the antibody of the present invention binds myostatin at neutral pH with an affinity 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 - fold higher than at acidic pH. In some embodiments, the antibody binds myostatin (e.g., latent myostatin or pro - peptide myostatin) at pH 7.4 with a higher affinity than at pH 5.8. In additional embodiments, the antibody binds myostatin at pH 7.4 with an affinity 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 - fold higher than at pH 5.8.

[0380] When the antigen is a soluble protein, the binding of an antibody to the antigen may result in an extended half - life of the antigen in the plasma (i.e., reduced clearance of the antigen from the plasma), because the half - life of the antibody in the plasma can be longer than that of the antigen itself and can act as a carrier for the antigen. This is due to the recycling of the antigen - antibody complex via FcRn through the endosomal pathway in cells (Roopenian, Nat. Rev. Immunol. 7(9): 715 - 725 (2007)). However, it is expected that an antibody having pH - dependent binding properties (which binds its antigen in the neutral extracellular environment and releases the antigen into the acidic endosomal compartment after entering the cell) will have better properties in terms of antigen neutralization and clearance compared to its counterpart that binds 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).

[0381] For the present disclosure, the "affinity" of an antibody for myostatin is represented by the KD of the antibody. The KD of an antibody refers to the equilibrium dissociation constant of the antibody-antigen interaction. The larger the KD value of an antibody binding to its antigen, the weaker its binding affinity for the specific antigen. Thus, as used herein, the statement "higher affinity at neutral pH than at acidic pH" (or the equivalent statement "pH-dependent binding") means that the KD of the antibody binding to myostatin at acidic pH is higher than the KD of the antibody binding to myostatin at neutral pH. For example, in the context of the present invention, if the KD of the antibody binding to myostatin at acidic pH is at least 2-fold higher than the KD of the antibody binding to myostatin at neutral pH, the antibody is considered to have a higher affinity for binding to myostatin at neutral pH than at acidic pH. Accordingly, the present invention encompasses antibodies wherein the KD of the antibody binding to myostatin at acidic pH 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-fold or more higher than the KD of the antibody binding to myostatin at neutral pH. In another embodiment, the KD value of the antibody at neutral pH can be 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or less. In another embodiment, the KD value of the antibody at acidic pH can be 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M or more.

[0382] In a further embodiment, if the KD of the antibody binding to myostatin at pH 5.8 is at least 2-fold higher than the KD of the antibody binding to myostatin at pH 7.4, the antibody is considered to have a higher affinity for binding to myostatin (e.g., latent myostatin or propeptide myostatin) at neutral pH than at acidic pH. In some embodiments, the provided antibody has a KD for binding to myostatin at pH 5.8 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, 10000-fold or more higher 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 can be 10 -7 M, 10-8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or less. In another embodiment, the KD value of the antibody at pH 5.8 can be 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M or greater.

[0383] The binding property of an antibody to a specific antigen can also be expressed as the kd of the antibody. The kd of an antibody refers to the dissociation rate constant of the antibody with respect to a specific antigen and is expressed in reciprocal seconds (i.e., sec -1 ). An increase in the kd value indicates weaker binding of the antibody to its antigen. The present invention thus includes such antibodies that have a higher kd value for binding myostatin at acidic pH than at neutral pH. The present invention includes such antibodies that have a kd for binding myostatin at acidic pH 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 times higher than the kd of the antibody for binding myostatin at neutral pH. In another embodiment, the kd value of the antibody at neutral pH can be 10 -2 1 / s, 10 -3 1 / s, 10 -4 1 / s, 10 -5 1 / s, 10 -6 1 / s or less. In another embodiment, the kd value of the antibody at acidic pH can be 10 -3 1 / s, 10 -2 1 / s, 10 -1 1 / s or greater. The present invention also includes such antibodies that have a higher kd value for binding myostatin (e.g., latent myostatin or propeptide myostatin) at pH 5.8 than at pH 7.4. The present invention includes such antibodies that have a kd for binding myostatin at pH 5.8 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, 10000 times higher than the kd of the antibody for binding myostatin at pH 7.4. In another embodiment, the kd value of the antibody at pH 7.4 can be 10 -2 1 / s, 10 -31 / s, 10 -4 1 / s, 10 -5 1 / s, 10 -6 Less than 1 / s. In another embodiment, the kd value of the antibody at pH 5.8 can be 10 -3 1 / s, 10 -2 1 / s, 10 -1 More than 1 / s.

[0384] In some cases, "the binding of the antibody to myostatin at acidic pH is reduced compared to its binding at neutral pH" is expressed as the ratio of the KD value of the antibody binding to myostatin at acidic pH to the KD value of the antibody binding to myostatin at neutral pH (or vice versa). For example, for the present invention, if the antibody shows an acidic / neutral KD ratio of 2 or more, it can be considered that the antibody shows "the binding of the antibody to myostatin at acidic pH is reduced compared to its binding at neutral pH". In some embodiments, the pH 5.8 / pH 7.4 KD ratio of the anti-myostatin antibody of the present invention is 2 or more. In some exemplary embodiments, the acidic / neutral KD ratio of the antibody of the present 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 can be 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 Less than M. In another embodiment, the KD value of the antibody at acidic pH can be 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 More than M. In other cases, if the antibody shows a pH 5.8 / pH 7.4 KD ratio of 2 or more, it can be considered that the antibody shows "the binding of the antibody to myostatin (e.g., latent myostatin) at acidic pH is reduced compared to its binding at neutral pH". In some exemplary embodiments, the pH 5.8 / pH 7.4 KD ratio of the 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 more. In another embodiment, the KD value of the antibody at pH 7.4 can be 10 -7M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 Less than M. In another embodiment, the KD value of the antibody at pH 5.8 can be 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 More than M.

[0385] In some cases, "the binding to myostatin at acidic pH is reduced compared to its binding at neutral pH" is expressed as the ratio of the kd value of the antibody binding to myostatin at acidic pH to the kd value of the antibody binding to myostatin at neutral pH (or vice versa). For example, for the present invention, if the antibody shows an acidic / neutral kd ratio of more than 2, it can be considered that the antibody shows "the binding to myostatin at acidic pH is reduced compared to its binding at neutral pH". In some exemplary embodiments, the pH5.8 / pH7.4 kd ratio of the antibody of the present invention is more than 2. In some exemplary embodiments, the acidic / neutral kd ratio of the antibody of the present 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 can be 10 -2 1 / s, 10 -3 1 / s, 10 -4 1 / s, 10 -5 1 / s, 10 -6 Less than 1 / s. In another embodiment, the kd value of the antibody at acidic pH can be 10 -3 1 / s, 10 -2 1 / s, 10 -1 More than 1 / s. In some exemplary embodiments, the pH5.8 / pH7.4 kd ratio of the antibody of the present 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 pH 7.4 can be 10 -2 1 / s, 10 -3 1 / s, 10 -4 1 / s, 10 -5 1 / s, 10-6 less than 1 / s. In another embodiment, the kd value of the antibody at pH 5.8 can be 10 -3 1 / s, 10 -2 1 / s, 10 -1 or more than 1 / s.

[0386] As used herein, the expression "acidic pH" refers to a pH of from 4.0 to 6.5. The expression "acidic pH" includes any pH value of 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4 and 6.5. In a particular aspect, "acidic pH" is 5.8.

[0387] As used herein, the expression "neutral pH" refers to a pH of from 6.7 to about 10.0. The expression "neutral pH" includes any pH value of 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 and 10.0. In a particular aspect, "neutral pH" is 7.4.

[0388] As indicated herein, the KD value and the kd value can be determined using a surface plasmon resonance-based biosensor to characterize the antibody-antigen interaction. (See, e.g., Example 7 herein). The KD value and the kd value can be determined at 25°C or 37°C.

[0389] In certain embodiments, the anti-myostatin antibodies of the invention bind myostatin from more than one species. In additional embodiments, the anti-myostatin antibodies bind myostatin from human and non-human animals. In additional embodiments, the anti-myostatin antibodies bind myostatin from human, mouse and monkey (e.g., cynomolgus monkey, rhesus monkey, marmoset, chimpanzee or baboon).

[0390] In certain embodiments, the anti-myostatin antibodies of the invention bind latent myostatin from more than one species. In additional embodiments, the anti-myostatin antibodies bind latent myostatin from human and non-human animals. In additional embodiments, the anti-myostatin antibodies bind latent myostatin from human, mouse and monkey.

[0391] In certain embodiments, the anti - myostatin antibodies of the invention bind pro - peptide myostatin from more than one species. In additional embodiments, the anti - myostatin antibodies bind pro - peptide myostatin from human and non - human animals. In further embodiments, the anti - myostatin antibodies bind pro - peptide myostatin from human, mouse, and monkey.

[0392] In another aspect, the invention provides anti - myostatin antibodies that form an immune complex (i.e., an antigen - antibody complex) with myostatin. In certain embodiments, more than two anti - myostatin antibodies bind more than two myostatin molecules to form an immune complex. This is possible because myostatin exists as a homodimer containing two myostatin molecules, and an antibody has two antigen - binding sites. The anti - myostatin antibodies can bind to the same epitope on the myostatin molecule or can bind to different epitopes on the myostatin molecule, much like a bispecific antibody. Generally, when more than two antibodies form an immune complex with more than two antigens, the resulting immune complex can bind strongly to Fc receptors present on the cell surface due to the avidity effect of the Fc regions of the antibodies in the complex and can then be internalized into the cell with high efficiency. Thus, the anti - myostatin antibodies capable of forming an immune complex containing more than two anti - myostatin antibodies and more than two myostatin molecules can lead to rapid clearance of myostatin from plasma in vivo due to strong binding to Fc receptors caused by the avidity effect.

[0393] In addition, antibodies with pH - dependent binding properties are believed to have superior properties in 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, it is expected that antibodies with the above two properties, i.e., antibodies with pH - dependent binding properties and that form an immune complex containing more than two antibodies with more than two antigens, have even better properties for highly accelerated elimination of the antigen from plasma (WO 2013 / 081143).

[0394] In another aspect, the present invention provides an anti - myostatin antibody comprising at least one, two, three, four, five or six HVRs selected from the following: (a) HVR - H1, wherein said HVR - H1 comprises the amino acid sequence of any one of SEQ ID NOs: 55 - 57, 114 - 115, 126; (b) HVR - H2, wherein said HVR - H2 comprises the amino acid sequence of any one of SEQ ID NOs: 58 - 60, 116 - 120, 127; (c) HVR - H3, wherein said HVR - H3 comprises the amino acid sequence of any one of SEQ ID NOs: 61 - 64, 121, 128; (d) HVR - L1, wherein said HVR - L1 comprises the amino acid sequence of any one of SEQ ID NOs: 65 - 69, 122 - 124, 129; (e) HVR - L2, wherein said HVR - L2 comprises the amino acid sequence of any one of SEQ ID NOs: 70 - 72, 125, 130; and (f) HVR - L3, wherein said HVR - L3 comprises the amino acid sequence of any one of SEQ ID NOs: 73 - 74, 131.

[0395] In another aspect, the present invention provides an anti - myostatin antibody comprising at least one, two, three, four, five or six HVRs selected from the following: (a) HVR - H1, wherein said HVR - H1 comprises the amino acid sequence of any one of SEQ ID NOs: 55 - 57; (b) HVR - H2, wherein said HVR - H2 comprises the amino acid sequence of any one of SEQ ID NOs: 58 - 60; (c) HVR - H3, wherein said HVR - H3 comprises the amino acid sequence of any one of SEQ ID NOs: 61 - 64; (d) HVR - L1, wherein said HVR - L1 comprises the amino acid sequence of any one of SEQ ID NOs: 65 - 69; (e) HVR - L2, wherein said HVR - L2 comprises the amino acid sequence of any one of SEQ ID NOs: 70 - 72; and (f) HVR - L3, wherein said HVR - L3 comprises the amino acid sequence of any one of SEQ ID NOs: 73 - 74.

[0396] 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 the following: (a) HVR - H1, wherein the HVR - H1 comprises the amino acid sequence of any one of SEQ ID NOs: 114 - 115; (b) HVR - H2, wherein the HVR - H2 comprises the amino acid sequence of any one of SEQ ID NOs: 116 - 120; (c) HVR - H3, wherein the HVR - H3 comprises the amino acid sequence of SEQ ID NO: 121; (d) HVR - L1, wherein the HVR - L1 comprises the amino acid sequence of any one of SEQ ID NOs: 122 - 124; (e) HVR - L2, wherein the HVR - L2 comprises the amino acid sequence of SEQ ID NO: 125; and (f) HVR - L3, wherein the HVR - L3 comprises the amino acid sequence of any one of SEQ ID NOs: 73 - 74.

[0397] In another aspect, the present invention provides an anti - myostatin antibody comprising at least one, two, three, four, five or six HVRs selected from the following: (a) HVR - H1, wherein the HVR - H1 comprises the amino acid sequence of SEQ ID NO: 114; (b) HVR - H2, wherein the HVR - H2 comprises the amino acid sequence of SEQ ID NO: 58; (c) HVR - H3, wherein the HVR - H3 comprises the amino acid sequence of SEQ ID NO: 63; (d) HVR - L1, wherein the HVR - L1 comprises the amino acid sequence of SEQ ID NO: 122; (e) HVR - L2, wherein the HVR - L2 comprises the amino acid sequence of SEQ ID NO: 71; and (f) HVR - L3, wherein the HVR - L3 comprises 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 the following: (a) HVR - H1, wherein the HVR - H1 comprises the amino acid sequence of SEQ ID NO: 114; (b) HVR - H2, wherein the HVR - H2 comprises the amino acid sequence of SEQ ID NO: 58; (c) HVR - H3, wherein the HVR - H3 comprises the amino acid sequence of SEQ ID NO: 63; (d) HVR - L1, wherein the HVR - L1 comprises the amino acid sequence of SEQ ID NO: 123; (e) HVR - L2, wherein the HVR - L2 comprises the amino acid sequence of SEQ ID NO: 71; and (f) HVR - L3, wherein the HVR - L3 comprises the amino acid sequence of SEQ ID NO: 74.

[0398] In another aspect, the present invention provides an anti-myostatin antibody comprising at least one, two, three, four, five or six HVRs selected from the following: (a) HVR-H1, said HVR-H1 comprising the amino acid sequence of SEQ ID NO: 126; (b) HVR-H2, said HVR-H2 comprising the amino acid sequence of SEQ ID NO: 127; (c) HVR-H3, said HVR-H3 comprising the amino acid sequence of SEQ ID NO: 128; (d) HVR-L1, said HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129; (e) HVR-L2, said HVR-L2 comprising the amino acid sequence of SEQ ID NO: 130; and (f) HVR-L3, said HVR-L3 comprising the amino acid sequence of SEQ ID NO: 131.

[0399] In one aspect, the present invention provides antibodies comprising at least one, at least two, or all three VH HVR sequences selected from the following: (a) HVR-H1, wherein HVR-H1 comprises the amino acid sequence of any one of SEQ ID NOs: 55-57, 114-115, 126; (b) HVR-H2, wherein HVR-H2 comprises the amino acid sequence of any one of SEQ ID NOs: 58-60, 116-120, 127; (c) HVR-H3, wherein HVR-H3 comprises the amino acid sequence of any one of SEQ ID NOs: 61-64, 121, 128. In one embodiment, the antibody comprises HVR-H3, wherein HVR-H3 comprises the amino acid sequence of any one of SEQ ID NOs: 61-64, 121, 128. In another embodiment, the antibody comprises HVR-H3, wherein HVR-H3 comprises the amino acid sequence of any one of SEQ ID NOs: 61-64, 121, 128; and HVR-L3, wherein HVR-L3 comprises the amino acid sequence of any one of SEQ ID NOs: 73-74, 131. In another embodiment, the antibody comprises HVR-H3, wherein HVR-H3 comprises the amino acid sequence of any one of SEQ ID NOs: 61-64, 121, 128; HVR-L3, wherein HVR-L3 comprises the amino acid sequence of any one of SEQ ID NOs: 73-74, 131; and HVR-H2, wherein HVR-H2 comprises the amino acid sequence of any one of SEQ ID NOs: 58-60, 116-120, 127. In another embodiment, the antibody comprises (a) HVR-H1, wherein HVR-H1 comprises the amino acid sequence of any one of SEQ ID NOs: 55-57, 114-115, 126; (b) HVR-H2, wherein HVR-H2 comprises the amino acid sequence of any one of SEQ ID NOs: 58-60, 116-120, 127; and (c) HVR-H3, wherein HVR-H3 comprises the amino acid sequence of any one of SEQ ID NOs: 61-64, 121, 128.

[0400] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the following: (a) HVR-H1, wherein HVR-H1 comprises the amino acid sequence of any one of SEQ ID NOs: 55-57; (b) HVR-H2, wherein HVR-H2 comprises the amino acid sequence of any one of SEQ ID NOs: 58-60; and (c) HVR-H3, wherein HVR-H3 comprises the amino acid sequence of any one of SEQ ID NOs: 61-64. In one embodiment, the antibody comprises HVR-H3, wherein HVR-H3 comprises the amino acid sequence of any one of SEQ ID NOs: 61-64. In another embodiment, the antibody comprises HVR-H3, wherein HVR-H3 comprises the amino acid sequence of any one of SEQ ID NOs: 61-64; and HVR-L3, wherein HVR-L3 comprises the amino acid sequence of any one of SEQ ID NOs: 73-74. In another embodiment, the antibody comprises HVR-H3, wherein HVR-H3 comprises the amino acid sequence of any one of SEQ ID NOs: 61-64; HVR-L3, wherein HVR-L3 comprises the amino acid sequence of any one of SEQ ID NOs: 73-74; and HVR-H2, wherein HVR-H2 comprises the amino acid sequence of any one of SEQ ID NOs: 58-60. In another embodiment, the antibody comprises (a) HVR-H1, wherein HVR-H1 comprises the amino acid sequence of any one of SEQ ID NOs: 55-57; (b) HVR-H2, wherein HVR-H2 comprises the amino acid sequence of any one of SEQ ID NOs: 58-60; and (c) HVR-H3, wherein HVR-H3 comprises the amino acid sequence of any one of SEQ ID NOs: 61-64.

[0401] In another aspect, the present invention provides an antibody comprising at least one, at least two or all three VH HVR sequences selected from the following: (a) HVR-H1, wherein HVR-H1 comprises the amino acid sequence of any one of SEQ ID NOs: 114-115; (b) HVR-H2, wherein HVR-H2 comprises the amino acid sequence of any one of SEQ ID NOs: 116-120; and (c) HVR-H3, wherein HVR-H3 comprises the amino acid sequence of SEQ ID NO: 121. In one embodiment, the antibody comprises HVR-H3, wherein HVR-H3 comprises the amino acid sequence of SEQ ID NO: 121. In another embodiment, the antibody comprises HVR-H3, wherein HVR-H3 comprises the amino acid sequence of SEQ ID NO: 121; and HVR-L3, wherein HVR-L3 comprises the amino acid sequence of any one of SEQ ID NOs: 73-74. In another embodiment, the antibody comprises HVR-H3, wherein HVR-H3 comprises the amino acid sequence of SEQ ID NO: 121; HVR-L3, wherein HVR-L3 comprises the amino acid sequence of any one of SEQ ID NOs: 73-74; and HVR-H2, wherein HVR-H2 comprises the amino acid sequence of any one of SEQ ID NOs: 116-120. In another embodiment, the antibody comprises (a) HVR-H1, wherein HVR-H1 comprises the amino acid sequence of any one of SEQ ID NOs: 114-115; (b) HVR-H2, wherein HVR-H2 comprises the amino acid sequence of any one of SEQ ID NOs: 116-120; and (c) HVR-H3, wherein HVR-H3 comprises the amino acid sequence of SEQ ID NO: 121.

[0402] In another aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the following: (a) HVR-H1, wherein HVR-H1 comprises the amino acid sequence of SEQ ID NO: 114; (b) HVR-H2, wherein HVR-H2 comprises the amino acid sequence of SEQ ID NO: 58; and (c) HVR-H3, wherein HVR-H3 comprises the amino acid sequence of SEQ ID NO: 63. In one embodiment, the antibody comprises HVR-H3, wherein HVR-H3 comprises the amino acid sequence of SEQ ID NO: 63. In another embodiment, the antibody comprises HVR-H3, wherein HVR-H3 comprises the amino acid sequence of SEQ ID NO: 63; and HVR-L3, wherein HVR-L3 comprises the amino acid sequence of SEQ ID NO: 74. In another embodiment, the antibody comprises HVR-H3, wherein HVR-H3 comprises the amino acid sequence of SEQ ID NO: 63; HVR-L3, wherein HVR-L3 comprises the amino acid sequence of SEQ ID NO: 74; and HVR-H2, wherein HVR-H2 comprises the amino acid sequence of SEQ ID NO: 58. In another embodiment, the antibody comprises (a) HVR-H1, wherein HVR-H1 comprises the amino acid sequence of SEQ ID NO: 114; (b) HVR-H2, wherein HVR-H2 comprises the amino acid sequence of SEQ ID NO: 58; and (c) HVR-H3, wherein HVR-H3 comprises the amino acid sequence of SEQ ID NO: 63.

[0403] In another aspect, the present invention provides antibodies that comprise at least one, at least two, or all three VH HVR sequences selected from the following: (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO: 126; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO: 127; and (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 128. In one embodiment, the antibody comprises HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 128. In another embodiment, the antibody comprises HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 128; and HVR-L3, which comprises the amino acid sequence of SEQ ID NO: 131. In another embodiment, the antibody comprises HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 128; HVR-L3, which comprises the amino acid sequence of SEQ ID NO: 131; and HVR-H2, which comprises the amino acid sequence of SEQ ID NO: 127. In another embodiment, the antibody comprises (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO: 126; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO: 127; and (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 128.

[0404] In another aspect, the present invention provides antibodies that comprise at least one, at least two, or all three of the VL HVR sequences selected from the following: (a) HVR-L1, which comprises the amino acid sequence of any one of SEQ ID NOs: 65-69, 122-124, 129; (b) HVR-L2, which comprises the amino acid sequence of any one of SEQ ID NOs: 70-72, 125, 130; and (c) HVR-L3, which comprises the amino acid sequence of any one of SEQ ID NOs: 73-74, 131. In one embodiment, the antibody comprises (a) HVR-L1, which comprises the amino acid sequence of any one of SEQ ID NOs: 65-69, 122-124, 129; (b) HVR-L2, which comprises the amino acid sequence of any one of SEQ ID NOs: 70-72, 125, 130; and (c) HVR-L3, which comprises the amino acid sequence of any one of SEQ ID NOs: 73-74, 131.

[0405] In another aspect, the present invention provides antibodies that comprise at least one, at least two, or all three of the VL HVR sequences selected from the following: (a) HVR-L1, which comprises the amino acid sequence of any one of SEQ ID NOs: 65-69; (b) HVR-L2, which comprises the amino acid sequence of any one of SEQ ID NOs: 70-72; and (c) HVR-L3, which comprises the amino acid sequence of any one of SEQ ID NOs: 73-74. In one embodiment, the antibody comprises (a) HVR-L1, which comprises the amino acid sequence of any one of SEQ ID NOs: 65-69; (b) HVR-L2, which comprises the amino acid sequence of any one of SEQ ID NOs: 70-72; and (c) HVR-L3, which comprises the amino acid sequence of any one of SEQ ID NOs: 73-74.

[0406] In another aspect, the invention provides antibodies comprising at least one, at least two, or all three of the VL HVR sequences selected from the following: (a) HVR-L1, which comprises the amino acid sequence of any one of SEQ ID NOs: 122-124; (b) HVR-L2, which comprises the amino acid sequence of SEQ ID NO: 125; and (c) HVR-L3, which comprises the amino acid sequence of any one of SEQ ID NOs: 73-74. In one embodiment, the antibody comprises (a) HVR-L1, which comprises the amino acid sequence of any one of SEQ ID NOs: 122-124; (b) HVR-L2, which comprises the amino acid sequence of SEQ ID NO: 125; and (c) HVR-L3, which comprises the amino acid sequence of any one of SEQ ID NOs: 73-74.

[0407] In another aspect, the invention provides antibodies comprising at least one, at least two, or all three of the VL HVR sequences selected from the following: (a) HVR-L1, which comprises the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2, which comprises the amino acid sequence of SEQ ID NO: 71; and (c) HVR-L3, which comprises the amino acid sequence of SEQ ID NO: 74. In one embodiment, the antibody comprises (a) HVR-L1, which comprises the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2, which comprises the amino acid sequence of SEQ ID NO: 71; and (c) HVR-L3, which comprises the amino acid sequence of SEQ ID NO: 74. In another aspect, the invention provides antibodies comprising at least one, at least two, or all three of the VL HVR sequences selected from the following: (a) HVR-L1, which comprises the amino acid sequence of SEQ ID NO: 123; (b) HVR-L2, which comprises the amino acid sequence of SEQ ID NO: 71; and (c) HVR-L3, which comprises the amino acid sequence of SEQ ID NO: 74. In one embodiment, the antibody comprises (a) HVR-L1, which comprises the amino acid sequence of SEQ ID NO: 123; (b) HVR-L2, which comprises the amino acid sequence of SEQ ID NO: 71; and (c) HVR-L3, which comprises the amino acid sequence of SEQ ID NO: 74.

[0408] In another aspect, the present invention provides an antibody comprising at least one, at least two or all three VL HVR sequences selected from the following: (a) HVR-L1, wherein HVR-L1 comprises the amino acid sequence of SEQ ID NO: 129; (b) HVR-L2, wherein HVR-L2 comprises the amino acid sequence of SEQ ID NO: 130; and (c) HVR-L3, wherein HVR-L3 comprises the amino acid sequence of SEQ ID NO: 131. In one embodiment, the antibody comprises (a) HVR-L1, wherein HVR-L1 comprises the amino acid sequence of SEQ ID NO: 129; (b) HVR-L2, wherein HVR-L2 comprises the amino acid sequence of SEQ ID NO: 130; and (c) HVR-L3, wherein HVR-L3 comprises the amino acid sequence of SEQ ID NO 131.

[0409] 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 the following: (i) HVR-H1, wherein HVR-H1 comprises the amino acid sequence of any one of SEQ ID NOs: 55-57, 114, 115, 126; (ii) HVR-H2, wherein HVR-H2 comprises the amino acid sequence of any one of SEQ ID NOs: 58-60, 116-120, 127; and (iii) HVR-H3, wherein HVR-H3 comprises the amino acid sequence of any one of SEQ ID NOs: 61-64, 121, 128; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from the following: (i) HVR-L1, wherein HVR-L1 comprises the amino acid sequence of any one of SEQ ID NOs: 65-69, 122-124, 129; (ii) HVR-L2, wherein HVR-L2 comprises the amino acid sequence of any one of SEQ ID NOs: 70-72, 125, 130; and (c) HVR-L3, wherein HVR-L3 comprises the amino acid sequence of any one of SEQ ID NOs: 73-74, 131.

[0410] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three of the VH HVR sequences selected from the following: (i) HVR-H1, which comprises an amino acid sequence of any one of SEQ ID NOs: 55-57; (ii) HVR-H2, which comprises an amino acid sequence of any one of SEQ ID NOs: 58-60; and (iii) HVR-H3, which comprises an amino acid sequence of any one of SEQ ID NOs: 61-64; and (b) a VL domain comprising at least one, at least two, or all three of the VL HVR sequences selected from the following: (i) HVR-L1, which comprises an amino acid sequence of any one of SEQ ID NOs: 65-69; (ii) HVR-L2, which comprises an amino acid sequence of any one of SEQ ID NOs: 70-72; and (c) HVR-L3, which comprises an amino acid sequence of any one of SEQ ID NOs: 73-74.

[0411] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three of the VH HVR sequences selected from the following: (i) HVR-H1, which comprises an amino acid sequence of any one of SEQ ID NOs: 114-115; (ii) HVR-H2, which comprises an amino acid sequence of any one of SEQ ID NOs: 116-120; and (iii) HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 121; and (b) a VL domain comprising at least one, at least two, or all three of the VL HVR sequences selected from the following: (i) HVR-L1, which comprises an amino acid sequence of any one of SEQ ID NOs: 122-124; (ii) HVR-L2, which comprises the amino acid sequence of SEQ ID NO: 125; and (c) HVR-L3, which comprises an amino acid sequence of any one of SEQ ID NOs: 73-74.

[0412] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three of the VH HVR sequences selected from: (i) HVR-H1, which comprises the amino acid sequence of SEQ ID NO: 114, (ii) HVR-H2, which comprises the amino acid sequence of SEQ ID NO: 58, and (iii) HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 63; and (b) a VL domain comprising at least one, at least two or all three of the VL HVR sequences selected from: (i) HVR-L1, which comprises the amino acid sequence of SEQ ID NO: 122, (ii) HVR-L2, which comprises the amino acid sequence of SEQ ID NO: 71, and (c) HVR-L3, which comprises the amino acid sequence of SEQ ID NO: 74. In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three of the VH HVR sequences selected from: (i) HVR-H1, which comprises the amino acid sequence of SEQ ID NO: 114, (ii) HVR-H2, which comprises the amino acid sequence of SEQ ID NO: 58, and (iii) HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 63; and (b) a VL domain comprising at least one, at least two or all three of the VL HVR sequences selected from: (i) HVR-L1, which comprises the amino acid sequence of SEQ ID NO: 123, (ii) HVR-L2, which comprises the amino acid sequence of SEQ ID NO: 71, and (c) HVR-L3, which comprises the amino acid sequence of SEQ ID NO: 74.

[0413] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three of the VH HVR sequences selected from: (i) HVR-H1, which comprises the amino acid sequence of SEQ ID NO: 126, (ii) HVR-H2, which comprises the amino acid sequence of SEQ ID NO: 127, and (iii) HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 128; and (b) a VL domain comprising at least one, at least two or all three of the VL HVR sequences selected from: (i) HVR-L1, which comprises the amino acid sequence of SEQ ID NO: 129, (ii) HVR-L2, which comprises the amino acid sequence of SEQ ID NO: 130, and (c) HVR-L3, which comprises the amino acid sequence of SEQ ID NO: 131.

[0414] In another aspect, the invention provides an antibody comprising (a) HVR-H1, which comprises any one of the amino acid sequences of SEQ ID NO: 55-57, 114-115, 126; (b) HVR-H2, which comprises any one of the amino acid sequences of SEQ ID NO: 58-60, 116-120, 127; (c) HVR-H3, which comprises any one of the amino acid sequences of SEQ ID NO: 61-64, 121, 128; (d) HVR-L1, which comprises any one of the amino acid sequences of SEQ ID NO: 65-69, 122-124, 129; (e) HVR-L2, which comprises any one of the amino acid sequences of SEQ ID NO: 70-72, 125, 130; and (f) HVR-L3, which comprises any one of the amino acid sequences of SEQ ID NO: 73-74, 131.

[0415] In another aspect, the present invention provides an antibody comprising (a) HVR-H1, which comprises the amino acid sequence of any one of SEQ ID NO: 55-57, 114-115; (b) HVR-H2, which comprises the amino acid sequence of any one of SEQ ID NO: 58-60, 116-120; (c) HVR-H3, which comprises the amino acid sequence of any one of SEQ ID NO: 61-64, 121; (d) HVR-L1, which comprises the amino acid sequence of any one of SEQ ID NO: 65-69, 122-124; (e) HVR-L2, which comprises the amino acid sequence of any one of SEQ ID NO: 70-72, 125; and (f) HVR-L3, which comprises the amino acid sequence of any one of SEQ ID NO: 73-74.

[0416] In another aspect, the present invention provides an antibody comprising (a) HVR-H1, which comprises the amino acid sequence of any one of SEQ ID NO: 114-115; (b) HVR-H2, which comprises the amino acid sequence of any one of SEQ ID NO: 116-120; (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 121; (d) HVR-L1, which comprises the amino acid sequence of any one of SEQ ID NO: 122-124; (e) HVR-L2, which comprises the amino acid sequence of SEQ ID NO: 125; and (f) HVR-L3, which comprises the amino acid sequence of any one of SEQ ID NO: 73-74.

[0417] In another aspect, the present invention provides an antibody comprising (a) HVR-H1, wherein HVR-H1 comprises the amino acid sequence of SEQ ID NO: 114; (b) HVR-H2, wherein HVR-H2 comprises the amino acid sequence of SEQ ID NO: 58; (c) HVR-H3, wherein HVR-H3 comprises the amino acid sequence of SEQ ID NO: 63; (d) HVR-L1, wherein HVR-L1 comprises the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2, wherein HVR-L2 comprises the amino acid sequence of SEQ ID NO: 71; and (f) HVR-L3, wherein HVR-L3 comprises the amino acid sequence of SEQ ID NO: 74. In another aspect, the present invention provides an antibody comprising (a) HVR-H1, wherein HVR-H1 comprises the amino acid sequence of SEQ ID NO: 114; (b) HVR-H2, wherein HVR-H2 comprises the amino acid sequence of SEQ ID NO: 58; (c) HVR-H3, wherein HVR-H3 comprises the amino acid sequence of SEQ ID NO: 63; (d) HVR-L1, wherein HVR-L1 comprises the amino acid sequence of SEQ ID NO: 123; (e) HVR-L2, wherein HVR-L2 comprises the amino acid sequence of SEQ ID NO: 71; and (f) HVR-L3, wherein HVR-L3 comprises the amino acid sequence of SEQ ID NO: 74.

[0418] In another aspect, the present invention provides an antibody comprising (a) HVR-H1, wherein HVR-H1 comprises the amino acid sequence of SEQ ID NO: 126; (b) HVR-H2, wherein HVR-H2 comprises the amino acid sequence of SEQ ID NO: 127; (c) HVR-H3, wherein HVR-H3 comprises the amino acid sequence of SEQ ID NO: 128; (d) HVR-L1, wherein HVR-L1 comprises the amino acid sequence of SEQ ID NO: 129; (e) HVR-L2, wherein HVR-L2 comprises the amino acid sequence of SEQ ID NO: 130; and (f) HVR-L3, wherein HVR-L3 comprises the amino acid sequence of SEQ ID NO: 131.

[0419] In certain embodiments, one or more amino acids of an anti - myostatin antibody are substituted at the following HVR positions: (a) in HVR - H1 (SEQ ID NO: 55), at positions 1 and 2; (b) in HVR - H2 (SEQ ID NO: 58), at positions 4, 7, 8, 10, 11, 12, and 16; (c) in HVR - H3 (SEQ ID NO: 61), at positions 5, 7, and 11; (d) in HVR - L1 (SEQ ID NO: 65), at positions 1, 2, 5, 7, 8, and 9; (e) in HVR - L2 (SEQ ID NO: 70), at positions 3 and 7; and (f) in HVR - L3 (SEQ ID NO: 73), at position 8.

[0420] In certain embodiments, one or more amino acid substitutions of the anti - myostatin antibody are conservative substitutions, as provided herein. In certain embodiments, any one or more of the following substitutions can 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, 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.

[0421] All possible combinations of the above substitutions are covered by the consensus sequences SEQ ID NO: 126, 127, 128, 129, 130, and 131 of HVR - H1, HVR - H2, HVR - H3, HVR - L1, HVR - L2, and HVR - L3, respectively.

[0422] In any of the above embodiments, the anti - myostatin antibody can be humanized. In one embodiment, the anti - myostatin antibody comprises HVRs as in any of the above embodiments, and further comprises a receptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti - myostatin antibody comprises HVRs as in any of the above embodiments, and further comprises a VH or VL containing FR sequences. In another embodiment, the anti - myostatin antibody comprises the following heavy - chain and / or light - chain variable domain FR sequences: for the heavy - chain variable domain, FR1 comprises 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 - 141, FR3 comprises the amino acid sequence of any one of SEQ ID NOs: 142 - 143, and FR4 comprises the amino acid sequence of SEQ ID NO: 144.

[0423] In one aspect, the invention provides an anti - myostatin antibody comprising at least one, two, three, four, five or six HVRs selected from the following: (a) HVR - H1, said HVR - H1 comprising the amino acid sequence of any one of SEQ ID NOs: 157 - 162; (b) HVR - H2, said HVR - H2 comprising the amino acid sequence of any one of SEQ ID NOs: 163 - 168; (c) HVR - H3, said HVR - H3 comprising the amino acid sequence of any one of SEQ ID NOs: 169 - 174; (d) HVR - L1, said HVR - L1 comprising the amino acid sequence of any one of SEQ ID NOs: 175 - 180; (e) HVR - L2, said HVR - L2 comprising the amino acid sequence of any one of SEQ ID NOs: 181 - 186; and (f) HVR - L3, said HVR - L3 comprising the amino acid sequence of any one of SEQ ID NOs: 187 - 192.

[0424] In one aspect, the present invention provides antibodies comprising at least one, at least two or all three VH HVR sequences selected from the following: (a) HVR-H1, wherein HVR-H1 comprises an amino acid sequence of any one of SEQ ID NOs: 157-162; (b) HVR-H2, wherein HVR-H2 comprises an amino acid sequence of any one of SEQ ID NOs: 163-168; and (c) HVR-H3, wherein HVR-H3 comprises an amino acid sequence of any one of SEQ ID NOs: 169-174. In one embodiment, the antibody comprises HVR-H3, wherein HVR-H3 comprises an amino acid sequence of any one of SEQ ID NOs: 169-174. In another embodiment, the antibody comprises HVR-H3, wherein HVR-H3 comprises an amino acid sequence of any one of SEQ ID NOs: 169-174, and HVR-L3, wherein HVR-L3 comprises an amino acid sequence of any one of SEQ ID NOs: 187-192. In another embodiment, the antibody comprises HVR-H3, wherein HVR-H3 comprises an amino acid sequence of any one of SEQ ID NOs: 169-174, HVR-L3, wherein HVR-L3 comprises an amino acid sequence of any one of SEQ ID NOs: 187-192; and HVR-H2, wherein HVR-H2 comprises an amino acid sequence of any one of SEQ ID NOs: 163-168. In another embodiment, the antibody comprises (a) HVR-H1, wherein HVR-H1 comprises an amino acid sequence of any one of SEQ ID NOs: 157-162; (b) HVR-H2, wherein HVR-H2 comprises an amino acid sequence of any one of SEQ ID NOs: 163-168; and (c) HVR-H3, wherein HVR-H3 comprises an amino acid sequence of any one of SEQ ID NOs: 169-174.

[0425] In another aspect, the invention provides an antibody comprising at least one, at least two or all three VL HVR sequences selected from the following: (a) HVR-L1, wherein HVR-L1 comprises an amino acid sequence of any one of SEQ ID NOs: 175-180; (b) HVR-L2, wherein HVR-L2 comprises an amino acid sequence of any one of SEQ ID NOs: 181-186; and (c) HVR-L3, wherein HVR-L3 comprises an amino acid sequence of any one of SEQ ID NOs: 187-192. In one embodiment, the antibody comprises (a) HVR-L1, wherein HVR-L1 comprises an amino acid sequence of any one of SEQ ID NOs: 175-180; (b) HVR-L2, wherein HVR-L2 comprises an amino acid sequence of any one of SEQ ID NOs: 181-186; and (c) HVR-L3, wherein HVR-L3 comprises an amino acid sequence of any one of SEQ ID NOs: 187-192.

[0426] In another aspect, the antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from the following: (i) HVR-H1, wherein HVR-H1 comprises an amino acid sequence of any one of SEQ ID NOs: 157-162, (ii) HVR-H2, wherein HVR-H2 comprises an amino acid sequence of any one of SEQ ID NOs: 163-168, and (iii) HVR-H3, wherein HVR-H3 comprises an amino acid sequence of any one of SEQ ID NOs: 169-174; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from the following: (i) HVR-L1, wherein HVR-L1 comprises an amino acid sequence of any one of SEQ ID NOs: 175-180, (ii) HVR-L2, wherein HVR-L2 comprises an amino acid sequence of any one of SEQ ID NOs: 181-186, and (c) HVR-L3, wherein HVR-L3 comprises an amino acid sequence of any one of SEQ ID NOs: 187-192.

[0427] In another aspect, the invention provides an antibody comprising (a) HVR-H1, wherein HVR-H1 comprises an amino acid sequence of any one of SEQ ID NOs: 157-162; (b) HVR-H2, wherein HVR-H2 comprises an amino acid sequence of any one of SEQ ID NOs: 163-168; (c) HVR-H3, wherein HVR-H3 comprises an amino acid sequence of any one of SEQ ID NOs: 169-174; (d) HVR-L1, wherein HVR-L1 comprises an amino acid sequence of any one of SEQ ID NOs: 175-180; (e) HVR-L2, wherein HVR-L2 comprises an amino acid sequence of any one of SEQ ID NOs: 181-186; and (f) HVR-L3, wherein HVR-L3 comprises an amino acid sequence of any one of SEQ ID NOs: 187-192.

[0428] 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 with an amino acid sequence of any one of SEQ ID NOs: 13, 16-30, 32-34 and 86-95. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, provided that the anti-myostatin antibody comprising the sequence retains the ability to bind myostatin. In certain embodiments, in any one of SEQ ID NOs: 13, 16-30 and 32-34, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-myostatin antibody comprises a VH sequence of any one of SEQ ID NOs: 13, 16-30 and 32-34, including post-translational modifications of the sequence. In particular embodiments, the VH comprises one, two or three HVRs selected from: (a) HVR-H1, wherein HVR-H1 comprises an amino acid sequence of any one of SEQ ID NOs: 55-57, (b) HVR-H2, wherein HVR-H2 comprises an amino acid sequence of any one of SEQ ID NOs: 58-60, and (c) HVR-H3, wherein HVR-H3 comprises an amino acid sequence of any one of SEQ ID NOs: 61-64.

[0429] In another aspect, the anti - myostatin antibody comprises a heavy chain variable domain (VH) sequence that has 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, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti - myostatin antibody comprising the sequence retains the ability to bind myostatin. In certain embodiments, in any one of SEQ ID NOs: 13, 16 - 30, 32 - 34, and 86 - 95, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., in the FRs). Optionally, the anti - myostatin antibody comprises a VH sequence of any one of SEQ ID NOs: 13, 16 - 30, 32 - 34, and 86 - 95, which includes post - translational modifications of the sequence. In particular embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR - H1, which comprises the amino acid sequence of any one of SEQ ID NOs: 55 - 57, 114 - 115, 126; (b) HVR - H2, which comprises the amino acid sequence of any one of SEQ ID NOs: 58 - 60, 116 - 120, 127; and (c) HVR - H3, which comprises the amino acid sequence of any one of SEQ ID NOs: 61 - 64, 121, 128.

[0430] In another aspect, the 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 of SEQ ID NOs: 13, 16 - 30, 32, 33, and 34. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but the anti - myostatin antibody comprising the sequence retains the ability to bind myostatin. In certain embodiments, in any of SEQ ID NOs: 13, 16 - 30, 32, 33 and 34, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., in the FRs). Optionally, the anti - myostatin antibody comprises a VH sequence of any of SEQ ID NOs: 13, 16 - 30, 32, 33 and 34, which includes post - translational modifications of the sequence. In particular embodiments, the VH comprises one, two or three HVRs selected from: (a) HVR - H1, which comprises the amino acid sequence of any of SEQ ID NOs: 55 - 57, (b) HVR - H2, which comprises the amino acid sequence of any of SEQ ID NOs: 58 - 60, and (c) HVR - H3, which comprises the amino acid sequence of any of SEQ ID NOs: 61 - 64.

[0431] In another aspect, the anti-myostatin antibody comprises a VH sequence that has 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, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-myostatin antibody comprising the sequence retains the ability to bind myostatin. In certain embodiments, in any one of SEQ ID NOs: 86-95, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., in the FRs). Optionally, the anti-myostatin antibody comprises a VH sequence of any one of SEQ ID NOs: 86-95, including post-translational modifications of the sequence. In particular embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1, which comprises the amino acid sequence of any one of SEQ ID NOs: 114-115, 126; (b) HVR-H2, which comprises the amino acid sequence of any one of SEQ ID NOs: 116-120, 127; and (c) HVR-H3, which comprises the amino acid sequence of any one of SEQ ID NOs: 121, 128.

[0432] In another aspect, the anti - myostatin antibody comprises a VH sequence that has 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, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti - myostatin antibody comprising the sequence retains the ability to bind myostatin. In certain embodiments, in SEQ ID NO: 86, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti - myostatin antibody comprises the VH sequence of SEQ ID NO: 86, which includes post - translational modifications of the sequence. In particular embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR - H1, which comprises the amino acid sequence of SEQ ID NO: 114, (b) HVR - H2, which comprises the amino acid sequence of SEQ ID NO: 58, and (c) HVR - H3, which comprises the amino acid sequence of SEQ ID NO: 63. In another aspect, the anti - myostatin antibody comprises a VH sequence that has 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, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti - myostatin antibody comprising the sequence retains the ability to bind myostatin. In certain embodiments, in SEQ ID NO: 92, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti - myostatin antibody comprises the VH sequence of SEQ ID NO: 92, which includes post - translational modifications of the sequence.In particular embodiments, the VH comprises one, two or three HVRs selected from: (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO: 114; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO: 58; and (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 63.

[0433] In another aspect, provided is an anti-myostatin antibody, wherein the antibody comprises a VL that has 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, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but the anti-myostatin antibody comprising the sequence retains the ability to bind myostatin. In certain embodiments, in any one of SEQ ID NOs: 15, 31, 35-38 and 96-99, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., in the FRs). Optionally, the anti-myostatin antibody comprises a VL sequence of any one of SEQ ID NOs: 15, 31, 35-38 and 96-99, which includes post-translational modifications of the sequence. In particular embodiments, the VL comprises one, two or three HVRs selected from: (a) HVR-L1, which comprises the amino acid sequence of any one of SEQ ID NOs: 65-69, 122-124, 129; (b) HVR-L2, which comprises the amino acid sequence of any one of SEQ ID NOs: 70-72, 125, 130; and (c) HVR-L3, which comprises the amino acid sequence of any one of SEQ ID NOs: 73-74, 131.

[0434] In another aspect, provided are anti - myostatin antibodies, wherein the antibody comprises a VL that has 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, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but the anti - myostatin antibody comprising the sequence retains the ability to bind myostatin. In certain embodiments, in any one of SEQ ID NOs: 15, 31, 35, 36, 37, and 38, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., in the FRs). Optionally, the anti - myostatin antibody comprises a VL sequence of any one of SEQ ID NOs: 15, 31, 35, 36, 37, and 38, which includes post - translational modifications of the sequence. In particular embodiments, the VL comprises one, two, or three HVRs selected from: (a) HVR - L1, which comprises the amino acid sequence of any one of SEQ ID NOs: 65 - 69; (b) HVR - L2, which comprises the amino acid sequence of any one of SEQ ID NOs: 70 - 72; and (c) HVR - L3, which comprises the amino acid sequence of any one of SEQ ID NOs: 73 - 74.

[0435] In another aspect, provided are anti - myostatin antibodies, wherein the antibody comprises a VL that has 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, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but the anti - myostatin antibody comprising the sequence retains the ability to bind myostatin. In certain embodiments, in any one of SEQ ID NOs: 96 - 99, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs. Optionally, the anti - myostatin antibody comprises a VL sequence of any one of SEQ ID NOs: 96 - 99, which includes post - translational modifications of the sequence. In particular embodiments, the VL comprises one, two, or three HVRs selected from: (a) HVR - L1, which comprises the amino acid sequence of any one of SEQ ID NOs: 122 - 124, 129; (b) HVR - L2, which comprises the amino acid sequence of any one of SEQ ID NOs: 125, 130; and (c) HVR - L3, which comprises the amino acid sequence of SEQ ID NO: 131.

[0436] In another aspect, provided are anti - myostatin antibodies, wherein the antibody comprises a VL that has 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, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but the anti - myostatin antibody comprising the sequence retains the ability to bind myostatin. In certain embodiments, in SEQ ID NO: 96, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs. Optionally, the anti - myostatin antibody comprises the VL sequence of SEQ ID NO: 96, which includes post - translational modifications of the sequence. In particular embodiments, the VL comprises one, two or three HVRs selected from: (a) HVR - L1, which comprises the amino acid sequence of SEQ ID NO: 122; (b) HVR - L2, which comprises the amino acid sequence of SEQ ID NO: 71; and (c) HVR - L3, which comprises the amino acid sequence of SEQ ID NO: 74. In another aspect, provided are anti - myostatin antibodies, wherein the antibody comprises a VL that has 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, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but the anti - myostatin antibody comprising the sequence retains the ability to bind myostatin. In certain embodiments, in SEQ ID NO: 97, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs. Optionally, the anti - myostatin antibody comprises the VL sequence of SEQ ID NO: 97, which includes post - translational modifications of the sequence.In particular embodiments, the VL comprises one, two or three HVRs selected from: (a) HVR-L1, which comprises the amino acid sequence of SEQ ID NO: 123; (b) HVR-L2, which comprises the amino acid sequence of SEQ ID NO: 71; and (c) HVR-L3, which comprises the amino acid sequence of SEQ ID NO: 74.

[0437] In another aspect, provided is an anti-myostatin antibody, wherein the antibody comprises a VH as in any of the above embodiments and a VL as in any of the above embodiments. In one embodiment, the antibody comprises a VH and a VL sequence selected from 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 the sequences. In one embodiment, the antibody comprises a VH and a VL sequence selected from 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 the sequences. In one embodiment, the antibody comprises a VH and a VL sequence selected from any one of SEQ ID NOs: 86-95 and any one of SEQ ID NOs: 96-99, respectively, including post-translational modifications of the sequences.

[0438] In another aspect, provided is an anti-myostatin antibody, wherein the antibody comprises a VH as in any of the above embodiments and a VL as in any of the above embodiments. In one embodiment, the antibody comprises a VH and a VL sequence selected from 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 the sequences. In one embodiment, the antibody comprises a VH and a VL sequence selected from 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 the sequences.

[0439] In another aspect, provided is an anti - myostatin antibody, wherein the antibody comprises a VH as in any of the above - mentioned embodiments and a VL as in any of the above - mentioned embodiments. In one embodiment, the antibody comprises a VH and a VL sequence, respectively, selected from any one of SEQ ID NOs: 86 - 95 and any one of SEQ ID NOs: 96 - 99, which includes post - translational modifications of the sequences. In one embodiment, the antibody comprises a VH and a VL sequence, respectively, selected from any one of SEQ ID NOs: 86 - 95 and any one of SEQ ID NOs: 96 - 99, which includes post - translational modifications of the sequences. In one embodiment, the antibody comprises a VH and a VL sequence, respectively, selected from any one of SEQ ID NOs: 86 - 95 and any one of SEQ ID NOs: 96 - 99, which includes post - translational modifications of the sequences. In another aspect, provided is an anti - myostatin antibody, wherein the antibody comprises a VH as in any of the above - mentioned embodiments and a VL as in any of the above - mentioned embodiments. In one embodiment, the antibody comprises a VH and a VL sequence, respectively, of SEQ ID NO: 86 and SEQ ID NO: 96, which includes post - translational modifications of the sequences. In one embodiment, the antibody comprises a VH and a VL sequence, respectively, of SEQ ID NO: 92 and SEQ ID NO: 97, which includes post - translational modifications of the sequences.

[0440] In another aspect, the anti-myostatin antibody comprises a VH sequence that has 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, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-myostatin antibody comprising the sequence retains the ability to bind myostatin. In certain embodiments, in any one of SEQ ID NOs: 12, 145 - 150, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., in the FRs). Optionally, the anti-myostatin antibody comprises a VH sequence of any one of SEQ ID NOs: 12, 145 - 150, including post-translational modifications of the sequence. In particular embodiments, the VH comprises one, two or three HVRs selected from: (a) HVR-H1, which comprises the amino acid sequence of any one of SEQ ID NOs: 55, 157 - 162, (b) HVR-H2, which comprises the amino acid sequence of any one of SEQ ID NOs: 58, 163 - 168, and (c) HVR-H3, which comprises the amino acid sequence of any one of SEQ ID NOs: 61, 169 - 174.

[0441] In another aspect, provided are anti-myostatin antibodies, wherein the antibody comprises a light chain variable domain (VL) that has 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, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but the anti-myostatin antibody comprising the sequence retains the ability to bind myostatin. In certain embodiments, in any one of SEQ ID NOs: 14, 151 - 156, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., in the FRs). Optionally, the anti-myostatin antibody comprises a VL sequence of any one of SEQ ID NOs: 14, 151 - 156, including post-translational modifications of the sequence. In particular embodiments, the VL comprises one, two, or three HVRs selected from: (a) HVR-L1, which comprises the amino acid sequence of any one of SEQ ID NOs: 65, 175 - 180; (b) HVR-L2, which comprises the amino acid sequence of any one of SEQ ID NOs: 70, 181 - 186; and (c) HVR-L3, which comprises the amino acid sequence of any one of SEQ ID NOs: 73, 187 - 192.

[0442] In another aspect, provided are anti-myostatin antibodies, wherein the antibody comprises a VH as in any of the above embodiments and a VL as in any of the above embodiments. In one embodiment, the antibody comprises a VH and a VL sequence 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 the sequences.

[0443] In certain embodiments, the anti-myostatin antibodies of the invention comprise a VH as in any of the above 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, 229 - 381. In certain embodiments, the anti-myostatin antibodies of the invention comprise a VL as in any of the above embodiments and a light chain constant region comprising the amino acid sequence of any one of SEQ ID NOs: 8 and 10.

[0444] In another aspect, the present invention provides antibodies that bind to the same epitope as the anti - myostatin antibodies provided herein. In another aspect, the present invention provides antibodies that bind to the same epitope as the antibodies described in Table 2a. In another aspect, the present invention provides antibodies that bind to the same epitope as the antibodies described in Table 11a or 13. In certain embodiments, antibodies are provided that bind to an epitope within 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.

[0445] In another aspect of the present invention, the anti - myostatin antibodies according to any of the above embodiments are monoclonal antibodies, including chimeric, humanized, or human antibodies. In one embodiment, the anti - myostatin antibody is an antibody fragment, e.g., Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full - length IgG antibody, e.g., a complete IgG1 or IgG4 antibody or other antibody classes or isotypes defined herein.

[0446] In another aspect, the anti - myostatin antibodies according to any of the above embodiments can bind any of the features described in Sections 1 - 7 below (individually or in combination).

[0447] 1. Antibody affinity

[0448] In certain embodiments, the dissociation constant (Kd) of the antibodies provided herein is 1 µM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M).

[0449] In one embodiment, the Kd is measured by a radiolabeled antigen - binding assay (RIA). In one embodiment, RIA is performed using the Fab form of the antibody of interest and its antigen. For example, the solution - binding affinity of the Fab for the antigen is measured by: in the presence of a titration series of unlabeled antigen with the minimum concentration of ( 125I) Label the antigen - balanced Fab, and then capture the bound antigen with a plate coated with anti - Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293:865 - 881 (1999)). To determine the assay conditions, a MICROTITER (registered trademark) multi - well plate (Thermo Scientific) was coated overnight with 5 μg / ml of the capture anti - Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then blocked with 2% (w / v) fetal bovine serum albumin in PBS for two to five hours at room temperature (about 23°C). In a non - adsorptive plate (Nunc #269620), 100 pM or 26 pM 125 I]- antigen was mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of the anti - VEGF antibody, Fab - 12 in Presta et al., Cancer Res. 57:4593 - 4599 (1997)). Then the Fab of interest was incubated overnight; however, the incubation can last longer (e.g., about 65 hours) to ensure equilibrium is achieved. Thereafter, the mixture was transferred to the capture plate for incubation at room temperature (e.g., for one hour). Then the solution was removed and the plate was washed eight times with 0.1% polysorbate 20 (TWEEN - 20 (registered trademark)) in PBS. When the plate was dry, 150 μl / well of scintillant (MICROSCINT - 20 TM ; Packard) was added, and the plate was counted for ten minutes on a TOPCOUNT TM γ - counter (Packard). The concentration of each Fab that resulted in less than or equal to 20% of the maximum binding was selected for the competitive binding assay.

[0450] According to another embodiment, Kd is measured using a BIACORE (registered trademark) surface plasmon resonance assay. For example, at 25°C, using an immobilized antigen CM5 chip at ~10 response units (RU), a BIACORE (registered trademark) - 2000 or BIACORE (Registered Trademark)-3000 assay of (BIACORE (Registered Trademark), Inc., Piscataway, NJ). In one embodiment, according to the instructions of the provider, the carboxymethylated dextran biosensor chip (CM5, BIACORE (Registered Trademark), Inc.) was activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The antigen was diluted to 5 µg / ml (~0.2 µM) with 10 mM sodium acetate pH 4.8 and then injected at a flow rate of 5 µl / min to achieve approximately 10 response units (RU) of the conjugated protein. After injecting the antigen, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, serial two-fold dilutions of Fab (0.78 nM to 500 nM) were injected at a flow rate of approximately 25 µl / min at 25 °C into PBS (PBST) with 0.05% polysorbate 20 (TWEEN-20 TM ) surfactant. The association rate (k on ) and dissociation rate (k off ) were calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE (Registered Trademark) Evaluation Software version 3.2). The equilibrium dissociation constant (Kd) was calculated as the ratio of k off / k on . See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the association rate measured by the above surface plasmon resonance assay exceeds 10 6 M -1 s -1 , the association rate can be determined by using fluorescence quenching techniques that measure the increase or decrease in the fluorescence emission intensity of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 at 25 °C in the presence of increasing concentrations of antigen, measured in a spectrometer such as a spectrophotometer equipped with a stop-flow device (Aviv Instruments) or an 8000-series SLM-AMINCO TM spectrophotometer (ThermoSpectronic) with a stirred cell (excitation = 295 nm; emission = 340 nm, 16 nm bandpass).

[0451] 2. Antibody Fragments

[0452] 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, and other fragments described below. For a review of certain antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also, WO 1993 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half-life, see U.S. Patent No. 5,869,046.

[0453] Diabodies are antibody fragments that have two antigen-binding sites and can be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetra-bodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0454] Single-domain antibodies are antibody fragments that contain all or part of the heavy-chain variable domain or all or part of the light-chain variable domain of an antibody. In certain embodiments, the single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516.

[0455] Antibody fragments can be prepared by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0456] 3. Chimeric and Humanized Antibodies

[0457] In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and 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 mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. In additional examples, a chimeric antibody is a "class-switch" antibody, where the class or subclass has been changed from that of the parental antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0458] In certain embodiments, the chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized to reduce their immunogenicity in humans while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., the CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. A humanized antibody optionally further comprises at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with the corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or enhance antibody specificity or affinity.

[0459] Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro, Front. Biosci. 13:1619-1633 (2008), and are further described, for example, in 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 specific determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); 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 "directed selection" methods for FR shuffling).

[0460] Human framework regions useful for humanization include, but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims et al., J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies having a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); and Presta et al., J. Immunol. 151:2623 (1993)); human mature (somatic 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 FR library screening (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0461] 4. Human Antibodies

[0462] In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be prepared using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-374 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0463] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce a complete human antibody or a complete antibody having a human variable region in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin locus, which replaces the endogenous immunoglobulin locus, or which is present extrachromosomally or randomly integrated into the chromosomes of the animal. In such transgenic mice, the endogenous immunoglobulin locus is typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE TMTechnologies; U.S. Patent No. 5,770,429, which describes HuMab® technology; U.S. Patent No. 7,041,870, which describes K-M MOUSE® technology, and U.S. Patent Application Publication No. US 2007 / 0061900, which describes VelociMOUSE® technology). The human variable regions from intact antibodies produced by such animals can be further modified, e.g., by combining with different human constant regions.

[0464] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma 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 prepared via human B-cell hybridoma technology have also been described in Li et al., Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006). Additional methods include those described in, for example, U.S. Patent No. 7,189,826 (describing the preparation of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, XiandaiMianyixue 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) has also been 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).

[0465] Human antibodies can also be produced by isolating Fv clone variable domain sequences from human-derived phage display libraries. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0466] 5. Antibodies from Libraries

[0467] The antibodies of the invention can be isolated by screening a combinatorial library of antibodies for one or more desired activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies with desired binding properties. Such methods are reviewed in, for example, Hoogenboom et al., Methods in Molecular Biology 178:1-37 (2000); O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and are further described in, for example, McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, 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).

[0468] In some phage display methods, VH and VL gene libraries are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, and the library can then be screened for phages that bind antigen, as described in Winter et al., Ann. Rev. Immunol. 12:433-455 (1994). Phages typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, a naive library (e.g., from humans) can be cloned to provide a single source of antibodies to a variety of non-self and self antigens without any immunization, as described in Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be synthetically prepared by cloning unrearranged V-gene segments from stem cells and using PCR primers containing random sequences to encode the hypervariable CDR3 region and effect rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol. 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Patent No. 5,750,373 and U.S. Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0469] As used herein, an antibody or antibody fragment isolated from a human antibody library is considered a human antibody or human antibody fragment.

[0470] 6. Multispecific Antibodies

[0471] In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. A multispecific antibody is a monoclonal antibody that has binding specificities for at least two different epitopes. In certain embodiments, one binding specificity is for myostatin and the other is for another antigen. In certain embodiments, the bispecific antibody can bind two different epitopes of myostatin. Bispecific antibodies can also be used to localize a cytotoxic agent to cells that express myostatin. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0472] Techniques for preparing 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)), WO1993 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Patent No. 5,731,168). Multispecific antibodies can also be prepared by engineering the electrostatic steering effect for the preparation of antibody Fc-heterodimer molecules (WO 2009 / 089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); using leucine zippers to prepare bispecific antibodies (see, e.g., Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)); using "diabody" technology for the preparation of bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol. 152:5368 (1994)); and the preparation of trispecific antibodies as described, for example, in Tutt et al., J. Immunol. 147:60 (1991).

[0473] Also included herein are engineered antibodies having three or more functional antigen-binding sites, including "octopus antibodies" (see, e.g., US 2006 / 0025576A1).

[0474] Antibodies or fragments herein also include "dual action FAb" or "DAF" which contain antigen-binding sites that bind myostatin as well as another different antigen (see, e.g., US 2008 / 0069820).

[0475] 7. Antibody Variants

[0476] In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it is desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from the antibody amino acid sequence, and / or insertions into and / or substitutions of residues within the antibody amino acid sequence. 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.

[0477] a. Substitution, insertion, and deletion variants

[0478] In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitution mutagenesis include HVRs and FRs. Conservative substitutions are shown under the heading of "preferred substitutions" in Table 1. More alterations are provided under the heading of "exemplary substitutions" in Table 1 and are further described below in terms of amino acid side chain classifications. Amino acid substitutions can be introduced into the target antibody and the product screened for the desired activity (e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

[0479] [Table 1]

[0480]

[0481] Amino acids can be grouped according to common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions entail the replacement of a member of one of these groups with a member of another group.

[0482] One type of substitution variant involves substituting one or more hypervariable region residues of a parental antibody (e.g., a humanized or human antibody). Generally, the resulting variant selected for further study will have an alteration (e.g., improvement) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parental antibody and / or will substantially retain certain biological properties of the parental antibody. Exemplary substitution variants are affinity matured antibodies, which can be routinely prepared, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0483] Alterations (e.g., substitutions) can be made in the HVRs, for example, to improve antibody affinity. Such alterations can be made in HVR "hotspots", i.e., residues encoded by codons that mutate at high frequency during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or residues that contact the antigen, and the binding affinity of the resulting variant VH or VL is tested. Affinity maturation by constructing a secondary library and rescreening therefrom has been described, e.g., in Hoogenboom et al., 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. The library is then screened to identify any antibody variants having the desired affinity. Another method of introducing diversity includes HVR-directed methods, in which several HVR residues (e.g., 4-6 residues simultaneously) are randomized. The HVR residues involved in antigen binding can be specifically identified, e.g., using alanine-scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are typically targeted.

[0484] In certain embodiments, substitutions, insertions, or deletions can occur within one or more HVRs, provided that such alterations do not significantly reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as described herein) that do not significantly reduce binding affinity can be made in the HVRs. Such alterations can be, for example, outside of the residues in the HVRs that contact the antigen. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unaltered or contains no more than one, two, or three amino acid substitutions.

[0485] A method that can be used to identify antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis", as described in Cunningham and Wells, Science 244:1081-1085 (1989). In this method, a residue or a set of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether it affects the interaction between the antibody and the antigen. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or additionally, the crystal structure of the antigen-antibody complex is determined to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or excluded as candidates for substitution. Variants can be screened to determine whether they have the desired properties.

[0486] Amino acid sequence insertions include amino-terminal and / or carboxyl-terminal fusions of polypeptides ranging in length from one residue to more than one hundred residues, as well as in-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N-terminus or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or polypeptide that increases the serum half-life of the antibody.

[0487] b. Glycosylation variants

[0488] In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree to which the antibody is glycosylated. Adding glycosylation sites to or removing glycosylation sites from an antibody can be readily accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0489] When the antibody contains an Fc region, the carbohydrate attached thereto can be altered. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are generally attached by N-linkage to Asn297 in the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharide can include a variety of carbohydrates, e.g., 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, the oligosaccharides in the antibodies of the invention can be modified to produce antibody variants with certain improved properties.

[0490] In one embodiment, antibody variants are provided that have a carbohydrate structure lacking fucose that is (directly or indirectly) linked to the Fc region. For example, the amount of fucose in such an antibody can be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the sum of all sugar structures (such as complex, hybrid and high mannose structures) linked to Asn 297, as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546, for example. Asn297 refers to the asparagine residue located near position 297 (Eu numbering of Fc region residues); however, due to small sequence variations in the antibody, Asn297 can also be located approximately + / - 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants can have enhanced ADCC function. See, for example, US Publication No. US 2003 / 0157108 (Presta, L.); US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Exemplary variants of "defucosylated" or "fucose-deficient" antibodies that are publicly available 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; US2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO2005 / 035778; WO 2005 / 053742; WO 2002 / 031140; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing afucosylated antibodies include the protein fucosylation-deficient Lec13 CHO cell line (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003 / 0157108 A1, Presta, L.; and WO 2004 / 056312, Adams et al., particularly Example 11), and knockout cell lines such as α-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).

[0491] Antibody variants having bisected oligosaccharides are also provided, e.g., wherein the branched oligosaccharides linked to the antibody Fc region are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or enhanced ADCC function. Examples of such antibody variants are described, e.g., in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharides linked to the Fc region are also provided. Such antibody variants may have enhanced CDC function. Such antibody variants are described, e.g., in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0492] c. Fc region variants

[0493] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants. The Fc region variants can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) that contains amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0494] In certain embodiments, the present invention contemplates antibody variants that have some but not all effector functions, making them desirable candidates for applications where the antibody in vivo half-life is important and certain effector functions, such as complement and ADCC, are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / elimination of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus likely lacks ADCC activity), but retains the ability to bind FcRn. The primary cells that mediate ADCC, NK cells, only express FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is outlined in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for assessing the ADCC activity of a target molecule are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom et al Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al, Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann et al, J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, e.g., ACTI for flow cytometry TMNon-radioactive cytotoxicity assays (CellTechnology, Inc., Mountain View, CA); and CytoTox 96® Non-radioactive Cytotoxicity Assay (Promega, Madison, WI). Effector cells useful in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, e.g., in an animal model as described in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody does not bind C1q and thus lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (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)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, e.g., Petkova et al., Int'l. Immunol. 18(12):1759-1769(2006)).

[0495] Antibodies with reduced effector function include antibodies having substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted to alanine (U.S. Patent No. 7,332,581).

[0496] Certain antibody variants with increased or decreased binding to FcR are described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604(2001).)

[0497] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that enhance ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0498] In some embodiments, alterations are made in the Fc region that result in altered (i.e., enhanced or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Patent No. 6,194,551, WO 1999 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0499] Antibodies having an increased half-life and enhanced binding to the neonatal Fc receptor (FcRn) responsible for transporting maternal IgG to the fetus (J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) are described in US2005 / 0014934A1 (Hinton et al.). The antibodies comprise an Fc region having one or more substitutions therein that enhance the binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the following 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., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826). See also, Duncan, Nature 322:738-40 (1988); U.S. Patent Nos. 5,648,260 and 5,624,821; and WO 1994 / 29351, which relates to other examples of Fc region variants.

[0500] d. Cysteine-engineered antibody variants

[0501] In certain embodiments, it may be desirable to prepare cysteine-engineered antibodies, e.g., "thioMAbs", in which one or more residues of the antibody are replaced with cysteine residues. In particular embodiments, the replaced residues occur at the access sites of the antibody. By replacing the residues with cysteine, reactive thiol groups are thereby placed at the access sites of the antibody and can be used to conjugate the antibody to other moieties, such as a drug moiety or a linker-drug moiety, thereby generating an immunoconjugate, as further described herein. In certain embodiments, any one or more of the following residues can be replaced with cysteine: V205 of the light chain (Kabat numbering); A118 of the heavy chain (EU numbering); and S400 of the heavy chain Fc region (EU numbering). Cysteine-engineered antibodies can be produced as described, for example, in U.S. Patent No. 7,521,541.

[0502] e. Antibody derivatives

[0503] In certain embodiments, the antibodies provided herein can be further modified to contain additional non-protein moieties known and readily available in the art. Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propanediol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-tri oxane, ethylene / maleic anhydride copolymer, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in preparation due to its stability in water. The polymers can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of derivatizing polymers can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative is to be used for therapy under defined conditions, and the like.

[0504] In another embodiment, conjugates of antibodies and non-protein moieties are provided that can be selectively heated by exposure to radiation. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation can have any wavelength and includes, but is not limited to, wavelengths that do not damage normal cells but heat the non-protein moiety to a temperature at which cells adjacent to the antibody-non-protein moiety are killed.

[0505] 8. Variant Fc region

[0506] In one aspect, the invention provides an isolated polypeptide comprising a variant Fc region having 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 variant Fc region comprises at least one amino acid residue alteration (e.g., substitution) compared to the corresponding sequence in the Fc region of a native or reference variant sequence (sometimes collectively referred to herein as the "parent" Fc region). In certain embodiments, the variant Fc region of the invention has enhanced binding activity to cynomolgus FcγRIIb compared to the parent Fc region. In a particular embodiment, the cynomolgus FcγRIIb is cynomolgus macaque FcγRIIb (SEQ ID NO: 223).

[0507] In certain embodiments, the ratio of [KD value of the parent Fc region for cynomolgus FcγRIIb] / [KD value of the variant Fc region for cynomolgus FcγRIIb] can be 2.0 or greater, 3.0 or greater, 4.0 or greater, 5.0 or greater, 6.0 or greater, 7.0 or greater, 8.0 or greater, 9.0 or greater, 10 or greater, 15 or greater, 20 or greater, 25 or greater, 30 or greater, 40 or greater, or 50 or greater. In additional embodiments, the variant Fc region has reduced binding activity to cynomolgus FcγRIIIa. In certain embodiments, the ratio of [KD value of the parent Fc region for cynomolgus FcγRIIIa] / [KD value of the variant Fc region for cynomolgus 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, the sequence of cynomolgus FcγRIIb is SEQ ID NO: 223 (cynomolgus macaque). In certain embodiments, the sequence of cynomolgus FcγRIIIa is SEQ ID NO: 224 (cynomolgus macaque).

[0508] In additional embodiments, the variant Fc region has increased binding activity to human FcγRIIb. In certain embodiments, the ratio of [KD value of the parental Fc region for human FcγRIIb] / [KD value of the variant 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 additional embodiments, the variant Fc region has decreased binding activity to human FcγRIIIa. In certain embodiments, [KD value of the parental Fc region for human FcγRIIIa] / [KD value of the variant 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, the sequence of human FcγRIIb is SEQ ID NO: 212, 213, or 214. In certain embodiments, the sequence of human FcγRIIIa is SEQ ID NO: 215, 216, 217, or 218.

[0509] In additional embodiments, the variant Fc region has decreased binding activity to human FcγRIIa (H type). In certain embodiments, the ratio of [KD value of the parental Fc region for human FcγRIIa (H type)] / [KD value of the variant 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 additional embodiments, the variant Fc region has decreased binding activity to human FcγRIIa (R type). In certain embodiments, the ratio of [KD value of the parental Fc region for human FcγRIIa (R type)] / [KD value of the variant 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, the sequence of human FcγRIIa (H type) is SEQ ID NO: 211. In certain embodiments, the sequence of human FcγRIIa (R type) is SEQ ID NO: 210.

[0510] In certain embodiments, [KD value of parental Fc region for monkey FcγRIIa] / [KD value of variant 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)).

[0511] In another embodiment, the KD value of the variant Fc region for monkey FcγRIIb can be 1.0x10 -6 M or less, 9.0x10 -7 M or less, 8.0x10 -7 M or less, 7.0x10 -7 M or less, 6.0x10 -7 M or less, 5.0x10 -7 M or less, 4.0x10 -7 M or less, 3.0x10 -7 M or less, 2.0x10 -7 M or less, or 1.0x10 -7 M or less. In another embodiment, the KD value of the variant Fc region for monkey FcγRIIIa can be 5.0x10 -7 M or more, 6.0x10 -7 M or more, 7.0x10 -7 M or more, 8.0x10 -7 M or more, 9.0x10 -7 M or more, 1.0x10 -6 M or more, 2.0x10 -6 M or more, 3.0x10 -6 M or more, 4.0x10 -6 M or more, 5.0x10 -6 M or more, 6.0x10 -6 M or more, 7.0x10 -6 M or more, 8.0x10 -6 M or more, 9.0x10 -6 M or more, or 1.0x10 -5M or more. In another embodiment, the KD value of the variant Fc region for human FcγRIIb can be 2.0x10 -6 M or less, 1.0x10 -6 M or less, 9.0x10 -7 M or less, 8.0x10 -7 M or less, 7.0x10 -7 M or less, 6.0x10 -7 M or less, 5.0x10 -7 M or less, 4.0x10 -7 M or less, 3.0x10 -7 M or less, 2.0x10 -7 M or less, or 1.0x10 -7 M or less. In another embodiment, the KD value of the variant Fc region for human FcγRIIIa can be 1.0x10 -6 M or more, 2.0x10 -6 M or more, 3.0x10 -6 M or more, 4.0x10 -6 M or more, 5.0x10 -6 M or more, 6.0x10 -6 M or more, 7.0x10 -6 M or more, 8.0x10 -6 M or more, 9.0x10 -6 M or more, 1.0x10 -5 M or more, 2.0x10 -5 M or more, 3.0x10 -5 M or more, 4.0x10 -5 M or more, or 5.0x10 -5 M or more. In another embodiment, the KD value of the variant Fc region for human FcγRIIa (H type) can be 1.0x10 -7 M or more, 2.0x10 -7 M or more, 3.0x10 -7 M or more, 4.0x10 -7 M or more, 5.0x10 -7 M or more, 6.0x10 -7 M or more, 7.0x10 -7 M or more, 8.0x10 -7 M or more, 9.0x10 -7 M or more, 1.0x10 -6 M or more, 2.0x10 -6 M or more, 3.0x10 -6M or more, 4.0x10 -6 M or more, or 5.0x10 -6 M or more. In another embodiment, the KD value of the variant Fc region for human FcγRIIa (R type) can be 2.0x10 -7 M or more, 3.0x10 -7 M or more, 4.0x10 -7 M or more, 5.0x10 -7 M or more, 6.0x10 -7 M or more, 7.0x10 -7 M or more, 8.0x10 -7 M or more, 9.0x10 -7 M or more, 1.0x10 -6 M or more, 2.0x10 -6 M or more, 3.0x10 -6 M or more, 4.0x10 -6 M or more, or 5.0x10 -6 M or more.

[0512] In another embodiment, the KD value of the variant Fc region for monkey FcγRIIa can be 1.0x10 -6 M or less, 9.0x10 -7 M or less, 8.0x10 -7 M or less, 7.0x10 -7 M or less, 6.0x10 -7 M or less, 5.0x10 -7 M or less, 4.0x10 -7 M or less, 3.0x10 -7 M or less, 2.0x10 -7 M or less, or 1.0x10 -7 M or less. In certain embodiments, monkey FcγRIIa can be selected from any one of monkey FcγRIIa1, monkey FcγRIIa2, and monkey FcγRIIa3.

[0513] When developing a drug product for treating human diseases, it is important to evaluate its efficacy and safety in monkeys because the biology of monkeys is similar to that of humans. From this perspective, the drug product to be developed preferably has cross-reactivity to both humans and monkeys in terms of target binding activity.

[0514] "Fcγ receptor" (referred to herein as Fcγ receptor, FcγR or FcgR) refers to a receptor that can bind to the Fc region of monoclonal antibodies of IgG1, IgG2, IgG3, and IgG4, and in fact refers to any member of the protein family encoded by the Fcγ receptor gene. In humans, this family includes FcγRI (CD64), including isotypes FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including isotypes FcγRIIa (including allotypes H131 (H type) and R131 (R type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), including isotypes FcγRIIIa (including allotypes V158 and F158), and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), and any human FcγR, FcγR isotypes or allotypes not yet discovered, but not limited to these. It has been reported that FcγRIIb1 and FcγRIIb2 are splicing variants of human FcγRIIb. In addition, a splicing variant called FcγRIIb3 has been reported (J Exp Med, 1989, 170: 1369-1385). In addition to these splicing variants, human FcγRIIb includes all splicing variants registered in NCBI, which are NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1, and NP_003992.3. In addition, human FcγRIIb includes each genetic polymorphism reported previously, as well as FcγRIIb (Arthritis Rheum. 48:3242-3252 (2003); Kono et al., Hum. Mol. Genet. 14:2881-2892 (2005); and Kyogoju et al., Arthritis Rheum. 46:1242-1254 (2002)), and each genetic polymorphism that will be reported in the future.

[0515] In FcγRIIa, there are two allotypes. In one allotype, the amino acid at position 131 of FcγRIIa is histidine (H type), and in the other allotype, the amino acid at position 131 is replaced by arginine (R type) (Warrmerdam, J. Exp. Med. 172:19-25 (1990)).

[0516] FcγRs include, but are not limited to, FcγRs from humans, mice, rats, rabbits, and monkeys, and can be from any organism. Mouse FcγRs include FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), and any mouse FcγR, or FcγR isotype, but are not limited thereto. Unless otherwise specified, the term "monkey FcγR" or variants 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).

[0517] The polynucleotide sequences of human FcγRI are shown in SEQ ID NO: 199 (NM_000566.3); the polynucleotide sequences of human FcγRIIa are shown in SEQ ID NO: 200 (BC020823.1) or SEQ ID NO: 201 (NM_001136219.1); the polynucleotide sequences of human FcγRIIb are shown in SEQ ID NO: 202 (BC146678.1) or SEQ ID NO: 203 (NM_004001.3); the polynucleotide sequences of human FcγRIIIa are shown in SEQ ID NO: 204 (BC033678.1) or SEQ ID NO: 205 (NM_001127593.1); the polynucleotide sequences of human FcγRIIIb are shown in SEQ ID NO: 206 (BC128562.1).

[0518] The amino acid sequence of human FcγRI is shown in SEQ ID NO: 207 (NP_000557.1); the amino acid sequence of human FcγRIIa is shown 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 shown in SEQ ID NO: 212 (AAI46679.1), SEQ ID NO: 213 or SEQ ID NO: 214; the amino acid sequence of human FcγRIIIa is shown in SEQ ID NO: 215 (AAH33678.1), SEQ ID NO: 216, SEQ ID NO: 217 or SEQ ID NO: 218; the amino acid sequence of human FcγRIIIb is shown in SEQ ID NO: 219 (AAI28563.1).

[0519] The amino acid sequence of cynomolgus macaque FcγRIIa is shown in SEQ ID NO: 220 (FcγRIIa1), SEQ ID NO: 221 (FcγRIIa2) or SEQ ID NO: 222 (FcγRIIa3); the amino acid sequence of cynomolgus macaque FcγRIIb is shown in SEQ ID NO: 223; the amino acid sequence of cynomolgus macaque FcγRIIIa is shown in SEQ ID NO: 224.

[0520] In one aspect, the present invention provides a polypeptide comprising a variant Fc region having enhanced FcγRIIb-binding activity compared to a corresponding reference FcγRIIb-binding polypeptide. In a further aspect, the polypeptide of the present invention comprises at least one amino acid alteration at at least one position selected from the group consisting of: 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 (according to EU numbering). In certain embodiments, FcγRIIb has the sequence of cynomolgus macaque FcγRIIb (SEQ ID NO: 223). In certain embodiments, FcγRIIb has the sequence of human FcγRIIb (e.g., SEQ ID NOS: 212, 213, or 214).

[0521] In one aspect, the present invention provides a polypeptide comprising a variant Fc region having enhanced FcγRIIb-binding activity, the variant Fc region comprising at least two amino acid alterations, the at least two amino acid alterations comprising: (a) one amino acid alteration at position 236, and (b) at least one amino acid alteration at at least one position selected from the group consisting of: 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 (according to 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).

[0522] In one aspect, the present invention provides a polypeptide comprising a variant Fc region having enhanced FcγRIIb-binding activity, the variant Fc region comprising an amino acid alteration at position 236 (according to EU numbering).

[0523] In one aspect, the present invention provides a polypeptide comprising a variant Fc region having enhanced FcγRIIb-binding activity, the variant Fc region comprising at least two amino acid alterations, the at least two amino acid alterations comprising: (a) one amino acid alteration at position 236, and (b) at least one amino acid alteration at at least one position selected from the group consisting of: 231, 232, 235, 239, 268, 295, 298, 326, 330, and 396 (according to EU numbering). In another embodiment, the variant Fc region comprises at least one amino acid alteration at at least one position selected from the group consisting of: 231, 232, 235, 239, 268, 295, 298, 326, 330, and 396 (according to EU numbering). In another embodiment, the variant Fc region comprises at least one amino acid alteration at at least one position selected from the group consisting of: 268, 295, 326, and 330 (according to 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).

[0524] In another aspect, the present invention provides a polypeptide comprising a variant Fc region having enhanced FcγRIIb-binding activity, said variant Fc region comprising an amino acid alteration of any one of the following (1)-(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, 268, 295 and 330; (7) positions 232, 236, 268 and 330; (8) positions 235, 236, 268, 295, 326 and 330; (9) positions 235, 236, 268, 295 and 330; (10) positions 235, 236, 268 and 330; (11) positions 235, 236, 268, 330 and 396; (12) positions 235, 236, 268 and 396; (13) positions 236, 239, 268, 295, 298 and 330; (14) positions 236, 239, 268, 295, 326 and 330; (15) positions 236, 239, 268, 295 and 330; (16) positions 236, 239, 268, 298 and 330; (17) positions 236, 239, 268, 326 and 330; (18) positions 236, 239, 268 and 330; (19) positions 236, 239, 268, 330 and 396; (20) positions 236, 239, 268 and 396; (21) positions 236 and 268; (22) positions 236, 268 and 295; (23) positions 236, 268, 295, 298 and 330; (24) positions 236, 268, 295, 326 and 330; (25) positions 236, 268, 295, 326, 330 and 396; (26) positions 236, 268, 295 and 330; (27) positions 236, 268, 295, 330 and 396; (28) positions 236, 268, 298 and 330; (29) positions 236, 268, 298 and 396; (30) positions 236, 268, 326 and 330; (31) positions 236, 268, 326, 330 and 396; (32) positions 236, 268 and 330; (33) positions 236, 268, 330 and 396; (34) positions 236, 268 and 396; (35) positions 236 and 295; (36) positions 236, 330 and 396; and (37) positions 236 and 396 (according to EU numbering).In certain embodiments, FcγRIIb has the sequence of cynomolgus macaque FcγRIIb (SEQ ID NO: 223). In certain embodiments, FcγRIIb has the sequence of human FcγRIIb (e.g., SEQ ID NO: 212, 213, or 214).

[0525] In another embodiment, a variant Fc region having 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, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr 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, 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) Thr at position 326; (s) Ile, Asn at position 327; (t) Thr at position 328; (u) Lys, Arg at position 330; (v) Glu at position 331; (w) Asp at position 332; (x) Asp, Ile, Met, Val, Tyr at position 334; and (y) Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 396 (according to 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).

[0526] In another embodiment, a variant Fc region having enhanced FcγRIIb-binding activity comprises at least one amino acid alteration (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 (according to EU numbering). In another embodiment, a variant Fc region having enhanced FcγRIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): Asn at position 236, Glu at position 268, Lys at position 330, and Met at position 396 (according to EU numbering). In another embodiment, a variant Fc region having enhanced FcγRIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): Asn at position 236, Asp at position 268, and Lys at position 330 (according to EU numbering). In another embodiment, a variant Fc region having enhanced FcγRIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): Asn at position 236, Asp at position 268, Leu at position 295, and Lys at position 330 (according to EU numbering). In another embodiment, a variant Fc region having enhanced FcγRIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): Thr at position 236, Asp at position 268, and Lys at position 330 (according to EU numbering). In another embodiment, a variant Fc region having enhanced FcγRIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): Asn at position 236, Asp at position 268, Leu at position 295, Thr at position 326, and Lys at position 330 (according to EU numbering). In another embodiment, a variant Fc region having enhanced FcγRIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): 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 (according to EU numbering).

[0527] In one aspect, the present invention provides a polypeptide comprising a variant Fc region having enhanced FcγRIIb-binding activity, said variant Fc region comprising an amino acid alteration at position 238 (according to EU numbering).

[0528] In one aspect, the present invention provides a polypeptide comprising a variant Fc region having enhanced FcγRIIb-binding activity, said variant Fc region comprising at least one amino acid alteration at at least one position selected from the group consisting of: 234, 238, 250, 264, 267, 307, and 330 (according to EU numbering). In additional embodiments, the polypeptide comprises at least one amino acid alteration at at least one position selected from the group consisting of: 234, 250, 264, 267, 307, and 330 (according to 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).

[0529] In another aspect, the present invention provides a polypeptide comprising a variant Fc region having enhanced FcγRIIb-binding activity, said variant Fc region comprising an amino acid alteration selected from any of (1)-(9) below: (1) positions 234, 238, 250, 307 and 330; (2) positions 234, 238, 250, 264, 307 and 330; (3) positions 234, 238, 250, 264, 267, 307 and 330; (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 (according to 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).

[0530] In another embodiment, a variant Fc region having enhanced FcγRIIb-binding activity comprises at least one amino acid alteration (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 (according to EU numbering). In another embodiment, a variant Fc region having enhanced FcγRIIb-binding activity comprises the following amino acid alteration (e.g., substitution): Asp at position 238 (according to EU numbering). In another embodiment, a variant Fc region having enhanced FcγRIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): Asp at position 238, Val at position 250, and Pro at position 307 (according to EU numbering). In another embodiment, a variant Fc region having enhanced FcγRIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): Asp at position 238, Val at position 250, Pro at position 307, and Lys at position 330 (according to EU numbering). In another embodiment, a variant Fc region having enhanced FcγRIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): Asp at position 238, Val at position 250, Ile at position 264, Pro at position 307, and Lys at position 330 (according to EU numbering). In another embodiment, a variant Fc region having enhanced FcγRIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): Asp at position 238, Val at position 250, Ala at position 267, Pro at position 307, and Lys at position 330 (according to EU numbering). In another embodiment, a variant Fc region having enhanced FcγRIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): Tyr at position 234, Asp at position 238, Val at position 250, Pro at position 307, and Lys at position 330 (according to EU numbering). In another embodiment, a variant Fc region having enhanced FcγRIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): 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 (according to EU numbering).In another embodiment, a variant Fc region having enhanced FcγRIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): 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 (according to EU numbering). In another embodiment, a variant Fc region having enhanced FcγRIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): 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 (according to EU numbering). In another embodiment, a variant Fc region having enhanced FcγRIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): 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 (according to 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).

[0531] In another aspect, the invention provides an isolated polypeptide comprising a variant Fc region having an increased isoelectric point (pI). In certain embodiments, the variant Fc region described herein comprises at least two amino acid alterations in the parental Fc region. In certain embodiments, each amino acid alteration results in an increase in the isoelectric point (pI) of the variant Fc region compared to the isoelectric point (pI) of the parental Fc region. This is based on the discovery that, for example, when an antibody is administered in vivo, an antibody having an increased pI by modifying at least two amino acid residues can promote the elimination of antigen from the plasma.

[0532] In the present invention, the pI can be a theoretical or experimentally determined pI. The pI value can be determined, for example, by isoelectric focusing known to those skilled in the art. The theoretical pI value can be calculated, for example, using gene and amino acid sequence analysis software (Genetyx, etc.).

[0533] In one embodiment, compared to before modification, the pI value can 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.

[0534] In certain embodiments, the amino acids that result in an increase in pI can be exposed on the surface of the variant Fc region. In the present invention, the amino acids that can be exposed on the surface generally refer to the amino acid residues located on the surface of the polypeptide constituting the variant Fc region. The amino acid residues located on the surface of the polypeptide refer to the amino acid residues having side chains that can contact solvent molecules (which are usually mostly water molecules). However, the side chains do not necessarily need to be in complete contact with the solvent molecules, and even when only a part of the side chain contacts the solvent molecules, the amino acid is defined as an "amino acid residue located on the surface". The amino acid residues located on the surface of the polypeptide also include the amino acid residues close to the surface and thus may be affected by the charge of another side chain or even an amino acid residue that only partially contacts the solvent molecules. For example, using commercially available software, those skilled in the art can prepare a homology model of the polypeptide. Alternatively, it is possible to use methods known to those skilled in the art, such as X-ray crystallography. For example, the amino acid residues that may be exposed on the surface are determined using the coordinates from a three-dimensional model using a computer program such as the InsightII program (Accelrys). The surface-exposed sites can be determined using algorithms known in the art (e.g., Lee and Richards (J. Mol. Biol. 55:379-400 (1971)); Connolly (J. Appl. Cryst. 16:548-558 (1983))). The surface-exposed sites can be determined using software suitable for protein modeling and three-dimensional structure information. Software that can be used for such purposes includes, for example, the SYBYL Biopolymer Module software (Tripos Associates). When the algorithm requires the user to input a size parameter, the "size" of the probe used for calculation can be set to a radius of about 1.4 Å or less. In addition, a method for determining the surface-exposed region using software for a personal computer has been described by Pacios (Comput. Chem. 18(4):377-386 (1994); J. Mol. Model. 1:46-53 (1995)). Based on the above information, suitable amino acid residues located on the surface of the polypeptide constituting the variant Fc region can be selected.

[0535] In certain embodiments, the polypeptide comprises both a variant Fc region and an antigen-binding domain. In additional embodiments, the antigen is a soluble antigen. In one embodiment, the antigen is present in a biological fluid of a subject (e.g., plasma, interstitial fluid, lymphatic fluid, ascitic fluid, and pleural fluid). The antigen can also be a membrane antigen.

[0536] In additional embodiments, the antigen-binding activity of the antigen-binding domain varies according to ionic concentration conditions. In one embodiment, the ionic concentration is not particularly limited and refers to the hydrogen ion concentration (pH) or the metal ion concentration. Herein, the metal ion refers to elements of Group I other than hydrogen such as alkali metals and elements of the copper group, elements of Group II such as alkaline earth metals and elements of the zinc group, elements of Group III other than boron, elements of Group IV other than carbon and silicon, elements of Group VIII such as the iron group and the platinum group, elements belonging to the A subgroups of Groups V, VI, and VII, and ions of metal elements such as antimony, bismuth, and polonium. In the present invention, the metal ion includes, for example, calcium ions, as described in WO 2012 / 073992 and WO 2013 / 125667. In one embodiment, the "ionic concentration conditions" can be conditions that focus on the difference in the biological performance of the antigen-binding domain between low and high ionic concentrations. Furthermore, "the antigen-binding activity of the antigen-binding domain changes according to ionic concentration conditions" means that the antigen-binding activity of the antigen-binding domain varies between low and high ionic concentrations (such an antigen-binding domain is referred to herein as an "ion concentration-dependent antigen-binding domain"). The antigen-binding activity of the antigen-binding domain under high ionic concentration conditions can be higher (stronger) or lower (weaker) than that under low ionic concentration conditions. In one embodiment, an ion concentration-dependent antigen-binding domain (such as a pH-dependent antigen-binding domain or a calcium ion concentration-dependent antigen-binding domain) can be obtained by known methods described, for example, in WO 2009 / 125825, WO 2012 / 073992, and WO 2013 / 046722.

[0537] In the present invention, the antigen-binding activity of the antigen-binding domain under high calcium ion concentration conditions can be higher than that under low calcium ion concentration conditions. The high calcium ion concentration is not particularly limited but can be a concentration selected between 100 μM and 10 mM, between 200 μM and 5 mM, between 400 μM and 3 mM, between 200 μM and 2 mM, between 400 μM and 1 mM, or between 500 μM and 2.5 mM, and the concentration is preferably close to the plasma (blood) concentration of calcium ions in vivo. At the same time, the low calcium ion concentration is not particularly limited but can be a concentration selected between 0.1 μM and 30 μM, between 0.2 μM and 20 μM, between 0.5 μM and 10 μM, between 1 μM and 5 μM, or between 2 μM and 4 μM, and the concentration is preferably close to the calcium ion concentration in early endosomes in vivo.

[0538] In one embodiment, the ratio of the 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 KD under high calcium ion concentration conditions, that is, KD (low calcium ion concentration conditions) / KD (high calcium ion concentration conditions), is 2 or greater, 10 or greater, or 40 or greater. The upper limit of the ratio can be 400, 1000, or 10000, as long as such an antigen-binding domain can be prepared by techniques known to those skilled in the art. 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, that is, kd (low calcium ion concentration conditions) / kd (high calcium ion concentration conditions), is 2 or greater, 5 or greater, 10 or greater, or 30 or greater. The upper limit of the ratio can be 50, 100, or 200, as long as the antigen-binding domain can be prepared based on the conventional technical knowledge of those skilled in the art.

[0539] In the present invention, the antigen-binding activity of the antigen-binding domain at a low hydrogen ion concentration (neutral pH) can be higher than that at a high hydrogen ion concentration (acidic pH). The acidic pH can be, for example, a pH selected from pH 4.0 to pH 6.5, selected from pH 4.5 to pH 6.5, selected from pH 5.0 to pH 6.5, or selected from pH 5.5 to pH 6.5, and the pH is preferably close to the pH in early endosomes in vivo. The acidic pH can also be, for example, pH 5.8 or pH 6.0. In a particular embodiment, the acidic pH is pH 5.8. Meanwhile, the neutral pH can be, for example, a pH selected from pH 6.7 to pH 10.0, selected from pH 6.7 to pH 9.5, selected from pH 7.0 to pH 9.0, or selected from pH 7.0 to pH 8.0, and the pH is preferably close to the pH in plasma (blood) in vivo. The neutral pH can also be, for example, pH 7.4 or pH 7.0. In a particular embodiment, the neutral pH is pH 7.4.

[0540] In one embodiment, the ratio of the antigen-binding activity under acidic pH conditions to that under neutral pH conditions is not limited, but the ratio of the dissociation constant (KD) under acidic pH conditions to the KD under neutral pH conditions, i.e., KD (acidic pH conditions) / KD (neutral pH conditions), is 2 or greater, 10 or greater, or 40 or greater. The upper limit of this ratio can be 400, 1000, or 10000, as long as such an antigen-binding domain can be prepared by techniques known to those skilled in the art. 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 greater, 5 or greater, 10 or greater, or 30 or greater. The upper limit of this ratio can be 50, 100, or 200, as long as the antigen-binding domain can be prepared based on the conventional technical knowledge of those skilled in the art.

[0541] In one embodiment, for example, at least one amino acid residue is replaced with an amino acid residue having a side chain pKa of 4.0 - 8.0, and / or at least one amino acid having a side chain pKa of 4.0 - 8.0 is inserted into the antigen-binding domain, as described in WO2009 / 125825. The amino acid can be replaced and / or inserted at any site as long as the antigen-binding activity of the antigen-binding domain is weaker at acidic pH conditions than at neutral pH conditions compared to before the replacement or insertion. When the antigen-binding domain has a variable region or CDR, the site can be within the variable region or CDR. The number of amino acids to be replaced or inserted can be appropriately determined by those skilled in the art; and the number can be more than one. Depending on the hydrogen ion concentration conditions, amino acids having a side chain pKa of 4.0 - 8.0 can be used to alter the antigen-binding activity of the antigen-binding domain. Such amino acids include, for example, natural amino acids such as His (H) and Glu (E), and 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 having a side chain pKa of 6.0 - 7.0 can also be used, which include, for example, His (H).

[0542] In another embodiment, preferred antigen-binding domains for variant Fc regions having an increased pI are described in Japanese Patent Applications JP2015 - 021371 and JP2015 - 185254 and can be obtained by the methods described therein.

[0543] In certain embodiments, the variant Fc region having an increased pI contains at least two amino acid alterations at at least two positions selected from the group consisting of: 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422, and 431 (according to EU numbering).

[0544] In additional embodiments, the variant Fc region having an increased pI contains at least two amino acid alterations at at least two positions selected from the group consisting of: 311, 341, 343, 384, 399, 400, 401, 402, and 413 (according to EU numbering).

[0545] In another aspect, the present invention provides a polypeptide comprising a variant Fc region having an increased pI, said variant Fc region comprising an amino acid alteration of any one of the following (1)-(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 (according to EU numbering).

[0546] Methods for increasing the pI of a protein are, for example, to reduce the number of amino acids having a negatively charged side chain (e.g., aspartic acid and glutamic acid) under neutral pH conditions and / or to increase the number of amino acids having a positively charged side chain (e.g., arginine, lysine, and histidine) under neutral pH conditions. Amino acids having a negatively charged side chain have a negative charge represented as -1 under pH conditions sufficiently higher than the pKa of their side chain, which is a theory 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 at pH 7.0). Conversely, amino acids having a positively charged side chain have a positive charge represented as +1 under pH conditions sufficiently lower than the pKa of their side chain. 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 at pH 7.0). At the same time, amino acids known to have no charge on their side chains under neutral pH conditions (e.g., in a solution at pH 7.0) include 15 types of natural amino acids, namely, alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, aspartic acid, proline, glutamine, serine, threonine, valine, tryptophan, and tyrosine. Of course, it is to be understood that the amino acids used to increase the pI can be non-natural amino acids.

[0547] From the above, a method for increasing the pI of a protein under neutral pH conditions (e.g., in a solution at pH 7.0) can impart a charge change of +1 to the protein of interest. For example, by substituting aspartic acid or glutamic acid (whose side chains have a negative charge of -1) with an amino acid having an uncharged side chain in the protein amino acid sequence. Additionally, for example, a charge change of +1 can be imparted to the protein by substituting an amino acid with an uncharged side chain with arginine or lysine (whose side chains have a positive charge of +1). Further, a charge change of +2 can be imparted to the protein at once by substituting aspartic acid or glutamic acid (whose side chains have a negative charge of -1) with arginine or lysine (whose side chains have a positive charge of +1). Alternatively, to increase the pI of a protein, an amino acid with an uncharged side chain and / or preferably an amino acid with a positively charged side chain can be added or inserted into the amino acid sequence of the protein, or an amino acid with an uncharged side chain and / or preferably an amino acid with a negatively charged side chain can be deleted from the amino acid sequence of the protein. It is understood that, for example, the N-terminal and C-terminal amino acid residues of a protein have charges from the main chain in addition to the charges from their side chains (the NH3 of the amino group at the N-terminal + and the COO of the carbonyl group at the C-terminal - ). Thus, the pI of a protein can also be increased by making some additions, deletions, substitutions, or insertions to the functional groups from the main chain.

[0548] Amino acid substitutions for increasing pI include, for example, in the amino acid sequence of the parental Fc region, substituting an amino acid with an uncharged side chain for an amino acid with a negatively charged side chain, substituting an amino acid with a positively charged side chain for an amino acid with an uncharged side chain, and substituting an amino acid with a positively charged side chain for an amino acid with a negatively charged side chain, which are carried out alone or in a suitable combination.

[0549] Amino acid insertions or additions for increasing pI include, for example, in the amino acid sequence of the parental Fc region, inserting or adding an amino acid with an uncharged side chain, and / or inserting or adding an amino acid with a positively charged side chain, which are carried out alone or in a suitable combination.

[0550] Amino acid deletions for increasing pI include, for example, in the amino acid sequence of the parental Fc region, deleting an amino acid with an uncharged side chain, and / or deleting an amino acid with a negatively charged side chain, which are carried out alone or in a suitable combination.

[0551] In one embodiment, the natural amino acids used to increase the pI can be classified as follows: (a) Amino acids with negatively charged side chains can be Glu (E) or Asp (D); (b) Amino acids with uncharged side chains 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 positively charged side chains can be His (H), Lys (K), or Arg (R). In one embodiment, the modified amino acid insertion or substitution is Lys (K) or Arg (R).

[0552] In another aspect, the invention provides an isolated polypeptide comprising a variant Fc region having enhanced FcγRIIb-binding activity and an increased pI. In certain embodiments, the variant Fc region described herein comprises at least two amino acid alterations in the parental Fc region.

[0553] In one aspect, the invention provides a polypeptide comprising a variant Fc region having enhanced FcγRIIb-binding activity and an increased pI, the variant Fc region comprising at least three amino acid alterations comprising: (a) at least one amino acid alteration at at least one position selected from the group consisting of 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 (according to EU numbering), and (b) at least two amino acid alterations at at least two positions selected from the group consisting of 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422, and 431 (according to EU numbering).

[0554] In one aspect, the present invention provides a polypeptide comprising a variant Fc region having enhanced FcγRIIb-binding activity and an increased pI, and the variant Fc region comprises at least three amino acid alterations comprising: (a) at least one amino acid alteration at at least one position selected from the group consisting of: 231, 232, 235, 236, 239, 268, 295, 298, 326, 330, and 396 (according to EU numbering), and (b) at least two amino acid alterations at at least two positions selected from the group consisting of: 311, 341, 343, 384, 399, 400, 401, 402, and 413 (according to EU numbering).

[0555] In another aspect, the present invention provides a polypeptide comprising a variant Fc region having enhanced FcγRIIb-binding activity and an increased pI, the variant Fc region comprising an amino acid alteration of any one of (1)-(9) below: (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, 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 (according to 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).

[0556] In one aspect, the present invention provides a polypeptide comprising a variant Fc region having enhanced FcγRIIb-binding activity and an increased pI, the variant Fc region comprising at least three amino acid alterations comprising: (a) at least one amino acid alteration at at least one position selected from the group consisting of: 234, 238, 250, 264, 267, 307, and 330, and (b) at least two amino acid alterations at at least two positions selected from the group consisting of: 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422, and 431 (according to EU numbering). In additional embodiments, the polypeptide comprises at least two amino acid alterations at at least two positions selected from the group consisting of: 311, 341, 343, 384, 399, 400, 401, 402, and 413 (according to 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).

[0557] In another aspect, the present invention provides a polypeptide comprising a variant Fc region having enhanced FcγRIIb-binding activity and an increased pI, the variant Fc region comprising an amino acid alteration selected from any of the following (1)-(16): (1) positions 234, 238, 250, 264, 307, 311, 330, and 343; (2) positions 234, 238, 250, 264, 307, 311, 330, and 413; (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 (according to EU numbering).

[0558] In another embodiment, the variant Fc region comprises an amino acid alteration selected from any single alteration, combination of single alterations, or combined alteration described in Table 14-30.

[0559] In some embodiments, the polypeptide comprises the variant Fc region of the present invention. In another embodiment, the polypeptide is an antibody heavy chain constant region. In another embodiment, the polypeptide is an antibody heavy chain. In another embodiment, the polypeptide is an antibody. In another embodiment, the polypeptide is an Fc fusion protein.

[0560] In another embodiment, the present invention provides a polypeptide comprising an amino acid sequence selected from any of SEQ ID NOs: 229-381.

[0561] As used herein, "parent Fc region" refers to the Fc region prior to introduction of the amino acid alterations described herein. Preferred examples of the parent Fc region include Fc regions derived from native antibodies. Antibodies include, for example, IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), and IgM, among others. Antibodies can be derived from human or monkey (e.g., cynomolgus monkey, rhesus monkey, marmoset, chimpanzee, or baboon). Native antibodies can also include naturally occurring mutations. The various allotypic sequences of IgG due to genetic polymorphisms are described in "Sequences of proteins of immunological interest", NIH publication No. 91-3242, and any of them can be used in the present invention. In particular, for human IgG1, the amino acid sequence at positions 356 to 358 (EU numbering) can be 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 the Fc region derived from the heavy chain constant region SG1 (SEQ ID NO: 9). In addition, the parent Fc region can be an Fc region generated by adding amino acid alterations different from those described herein to an Fc region derived from a native antibody.

[0562] In addition, amino acid changes made for other purposes can be incorporated into the variant Fc regions described herein. For example, amino acid substitutions that enhance FcRn-binding activity can be added (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); WO 2006 / 019447; WO 2006 / 053301; and WO 2009 / 086320), as well as amino acid substitutions for enhancing antibody heterogeneity or stability (WO 2009 / 041613). Alternatively, polypeptides having properties that promote antigen clearance described in WO 2011 / 122011, WO2012 / 132067, WO 2013 / 046704 or WO 2013 / 180201, polypeptides having properties that specifically bind to target tissues described in WO 2013 / 180200, polypeptides having properties that repeatedly bind to multiple antigen molecules described in WO 2009 / 125825, WO2012 / 073992 or WO 2013 / 047752 can be combined with the variant Fc regions described herein. Alternatively, amino acid changes disclosed in EP1752471 and EP1772465 can be incorporated into the CH3 of the variant Fc regions described herein in order to confer binding ability to other antigens. Alternatively, amino acid changes that lower the pI of the constant region (WO 2012 / 016227) can be incorporated into the variant Fc regions described herein in order to increase plasma retention. Alternatively, amino acid changes that increase the pI of the constant region (WO 2014 / 145159) can be incorporated into the variant Fc regions described herein in order to facilitate uptake into cells. Alternatively, amino acid changes that increase the pI of the constant region (Japanese Patent Application Nos. JP2015-021371 and JP2015-185254) can be incorporated into the variant Fc regions described herein in order to promote elimination of the target molecule from the plasma. In one embodiment, such changes can include, for example, substitutions at at least one position selected from the following: 311, 343, 384, 399, 400, and 413 (according to EU numbering). In another embodiment, such substitutions can be replacement of the amino acid with Lys or Arg at each position.

[0563] Amino acid alterations that enhance human FcRn-binding activity at acidic pH can also be incorporated into the variant Fc regions described herein. Specifically, such alterations can include, for example, a Leu substitution for Met at position 428 and a Ser substitution for Asn at position 434 according to EU numbering (Zalevsky et al., Nat. Biotechnol. 28:157-159 (2010)); an Ala substitution for Asn at position 434 (Deng et al., Metab. Dispos. 38(4):600-605 (2010)); a Tyr substitution for Met at position 252, a Thr substitution for Ser at position 254, and a Glu substitution for Thr at position 256 (Dall'Acqua et al., J. Biol. Chem. 281:23514-23524 (2006)); a Gln substitution for Thr at position 250 and a Leu substitution for Met at position 428 (Hinton et al., J. Immunol. 176(1):346-356 (2006)); a His substitution for Asn at position 434 (Zheng et al., Clin. Pharmacol. Ther. 89(2):283-290 (2011), as well as the alterations described in WO 2010 / 106180, WO 2010 / 045193, WO 2009 / 058492, WO 2008 / 022152, WO 2006 / 050166, WO 2006 / 053301, WO 2006 / 031370, WO 2005 / 123780, WO2005 / 047327, WO 2005 / 037867, WO 2004 / 035752, or WO 2002 / 060919. Such alterations can include, for example, at least one alteration selected from: a Leu substitution for Met at position 428, an Ala substitution for Asn at position 434, and a Thr substitution for Tyr at position 436. The alterations can also include an Arg substitution for Gln at position 438 and / or a Glu substitution for Ser at position 440 (Japanese Patent Application Nos. JP2015-021371 and JP2015-185254).

[0564] Two or more polypeptides comprising the variant Fc regions described herein can be included in a molecule, wherein two polypeptides comprising the variant Fc regions are associated, much like in an antibody. The type of antibody is not restricted, and IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), and IgM, etc. can be used.

[0565] The two combined polypeptides comprising variant Fc regions can be polypeptides comprising variant Fc regions in which the same amino acid changes have been introduced (hereinafter referred to as homologous variant Fc regions), or polypeptides comprising variant Fc regions in which different amino acid changes have been introduced, or alternatively polypeptides comprising variant Fc regions in which the amino acid changes have been introduced only into one Fc region (hereinafter referred to as heterologous polypeptides comprising variant Fc regions). One of the preferred amino acid changes is an alteration in the loop structure at positions 233 to 239 (EU numbering) in the CH2 domain of the Fc region that is involved in binding to FcγRIIb and FcγRIIa. Preferably, one change that enhances FcγRIIb-binding activity and / or selectivity is introduced into the loop structure of the CH2 domain of one Fc region, while another destabilizing change is introduced into the loop structure of the CH2 domain of the other Fc region. Examples of amino acid changes that can destabilize the loop structure of the CH2 domain can be at least one amino acid substitution of an amino acid selected from positions 235, 236, 237, 238, and 239 with another amino acid. Specifically, it can be destabilized, for example, by changing the amino acid at position 235 to Asp, Gln, Glu, or Thr, the amino acid at position 236 to Asn, the amino acid at position 237 to Phe or Trp, the amino acid at position 238 to Glu, Gly, or Asn, and the amino acid at position 239 to Asp or Glu (according to EU numbering).

[0566] To bind heterologous polypeptides comprising variant Fc regions, techniques can be applied that inhibit the binding of unwanted homologous polypeptides comprising variant Fc regions by introducing electrostatic repulsion at the interface of the CH2 or CH3 domain of the Fc region, as described in WO2006 / 106905.

[0567] Examples of amino acid residues that come into contact at the interface of the CH2 or CH3 domain of the Fc region include, in the CH3 domain, the residue at position 356 (EU numbering), the residue at position 439 (EU numbering), the residue at position 357 (EU numbering), the residue at position 370 (EU numbering), the residue at position 399 (EU numbering), and the residue at position 409 (EU numbering).

[0568] More specifically, for example, an Fc region can be prepared in which one to three pairs of amino acid residues selected from the following shown (1) to (3) have the same charge: (1) the amino acid residues at positions 356 and 439 (EU numbering) in the CH3 domain; (2) the amino acid residues at positions 357 and 370 (EU numbering) in the CH3 domain; and (3) the amino acid residues at positions 399 and 409 (EU numbering) in the CH3 domain.

[0569] In addition, heterologous polypeptides comprising variant Fc regions can be prepared, wherein one to three pairs of amino acid residues selected from (1) to (3) as shown above have the same charge in the CH3 domain of the first Fc region, and the pair of amino acid residues selected in the first Fc region also have the same charge in the CH3 domain of the second Fc region, provided that the charges in the first and second Fc regions are opposite.

[0570] In the Fc regions mentioned above, 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).

[0571] In addition, other known techniques can be used for the heterologous binding of polypeptides comprising variant Fc regions. Specifically, such techniques are carried out by replacing the amino acid side chain present in one Fc region with a larger side chain (knob; which refers to a "bulging part"), and replacing the amino acid side chain present in the Fc region with a smaller side chain (hole; which refers to an "empty part") to place the knob inside the hole. This can promote the effective binding of Fc region-containing polypeptides having different amino acid sequences to each other (WO 1996 / 027011; Ridgway et al., Prot. Eng. 9:617-621 (1996); Merchant et al., Nat. Biotech. 16, 677-681 (1998)).

[0572] In addition, other known techniques can also be used for the heterologous binding of polypeptides comprising variant Fc regions. The binding of Fc region-containing polypeptides can be effectively introduced using a chain-exchanged engineered domain CH3 heterodimer (Davis et al., Prot. Eng. Des. & Sel., 23:195-202 (2010)). This technique can also be used to effectively introduce the binding between Fc region-containing polypeptides having different amino acid sequences.

[0573] In addition, the heterodimeric antibody preparation technique described in WO 2011 / 028952 that utilizes the binding of antibody CH1 and CL and the binding of VH and VL can also be used.

[0574] Regarding the methods described in WO 2008 / 119353 and WO 2011 / 131746, it is also possible to use the technique for preparing heterodimeric antibodies by preparing two types of homodimeric antibodies in advance, incubating the antibodies under reducing conditions to dissociate them, and allowing them to bind again.

[0575] Regarding the method described in Strop (J. Mol. Biol. 420:204-219 (2012)), it is also possible to use techniques for preparing heterodimeric antibodies by introducing electrostatic repulsion into the CH3 domain by introducing charged residues such as Lys, Arg, Glu, and Asp.

[0576] In addition, regarding the method described in WO 2012 / 058768, it is also possible to use techniques for preparing heterodimeric antibodies by adding modifications to the CH2 and CH3 domains.

[0577] When two polypeptides each containing a variant Fc region with a different amino acid sequence are co-expressed to prepare a polypeptide containing a heterologous variant Fc region, polypeptides containing a homologous variant Fc region are also typically produced as impurities. In such cases, the polypeptide containing the heterologous variant Fc region can be effectively obtained by separating it from the polypeptide containing the homologous variant Fc region using known techniques. Methods for effectively separating and purifying heterodimeric antibodies from homologous dimeric antibodies by using ion exchange chromatography and introducing amino acid changes into the variable regions of the two types of antibody heavy chains to create an isoelectric point difference between the homologous dimeric antibody and the heterodimeric antibody have been reported (WO 2007 / 114325). Another method for purifying heterodimeric antibodies by using protein A chromatography by constructing a heterodimeric antibody containing two types of heavy chains derived from mouse IgG2a that binds to protein A and rat IgG2b that does not bind to protein A has been reported (WO 1998 / 050431 and WO 1995 / 033844).

[0578] In addition, heterodimeric antibodies can be effectively purified using protein A chromatography by replacing the amino acid residues at positions 435 and 436 (EU numbering) in the protein A binding site of the antibody heavy chain with amino acids such as Tyr or His to create different protein A binding affinities.

[0579] In the present invention, an amino acid change means any one of substitution, deletion, addition, insertion, and modification, or a combination thereof. In the present invention, an amino acid change may be expressed as an amino acid mutation.

[0580] When substituting an amino acid residue, the substitution with a different amino acid residue may be carried out to change aspects such as (a)-(c) below: (a) the polypeptide backbone structure in a beta-sheet structure or a helical structure region; (b) the charge or hydrophobicity at the target site; or (c) the size of the side chain.

[0581] Amino acid residues are classified into the following groups based on their overall 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.

[0582] Amino acid alterations are generated by a variety of methods known to those of skill in the art. Such methods include 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, but are not limited thereto.

[0583] The number of amino acid alterations introduced into the Fc region is not limited. In certain embodiments, it can 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.

[0584] Amino acid modifications include post-translational modifications. Specific post-translational modifications can be the addition or deletion of sugar chains. For example, the amino acid residue at position 297 (EU numbering) in the IgG1 constant region can be glycosylation-modified. The sugar chain structure for modification is not limited. For example, sialic acid can be added to the sugar chain 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, it is known that some types of sugar chains are commonly added to antibodies expressed in cells such as antibody-producing cells of naturally occurring mammals or eukaryotic cells transformed with an expression vector containing DNA encoding an antibody.

[0585] The eukaryotic cells shown herein include yeast and animal cells. For example, CHO cells and HEK293 cells are representative animal cells used for transformation with an expression vector containing DNA encoding an antibody. On the other hand, constant regions that are not glycosylated are also included in the present invention. Antibodies having a non-glycosylated constant region can be obtained by expressing an antibody-encoding gene in a prokaryotic cell such as Escherichia coli.

[0586] In addition, polypeptides comprising the variant Fc region of the present invention can be chemically modified with a variety of molecules such as polyethylene glycol (PEG) and cytotoxic substances. Methods for such chemical modification of polypeptides are well established in the art.

[0587] In one aspect, the present invention provides an isolated polypeptide comprising a variant Fc region having enhanced FcγRIIb-binding activity. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In one aspect, the present invention provides an isolated polypeptide comprising a variant Fc region having enhanced FcγRIIb-binding activity. In some aspects, the polypeptide is an antibody. In certain embodiments, the antibody is a chimeric antibody or a humanized antibody. The source 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.

[0588] The variable region of an antibody comprising the variant Fc region provided herein and the protein-binding motif of an Fc fusion protein comprising the variant Fc region can recognize any antigen. Examples of antigens that can be bound by such antibodies and fusion proteins include, but are not limited to, ligands (cytokines, chemokines, etc.), receptors, cancer antigens, MHC antigens, differentiation antigens, immunoglobulins, and immune complexes containing immunoglobulins in part.

[0589] Examples of cytokines that can be bound by an antibody or fusion protein comprising the variant Fc region of the present invention and / or recombinantly fused with a polypeptide comprising the disclosed variant Fc region include, but are not limited to, interleukin 1 to 18, colony stimulating factors (G-CSF, M-CSF, GM-CSF, etc.), interferons (IFN-α, IFN-β, IFN-γ, etc.), growth factors (EGF, FGF, IGF, NGF, PDGF, TGF, HGF, etc.), tumor necrosis factors (TNF-α and TNF-β), lymphotoxin, erythropoietin, leptin, SCF, TPO, MCAF, and BMP.

[0590] Examples of chemokines that can be bound by an antibody or fusion protein containing a variant Fc region of the present invention and / or recombinantly fused to a polypeptide comprising the disclosed variant Fc region include, but are not limited to, CC chemokines such as CCL1 to CCL28, CXC chemokines such as CXCL1 to CXCL17, C chemokines such as XCL1 to XCL2, and CX3C chemokines such as CX3CL1.

[0591] Examples of receptors that can be bound by an antibody or fusion protein containing a variant Fc region of the present invention and / or recombinantly fused to a polypeptide comprising the disclosed variant Fc region include, but are not limited to, receptors belonging to receptor families such as the hematopoietic growth factor receptor family, cytokine receptor family, tyrosine kinase type receptor family, serine / threonine kinase type receptor family, TNF receptor family, G protein-coupled receptor family, GPI-anchored type receptor family, tyrosine phosphatase type receptor family, adhesion factor family, and hormone receptor family. Receptors belonging to these receptor families and their characteristics have been described in many documents, such as 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. (Cell 76(6):959-962 (1994)); and Flower (Biochim. Biophys. Acta 1422(3): 207-234 (1999)).

[0592] Examples of specific receptors belonging to the receptor families mentioned above include the human or murine erythropoietin (EPO) receptor (Jones et al., Blood 76(1):31-35 (1990); D'Andrea et al., Cell 57(2):277-285 (1989)), the human or murine granulocyte colony-stimulating factor (G-CSF) receptor (Fukunaga et al., Proc. Natl. Acad. Sci. USA 87(22):8702-8706 (1990), mG-CSFR; Fukunaga et al., Cell 61(2): 341-350 (1990)), the human or murine thrombopoietin (TPO) receptor (Vigon et al., Proc. Natl. Acad. Sci. USA. 89(12):5640-5644 (1992); Skoda et al., EMBO J. 12(7):2645-2653 (1993)), the human or murine insulin receptor (Ullrich et al., Nature 313(6005):756-761 (1985)), the human or murine Flt-3 ligand receptor (Small et al., Proc. Natl. Acad. Sci. USA. 91(2):459-463 (1994)), the human or murine platelet-derived growth factor (PDGF) receptor (Gronwald et al., Proc. Natl. Acad. Sci. USA. 85(10):3435-3439 (1988)), the human or murine interferon (IFN)-α and β receptor (Uze et al., Cell 60(2):225-234 (1990); Novick et al., Cell 77(3):391-400 (1994)), the human or murine leptin receptor, the human or murine growth hormone (GH) receptor, the human or murine interleukin (IL)-10 receptor, the human or murine insulin-like growth factor (IGF)-I receptor, the human or murine leukemia inhibitory factor (LIF) receptor, and the human or murine ciliary neurotrophic factor (CNTF) receptor.

[0593] Cancer antigens are antigens that are expressed when cells become malignant, and they are also referred to as tumor-specific antigens. Abnormal sugar chains that appear on the cell surface or protein molecules when cells become cancer cells are also cancer antigens, and they are also referred to as carbohydrate cancer antigens. Examples of cancer antigens that can be bound by an antibody or fusion protein comprising a variant Fc region of the present invention include, but are not limited to, GPC3, which is a receptor belonging to the GPI-anchored receptor family mentioned above and is also expressed in various cancers including liver cancer (Midorikawa et al., Int. J. Cancer 103(4):455-465 (2003)), and EpCAM, which is expressed in various 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).

[0594] MHC antigens are roughly classified into MHC class I antigens and MHC class II antigens. MHC class I antigens include HLA-A, -B, -C, -E, -F, -G, and -H, and MHC class II antigens include HLA-DR, -DQ, and -DP.

[0595] Examples of differentiation antigens that can be bound by an antibody or fusion protein comprising a variant Fc region of the present invention and / or recombinantly fused with a polypeptide comprising the disclosed variant Fc region include, but are not limited to, 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.

[0596] Immunoglobulins include IgA, IgM, IgD, IgG, and IgE. Immune complexes include components of at least any immunoglobulin.

[0597] Other examples of antigens that can be bound by antibodies or fusion proteins containing a variant Fc region of the present invention and / or recombinantly fused to a polypeptide comprising the disclosed variant Fc region include, but are not limited to, 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activation protein, activation protein A, activation protein AB, activation protein B, activation protein C, activation protein RIA, activation protein RIA ALK-2, activation protein RIB ALK-4, activation protein RIIA, activation protein RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, 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, artemin, anti-Id, ASPARTIC, atrial natriuretic peptide, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 osteogenin, BMP-4, BMP-2b, BMP-5, BMP-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, calciton, 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, C...

Claims

1. An isolated antibody that binds latent myostatin, wherein the antibody inhibits the activation of myostatin.

2. The antibody of claim 1, wherein the antibody: (a) blocks the release of mature myostatin from latent myostatin; (b) blocks the proteolytic release of mature myostatin; (c) blocks the spontaneous release of mature myostatin; or (d) does not bind mature myostatin; or binds to an epitope within a fragment consisting of amino acids 21-100 of the myostatin propeptide (SEQ ID NO:78).

3. The antibody of claim 1 or 2, wherein the antibody competes with an antibody comprising a VH and VL pair as set forth in Table 2a, 11a or 13 for binding to latent myostatin, or binds to the same epitope as an antibody comprising a VH and VL pair as set forth in Table 2a, 11a or 13.

4. The antibody of any one of claims 1 to 3, which has a higher affinity for binding latent myostatin at neutral pH than at acidic pH, or which has a higher affinity for binding latent myostatin at pH 7.4 than at pH 5.

8.

5. The antibody of any one of claims 1 to 4, which 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 myostatin.

6. The antibody of any one of claims 1 to 5, wherein the antibody comprises: (a) (i) HVR-H3, which comprises the amino acid sequence GVPAX1SX2GGDX3, wherein X1 is Y or H, X2 is T or H, X3 is L or K (SEQ ID NO: 128), (ii) HVR-L3, which comprises the amino acid sequence AGGYGGGX1YA, wherein X1 is L or R (SEQ ID NO: 131), and (iii) HVR-H2, which comprises 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, X7 is G or K (SEQ ID NO: 127); (b) (i) HVR-H1, wherein the HVR-H1 comprises the amino acid sequence X1X2DIS, where X1 is S or H, and X2 is Y, T, D, or E (SEQ ID NO: 126); (ii) HVR-H2, wherein the HVR-H2 comprises the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7, where X1 is Y or H, X2 is S or K, X3 is T, M, or K, X4 is Y or K, X5 is A, M, or E, X6 is S or E, and X7 is G or K (SEQ ID NO: 127); and (iii) HVR-H3, wherein the HVR-H3 comprises the amino acid sequence GVPAX1SX2GGDX3, where X1 is Y or H, X2 is T or H, and X3 is L or K (SEQ ID NO: 128); (c) (i) HVR-H1, wherein the HVR-H1 comprises the amino acid sequence X1X2DIS, where X1 is S or H, and X2 is Y, T, D, or E (SEQ ID NO: 126); (ii) HVR-H2, wherein the HVR-H2 comprises the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7, where X1 is Y or H, X2 is S or K, X3 is T, M, or K, X4 is Y or K, X5 is A, M, or E, X6 is S or E, and X7 is G or K (SEQ ID NO: 127); (iii) HVR-H3, wherein the HVR-H3 comprises the amino acid sequence GVPAX1SX2GGDX3, where X1 is Y or H, X2 is T or H, and X3 is L or K (SEQ ID NO: 128); (iv) HVR-L1, wherein the HVR-L1 comprises the amino acid sequence X1X2SQX3VX4X5X6NWLS, where X1 is Q or T, X2 is S or T, X3 is S or E, X4 is Y or F, X5 is D or H, and X6 is N, D, A, or E (SEQ ID NO: 129); (v) HVR-L2, wherein the HVR-L2 comprises the amino acid sequence WAX1TLAX2, where X1 is S or E, and X2 is S, Y, F, or W (SEQ ID NO: 130); and (vi) HVR-L3, wherein the HVR-L3 comprises the amino acid sequence AGGYGGGX1YA, where X1 is L or R (SEQ ID NO: 131); or (d) (i) HVR-L1, which comprises the amino acid sequence X1X2SQX3VX4X5X6NWLS, where X1 is Q or T, X2 is S or T, X3 is S or E, X4 is Y or F, X5 is D or H, and X6 is N, D, A or E (SEQ ID NO: 129); (ii) HVR-L2, which comprises the amino acid sequence WAX1TLAX2, where X1 is S or E and X2 is S, Y, F or W (SEQ ID NO: 130); and (iii) HVR-L3, which comprises the amino acid sequence AGGYGGGX1YA, where X1 is L or R (SEQ ID NO: 131).

7. The antibody of claim 6(b), which further comprises: a heavy chain variable domain framework FR1, which comprises the amino acid sequence of any one of SEQ ID NOs: 132-134; FR2, which comprises the amino acid sequence of any one of SEQ ID NOs: 135-136; FR3, which comprises the amino acid sequence of SEQ ID NO: 137; and FR4, which comprises the amino acid sequence of SEQ ID NO:

138.

8. The antibody of claim 6(d), which further comprises: a light chain variable domain framework FR1, which comprises the amino acid sequence of SEQ ID NO: 139; FR2, which comprises the amino acid sequence of any one of SEQ ID NOs: 140-141; FR3, which comprises the amino acid sequence of any one of SEQ ID NOs: 142-143; and FR4, which comprises the amino acid sequence of SEQ ID NO:

144.

9. The antibody of any one of claims 1 to 5, which comprises (a) a VH sequence having at least 95% sequence identity with 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 with the amino acid sequence of any one of SEQ ID NOs: 15, 31, 35-38 and 96-99; or (c) 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.

10. An isolated nucleic acid encoding the antibody of any one of claims 1 to 9.

11. A host cell comprising the nucleic acid of claim 10.

12. A method for preparing an anti-myostatin antibody, the method comprising: (a) culturing the host cell of claim 11 to prepare the antibody; or (b) immunizing an animal with a polypeptide, wherein the polypeptide comprises a region corresponding to the amino acids at positions 21 to 100 of the myostatin propeptide (SEQ ID NO: 78).

13. A pharmaceutical preparation, which comprises the antibody of any one of claims 1 to 9 and a pharmaceutical carrier.

14. A polypeptide, which comprises a variant Fc region, and the variant Fc region comprises at least one amino acid alteration in the parental Fc region, wherein the ratio of [KD value of the parental Fc region for simian FcγRIIb] / [KD value of the variant Fc region for simian FcγRIIb] is 2.0 or greater, and the ratio of [KD value of the parental Fc region for simian FcγRIIIa] / [KD value of the variant Fc region for simian FcγRIIIa] is 0.5 or less.

15. The polypeptide of claim 14, wherein further, the ratio of [KD value of the parental Fc region for human FcγRIIb] / [KD value of the variant Fc region for human FcγRIIb] is 2.0 or greater, and the ratio of [KD value of the parental Fc region for human FcγRIIIa] / [KD value of the variant Fc region for human FcγRIIIa] is 0.5 or less.

16. The polypeptide of claim 15, wherein further, the ratio of [KD value of the parental Fc region for human FcγRIIa (H type)] / [KD value of the variant Fc region for human FcγRIIa (H type)] is 5.0 or less.

17. The polypeptide of claim 16, wherein further, the ratio of [KD value of the parental Fc region for human FcγRIIa (R type)] / [KD value of the variant Fc region for human FcγRIIa (R type)] is 5.0 or less.

18. The polypeptide of claim 14, wherein the variant Fc region has a KD value for monkey FcγRIIb of 1.0x10 -6 M or less, and the variant Fc region has a KD value for monkey FcγRIIIa of 5.0x10 -7 M or more.

19. The polypeptide of claim 15, wherein the variant Fc region has a KD value for human FcγRIIb of 2.0x10 -6 M or less, and the variant Fc region has a KD value for human FcγRIIIa of 1.0x10 -6 M or more.

20. The polypeptide of claim 16, wherein the variant Fc region has a KD value for human FcγRIIa (H-type) of 1.0 x 10 -7 M or greater.

21. The polypeptide of claim 17, wherein the variant Fc region has a KD value for human FcγRIIa (R form) of 2.0 x 10 -7 M or greater. The polypeptide according to any one of claims 14 to 21, wherein, According to the EU numbering, the variant Fc region comprises at least one amino acid alteration at at least one position selected from the group consisting of: 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.

23. The polypeptide of claim 22, wherein, According to the EU numbering, the variant Fc region comprises at least two amino acid alterations, and the at least two amino acid alterations comprise: (a) one amino acid alteration at position 236, and (b) at least one amino acid alteration 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.

24. The polypeptide of claim 22 or 23, wherein, According to the EU numbering, the variant Fc region 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, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr 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, 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) Thr at position 326, (s) Ile, Asn at position 327, (t) Thr at position 328, (u) Lys, Arg at position 330, (v) Glu at position 331, (w) Asp at position 332, (x) Asp, Ile, Met, Val, Tyr at position 334, and (y) Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 396.

25. The polypeptide of claim 24, wherein, According to the EU numbering, the variant Fc region 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.

26. A polypeptide, said polypeptide comprising a variant Fc region, said variant Fc region comprising at least two amino acid alterations in a parental Fc region, wherein each said amino acid alteration results in an increase in the isoelectric point (pI) of the variant Fc region as compared to the isoelectric point of the parental Fc region.

27. The polypeptide of claim 26, wherein the amino acid alterations are exposed on the surface of the variant Fc region.

28. The polypeptide of claim 26 or 27, which further comprises an antigen-binding domain.

29. The polypeptide of claim 28, wherein the antigen-binding activity of the antigen-binding domain varies according to ionic concentration conditions. The polypeptide according to any one of claims 26 to 29, wherein, According to EU numbering, the variant Fc region comprises at least two amino acid alterations at at least two positions selected from the group consisting of: 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422 and 431.

31. The polypeptide of claim 30, wherein the variant Fc region comprises Arg or Lys at each of the selected positions.

32. A polypeptide, said polypeptide comprising a variant Fc region, said variant Fc region comprising the amino acid alterations described in Table 14 - 30.

33. The polypeptide of any one of claims 14 to 32, wherein the parental Fc region is derived from human IgG1.

34. The polypeptide of any one of claims 14 to 33, wherein the polypeptide is an antibody or an Fc fusion protein.

35. A polypeptide, said polypeptide comprising an amino acid sequence of any one of SEQ ID NO: 229 - 381.

36. An isolated nucleic acid, said isolated nucleic acid encoding the polypeptide of any one of claims 14 to 35.

37. A host cell, said host cell comprising the nucleic acid of claim 36.

38. A method for preparing a polypeptide comprising a variant Fc region, said method comprising culturing the host cell of claim 37 to prepare said polypeptide.

39. A pharmaceutical formulation, said pharmaceutical formulation comprising the polypeptide of any one of claims 14 to 35 and a pharmaceutical carrier.

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