CA3002422A1 - Anti-myostatin antibodies, polypeptides containing variant fc regions, and methods of use - Google Patents

Abstract

The disclosure provides anti-myostatin antibodies and methods of making and using the same. Nucleic acids encoding the anti-myostatin antibodies and host cells comprising the nucleic acids are also provided. The disclosure also provides polypeptides containing a variant Fc region and methods of making and using the same. Nucleic acids encoding the polypeptides and host cells comprising the nucleic acids are also provided.

Description

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Description Title of Invention: ANTI-MYOSTATIN ANTIBODIES, POLYPEPTIDES CONTAINING VARIANT FC REGIONS, AND
METHODS OF USE
Technical Field [0001] The present invention relates to anti-myostatin antibodies and methods of using the same. The present invention also relates to polypeptides containing variant Fc regions and methods of using the same.
Background Art

[0002] Myostatin, also referred to as growth differentiation factor-8 (GDF8), is a secreted protein and is a member of the transforming growth factor- beta (TGF-beta) su-perfamily of proteins. Members of this superfamily possess growth-regulatory and morphogenetic properties (see, e.g., NPL 1, NPL 2, and PTL 1). Myostatin is expressed primarily in the developing and adult skeletal muscle and functions as a negative regulator of muscle growth. Systemic overexpression of myostatin in adult mice leads to muscle wasting (see, e.g., NPL 3) while, conversely, a myostatin knockout mouse is characterized by hypertrophy and hyperplasia of the skeletal muscle resulting in two- to threefold greater muscle mass than their wild type littermates (see, e.g., NPL 4).

[0003] Like other members of the TGF-beta family, myostatin is synthesized as a large precursor protein containing an N-terminal propeptide domain, and a C-terminal domain considered as the active molecule (see, e.g., NPL 5; PTL 2). Two molecules of myostatin precursor are covalently linked via a single disulfide bond present in the C-terminal growth factor domain. Active mature myostatin (disulfide-bonded homodimer consisting of the C-terminal growth factor domain) is liberated from myostatin precursor through multiple steps of proteolytic processing. In the first step of the myostatin activation pathway, a 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 in both chains of the homodimeric precursor.
But the resulting three peptides (two propeptides and one mature myostatin (i.e., a disulfide-bonded homodimer consisting of the growth factor domains)) remain associated, forming a noncovalent inactive complex that is referred to as "latent myostatin."
Mature myostatin can then be liberated from latent myostatin through degradation of the propeptide. Members of the bone morphogenetic protein 1 (BMP1) family of met-alloproteinases cleave a single peptide bond within the propeptide, Arg98-Asp99, with concomitant release of mature, active myostatin, a homodimer (see, e.g., NPL
6).

Moreover, the latent myostatin can be activated in vitro by dissociating the complex with either acid or heat treatment as well (see, e.g., NPL 7).

[0004] Myostatin exerts its effects through a transmembrane serine/threonine kinase het-erotetramer receptor family, activation of which enhances receptor transphospho-rylation, leading to the stimulation of serine/threonine kinase activity. It has been shown that the myostatin pathway involves an active myostatin dimer binding to the activin receptor type JIB (ActRIIB) with high affinity, which then recruits and activates the transphosphorylation of the low affinity receptor, the activin-like kinase 4 (ALK4) or activin-like kinase 5 (ALK5). It has also been shown that the proteins Smad 2 and Smad 3 are subsequently activated and form complexes with Smad 4, which are then translocated to the nucleus for the activation of target gene transcription. It has been demonstrated that ActRIIB is able to mediate the influence of myostatin in vivo, as expression of a dominant negative form of ActRIIB in mice mimics myostatin gene knockout (see, e.g., NPL 8).

[0005] A number of disorders or conditions are associated with muscle wasting (i.e., loss of or functional impairment of muscle tissue), such as muscular dystrophy (MD;
including Duchenne muscular dystrophy), amyotrophic lateral sclerosis (ALS), muscle atrophy, organ atrophy, frailty, congestive obstructive pulmonary disease (COPD), sarcopenia, and cachexia resulting from cancer or other disorders, as well as renal disease, cardiac failure or disease, and liver disease. Patients will benefit from an increase in muscle mass and/or muscle strength; however, there are presently limited treatments available for these disorders. Thus, due to its role as a negative regulator of skeletal muscle growth, myostatin becomes a desirable target for therapeutic or pro-phylactic intervention for such disorders or conditions, or for monitoring the pro-gression of such disorders or conditions. In particular, agents that inhibit the activity of myostatin may be therapeutically beneficial.

[0006] Inhibition of myostatin expression leads to both muscle hypertrophy and hyperplasia (NPL 4). Myostatin negatively regulates muscle regeneration after injury and lack of myostatin in myostatin null mice results in accelerated muscle regeneration (see, e.g., NPL 9). Anti-myostatin (GDF8) antibodies described in, e.g., PTL 3, PTL 4, PTL
5, PTL 6, and PTL 7, and PTL 8, PTL 9, and PTL 10 have been shown to bind to myostatin and inhibit myostatin activity 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 the skeletal muscle of wild type mice (see, e.g., NPL 10) and the mdx mice, a model for muscular dystrophy (see, e.g., NPL 11; NPL 12). However, these prior art antibodies are all specific for mature myostatin but not for latent myostatin, and the strategies described for inhibiting myostatin activity have utilized antibodies that can bind to and neutralize mature myostatin.

[0007] Antibodies are drawing attention as pharmaceuticals since they are highly stable in blood and have few side effects (see, e.g., NPL 13 and NPL 14). Almost all therapeutic antibodies currently on the market are antibodies of the human IgG1 subclass.
One of the known functions of IgG class antibodies is antibody-dependent cell-mediated cyto-toxicity (hereinafter denoted as ADCC activity) (see, e.g., NPL 15). For an antibody to exhibit ADCC activity, the antibody Fc region must bind to an Fc gamma receptor (hereinafter denoted as Fc gamma R) which is an antibody-binding receptor present on the surface of effector cells such as killer cells, natural killer cells, and activated macrophages.

[0008] In humans, the Fc gamma RIa (CD64A), Fc gamma RIIa (CD32A), Fc gamma Ruth (CD32B), Fc gamma RIIIa (CD16A), and Fc gamma RIIIb (CD16B) isoforms have been reported as the Fc gamma R protein family, and the respective allotypes have also been reported (see, e.g., NPL 16). Fc gamma RIa, Fc gamma RIIa, and Fc gamma RIIIa are called activating Fc gamma R since they have immunologically active functions, and Fc gamma Ruth is called inhibitory Fc gamma R since it has immuno-suppressive functions (see, e.g., NPL 17).

[0009] In the binding between the Fc region and Fc gamma R, several amino acid residues in the antibody hinge region and CH2 domain, and a sugar chain attached to Asn at position 297 (EU numbering) bound to the CH2 domain have been shown to be important (see, e.g., NPL 18, NPL 19, and NPL 20). Various variants having Fc gamma R-binding properties, mainly antibodies with mutations introduced into these sites, have been studied so far; and Fc region variants having higher binding activities towards activating Fc gamma R have been obtained (see, e.g., PTL 11, PTL 12, PTL
13, and PTL 14).

[0010] When an activating Fc gamma R is cross-linked with an immune complex, it phos-phorylates immunoreceptor tyrosine-based activating motifs (ITAMs) contained in the intracellular domain or FcR common gamma-chain (an interaction partner), activates a signal transducer SYK, and triggers an inflammatory immune response by initiating an activation signal cascade (see, e.g., NPL 21).

[0011] Fc gamma RIM is the only Fc gamma R expressed on B cells (see, e.g., NPL 22). In-teraction of the antibody Fc region with Fc gamma RIIb has been reported to suppress the primary immune response of B cells (see, e.g., NPL 23). Furthermore, it is reported that when Fc gamma RIIb on B cells and B cell receptor (BCR) are cross-linked via an immune complex in blood, B cell activation and antibody production by B cells is suppressed (see, e.g., NPL 24). In this immunosuppressive signal transduction mediated by BCR and Fc gamma RIIb, the immunoreceptor tyrosine-based inhibitory motif (ITIM) contained in the intracellular domain of Fc gamma RIIb is necessary (see, e.g., NPL 25 and NPL 26). When ITIM is phosphorylated upon signaling, SH2-containing inositol polyphosphate 5-phosphatase (SHIP) is recruited, transduction of other activating Fc gamma R signal cascades is inhibited, and inflammatory immune response is suppressed (see, e.g., NPL 27). Furthermore, aggregation of Fc gamma RIIb alone has been reported to transiently suppress calcium influx due to BCR
cross-linking and B cell proliferation in a BCR-independent manner without inducing apoptosis of IgM-producing B cells (see, e.g., NPL 28).

[0012] Fc gamma RIM is also expressed on dendritic cells, macrophages, activated neu-trophils, mast cells, and basophils. Fc gamma RIIb inhibits the functions of activating Fc gamma R such as phagocytosis and release of inflammatory cytokines in these cells, and suppresses inflammatory immune responses (see, e.g., NPL 17).

[0013] The importance of immunosuppressive functions of Fc gamma RIIb has been elucidated so far through studies using Fc gamma RIIb knockout mice. There are reports that in Fc gamma RIIb knockout mice, humoral immunity is not appropriately regulated (see, e.g., NPL 29), sensitivity towards collagen-induced arthritis (CIA) is increased (see, e.g., NPL 30), lupus-like symptoms are presented, and Goodpasture's syndrome-like symptoms are presented (see, e.g., NPL 31).

[0014] Additionally, regulatory inadequacy of Fc gamma RIM has been reported to be related to human autoimmnue diseases. For example, the relationship between genetic polymorphism in the transmembrane region and promoter region of Fc gamma RIM, and the frequency of development of systemic lupus erythematosus (SLE) (see, e.g., NPL 32, NPL 33, NPL 34, NPL 35, and NPL 36), and decrease of Fc gamma RIIb ex-pression on the surface of B cells in SLE patients (see, e.g., NPL 37 and NPL
38) have been reported.

[0015] From mouse models and clinical findings as such, Fc gamma RIM is considered to play the role of controlling autoimmune diseases and inflammatory diseases through involvement in particular with B cells, and it is a promising target molecule for con-trolling autoimmune diseases and inflammatory diseases.

[0016] IgGl, mainly used as a commercially available therapeutic antibody, is known to bind not only to Fc gamma RIM, but also strongly to activating Fc gamma R
(see, e.g., NPL 39). It may be possible to develop therapeutic antibodies having greater immuno-suppressive properties compared with those of IgGl, by utilizing an Fc region with enhanced Fc gamma RIIb binding, or improved Fc gamma RIIb-binding selectivity compared with activating Fc gamma R. For example, it has been suggested that the use of an antibody having a variable region that binds to BCR and an Fc with enhanced Fc gamma RIIb binding may inhibit B cell activation (see, e.g., NPL 40). It has been reported that crosslinking Fc gamma RIIb on B cells and IgE bound to a B-cell receptor suppresses differentiation of B cells into plasma cells, which as a result causes suppression of IgE production; and in human PBMC-transplanted mice, human IgG
and IgM concentrations are maintained whereas the human IgE concentration is decreased (see, e.g., NPL 41). Besides IgE, it has been reported that when Fc gamma RIIB and CD79b which is a constituent molecule of a B-cell receptor complex are cross-linked by an antibody, B cell proliferation is suppressed in vitro, and arthritis symptoms are alleviated in the collagen arthritis model (see, e.g., NPL 42).

[0017] Besides B cells, it has been reported that crosslinking of Fc epsilon RI and Fc gamma RIIb on mast cells using molecules, in which the Fc portion of an IgG with enhanced Fc gamma RIIb binding is fused to the Fc portion of IgE that binds to an IgE
receptor Fc epsilon RI, causes phosphorylation of Fc gamma RIIb, thereby suppressing Fc epsilon RI-dependent calcium influx. This suggests that inhibition of degranulation via Fc gamma RIIb stimulation is possible by enhancing Fc gamma RIIb binding (see, e.g., NPL 43).

[0018] Accordingly, an antibody having an Fc with improved Fc gamma RIIb-binding activity is suggested to be promising as a therapeutic agent for inflammatory diseases such as an autoimmune disease.

[0019] Furthermore, it has been reported that activation of macrophages and dendritic cells via Toll-like receptor 4 due to LPS stimulation is suppressed in the presence of an antibody-antigen immune complex, and this effect is also suggested to be actions of the immune complex via Fc gamma RIIb (see, e.g., NPL 44 and NPL 45). Therefore, use of antibodies with enhanced Fc gamma RIIb binding is expected to enable en-hancement of TLR-mediated activation signal-suppressing actions; thus such an-tibodies have been suggested as being promising as therapeutic agents for in-flammatory diseases such as autoimmune diseases.

[0020] Additionally, mutants with enhanced Fc gamma RIIb binding have been suggested to be promising therapeutic agents for cancer, as well as therapeutic agents for in-flammatory diseases such as autoimmune diseases. So far, Fc gamma RIIb has been found to play an important role in the agonistic activity of agonist antibodies against the anti-TNF receptor superfamily. Specifically, it has been suggested that interaction with Fc gamma RIIb is required for the agonistic activity of antibodies against CD40, DR4, DR5, CD30, and CD137, which are included in the TNF receptor family (see, e.g., NPL 46, NPL 47, NPL 48, NPL 49, NPL 50, NPL 51 and NPL 52). NPL 46 shows that the use of antibodies with enhanced Fc gamma RIM binding enhances the anti-tumor effect of anti-CD40 antibodies. Accordingly, antibodies with enhanced Fc gamma RIM are expected to have an effect of enhancing agonistic activity of agonist antibodies including antibodies against the anti-TNF receptor superfamily.

[0021] In addition, it has been shown that cell proliferation is suppressed when using an antibody that recognizes Kit, a type of receptor tyrosine kinase (RTK), to crosslink Fc gamma Ruth and Kit on Kit-expressing cells. Similar effects have been reported even in cases where this Kit is constitutively activated and has mutations that cause oncogenesis (see, e.g., NPL 53). Therefore, it is expected that use of antibodies with enhanced Fc gamma Ruth binding may enhance inhibitory effects on cells expressing RTK having constitutively activated mutations.

[0022] Antibodies having an Fc with improved Fc gamma Ruth-binding activity have been reported (see, e.g., NPL 40). In this Literature, Fc gamma Rllb-binding activity was improved by adding alterations such as 5267E/L328F, G236D/5267E, and 5239D/5267E to an antibody Fc region. Among them, the antibody introduced with the 5267E/L328F mutation most strongly binds to Fc gamma RHb, and maintains the same level of binding to Fc gamma RIa and Fc gamma RIIa type H in which a residue at position 131 of Fc gamma RIIa is His as that of a naturally-occurring IgGl.

However, another report shows that this alteration enhances the binding to Fc gamma RIIa type R in which a residue at position 131 of Fc gamma RIIa is Arg several hundred times to the same level of Fc gamma Ruth binding, which means the Fc gamma Ruth-binding selectivity is not improved in comparison with type-R Fc gamma RIIa (see, e.g., PTL 15).

[0023] Only the effect of enhancing Fc gamma RIIa binding and not the enhancement of Fc gamma Ruth binding is considered to have influence on cells such as platelets which express Fc gamma RIIa but do not express Fc gamma Ruth (see, e.g., NPL 17).
For example, the group of patients who were administered bevacizumab, an antibody against VEGF, is known to have an increased risk for thromboembolism (see, e.g., NPL 54). Furthermore, thromboembolism has been observed in a similar manner in clinical development tests of antibodies against the CD40 ligand, and the clinical study was discontinued (see, e.g., NPL 55). In both cases of these antibodies, later studies using animal models and such have suggested that the administered antibodies aggregate platelets via Fc gamma RIIa binding on the platelets, and form blood clots (see, e.g., NPL 56 and NPL 57). In systemic lupus erythematosus which is an au-toimmune disease, platelets are activated via an Fc gamma RIIa-dependent mechanism, and platelet activation has been reported to correlate with the severity of symptoms (see, e.g., NPL 58). Administering an antibody with enhanced Fc gamma RIIa binding to such patients who already have a high risk for developing thromboembolism will increase the risk for developing thromboembolism, thus is extremely dangerous.

[0024] Furthermore, antibodies with enhanced Fc gamma RIIa binding have been reported to enhance macrophage-mediated antibody dependent cellular phagocytosis (ADCP) (see, e.g., NPL 59). When antigens to be bound by the antibodies are phagocytized by macrophages, antibodies themselves are considered to be also phagocytized at the same time. When antibodies are administered as pharmaceuticals, it is supposed that peptide fragments derived from the administered antibodies are likely to be also presented as an antigen, thereby increasing the risk of production of antibodies against therapeutic antibodies (anti-therapeutic antibodies). More specifically, enhancing Fc gamma RIIa binding will increase the risk of production of antibodies against the therapeutic antibodies, and this will remarkably decrease their value as pharma-ceuticals. Furthermore, Fc gamma RHb on dendritic cells have been suggested to contribute to peripheral tolerance by inhibiting dendritic cell activation caused by immune complexes formed between antigens and antibodies, or by suppressing antigen presentation to T cells via activating Fc gamma receptors (see, e.g., NPL 60).
Since Fc gamma RIIa is also expressed on dendritic cells, when antibodies having an Fc with enhanced selective binding to Fc gamma Ruth are used as pharmaceuticals, antigens are not readily presented by dendritic cells and such due to enhanced selective binding to Fc gamma Ruth, and risk of anti-drug antibody production can be relatively decreased. Such antibodies may be useful in that regard as well.

[0025] More specifically, the value as pharmaceuticals will be considerably reduced when Fc gamma RIIa binding is enhanced, which leads to increased risk of thrombus formation via platelet aggregation and increased risk of anti-therapeutic antibody production due to an increased immunogenicity.

[0026] From such a viewpoint, the aforementioned Fc variant with enhanced Fc gamma Ruth binding shows significantly enhanced type-R Fc gamma RIIa binding compared with that of a naturally-occurring IgG 1. Therefore, its value as a pharmaceutical for patients carrying type-R Fc gamma RIIa is considerably reduced. Types H and R
of Fc gamma RIIa are observed in Caucasians and African-Americans with approximately the same frequency (see, e.g., NPL 61 and NPL 62). Therefore, when this Fc variant was used for treatment of autoimmune diseases, the number of patients who can safely use it while enjoying its effects as a pharmaceutical will be limited.

[0027] Furthermore, in dendritic cells deficient in Fc gamma Ruth or dendritic cells in which the interaction between Fc gamma RHb and the antibody Fc portion is inhibited by an anti-Fc gamma Ruth antibody, dendritic cells have been reported to mature (see, e.g., NPL 63 and NPL 64). This report suggests that Fc gamma Ruth is actively suppressing maturation of dendritic cells in a steady state where inflammation and such are not taking place and activation does not take place. Fc gamma RIIa is expressed on the dendritic cell surface in addition to Fc gamma Ruth; therefore, even if binding to in-hibitory Fc gamma RIIb is enhanced and if binding to activating Fc gamma R
such as Fc gamma RIIa is also enhanced, maturation of dendritic cells may be promoted as a result. More specifically, improving not only the Fc gamma RIIb-binding activity but also the ratio of Fc gamma RIIb-binding activity relative to Fc gamma RIIa-binding activity is considered to be important in providing antibodies with an immunosup-pres sive action.

[0028] Therefore, when considering the generation of a pharmaceutical that utilizes the Fc gamma Ruth binding-mediated immunosuppressive action, there is a need for an Fc variant that not only has enhanced Fc gamma RHb-binding activity, but also has binding to both Fc gamma RIIa types H and R allotypes, which is maintained at a similar level or is weakened to a lower level than that of a naturally-occurring IgGl.

[0029] Meanwhile, cases where amino acid alterations were introduced into the Fc region to increase the Fc gamma Ruth-binding selectivity have been reported so far (see, e.g., NPL 65). However, all variants said to have improved Fc gamma Ruth selectivity as reported in this literature showed decreased Fc gamma Ruth binding compared with that of a naturally-occurring IgGl. Therefore, it is considered to be difficult for these variants to actually induce an Fc gamma Ruth-mediated immunosuppressive reaction more strongly than IgGl.

[0030] Furthermore, since Fc gamma Ruth plays an important role in the agonist antibodies mentioned above, enhancing their binding activity is expected to enhance the agonistic activity. However, when Fc gamma RIIa binding is similarly enhanced, unintended ac-tivities such as ADCC activity and ADCP activity will be exhibited, and this may cause side effects. Also from such viewpoint, it is preferable to be able to selectively enhance Fc gamma RHb-binding activity.

[0031] From these results, in producing therapeutic antibodies to be used for treating au-toimmune diseases and cancer utilizing Fc gamma Ruth, it is important that compared with those of a naturally-occurring IgG, the activities of binding to both Fc gamma RIIa allotypes are maintained or decreased, and Fc gamma Ruth binding is enhanced.
However, Fc gamma RHb shares 93% sequence identity in the extracellular region with that of Fc gamma RIIa which is one of the activating Fc gamma Rs, and they are very similar structurally. There are allotypes of Fc gamma RIIa, H type and R
type, in which the amino acid at position 131 is His (type H) or Arg (type R), and yet each of them reacts differently with the antibodies (see, e.g., NPL 66). Therefore, the difficult problem may be producing an Fc region variant with enhanced selective Fc gamma RHb binding as compared to each allotype of Fc gamma RIIa, which involves distin-guishing highly homologous sequences between Fc gamma RIIa and Fc gamma RIM.
In spite of those difficulties, several Fc region variants have been identified so far, which has selective binding activity to Fc gamma RIIb as compared to Fc gamma RIIa, by conducting comprehensive amino acid modification analysis in the Fc region (see, e.g., PTL 16, PTL 17, PTL 18, PTL 19 and PTL 20).

[0032] There has been a report on an Fc region variant with binding selectivity for Fc gamma RIIb in relation to human Fc gamma R so far, whereas there has been no report on an Fc region variant with binding selectivity for Fc gamma RIIb in relation to monkey Fc gamma R. Owing to the absence of such an Fc variant, the effects of the Fc variant selectively binding to Fc gamma RIM have not been thoroughly tested yet in monkey.

[0033] Apart from the above, it is reported that by modifying the charge of amino acid residues which may be exposed on the surface of an antibody so as to increase or decrease the isoelectric point (pI) of the antibody, it is possible to regulate the half-life of the antibody in blood (see, e.g., PTL 21 and PTL 22). They show that it is possible to prolong the plasma half-life of an antibody by reducing the antibody's pI
and vice versa.

[0034] Further, it is reported that incorporation of an antigen into cells can be promoted by modifying the charge of specified amino acid residues particularly in its CH3 domain to increase the antibody's pI (see, e.g., PTL 23). Also, it has been reported that modifying the charge of amino acid residues in the constant region (mainly CH1 domain) of an antibody to reduce pI can prolong the half-life of the antibody in plasma (see, e.g., PTL 24).
Citation List Patent Literature

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[0037] An objective of the invention is to provide anti-myostatin antibodies, polypeptides containing variant Fc regions, and methods of using the same.

Solution to Problem

[0038] The invention provides anti-myostatin antibodies and methods of using the same.
The invention also provides proteins containing a variant Fc region and methods of using the same.

[0039] In some embodiments, an isolated anti-myostatin antibody of the present invention binds to latent myostatin. In further embodiments, the antibody binds to an epitope within a fragment consisting of amino acids 21-100 of myostatin propeptide (SEQ ID
NO: 78). In some embodiments, an isolated anti-myostatin antibody of the present invention inhibits activation of myostatin. In further embodiments, the antibody blocks the release of mature myostatin from latent myostatin. In further embodiments, the antibody blocks the proteolytic release of mature myostatin. In further embodiments, the antibody blocks the spontaneous release of mature myostatin. In further em-bodiments, the antibody does not bind to mature myostatin. In further embodiments, the antibody binds to the same epitope as an antibody described in Table 13.
In further embodiments, the antibody binds to the same epitope as an antibody comprising a VH
and a VL pair described in Table 13. In further embodiments, the antibody binds to the same epitope as an antibody described in Table 2a. In further embodiments, the antibody binds to the same epitope as an antibody comprising a VH and a VL
pair described in Table 2a. In further embodiments, the antibody binds to the same epitope as an antibody described in Table 1 la. . In further embodiments, the antibody binds to the same epitope as an antibody comprising a VH and a VL pair described in Table 11a. In further embodiments, the antibody binds to the same epitope as an antibody described in Table 2a, 11a, or 13. In further embodiments, the antibody binds to the same epitope as an antibody comprising a VH and a VL pair described in Table 2a, 11a, or 13.

[0040] In some embodiments, an isolated anti-myostatin antibody of the present invention binds to latent myostatin with higher affinity at neutral pH than at acidic pH. In some embodiments, the anti-myostatin antibody binds to latent myostatin with higher affinity at pH7.4 than at pH5.8. In some embodiments, an isolated anti-myostatin antibody of the present invention binds to a polypeptide fragment consisting of amino acids 21-100 of myostatin propeptide (SEQ ID NO: 78) with higher affinity at pH7.4 than at pH5.8. In some embodiments, the antibody binds to the same myostatin epitope as an antibody described in Table 13 with a higher affinity at neutral pH than at acidic pH. In additional embodiments, an anti-myostatin antibody binds to the same epitope as an antibody described in Table 13 with a higher affinity at pH7.4 than at pH5.8. In further embodiments, the antibody binds to the same epitope as an antibody comprising a VH and a VL pair described in Table 13 with a higher affinity at pH7.4 than at pH5.8. In some embodiments, the antibody binds to the same myostatin epitope as an antibody described in Table 2a with a higher affinity at neutral pH than at acidic pH. In some embodiments, the antibody binds to the same myostatin epitope as an antibody described in Table 2a with a higher affinity at pH7.4 than at pH5.8.
In further embodiments, the antibody binds to the same epitope as an antibody comprising a VH
and a VL pair described in Table 2a with a higher affinity at pH7.4 than at pH5.8. In additional embodiments, an anti-myostatin antibody binds to the same epitope as an antibody described in Table lla with a higher affinity at neutral pH than at acidic pH.
In further embodiments, the antibody binds to the same myostatin epitope as an antibody described in Table lla with a higher affinity at pH7.4 than at pH5.8.
In further embodiments, the antibody binds to the same epitope as an antibody comprising a VH and a VL pair described in Table lla with a higher affinity at pH7.4 than at pH5.8. In additional embodiments, an anti-myostatin antibody binds to the same epitope as an antibody described in Table 2a, 11a, or 13 with a higher affinity at neutral pH than at acidic pH. In further embodiments, the antibody binds to the same myostatin epitope as an antibody described in Table 2a, 11a, or 13 with a higher affinity at pH7.4 than at pH5.8. In further embodiments, the antibody binds to the same epitope as an antibody comprising a VH and a VL pair described in Table 2a, 11a, or 13 with a higher affinity at pH7.4 than at pH5.8.

[0041] In some embodiments, an isolated anti-myostatin antibody of the present invention competes for binding latent myostatin with an antibody provided herein.In some em-bodiments, an isolated anti-myostatin antibody of the present invention competes for binding latent myostatin with an antibody described in Table 13. In some em-bodiments, an isolated anti-myostatin antibody of the present invention competes for binding latent myostatin with an antibody comprising a VH and VL pair described in Table 13. In some embodiments, the antibody competes for binding latent myostatin with an antibody described in Table 2a. In some embodiments, an isolated anti-myostatin antibody of the present invention competes for binding latent myostatin with an antibody comprising a VH and VL pair described in Table 2a. In some em-bodiments, the antibody competes for binding latent myostatin with an antibody described in Table 1 la. In some embodiments, an isolated anti-myostatin antibody of the present invention competes for binding latent myostatin with an antibody comprising a VH and VL pair described in Table lla. In additional embodiments, an anti-myostatin antibody competes for binding latent myostatin with an antibody described in Table 2a, 11a, or 13. In additional embodiments, an anti-myostatin antibody competes for binding latent myostatin with an antibody comprising a VH and VL pair described in Table 2a, 11a, or 13. In further embodiments, the anti-myostatin antibody binds to latent myostatin with a higher affinity at neutral pH than at acidic pH. In further embodiments, the anti-myostatin antibody binds to latent myostatin with a higher affinity at pH7.4 than at pH5.8. In further embodiments, the anti-myostatin antibody binds to a polypeptide fragment consisting of amino acids 21-100 of myostatin propeptide (SEQ ID NO: 78) with higher affinity at pH7.4 than at pH5.8.
Methods for assessing the ability of an antibody to compete with a reference antibody for binding latent myostatin are described herein and known in the art.

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

[0043] In some embodiments, a anti-myostatin antibody of the invention comprises:
(a) (i) a HVR-H3 comprising the amino acid sequence GVPAXISX2GGDX3, wherein X1 is Y or H, X2 is T or H, X3 is L or K (SEQ ID NO: 128), (ii) a HVR-L3 comprising the amino acid sequence AGGYGGGX1YA, wherein X1 is L or R (SEQ ID NO: 131), and (iii) a HVR-H2 comprising the amino acid sequence IISX1AGX2X3YX4X5X6 WAKX7, 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) a HVR-H1 comprising the amino acid sequence X1X2DIS, wherein X1 is S
or H, X2 is Y, T, D or E (SEQ ID NO: 126), (ii) a HVR-H2 comprising the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7, wherein X1 is Y or H, X2 is S or K, X3 is T, M or K, X4 is Y or K, X5 is A, M or E, X6 is S or E, X7 is G or K (SEQ ID NO:
127), and (iii) a HVR-H3 comprising the amino acid sequence GVPAXISX2GGDX3, wherein X1 is Y or H, X2 is T or H, X3 is L or K (SEQ ID NO: 128);
(c) (i) a HVR-H1 comprising the amino acid sequence X1X2DIS, wherein X1 is S
or H, X2 is Y, T, D or E (SEQ ID NO: 126), (ii) a HVR-H2 comprising the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7, wherein X1 is Y or H, X2 is S or K, X3 is T, M or K, X4 is Y or K, X5 is A, M or E, X6 is S or E, X7 is G or K (SEQ ID NO:
127), (iii) a HVR-H3 comprising the amino acid sequence GVPAXISX2GGDX3, wherein X1 is Y or H, X2 is T or H, X3 is L or K (SEQ ID NO: 128), (iv) a HVR-L1 comprising the amino acid sequence X1X2SQX3VX4X5X6NWLS, wherein X1 is Q or T, X2 is S or T, X

3 is S or E, X4 is Y or F, X5 is D or H, X6 is N, D, A or E (SEQ ID NO: 129);
(v) a HVR-L2 comprising the amino acid sequence WAXITLAX2, wherein X1 is S or E, X2 is S, Y, F or W (SEQ ID NO: 130); and (vi) a HVR-L3 comprising the amino acid sequence AGGYGGGX1YA, wherein X1 is L or R (SEQ ID NO: 131);
(d) (i) a HVR-L1 comprising the amino acid sequence X1X2SQX3VX4X5X6NWLS, wherein X1 is Q or T, X2 is S or T, X3 is S or E, X4 is Y or F, X5 is D or H, X6 is N, D, A or E (SEQ ID NO: 129); (ii) a HVR-L2 comprising the amino acid sequence WAX1 TLAX2, wherein X1 is S or E, X2 is S, Y, F or W (SEQ ID NO: 130); and (iii) a HVR-L3 comprising the amino acid sequence AGGYGGGX1YA, wherein X1 is L or R (SEQ
ID NO: 131). In some embodiments the antibody of (b) further comprises a heavy chain variable domain framework FR1 comprising the amino acid sequence of any one of SEQ ID NOs: 132-134; FR2 comprising the amino acid sequence of any one of SEQ
ID NOs: 135-136; FR3 comprising the amino acid sequence of SEQ ID NO: 137; and FR4 comprising the amino acid sequence of SEQ ID NO: 138. In some embodiments, the antibody of (d), further comprises a light chain variable domain framework comprising the amino acid sequence of SEQ ID NO: 139; FR2 comprising the amino acid sequence of any one of SEQ ID NOs: 140-141; FR3 comprising the amino acid sequence of any one of SEQ ID NOs: 142-143; and FR4 comprising the amino acid sequence of SEQ ID NO: 144.

[0044] In some embodiments, an isolated anti-myostatin antibody of the present invention comprises (a) a HVR-H3 comprising the amino acid sequence GVPAXISX2GGDX3, wherein X1 is Y or H, X2 is T or H, X3 is L or K (SEQ ID NO: 128), (b) a HVR-comprising the amino acid sequence AGGYGGGX1YA, wherein X1 is L or R (SEQ ID
NO: 131), and (c) a HVR-H2 comprising the amino acid sequence IISX1AGX2X3YX4X
5X6WAKX7, 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).

[0045] In some embodiments, an isolated anti-myostatin antibody of the present invention comprises (a) a HVR-H1 comprising the amino acid sequence X1X2DIS, wherein X1 is S or H, X2 is Y, T, D or E (SEQ ID NO: 126), (b) a HVR-H2 comprising the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7, wherein X1 is Y or H, X2 is S or K, X3 is T, M or K, X4 is Y or K, X5 is A, M or E, X6 iS S or E, X7 is G or K (SEQ
ID NO:
127), and (c) a HVR-H3 comprising the amino acid sequence GVPAXISX2GGDX3, wherein X1 is Y or H, X2 is T or H, X3 is L or K (SEQ ID NO: 128). In further em-bodiments, the antibody comprises a heavy chain variable domain framework FR1 comprising the amino acid sequence of any one of SEQ ID NOs: 132-134; FR2 comprising the amino acid sequence of any one of SEQ ID NOs: 135-136; FR3 comprising the amino acid sequence of SEQ ID NO: 137; and FR4 comprising the amino acid sequence of SEQ ID NO:138. In further embodiments, the antibody addi-tionally comprises (a) a HVR-Li comprising the amino acid sequence X1X2SQX3VX4 X5X6NWLS, wherein X1 is Q or T, X2 is S or T, X3 is S or E, X4 is Y or F, X5 is D or H, X6 is N, D, A or E (SEQ ID NO: 129); (b) a HVR-L2 comprising the amino acid sequence WAXITLAX2, wherein X1 is S or E, X2 is S, Y, F or W (SEQ ID NO: 130);

and (c) a HVR-L3 comprising the amino acid sequence AGGYGGGX1YA, wherein X1 is L or R (SEQ ID NO: 131).

[0046] In some embodiments, an isolated anti-myostatin antibody of the present invention comprises (a) a HVR-L1 comprising the amino acid sequence X1X2SQX3VX4X5X6 NWLS, wherein X1 is Q or T, X2 is S or T, X3 is S or E, X4 is Y or F, X5 is D
or H, X6 is N, D, A or E (SEQ ID NO: 129); (b) a HVR-L2 comprising the amino acid sequence WAXITLAX2, wherein X1 is S or E, X2 is S, Y, F or W (SEQ ID NO: 130); and (c) a HVR-L3 comprising the amino acid sequence AGGYGGGX1YA, wherein X1 is L or R
(SEQ ID NO: 131). In a further embodiment, the antibody further comprises a light chain variable domain framework FR1 comprising the amino acid sequence of SEQ
ID
NO: 139; FR2 comprising the amino acid sequence of any one of SEQ ID NOs:
140-141; FR3 comprising the amino acid sequence of any one of SEQ ID NOs:
142-143; and FR4 comprising the amino acid sequence of SEQ ID NO: 144.

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

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

sequence of any one of SEQ ID NOs: 15, 31, 35-38, and 96-99.

[0049] The invention also provides isolated nucleic acids encoding an anti-myostatin antibody of the present invention. The invention also provides host cells comprising a nucleic acid of the present invention. The invention also provides a method of producing an antibody comprising culturing a host cell of the present invention so that the antibody is produced.

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

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

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

[0053] Anti-myostatin antibodies of the present invention may be for use as a medicament.
In some embodiments, the antibody is used in the manufacture of a medicament for:
(a) treatment of a muscle wasting disease; (b) increasing mass of muscle tissue; (c) in-creasing strength of muscle tissue; or (d) reducing body fat accumulation. In some em-bodiments, anti-myostatin antibodies of the present invention may be for use in treating a muscle wasting disease. Anti-myostatin antibodies of the present invention may be for use in increasing mass of muscle tissue. Anti-myostatin antibodies of the present invention may be for use in increasing strength of muscle tissue. Anti-myostatin antibodies of the present invention may be for use in reducing body fat accu-mulation.

[0054] In some embodiments, an anti-myostatin antibody provided herein has use in: (a) treating a muscle wasting disease; (b) increasing mass of muscle tissue; (c) increasing strength of muscle tissue; or (d) reducing body fat accumulation.

[0055] Anti-myostatin antibodies of the present invention may be used in the manufacture of a medicament. In some embodiments, the antibody is used in the manufacture of a medicament for: (a) treatment of a muscle wasting disease; (b) increasing mass of muscle tissue; (c) increasing strength of muscle tissue; or (d) reducing body fat accu-mulation. In some embodiments, the medicament is for treatment of a muscle wasting disease. In some embodiments, the medicament is for increasing mass of muscle tissue.
In some embodiments, the medicament is for increasing strength of muscle tissue. In some embodiments, the medicament is for reducing body fat accumulation.

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

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

[0058] In one embodiment, the invention provides Fc gamma RIB-binding polypeptides comprising variant Fc regions and methods of using the same. In some embodiments, a variant Fc region with enhanced Fc gamma RHb-binding activity of the present invention comprises at least one amino acid alteration in a parent Fc region.
In further embodiments, the ratio of [KD value of the parent Fc region for monkey Fc gamma RIIb]/[KD value of the variant Fc region for monkey Fc gamma RIIb] is 2.0 or greater.
In further embodiments, the ratio of [KD value of the parent Fc region for monkey Fc gamma RIIIa]/[KD value of the variant Fc region for monkey Fc gamma RIIIa] is 0.5 or smaller. In further embodiments, the ratio of [KD value of the parent Fc region for human Fc gamma RI1b1/[KD value of the variant Fc region for human Fc gamma RIIb]
is 2.0 or greater. In further embodiments, the ratio of [KD value of the parent Fc region for human Fc gamma RIIIaNKD value of the variant Fc region for human Fc gamma RIIIa] is 0.5 or smaller. In further embodiments, the ratio of [KD value of the parent Fc region for human Fc gamma RIIa (type H)1/[KD value of the variant Fc region for human Fc gamma RIIa (type H)] is 5.0 or smaller. In further embodiments, the ratio of [KD value of the parent Fc region for human Fc gamma RIIa (type R)1/[KD value of the variant Fc region for human Fc gamma RIIa (type R)] is 5.0 or smaller. In another embodiment, the KD value of the variant Fc region for monkey Fc gamma Ruth is 1.0x106 M or smaller. In another embodiment, the KD value of the variant Fc region for monkey Fc gamma RIIIa is 5.0x107 M or greater. In another embodiment, the KD
value of the variant Fc region for human Fc gamma RHb is 2.0x106 M or smaller.
In another embodiment, the KD value of the variant Fc region for human Fc gamma RIIIa is 1.0x10 6 M or greater. In another embodiment, the KD value of the variant Fc region for human Fc gamma RIIa (type H) is 1.0x10 7 M or greater. In another embodiment, the KD value of the variant Fc region for human Fc gamma RIIa (type R) is 2.0x107 M
or greater.

[0059] In some embodiments, a variant Fc region with enhanced Fc gamma Ruth-binding activity of the present invention comprises at least one amino acid alteration of 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.

[0060] In further embodiments, the variant Fc region with enhanced Fc gamma Ruth-binding activity comprises at least two amino acid alterations comprising: (a) one amino acid alteration at position 236, and (b) at least one amino acid alteration of at least one position selected from the group consisting of: (i) position 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) position: 231, 232, 235, 239, 268, 295, 298, 326, 330, and 396; or (iii) position 268, 295, 326, and 330; according to EU numbering.

[0061] In further embodiments, the variant Fc region with enhanced Fc gamma Ruth-binding activity comprises at least two amino acid alterations comprising: (a) one amino acid alteration at position 236, and (b) at least one amino acid alteration of 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.

[0062] In further embodiments, the variant Fc region with enhanced Fc gamma Ruth-binding activity comprises at least two amino acid alterations comprising: (a) one amino acid alteration at position 236, and (b) at least one amino acid alteration of 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.

[0063] In further embodiments, the variant Fc region with enhanced Fc gamma Ruth-binding activity comprises at least two amino acid alterations comprising: (a) one amino acid alteration at position 236, and (b) at least one amino acid alteration of at least one position selected from the group consisting of: 268, 295, 326, and 330 according to EU
numbering.

[0064] In some embodiments, a variant Fc region with enhanced Fc gamma Ruth-binding activity of the present invention 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, Gin, Tyr at position 238; (i) Ile, Leu, Asn, Pro, Val at position 239; (j) Ile at position 264; (k) Phe at position 266; (1) 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, Gin, Arg, Ser, Thr, Val, Trp, Tyr at position 396; according to EU numbering.

[0065] In further embodiments, the variant Fc region with enhanced Fc gamma Ruth-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.

[0066] In another embodiment, the invention provides a polypeptide comprising an iso-electric point (p1)-increased variant Fc region and a method of using the same. In some embodiments, a polypeptide comprising a variant Fc region with increased pI
comprises at least two amino acid alterations in a parent Fc region. In further em-bodiments, each of the amino acid alterations increases the isoelectric point (pI) of the variant Fc region compared with that of the parent Fc region. In further embodiments, the amino acid can be exposed on the surface of the variant Fc region. In further em-bodiments, a polypeptide comprises the variant Fc region and an antigen-binding domain. In further embodiments, antigen-binding activity of the antigen-binding domain changes according to ion concentration conditions. In further embodiments, the variant Fc region with increased pI of the present invention comprises at least two amino acid alterations of 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 further embodiments, the variant Fc region with increased pI comprises Arg or Lys at each of the positions selected.

[0067] In some embodiments, a variant Fc region of the present invention comprises amino acid alterations described in Tables 14-30.

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

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

[0070] The invention also provides isolated nucleic acid encoding the polypeptide comprising a variant Fc region of the present invention. The invention also provides a host cell comprising the nucleic acid of the present invention. The invention also provides a method of producing a polypeptide comprising a variant Fc region comprising culturing the host of the present invention so that the polypeptide is produced.

[0071] The invention further provides a pharmaceutical formulation comprising the polypeptide comprising a variant Fc region of the present invention and a pharma-ceutically acceptable carrier.
Brief Description of Drawings

[0072] [fig.11Figure 1 illustrates inhibition of proteolytic activation of latent myostatin by anti-latent myostatin antibody, as described in Example 3. The activity of active myostatin released from latent myostatin by BMP1 protease was measured in the presence of anti-latent myostatin antibody, using HEK Blue Assay.
[fig.21Figure 2 illustrates inhibition of spontaneous activation of latent myostatin by anti-latent myostatin antibody, as described in Example 4. The activity of active myostatin released from latent myostatin by 37 degrees C incubation was measured in the presence of anti-latent myostatin antibody, using HEK Blue Assay.
[fig.31Figure 3 illustrates binding of anti-latent myostatin antibody to propeptide domain, as described in Example 5.
[fig.41Figure 4 illustrates Western Blot analysis against myostatin propeptide, as described in Example 6. Proteolytic cleavage of myostatin propeptide by BMP1 was assessed in the presence and absence of anti-latent myostatin antibody.
[fig.51Figures 5A-5C illustrate BIACORE (registered trademark) sensorgrams of anti-latent myostatin antibody MST1032-Glm towards human latent myostatin (A), cynomolgus monkey latent myostatin (B), and mouse latent myostatin (C), as described in Example 7.
[fig.6A1Figure 6A illustrates in vivo efficacy of anti-latent myostatin antibody and anti-mature myostatin antibody on muscle mass and fat mass, as described in Example 8. Anti-latent myostatin antibody (MST1032-Glm; described as MST1032 in the Figures) or anti-mature myostatin antibody (41C1E4) was administered to SCID
mice, and full body lean mass (or lean body mass: LBM) was measured.
[fig.6B1Figure 6B illustrates in vivo efficacy of anti-latent myostatin antibody and anti-mature myostatin antibody on muscle mass and fat mass, as described in Example 8.
Anti-latent myostatin antibody (MST1032-Glm; described as MST1032 in the Figures) or anti-mature myostatin antibody (41C1E4) was administered to SCID
mice, and change of full body fat mass from Day 0 to Day 14 was measured.

[fig.6C1Figure 6C illustrates in vivo efficacy of anti-latent myostatin antibody and anti-mature myostatin antibody on muscle mass and fat mass, as described in Example 8.
Anti-latent myostatin antibody (MST1032-Glm; described as MST1032 in the Figures) or anti-mature myostatin antibody (41C1E4) wasadministered to SCID
mice, and gastrocnemius and quadriceps muscle mass was measured.
[fig.7A1Figure 7A illustrates comparison of in vivo efficacy among several anti-myostatin antibodies, as described in Example 9. Anti-latent myostatin antibody (MST1032-Glm; described as MST1032 in the Figures) or anti-mature myostatin antibody (41C1E4, REGN, OGD, or MY0-029) was administered to SCID mice, and full body lean mass was measured.
[fig.7B1Figure 7B illustrates comparison of in vivo efficacy among several anti-myostatin antibodies, as described in Example 9. Anti-latent myostatin antibody (MST1032-Glm; described as MST1032 in the Figures) or anti-mature myostatin antibody (41C1E4, REGN, OGD, or MY0-029) was administered to SCID mice, and grip strength was measured.
[fig.7C1Figure 7C illustrates comparison of in vivo efficacy among several anti-myostatin antibodies, as described in Example 9. Anti-latent myostatin antibody (MST1032-Glm; described as MST1032 in the Figures) or anti-mature myostatin antibody (41C1E4, REGN, OGD, or MY0-029) was administered to SCID mice, and change of full body fat mass from Day 0 to Day 14 was measured.
[fig.81Figure 8 illustrates inhibition of proteolytic and spontaneous activation of latent myostatin by humanized anti-latent myostatin antibody, as described in Example 10.
The activity of active myostatin released from latent myostatin by BMP1 protease (proteolytic) or by 37 degrees C incubation without BMP1 (spontaneous) was measured in the presence of anti-latent myostatin antibody, using HEK Blue Assay.
[fig.91Figure 9 illustrates BIACORE (registered trademark) sensorgrams of histidine substituted variants of anti-latent myostatin antibody, as described in Example 11. The antibody/antigen complexes were allowed to dissociate at pH7.4, followed by ad-ditional dissociation at pH5.8 (pointed by an arrow) to assess the pH-dependent in-teractions. Antibodies tested in this experiment are: Ab001 (black solid curve), Ab002 (black short-dashed curve), Ab003 (black dotted curve), Ab004 (gray short-dashed curve), Ab005 (gray solid curve), Ab006 (gray long-dashed curve), and Ab007 (black long-dashed curve).
[fig.101Figure 10 illustrates inhibition of proteolytic and spontaneous activation of latent myostatin by pH-dependent anti-latent myostatin antibodies, as described in Example 13. The activity of active myostatin released from latent myostatin by protease (proteolytic) or by 37 degrees C incubation without BMP1 (spontaneous) was measured in the presence of anti-latent myostatin antibodies, using HEK Blue Assay.

Antibodies MS1032L001-SG1, MS1032L002-SG1, MS1032L003-SG1, and M51032L004-SG1 are described respectively as MSLO-01, MSLO-02, MSLO-03, and MSLO-04 in the Figure. Comparable inhibition of proteolytic and spontaneous ac-tivation of latent myostatin to M51032L000-SG1 was achieved by MS1032L001-SG1, MS1032L002-SG1, MS1032L003-SG1, and MS1032L004-SG1.
[fig.111Figures 11A-11F illustrate BIACORE (registered trademark) sensorgrams of pH-dependent anti-latent myostatin antibody, as described in Example 14.
Kinetic pa-rameters of M5T1032-SG1 (A), M51032L000-SG1 (B), M51032L001-SG1 (C), M51032L002-SG1 (D), M51032L003-SG1 (E), and M51032L004-SG1 (F), were measured at neutral pH and acidic pH.
[fig.121Figure 12 illustrates the time course of plasma myostatin concentration after in-travenous administration of an anti-myostatin antibody in mice, as described in Example 15. The effects of Fc gamma R-mediated cellular uptake of antibody/antigen complexes on myostatin clearance in vivo was assessed by comparing anti-myostatin antibodies with Fc gamma R binding (M51032L000-SG1) and anti-myostatin an-tibodies with abolished Fc gamma R binding (MS1032L000-F760).
[fig.131Figure 13 illustrates the time course of plasma myostatin concentration after in-travenous administration of an anti-myostatin antibody in mice, as described in Example 16. The effects of pH-dependent binding of anti-myostatin antibody on myostatin clearance in vivo was assessed by comparing pH-dependent anti-myostatin antibody (M51032L001-SG1 or M51032L001-F760) and non pH-dependent anti-myostatin antibody (MS1032L000-SG1 or MS1032L000-F760).
[fig.141Figures 14A-14E illustrate in vivo efficacy of pH-dependent and non pH-dependent anti-latent myostatin antibodies, as described in Example 17. pH-dependent anti-latent myostatin antibody (M51032L001-SG1; described as MSLO1 in the Figure) or non pH-dependent anti-latent myostatin antibody (M51032L000-SG1;
described as MSLOO in the Figure) was administered to SCID mice, and full body lean mass (A), full body fat mass (B), quadriceps muscle mass (C), gastrocnemius muscle mass (D), and grip strength (E) were measured.
[fig.151Figure 15 illustrates binding activity of anti-latent myostatin antibody MST1032 to latent myostatin and GDF11, as described in Example 19.
[fig.161Figure 16 illustrates the inhibitory activity of anti-latent myostatin antibody MST1032 against proteolytic and spontaneous activation of GDF11, as described in Example 20. The activity of active GDF11 released by BMP1 protease (proteolytic) or by 37 degrees C incubation without BMP1 (spontaneous) was measured in the presence of anti-latent myostatin antibody, using HEK Blue Assay.
[fig.171Figure 17 illustrates inhibition of proteolytic activation of latent myostatin by anti-latent myostatin antibody, as described in Example 22. The activity of active myostatin released from latent myostatin by BMP1 protease was measured in the presence of anti-latent myostatin antibody, using HEK Blue Assay.
[fig.181Figure 18 illustrates the time course of plasma myostatin concentration after in-travenous administration of anti-latent myostatin antibodies in mice, as described in Example 23. The effects of pH dependency on myostatin clearance in vivo was assessed by comparing non-pH dependent anti-latent myostatin antibody (MS1032L000-SG1) and different pH-dependent anti-latent myostatin antibodies (MS1032L001-SG1, M51032L006-SG1, MS1032L011-SG1, M51032L018-SG1, MS1032L019-SG1, MS1032L021-SG1 and MS1032L025-SG1).
[fig.191Figures 19A and 19B illustrate the time course of plasma myostatin con-centration after intravenous administration of anti-latent myostatin antibodies in cynomolgus monkey, as described in Example 24. (A) The effect of pH-dependency and Fc engineering on myostatin clearance in vivo was assessed by comparing non pH-dependent anti-latent myostatin antibody (MS1032L000-SG1) and pH-dependent anti-latent myostatin antibodies with Fc engineering (M51032L006-5G1012, MS1032L006-SG1016, MS1032L006-SG1029, MS1032L006-5G1031, M51032L006-5G1033, M51032L006-5G1034). (B) The effect of Fc engineering on myostatin clearance in vivo was assessed by comparing anti-latent myostatin an-tibodies (MS1032L019-SG1079, MS1032L019-SG1071, MS1032L019-SG1080, MS1032L019-SG1074, MS1032L019-5G1081, and M51032L019-5G1077).
[fig.20A1Figure 20A, together with Figures 20B-201, illustrates in vivo efficacy of anti-latent myostatin antibodies (MS1032 variants) on lean body mass (LBM), grip strength, and body fat mass, as described in Example 25. M51032L006-SG1, M51032L011-SG1, and M51032L018-SG1 were administered to Scid mice, and lean body mass (A)was measured.
[fig.20B1Figure 20B, together with Figures 20A, 20C-201, illustrates in vivo efficacy of anti-latent myostatin antibodies (MS1032 variants) on lean body mass (LBM), grip strength, and body fat mass, as described in Example 25. M51032L006-SG1, M51032L019-SG1, and M51032L025-SG1 were administered to Scid mice, and lean body mass (B) was measured.
[fig.20C1Figure 20C, together with Figures 20A-B, 20D-I, illustrates in vivo efficacy of anti-latent myostatin antibodies (MS1032 variants) on lean body mass (LBM), grip strength, and body fat mass, as described in Example 25. M51032L001-SG, M51032L006-SG1, and M51032L011-SG1 were administered to Scid mice, and lean body mass (C) was measured.
[fig.20D1Figure 20D, together with Figures 20A-C, 20E-I, illustrates in vivo efficacy of anti-latent myostatin antibodies (MS1032 variants) on lean body mass (LBM), grip strength, and body fat mass, as described in Example 25. MS1032L006-SG1, MS1032L011-SG1, and MS1032L018-SG1 were administered to Scid mice, and grip strength (D) was measured.
[fig.20E1Figure 20E, together with Figures 20A-D, 20E-20I, illustrates in vivo efficacy of anti-latent myostatin antibodies (MS1032 variants) on lean body mass (LBM), grip strength, and body fat mass, as described in Example 25. M51032L006-SG1, M51032L019-SG1, and M51032L025-SG1 were administered to Scid mice, and grip strength (E) was measured.
[fig.20F1Figure 20F, together with Figures 20A-E, 20G-I, illustrates in vivo efficacy of anti-latent myostatin antibodies (MS1032 variants) on lean body mass (LBM), grip strength, and body fat mass, as described in Example 25. MS1032L001-SG, MS1032L006-SG1, and MS1032L011-SG1 were administered to Scid mice, and grip strength (F) was measured.
[fig.20G1Figure 20G, together with Figures 20A-F, 20H-I, illustrates in vivo efficacy of anti-latent myostatin antibodies (MS1032 variants) on lean body mass (LBM), grip strength, and body fat mass, as described in Example 25. M51032L006-SG1, M51032L011-SG1, and M51032L018-SG1 were administered to Scid mice, and body fat mass (G) was measured.
[fig.20H1Figure 20H, together with Figures 20A-G, 201, illustrates in vivo efficacy of anti-latent myostatin antibodies (MS1032 variants) on lean body mass (LBM), grip strength, and body fat mass, as described in Example 25. M51032L006-SG1, M51032L019-SG1, and M51032L025-SG1 were administered to Scid mice, and body fat mass (H) was measured.
[fig.20I1Figure 201, together with Figures 20A-H, illustrates in vivo efficacy of anti-latent myostatin antibodies (MS1032 variants) on lean body mass (LBM), grip strength, and body fat mass, as described in Example 25. M51032L001-SG, M51032L006-SG1, and M51032L011-SG1 were administered to Scid mice, and body fat mass (I) was measured.
[fig.211Figure 21 illustrates inhibitory activity on latent myostatin activation by anti-latent myostatin antibodies, as described in Example 26. The amounts of mature myostatin released from latent myostatin by BMP1 protease were measured in the presence of anti-latent myostatin antibodies (M5T1032, M5T1504, M5T1538, M5T1551, M5T1558, M5T1572, and M5T1573).
[fig.22A1Figure 22A illustrates a schematic diagram of latent myostatin fragments of 100 amino acid each, designed for epitope mapping of anti-latent myostatin antibodies, as described in Example 26.
[fig.22B1Figure 22B illustrates Western blotting analysis against GST tagged human latent myostatin fragments (GST-hMSTN) by an anti-GST antibody, as described in Example 26. 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.
[fig.22C1Figure 22C illustrates Western blotting analysis against GST tagged human latent myostatin fragments (GST-hMSTN) by anti-latent myostatin antibodies (MST1032, MST1538, MST1572, and MST1573), as described in Example 26. 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 (10Ong).
[fig.22D1Figure 22D illustrates summarized results of Western blotting analysis and deduced epitope positions for anti-latent myostatin antibodies (MST1032, MST1538, MST1572, and MST1573), as described in Example 26.
[fig.231Figure 23 illustrates an alignment of amino acid sequences of cynomolgus (cyno) Fc gamma RIIal, Fc gamma RIIa2, Fc gamma RIIa3, Fc gamma Ruth, human Fc gamma RIIaH, Fc gamma RIIaR, and Fc gamma RIM. Squared regions indicate putative residues which interact with Fc domain.
[fig.241Figure 24 illustrates the time course of total myostatin concentration in plasma after intravenous administration of an anti-myostatin antibody with an Fc gamma RIM-enhanced Fc variant in all human Fc gamma R transgenic mice, as described in Example 28. The effects of Fc gamma Ruth-enhanced Fc variants on antigen elimination through human Fc gamma RIM were evaluated.
[fig.251Figure 25 illustrates the time course of antibody concentration in plasma after intravenous administration of an anti-myostatin antibody with an Fc gamma RIM-enhanced Fc variant in all human Fc gamma R transgenic mice, as described in Example 28. The effects of Fc gamma Ruth-enhanced Fc variants on antibody pharma-cokinetics were evaluated.
[fig.261Figures 26A and 26B illustrate the time course of plasma myostatin con-centration after intravenous administration of anti-latent myostatin antibodies in cynomolgus monkey, as described in Example 29. (A) The effect of pH-dependency and Fc engineering on myostatin clearance in vivo was assessed by comparing non pH-dependent anti-latent myostatin antibody (MS1032L000-SG1) and pH-dependent anti-latent myostatin antibodies with Fc engineering (M51032L006-5G1012, MS1032L006-SG1016, MS1032L006-SG1029, MS1032L006-5G1031, M51032L006-5G1033, M51032L006-5G1034). (B) The effect of Fc engineering on myostatin clearance in vivo was assessed by comparing anti-latent myostatin an-tibodies (MS1032L019-SG1079, MS1032L019-SG1071, MS1032L019-SG1080, MS1032L019-SG1074, MS1032L019-5G1081, and M51032L019-5G1077).
[fig.27A1Figure 27A illustrates the time course of total myostatin concentration in plasma after intravenous administration of an anti-myostatin antibody with a p1-increased Fc variant in human FcRn transgenic mice, as described in Example 30. The effects of p1-increased Fc variants on antigen elimination were evaluated.
[fig.27B1Figure 27B illustrates the time course of antibody concentration in plasma after intravenous administration of an anti-myostatin antibody with a p1-increased Fc variant in human FcRn transgenic mice, as described in Example 30. The effects of p1-increased Fc variants on antibody pharmacokinetics were evaluated.
[fig.28A1Figure 28A illustrates the time course of total myostatin concentration in plasma after intravenous administration of an anti-myostatin antibody with a p1-increased Fc variant in human FcRn transgenic mice, as described in Example 30. The effects of p1-increased Fc variants on antigen elimination were evaluated. In this assay, an excess of human normal immunogloblin were co-administered with the anti-myostatin antibody in order to mimic the situation of human plasma.
[fig.28B1Figure 28B illustrates the time course of antibody concentration in plasma after intravenous administration of an anti-myostatin antibody with a p1-increased Fc variant in human FcRn transgenic mice, as described in Example 30. The effects of p1-increased Fc variants on antibody pharmacokinetics were evaluated. In this assay, an excess of human normal immunogloblin were co-administered with the anti-myostatin antibody in order to mimic the situation of human plasma.
[fig.291Figure 29 illustrates the time course of total myostatin concentration in plasma after intravenous administration of an anti-myostatin antibody with an Fc gamma RIM-enhanced Fc variant in human Fc gamma RIM transgenic mice, as described in Example 31. The effects of Fc gamma Ruth-enhanced Fc variants on antigen elimination through human Fc gamma RIM were evaluated.
[fig.30]Figure 30 illustrates the time course of antibody concentration in plasma after intravenous administration of an anti-myostatin antibody with an Fc gamma RIM-enhanced Fc variant in human Fc gamma RIM transgenic mice, as described in Example 31. The effects of Fc gamma Ruth-enhanced Fc variants on antibody pharma-cokinetics were evaluated.
[fig.31]Figure 31 illustrates results of cell imaging analysis of anti-myostatin an-tibodies with an Fc gamma Ruth-enhanced Fc variant, as described in Example 33.
Each antibody was complexed with fluorescence-labelled myostatin and intracellular uptake of the antigen-antibody complex into cells expressing human Fc gamma RIM
was measured.
Description of Embodiments

[0073] The techniques and procedures described or referenced herein are generally well un-derstood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (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. Cabs, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Im-munology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); An-tibodies (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).
I. DEFINITIONS

[0074] Unless defined otherwise, technical and scientific terms used herein have the same meaning 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 skilled in the art with a general guide to many of the terms used in the present application. All references cited herein, including patent ap-plications and publications, are incorporated by reference in their entirety.

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

[0076] An "acceptor human framework" for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

[0077] "Affinity" refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity"
refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and 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 common methods known in the art, including those described herein. Specific il-lustrative and exemplary embodiments for measuring binding affinity are described in the following.

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

[0079] The terms "anti-myostatin antibody" and "an antibody that binds to myostatin" refer to an antibody that is capable of binding myostatin with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting myostatin. In one embodiment, the extent of binding of an anti-myostatin antibody to an unrelated, non-myostatin protein is less than about 10% of the binding of the antibody to myostatin as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that binds to myostatin has a dissociation constant (Kd) of 1 micro M 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., 108M or less, e.g., from 108M to 1013M, e.g., from 10 9 M to 1013M). In certain embodiments, an anti myostatin antibody binds to an epitope of myostatin that is conserved among myostatin from different species.

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

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

[0082] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay, and/or conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay. An exemplary competition assay is provided herein.

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

[0084] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGI, IgG2, IgG3, IgG4, IgAi, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

[0085] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction.
Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At211, I131, 1125, Y9O, Re186, Rem sm153, Bi212, p32, pb212 and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; 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 the various antitumor or anticancer agents disclosed below.

[0086] "Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC);
phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B
cell activation.

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

[0088] The term "epitope" includes any determinant capable of being bound by an antibody.
An epitope is a region of an antigen that is bound by an antibody that targets that antigen, and includes specific amino acids that directly contact the antibody.
Epitope determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three dimensional structural characteristics, and/or specific charge characteristics.
Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and/or macromolecules.

[0089] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, an FcR is a native human FcR. In some embodiments, an FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the Fc gamma RI, Fc gamma RII, and Fc gamma RIII subclasses, including allelic variants and alternatively spliced forms of those receptors. Fc gamma RII
receptors include Fc gamma RIIA (an "activating receptor") and Fc gamma RIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cy-toplasmic domains thereof. Activating receptor Fc gamma RIIA contains an im-munoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
In-hibiting receptor Fc gamma RIIB contains an immunoreceptor tyrosine-based in-hibition motif (ITIM) in its cytoplasmic domain. (See, e.g., Daeron, Annu.
Rev.
Immunol. 15:203-234 (1997).) FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994);
and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein.

[0090] The term "Fc receptor" or "FcR" also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J.
Immunol.
117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and regulation of homeostasis of immunoglobulins. Methods of 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.). Binding to human FcRn in vivo and serum half life of human FcRn high affinity binding polypeptides can be assayed, e.g., in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which the polypeptides with a variant Fc region are ad-ministered. WO 2000/42072 (Presta) describes antibody variants with improved or di-minished binding to FcRs. See also, e.g., Shields et al., J. Biol. Chem.
9(2):6591-6604 (2001).

[0091] The term "Fc region" herein is used to define a C-terminal region of an im-munoglobulin 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, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (residues 446-447) of the Fc region may or may not be present.
Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU
index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0092] The term "Fc region-comprising antibody" refers to an antibody that comprises an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) or C-terminal glycine-lysine (residues 446-447) of the Fc region may be removed, for example, during purification of the antibody or by recombinant engineering of the nucleic acid encoding the antibody. Accordingly, a composition comprising an antibody having an Fc region according to this invention can comprise an antibody with G446-K447, with G446 and without K447, with all G446-K447 removed, or a mixture of three types of antibodies described above.

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

[0094] The terms "full length antibody," "intact antibody," and "whole antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0095] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Exemplary "effector functions" include Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis; down regulation 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 assessed using various assays as disclosed, for example, in definitions herein.

[0096] The terms "host cell," "host cell line," and "host cell culture" are used inter-changeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations.
Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0097] A "human antibody" is one which possesses an amino acid sequence which cor-responds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

[0098] A "human consensus framework" is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL
or VH framework sequences. Generally, the selection of human immunoglobulin VL

or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Im-munological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD
(1991), vols. 1-3. In one embodiment, for the VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for the VH, the subgroup is subgroup III as in Kabat et al., supra.

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

[0100] The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence ("complementarity determining regions" or "CDRs") and/or form structurally defined loops ("hypervariable loops") and/or contain the antigen-contacting residues ("antigen contacts"). Generally, antibodies comprise six HVRs: three in the VH (H1, H2, H3), and three in the 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 Im-munological Interest, 5th Ed. Public Health Service, NIH, Bethesda, MD
(1991)); (c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262:

732-745 (1996)); and (d) 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).

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

[0102] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.

[0103] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated 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.

[0104] An "isolated" antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0105] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily 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.

[0106] "Isolated nucleic acid encoding an anti-myostatin antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

[0107] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous 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 production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

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

[0109] "Native antibodies" refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glyco-proteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3).
Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (kappa) and lambda (lambda), based on the amino acid sequence of its constant domain.

[0110] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include a 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 as well as naturally occurring variants thereof.

[0111] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the in-dications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

[0112] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative sub-stitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX
(registered trademark) (Genetyx Co., Ltd.). Those skilled in the art can determine ap-propriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the US Copyright Office, Washington D.C., 20559, where it is registered under US Copyright Reg-istration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0113] In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y; where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be ap-preciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the im-mediately preceding paragraph using the ALIGN-2 computer program.

[0114] The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

[0115] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
A pharma-ceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0116] The term "myostatin", as used herein, may refer to any native myostatin from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). Unless otherwise indicated, the term "myostatin "refers to a human myostatin protein having the amino acid sequence shown in SEQ ID NO: 1 and containing the terminal propeptide domain of human myostatin as shown in SEQ
ID
NO: 75 or 78. The term encompasses "full-length", unprocessed myostatin as well as any form of myostatin that results from processing in the cell. The term also en-compasses naturally occurring variants of myostatin, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human myostatin (promyostatin) is shown in SEQ ID NO: 1. The amino acid sequence of an exemplary C-terminal growth factor domain of human myostatin is shown in SEQ ID NO: 2. The amino acid sequence of an exemplary N-terminal propeptide domain of human myostatin is shown in SEQ ID NO: 75 or 78. Active mature myostatin is a disulfide-bonded homodimer consisting of two C-terminal growth factor domains. Inactive latent myostatin is a non-covalently-associated complex of two propeptides and the mature myostatin. As disclosed herein, the antibodies of the invention bind inactive latent myostatin, but do not bind the mature active myostatin homodimer. In some embodiments, the antibodies of the invention bind an epitope within a fragement consisting of amino acids of myostatin propeptide (SEQ ID NO:78), but do not bind the mature active myostatin homodimer.The amino acid sequence of an exemplary cynomolgus monkey and murine myostatin (promyostatin) are shown in SEQ ID NO: 3 and 5, respectively.
The amino acid sequence of an exemplary C-terminal growth factor domain of cynomolgus monkey and murine myostatin are shown in SEQ ID NO: 4 and 6, respectively. The amino acid sequence of an exemplary N-terminal propeptide domain of cynomolgus monkey and murine myostatin are shown in SEQ ID NO: 76 or 79, and 77 or 80, re-spectively. GDF-11 (BMP-11) is a closely related molecule to myostatin, both of which are members of TGF-beta superfamily. Similarly to myostatin, GDF11 is syn-thesized as a precursor polypeptide first, and then cleaved into an N-terminal prodomain and 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 an N-terminal prodomain of human GDF11 is shown in SEQ ID NO: 83 or 84. Amino acid sequences of SEQ ID NOs: 1, 3, 5, 78, 79, 80, 81, and 84 include a signal sequence.
Amino acids 1-24 of them correspond to it and they are removed during processing in the cell.

[0117] As used herein, "treatment" (and grammatical variations thereof such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, di-minishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some em-bodiments, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.

[0118] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (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, 6th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity.
Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of com-plementary VL or VH domains, respectively. See, e.g., Portolano et al., J.
Immunol.
150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

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

[0120] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors".
II. COMPOSITIONS AND METHODS

[0121] In one aspect, the invention is based, in part, on anti-myostatin antibodies and uses thereof. In certain embodiments, antibodies that bind to myostatin are provided. An-tibodies of the invention are useful, e.g., for the diagnosis or treatment of a muscle wasting disease.

[0122] In another aspect, the invention is based, in part, on polypeptides comprising variant Fc regions and uses thereof. In one embodiment, polypeptides comprising variant Fc regions with enhanced Fc gamma RIIb-binding activity are provided. In another em-bodiment, polypeptides comprising variant Fc regions with increased pI are provided.
In particular embodiments, the polypeptides of the invention are antibodies.
Polypeptides comprising a variant Fc region of the invention are useful, e.g., for the diagnosis or treatment of diseases.
A. Exemplary Anti-myostatin Antibodies and Polypeptides comprising variant Fc regions

[0123] In one aspect, the invention provides isolated antibodies that bind to myostatin. In certain embodiments, an anti-myostatin antibody of the present invention binds to latent myostatin. In further embodiments, an 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 further embodiments, the antibody binds to an epitope within a fragment consisting of amino acids 21-100 of myostatin propeptide (SEQ ID NO: 78). Propeptide is contained in latent myostatin as one of the components, as described above. In certain em-bodiments, an anti-myostatin antibody of the present invention inhibits 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 may 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 access of a protease to latent myostatin (especially, to the proteolytic cleavage site (Arg98-Asp99) of latent myostatin). In further embodiments, the protease may be a BMP1/TLD family metallo-protease 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. The non-proteolytic release as used herein means a spontaneous release of mature myostatin from latent myostatin, which is not accompanied by the proteolytic cleavage of latent myostatin.
The non-proteolytic release includes, for example, a release of mature myostatin by in-cubating latent myostatin e.g., at 37 degrees C in the absence of a protease that cleaves the latent myostatin. In certain embodiments, the anti-myostatin antibody of the present invention does not bind to mature myostatin. In some embodiments, the anti-myostatin antibody binds to the same epitope as an antibody described in Table 2a. In some embodiments, the anti-myostatin antibody competes for binding latent myostatin with an antibody described in Table 2a. In additional embodiments, an anti-myostatin antibody competes for binding latent myostatin with an antibody comprising a VH and VL pair described in Table 2a. In some embodiments, the anti-myostatin antibody competes for binding a fragment consisting of amino acids 21-100 of myostatin propeptide (SEQ ID NO: 78) with an antibody described in Table 2a. In further em-bodiments, the anti-myostatin antibody binds to the same epitope as an antibody described in Table lla or 13. In some embodiments, the anti-myostatin antibody competes for binding latent myostatin with an antibody described in Table lla or 13.
In some embodiments, the anti-myostatin antibody competes for binding a fragment consisting of amino acids 21-100 of myostatin propeptide (SEQ ID NO: 78) with an antibody described in Table lla or 13.
In some embodiments, an anti-myostatin antibody of the present invention binds to latent myostatin and inhibits the activation of myostatin. In further 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 myostatin propeptide (SEQ ID
NO: 78). In further embodiments, the antibody competes for binding latent myostatin with, or binds the same epitope as, an antibody comprising a VH and VL pair described in Tables 2a, 11a, or 13. In further embodiments, the antibody binds to latent myostatin with higher affinity at neutral pH (e.g., pH7.4) than at acidic pH
(e.g., pH5.8). In further embodiments, the antibody is (a) a monoclonal antibody, (b) a human, humanized, or chimeric antibody; (c) a full length IgG antibody or (d) an antibody fragment that binds to latent myostatin or myostatin propeptide.

[0124] In another embodiment, an anti-myostatin antibody of the present invention does not bind to GDF11. In certain embodiments, an anti-myostatin antibody of the present invention does not inhibit 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 either proteolytic or non-proteolytic release of mature GDF11 from latent GDF11. In certain em-bodiments, the anti-myostatin antibody does not block the proteolytic cleavage of latent GDF11. In certain embodiments, the anti-myostatin antibody does not block access of a protease to latent GDF11 (especially, to the proteolytic cleavage site of latent GDF11). In further embodiments, the protease may be a BMP1/TLD family met-alloprotease such as BMP1, TED, tolloid-like protein-1 (TLL-1), or tolloid-like protein-2 (TLL-2). The non-proteolytic release as used herein means a spontaneous release of mature GDF11 from latent GDF11, which is not accompanied by the pro-teolytic cleavage of latent GDF11. The non-proteolytic release includes, for example, a release of mature GDF11 by incubating latent GDF11 e.g., at 37 degrees C in the absence of a protease that cleaves the latent GDF11. Most of the anti-myostatin an-tibodies known to date were not specific for myostatin. These antibodies have high affinity for other members of the TGF-beta superfamily, such as GDF11 and neutralize the biological activities of them. GDF11 plays an important role during em-bryogenesis, and is responsible for homeotic transformation of the axial skeleton. Ho-mozygous GDF11 knockout mice are perinatal lethal, mice with one wild type copy of the GDF11 gene are viable but have skeletal defects. Since GDF11 plays an important role during embryogenesis, an antagonist that inhibits GDF11 poses theoretical safety risks that could present either as toxicity in treated patients or as reproductive toxicity in, e.g., women of childbearing potential. Thus, there is a need for specific inhibition of myostatin activity in treatments of myostatin-associated disorders for which it is desirable to increase muscle mass, size, strength, etc., particularly in women with childbearing potential.

[0125] In another aspect, the invention provides anti-myostatin antibodies that exhibit pH-dependent binding characteristics. As used herein, the expression "pH-dependent binding" means that the antibody exhibits "reduced binding to myostatin at acidic pH
as compared to its binding at neutral pH" (for purposes of the present disclosure, both expressions may be used interchangeably). For example, antibodies "with pH-dependent binding characteristics" include antibodies that bind to myostatin with higher affinity at neutral pH than at acidic pH. In certain embodiments, the antibodies of the present invention bind to myostatin with at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more times higher affinity at neutral pH than at acidic pH. In some embodiments, the an-tibodies bind to myostatin (e.g., latent myostatin or propeptide myostatin) with higher affinity at pH7.4 than at pH5.8. In further embodiments, the antibodies bind to myostatin with at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more times higher affinity at pH7.4 than at pH5.8.

[0126] When an antigen is a soluble protein, the binding of an antibody to the antigen can result in an extended half-life of the antigen in plasma (i.e., reduced clearance of the antigen from plasma), since the antibody can have a longer half-life in plasma than the antigen itself and may serve as a carrier for the antigen. This is due to the recycling of the antigen-antibody complex by FcRn through the endosomal pathway in cell (Roopenian, Nat. Rev. Immunol. 7(9): 715-725 (2007)). However, an antibody with pH-dependent binding characteristics, which binds to its antigen in neutral extra-cellular environment while releasing the antigen into acidic endosomal compartments following its entry into cells, is expected to have superior properties in terms of antigen neutralization and clearance relative 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).

[0127] The "affinity" of an antibody for myostatin, for purposes of the present disclosure, is expressed in terms of the KD of the antibody. The KD of an antibody refers to the equilibrium dissociation constant of an antibody-antigen interaction. The greater the KD value is for an antibody binding to its antigen, the weaker its binding affinity is for that particular antigen. Accordingly, as used herein, the expression "higher affinity at neutral pH than at acidic pH" (or the equivalent expression "pH-dependent binding") means that the KD of the antibody binding to myostatin at acidic pH is greater than the KD of the antibody binding to myostatin at neutral pH. For example, in the context of the present invention, an antibody is considered to bind to myostatin with higher affinity at neutral pH than at acidic pH if the KD of the antibody binding to myostatin at acidic pH is at least 2 times greater than the KD of the antibody binding to myostatin at neutral pH. Thus, the present invention includes antibodies that bind to myostatin at acidic pH with a KD that is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more times greater than the KD of the antibody binding to myostatin at neutral pH. In another embodiment, the KD
value of the antibody at neutral pH can be 10 7 M, 108 M, 10 9 M, 10 1 M, 101 M, 10-12 M, or less. In another embodiment, the KD value of the antibody at acidic pH can be 9 M, 10 8M, 10 7 M, 106 M, or greater.

[0128] In further embodiments an antibody is considered to bind to myostatin (e.g., latent myostatin or propeptide myostatin) with a higher affinity at neutral pH than at acidic pH if the KD of the antibody binding to myostatin at pH5.8 is at least 2 times greater than the KD of the antibody binding to myostatin at pH7.4. In some embodiments the provided antibodies bind to myostatin at pH5.8 with a KD that is at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more times greater than the KD of the antibody binding to myostatin at pH7.4. In another embodiment, the KD value of the antibody at pH7.4 can be 10 7 M, 10 8M, 10 9 M, 10 10M, 10 " M, 10 12 M, or less. In another embodiment, the KD
value of the antibody at pH5.8 can be 10 9 M, 10 8M, 10 7 M, 10-6 M, or greater.

[0129] The binding properties of an antibody for a particular antigen may also be expressed in terms of the kd of the antibody. The kd of an antibody refers to the dissociation rate constant of the antibody with respect to a particular antigen and is expressed in terms of reciprocal seconds (i.e., sec 1). An increase in kd value signifies weaker binding of an antibody to its antigen. The present invention therefore includes antibodies that bind to myostatin with a higher kd value at acidic pH than at neutral pH. The present invention includes antibodies that bind to myostatin at acidic pH with a kd that is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more times greater than the kd of the antibody binding to myostatin at neutral pH. In another embodiment, the kd value of the antibody at neutral pH 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/s, or greater.
The invention also includes antibodies that bind to myostatin (e.g., latent myostatin or propeptide myostatin) with a higher kd value at pH5.8 than at pH7.4. The invention includes antibodies that bind to myostatin at pH5.8 with a kd that is at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more times greater than the kd of the antibody binding to myostatin at pH7.4. In another embodiment, the kd value of the antibody at pH7.4 can be 10-2 1/s, 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 pH5.8 can be 10 3 1/s, 10-2 1/s, 10 1/s, or greater.

[0130] In certain instances, a "reduced binding to myostatin at acidic pH
as compared to its binding at neutral pH" is expressed in terms of 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, an antibody may be regarded as exhibiting "reduced binding to myostatin at acidic pH as compared to its binding at neutral pH", for purposes of the present invention, if the antibody exhibits an acidic/
neutral KD ratio of 2 or greater. In certain embodiments, the pH5.8/pH7.4 KD
ratio for an anti-myostatin antibody of the present invention is 2 or greater. In certain exemplary embodiments, the acidic/neutral KD ratio for an antibody of the 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 greater. In another embodiment, the KD value of the antibody at neutral pH can be 10 7 M, 10 8M, 10 9 M, 10 1 M, 10 " M, 10i2 M, or less. In another embodiment, the KD value of the antibody at acidic pH can be 10 9 M, 10 8M, 10 7 M, 10-6 M, or greater. In further instances an antibody may be regarded as exhibiting "reduced binding to myostatin (e.g., latent myostatin) at acidic pH as compared to its binding at neutral pH", if the antibody exhibits an pH5.8/pH7.4 KD ratio of 2 or greater. In certain exemplary embodiments, the pH5.8/pH7.4 KD ratio for 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 greater. In another embodiment, the KD value of the antibody at pH7.4 can be 10 7 M, 10 8M, 10 9 M, 10 1 M, 10 " M, 10i2 M, or less. In another embodiment, the KD value of the antibody at pH5.8 can be 10 9 M, 10 8M, 10 7 M, 10-6 M, or greater.

[0131] In certain instances, a "reduced binding to myostatin at acidic pH
as compared to its binding at neutral pH" is expressed in terms of 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, an antibody may be regarded as exhibiting "reduced binding to myostatin at acidic pH as compared to its binding at neutral pH", for purposes of the present invention, if the antibody exhibits an acidic/neutral kd ratio of 2 or greater. In certain exemplary embodiments, the pH5.8/pH7.4 kd ratio for an antibody of the present invention is 2 or greater. In certain exemplary embodiments, the acidic/neutral kd ratio for an 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 greater. 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/s, or greater. In certain exemplary embodiments, the pH5.8/pH7.4 kd ratio for an 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 greater. In another embodiment, the kd value of the antibody at pH7.4 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 pH5.8 can be 10 3 1/s, 10-2 1/s, 10_i 1/s, or greater.

[0132] As used herein, the expression "acidic pH" means a pH of 4.0 to 6.5.
The expression "acidic pH" includes pH values of any one 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 particular aspects, the "acidic pH" is 5.8.

[0133] As used herein, the expression "neutral pH" means a pH of 6.7 to about 10Ø The ex-pression "neutral pH" includes pH values of any one 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 10Ø In particular aspects, the "neutral pH" is 7.4.

[0134] KD values, and kd values, as expressed herein, may be determined using a surface plasmon resonance-based biosensor to characterize antibody-antigen interactions. (See, e.g., Example 7, herein.) KD values, and kd values can be determined at 25 degrees C
or 37 degrees C.

[0135] In certain embodiments, an anti-myostatin antibody of the present invention binds to myostatin from more than one species. In further embodiments, the anti-myostatin antibody binds to myostatin from a human and non-human animal. In further em-bodiments, the anti-myostatin antibody binds to myostatin from human, mouse, and monkey (e.g., cynomolgus, rhesus macaque, marmoset, chimpanzee, or baboon).

[0136] In certain embodiments, an anti-myostatin antibody of the present invention binds to latent myostatin from more than one species. In further embodiments, the anti-myostatin antibody binds to latent myostatin from a human and non-human animal. In further embodiments, the anti-myostatin antibody binds to latent myostatin from human, mouse, and monkey.

[0137] In certain embodiments, an anti-myostatin antibody of the present invention binds to propeptide myostatin from more than one species. In further embodiments, the anti-myostatin antibody binds to propeptide myostatin from a human and non-human animal. In further embodiments, the anti-myostatin antibody binds to propeptide myostatin from human, mouse, and monkey.

[0138] In a further aspect, the invention provides an anti-myostatin antibody that forms an immune complex (i.e. antigen-antibody complex) with myostatin. In certain em-bodiments, two or more anti-myostatin antibodies bind to two or more myostatin molecules to form an immune complex. This is possible because myostatin exists as a homodimer containing two myostatin molecules while an antibody has two antigen-binding sites. The anti-myostatin antibody may bind to the same epitope on a myostatin molecule or may bind to different epitopes on a myostatin molecule, much like a bispecific antibody. Generally speaking, when two or more antibodies form an immune complex with two or more antigens, the resulting immune complex can strongly bind to Fc receptors existing on cell surfaces due to avidity effects through the Fc regions of the antibodies in the complex and can then be taken up into the cell with high efficiency. Thus, the above-mentioned anti-myostatin antibody capable of forming an immune complex containing two or more anti-myostatin antibodies and two or more myostatin molecules can lead to a rapid clearance of myostatin from plasma in a living body, via the strong binding to Fc receptors due to avidity effects.

[0139] Furthermore, an antibody with pH-dependent binding characteristics is thought to have superior properties in terms of antigen neutralization and clearance relative to its counterpart that binds 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, an antibody having both properties above, that is, an antibody which has pH-dependent binding characteristics and which forms an immune complex containing two or more antibodies with two or more antigens, is expected to have even more superior properties for highly accelerated elimination of antigens from plasma (WO 2013/081143).

[0140] In another aspect, the invention provides an anti-myostatin antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) a HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 55-57, 114-115, 126; (b) a HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 58-60, 116-120, 127; (c) a HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs:

61-64, 121, 128; (d) a HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 65-69, 122-124, 129; (e) a HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 70-72, 125, 130; and (f) a HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 73-74, 131.

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

[0142] In one aspect, the invention provides an anti-myostatin antibody comprising at least one, two, three, four, five, or six hypervariable regions (HVRs) selected from (a) a HVR-Hl comprising the amino acid sequence of any one of SEQ ID NOs:114-115;
(b) a HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 116-120;

(c) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) a HVR-comprising the amino acid sequence of any one of SEQ ID NOs: 122-124; (e) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 125; and (f) a HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 73-74.

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

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

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

NOs: 55-57, 114-115, 126; (b) a HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 58-60, 116-120, 127; and (c) a HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 61-64, 121, 128.

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

ID NOs: 73-74, and HVR-H2 comprising the amino acid sequence of any one of SEQ

ID NOs: 58-60. In a further embodiment, the antibody comprises (a) a HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 55-57; (b) a HVR-comprising the amino acid sequence of any one of SEQ ID NOs: 58-60; and (c) a HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 61-64.

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

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

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

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

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

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

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

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

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

[0155] In another aspect, an antibody of the invention comprises (a) a VH
domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) a HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 114-115, (ii) a HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs:

116-120, and (iii) a HVR-H3 comprising the amino acid sequence of SEQ ID NO:
121;
and (b) a VL domain comprising at least one, at least two, or all three VL HVR

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

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

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

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

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

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

[0161] In certain embodiments, any one or more amino acids of an anti-myostatin antibody as provided above are substituted at the following HVR positions: (a) in HVR-(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.

[0162] In certain embodiments, the one or more amino acid substitutions of an anti-myostatin antibody are conservative substitutions, as provided herein. In certain em-bodiments, any one or more of the following substitutions may be made in any com-bination: (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; YlOK; Al 1M or E; 512E; G16K; (c) in HVR-H3 (SEQ ID NO: 61), Y5H; T7H; L11K; (d) in HVR-L1 (SEQ ID NO: 65), Q1T; 52T;
55E; Y7F; D8H; N9D or A or E; (e) in HVR-L2 (SEQ ID NO: 70), 53E; 57Y, F or W;

and (f) in HVR-L3 (SEQ ID NO: 73), L8R.

[0163] All possible combinations of the above substitutions are encompassed by the consensus sequences of SEQ ID NOs: 126, 127, 128, 129, 130, and 131 for HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, respectively.

[0164] In any of the above embodiments, an anti-myostatin antibody can be humanized. In one embodiment, an anti-myostatin antibody comprises HVRs as in any of the above embodiments, and further comprises an acceptor human framework, e.g., a human im-munoglobulin framework or a human consensus framework. In another embodiment, an anti-myostatin antibody comprises HVRs as in any of the above embodiments, and further comprises a VH or VL comprising an FR sequence. In a further embodiment, the anti-myostatin antibody comprises the following heavy chain and/or light chain variable domain FR sequences: For the heavy chain variable domain, the 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, 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, FR4 comprises the amino acid sequence of SEQ ID
NO:
144.

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

187-192.

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

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

[0168] In another aspect, an antibody of the invention comprises (a) a VH
domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) a HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 157-162, (ii) a HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs:
163-168, and (iii) a HVR-H3 comprising the 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 (i) a HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 175-180, (ii) a HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 181-186, and (iii) a HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 187-192.

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

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

[0171] In another aspect, an anti-myostatin antibody comprises a VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 13, 16-30, 32, 33,and 34. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-myostatin antibody comprising that sequence retains the ability to bind to myostatin. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in any one of SEQ ID NOs: 13, 16-30, 32, 33 and34. In certain em-bodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-myostatin antibody comprises the VH
sequence in any one of SEQ ID NOs: 13, 16-30, 32, 33, and 34, including post-translational modifications of that sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) a HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 55-57, (b) a HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 58-60, and (c) a HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 61-64. Post-translational modifications include but are not limited to a modification of glutamine or glutamate in N-terminal of heavy chain or light chain to pyroglutamic acid by pyroglutamylation.

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

[0173] In another aspect, an anti-myostatin antibody is provided, wherein the antibody comprises a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID

NOs: 15, 31, 35-38, and 96-99. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains sub-stitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-myostatin antibody comprising that sequence retains the ability to bind to myostatin. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in any one of SEQ ID NOs: 15, 31, 35-38, and 96-99. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-myostatin antibody comprises the VL sequence in any one of SEQ ID NOs: 15, 31, 35-38, and 96-99, including post-translational modifications of that sequence. In a particular em-bodiment, the VL comprises one, two or three HVRs selected from (a) a HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 65-69, 122-124, 129;

(b) a HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 70-72, 125, 130; and (c) a HVR-L3 comprising the amino acid sequence of any one of SEQ
ID NOs: 73-74, 131. Post-translational modifications include but are not limited to a modification of glutamine or glutamate in N-terminal of heavy chain or light chain to pyroglutamic acid by pyroglutamylation.

[0174] In another aspect, an anti-myostatin antibody is provided, wherein the antibody comprises a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID

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

[0175] In another aspect, an anti-myostatin antibody is provided, wherein the antibody comprises a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID

NOs: 96-99. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., con-servative substitutions), insertions, or deletions relative to the reference sequence, but an anti-myostatin antibody comprising that sequence retains the ability to bind to myostatin. In certain embodiments, a total of 1 to 10 amino acids have been sub-stituted, inserted and/or deleted in any one of SEQ ID NOs: 96-99. In certain em-bodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs. Optionally, the anti-myostatin antibody comprises the VL sequence in any one of SEQ ID NOs: 96-99, including post-translational modifications of that sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from (a) a HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 122-124, 129; (b) a HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs:

71, 125, 130; and (c) a HVR-L3 comprising the amino acid sequence of SEQ ID
NO:
74, 131. Post-translational modifications include but are not limited to a modification of glutamine or glutamate in N-terminal of heavy chain or light chain to pyroglutamic acid by pyroglutamylation.
In another aspect, an anti-myostatin antibody is provided, wherein the antibody comprises a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 96. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative sub-stitutions), insertions, or deletions relative to the reference sequence, but an anti-myostatin antibody comprising that sequence retains the ability to bind to myostatin. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 96. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs. Optionally, the anti-myostatin antibody comprises the VL sequence in SEQ ID NO: 96, including post-translational modifications of that sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from (a) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (b) a HVR-L2 comprising the amino acid sequence of SEQ ID NO:
71; and (c) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74. Post-translational modifications include but are not limited to a modification of glutamine or glutamate in N-terminal of heavy chain or light chain to pyroglutamic acid by pyroglu-tamylation. In another aspect, an anti-myostatin antibody is provided, wherein the antibody comprises a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:
97.
In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative sub-stitutions), insertions, or deletions relative to the reference sequence, but an anti-myostatin antibody comprising that sequence retains the ability to bind to myostatin. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 97. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs. Optionally, the anti-myostatin antibody comprises the VL sequence in SEQ ID NO: 97, including post-translational modifications of that sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from (a) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 123; (b) a HVR-L2 comprising the amino acid sequence of SEQ ID NO:
71; and (c) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74. Post-translational modifications include but are not limited to a modification of glutamine or glutamate in N-terminal of heavy chain or light chain to pyroglutamic acid by pyroglu-tamylation.

[0176] In another aspect, an anti-myostatin antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH
and VL sequences in any one of SEQ ID NOs: 13, 16-30, 32-34, and 86-95 and any one of SEQ ID NOs: 15, 31, 35-38, and 96-99, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in any one of SEQ ID NOs: 13, 16-30, and 32-and any one of SEQ ID NOs: 15, 31, and 35-38, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in any one of SEQ ID NOs: 86-95 and any one of SEQ ID NOs: 96-99, respectively, including post-translational modifications of those sequences. Post-translational modifications include but are not limited to a modi-fication of glutamine or glutamate in N-terminal of heavy chain or light chain to py-roglutamic acid by pyroglutamylation.

[0177] In another aspect, an anti-myostatin antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH
and VL sequences in SEQ ID NO: 86 and SEQ ID NO: 96, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 92 and SEQ ID NO: 97, re-spectively, including post-translational modifications of those sequences.
Post-translational modifications include but are not limited to a modification of glutamine or glutamate in N-terminal of heavy chain or light chain to pyroglutamic acid by pyroglu-tamylation.

[0178] In another aspect, an anti-myostatin antibody comprises a VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 12, 145-150. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), in-sertions, or deletions relative to the reference sequence, but an anti-myostatin antibody comprising that sequence retains the ability to bind to myostatin. In certain em-bodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in any one of SEQ ID NOs: 12, 145-150. In certain embodiments, substitutions, in-sertions, or deletions occur in regions outside the HVRs (i.e., in the FRs).
Optionally, the anti-myostatin antibody comprises the VH sequence in any one of SEQ ID
NOs:

12, 145-150, including post-translational modifications of that sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) a HVR-comprising the amino acid sequence of any one of SEQ ID NOs: 55, 157-162, (b) a HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 58, 163-168, and (c) a HVR-H3 comprising the amino acid sequence of any one of SEQ

ID NOs: 61, 169-174. Post-translational modifications include but are not limited to a modification of glutamine or glutamate in N-terminal of heavy chain or light chain to pyroglutamic acid by pyroglutamylation.

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

[0180] In another aspect, an anti-myostatin antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH
and VL sequences in any one of SEQ ID NOs: 12, 145-150 and any one of SEQ ID
NOs: 14, 151-156, respectively, including post-translational modifications of those sequences. Post-translational modifications include but are not limited to a modi-fication of glutamine or glutamate in N-terminal of heavy chain or light chain to py-roglutamic acid by pyroglutamylation.

[0181] In certain embodiments, an anti-myostatin antibody of the present invention comprises a VH as in any of the embodiments provided above 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, an anti-myostatin antibody of the present invention comprises a VL as in any of the embodiments provided above, and a light chain constant region comprising the amino acid sequence of any one of SEQ ID NOs: 8 and 10.

[0182] In a further aspect, the invention provides an antibody that binds to the same epitope as an anti-myostatin antibody provided herein. In a further aspect, the invention provides an antibody that binds to the same epitope as an antibody described in Table 2a. In a further aspect, the invention provides an antibody that binds to the same epitope as an antibody described in Tables 1 la or 13. In certain embodiments, an antibody is provided that binds to an epitope within a fragment of myostatin propeptide consisting of amino acids 21-100 of SEQ ID NO: 78. Alternatively, the antibody binds a myostatin propeptide fragment consisting of amino acids 21-80, 41-100, 21-60, 41-80, 61-100, 21-40, 41-60, 61-80, or 81-100, of SEQ ID NO: 78.

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

[0184] In a further aspect, an anti-myostatin antibody according to any of the above em-bodiments may incorporate any of the features, singly or in combination, as described in Sections 1-7 below.
1. Antibody Affinity

[0185] In certain embodiments, an antibody provided herein has a dissociation constant (Kd) of 1 micro M 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., 108M or less, e.g., from 108M to 1013M, e.g., from 10 9 M to 1013M).

[0186] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA).
In one embodiment, an RIA is performed with the Fab version of an antibody of interest and its antigen. For example, solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of (125I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.
293:865-881 (1999)). To establish conditions for the assay, MICROTITER (registered trademark) multi-well plates (Thermo Scientific) are coated overnight with 5 micro g/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23 degrees C). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [125I1-antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1% polysorbate 20 (TWEEN-20 (registered trademark)) in PBS. When the plates have dried, 150 micro 1/
well of scintillant (MICROSCINT-20 TM; Packard) is added, and the plates are counted on a TOPCOUNT TM gamma counter (Packard) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in com-petitive binding assays.

[0187] According to another embodiment, Kd is measured using a BIACORE
(registered trademark) surface plasmon resonance assay. For example, an assay using a BIACORE
(registered trademark)-2000 or a BIACORE (registered trademark)-3000 (BIACORE
(registered trademark), Inc., Piscataway, NJ) is performed at 25 degrees C
with im-mobilized antigen CM5 chips at ¨10 response units (RU). In one embodiment, car-boxymethylated dextran biosensor chips (CM5, BIACORE (registered trademark), Inc.) are activated with N-ethyl-N'- (3-dimethylaminopropy1)-carbodiimide hy-drochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's in-structions. Antigen is diluted with 10 mM sodium acetate, pH4.8, to 5 micro g/m1 (-0.2 micro M) before injection at a flow rate of 5 micro I/minute to achieve ap-proximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic mea-surements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS
with 0.05% polysorbate 20 (TWEEN-20Tm) surfactant (PBST) at 25 degrees C at a flow rate of approximately 25 micro 1/min. Association rates (kon) and dissociation rates (koff) are calculated using a simple one-to-one Langmuir binding model (BIACORE (registered trademark) Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio koff/kon See, e.g., Chen et al., J.
Mol. Biol.
293:865-881 (1999). If the on-rate exceeds 106 M1 s 1 by the surface plasmon resonance assay above, then the on-rate can be determined using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm band-pass) at 25 degrees C
of a 20 nM anti-antigen antibody (Fab form) in PBS, pH7.2, in the presence of in-creasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophotometer (Aviv Instruments) or a 8000-series SLM-AMINCO TM
spectrophotometer (ThermoSpectronic) with a stirred cuvette.
2. Antibody Fragments

[0188] In certain embodiments, an antibody provided herein is an antibody fragment.
Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(abt)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, in The Pharmacology of Monoclonal Antibodies, vol.
113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994);
see also, WO 1993/16185; and US Patent Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(abt)2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see US Patent No. 5,869,046.

[0189] Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, 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 tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0190] Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., US Patent No. 6,248,516 B1).

[0191] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g., E. coli or phage), as described herein.
3. Chimeric and Humanized Antibodies

[0192] In certain embodiments, an antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, e.g., in US 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 a further example, a chimeric antibody is a "class switched"
antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0193] In certain embodiments, a chimeric antibody is a humanized antibody.
Typically, a non-human antibody is humanized to reduce immunogenicity to 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 HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region.
In some embodiments, some FR residues in a humanized antibody are substituted with corre-sponding residues from a non-human antibody (e.g., the antibody from which the HVR
residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0194] Humanized antibodies and methods of making them are reviewed, e.g., in Almagro, Front. Biosci. 13:1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA
86:10029-10033 (1989); US Patent Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity de-termining 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 the "guided selection" approach to FR
shuffling).

[0195] Human framework regions that may be used 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 of 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 (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front.
Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR
libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).
4. Human Antibodies

[0196] In certain embodiments, an antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human an-tibodies are described generally in van Dijk and van de Winkel, Curr. Opin.
Pharmacol. 5:368-374 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0197] Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the en-dogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic mice, the endogenous im-munoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech.

23:1117-1125 (2005). See also, e.g., US Patent Nos. 6,075,181 and 6,150,584 de-scribing XENOMOUSETm technology; US Patent No. 5,770,429 describing HUMAB
(registered trademark) technology; US Patent No. 7,041,870 describing K-M
MOUSE
(registered trademark) technology, and US Patent Application Publication No.
US
2007/0061900, describing VELOCIMOUSE (registered trademark) technology).
Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.

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

[0199] Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain.
Techniques for selecting human antibodies from antibody libraries are described below.
5. Library-Derived Antibodies

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

[0201] In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol. 12:433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments.
Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syn-thetically by cloning unrearranged V-gene segments from stem cells, and using PCR
primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J.
Mol. Biol. 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: US Patent No. 5,750,373, and US Publ. Nos.
2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

[0202] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.
6. Multispecific Antibodies

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

[0204] Techniques for making multispecific antibodies include, but are not limited to, re-combinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see, Milstein and Cuello, Nature 305:537 (1983)), WO
1993/08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole" en-gineering (see, e.g., US Patent No. 5,731,168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)); using "diabody" technology for making 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 preparing trispecific antibodies as described, e.g., in Tutt et al., J. Immunol. 147:60 (1991).

[0205] Engineered antibodies with three or more functional antigen binding sites, including "Octopus antibodies," are also included herein (see, e.g., US 2006/0025576A1).

[0206] The antibody or fragment herein also includes a "Dual Acting FAb" or "DAF"
comprising an antigen binding site that binds to myostatin as well as another, different antigen (see, US 2008/0069820, for example).
7. Antibody Variants

[0207] In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modi-fications include, for example, deletions from, and/or insertions into and/or sub-stitutions of residues within the amino acid sequences of the antibody. Any com-bination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.
a. Substitution, Insertion, and Deletion Variants

[0208] In certain embodiments, antibody variants having one or more amino acid sub-stitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions." More substantial changes are provided in Table 1 under the heading of "exemplary substitutions," and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/
improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

[Table 1]
Original Residue Exemplary Substitutions Preferred Substitution Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu. Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

[0209] Amino acids may be grouped according to common side-chain properties: (1) hy-drophobic: 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 sub-stitutions will entail exchanging a member of one of these groups for a member of another group.

[0210] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody).
Generally, the resulting variant(s) selected for further study will have modifications (e.g., im-provements) in certain biological properties (e.g., increased affinity, reduced immuno-genicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein.
Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0211] Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody affinity. Such alterations may be made in HVR "hotspots," i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or residues that contact antigen, with the resulting variant VH or VL being tested for binding affinity.
Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001).) In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen 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 created.
The library is then screened to identify any antibody variants with the desired affinity.
Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mu-tagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

[0212] In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative sub-stitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may, for example, be outside of antigen contacting residues in the HVRs. In certain embodiments of the variant VH and VL
sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.

[0213] A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demon-strating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex may be analyzed to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution.
Variants may be screened to determine whether they contain the desired properties.

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

[0215] In certain embodiments, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of gly-cosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

[0216] Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH
15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (G1cNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure.
In some embodiments, modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.

[0217] In one embodiment, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may 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 glycostructures attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO
2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about +/- 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies.
Such fucosylation variants may have improved ADCC function. See, e.g., US
Publ.Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated" or "fucose-deficient"
antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US
2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US
2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO
2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; 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 defu-cosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US
2003/0157108 Al, Presta, L; and WO 2004/056312, Adams et al., especially at Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng.
87:614 (2004); Kanda et al., Biotechnol. Bioeng. 94(4):680-688 (2006); and WO
2003/085107).

[0218] Antibodies variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.);
and US 2005/0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
c. Fc region variants

[0219] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0220] In certain embodiments, the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/
depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks Fc gamma R binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express Fc gamma RIII only, whereas monocytes express Fc gamma RI, Fc gamma RII and Fc gamma RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.
Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC
activity of a molecule of interest is described in US 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 methods may be employed (see, for example, ACT 1TM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox (registered trademark) non-radioactive cytotoxicity assay (Promega, Madison, WI)).
Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC
activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998).
Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO
2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol.
Methods 202:163 (1996); Cragg et al., Blood 101:1045-1052 (2003); and Cragg, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova et al., Int'l.
Immunol. 18(12):1759-1769 (2006)).

[0221] Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (US Patent No.

6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No.
7,332,581).

[0222] Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., US Patent No. 6,737,056; WO 2004/056312, and Shields et al., J.
Biol. Chem. 9(2): 6591-6604 (2001).)

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

[0224] In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cyto-toxicity (CDC), e.g., as described in US Patent No. 6,194,551, WO 1999/51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0225] Antibodies with increased half lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in U52005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (US

Patent No. 7,371,826). See also, Duncan, Nature 322:738-40 (1988); US Patent Nos.
5,648,260 and 5,624,821; and WO 1994/29351 concerning other examples of Fc region variants.
d. Cysteine engineered antibody variants

[0226] In certain embodiments, it may be desirable to create cysteine engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at ac-cessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein. In certain em-bodiments, any one or more of the following residues may be substituted with cysteine:
V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain;
and S400 (EU numbering) of the heavy chain Fc region. Cysteine engineered an-tibodies may be generated as described, e.g., in US Patent No. 7,521,541.
e. Antibody Derivatives

[0227] In certain embodiments, an antibody provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody 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), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, polypropylene glycol ho-mopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
The polymer may be of any molecular weight, and may be branched or unbranched.

The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

[0228] In another embodiment, conjugates of an antibody and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided. In one embodiment, the nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl.
Acad. Sci.

USA 102:11600-11605 (2005)). The radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the nonproteinaceous moiety to a temperature at which cells proximal to the antibody-nonproteinaceous moiety are killed.
8. Variant Fc regions

[0229] In one aspect, the invention provides an isolated polypeptide comprising a variant Fc region with enhanced Fc gamma Ruth-binding activity. In some apsects, the polypeptide is an antibody. In some apsects, 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 a "parent" Fc region). In certain embodiments, the variant Fc region of the invention has enhanced binding activity for monkey Fc gamma RIth compared to the parent Fc region. In particular embodiments, the monkey Fc gamma RIth is cynomolgus monkey Fc gamma Ruth (SEQ ID NO: 223).

[0230] In certain embodiments, the ratio of [KD value of a parent Fc region for monkey Fc gamma RIIb1/[KD value of a variant Fc region for monkey Fc gamma RIIb] 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 further embodiments, the variant Fc region has decreased binding activity for monkey Fc gamma RIIIa. In certain embodiments, the ratio of [KD value of a parent Fc region for monkey Fc gamma RIIIaNKD
value of a variant Fc region for monkey Fc gamma RIIIa] can be 0.50 or smaller, 0.40 or smaller, 0.30 or smaller, 0.20 or smaller, 0.10 or smaller, 0.09 or smaller, 0.08 or smaller, 0.07 or smaller, 0.06 or smaller, 0.05 or smaller, 0.04 or smaller, 0.03 or smaller, 0.02 or smaller, or 0.01 or smaller. In certain embodiments, the monkey Fc gamma RIth has the sequence of SEQ ID NO:223 (cynomolgus monkey). In certain embodiments, the monkey Fc gamma RIIIa has the sequence of SEQ ID NO:224 (cynomolgus monkey).

[0231] In further embodiments, the variant Fc region has enhanced binding activity for human Fc gamma RIIb. In certain embodiments, the ratio of [KD value of a parent Fc region for human Fc gamma RIIb1/[KD value of a variant Fc region for human Fc gamma RIIb] 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 further em-bodiments, the variant Fc region has decreased binding activity for human Fc gamma RIIIa. In certain embodiments, the ratio of [KD value of a parent Fc region for human Fc gamma RIIIaNKD value of a variant Fc region for human Fc gamma RIIIa] can be 0.50 or smaller, 0.40 or smaller, 0.30 or smaller, 0.20 or smaller, 0.10 or smaller, 0.09 or smaller, 0.08 or smaller, 0.07 or smaller, 0.06 or smaller, 0.05 or smaller, 0.04 or smaller, 0.03 or smaller, 0.02 or smaller, or 0.01 or smaller. In certain embodiments, the human Fc gamma Ruth has the sequence of SEQ ID NOS:212, 213, or 214. In certain embodiments, the human Fc gamma RIIIa has the sequence of SEQ ID
NO:215, 216, 217, or 218.

[0232] In further embodiments, the variant Fc region has decreased binding activity for human Fc gamma RIIa (type H) than for human Fc gamma RIIb. In certain em-bodiments, the ratio of [KD value of a parent Fc region for human Fc gamma RIIa (type H)1/[KD value of a variant Fc region for human Fc gamma RIIa (type H)]
can be 5.0 or smaller, 4.0 or smaller, 3.0 or smaller, 2.0 or smaller, 1.0 or smaller, 0.9 or smaller, 0.8 or smaller, 0.7 or smaller, 0.6 or smaller, 0.5 or smaller, 0.4 or smaller, 0.3 or smaller, 0.2 or smaller, or 0.1 or smaller. In further embodiments, the variant Fc region has decreased binding activity for human Fc gamma RIIa (type R) than for human Fc gamma RIIb. In certain embodiments, the ratio of [KD value of a parent Fc region for human Fc gamma RIIa (type R)]/[KD value of a variant Fc region for human Fc gamma RIIa (type R)] can be 5.0 or smaller,4.0 or smaller, 3.0 or smaller, 2.0 or smaller, 1.0 or smaller, 0.9 or smaller, 0.8 or smaller, 0.7 or smaller, 0.6 or smaller, 0.5 or smaller, 0.4 or smaller, 0.3 or smaller, 0.2 or smaller, or 0.1 or smaller.
In certain embodiments, the human Fc gamma RIIa (type H) has the sequence of SEQ
ID NO:211. In certain embodiments, the human Fc gamma RIIa (type R) has the sequence of SEQ ID NO:210.

[0233] In certain embodiments, the ratio of [KD value of a parent Fc region for monkey Fc gamma RIIa1/[KD value of a variant Fc region for monkey Fc gamma RIIa] 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 certain embodiments, monkey Fc gamma RIIa is selected from monkey Fc gamma RIIal (e.g., cynomolgus Fc gamma RIIal (SEQ ID NO:220)), monkey Fc gamma RIIa2 (e.g., cynomolgus Fc gamma RIIa2 (SEQ ID NO:221)), and monkey Fc gamma RIIa3 (e.g., cynomolgus Fc gamma RIIa3 (SEQ ID NO:222).

[0234] In another embodiment, the KD value of the variant Fc region for monkey Fc gamma Ruth can be 1.0x106 M or smaller, 9.0x107 M or smaller, 8.0x107 M or smaller, 7.0x107 M or smaller, 6.0x107 M or smaller, 5.0x107 M or smaller, 4.0x107 M or smaller, 3.0x107 M or smaller, 2.0x107 M or smaller, or 1.0x107 M or smaller.
In another embodiment, the KD value of the variant Fc region for monkey Fc gamma RIIIa can be 5.0x107 M or greater, 6.0x107 M or greater, 7.0x107 M or greater, 8.0x10 7 M or greater, 9.0x107 M or greater, 1.0x106 M or greater, 2.0x106 M or greater, 3.0x106 M or greater, 4.0x106 M or greater, 5.0x106 M or greater, 6.0x106 M or greater, 7.0x106 M or greater, 8.0x106 M or greater, 9.0x106 M or greater, or 1.0x105 M or greater. In another embodiment, the KD value of the variant Fc region for human Fc gamma Ruth can be 2.0x106 M or smaller, 1.0x106 M or smaller, 9.0x107 M or smaller, 8.0x10 7 M or smaller, 7.0x107 M or smaller, 6.0x107 M or smaller, 5.0x107 M or smaller, 4.0x107 M or smaller, 3.0x107 M or smaller, 2.0x107 M or smaller, or 1.0x107 M or smaller. In another embodiment, the KD value of the variant Fc region for human Fc gamma RIIIa can be 1.0x106 M or greater, 2.0x106 M or greater, 3.0x10 6 M or greater, 4.0x106 M or greater, 5.0x106 M or greater, 6.0x106 M or greater, 7.0x106 M or greater, 8.0x106 M or greater, 9.0x106 M or greater, 1.0x105 M or greater, 2.0x10 5 M or greater, 3.0x105 M or greater, 4.0x105 M or greater, or 5.0x105 M or greater. In another embodiment, the KD value of the variant Fc region for human Fc gamma RIIa (type H) can be 1.0x107 M or greater, 2.0x107 M or greater, 3.0x107 M or greater, 4.0x107 M or greater, 5.0x107 M or greater, 6.0x107 M or greater, 7.0x107 M or greater, 8.0x107 M or greater, 9.0x107 M or greater, 1.0x106 M or greater, 2.0x106 M or greater, 3.0x106 M or greater, 4.0x106 M or greater, or 5.0x106 M or greater. In another embodiment, the KD value of the variant Fc region for human Fc gamma RIIa (type R) can be 2.0x107 M or greater, 3.0x107 M or greater, 4.0x107 M or greater, 5.0x107 M or greater, 6.0x107 M or greater, 7.0x107 M or greater, 8.0x107 M or greater, 9.0x107 M or greater, 1.0x106 M or greater, 2.0x106 M or greater, 3.0x106 M or greater, 4.0x106 M or greater, or 5.0x106 M or greater.

[0235] In another embodiment, the KD value of the variant Fc region for monkey Fc gamma RIIa can be 1.0x106 M or smaller, 9.0x107 M or smaller, 8.0x107 M or smaller, 7.0x107 M or smaller, 6.0x107 M or smaller, 5.0x107 M or smaller, 4.0x107 M or smaller, 3.0x107 M or smaller, 2.0x107 M or smaller, or 1.0x107 M or smaller.
In certain embodiments, monkey Fc gamma RIIa can be selected from any one of monkey Fc gamma RIIal, monkey Fc gamma RIIa2, and monkey Fc gamma RIIa3.

[0236] When we develop a pharmaceutical product for the treatment of human diseases, evaluation of its efficacy and safety in monkey are important because of its biological proximity to human. From such viewpoints, a pharmaceutical product to be developed is preferable to have cross-reactivity to both human and monkey in the target binding activity.

[0237] "Fc gamma receptors" (herein, referred to as Fc gamma receptors, Fc gamma R or FcgR) refers to receptors that may bind to the Fc region of IgG 1, IgG2, IgG3, and IgG4 monoclonal antibodies, and practically means any member of the family of proteins encoded by the Fc gamma receptor genes. In humans, this family includes Fc gamma RI (CD64) including isoforms Fc gamma RIa, Fc gamma RIb, and Fc gamma RIc; Fc gamma RII (CD32) including isoforms Fc gamma RIIa (including allotypes H131 (type H) and R131 (type R)), Fc gamma Ruth (including Fc gamma Rllb-1 and Fc gamma RIIb-2), and Fc gamma RIIc; and Fc gamma RIII (CD16) including isoforms Fc gamma RIIIa (including allotypes V158 and F158), and Fc gamma RIIIb (including allotypes Fc gamma RIIIb-NA1 and Fc gamma RIIIb-NA2), and any human Fc gamma Rs, Fc gamma R isoforms or allotypes yet to be discovered, but is not limited thereto. Fc gamma RIIbl and Fc gamma RIIb2 have been reported as splicing variants of human Fc gamma RHb. In addition, a splicing variant named Fc gamma RIIb3 has been reported (J Exp Med, 1989, 170: 1369-1385). In addition to these splicing variants, human Fc gamma RHb 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. Furthermore, human Fc gamma Ruth includes every previously-reported genetic polymorphism, as well as Fc gamma RHb (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 every genetic poly-morphism that will be reported in the future.

[0238] In Fc gamma RIIa, there are two allotypes, one where the amino acid at position 131 of Fc gamma RIIa is histidine (type H) and the other where the amino acid at position 131 is substituted with arginine (type R) (Warrmerdam, J. Exp. Med. 172:19-25 (1990)).

[0239] The Fc gamma R includes human, mouse, rat, rabbit, and monkey-derived Fc gamma Rs but is not limited thereto, and may be derived from any organism. Mouse Fc gamma Rs include Fc gamma RI (CD64), Fc gamma RII (CD32), Fc gamma RIII
(CD16), and Fc gamma RIII-2 (CD16-2), and any mouse Fc gamma Rs, or Fc gamma R isoforms, but are not limited thereto. Unless otherwise specified, the term "monkey Fc gamma R" or variation thereof, refers to cynomolgus Fc gamma RIIal (SEQ ID
NO:220), Fc gamma RIIa2 (SEQ ID NO:221), Fc gamma RIIa3 (SEQ ID NO:222), Fc gamma RIM (SEQ ID NO:223), or Fc gamma RIIIaS (SEQ ID NO:224).

[0240] The polynucleotide sequence of human Fc gamma RI is set forth in SEQ
ID NO: 199 (NM 000566.3); the polynucleotide sequence of human Fc gamma RIIa is set forth in SEQ ID NO: 200 (BCO20823.1) or SEQ ID NO:201 (NM 001136219.1); the polynu-cleotide sequence of human Fc gamma RIM is set forth in SEQ ID NO: 202 (BC146678.1) or SEQ ID NO:203 (NM 004001.3); the polynucleotide sequence of human Fc gamma RIIIa is set forth in SEQ ID NO: 204 (BC033678.1) or SEQ ID
NO:205 (NM 001127593.1); and the polynucleotide sequence of human Fc gamma RIIIb is set forth in SEQ ID NO: 206 (BC128562.1).

[0241] The amino acid sequence of human Fc gamma RI is set forth in SEQ ID NO:

(NP 000557.1); the amino acid sequence of human Fc gamma RIIa is set forth in SEQ
ID NO: 208 (AAH20823.1), SEQ ID NO:209, SEQ ID NO:210 or SEQ ID NO:211;

the amino acid sequence of human Fc gamma RIth is set forth in SEQ ID NO: 212 (AAI46679.1), SEQ ID NO: 213 or SEQ ID NO:214; the amino acid sequence of human Fc gamma RIIIa is set forth in SEQ ID NO: 215 (AAH33678.1), SEQ ID
NO:216, SEQ ID NO:217 or SEQ ID NO:218; and the amino acid sequence of human Fc gamma RIIIb is set forth in SEQ ID NO: 219 (AAI28563.1).

[0242] The amino acid sequence of cynomolgus monkey Fc gamma RIIa is set forth in SEQ
ID NO: 220 (Fc gamma RIIal), SEQ ID NO:221 (Fc gamma RIIa2) or SEQ ID
NO :222 (Fc gamma RIIa3); the amino acid sequence of cynomolgus Fc gamma RIth is set forth in SEQ ID NO: 223; and the amino acid sequence of cynomolgus monkey Fc gamma RIIIa is set forth in SEQ ID NO: 224.

[0243] In one aspect, the invention provides polypeptides containing variant Fc regions with enhanced Fc gamma Ruth-binding activity compared to a corresponding reference Fc gamma Ruth-binding polypeptide. In further aspects, the polypeptide of the invention comprises at least one amino acid alteration of 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, the Fc gamma RIth has the sequence of cynomolgus monkey Fc gamma Ruth (SEQ ID NO:223). In certain embodiments, the Fc gamma Ruth has the sequence of human Fc gamma RIth (e.g., SEQ ID NOS:212, 213, or 214).

[0244] In one aspect, the invention provides polypeptides comprising variant Fc regions with enhanced Fc gamma Ruth-binding activity comprising at least two amino acid al-terations comprising: (a) one amino acid alteration at position 236, and (b) at least one amino acid alteration of 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 em-bodiments, the Fc gamma RIth has the sequence of cynomolgus monkey Fc gamma Ruth (SEQ ID NO:223). In certain embodiments, the Fc gamma Ruth has the sequence of human Fc gamma RIth (e.g., SEQ ID NOS:212, 213, or 214).

[0245] In one aspect, the invention provides polypeptides comprising a variant Fc region with enhanced Fc gamma Ruth-binding activity comprising an amino acid alteration at position 236 according to EU numbering.

[0246] In one aspect, the invention provides polypeptides comprising a variant Fc region with enhanced Fc gamma RIM-binding activity comprising at least two amino acid al-terations comprising: (a) one amino acid alteration at position 236, and (b) at least one amino acid alteration of 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 a further embodiment, the variant Fc region comprises an amino acid alteration of 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 a further embodiment, the variant Fc region comprises an amino acid alteration of at least one position selected from the group consisting of: 268, 295, 326, and 330, according to EU
numbering. In certain embodiments, the Fc gamma Ruth has the sequence of cynomolgus monkey Fc gamma RIth (SEQ ID NO:223). In certain embodiments, the Fc gamma RIth has the sequence of human Fc gamma Ruth (e.g., SEQ ID NOS:212, 213, or 214).

[0247] In another aspect, the invention provides polypeptides comprising variant Fc regions with enhanced Fc gamma Ruth-binding activity comprising amino acid alterations 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, the Fc gamma RIth has the sequence of cynomolgus monkey Fc gamma Ruth (SEQ ID NO:223). In certain embodiments, the Fc gamma Ruth has the sequence of human Fc gamma RIth (e.g., SEQ ID NOS:212, 213, or 214).

[0248] In a further embodiment, the variant Fc region with enhanced Fc gamma Ruth-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; (1) 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, Gin, Arg, Ser, Thr, Val, Trp, Tyr at position 396;
according to EU numbering. In certain embodiments, the Fc gamma Ruth has the sequence of cynomolgus monkey Fc gamma Ruth (SEQ ID NO:223). In certain em-bodiments, the Fc gamma Ruth has the sequence of human Fc gamma Ruth (e.g., SEQ
ID NOS:212, 213, or 214).

[0249] In a further embodiment, the variant Fc region with enhanced Fc gamma Ruth-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 a further embodiment, the variant Fc region with enhanced Fc gamma Ruth-binding activity comprises amino acid alterations (e.g., substitutions) of:
Asn at position 236, Glu at position 268, Lys at position 330, and Met at position 396;
according to EU numbering. In a further embodiment, the variant Fc region with enhanced Fc gamma Rllb-binding activity comprises amino acid alterations (e.g., sub-stitutions) of: Asn at position 236, Asp at position 268, and Lys at position 330;
according to EU numbering. In a further embodiment, the variant Fc region with enhanced Fc gamma Rllb-binding activity comprises amino acid alterations (e.g., sub-stitutions) of: Asn at position 236, Asp at position 268, Leu at position 295, and Lys at position 330; according to EU numbering. In a further embodiment, the variant Fc region with enhanced Fc gamma Ruth-binding activity comprises amino acid al-terations (e.g., substitutions) of: Thr at position 236, Asp at position 268, and Lys at position 330; according to EU numbering. In a further embodiment, the variant Fc region with enhanced Fc gamma Ruth-binding activity comprises amino acid al-terations (e.g., substitutions) of: 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 a further embodiment, the variant Fc region with enhanced Fc gamma RHb-binding activity comprises amino acid alterations (e.g., substitutions) of: 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.

[0250] In one aspect, the invention provides polypeptides comprising variant Fc regions with enhanced Fc gamma Ruth-binding activity comprising an amino acid alteration at position 238 according to EU numbering.

[0251] In one aspect, the invention provides polypeptides comprising variant Fc regions with enhanced Fc gamma Ruth-binding activity comprising at least one amino acid al-teration of at least one position selected from the group consisting of: 234, 238, 250, 264, 267, 307, and 330 according to EU numbering. In further embodiments, the polypeptide comprises at least one amino acid alteration of at least one position selected from the group consisting of: 234, 250, 264, 267, 307, and 330 according to EU numbering. In certain embodiments, the Fc gamma Ruth has the sequence of cynomolgus monkey Fc gamma Ruth (SEQ ID NO:223). In certain embodiments, the Fc gamma Ruth has the sequence of human Fc gamma Ruth (e.g., SEQ ID NOS:212, 213, or 214).

[0252] In another aspect, the invention provides polypeptides comprising variant Fc regions with enhanced Fc gamma Ruth-binding activity comprising amino acid alterations of any one of the following (1)-(9): (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, the Fc gamma Ruth has the sequence of cynomolgus monkey Fc gamma Ruth (SEQ ID NO:223). In certain em-bodiments, the Fc gamma Ruth has the sequence of human Fc gamma Ruth (e.g., SEQ
ID NOS:212, 213, or 214).

[0253] In a further embodiment, the variant Fc region with enhanced Fc gamma Ruth-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 a further em-bodiment, the variant Fc region with enhanced Fc gamma RHb-binding activity comprises amino acid alterations (e.g., substitutions) of: Asp at position 238; according to EU numbering. In a further embodiment, the variant Fc region with enhanced Fc gamma Ruth-binding activity comprises amino acid alterations (e.g., substitutions) of:
Asp at position 238, Val at position 250, and Pro at position 307; according to EU
numbering. In a further embodiment, the variant Fc region with enhanced Fc gamma Rllb-binding activity comprises amino acid alterations (e.g., substitutions) of: Asp at position 238, Val at position 250, Pro at position 307, and Lys at position 330;
according to EU numbering. In a further embodiment, the variant Fc region with enhanced Fc gamma RHb-binding activity comprises amino acid alterations (e.g., sub-stitutions) of: 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 a further em-bodiment, the variant Fc region with enhanced Fc gamma RHb-binding activity comprises amino acid alterations (e.g., substitutions) of: 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 a further embodiment, the variant Fc region with enhanced Fc gamma RHb-binding activity comprises amino acid alterations (e.g., sub-stitutions) of: 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 a further em-bodiment, the variant Fc region with enhanced Fc gamma RHb-binding activity comprises amino acid alterations (e.g., substitutions) of: 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 a further embodiment, the variant Fc region with enhanced Fc gamma Ruth-binding activity comprises amino acid al-terations (e.g., substitutions) of: 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 a further embodiment, the variant Fc region with enhanced Fc gamma Rllb-binding activity comprises amino acid alterations (e.g., substitutions) of: 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 a further em-bodiment, the variant Fc region with enhanced Fc gamma RHb-binding activity comprises amino acid alterations (e.g., substitutions) of: 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 em-bodiments, the Fc gamma Ruth has the sequence of cynomolgus monkey Fc gamma Ruth (SEQ ID NO:223). In certain embodiments, the Fc gamma Ruth has the sequence of human Fc gamma RIM (e.g., SEQ ID NOS:212, 213, or 214).

[0254] In another aspect, the invention provides isolated polypeptides comprising variant Fc regions with increased isoelectric point (pI). In certain embodiments, a variant Fc region described herein comprises at least two amino acid alterations in a parent Fc region. In certain embodiments, each of the amino acid alterations increases the iso-electric point (pI) of the variant Fc region compared with that of the parent Fc region.
They are based on the findings that antigen elimination from plasma can be promoted with an antibody whose pI has been increased by modification of at least two amino acid residues, for example when the antibody is administered in vivo.

[0255] In the present invention, pI may be either a theoretical or an experimentally de-termined pI. The value of pI can be determined, for example, by isoelectric focusing known to those skilled in the art. The value of a theoretical pI can be calculated, for example, using gene and amino acid sequence analysis software (Genetyx, etc.).

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

[0257] In certain embodiments, the amino acid for increased pI can be exposed on the surface of the variant Fc region. In the present invention, an amino acid that can be exposed on the surface generally refers to an amino acid residue located on the surface of a polypeptide constituting a variant Fc region. An amino acid residue located on the surface of a polypeptide refers to an amino acid residue whose side chain can be in contact with solvent molecules (which in general are mostly water molecules).
However, the side chain does not necessarily have to be wholly in contact with solvent molecules, and when even a portion of the side chain is in contact with 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 a polypeptide also include amino acid residues located close to the surface and thereby can have an electric charge influence from another amino acid residue whose side chain, even partly, is in contact with the solvent molecules. Those skilled in the art can prepare a homology model of a polypeptide for example, using commercially available softwares.
Alternatively, it is possible to use methods known to those skilled in the art, such as X-ray crystal-lography. The amino acid residues that can be exposed on the surface are determined, for example, using coordinates from a three-dimensional model using a computer program such as InsightII program (Accelrys). Surface-exposable sites may be de-termined using algorithms known in the technical field (for example, Lee and Richards (J. Mol. Biol. 55:379-400 (1971)); Connolly (J. Appl. Cryst. 16:548-558 (1983)).
Surface-exposable sites can be determined using software suitable for protein modeling and three-dimensional structure information. Software available for such purposes includes, for example, the SYBYL Biopolymer Module software (Tripos Associates).
When an algorithm requires a user input size parameter, the "size" of a probe which is used in the calculation may be set to about 1.4 Angstrom (A) or less in radius. Fur-thermore, methods for determining surface-exposable regions using software for personal computers have been described by Pacios (Comput. Chem. 18(4):377-386 (1994); J. Mol. Model. 1:46-53 (1995)). Based on such information as described above, appropriate amino acid residues located on the surface of a polypeptide that constitutes a variant Fc region can be selected.

[0258] In certain embodiments, a polypeptide comprises both the variant Fc region and an antigen-binding domain. In further embodiments, the antigen is a soluble antigen. In one embodiment, the antigen is present in biological fluids (for example, plasma, in-terstitial fluid, lymphatic fluid, ascitic fluid, and pleural fluid) of subjects. The antigen may also be a membrane antigen.

[0259] In further embodiments, antigen-binding activity of the antigen-binding domain changes according to ion concentration conditions. In one embodiment, ion con-centration is not particularly limited and refers to hydrogen ion concentration (pH) or metal ion concentration. Herein, metal ions refer to ions of group I elements except hydrogen, such as alkaline metals and the copper group elements, group II
elements such as alkaline earth metals and zinc group elements, group III elements except boron, group IV elements except carbon and silicon, group VIII elements such as iron group and platinum group elements, elements belonging to subgroup A of groups V, VI, and VII, and metal elements such as antimony, bismuth, and polonium. In the present invention, metal ions include, for example, calcium ion, as described in WO
2012/073992 and WO 2013/125667. In one embodiment, "ion concentration condition" may be a condition that focuses on differences in the biological behavior of an antigen-binding domain between a low ion concentration and a high ion con-centration. Furthermore, "antigen-binding activity of an antigen-binding domain changes according to ion concentration conditions" means that the antigen-binding activity of an antigen-binding domain changes between a low ion concentration and a high ion concentration (such an antigen-binding domain is referred to herein as "ion concentration-dependent antigen-binding domain"). The antigen-binding activity of an antigen-binding domain under a high ion concentration condition may be higher (stronger) or lower (weaker) than that under a low ion concentration condition. In one embodiment, ion concentration-dependent antigen-binding domains (such as pH-dependent antigen-binding domains or calcium ion concentration-dependent antigen-binding domains) can be obtained by known methods, for example, described in WO
2009/125825, WO 2012/073992, and WO 2013/046722.

[0260] In the present invention, the antigen-binding activity of an antigen-binding domain under a high calcium ion concentration condition may be higher than under a low calcium ion concentration condition. The high calcium ion concentration is not par-ticularly limited to but may be a concentration selected between 100 micro M
and 10 mM, between 200 micro M and 5 mM, between 400 micro M and 3 mM, between 200 micro M and 2 mM, between 400 micro M and 1 mM, or between 500 micro M and 2.5 mM, which is preferable to be close to the plasma (blood) concentration of calcium ion in vivo. Meanwhile, the low calcium ion concentration is not particularly limited to but may be a concentration selected between 0.1 micro M and 30 micro M, between 0.2 micro M and 20 micro M, between 0.5 micro M and 10 micro M, between 1 micro M and 5 micro M, or between 2 micro M and 4 micro M, which is preferable to be close to the concentration of calcium ion in early endosomes in vivo.

[0261] In one embodiment, the ratio between the antigen-binding activities under a low calcium ion concentration condition and a high calcium ion concentration condition is not limited but the ratio of the dissociation constant (KD) under a low calcium ion con-centration condition to the KD under a high calcium ion concentration condition, i.e., KD (low calcium ion concentration condition)/KD (high calcium ion concentration condition), is 2 or more, 10 or more, or 40 or more. The upper limit of the ratio may be 400, 1000, or 10000, as long as such an antigen-binding domain can be produced by techniques known to those skilled in the art. Alternatively, for example, the dis-sociation rate constant (kd) can be used instead of the KD. In this case, the ratio of the kd under a low calcium ion concentration condition to the kd under a high calcium ion concentration condition, i.e., kd (low calcium ion concentration condition)/kd (high calcium ion concentration condition), is 2 or more, 5 or more, 10 or more, or 30 or more. The upper limit of the ratio may be 50, 100, or 200, as long as the antigen-binding domain can be produced based on the common technical knowledge of those skilled in the art.

[0262] In the present invention, the antigen-binding activity of an antigen-binding domain under a low hydrogen ion concentration (neutral pH) may be higher than under a high hydrogen ion concentration (acidic pH). The acidic pH may be, for example, a pH
selected from pH4.0 to pH6.5, selected from pH4.5 to pH6.5, selected from pH5.0 to pH6.5, or selected from pH5.5 to pH6.5, which is preferable to be close to the in vivo pH in early endosomes. The acidic pH may also be, for example, pH5.8 or pH6Ø
In particular embodiments, the acidic pH is pH5.8. Meanwhile, the neutral pH may be, for example, a pH selected from pH6.7 to pH10.0, selected from pH6.7 to pH9.5, selected from pH7.0 to pH9.0, or selected from pH7.0 to pH8.0, which is preferable to be close to the in vivo pH in plasma (blood). The neutral pH may also be, for example, pH7.4 or pH7Ø In particular embodiments, the neutral pH is pH7.4.

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

[0264] In one embodiment, for example, at least one amino acid residue is substituted with an amino acid residue with a side-chain pKa of 4.0-8.0, and/or at least one amino acid with a side-chain pKa of 4.0-8.0 is inserted in the antigen-binding domain, as described in WO 2009/125825. The amino acid may be substituted and/or inserted at any site as long as the antigen-binding activity of the antigen-binding domain becomes weaker under an acidic pH condition than under a neutral pH condition as compared to before the substitution or insertion. When the antigen-binding domain has a variable region or CDR, the site may be within the variable region or CDR. The number of amino acids that are substituted or inserted can be appropriately determined by those skilled in the art; and the number may be one or more. Amino acids with a side-chain pKa of 4.0-8.0 can be used to change the antigen-binding activity of the antigen-binding domain according to the hydrogen ion concentration condition. Such amino acids include, for example, natural amino acids such as His (H) and Glu (E), and unnatural amino acids such as histidine analogs (U52009/0035836), m-NO2-Tyr (pKa 7.45), 3,5-Br2-Tyr (pKa 7.21), and 3,5-I2-Tyr (pKa 7.38) (Hey' et al., Bioorg. Med. Chem.
11(17):3761-3768 (2003)). Amino acids with a side-chain pKa of 6.0-7.0 can also be used, which include, e.g., His (H).

[0265] In another embodiment, preferable antigen-binding domains for the variant Fc region with increased pI are described and can be obtained by methods described in Japanese patent applications JP2015-021371 and JP2015-185254.

[0266] In certain embodiments, the variant Fc region with increased pI
comprises at least two amino acid alterations of 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.

[0267] In further embodiments, the variant Fc region with increased pI
comprises at least two amino acid alterations of at least two positions selected from the group consisting of: 311, 341, 343, 384, 399, 400, 401, 402, and 413, according to EU
numbering.

[0268] In another aspect, the invention provides polypeptides comprising variant Fc regions with increased pI comprising amino acid alterations 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.

[0269] A method for increasing the pI of a protein is, for example, to reduce the number of amino acids with a negatively charged side chain (for example, aspartic acid and glutamic acid) and/or to increase the number of amino acids with a positively charged side chain (for example, arginine, lysine and histidine) at a neutral pH
condition.
Amino acids with a negatively charged side chain have a negative charge represented as -1 at a pH condition that is sufficiently higher than their side chain pKa, which is a theory well known to those skilled in the art. For example, the theoretical pKa for the side chain of aspartic acid is 3.9, and the side chain has a negative charge represented as -1 at a neutral pH condition (for example, in a solution of pH7.0).
Conversely, amino acids with a positively charged side chain have a positive charge represented as +1 at a pH condition that is sufficiently lower than their side chain pKa. For example, the theoretical pKa for the side chain of arginine is 12.5, and the side chain has a positive charge represented as +1 at a neutral pH condition (for example, in a solution of pH7.0). Meanwhile, amino acids whose side chain has no charge at a neutral pH
condition (for example, in a solution of pH7.0) are known to include 15 types of natural amino acids, i.e., alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan, and tyrosine. As a matter of course, it is understood that amino acids for increasing the pI
may be unnatural amino acids.

[0270] From the above, a method for increasing the pI of a protein at a neutral pH condition (for example, in a solution of pH7.0) can confer a charge alteration of +1 to a protein of interest, for example, by substituting amino acids with non-charged side chains for aspartic acid or glutamic acid (whose side chain has a negative charge of -1) in the amino acid sequence of the protein. Furthermore, a charge alteration of +1 can be conferred to the protein, for example, by substituting arginine or lysine (whose side chain has a positive charge of +1) for amino acids whose side chain has no charge.
Moreover, a charge alteration of +2 can be conferred at a time to the protein by sub-stituting arginine or lysine (whose side chain has a positive charge of +1) for aspartic acid or glutamic acid (whose side chain has a negative charge of -1).
Alternatively, to increase the pI of a protein, amino acids with a side chain having no charge and/or preferably amino acids having a positively charged side chain can be added or inserted into the amino acid sequence of the protein, or amino acids with a side chain having no charge and/or preferably amino acids with a negatively charged side chain present in the amino acid sequence of the protein can be deleted. It is understood that, for example, the N-terminal and C-terminal amino acid residues of a protein have a main chain-derived charge (NH3+ of the amino group at the N-terminus and COO of the carbonyl group at the C-terminus) in addition to their side chain-derived charges. Thus, the pI of a protein can also be increased by performing to the main chain-derived functional groups some addition, deletion, substitution, or insertion.

[0271] The substitution of an amino acid to increase the pI includes, for example, sub-stitution of an amino acid whose side chain has no charge for an amino acid having a negatively charged side chain, substitution of an amino acid having a positively charged side chain for an amino acid whose side chain has no charge, and substitution of an amino acid having a positively charged side chain for an amino acid having a negatively charged side chain in the amino acid sequence of a parent Fc region, which are performed alone or in appropriate combinations.

[0272] The insertion or addition of an amino acid to increase the pI
includes, for example, insertion or addition of an amino acid whose side chain has no charge, and/or insertion or addition of an amino acid having a positively charged side chain in the amino acid sequence of a parent Fc region, which are performed alone or in appropriate com-binations.

[0273] The deletion of an amino acid to increase the pI includes, for example, deletion of an amino acid whose side chain has no charge, and/or deletion of an amino acid having a negatively charged side chain in the amino acid sequence of a parent Fc region, which are performed alone or in appropriate combinations.

[0274] In one embodiment, natural amino acids used for increasing pI can be classified as follows: (a) an amino acid with a negatively charged side chain can be Glu (E) or Asp (D); (b) an amino acid whose side chain has no charge 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) an amino acid with a positively charged side chain can be His (H), Lys (K), or Arg (R). In one embodiment, the amino acid insertion or substitution after modification is Lys (K) or Arg (R).

[0275] In another aspect, the invention provides isolated polypeptides comprising variant Fc regions with enhanced Fc gamma Ruth-binding activity and increased pI. In certain embodiments, a variant Fc region described herein comprises at least two amino acid alterations in a parent Fc region.

[0276] In one aspect, the invention provides polypeptides comprising variant Fc regions with enhanced Fc gamma Ruth-binding activity and increased pI comprising at least three amino acid alterations comprising: (a) at least one amino acid alteration of 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 of 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.

[0277] In one aspect, the invention provides polypeptides comprising variant Fc regions with enhanced Fc gamma RIM-binding activity and increased pI, and that comprise at least three amino acid alterations comprising: (a) at least one amino acid alteration of 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 of at least two positions selected from the group consisting of:

311, 341, 343, 384, 399, 400, 401, 402, and 413, according to EU numbering.

[0278] In another aspect, the invention provides polypeptides comprising variant Fc regions with enhanced Fc gamma Ruth-binding activity and increased pI comprising amino acid alterations of any one of the following (1)-(9): (1) positions 235, 236, 268, 295, 311, 326, 330 and 343; (2) positions 236, 268, 295, 311, 326, 330 and 343; (3) positions 236, 268, 295, 311, 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, the Fc gamma Ruth has the sequence of cynomolgus monkey Fc gamma Ruth (SEQ ID NO:223). In certain embodiments, the Fc gamma Ruth has the sequence of human Fc gamma Ruth (e.g., SEQ ID NOS:212, 213, or 214).

[0279] In one aspect, the invention provides polypeptides comprising variant Fc regions with enhanced Fc gamma Ruth-binding activity and increased pI comprising at least three amino acid alterations comprising: (a) at least one amino acid alteration of 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 of 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 further embodiments, the polypeptides comprise at least two amino acid alterations of 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 em-bodiments, the Fc gamma Ruth has the sequence of cynomolgus monkey Fc gamma RHb (SEQ ID NO:223). In certain embodiments, the Fc gamma RHb has the sequence of human Fc gamma RIM (e.g., SEQ ID NOS:212, 213, or 214).

[0280] In another aspect, the invention provides polypeptides comprising variant Fc regions with enhanced Fc gamma Ruth-binding activity and increased pI comprising amino acid alterations of any one 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.

[0281] In a further embodiment, the variant Fc region comprises amino acid alterations selected from any single alteration, combination of single alterations, or combination alterations described in Tables 14-30.

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

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

[0284] A "parent Fc region" as used herein refers to an Fc region prior to introduction of amino acid alteration(s) described herein. Preferred examples of the parent Fc region include Fc regions derived from native antibodies. Antibodies include, for example, IgA (IgAl, IgA2), IgD, IgE, IgG (IgG 1, IgG2, IgG3, IgG4), and IgM, or such.
An-tibodies may be derived from human or monkey (e.g., cynomolgus, rhesus macaque, marmoset, chimpanzee, or baboon). Native antibodies may also include naturally-occurring mutations. A plurality of allotype sequences of IgGs due to genetic poly-morphism are described in "Sequences of proteins of immunological interest", NIH
Publication No. 91-3242, and any of them may be used in the present invention.
In particular, for human IgG 1, the amino acid sequence at positions 356 to 358 (EU
numbering) may be either DEL or EEM. Preferred examples of the parent Fc region include Fc regions derived from a heavy chain constant region of human IgG1 (SEQ
ID NO: 195), human IgG2 (SEQ ID NO: 196), human IgG3 (SEQ ID NO: 197), and human IgG4 (SEQ ID NO: 198). Another preferred example of the parent Fc region is an Fc region derived from a heavy chain constant region SG1 (SEQ ID NO: 9).
Fur-thermore, the parent Fc region may be an Fc region produced by adding an amino acid alteration(s) other than the amino acid alteration(s) described herein to an Fc region derived from a native antibody.

[0285] In addition, amino acid alterations performed for other purpose(s) can be combined in a variant Fc region described herein. For example, amino acid substitutions that improve FcRn-binding activity (Hinton et al., J. Immunol. 176(1):346-356 (2006);
Dall'Acqua et al., J. Biol. Chem. 281(33):23514-23524 (2006); Petkova et al., Intl.
Immunol. 18(12):1759-1769 (2006); Zalevsky et al., Nat. Biotechnol. 28(2):157-(2010); WO 2006/019447; WO 2006/053301; and WO 2009/086320), and amino acid substitutions for improving antibody heterogeneity or stability (WO
2009/041613) may be added. Alternatively, polypeptides with the property of promoting antigen clearance, which are described in WO 2011/122011, WO 2012/132067, WO
2013/046704 or WO 2013/180201, polypeptides with the property of specifc binding to a target tissue, which are described in WO 2013/180200, polypeptides with the property for repeated binding to a plurality of antigen molecules, which are described in WO 2009/125825, WO 2012/073992 or WO 2013/047752, can be combined with a variant Fc region described herein. Alternatively, with the objective of conferring binding ability to other antigens, the amino acid alterations disclosed in and EP1772465 may be combined in CH3 of a variant Fc region described herein.
Al-ternatively, with the objective of increasing plasma retention, amino acid alterations that decrease the pI of the constant region (WO 2012/016227) may be combined in a variant Fc region described herein. Alternatively, with the objective of promoting uptake into cells, amino acid alterations that increase the pI of the constant region (WO
2014/145159) may be combined in a variant Fc region described herein.
Alternatively, with the objective of promoting elimination of a target molecule from plasma, amino acid alterations that increase the pI of the constant region (Japanese patent application numbers JP2015-021371 and JP2015-185254) may be combined in a variant Fc region described herein. In one embodiment, such alteration may include, for example, sub-stitution at al least one position selected from the group consisting of 311, 343, 384, 399, 400, and 413 according to EU numbering. In a further embodiment, such sub-stitution may be a replacement of an amino acid with Lys or Arg at each position.

[0286] Amino acid alterations of enhancing human FcRn-binding activity under acidic pH
can also be combined in a variant Fc region described herein. Specifically, such al-terations may include, for example, substitution of Leu for Met at position 428 and substitution of Ser for Asn at position 434, according to EU numbering (Zalevsky et al., Nat. Biotechnol. 28:157-159 (2010)); substitution of Ala for Asn at position 434 (Deng et al., Metab. Dispos. 38(4):600-605 (2010)); substitution of Tyr for Met at position 252, substitution of Thr for Ser at position 254 and substitution of Glu for Thr at position 256 (Dall'Acqua et al., J. Biol. Chem. 281:23514-23524 (2006));
sub-stitution of Gln for Thr at position 250 and substitution of Leu for Met at position 428 (Hinton et al., J. Immuno1.176(1):346-356 (2006)); substitution of His for Asn at position 434 (Zheng et al., Clin. Pharmacol. Ther. 89(2):283-290 (2011), and al-terations 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, WO 2005/047327, WO 2005/037867, WO 2004/035752, or WO
2002/060919. Such alterations may include, for example, at least one alteration selected from the group consisiting of substitution of Leu for Met at position 428, sub-stitution of Ala for Asn at position 434 and substitution of Thr for Tyr at position 436.
Those alterations may further include substitution of Arg for Gln at position 438 and/or substitution of Glu for Ser at position 440 (Japanese patent application numbers JP2015-021371 and JP2015-185254).

[0287] Two or more polypeptides comprising a variant Fc region described herein can be included in one molecule, wherein two polypeptides comprising variant Fc regions are associated, much like in an antibody. The type of antibody is not limited, and IgA
(IgAl, IgA2), IgD, IgE, IgG (IgGl, IgG2, IgG3, IgG4), and IgM, or such can be used.

[0288] The two associated polypeptides comprising variant Fc regions may be polypeptides comprising variant Fc regions into which the same amino acid alteration(s) have been introduced (hereinafter, referred to as homologous variant Fc regions), or polypeptides comprising variant Fc regions into which different amino acid alteration(s) have been introduced, or alternatively polypeptides comprising variant Fc regions where amino acid alteration(s) have been introduced into only one of the Fc regions (hereinafter, referred as a heterologous polypeptides comprising variant Fc regions). One of the preferable amino acid alterations is an alteration in the loop structure from positions 233 to 239 (EU numbering) in the CH2 domain of the Fc region, which is involved in binding with Fc gamma RIM and Fc gamma RIIa. Preferably, an alteration is in-troduced in the loop structure of the CH2 domain of one of the Fc regions that enhances Fc gamma Ruth-binding activity and/or selectivity, and another alteration is introduced in the loop structure of the CH2 domain of the other Fc region that destabilizes it. Examples of amino acid alterations that can destabilize the loop structure of the CH2 domain may be substitution of at least one amino acid selected from amino acids at positions 235, 236, 237, 238, and 239 to another amino acid.
Specifically, it can be destabilized, for example, by altering the amino acid at position 235 to Asp, Gln, Glu, or Thr, altering the amino acid at position 236 to Asn, altering the amino acid at position 237 to Phe or Trp, altering the amino acid at position 238 to Glu, Gly, or Asn, and altering the amino acid at position 239 to Asp or Glu, according to EU numbering.

[0289] For association of heterologous polypeptides comprising variant Fc regions, a technique of suppressing unintended association of homologous polypeptides comprising variant Fc regions by introducing electrostatic repulsion into the interface of the CH2 or CH3 domain of the Fc region can be applied, as described in WO
2006/106905.

[0290] Examples of amino acid residues in contact at the interface of the CH2 or CH3 domain of the Fc region include 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) in the CH3 domain.

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

[0292] Furthermore, heterologous polypeptides comprising variant Fc regions can be produced, wherein one to three pairs of amino acid residues selected from (1) to (3) indicated above have the same charge in the CH3 domain of the first Fc region, and the pairs of amino acid residues selected in the aforementioned 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.

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

[0294] Other known techniques can be used additionally for association of heterologous polypeptides comprising variant Fc regions. Specifically, such a technique is conducted by substituting an amino acid side chain present in one of the Fc regions with a larger side chain (knob; which means "bulge"), and substituting an amino acid side chain present in the Fc region with a smaller side chain (hole; which means "void"), to place the knob within the hole. This can promote efficient association between Fc-region-containing polypeptides having different amino acid sequences from each other (WO 1996/027011; Ridgway et al., Prot. Eng. 9:617-621 (1996);
Merchant et al., Nat.Biotech. 16, 677-681 (1998)).

[0295] In addition, other known techniques can also be used for heterologous association of polypeptides comprising variant Fc regions. Association of polypeptides comprising an Fc region can be induced efficiently using strand-exchange engineered domain heterodimers (Davis et al., Prot. Eng. Des. & Sel., 23:195-202 (2010)). This technique can also be used to efficiently induce association between Fc region-containing polypeptides having different amino acid sequences.

[0296] In addition, heterodimerized antibody production techniques that use association of antibody CH1 and CL, and association of VH and VL, which are described in WO
2011/028952, can also be used.

[0297] As with the method described in WO 2008/119353 and WO 2011/131746, it is also possible to use the technique of producing heterodimerized antibodies by producing two types of homodimerized antibodies in advance, incubating the antibodies under reducing conditions to dissociate them, and allowing them to associate again.

[0298] As with the method described in Strop (J. Mol. Biol. 420:204-219 (2012)), it is also possible to use the technique of producing heterodimerized antibodies by introducing charged residues such as Lys, Arg, Glu, and Asp so that electrostatic repulsion is in-troduced into CH3 domains.

[0299] Furthermore, as with the method described in WO 2012/058768, it is also possible to use the technique of producing heterodimerized antibodies by adding alterations to the CH2 and CH3 domains.

[0300] When simultaneously expressing two polypeptides comprising a variant Fc region which have different amino acid sequences, in order to produce polypeptides comprising heterologous variant Fc regions, polypeptides comprising homologous variant Fc regions are also usually produced as impurities. In such cases, polypeptides comprising heterologous variant Fc regions can be efficiently obtained by separating and purifying them from polypeptides comprising homologous variant Fc regions using known technologies. A method has been reported to efficiently separate and purify heterodimerized antibodies from a homodimerized antibodies using ion exchange chromatography, by introducing amino acid alterations into the variable regions of the two types of antibody heavy chains to create a difference in isoelectric points between the homodimerized antibodies and the heterodimerized antibodies (WO
2007/114325). Another method has been reported to purify heterodimerized antibodies using Protein A chromatography, by constructing a heterodimerized antibody comprising two types of heavy chains derived from mouse IgG2a that binds to Protein A and rat IgG2b that does not bind to Protein A (WO 1998/050431 and WO
1995/033844).

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

[0302] In the present invention, amino acid alteration means any of substitution, deletion, addition, insertion, and modification, or a combination thereof. In the present invention, amino acid alteration may be rephrased as amino acid mutation.

[0303] When substituting amino acid residues, substitution to a different amino acid residue can be carried out with the objective of altering aspects such as (a)-(c) described below: (a) polypeptide backbone structure in the sheet-structure or helical-structure region; (b) electric charge or hydrophobicity at the target site; or (c) size of the side chain.

[0304] Amino acid residues are classified into the following groups based on their general side chain properties: (a) hydrophobic: Norleucine, Met, Ala, Val, Leu, and Ile; (b) neutral hydrophilic: Cys, Ser, Thr, Asn, and Gln; (c) acidic: Asp and Glu; (d) basic:

His, Lys, and Arg; (e) residues that affect the chain orientation: Gly and Pro; and (f) aromatic: Trp, Tyr, and Phe.

[0305] Amino acid alterations are produced by various methods known to those skilled in the art. Such methods include the site-directed mutagenesis method (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-(1985)), the PCR mutation method, and the cassette mutation method, but are not limited thereto.

[0306] The number of amino acid alterations introduced into an 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.

[0307] Amino acid modification includes post-translational modification. A
specific post-translational modification may be addition or deletion of a sugar chain. For example, the amino acid residue at position 297 (EU numbering) in the IgG1 constant region may be sugar chain-modified. The sugar chain structure for the modification is not limited. For example, sialic acid may be added to the sugar chain of an Fc region (MAbs 2010 Sep-Oct, 2(5): 519-527). Generally, antibodies expressed in eukaryotic cells comprise glycosylation in the constant region. For example, it is known that some type of sugar chain are normally added to antibodies expressed in cells such as naturally-occurring antibody-producing cells of mammals or eukaryotic cells transformed with an expression vector comprising a DNA encoding an antibody.

[0308] Eukaryotic cells shown here include yeast and animal cells. For example, CHO cells and HEK293 cells are representative animal cells used in transformation with an ex-pression vector comprising an antibody-encoding DNA. On the other hand, constant regions without glycosylation are also included in the present invention.
Antibodies whose constant region is not glycosylated can be obtained by expressing an antibody-encoding gene in prokaryotic cells such as Escherichia coli.

[0309] Furthermore, a polypeptide comprising a variant Fc region of the present invention may be chemically modified with various molecules such as polyethylene glycol (PEG) and cytotoxic substances. Methods for such chemical modification of a polypeptide are established in the art.

[0310] In one aspect, the invention provides an isolated polypeptide comprising a variant Fc region with enhanced Fc gamma RIM-binding activity. In some apsects, the polypeptide is an antibody. In some aspsects, the polypeptide is an Fc fusion protein.
In certain embodiments, an antibody is a chimeric antibody, or a humanized antibody.
The origin of an antibody is not particularly limited, but examples include a human antibody, a mouse antibody, a rat antibody, and a rabbit antibody. In some apsects, the polypeptide is an Fc fusion protein.

[0311] The variable regions of antibodies that comprise a variant Fc region provided herein and the protein binding motifs of Fc fusion proteins comprising a 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, and such), receptors, cancer antigens, MHC antigens, differentiation antigens, im-munoglobulins, and immune complexes partly containing immunoglobulins.

[0312] Examples of cytokines that can be bound by an antibody or fusion protein containing a variant Fc region of the invention, and/or recombinantly fused with a polypeptide comprising a disclosed variant Fc region include but are not limited to, interleukins 1 to 18, colony stimulating factors (G-CSF, M-CSF, GM-CSF, etc.), interferons (IFN-alpha, IFN-beta, IFN-gamma, etc.), growth factors (EGF, FGF, IGF, NGF, PDGF, TGF, HGF, etc.), tumor necrosis factors (TNF-alpha and TNF-beta), lym-photoxin, erythropoietin, leptin, SCF, TPO, MCAF, and BMP.

[0313] Examples of chemokines that can be bound by an antibody or fusion protein containing a variant Fc region of the invention, and/or recombinantly fused with a polypeptide comprising a 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.

[0314] Examples of receptors that can be bound by an antibody or fusion protein containing a variant Fc region of the invention, and/or recombinantly fused with a polypeptide comprising a 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
anchor-type receptor family, tyrosine phosphatase-type receptor family, adhesion factor family, and hormone receptor family. The 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-(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)).

[0315] Examples of specific receptors belonging to the above-mentioned receptor families include human or mouse erythropoietin (EPO) receptors (Jones et al., Blood 76(1):31-35 (1990); D'Andrea et al., Cell 57(2):277-285 (1989)), human or mouse granulocyte-colony stimulating factor (G-CSF) receptors (Fukunaga et al., Proc. Natl.
Acad. Sci. USA 87(22):8702-8706 (1990), mG-CSFR; Fukunaga et al., Cell 61(2):
341-350 (1990)), human or mouse thrombopoietin (TPO) receptors (Vigon et al., Proc.
Natl. Acad. Sci. USA. 89(12):5640-5644 (1992); Skoda et al., EMBO J.
12(7):2645-2653 (1993)), human or mouse insulin receptors (Ullrich et al., Nature 313(6005):756-761 (1985)), human or mouse Flt-3 ligand receptors (Small et al., Proc.
Natl. Acad. Sci. USA. 91(2):459-463 (1994)), human or mouse platelet-derived growth factor (PDGF) receptors (Gronwald et al., Proc. Natl. Acad. Sci. USA.
85(10):3435-3439 (1988)), human or mouse interferon (IFN)-alpha and beta receptors (Uze et al., Cell 60(2): 225-234 (1990); Novick et al., Cell 77(3):391-400 (1994)), human or mouse leptin receptors, human or mouse growth hormone (GH) receptors, human or mouse interleukin (IL)-10 receptors, human or mouse insulin-like growth factor (IGF)-I receptors, human or mouse leukemia inhibitory factor (LIF) receptors, and human or mouse ciliary neurotrophic factor (CNTF) receptors.

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

[0317] MHC antigens are 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.

[0318] Examples of differentiation antigens that can be bound by an antibody or fusion protein containing a variant Fc region of the invention, and/or recombinantly fused with a polypeptide comprising a 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.

[0319] Immunoglobulins include IgA, IgM, IgD, IgG, and IgE. Immune complexes include a component of at least any of the immunoglobulins.

[0320] Other examples of antigens that can be bound by an antibody or fusion protein containing a variant Fc region of the invention, and/or recombinantly fused with a polypeptide comprising a disclosed variant Fc region include but are not limited to, 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, Al adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V/beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic peptide, av/b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulating factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bc1, 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 (0P-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 neu-rotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer associated antigen, cathepsin A, cathepsin B, cathepsin C/DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin 0, cathepsin S, cathepsin V, cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR11, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD3OL, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD4OL, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6,cytokeratin tumor associated antigen, DAN, DCC, DcR3, DC-SIGN, complement regulatory factor (Decay accelerating factor), des(1-3)-IGF-I
(brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-Al, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2/EphB4, EPO, ERCC, E-selectin, ET-1, factor Ha, factor VII, factor VIIIc, factor IX, fibroblast ac-tivation protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GFAP, GFRa-1, GFR-alphal, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut4, glycoprotein Hb/IIIa (GPIIb/IIIa), GM-CSF, gp130, gp72, GRO, growth hormone releasing hormone, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope gly-coprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-as-sociated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cy-tomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (IFN)-alpha, IFN-beta, IFN-gamma, inhibin, iNOS, insulin A chain, insulin B
chain, insulin-like growth factorl, integrin alpha2, integrin alpha3, integrin alpha4, integrin alpha4/beta1, integrin alpha4/beta7, integrin alpha5 (alpha V), integrin alpha5/beta1, integrin alpha5/beta3, integrin alpha6, integrin beta 1, integrin beta2, interferon gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein Li, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1 bpi, LBP, LDGF, LECT2, lefty, Lewis-Y antigen, Lewis-Y associated antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface, luteinizing hormone, lym-photoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES, MGDF receptor, MGMT, MHC
(HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Mud), MUC18, Mullerian-inhibiting substance, Mug, MuSK, NAIP, NAP, NCAD, N-Cadherin, NCA

90, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX4OL, OX4OR, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T-cell receptor (for example, T-cell receptor alpha/beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testis PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific, TGF-betaRI (ALK-5), TGF-betaRII, TGF-betaRIIb, TGF-betaRIII, TGF-betal, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, thrombin, thymus Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alphabeta, TNF-beta2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF1OB (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C
(TRAIL R3 DcR1, LIT, TRID), TNFRSF1OD (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60), TNFRSF1B (TNF RII
CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (0X40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE/RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A/VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-a Conectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (0X40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor associated antigen CA125, tumor associated antigen expressing Lewis-Y associated carbohydrates, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (fit-1), VEGF, VEGFR, VEGFR-3 (fit-4), VEGI, VIM, virus antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, A beta, CD81, CD97, CD98, DDR1, DKK1, EREG, Hsp90, IL-17/IL-17R, IL-20/IL-20R, oxidized LDL, PCSK9, prekallikrein, RON, TMEM16F, SOD1, Chromogranin A, Chro-mogranin B, tau, VAP1, high molecular weight kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, Cl, Clq, Clr, Cis, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, factor B, factor D, factor H, properdin, sclerostin, fibrinogen, fibrin, prothrombin, thrombin, tissue factor, factor V, factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X, factor Xa, factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, Syndecan-1, Syndecan-2, Syndecan-3, Syndecan-4, LPA, and S1P; and receptors for hormone and growth factors.

[0321] As discussed herein, one or more amino acid residue alterations are allowed in the amino acid sequences constituting the variable regions as long as their antigen-binding activities are maintained. When altering a variable region amino acid sequence, there is no particularly limitation on the site of alteration and number of amino acids altered.
For example, amino acids present in CDR and/or FR can be altered appropriately.
When altering amino acids in a variable region, the binding activity is preferably maintained without particular limitation; and for example, as compared to before al-teration, the binding activity may be 50% or more, 80% or more, and 100% or more.
Furthermore, the binding activity may be increased by amino acid alterations.
For example, the binding activity may be 2-, 5-, 10-times higher or such than that before alteration. Alteration of amino acid sequence may be at least one of amino acid residue substitution, addition, deletion, and modification.

[0322] For example, the modification of the N-terminal glutamine of a variable region into pyroglutamic acid by pyroglutamylation is a modification well known to those skilled in the art. Thus, when the heavy-chain N terminus is glutamine, antibodies described herein may comprise the variable regions in which the glutamine is modified to py-roglutamic acid.

[0323] Antibody variable regions described herein may have any sequences, and they may be antibody variable regions of any origin, such as mouse antibodies, rat antibodies, rabbit antibodies, goat antibodies, camel antibodies, humanized antibodies produced by humanizing these non-human antibodies, and human antibodies. Furthermore, these antibodies may have various amino acid substitutions introduced into their variable regions to improve their antigen binding, pharmacokinetics, stability, and immuno-genicity. Variable regions may be able to bind antigens repeatedly due to their pH de-pendency in antigen binding (WO 2009/125825).

[0324] kappa chain and lambda chain are present in antibody light-chain constant regions, and either one is acceptable. Furthermore, they may have some amino acid alterations such as substitutions, deletions, additions, and/or insertions.

[0325] Furthermore, polypeptides comprising variant Fc regions described herein may be made into Fc fusion proteins by linking to other proteins such as physiologically active peptides. Such fusion proteins may be a multimer of at least two polypeptides comprising the variant Fc regions. Examples of the other proteins include receptors, adhesion molecules, ligands, and enzymes, but are not limited thereto.

[0326] Examples of Fc fusion proteins include proteins fused with an Fc region to a receptor that binds to a target molecule, including TNFR-Fc fusion protein, IL1R-Fc fusion protein, VEGFR-Fc fusion protein, and CTLA4-Fc fusion protein (Economides et al,.
Nat. Med. 9(1):47-52 (2003); Dumont et al., BioDrugs. 20(3):151-60 (2006)).
Fur-thermore, proteins to be fused may be other molecules that have a target-binding activity, for example, scFvs (WO 2005/037989), single-domain antibodies (WO
2004/058821; WO 2003/002609), antibody-like molecules (Davinder, Curr. Op.
Biotech. 17:653-658 (2006); Current Opinion in Biotechnology 18:1-10 (2007);
Nygren et al., Curr. Op. Struct. Biol. 7:463-469 (1997); and Hoss. Protein Science 15:14-27 (2006)) such as DARPins (WO 2002/020565), Affibody (WO 1995/001937), Avimer (WO 2004/044011; WO 2005/040229), and Adnectin (WO 2002/032925).
Furthermore, antibodies and Fc fusion proteins may be multispecific and may bind to multiple types of target molecules or epitopes.
B. Recombinant Methods and Compositions

[0327] Antibodies may be produced using recombinant methods and compositions, e.g., as described in US Patent No. 4,816,567. In one embodiment, isolated nucleic acid encoding an anti-myostatin antibody described herein is provided. In another em-bodiment, isolated nucleic acid encoding a polypeptide comprising a variant Fc region or a parent Fc region described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one embodiment, a host cell comprises (e.g., has been transformed with):
(1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., a YO, NSO, and Sp20 cell). In one embodiment, a method of making an anti-myostatin antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally re-covering the antibody from the host cell (or host cell culture medium). In another em-bodiment, a method of making a polypeptide comprising a variant Fc region or a parent Fc region is provided, wherein the method comprises culturing a host cell comprising the nucleic acid(s) encoding a polypeptide such as, an antibody, Fc region, or variant Fc region, as provided above, under conditions suitable for expression of the polypeptide, and optionally recovering the polypeptide from the host cell (or host cell culture medium).

[0328] For recombinant production of an anti-myostatin antibody, nucleic acids encoding an antibody, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. For recombinant production of an Fc region, nucleic acid encoding an Fc region is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., using oligonu-cleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).

[0329] Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., US Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also, Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp.
245-254, describing expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0330] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized,"
resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech.
24:210-215 (2006).

[0331] Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0332] Plant cell cultures can also be utilized as hosts. See, e.g., US
Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIESTm technology for producing antibodies in transgenic plants).

[0333] Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by 5V40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A);
human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT
060562); TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad.
Sci.
383:44-68 (1982); MRC 5 cells; and F54 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as YO, NSO and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0334] Antibodies with pH-dependent characteristics may be obtained using screening methods and/or mutagenesis methods e.g., as described in WO 2009/125825. The screening methods may comprise any process by which an antibody having pH-dependent binding characteristics is identified within a population of antibodies specific for a particular antigen. In certain embodiments, the screening methods may comprise measuring one or more binding parameters (e.g., KD or kd) of individual an-tibodies within an initial population of antibodies both at acidic and neutral pH. The binding parameters of the antibodies may be measured using, e.g., surface plasmon resonance, or any other analytic method that allows for the quantitative or qualitative assessment of the binding characteristics of an antibody to a particular antigen. In certain embodiments, the screening methods may comprise identifying an antibody that binds to an antigen with an acidic/neutral KD ratio of 2 or greater. In further em-bodiments, the screening methods may comprise identifying an antibody that binds to an antigen with a pH5.8/pH7.4 KD ratio of 2 or greater. Alternatively, the screening methods may comprise identifying an antibody that binds to an antigen with an acidic/
neutral kd ratio of 2 or greater. In further embodiments, the screening methods may comprise identifying an antibody that binds to an antigen with a pH5.8/pH7.4 kd ratio of 2 or greater.

[0335] In another embodiment, the mutagenesis methods may comprise incorporating a deletion, substitution, or addition of an amino acid within the heavy and/or light chain of the antibody to enhance the pH-dependent binding of the antibody to an antigen. In certain embodiments, the mutagenesis may be carried out within one or more variable domains of the antibody, e.g., within one or more HVRs (e.g., CDRs). For example, the mutagenesis may comprise substituting an amino acid within one or more HVRs (e.g., CDRs) of the antibody with another amino acid. In certain embodiments, the mu-tagenesis may comprise substituting one or more amino acids in at least one HVR
(e.g., CDR) of the antibody with a histidine. In certain embodiments, "enhanced pH-dependent binding" means that the mutated version of the antibody exhibits a greater acidic/neutral KD ratio, or a greater acidic/neutral kd ratio, than the original "parent"
(i.e., the less pH-dependent) version of the antibody prior to mutagenesis. In certain embodiments, the mutated version of the antibody has an acidic/neutral KD
ratio of 2 or greater. In certain embodiments, the mutated version of the antibody has a pH5.8/pH7.4 KD ratio of 2 or greater. Alternatively, the mutated version of the antibody has an acidic/neutral kd ratio of 2 or greater. In further embodiments, the mutated version of the antibody has a pH5.8/pH7.4 kd ratio of 2 or greater.

[0336] Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thy-roglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOC12, or RiN,C=NR, where R and R1 are different alkyl groups.

[0337] Animals (usually non-human mammals) are immunized against the antigen, im-munogenic conjugates, or derivatives by combining, e.g., 100 micro g or 5 micro g of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites.
One month later the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites.
Seven to 14 days later the animals are bled and the serum is assayed for antibody titer.
Animals are boosted until the titer plateaus. Preferably, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.

[0338] Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.

[0339] For example, the monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature 256(5517):495-497 (1975). In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as hereinabove described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization.
Alter-natively, lymphocytes may be immunized in vitro.

[0340] The immunizing agent will typically include the antigenic protein or a fusion variant thereof. Generally either peripheral blood lymphocytes (PBLs) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103).

[0341] Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which are substances that prevent the growth of HGPRT-deficient cells.

[0342] Preferred immortalized myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 cells (and derivatives thereof, e.g., X63-Ag8-653) available from the American Type Culture Collection, Manassas, Virginia USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor et al. J. Immunol. 133(6):3001-3005 (1984);
Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, pp. 51-63 (1987)).

[0343] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunopre-cipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. For example, binding affinity may be determined by the Scatchard analysis of Munson, Anal. Biochem. 107(1):220-239 (1980).

[0344] After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as tumors in a mammal.

[0345] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxyapatite chro-matography, gel electrophoresis, dialysis, or affinity chromatography.

[0346] Antibodies may be produced by immunizing an appropriate host animal against an antigen. In one embodiment, the antigen is a polypeptide comprising a full-length myostatin. In one embodiment, the antigen is a polypeptide comprising latent myostatin. In one embodiment, the antigen is a polypeptide comprising a myostatin propeptide. In one embodiment, the antigen is a polypeptide comprising the region cor-responding to the amino acids at positions 21 to 100 of myostatin propeptide (SEQ ID
NO: 78). In one embodiment, the antigen is a polypeptide comprising amino acids at positions 21-80, 41-100, 21-60, 41-80, 61-100, 21-40, 41-60, 61-80, or 81-100 of myostatin propeptide (SEQ ID NO: 78). Also included in the present invention are an-tibodies produced by immunizing an animal against the antigen. The antibodies may incorporate any of the features, singly or in combination, as described in "Exemplary Anti-myostatin Antibodies" above.

[0347] An Fc region may be obtained by re-eluting the fraction adsorbed onto protein A
column after partially digesting IgGl, IgG2, IgG3, IgG4 monoclonal antibodies or such using a protease such as pepsin. The protease is not particularly limited as long as it can digest a full-length antibody so that Fab and F(ab')2 will be produced in a re-strictive manner by appropriately setting the enzyme reaction conditions such as pH, and examples include pepsin and papain.

[0348] Furthermore, the present invention provides a method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma Ruth-binding activity in comparison with a polypeptide comprising a parent Fc region, which comprises in-troducing at least one amino acid alteration to the parent Fc region. In some apsects, the produced polypeptide is an antibody. In certain embodiments, an antibody is a chimeric antibody, or a humanized antibody. In some apsects, the produced polypeptide is an Fc fusion protein.

[0349] In certain embodiments, the production method comprises the following steps: (a) preparing a polypeptide comprising a parent Fc region; (b) introducing at least one amino acid alteration to the parent Fc region within the polypeptide; (c) measuring the monkey Fc gamma Ruth-binding activities of the polypeptide comprising the parent Fc region and the polypeptide comprising the Fc region in step (b); and (d) selecting a polypeptide comprising a variant Fc region with enhanced monkey Fc gamma RHb-binding activity in comparison with the polypeptide comprising the parent Fc region.

[0350] In certain embodiments, the production method comprises the following steps: (a) preparing a polypeptide comprising a parent Fc region; (b) introducing at least one amino acid alteration to the parent Fc region within the polypeptide; (c) measuring the monkey Fc gamma RIIIa-binding activities of the polypeptide comprising the parent Fc region and the polypeptide comprising the Fc region in step (b); and (d) selecting a polypeptide comprising a variant Fc region with decreased monkey Fc gamma RIIIa-binding activity in comparison with the polypeptide comprising the parent Fc region.

[0351] In certain embodiments, the production method comprises the following steps: (a) preparing a polypeptide comprising a parent Fc region; (b) introducing at least one amino acid alteration to the parent Fc region within the polypeptide; (c) measuring the human Fc gamma Ruth-binding activities of the polypeptide comprising the parent Fc region and the polypeptide comprising the Fc region in step (b); and (d) selecting a polypeptide comprising a variant Fc region with enhanced human Fc gamma RHb-binding activity in comparison with the polypeptide comprising the parent Fc region.

[0352] In certain embodiments, the production method comprises the following steps: (a) preparing a polypeptide comprising a parent Fc region; (b) introducing at least one amino acid alteration to the parent Fc region within the polypeptide; (c) measuring the human Fc gamma RIIIa-binding activities of the polypeptide comprising the parent Fc region and the polypeptide comprising the Fc region in step (b); and (d) selecting a polypeptide comprising a variant Fc region with decreased human Fc gamma RIIIa-binding activity in comparison with the polypeptide comprising the parent Fc region.

[0353] In certain embodiments, the production method comprises the following steps: (a) preparing a polypeptide comprising a parent Fc region; (b) introducing at least one amino acid alteration to the parent Fc region within the polypeptide; (c) measuring the human Fc gamma RIIa (type H)-binding activities of the polypeptide comprising the parent Fc region and the polypeptide comprising the Fc region in step (b); and (d) selecting a polypeptide comprising a variant Fc region with decreased human Fc gamma RIIa (type H)-binding activity in comparison with the polypeptide comprising the parent Fc region.

[0354] In certain embodiments, the production method comprises the following steps: (a) preparing a polypeptide comprising a parent Fc region; (b) introducing at least one amino acid alteration to the parent Fc region within the polypeptide; (c) measuring the human Fc gamma RIIa (type R)-binding activities of the polypeptide comprising the parent Fc region and the polypeptide comprising the Fc region in step (b); and (d) selecting a polypeptide comprising a variant Fc region with decreased human Fc gamma RIIa (type R)-binding activity in comparison with the polypeptide comprising the parent Fc region.

[0355] In one aspect, at least one amino acid is altered in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma RHb-binding activity, of 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 em-bodiments, the Fc gamma Ruth has the sequence of cynomolgus monkey Fc gamma Ruth (SEQ ID NO:223). In certain embodiments, the Fc gamma Ruth has the sequence of human Fc gamma Ruth (e.g., SEQ ID NOS:212, 213, or 214).

[0356] In another aspect, one amino acid is altered in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma Rllb-binding activity, at position 236.

[0357] In another aspect, at least two amino acids are altered in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma Ruth-binding activity, the alterations comprising: (a) one amino acid alteration at position 236, and (b) at least one amino acid alteration of 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, the Fc gamma RHb has the sequence of cynomolgus monkey Fc gamma Ruth (SEQ ID NO:223). In certain em-bodiments, the Fc gamma RIM has the sequence of human Fc gamma Ruth (e.g., SEQ

ID NOS:212, 213, or 214).

[0358] In another aspect, at least two amino acids are altered in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma Ruth-binding activity, the alterations comprising: (a) one amino acid alteration at position 236, and (b) at least one amino acid alteration of 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 certain embodiments, the Fc gamma Ruth has the sequence of cynomolgus monkey Fc gamma Ruth (SEQ ID NO:223). In certain em-bodiments, the Fc gamma Ruth has the sequence of human Fc gamma Ruth (e.g., SEQ
ID NOS:212, 213, or 214).

[0359] In another aspect, at least two amino acids are altered in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma Ruth-binding activity, the alterations comprising: (a) one amino acid alteration at position 236, and (b) at least one amino acid alteration of at least one position selected from the group consisting of: 268, 295, 326, and 330, according to EU

numbering. In certain embodiments, the Fc gamma Ruth has the sequence of cynomolgus monkey Fc gamma Ruth (SEQ ID NO:223). In certain embodiments, the Fc gamma Ruth has the sequence of human Fc gamma Ruth (e.g., SEQ ID NOS:212, 213, or 214).

[0360] In another aspect, amino acids are altered in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma RHb-binding activity, at positions of any one of the following (1)-(37): (1) at positions 231, 236, 239, 268 and 330; (2) at positions 231, 236, 239, 268, 295 and 330;
(3) at positions 231, 236, 268 and 330; (4) at positions 231, 236, 268, 295 and 330;
(5) at positions 232, 236, 239, 268, 295 and 330; (6) at positions 232, 236, 268, 295 and 330;
(7) at positions 232, 236, 268 and 330; (8) at positions 235, 236, 268, 295, 326 and 330; (9) at positions 235, 236, 268, 295 and 330; (10) at positions 235, 236, 268 and 330; (11) at positions 235, 236, 268, 330 and 396; (12) at positions 235, 236, 268 and 396; (13) at positions 236, 239, 268, 295, 298 and 330; (14) at positions 236, 239, 268, 295, 326 and 330; (15) at positions 236, 239, 268, 295 and 330; (16) at positions 236, 239, 268, 298 and 330; (17) at positions 236, 239, 268, 326 and 330; (18) at positions 236, 239, 268 and 330; (19) at positions 236, 239, 268, 330 and 396; (20) at positions 236, 239, 268 and 396; (21) at positions 236 and 268; (22) at positions 236, 268 and 295; (23) at positions 236, 268, 295, 298 and 330; (24) at positions 236, 268, 295, 326 and 330; (25) at positions 236, 268, 295, 326, 330 and 396; (26) at positions 236, 268, 295 and 330; (27) at positions 236, 268, 295, 330 and 396; (28) at positions 236, 268, 298 and 330; (29) at positions 236, 268, 298 and 396; (30) at positions 236, 268, 326 and 330; (31) at positions 236, 268, 326, 330 and 396; (32) at positions 236, 268 and 330; (33) at positions 236, 268, 330 and 396; (34) at positions 236, 268 and 396; (35) at positions 236 and 295; (36) at positions 236, 330 and 396; and (37) at positions 236 and 396; according to EU numbering. In certain embodiments, the Fc gamma Ruth has the sequence of cynomolgus monkey Fc gamma Ruth (SEQ ID NO:223). In certain embodiments, the Fc gamma Ruth has the sequence of human Fc gamma RHb (e.g., SEQ ID NOS:212, 213, or 214).

[0361] In a further aspect, an amino acid alteration in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma RHb-binding activity, is selected at each position 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; (1) 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, the Fc gamma Ruth has the sequence of cynomolgus monkey Fc gamma RHb (SEQ ID NO:223). In certain embodiments, the Fc gamma Ruth has the sequence of human Fc gamma Ruth (e.g., SEQ ID NOS:212, 213, or 214).

[0362] In a further aspect, an amino acid alteration in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma RHb-binding activity, is selected at each position 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 certain embodiments, the Fc gamma Ruth has the sequence of cynomolgus monkey Fc gamma Ruth (SEQ ID
NO:223). In certain embodiments, the Fc gamma RIM has the sequence of human Fc gamma RIM (e.g., SEQ ID NOS:212, 213, or 214).
In a further aspect, amino acid alterations in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma RHb-binding activity, are: Asn at position 236, Glu at position 268, Lys at position 330, and Met at position 396; according to EU numbering. In a further aspect, amino acid alterations in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma Ruth-binding activity, are:
Asn at position 236, Asp at position 268, and Lys at position 330; according to EU
numbering. In a further aspect, amino acid alterations in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma Rllb-binding activity, are: Asn at position 236, Asp at position 268, Leu at position 295, and Lys at position 330; according to EU numbering. In a further aspect, amino acid alterations in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma Ruth-binding activity, are:
Thr at position 236, Asp at position 268, and Lys at position 330; according to EU
numbering. In a further aspect, amino acid alterations in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma Rllb-binding activity, are: 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 a further aspect, amino acid alterations in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma RHb-binding activity, are: 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.

[0363] In one aspect, at least one amino acid is altered in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma RHb-binding activity, of at least one position selected from the group consisting of:
234, 238, 250, 264, 267, 307, and 330 according to EU numbering. In certain em-bodiments, the Fc gamma Ruth has the sequence of cynomolgus monkey Fc gamma Ruth (SEQ ID NO:223). In certain embodiments, the Fc gamma Ruth has the sequence of human Fc gamma Ruth (e.g., SEQ ID NOS:212, 213, or 214).

[0364] In another aspect, one amino acid is altered in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma Rllb-binding activity, at position 238.

[0365] In another aspect, at least two amino acids are altered in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma Ruth-binding activity, the alterations comprising: (i) one amino acid alteration at position 238, and (ii) at least one amino acid alteration of at least one position selected from the group consisting of: 234, 250, 264, 267, 307, and 330 according to EU numbering. In certain embodiments, the Fc gamma RIIb has the sequence of cynomolgus monkey Fc gamma Ruth (SEQ ID NO:223). In certain embodiments, the Fc gamma Ruth has the sequence of human Fc gamma RIIb (e.g., SEQ ID NOS:212, 213, or 214).

[0366] In another aspect, amino acids are altered in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma Rllb-binding activity, at positions of any one of the following (1)-(9): (1) at positions 234, 238, 250, 307 and 330; (2) at positions 234, 238, 250, 264, 307 and 330;
(3) at positions 234, 238, 250, 264, 267, 307 and 330; (4) at positions 234, 238, 250, 267, 307 and 330; (5) at positions 238, 250, 264, 307 and 330; (6) at positions 238, 250, 264, 267, 307 and 330; (7) at positions 238, 250, 267, 307 and 330; (8) at positions 238, 250 and 307; and (9) at positions 238, 250, 307 and 330; according to EU
numbering.

[0367] In a further aspect, an amino acid alteration in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma RHb-binding activity, is selected at each position 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 a further aspect, amino acid alterations in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma Ruth-binding activity, are: Asp at position 238; according to EU
numbering. In a further aspect, amino acid alterations in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma RHb-binding activity, are: Asp at position 238, Val at position 250, and Pro at position 307;
according to EU numbering. In a further aspect, amino acid alterations in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma Rllb-binding activity, are: Asp at position 238, Val at position 250, Pro at position 307, and Lys at position 330; according to EU numbering.
In a further aspect, amino acid alterations in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma RHb-binding activity, are: 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 a further aspect, amino acid alterations in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma Ruth-binding activity, are:
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 a further aspect, amino acid al-terations in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma RHb-binding activity, are: 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 a further aspect, amino acid alterations in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma RIIb-binding activity, are: 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 a further aspect, amino acid alterations in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma Ruth-binding activity, are: 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 a further aspect, amino acid alterations in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma Ruth-binding activity, are: 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 a further aspect, amino acid alterations in the above-mentioned method for producing a polypeptide comprising a variant Fc region with enhanced Fc gamma Ruth-binding activity, are: 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 em-bodiments, the Fc gamma Ruth has the sequence of cynomolgus monkey Fc gamma RHb (SEQ ID NO:223). In certain embodiments, the Fc gamma RHb has the sequence of human Fc gamma Ruth (e.g., SEQ ID NOS:212, 213, or 214).

[0368] Furthermore, the present invention provides a method for producing a polypeptide comprising a variant Fc region with increased pI in comparison with a polypeptide comprising a parent Fc region, which comprises introducing at least two amino acid al-terations to the parent Fc region.

[0369] In certain embodiments, the production method for producing a polypeptide comprising a variant Fc region provided herein comprises the following steps:
(a) preparing a polypeptide comprising a parent Fc region; (b) introducing at least two amino acid alterations to the parent Fc region within the polypeptide; (c) measuring the pI's of the polypeptide comprising the parent Fc region and the polypeptide comprising the Fc region in step (b); and (d) selecting a polypeptide comprising a variant Fc region with increased pI in comparison with the corresponding polypeptide comprising the parent Fc region.

[0370] In one aspect, at least two amino acids are altered in the above-mentioned method for producing a polypeptide comprising a variant Fc region with increased pI, of 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 a further aspect, the produced polypeptide comprises a variant Fc region with increased pI, which comprises at least two amino acid al-terations of at least two positions selected from the group consisting of:
311, 341, 343, 384, 399, 400, 401, 402, and 413, according to EU numbering.

[0371] In another aspect, amino acids are altered in the above-mentioned method for producing a polypeptide comprising a variant Fc region with increased pI, at positions of any one of the following (1)-(10): (1) at positions 311 and 341; (2) at positions 311 and 343; (3) at positions 311, 343 and 413; (4) at positions 311, 384 and 413;
(5) at positions 311 and 399; (6) at positions 311 and 401; (7) at positions 311 and 413; (8) at positions 400 and 413; (9) at positions 401 and 413; and (10) at positions 402 and 413;
according to EU numbering.
In a further aspect, amino acid alterations in the above-mentioned method for producing a polypeptide comprising a variant Fc region with increased pI, are:
Arg at position 400 and Lys at position 413; according to EU numbering. In a further aspect, amino acid alterations in the above-mentioned method for producing a polypeptide comprising a variant Fc region with increased pI, are: Arg at position 311 and Lys at position 413; according to EU numbering. In a further aspect, amino acid alterations in the above-mentioned method for producing a polypeptide comprising a variant Fc region with increased pI, are: Arg at position 311 and Arg at position 399;
according to EU numbering. In a further aspect, amino acid alterations in the above-mentioned method for producing a polypeptide comprising a variant Fc region with increased pI, are: Arg at position 311 and Arg at position 343; according to EU numbering.
In a further aspect, amino acid alterations in the above-mentioned method for producing a polypeptide comprising a variant Fc region with increased pI, are: Arg at position 311 and Arg at position 413; according to EU numbering. In a further aspect, amino acid alterations in the above-mentioned method for producing a polypeptide comprising a variant Fc region with increased pI, are: Arg at position 311, Arg at position 343, and Arg at position 413; according to EU numbering. In a further aspect, amino acid al-terations in the above-mentioned method for producing a polypeptide comprising a variant Fc region with increased pI, are: Arg at position 311, Arg at position 384, and Arg at position 413; according to EU numbering.

[0372] In a further aspect, the amino acid alterations in the above-mentioned method for producing a polypeptide comprising a variant Fc region with increased pI, are selected from Lys or Arg at each position.

[0373] In a further aspect, the amino acid alterations in the above-mentioned production methods are selected from any single alteration, combination of single alterations, or combination alterations described in Tables 14-30.

[0374] Optionally, the following additional steps can be included in the above-mentioned methods for producing a polypeptide comprising a variant Fc region, when the polypeptide further comprises an antigen-binding domain: (e) assessing pharma-cokinetic of the antigen in plasma, after administration of the polypeptide comprising the parent Fc region and the polypeptide comprising the variant Fc region in animals such as mice, rats, rabbits, dogs, monkeys, and humans; and (f) selecting a polypeptide comprising a variant Fc region with enhanced ability of antigen elimination from plasma in comparison with the polypeptide comprising the parent Fc region.

[0375] Polypeptides comprising a variant Fc region produced by any of the above-mentioned methods or other methods know in the art are included in the present invention.
C. Assays

[0376] Anti-myostatin antibodies provided herein may be identified, screened for, or char-acterized for their physical/chemical properties and/or biological activities by various assays known in the art.

[0377] Variant Fc regions provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays described herein or otherwise known in the art.
1. Binding assays and other assays

[0378] In one aspect, an antibody of the invention is tested for its antigen binding activity, by known methods such as ELISA, Western blot, BIACORE (registered trademark), etc. In one aspect, a polypeptide comprising an variant Fc region of the invention is tested for its Fc receptor binding activity, by known methods such as BIACORE
(registered trademark), etc.

[0379] In another aspect, competition assays may be used to identify an antibody that competes for binding to myostatin with any anti-myostatin antibody described herein.
In certain embodiments, when such a competing antibody is present in excess, it blocks (e.g., reduces) the binding of a reference antibody to myostatin by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more. In some instances, binding is inhibited by at least 80%, 85%, 90%, 95%, or more. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear or a conformational epitope) that is bound by an anti-myostatin antibody described herein (e.g., an anti-myostatin antibody described in Tables 2a, 11a, or 13). In further aspects, the reference antibody has a VH and a VL pair described in Table 2a, 11a, or 13.
Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0380] In an exemplary competition assay, immobilized latent myostatin or myostatin propeptide is incubated in a solution comprising a first labeled antibody that binds to the myostatin and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to the myostatin. The second antibody may be present in a hybridoma supernatant. As a control, immobilized myostatin is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the first antibody to the myostatin, excess unbound antibody is removed, and the amount of label associated with immobilized myostatin is measured. If the amount of label as-sociated with immobilized myostatin is substantially reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to the myostatin. See, Harlow and Lane (1988) An-tibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0381] Assays for determining the binding activity of a polypeptide containing a variant Fc region towards one or more Fc gamma R family members are described herein or otherwise known in the art. Such binding assays include but are not limited to BIACORE (registered trademark) analysis, which utilizes the surface plasmon resonance (SPR) phenomena, Amplified Luminescent Proximity Homogeneous Assay (ALPHA) screening, ELISA, and fluorescence activated cell sorting (FACS) (Lazar et al., Proc. Natl. Acad. Sci. USA (2006) 103(11): 4005-4010).

[0382] In one embodiment, BIACORE (registered trademark) analysis can be used to evaluate whether the binding activity of a polypeptide comprising a variant Fc region is enhanced, or maintained or decreased with respect to a particular Fc gamma R
family member, for example, by observing whether there is a decrease or an increase in the dissociation constant (KD) value obtained from sensorgram analysis, where various Fc gamma Rs are subjected to interaction as an analyte with polypeptides comprising a variant Fc region immobilized or captured onto the sensor chip using known methods and reagents (such as Protein A, Protein L, Protein A/G, Protein G, anti-lamda chain antibodies, anti-kappa chain antibodies, antigenic peptides, antigenic proteins). Al-terations in binding activity can also be determined by comparing changes in the resonance unit (RU) value on the sensorgram before and after the one or more types of Fc gamma Rs are subjected to interaction as analytes with the captured polypeptides comprising the variant Fc region. Alternatively, Fc gamma R can be immobilized or captured onto the sensor chips, and the polypeptides comprising the variant Fc region are used as an analyte.

[0383] In BIACORE (registered trademark) analysis, one of the substances (the ligand) in observation of an interaction is immobilized onto a gold thin film on a sensor chip, and by shining light from the reverse side of the sensor chip so that total reflection takes place at the interface between the gold thin film and glass, a portion of reduced re-flection intensity is formed in part of the reflected light (SPR signal). When the other one of the substances (the analyte) in observation of an interaction is made to flow on the sensor chip surface and the ligand binds to the analyte, the mass of the immobilized ligand molecule increases and the refractive index of the solvent on the sensor chip surface changes. The position of the SPR signal shifts as a result of this change in re-fractive index (on the other hand, the signal position returns when this binding dis-sociates). The BIACORE (registered trademark) system indicates the amount of shift mentioned above, or more specifically the time variable of mass by plotting the change in mass on the sensor chip surface on the ordinate as the measurement data (sensorgram). The amount of analyte bound to the ligand trapped on the sensor chip surface is determined from the sensorgram. Kinetic parameters such as association rate constants (ka) and dissociation rate constants (kd) are determined from the curves of the sensorgram, and the dissociation constants (KD) are determined from the ratio of these constants. In the BIACORE (registered trademark) method, a method for measuring inhibition is preferably used. An example of the method for measuring in-hibition is described in Lazar et al., Proc. Natl. Acad. Sci. USA 103(11):4005-(2006).

[0384] ALPHA screening is performed by ALPHA technology which uses two beads, a donor and an acceptor, based on the following principles. Luminescent signals are detected only when molecules bound to donor beads physically interact with molecules bound to the acceptor beads, and the two beads are in close proximity to each other.
Laser-excited photosensitizer in the donor beads converts ambient oxygen to excited-state singlet oxygen. Singlet oxygen is dispersed around the donor beads, and when it reaches the adjacent acceptor beads, chemiluminescent reaction is induced in the beads, and light is ultimately emitted. When the molecules bound to the donor beads do not interact with the molecules bound to the acceptor beads, the chemiluminescent reaction does not take place because singlet oxygen produced by the donor beads does not reach the acceptor beads.

[0385] For example, a biotinylated polypeptide complex is bound to the donor beads, and Fc gamma receptor tagged with glutathione S transferase (GST) is linked to the acceptor beads. In the absence of a competing polypeptide complex comprising a variant Fc region, the polypeptide complex comprising a parent Fc region interacts with the Fc gamma receptor and produces 520-620 nm signals. The polypeptide complex comprising an untagged variant Fc region competes with the polypeptide complex comprising a parent Fc region for interaction with the Fc gamma receptor.
Relative binding activities can be determined by quantifying the decrease in fluorescence observed as a result of the competition. Biotinylation of polypeptide complexes such as antibodies using Sulfo-NHS-biotin and such is well known. The method of expressing the Fc gamma receptor and GST in a cell carrying a fusion gene produced by fusing a polynucleotide encoding the Fc gamma receptor in frame with a polynucleotide encoding GST in an expressible vector, and performing purification using a glutathione column is appropriately adopted as a method for tagging an Fc gamma receptor with GST. The obtained signals are preferably analyzed, for example, by fitting them to a one-site competition model which uses a non-linear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).

[0386] A variant Fc region with decreased Fc gamma R-binding activity refers to an Fc region which binds to Fc gamma R with essentially weaker binding activity than a parent Fc region when assays are performed using substantially the same amount of a corresponding parent Fc region and a variant Fc region. Furthermore, a variant Fc region with enhanced Fc gamma R-binding activity refers to an Fc region which binds to Fc gamma R with essentially stronger binding activity than a corresponding parent Fc region when assays are performed using substantially the same amount of a parent Fc region and a variant Fc region. A variant Fc region with maintained Fc gamma R-binding activity refers to an Fc region that binds to Fc gamma R with binding activity equivalent to or essentially not different from that of a parent Fc region when assays are performed using substantially the same amount of the corresponding parent Fc region and the polypeptide containing the variant Fc region.

[0387] Whether or not the binding activities of an Fc region towards various Fc gamma Rs were enhanced or decreased can be determined from the increase or decrease in the amount of binding of the various Fc gamma Rs to the Fc region, which were de-termined according to the above-mentioned measurement method. Here, the amount of binding of the various Fc gamma Rs to the Fc region can be evaluated as a value obtained by dividing the difference in the RU values of sensorgrams that changed before and after interaction of various Fc gamma Rs as the analyte with the Fc region, by the difference in the RU values of sensorgrams that changed before and after capturing the Fc regions to the sensor chips.

[0388] In the present invention, enhanced Fc gamma RIM-binding activity preferably means, for example, that the ratio of [KD value of a parent Fc region for Fc gamma RIIb1/[KD value of a variant Fc region for Fc gamma RIIb] in the KD values measured by the above-mentioned measurement method preferably becomes 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, 35 or greater, 40 or greater, 45 or greater, or even 50 or greater, 55 or greater, 60 or greater, 65 or greater, 70 or greater, 75 or greater, 80 or greater, 85 or greater, 90 or greater, 95 or greater, or 100 or greater.

[0389] The KD value (mol/L) of a variant Fc region decribed herein for Fc gamma RIIb is preferably less than that of a parent Fc region for Fc gamma RIIb, and may be, for example, 2.0x10 6 M or smaller, 1.0x106 M or smaller, 9.0x107 M or smaller, 8.0x107 M or smaller, 7.0x107 M or smaller, 6.0x107 M or smaller, 5.0x107 M or smaller, 4.0x107 M or smaller, 3.0x107 M or smaller, 2.0x107 M or smaller, 1.0x107 M or smaller, 9.0x108 M or smaller, 8.0x10 8M or smaller, 7.0x10 8M or smaller, 6.0x108 M or smaller, 5.0x108 M or smaller.

[0390] Furthermore, a variant Fc region with enhanced binding selectivity to Fc gamma RHb compared to Fc gamma RIIa refers to an Fc region where: (a) Fc gamma Ruth-binding activity is enhanced, and Fc gamma RIIa-binding activity is maintained or decreased; (b) Fc gamma Ruth-binding activity is enhanced and Fc gamma RIIa-binding activity is also enhanced, but the degree of enhancement of Fc gamma RIIa-binding activity is lower than the degree of enhancement of Fc gamma Rllb-binding activity; or (c) Fc gamma Rllb-binding activity is decreased and Fc gamma RIIa-binding activity is also decreased, but the degree of decrease of Fc gamma Ruth-binding activity is less than the degree of decrease of Fc gamma RIIa-binding activity.
Whether or not a variant Fc region has improved binding selectivity for Fc gamma Ruth rather than for Fc gamma RIIa can be determined, for example, by comparing the ratio of the KD value for Fc gamma RIIa to the KD value for Fc gamma Ruth of the variant Fc region (the ratio of [KD value of the variant Fc region for Fc gamma RIIa]/[KD value of the variant Fc region for Fc gamma RIIbp, with the ratio of the KD
value for Fc gamma RIIa to the KD value for Fc gamma RHb of the parent Fc region (the ratio of [KD value of the parent Fc region for Fc gamma RIIa1/[KD value of the parent Fc region for Fc gamma RI1b1), which were determined according to the above-mentioned examples. Specifically, when the KD ratio for the variant Fc region is greater than that of the parent Fc region, the variant Fc region can be determined to have an improved binding selectivity for Fc gamma Ruth rather than for Fc gamma RIIa in comparison with the parent Fc region. Especially in human, the Fc gamma RHb-binding activity is likely to correlate with binding activity to Fc gamma RIIa (type R) than to Fc gamma RIIa (type H) since the amino acid sequence of Fc gamma RHb shares higher identity with Fc gamma RIIa (type R) than Fc gamma RIIa (type H). Therefore, finding amino acid alteration(s) that can enhance binding selectivity to human Fc gamma Ruth compared to human Fc gamma RIIa (type R) is important for enhancing binding selectivity to Fc gamma RHb compared to Fc gamma RIIa in humans.

[0391] When a variant Fc region of the present invention has higher binding activity against Fc gamma RHb and lower binding activity against Fc gamma RIII (such as Fc gamma RIIIa or Fc gamma RIIIb) compared to those of a parent Fc region, the variant Fc region can be said to be Fc gamma RIIb-specific.
2. Activity assays

[0392] In one aspect, assays are provided for identifying anti-myostatin antibodies having biological activity. Biological activity may include, e.g., inhibiting the activation of myostatin, blocking the release of mature myostatin from latent myostatin, inhibiting the proteolytic cleavage of latent myostatin, blocking the access of a protease to latent myostatin, etc. Antibodies having such biological activity in vivo and/or in vitro are also provided. In certain embodiments, an antibody of the invention is tested for such biological activity.

[0393] In certain embodiments, whether a test antibody inhibits the cleavage of latent myostatin is determined by detecting the cleavage product of latent myostatin (myostatin) using a method known in the art such as electrophoresis, chromatography, immunoblot analysis, an enzyme-linked immunosorbent assay (ELISA), or mass spec-trometry, after a protease that can cleave latent myostatin is contacted with the latent myostatin in the presence or absence of the test antibody (see, for example, Thies et al., Growth Factors 18(4):251-259 (2001)). Where a decreased amount of the cleavage product of latent myostatin (e.g., human myostatin propeptide) is detected in the presence of (or following contact with) the test antibody, the test antibody is identified as an antibody that can inhibit the cleavage of latent myostatin. In certain em-bodiments, whether a test antibody blocks access of a protease to latent myostatin is determined by methods for the detection of protein interactions between the protease and latent myostatin, e.g., ELISAs or BIACORE (registered trademark). Where a decreased interaction between the protease and latent myostatin is detected in the presence of (or following contact with) the test antibody, the test antibody is identified as an antibody that can block access of the protease to latent myostatin.

[0394] In certain embodiments, whether a test antibody blocks the release of mature myostatin from latent myostatin is determined by detecting mature myostatin activity, for example, the activity of binding to a myostatin receptor, or the activity of mediating signal transduction in a cell expressing a myostatin receptor (e.g., ActRIIb).
Cells useful for such an assay can be those that express an endogenous myostatin receptor, for example, L6 myocytes, or can be those that are genetically modified, transiently or stably, to express a transgene encoding a myostatin receptor, for example, an activin receptor such as an activin type II receptor (Thies et al, Supra). Binding of myostatin to a myostatin receptor can be detected using a receptor binding assay. Myostatin mediated signal transduction can be detected at any level in the signal transduction pathway, for example, by examining phosphorylation of a Smad polypeptide, examining expression of a myostatin regulated gene including a reporter gene, or measuring proliferation of a myostatin-dependent cell. Where a decreased mature myostatin activity is detected in the presence of (or following contact with) the test antibody, the test antibody is identified as an antibody that can block the release of mature myostatin from latent myostatin.

[0395] Inhibition of myostatin activation can also be detected and/or measured using the methods set forth and exemplified in the working examples. Using assays of these or other suitable types, test antibodies can be screened for those capable of inhibiting the activation of myostatin. In certain embodiments, inhibition of myostatin activation includes at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% or greater decrease in myostatin activation in the assay as compared to a negative control under similar conditions. In some embodiments, it refers to the inhibition of myostatin activation of at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or greater.

[0396] In another aspect, assays for identifying anti-myostatin antibodies that form an immune complex (i.e., an antigen-antibody complex) with myostatin are provided. An-tibodies having such biological activity in vivo and/or in vitro are also provided.

[0397] In certain embodiments, an antibody of the invention is tested for such biological activity.

[0398] In certain embodiments, the formation of an immune complex is evaluated by a method such as size exclusion (gel filtration) chromatography, ultracentrifugation, light scattering, electron microscope, or mass spectrometry (Mol. Immunol.
39:77-84 (2002), Mol. Immunol. 47:357-364 (2009)). These methods make use of the property that an immune complex is a larger molecule than an antibody alone, or an antigen alone. Where a large complex containing two or more antibodies and two or more antigens (e.g., myostatin molecules) is detected in the presence of a test antibody and an antigen, the test antibody is identified as an antibody that can form an immune complex containing two or more antibodies and two or more molecules of myostatin.
In another embodiment, formation of an immune complex is evaluated by a method such as, ELISA, FACS, or SPR (surface plasmon resonance assay; for example, using BIACORE (registered trademark)) (Shields et al., J. Biol. Chem. 276(9):6591-(2001); Singh et al., J. Immunol. Methods 50:109-114 (1982); Suzuki et al., J.

Immunol. 184(4):1968-1976 (2010); Luo et al., mAbs 1(5):491-504 (2009)). These methods make use of the property that an immune complex containing two or more an-tibodies and two or more antigens can bind more strongly to an Fc receptor or a complement component than an antibody or antigen alone can. Where an increased binding to an Fc receptor or a complement component is detected in the presence of both a test antibody and an antigen compared to in the presence of an antibody alone, the test antibody is identified as an antibody that can form an immune complex containing two or more antibodies and two or more molecules of myostatin. In another embodiment, formation of an immune complex is evaluated by administering a test antibody to an animal (e.g., a mouse) and measuring the clearance of antigen from plasma. As described above, an antibody which forms an immune complex containing two or more antibodies with two or more antigens is expected to accelerate the elimination of antigens from plasma. Therefore, where an accelerated elimination of myostatin from plasma is observed in a test antibody-administered mouse compared to in a reference antibody-administered mouse, the test antibody is identified as an antibody that can form an immune complex containing two or more antibodies and two or more molecules of myostatin more efficiently than the reference antibody.
D. Immunoconjugates

[0399] In some embodiments, the invention provides immunoconjugates comprising an anti-myostatin antibody herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.

[0400] In some embodiments, the invention provides immunoconjugates comprising a polypeptide comprising a variant Fc region herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.

[0401] In one embodiment, an immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, including but not limited to a maytansinoid (see US Patent Nos. 5,208,020, 5,416,064 and EP Patent EP 0 425 B1); an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE
and MMAF) (see US Patent Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or derivative thereof (see US Patent Nos.
5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296;
Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res.
58:2925-2928 (1998)); an anthracycline such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic &
Med.
Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005);
Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg.
& Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-(2002); and US Patent No. 6,630,579); methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene;
and CC1065.

[0402] In another embodiment, an immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and a tricothecene.

[0403] In another embodiment, an immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of ra-dioactive isotopes are available for the production of radioconjugates.
Examples include At211, I131, 1125, Y9O, Re186, Rem', Sm153, Bi212, p32, pb212 and radioactive isotopes of Lu. When the radioconjugate is used for detection, it may comprise a radioactive atom for scintigraphic studies, for example tc99m or 1123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese and iron.

[0404] Conjugates of an antibody and cytotoxic agent may be made using a variety of bi-functional protein coupling agents such as N-succinimidy1-3-(2-pyridyldithio) propionate (SPDP), succinimidy1-4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoy1)-ethylenediamine), diiso-cyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzy1-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO 1994/11026. The linker may be a "cleavable linker" facilitating release of a cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); US Patent No. 5,208,020) may be used.

[0405] The immunoconjugates or ADCs herein expressly contemplate, but are not limited to such conjugates prepared with cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, STAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidy1-(4-vinylsulfone)benzoate) which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A).
E. Methods and Compositions for Diagnostics and Detection

[0406] In certain embodiments, any of the anti-myostatin antibodies provided herein is useful for detecting the presence of myostatin in a biological sample. The term "detecting" as used herein encompasses quantitative or qualitative detection.
In certain embodiments, a biological sample comprises a cell or tissue, such as serum, whole blood, plasma, biopsy sample, tissue sample, cell suspension, saliva, sputum, oral fluid, cerebrospinal fluid, amniotic fluid, ascites fluid, milk, colostrum, mammary gland secretion, lymph, urine, sweat, lacrimal fluid, gastric fluid, synovial fluid, peritoneal fluid, ocular lens fluid or mucus.

[0407] In one embodiment, an anti-myostatin antibody for use in a method of diagnosis or detection is provided. In a further aspect, a method of detecting the presence of latent myostatin or myostatin propeptide in a biological sample is provided. In certain em-bodiments, the method comprises contacting the biological sample with an anti-myostatin antibody as described herein under conditions permissive for binding of the anti-myostatin antibody to myostatin, and detecting whether a complex is formed between the anti-myostatin antibody and myostatin. Such method may be an in vitro or in vivo method. In one embodiment, an anti-myostatin antibody is used to select subjects eligible for therapy with an anti-myostatin antibody, e.g., where myostatin is a biomarker for selection of patients.

[0408] Exemplary disorders that may be diagnosed using an antibody of the invention include but are not limited to, muscular dystrophy (MD; including Duchenne muscular dystrophy), amyotrophic lateral sclerosis (ALS), muscle atrophy, organ atrophy, carpal tunnel syndrome, frailty, congestive obstructive pulmonary disease (COPD), sarcopenia, cachexia, muscle wasting syndromes, HIV-induced muscle wasting, type 2 diabetes, impaired glucose tolerance, metabolic syndrome (including syndrome X), insulin resistance (including resistance induced by trauma, e.g., bums or nitrogen imbalance), adipose tissue disorders (e.g., obesity, dyslipidemia, nonalcoholic fatty liver disease, etc.), osteoporosis, osteopenia, osteoarthritis, and metabolic bone disorders (including low bone mass, premature gonadal failure, androgen suppression, vitamin D deficiency, secondary hyperparathyroidism, nutritional deficiencies, and anorexia nervosa).

[0409] In certain embodiments, labeled anti-myostatin antibodies are provided. Labels include, but are not limited to, labels or moieties that are detected directly (such as flu-orescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, the radioisotopes 32P, 14C, 125I, 3H, and "'I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, um-belliferone, luceriferases, e.g., firefly luciferase and bacterial luciferase (US Patent No.
4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehy-drogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lac-toperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like.
F. Pharmaceutical Formulations

[0410] Pharmaceutical formulations of an anti-myostatin antibody as described herein are prepared by mixing such antibody having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.

[0411] Pharmaceutical formulations of a polypeptide comprising a variant Fc region as described herein are prepared by mixing such polypeptide having the desired degree of purity with one or more optional pharmaceutically acceptable carriers in the form of lyophilized formulations or aqueous solutions.

[0412] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to:
buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hex-amethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol;
resorcinol;
cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX (registered trademark), Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Publ. Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

[0413] Exemplary lyophilized antibody formulations are described in US
Patent No.
6,267,958. Aqueous antibody formulations include those described in US Patent No.
6,171,586 and WO 2006/044908, the latter formulations including a histidine-acetate buffer.

[0414] The formulation herein may also contain more than one active ingredients as necessary for the particular indication being treated, preferably those with com-plementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

[0415] Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethyl-cellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, re-spectively, in colloidal drug delivery systems (for example, liposomes, albumin mi-crospheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0416] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0417] The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
G. Therapeutic Methods and Compositions

[0418] Any of the anti-myostatin antibodies provided herein may be used in therapeutic methods. Likewise, any of the polypeptpides comprising an variant Fc region provided herein may be used in therapeutic methods.

[0419] In one aspect, an anti-myostatin antibody for use as a medicament is provided. In further aspects, an anti-myostatin antibody for use in treating a muscle wasting disease is provided. In certain embodiments, an anti-myostatin antibody for use in a method of treatment is provided. In certain embodiments, the invention provides an anti-myostatin antibody for use in a method of treating an individual having a muscle wasting disease comprising administering to the individual an effective amount of the anti-myostatin antibody. In one embodiment, the method further comprises admin-istering to the individual an effective amount of at least one additional therapeutic agent. In further embodiments, the invention provides an anti-myostatin antibody for use in increasing mass of muscle tissue. In certain embodiments, the invention provides an anti-myostatin antibody for use in a method of increasing mass of muscle tissue in an individual comprising administering to the individual an effective amount of the anti-myostatin antibody to increase mass of muscle tissue. In further em-bodiments, the invention provides an anti-myostatin antibody for use in increasing strength of muscle tissue. In certain embodiments, the invention provides an anti-myostatin antibody for use in a method of increasing strength of muscle tissue in an in-dividual comprising administering to the individual an effective amount of the anti-myostatin antibody to increase strength of muscle tissue. In further embodiments, the invention provides an anti-myostatin antibody for use in reducing body fat accu-mulation. In certain embodiments, the invention provides an anti-myostatin antibody for use in a method of reducing body fat accumulation in an individual comprising ad-ministering to the individual an effective amount of the anti-myostatin antibody to reduce body fat accumulation. An "individual" according to any of the above em-bodiments is preferably a human.

[0420] An anti-myostatin antibody of the present invention may exhibit pH-dependent binding characteristics. In further embodiments, the invention provides an anti-myostatin antibody for use in enhancing the clearance of myostatin from plasma. In certain embodiments, the invention provides an anti-myostatin antibody for use in a method of enhancing the clearance of myostatin from plasma in an individual comprising administering to the individual an effective amount of the anti-myostatin antibody to enhance the clearance of myostatin from plasma. In one embodiment, an anti-myostatin antibody with pH-dependent binding characteristics enhances the clearance of myostatin from plasma, compared to a conventional anti-myostatin antibody which does not have pH-dependent binding characteristics. In a further em-bodiment, an anti-myostatin antibody with a pH-dependent binding characteristic between binding at pH5.8 and pH7.4 enhances the clearance of myostatin from plasma, compared to a conventional anti-myostatin antibody which does not have pH-dependent binding characteristics. An "individual" according to any of the above em-bodiments is preferably a human.

[0421] In a further aspect, the invention provides the use of an anti-myostatin antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treatment of a muscle wasting disease. In a further embodiment, the medicament is for use in a method of treating a muscle wasting disease comprising administering to an individual having a muscle wasting disease an effective amount of the medicament.
In one embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In a further embodiment, the medicament is for increasing mass of muscle tissue. In a further embodiment, the medicament is for use in a method of increasing mass of muscle tissue in an individual comprising administering to the individual an effective amount of the medicament to increase mass of muscle tissue. In a further embodiment, the medicament is for in-creasing strength of muscle tissue. In a further embodiment, the medicament is for use in a method of increasing strength of muscle tissue in an individual comprising admin-istering to the individual an effective amount of the medicament to increase strength of muscle tissue. In a further embodiment, the medicament is for reducing body fat accu-mulation. In a further embodiment, the medicament is for use in a method of reducing body fat accumulation in an individual comprising administering to the individual an effective amount of the medicament to reduce body fat accumulation. An "individual"
according to any of the above embodiments may be a human.

[0422] An anti-myostatin antibody of the present invention may exhibit pH-dependent binding characteristics. In a further embodiment, the medicament is for enhancing the clearance of myostatin from plasma. In a further embodiment, the medicament is for use in a method of enhancing the clearance of myostatin from plasma in an individual comprising administering to the individual an effective amount of the medicament to enhance the clearance of myostatin from plasma. In one embodiment, an anti-myostatin antibody with pH-dependent binding characteristics enhances the clearance of myostatin from plasma, compared to a conventional anti-myostatin antibody which does not have pH-dependent binding characteristics. In a further embodiment, the anti-myostatin antibody exhibits different pH-dependent binding characteristics between pH5.8 and pH7.4. An "individual" according to any of the above embodiments is preferably a human.

[0423] In another aspect, the invention provides a method for treating a muscle wasting disease. In one embodiment, the method comprises administering to an individual having such a muscle wasting disease an effective amount of an anti-myostatin antibody provided herein. In one embodiment, the method further comprises admin-istering to the individual an effective amount of at least one additional therapeutic agent. The "individual" according to any of the above embodiments may be a human.

[0424] In another aspect, the invention provides a method for increasing mass of muscle tissue in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-myostatin antibody provided herein to increase mass of muscle tissue. In one embodiment, the "individual" is a human.

[0425] In another aspect, the invention provides a method for increasing strength of muscle tissue in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-myostatin antibody provided herein to increase strength of muscle tissue. In one embodiment, the "individual" is a human.

[0426] In another aspect, the invention provides a method for reducing body fat accu-mulation in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-myostatin antibody provided herein to reduce body fat accumulation. In one embodiment, the "individual" is a human.

[0427] An anti-myostatin antibody of the present invention may exhibit pH-dependent binding characteristics. In a further embodiment, the invention provides a method for enhancing the clearance of myostatin from plasma in an individual. In one em-bodiment, the method comprises administering to the individual an effective amount of an anti-myostatin antibody provided herein to enhance the clearance of myostatin from plasma. In one embodiment, an anti-myostatin antibody with pH-dependent binding characteristics enhances the clearance of myostatin from plasma, compared to a con-ventional anti-myostatin antibody which does not have pH-dependent binding charac-teristics. In a further embodiment, the anti-myostatin antibody exhibits different pH-dependent binding characteristics between pH5.8 and pH7.4. In one embodiment, an "individual" is a human.

[0428] In a further aspect, the invention provides pharmaceutical formulations comprising any of the anti-myostatin antibodies provided herein, e.g., for use in any of the above therapeutic methods. In one embodiment, a pharmaceutical formulation comprises any of the anti-myostatin antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical formulation comprises any of the anti-myostatin antibodies provided herein and at least one additional therapeutic agent.

[0429] In a further aspect, the pharmaceutical formulation is for treatment of a muscle wasting disease. In a further embodiment, the pharmaceutical formulation is for in-creasing mass of muscle tissue. In a further embodiment, the pharmaceutical for-mulation is for increasing strength of muscle tissue. In a further embodiment, the phar-maceutical formulation is for reducing body fat accumulation. An anti-myostatin antibody of the present invention may exhibit pH-dependent binding characteristics. In a further embodiment, the pharmaceutical formulation is for enhancing the clearance of myostatin from plasma. In one embodiment, the pharmaceutical formulation is ad-ministered to an individual having a muscle wasting disease. An "individual"
according to any of the above embodiments is preferably a human.

[0430] In a further aspect, the invention provides methods for preparing a medicament or a pharmaceutical formulation, comprising mixing any of the anti-myostatin antibodies provided herein with a pharmaceutically acceptable carrier, e.g., for use in any of the above therapeutic methods. In one embodiment, the methods for preparing a medicament or a pharmaceutical formulation further comprise adding at least one ad-ditional therapeutic agent to the medicament or pharmaceutical formulation.

[0431] In certain embodiments, a muscle wasting disease is selected from the group consisting of muscular dystrophy (MD; including Duchenne muscular dystrophy), amyotrophic lateral sclerosis (ALS), muscle atrophy, organ atrophy, carpal tunnel syndrome, frailty, congestive obstructive pulmonary disease (COPD), sarcopenia, cachexia, muscle wasting syndromes, HIV-induced muscle wasting, type 2 diabetes, impaired glucose tolerance, metabolic syndrome (including syndrome X), insulin re-sistance (including resistance induced by trauma, e.g., burns or nitrogen imbalance), adipose tissue disorders (e.g., obesity, dyslipidemia, nonalcoholic fatty liver disease, etc.), osteoporosis, osteopenia, osteoarthritis, and metabolic bone disorders (including low bone mass, premature gonadal failure, androgen suppression, vitamin D de-ficiency, secondary hyperparathyroidism, nutritional deficiencies, and anorexia nervosa).

[0432] Any of the polypeptide comprising a variant Fc region provided herein may be used in therapeutic methods. In a further aspect, the invention provides pharmaceutical for-mulations comprising a polypeptide comprising any of the polypeptides comprising variant Fc regions provided herein, e.g., for use in therapeutic methods. In one em-bodiment, a pharmaceutical formulation comprises a polypeptide comprising any of the polypeptides comprising variant Fc regions provided herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical formulation comprises a polypeptide comprising any of the variant Fc regions provided herein and at least one additional therapeutic agent.

[0433] In one aspect, a polypeptide comprising a variant Fc region for use as a medicament is provided. In further aspects, a polypeptide comprising a variant Fc region for use in treating a disorder is provided. In certain embodiments, a polypeptide comprising a variant Fc region for use in a method of treatment is provided. In certain embodiments, the invention provides a polypeptide comprising a variant Fc region for use in a method of treating an individual having a disorder comprising administering to the in-dividual an effective amount of the polypeptide comprising a variant Fc region provided herein. In one embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In one embodiment, the "individual" is a human.

[0434] In a further aspect, the invention provides the use of a polypeptide comprising a variant Fc region in the manufacture or preparation of a medicament. In one em-bodiment, the medicament is for treatment of a disorder. In some apsects, the polypeptide is an antibody. In some aspsects, the polypeptide is an Fc fusion protein.
In a further embodiment, the medicament is for use in a method of treating a disorder comprising administering to an individual having the disorder to be treated an effective amount of the medicament. In one embodiment, the method further comprises admin-istering to the individual an effective amount of at least one additional therapeutic agent. In one embodiment, the "individual" is a human.

[0435] In a further aspect, the invention provides a method for treating a disorder. In one embodiment, the method comprises administering to an individual having such a disorder an effective amount of a polypeptide comprising a variant Fc region.
In one embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In one embodiment, the "in-dividual" is a human.

[0436] In a further aspect, the invention provides pharmaceutical formulations comprising a polypeptide comprising a variant Fc region provided herein, for use in a therapeutic method such as any of the therapeutic methods described herein. In one embodiment, a pharmaceutical formulation comprises a polypeptide comprising a variant Fc region provided herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical formulation comprises a polypeptide comprising a variant Fc region provided herein and at least one additional therapeutic agent.

[0437] In a further aspect, the pharmaceutical formulation is for treatment of a disorder. In one embodiment, the pharmaceutical formulation is administered to an individual having a disorder. In one embodiment, the "individual" is a human.

[0438] In further embodiments, the invention provides a polypeptide comprising a variant Fc region for use in suppressing the activation of B cells, mast cells, dendritic cells, and/or basophils. In certain embodiments, the invention provides a polypeptide comprising a variant Fc region for use in a method of suppressing the activation of B
cells, mast cells, dendritic cells, and/or basophils in an individual comprising administering to the individual an effective amount of the polypeptide comprising a variant Fc region to suppress the activation of B cells, mast cells, dendritic cells, and/or basophils. In one embodiment, the "individual" is a human.

[0439] In a further aspect, the invention provides the use of a polypeptide comprising a variant Fc region in the manufacture or preparation of a medicament. In some apsects, the polypeptide is an antibody. In some aspsects, the polypeptide is an Fc fusion protein. In a further embodiment, the medicament is for suppressing the activation of B
cells, mast cells, dendritic cells, and/or basophils. In a further embodiment, the medicament is for use in a method of suppressing the activation of B cells, mast cells, dendritic cells, and/or basophils in an individual comprising administering to the in-dividual an effective amount of the medicament to suppress the activation of B
cells, mast cells, dendritic cells, and/or basophils. In one embodiment, the "individual" is a human.

[0440] In a further aspect, the invention provides a method for suppressing the activation of B cells, mast cells, dendritic cells, and/or basophils in an individual. In one em-bodiment, the method comprises administering to the individual an effective amount of a polypeptide comprising a variant Fc region to suppress the activation of B
cells, mast cells, dendritic cells, and/or basophils. In one embodiment, the "individual"
is a human.

[0441] In a further aspect, the invention provides pharmaceutical formulations comprising a polypeptide comprising a variant Fc region. In a further embodiment, the pharma-ceutical formulation is for suppressing the activation of B cells, mast cells, dendritic cells, and/or basophils.

[0442] Polypeptides comprising a variant Fc region of the present invention can suppress the activation of B cells, mast cells, dendritic cells, and/or basophils. Without wishing to be bound by theory, it is believed that this suppressed activation is the result of the selective binding of the provided variant Fc regions to Fc gamma RHb without ac-tivating activating Fc gamma R. As used herein, "B cell activation" includes pro-liferation, IgE production, IgM production, and IgA production. Without wishing to be bound by theory, it is believed that the suppressed activation of B cells is the result of the ability of the provided variant Fc regions on polypeptides comprising a variant Fc region of the present invention to crosslink Fc gamma Ruth with IgE to suppress IgE
production of B cells, with IgM to suppress IgM production of B cells, and with IgA to suppress IgA production. Other than the above, suppressive effects similar to those mentioned above are exhibited by directly or indirectly cross-linking Fc gamma Ruth with molecules that are expressed on B cells and comprise the ITAM domain inside the cell or interact with the ITAM domain such as BCR, CD19, and CD79b. As used herein, "activation of mast cells" includes proliferation, activation by IgE, and de-granulation. Without wishing to be bound by theory, it is believed that the suppressed activation of mast cells is the result of the ability of the provided variant Fc regions on polypeptides comprising a variant Fc region of the present invention to directly or in-directly cross-link Fc gamma RHb with IgE receptor molecules that are expressed on mast cells and comprise the ITAM domain or interact with the ITAM domain such as Fc epsilon RI, DAP12, and CD200R3. As used herein, "activation of basophils"
includes proliferation and degranulation of basophils. Without wishing to be bound by theory, it is believed that the suppressed activation of basophils is the result of the ability of the provided variant Fc regions on polypeptides comprising a variant Fc region of the present invention to directly or indirectly cross-link Fc gamma RIIb with molecules on the cell membrane, which comprise the ITAM domain inside the cell or interact with the ITAM domain. As used herein, "activation of dendritic cells"
includes proliferation and degranulation of dendritic cells. Without wishing to be bound by theory, it is believed that the suppressed activation of dendritic cells is the result of the ability of the provided variant Fc regions on polypeptides comprising a variant Fc region of the present invention to directly or indirectly cross-link Fc gamma RIIb with molecules on the cell membrane, which comprise the ITAM domain inside the cell or interact with the ITAM domain.

[0443] In further embodiments, the invention provides a polypeptide comprising a variant Fc region provided herein for use in treating an immunological inflammatory disease. In certain embodiments, the invention provides a polypeptide comprising a variant Fc region for use in a method of treating an immunological inflammatory disease in an in-dividual comprising administering to the individual an effective amount of the polypeptide comprising a variant Fc region to treat an immunological inflammatory disease. In one embodiment, the "individual" is a human.

[0444] In a further aspect, the invention provides the use of a polypeptide comprising a variant Fc region provided herein in the manufacture or preparation of a medicament.
In some apsects, the polypeptide is an antibody. In some aspsects, the polypeptide is an Fc fusion protein. In a further embodiment, the medicament is for treating an im-munological inflammatory disease. In a further embodiment, the medicament is for use in a method of treating an immunological inflammatory disease in an individual comprising administering to the individual an effective amount of the medicament to treat immunological inflammatory diseases. An "individual" according to any of the above embodiments may be a human.

[0445] In a further aspect, the invention provides a method for treating an immunological in-flammatory disease in an individual. In one embodiment, the method comprises admin-istering to the individual an effective amount of a polypeptide comprising a variant Fc region to treat immunological inflammatory diseases. In one embodiment, an "in-dividual" is a human.

[0446] In a further aspect, the invention provides pharmaceutical formulations comprising any of the polypeptides comprising a variant Fc region provided herein. In a further embodiment, the pharmaceutical formulation is for treating an immunological in-flammatory disease.

[0447] As described above, since polypeptides comprising a variant Fc region of the present invention can suppress activation of B cells, mast cells, dendritic cells and/or basophils, administration of the polypeptides comprising a variant Fc region of the present invention as a result can treat or prevent immunological inflammatory diseases.

[0448] In certain embodiments, the treated immunological inflammatory disease is selected from the group consisting of rheumatoid arthritis, autoimmune hepatitis, autoimmune thyroiditis, autoimmune blistering diseases, autoimmune adrenocortical disease, au-toimmune hemolytic anemia, autoimmune thrombocytopenic purpura, megalocytic anemia, autoimmune atrophic gastritis, autoimmune neutropenia, autoimmune orchitis, autoimmune encephalomyelitis, autoimmune receptor disease, autoimmune infertility, chronic active hepatitis, glomerulonephritis, interstitial pulmonary fibrosis, multiple sclerosis, Paget's disease, osteoporosis, multiple myeloma, uveitis, acute and chronic spondylitis, gouty arthritis, inflammatory bowel disease, adult respiratory distress syndrome (ARDS), psoriasis, Crohn's disease, Basedow's disease, juvenile diabetes, Addison's disease, myasthenia gravis, lens-induced uveitis, systemic lupus ery-thematosus, allergic rhinitis, allergic dermatitis, ulcerative colitis, hypersensitivity, muscle degeneration, cachexia, systemic scleroderma, localized scleroderma, Sjogren's syndrome, Behchet's disease, Reiter's syndrome, type I and type II diabetes, bone re-sorption disorder, graft-versus-host reaction, ischemia-reperfusion injury, atherosclerosis, brain trauma, cerebral malaria, sepsis, septic shock, toxic shock syndrome, fever, malgias due to staining, aplastic anemia, hemolytic anemia, id-iopathic thrombocytopenia, Goodpasture's syndrome, Guillain-Barre syndrome, Hashimoto's thyroiditis, pemphigus, IgA nephropathy, pollinosis, antiphospholipid antibody syndrome, polymyositis, Wegener's granulomatosis, arteritis nodosa, mixed connective tissue disease, fibromyalgia, asthma, atopic dermatitis, chronic atrophic gastritis, primary biliary cirrhosis, primary sclerosing cholangitis, autoimmune pan-creatitis, aortitis syndrome, rapidly progressive glomerulonephritis, megaloblastic anemia, idiopathic thrombocytopenic purpura, primary hypothyroidism, idiopathic Addison's disease, insulin-dependent diabetes mellitus, chronic discoid lupus ery-thematosus, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, epi-dermolysis bullosa acquisita, alopecia areata, vitiligo vulgaris, leukoderma acquisitum centrifugum of Sutton, Harada's disease, autoimmune optic neuropathy, idiopathic azoospermia, habitual abortion, hypoglycemia, chronic urticaria, ankylosing spondylitis, psoriatic arthritis, enteropathic arthritis, reactive arthritis, spondy-loarthropathy, enthesopathy, irritable bowel syndrome, chronic fatigue syndrome, der-matomyositis, inclusion body myositis, Schmidt's syndrome, Graves' disease, pernicious anemia, lupoid hepatitis, presenile dementia, Alzheimer's disease, de-myelinating disorder, amyotrophic lateral sclerosis, hypoparathyroidism, Dressler's syndrome, Eaton-Lambert syndrome, dermatitis herpetiformis, alopecia, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), sarcoidosis, rheumatic fever, erythema multiforme, Cushing's syndrome, transfusion reaction, Hansen's disease, Takayasu arteritis, polymyalgia rheumatica, temporal arteritis, giant cell arthritis, eczema, lym-phomatoid granulomatosis, Kawasaki disease, endocarditis, endomyocardial fibrosis, endophthalmitis, fetal erythroblastosis, eosinophilic fasciitis, Felty syndrome, Henoch-Schonlein purpura, transplant rejection, mumps, cardiomyopathy, purulent arthritis, familial Mediterranean fever, Muckle-Wells syndrome, and hyper-IgD syndrome.

[0449] In further embodiments, the invention provides a polypeptide comprising a variant Fc region for use in treating or preventing an autoimmune diseases which may be caused by or associated with the production of antibodies against autoantigens (autoantibodies). In certain embodiments, the invention provides a polypeptide comprising a variant Fc region for use in a method of treating or preventing an au-toimmune disease which may be caused by, or associated with, the production of an-tibodies against an autoantigen (autoantibody) in an individual comprising admin-istering to the individual an effective amount of the polypeptide comprising a variant Fc region to treat or prevent the autoimmune disease. An "individual"
according to any of the above embodiments is preferably a human.

[0450] In a further aspect, the invention provides the use of a polypeptide comprising a variant Fc region in the manufacture or preparation of a medicament. In some apsects, the polypeptide is an antibody. In some aspsects, the polypeptide is an Fc fusion protein. In a further embodiment, the medicament is for treating or preventing an au-toimmune disease which may be caused by or associated with production of antibodies against autoantigens (autoantibodies). In a further embodiment, the medicament is for use in a method of treating or preventing an autoimmune disease which may be caused by or associated with production of antibodies against autoantigens (autoantibodies) in an individual comprising administering to the individual an effective amount of the medicament to treat or prevent the autoimmune disease. An "individual"
according to any of the above embodiments may be a human.

[0451] In a further aspect, the invention provides a method for treating or preventing an au-toimmune disease which may be caused by or associated with production of antibodies against autoantigens (autoantibodies) in an individual. In one embodiment, the method comprises administering to the individual an effective amount of a polypeptide comprising a variant Fc region to treat or prevent the autoimmune disease. In one em-bodiment, an "individual" is a human.

[0452] In a further aspect, the invention provides pharmaceutical formulations comprising any of the polypeptides comprising a variant Fc region provided herein. In a further embodiment, the pharmaceutical formulation is for treating or preventing an au-toimmune disease which may be caused by or associated with production of antibodies against an autoantigen (autoantibodies).

[0453] The polypeptides comprising a variant Fc region of the present invention can treat or prevent an autoimmune disease which may be caused by or associated with production of antibodies against an autoantigen (autoantibodies) by suppressing production of those autoantibodies. Use of a fusion molecule of an antibody Fc portion with AchR
(an autoantigen of myasthenia gravis) has been reported to suppress proliferation of B
cells which express AchR-recognizing BCR, and induce apoptosis (J.
Neuroimmunol 227: 35-43 (2010)). Use of a fusion protein formed between a variant Fc region of the present invention and an antigen recognized by an autoantibody enables crosslinking of Fc gamma RHb with BCR for that autoantigen on a B cell, which causes sup-pression of proliferation of the B cells and/or causes induction of apoptosis of the B
cells.

[0454] In certain embodiments, an autoimmune disease that can be treated or or prevented is selected from the group consisting of Guillain-Barre syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, autoimmune pancreatitis, aortitis syndrome, Goodpasture's syndrome, rapidly progressive glomerulonephritis, megaloblastic anemia, autoimmune hemolytic anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Basedow's disease, Hashimoto's thyroiditis, primary hypothyroidism, idiopathic Addison's disease, insulin-dependent diabetes mellitus, chronic discoid lupus ery-thematosus, localized scleroderma, pemphigus, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, epidermolysis bullosa acquisita, alopecia areata, vitiligo vulgaris, leukoderma acquisitum centrifugum of Sutton, Harada's disease, autoimmune optic neuropathy, idiopathic azoospermia, habitual abortion, type II diabetes, hypo-glycemia, and chronic urticarial.

[0455] In further embodiments, the invention provides a polypeptide comprising a variant Fc region for use in treating a disease associated with a deficiency of a biologically essential protein. In certain embodiments, the invention provides a polypeptide comprising a variant Fc region for use in a method of treating a disease associated with a deficiency of a biologically essential protein in an individual comprising admin-istering to the individual an effective amount of the polypeptide comprising a variant Fc region to treat the disease. An "individual" according to any of the above em-bodiments is preferably a human.

[0456] In a further aspect, the invention provides the use of a polypeptide comprising a variant Fc region in the manufacture or preparation of a medicament. In some apsects, the polypeptide is an antibody. In some aspsects, the polypeptide is an Fc fusion protein. In a further embodiment, the medicament is for treating a disease associated with a deficiency of a biologically essential protein. In a further embodiment, the medicament is for use in a method of treating a disease associated with a deficiency of a biologically essential protein in an individual comprising administering to the in-dividual an effective amount of the medicament to treat the disease. An "individual"
according to any of the above embodiments may be a human.

[0457] In a further aspect, the invention provides a method of treating a disease associated with a deficiency of a biologically essential protein in an individual. In one em-bodiment, the method comprises administering to the individual an effective amount of a polypeptide comprising a variant Fc region to treat the disease. In one embodiment, an "individual" is a human.

[0458] In a further aspect, the invention provides pharmaceutical formulations comprising any of the polypeptides comprising a variant Fc region provided herein. In a further embodiment, the pharmaceutical formulation is for treating a disease associated with a deficiency of a biologically essential protein.

[0459] For diseases with deficiency of a biologically essential protein, therapeutic methods that administer and supplement the protein as a pharmaceutical agent are used.

However, since the patient lacks the protein from the beginning, the externally sup-plemented protein is recognized as a foreign substance and antibodies against that protein are produced. As a result, the protein becomes easily removed, and the effect as a pharmaceutical is reduced. Use of a fusion protein comprising such a protein and a variant Fc region of the present invention enables cros slinking between Fc gamma RHb and BCR that recognizes the protein on B cells, which causes suppression of antibody production against the protein.

[0460] In certain embodiments, the protein to be supplemented is selected from the group consisting of Factor VIII, Factor IX, TPO, EPO, alpha-iduronidase, iduronate sulfatase, A-type heparan N-sulfatase, B type alpha-N-acetylglucosaminidase, C type acetyl CoA: alpha-glucosaminidase acetyltransferase, D type N-acetylglucosamine 6-sulfatase, galactose 6-sulfatase, N-acetylgalactosamine 4-sulfatase, beta-glucuronidase, alpha-galactosidase, acidic alpha-galactosidase, and glucocere-brosidase. These proteins may be supplemented for diseases such as hemophilia, id-iopathic thrombocytopenic purpura, renal anemia, and lysosomal disease (mucopolysaccharidosis, Fabry's disease, Pompe disease, and Gaucher's disease), without being limited thereto.

[0461] In further embodiments, the invention provides a polypeptide comprising a variant Fc region for use in treating a viral infection. In certain embodiments, the invention provides a polypeptide comprising a variant Fc region for use in a method of treating a viral infection in an individual comprising administering to the individual an effective amount of the polypeptide comprising a variant Fc region to treat a viral infection. An "individual" according to any of the above embodiments is preferably a human.

[0462] In a further aspect, the invention provides the use of a polypeptide comprising a variant Fc region provided herein in the manufacture or preparation of a medicament.
In some apsects, the polypeptide is an antibody. In some aspsects, the polypeptide is an Fc fusion protein. In a further embodiment, the medicament is for treating a viral infection. In a further embodiment, the medicament is for use in a method of treating a viral infection in an individual comprising administering to the individual an effective amount of the medicament to treat a viral infection. An "individual" according to any of the above embodiments may be a human.

[0463] In a further aspect, the invention provides a method for treating a viral infection in an individual. In one embodiment, the method comprises administering to the individual an effective amount of a polypeptide comprising a variant Fc region to treat a viral infection. In one embodiment, an "individual" is a human.

[0464] In a further aspect, the invention provides pharmaceutical formulations comprising any of the polypeptides comprising a variant Fc region provided herein. In a further embodiment, the pharmaceutical formulation is for treating a viral infection.

[0465] Anti-virus antibodies that comprise a variant Fc region of the present invention can suppress antibody-dependent enhancement observed with conventional anti-virus an-tibodies. Antibody-dependent enhancement is a phenomenon where a virus bound to an antibody is phagocytosed via activating Fc gamma Rs so that infection of the virus to a cell is enhanced. Binding of anti-dengue-virus antibodies to Fc gamma RIIb has been reported to play an important role in suppressing antibody-dependent en-hancement (Proc. Natl. Acad. Sci. USA 108:12479-12484, (2011)). Crosslinking Fc gamma Ruth molecules by an immune complex of the anti-dengue-virus antibodies and dengue virus, inhibits Fc gamma R-mediated phagocytosis, resulting in the suppression of antibody-dependent enhancement. Examples of such viruses include dengue virus (DENV1, DENV2, DENV3, and DENV4) and HIV, but are not limited thereto.

[0466] In further embodiments, the invention provides a polypeptide comprising a variant Fc region for use in preventing or treating arteriosclerosis. In certain embodiments, the invention provides a polypeptide comprising a variant Fc region for use in a method of preventing or treating arteriosclerosis in an individual comprising administering to the individual an effective amount of the polypeptide comprising a variant Fc region to prevent or treat arteriosclerosis. In some apsects, the polypeptide is an antibody. In some aspsects, the polypeptide is an Fc fusion protein. An "individual"
according to any of the above embodiments is preferably a human.

[0467] In a further aspect, the invention provides the use of a polypeptide comprising a variant Fc region in the manufacture or preparation of a medicament. In some apsects, the polypeptide is an antibody. In some aspsects, the polypeptide is an Fc fusion protein. In a further embodiment, the medicament is for preventing or treating arte-riosclerosis. In a further embodiment, the medicament is for use in a method of preventing or treating arteriosclerosis in an individual comprising administering to the individual an effective amount of the medicament to prevent or treat arteriosclerosis.
An "individual" according to any of the above embodiments may be a human.

[0468] In a further aspect, the invention provides a method for preventing or treating arte-riosclerosis in an individual. In one embodiment, the method comprises administering to the individual an effective amount of a polypeptide comprising a variant Fc region to prevent or treat arteriosclerosis. In one embodiment, an "individual" is a human.

[0469] In a further aspect, the invention provides pharmaceutical formulations comprising any of the polypeptides comprising a variant Fc region provided herein. In some apsects, the polypeptide is an antibody. In some aspsects, the polypeptide is an Fc fusion protein. In a further embodiment, the pharmaceutical formulation is for preventing or treating arteriosclerosis.

[0470] Antibodies against oxidized LDL, i.e., a cause for arteriosclerosis, comprising a variant Fc region of the present invention can prevent Fc gamma RIIa-dependent adhesion of inflammatory cells. It has been reported that while anti-oxidized LDL an-tibodies inhibit the interaction between oxidized LDL and CD36, anti-oxidized LDL
antibodies bind to endothelial cells, and monocytes recognize their Fc portion in an Fc gamma RIIa-dependent or Fc gamma RI-dependent manner (Immunol. Lett.
108:52-61, (2007)). Using antibodies comprising a variant Fc region of the present invention may inhibit Fc gamma RIIa-dependent binding and suppress monocyte adhesion by Fc gamma Ruth-mediated inhibitory signals.

[0471] In further embodiments, the invention provides a polypeptide comprising a variant Fc region for use in preventing or treating cancer. In certain embodiments, the invention provides a polypeptide comprising a variant Fc region for use in a method of preventing or treating cancer in an individual comprising administering to the in-dividual an effective amount of the polypeptide comprising a variant Fc region to prevent or treat cancer. An "individual" according to any of the above embodiments is preferably a human.

[0472] In a further aspect, the invention provides the use of a polypeptide comprising a variant Fc region provided herein in the manufacture or preparation of a medicament.
In some apsects, the polypeptide is an antibody. In some aspsects, the polypeptide is an Fc fusion protein. In a further embodiment, the medicament is for preventing or treating cancer. In a further embodiment, the medicament is for use in a method of preventing or treating cancer in an individual comprising administering to the in-dividual an effective amount of the medicament to prevent or treat cancer. An "in-dividual" according to any of the above embodiments may be a human.

[0473] In a further aspect, the invention provides a method for preventing or treating cancer in an individual. In one embodiment, the method comprises administering to the in-dividual an effective amount of a polypeptide comprising a variant Fc region to prevent or treat cancer. In some apsects, the polypeptide is an antibody. In some aspsects, the polypeptide is an Fc fusion protein. In one embodiment, an "individual" is a human.

[0474] In a further aspect, the invention provides pharmaceutical formulations comprising any of the polypeptides comprising a variant Fc region provided herein. . In some apsects, the polypeptide is an antibody. In some aspsects, the polypeptide is an Fc fusion protein. In a further embodiment, the pharmaceutical formulation is for preventing or treating cancer.

[0475] As described above, it is known that enhancing the Fc gamma RIM
binding increases the agonistic activity of an agonist antibody, and enhances the antitumor effect of the antibody. Therefore, agonist antibodies comprising a variant Fc region of the present invention are useful for treatment or prevention of cancer. Specifically, a variant Fc region of the present invention enhances the agonistic activity of antibodies against, for example, receptors of the TNF receptor family such as, CD120a, CD120b, Lym-photoxin beta receptor, CD134, CD40, FAS, TNFRSF6B, CD27, CD30, CD137, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, RANK, Osteoprotegerin, TNFRSF12A, TNFRSF13B, TNFRSF13C, TNFRSF14, Nerve growth factor receptor, TNFRSF17, TNFRSF18, TNFRSF19, TNFRSF21, TNFRSF25, and Ectodysplasin A2 receptor, and can be used for treating or preventing cancer. Furthermore, agonistic activity is also enhanced for antibodies against other molecules, which exhibit agonistic activity through interaction with Fc gamma RIM. In addition, by incor-porating a variant Fc region of the present invention into an antibody against a receptor tyrosine kinase (RTK) such as Kit, which suppresses cell proliferation upon crosslinking with Fc gamma Ruth, the inhibitory effect of the antibody against cell pro-liferation may be enhanced.

[0476] In some embodiments, the invention provides a method for preventing or treating a cancer including but not limited to a member seclected from: small cell lung cancer, non-small cell lung cancer, pulmonary adenocarcinoma, and squamous cell carcinoma of the lung), large intestine cancer, rectal cancer, colon cancer, breast cancer, liver cancer, gastric cancer, pancreatic cancer, renal cancer, prostate cancer, ovarian cancer, thyroid cancer, cholangiocarcinoma, peritoneal cancer, mesothelioma, squamous cell carcinoma, cervical cancer, endometrial cancer, bladder cancer, esophageal cancer, head and neck cancer, nasopharyngeal cancer, salivary gland tumor, thymoma, skin cancer, basal cell tumor, malignant melanoma, anal cancer, penile cancer, testicular cancer, Wilms' tumor, acute myeloid leukemia (including acute myeloleukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, and acute monocytic leukemia), chronic myelogenous leukemia, acute lym-phoblastic leukemia, chronic lymphatic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma (Burkitt's lymphoma, chronic lymphocytic leukemia, mycosis fungoides, mantle cell lymphoma, follicular lymphoma, diffuse large-cell lymphoma, marginal zone lymphoma, pilocytic leukemia plasmacytoma, peripheral T-cell lymphoma, and adult T cell leukemia/lymphoma), Langerhans cell histiocytosis, multiple myeloma, myelodysplastic syndrome, brain tumor (including glioma, as-troglioma, glioblastoma, meningioma, and ependymoma), neuroblastoma, retinoblastoma, osteosarcoma, Kaposi's sarcoma, Ewing's sarcoma, angiosarcoma, and hemangiopericytoma.

[0477] Antibodies which have been modified to have enhanced binding activity to Fc gamma Rs including Fc gamma RHb by modification of at least one amino acid residue can promote antigen elimination from the plasma, as described or suggested in, for example, WO 2013/047752, WO 2013/125667, WO 2014/030728, or WO
2014/163101.

[0478] Without being bound by a particular theory, an antibody having an increased Fc gamma R-binding activity under a neutral pH condition is rapidly taken up into cells together with its antigen complexed with the antibody, and as a result, antigen elimination from plasma can be promoted when the antibody is administered in vivo.

[0479] Antibodies which have been modified to have an increased pI by modification of at least one amino acid residue that can be exposed on the antibody surface can be in-corporated more rapidly into cells or can promote antigen elimination from the plasma, as described or suggested in, for example, WO 2007/114319, WO 2009/041643, WO
2014/145159, or WO 2012/016227.

[0480] Without being bound by a particular theory, it is believed that the pH of biological fluids (for example, plasma) is in a neutral pH range. In biological fluids, the net positive charge of a p1-increased antibody is increased due to the increased pI, and as a result the antibody is more strongly attracted by physicochemical Coulomb interaction to the endothelial cell surface that has a net negative charge compared to an antibody not having an increased pI. That means, via such non-specific binding, uptake into cells of the antibody complexed with its antigen is enhanced, which results in en-hancement of antigen elimination from plasma. These phenomena are expected to occur commonly in vivo, regardless of cell type, tissue type, organ type, etc.

[0481] Herein, enhancement of antigen elimination from plasma means, for example, that the rate of antigen elimination from plasma is increased as compared to that when a polypeptide comprising a corresponding Fc region that has not been so modified has been administered. The increase of antigen elimination from plasma can be assessed, for example, by measuring the concentration of the antigen in plasma. Various methods for measuring antigen concentration in plasma are known in the art.

[0482] In further embodiments, the invention provides a polypeptide comprising a variant Fc region for use in promoting elimination of an antigen from plasma. In certain em-bodiments, the invention provides a polypeptide comprising a variant Fc region for use in a method of promoting elimination of an antigen from plasma in an individual comprising administering to the individual an effective amount of the polypeptide comprising a variant Fc region to promote elimination of an antigen from plasma. In those embodiments, it is preferable that the polypeptide further comprises an antigen-binding domain. An "individual" according to any of the above embodiments is preferably a human.

[0483] In a further aspect, the invention provides the use of a polypeptide comprising a variant Fc region in the manufacture or preparation of a medicament. In some apsects, the polypeptide is an antibody. In some aspsects, the polypeptide is an Fc fusion protein. In a further embodiment, the medicament is for promoting elimination of an antigen from plasma. In a further embodiment, the medicament is for use in a method of promoting elimination of an antigen from plasma in an individual comprising ad-ministering to the individual an effective amount of the medicament to promote elimination of an antigen from plasma. In those embodiments, it is preferable that the polypeptide further comprises an antigen-binding domain. An "individual"
according to any of the above embodiments may be a human.

[0484] In a further embodiment, the invention provides a method for promoting elimination of an antigen from plasma in an individual. In one embodiment, the method comprises administering to the individual an effective amount of a polypeptide comprising a variant Fc region to promote elimination of an antigen from plasma. In those em-bodiments, it is preferable that the polypeptide further comprises an antigen-binding domain. In one embodiment, an "individual" is a human.

[0485] In a further aspect, the invention provides pharmaceutical formulations comprising any of the polypeptides comprising a variant Fc region provided herein. In a further embodiment, the pharmaceutical formulation is for promoting elimination of an antigen from plasma. In that embodiment, it is preferable that the polypeptide further comprises an antigen-binding domain.

[0486] In one embodiment, when compared to before amino acid modification, an antibody having a variant Fc region of the present invention enhances antigen elimination from plasma, for example, by at least 1.1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2.0-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3.0-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4.0-fold, 4.25-fold, 4.5-fold, 4.75-fold, 5.0-fold, 5.5-fold, 6.0-fold, 6.5-fold, 7.0-fold, 7.5-fold, 8.0-fold, 8.5-fold, 9.0-fold, 9.5-fold, or 10.0-fold or more, when the antibody is ad-ministered in vivo.

[0487] In a further aspect, the invention provides methods for preparing a medicament or a pharmaceutical formulation, comprising mixing any of the polypeptides comprising a variant Fc region provided herein with a pharmaceutically acceptable carrier, e.g., for use in any of the above therapeutic methods. In one embodiment, the methods for preparing a medicament or a pharmaceutical formulation further comprise adding at least one additional therapeutic agent to the medicament or pharmaceutical for-mulation.

[0488] Antibodies of the invention can be used either alone or in combination with other agents in a therapy. For instance, an antibody of the invention may be co-administered with at least one additional therapeutic agent.

[0489] Likewise, polypeptides comprising a variant Fc region of the invention can be used either alone or in combination with other agents in a therapy. For instance, a polypeptide comprising a variant Fc region of the invention may be co-administered with at least one additional therapeutic agent.

[0490] Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the antibody or polypeptide comprising a variant Fc region of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent or agents. In one embodiment, the administration of the anti-myostatin antibody and the administration of an additional therapeutic agent occur within about one month, or within about one, two or three weeks, or within about one, two, three, four, five, or six days, of each other.

[0491] In another embodiment, the administration of the polypeptide comprising the variant Fc region and the administration of an additional therapeutic agent occur within about one month, or within about one, two or three weeks, or within about one, two, three, four, five, or six days, of each other.

[0492] An antibody or a polypeptide comprising a variant Fc region of the invention (and optionally any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, in-travenous, intraarterial, intraperitoneal, or subcutaneous administration.
Dosing can be by any suitable route, e.g., by injections, such as intravenous or subcutaneous in-jections, depending in part on whether the administration is brief or chronic.
Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

[0493] Antibodies or polypeptides comprising a variant Fc region of the invention can be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of ad-ministration, the scheduling of administration, and other factors known to medical practitioners. The antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.

[0494] For the prevention or treatment of disease, the appropriate dosage of an antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the type of polypeptide comprising a variant Fc region, the severity and course of the disease, whether the antibody or polypeptide comprising the variant Fc region, is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody or polypeptide comprising the variant Fc region, and the discretion of the attending physician. The antibody or polypeptide comprising a variant Fc region of the invention is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 micro g/kg to 15 mg/kg (e.g., 0.1mg/kg-10mg/kg) of antibody can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 micro g/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the antibody or polypeptide comprising the variant Fc region would be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g., every week or every three weeks (e.g., such that the patient receives from about two to about twenty, or e.g., about six doses of the antibody or polypeptide comprising the variant Fc region). An initial higher loading dose, followed by one or more lower doses may be administered.
The progress of this therapy is easily monitored by conventional techniques and assays.

[0495] It is understood that any of the above formulations or therapeutic methods may be carried out using an immunoconjugate of the invention in place of or in addition to an anti-myostatin antibody.

[0496] It is likewise understood that any of the above formulations or therapeutic methods may be carried out using an immunoconjugate of the invention in place of or in addition to a polypeptide comprising a variant Fc region provided herein.
H. Articles of Manufacture

[0497] In another aspect of the invention, an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the com-position comprises an antibody of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of man-ufacture may further comprise a second (or third) container comprising a pharma-ceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0498] It is understood that any of the above articles of manufacture may include an im-munoconjugate of the invention in place of or in addition to an anti-myostatin antibody.
Examples

[0499] The following are examples of methods and compositions of the invention. It is un-derstood that various other embodiments may be practiced, given the general de-scription provided above.
Example 1

[0500] Expression and purification of human. cynomolgus monkey. and mouse myostatin latent and mature form Human latent myostatin (also described herein as human myostatin latent form) (SEQ ID NO: 1) was expressed transiently using FreeStyle293-F cells (F5293-F
cells) (Thermo Fisher, Carlsbad, CA, USA). Conditioned media containing expressed human myostatin latent form was acidified to pH6.8 and diluted with 1/2 vol of milliQ water, followed by application to a Q-sepharose FF anion exchange column (GE
healthcare, Uppsala, Sweden). The flow-through fraction was adjusted to pH5.0 and applied to a SP-sepharose HP cation exchange column (GE healthcare, Uppsala, Sweden), and then eluted with a NaC1 gradient. Fractions containing the human myostatin latent form were collected and subsequently subjected to a Superdex 200 gel filtration column (GE
healthcare, Uppsala, Sweden) equilibrated with lx PBS. Fractions containing the human myostatin latent form were then pooled and stored at -80 degrees C.

[0501] Human mature myostatin (also described herein as human myostatin mature form) (SEQ ID NO: 2) was purified from the purified human myostatin latent form. The human myostatin latent form was acidified by addition of 0.1% trifluoroacetic acid (TFA) and applied to a Vydac 214TP C4 reverse phase column (Grace, Deerfield, IL, USA) and eluted with a TFA/CH3CN gradient. Fractions containing human mature myostatin were pooled, dried and stored at -80 degrees C. To reconstitute, human mature myostatin was dissolved in 4mM HC1.

[0502] Expression and purification of myostatin latent and mature form from cynomolgus monkey (cynomolgus or cyno) (SEQ ID NOs: 3 and 4, respectively) and mouse (SEQ

ID NOs: 5 and 6, respectively) were all performed exactly the same way as the human counterpart. The sequence homology of mature form among human, cyno, and mouse are 100% identical, therefore, any of mature myostatin regardless of species were used as mature myostatin in all the necessary experiments.
Example 2

[0503] Identification of anti-latent myostatin antibody Anti-latent myostatin antibodies were prepared, selected, and assayed as follows.

[0504] Twelve to sixteen week old NZW rabbits were immunized intradermally with mouse latent myostatin and/or human latent myostatin (50-100 micro g/dose/rabbit).
This dose was repeated 3-4 times over a one month period. One week after the final immu-nization, the spleen and blood from the immunized rabbit were collected.
Antigen-specific B-cells were stained with labelled antigen, sorted with FCM cell sorter (FACS
aria III, BD), and plated in 96-well plates at a one cell/well density together with 25,000 cells/well of EL4 cells (European Collection of Cell Cultures) and with rabbit T-cell conditioned medium diluted 20 times, and were cultured for 7-12 days.

cells were treated with mitomycin C (Sigma) for 2 hours and washed 3 times in advance. The rabbit T-cell conditioned medium was prepared by culturing rabbit thymocytes in RPMI-1640 containing Phytohemagglutinin-M (Roche), phorbol 12-myristate 13-acetate (Sigma) and 2% FBS. After cultivation, B-cell culture su-pernatants were collected for further analysis and pellets were cryopreserved.

[0505] An ELISA assay was used to test the specificity of antibodies in a B-cell culture su-pernatant. Streptavidin (GeneScript) was coated onto a 384-well MAXISorp (Nunc) at 50nM in PBS for 1 hour at room temperature. Plates were then blocked with Blocking One (Nacalai Tesque) diluted 5 times. Human or mouse latent myostatin was labelled with NHS-PEG4-Biotin (PIERCE) and was added to the blocked ELISA plates, incubated for 1 hour, and washed with Tris-buffered saline with 0.05% Tween-20 (TBS-T). B-cell culture supernatants were added to the ELISA plates, incubated for lhour, and washed with TBS-T. Binding was detected by goat anti-rabbit IgG-horseradish peroxidase (BETHYL) followed by the addition of ABTS (KPL).

[0506] A total of 17,818 B-cell lines were screened for binding specificity to mouse and/or human latent myostatin and 299 lines were selected and designated M5T0255-287, 630-632, 677-759, 910, 932-1048, 1050-1055, 1057-1066, 1068, 1070-1073, 1075-1110, 1113-1119. RNA was purified from corresponding cell pellets using Quick-RNA kits (ZYMO RESEARCH).

[0507] DNA encoding the variable regions of the heavy and light chain of each selected cell line was amplified by reverse transcription PCR and cloned into expression vectors with the heavy chain constant region Glm sequence (SEQ ID NO: 50 (the amino acid sequence is shown in SEQ ID NO: 7)) and with the light chain constant region kOMTC
or kOMC sequence (SEQ ID NO: 53 or 194 (the amino acid sequence (both are the same) is shown in SEQ ID NO: 8)), respectively. Recombinant antibodies were expressed transiently using the FreeStyle F5293-F cells and 293fectin (Life tech-nologies), according to the manufacturer's instructions. Culture supernatant or re-combinant antibodies were used for screening. Recombinant antibodies were purified with protein A (GE Healthcare) and eluted in D-PBS or His buffer (20mM
Histidine, 150mM NaC1, pH6.0). Size exclusion chromatography was further conducted to remove high molecular weight and/or low molecular weight component, if necessary.
Example 3

[0508]
Characterization of anti-latent myostatin antibody (HEK Blue Assay (BMP1 ac-tivation)) A reporter gene assay was used to assess the biological activity of active myostatin in vitro. HEK-B1ueTM TGF-beta cells (Invivogen) which express a Smad3/4-binding elements (SBE)-inducible SEAP reporter genes, allow the detection of bioactive myostatin by monitoring the activation of the activin type 1 and type 2 receptors.
Active myostatin stimulates the production of SEAP which is secreted into the cell su-pernatant. The quantity of SEAP secreted is then assessed using QUANTIBlueTm (Invivogen).

[0509] HEK-B1ueTM TGF-beta cells were maintained in DMEM medium (Gibco) sup-plemented with 10% fetal bovine serum, 50 micro g/mL streptomycin, 50 U/mL
penicillin, 100 micro g/mL NormocinTM, 30 micro g/mL of Blasticidin, 200 micro g/
mL of HygroGoldTM and 100 micro g/mL of ZeocinTM. During the functional assay, cells were changed to assay medium (DMEM with 0.1% bovine serum albumin, streptomycin, penicillin and NormocinTM) and seeded to a 96-well plate. Human, cynomolgus, or mouse latent myostatin was incubated with recombinant human (R&D Systems) and anti-latent antibody at 37degrees C overnight. The sample mixtures were transferred to cells. After 24-hour incubation, the cell supernatants were mixed with QUANTIBlueTm and the optical density at 620 nm was measured in a col-orimetric plate reader. As shown as Figure 1, mAb M5T1032-Glm (see Table 2a for amino acid and nucleotide sequences) prevented protease-mediated activation of human, cynomolgus, and mouse latent myostatin, as reflected by lowered concen-trations of secreted SEAP. M5T1032-Glm showed almost comparable inhibition activity against human, cynomolgus, and mouse latent myostatin.

t,..) =
Anti-latent myostatin antibodies and their DNA and amino acid sequences (shown as SEQ ID NOs) P

Variable region Constant region oe Heavy Light Heavy Light P W
Antibody name Abbreviation DNA PROTEIN DNA
PROTEIN DNA PROTEIN DNA PROTEIN
MS_MST1032Ha-G1m/MST1032La-k0MC MST1032-G1m 039 012 041 MS_MST1032Ha-F760/MST1032La-kOMC MST1032-F760 039 012 041 MS_M103205H000-SG1/M103202L000-SK1 MS1032L000-SG1 or Ab001 MS_M 103205 H000-F760/M 103202L000-SK1 MS1032L000-F760 040 MS_M103205H714-SG1/M103202L861-SK1 MS1032L001-SG1 043 032 046 MS_M103205H781-SG1/M103202L719-SK1 MS1032L002-SG1 044 033 047 P
MS_M103205H781-SG1/M103202L813-SK1 MS1032L003-SG1 044 033 048 037 052 009 054 010 .
L.
MS_M103205H707-SG1/M103202L802-SK1 MS1032L004-SG1 045 034 049 038 052 009 054 010 .
.
N, MS_M103205H714-F760/M103202L861-SK1 MS1032L001-F760 043 032 046 035 051 011 054 010 .
r., r., MS_M103205H781-F760/M103202L719-SK1 MS1032L002-F760 044 MS_M103205H781-F760/M103202L813-SK1 MS1032L003-F760 044 , MS_M103205H707-F760/M103202L802-SK1 MS1032L004-F760 045 034 049 038 051 011 054 010 .
, , IV
n ,-i =
c7, 7:-:--, oe --.1 .6.
oe --.1 t..) HVR amino acid sequences of anti-latent myostatin antibodies (shown as SEQ ID
NOs) Hyper Variable region (HVR) oe Antibody name Abbreviation H1 H2 H3 MS_MST1032Ha-G1m/MST1032La-kOMC MST1032-G1m 055 058 MS_MST1032Ha-F760/MST1032La-kOMC MST1032-F760 055 058 MS M103205H000-SG1/M103202L000-SK1 MS1032L000-SG1 or Ab001 055 058 _ MS_M 103205H 000-F760/M 103202L000-SK1 MS1032L000-F760 055 058 MS_M103205H714-SG1/M103202L861-SK1 MS1032L001-SG1 057 058 MS_M103205H781-SG1/M103202L719-SK1 MS1032L002-SG1 057 060 MS_M103205H781-SG1/M103202L813-SK1 MS1032L003-SG1 057 060 063 068 070 074 .
MS_M103205H707-SG1/M103202L802-SK1 MS1032L004-SG1 056 058 r., r., MS_M 103205H 714-F760/M 103202L861-SK1 MS1032L001-F760 057 058 MS_M 103205H 781-F760/M 103202L719-SK1 MS1032L002-F760 057 060 MS_M 103205H 781-F760/M 103202L813-SK1 MS1032L003-F760 057 060 063 068 070 074 .
, , , MS_M 103205H 707-F760/M 103202L802-SK1 MS1032L004-F760 056 058 Iv n ,-i k....-) c7, -a oe .6.
oe Example 4 [05101 Characterization of anti-latent myostatin antibody (HEK Blue Assay (Spontaneous activation)) Human, cynomolgus, or mouse latent myostatin was incubated with the anti-latent myostatin antibody at 37 degrees C overnight and the sample mixtures were transferred to HEKBlueTM TGF-beta cells. After 24-hour incubation, cell supernatant was mixed with QUANTIBlueTm and the optical density at 620 nm was measured in a colorimetric plate reader. As shown as Figure 2, the liberation of active myostatin from its latent form was detected after incubation at 37 degrees C. In the presence of mAb M5T1032-Glm, myostatin activation was inhibited and thus a lower SEAP level was detected in the cell supernatant. M5T1032-Glm inhibited spontaneous activation of latent myostatin, and showed almost comparable inhibition activity against human, cynomolgus, and mouse latent myostatin.
Example 5 [05111 Characterization of anti-latent myostatin antibody (ELISA) Uncoated ELISA plates (NUNC-IMMUNO plate MAXISORP surface, Nalge Nunc International) were coated with 20 micro L of 50nM streptavidin (GenScript) for 1 hour at room temperature. Plates were then washed with PBST three times and blocked with 50 micro L 20% Blocking One (Nacalai Tesque) overnight. On the next day, each well of each plate was incubated with biotinylated mature myostatin, human or mouse biotinylated latent myostatin, or biotinylated recombinant mouse myostatin propeptide-Fc chimera protein (R&D Systems) at 4nM/wel1/20 micro L for 2 hours. After washing, 20 micro L of antibody sample was added to the wells and the plates were left for 1 hour. The plates were washed and 20 micro L of anti-human IgG-horseradish peroxidase (HRP) (Abcam) diluted in HEPES-buffered saline was added, and the plates were left for another hour. The plates were then washed again, and then micro L ABTS (KPL) was added to each well, and the plates were incubated for 1 hour. The signal was detected at 405 nm in a colorimetric plate reader. The results of the binding experiment are shown in Figure 3. M5T1032-Glm bound to latent myostatin (i.e., the non-covalent complex of mature myostatin and propeptides) and propeptide, but did not bind to mature myostatin. These results show that M5T1032-Glm specifically binds to the myostatin propeptide (e.g., the prppeptide portion), but not to active myostatin (e.g., mature region).
Example 6 [05121 Characterization of anti-latent myostatin antibody (Western Blot) Mouse latent myostatin was incubated with recombinant human BMP1 (R&D
Systems), with or without M5T1032-Glm at 37 degrees C overnight. The samples were then mixed with 4x reducing SDS-PAGE sample buffer (Wako) and heated at degrees C for 5 minutes and then loaded for SDS gel electrophoresis. Proteins were transferred to membrane by Trans-Blot (registered trademark) TurboTm Transfer System (Bio-rad). Myostatin propeptide was detected using a sheep anti-mouse propeptide antibody (R&D Systems), which was then detected by anti-sheep IgG-HRP
(Santa Cruz). The membrane was incubated with ECL substrate, and imaged using an ImageQuant LAS 4000 (GE Healthcare). As shown in Figure 4, propeptide cleavage by BMP1 was inhibited by M5T1032-Glm.
Example 7 [0513] Characterization of anti-latent myostatin antibody (BIACORE
(registered trademark)) The kinetic parameters of anti-latent myostatin antibodies against human, cynomolgus monkey (cyno), and mouse latent myostatin were assessed at 37 degrees C
at pH7.4 using a BIACORE (registered trademark) T200 instrument (GE
Healthcare).
ProA/G (Pierce) was immobilized onto all flow cells of a CM4 chip using amine coupling kit (GE Healthcare). Anti-latent myostatin antibody and analytes were prepared in ACES pH7.4 (20 mM ACES, 150 mM NaC1, 1.2 mM CaC12, 0.05% Tween 20, 0.005% NaN3). Antibody was captured onto the sensor surface by ProA/G.
Antibody capture levels were typically 150 to 220 resonance units (RU). Then human, cyno, or mouse latent myostatin was injected at 3.125 to 50 nM prepared by two-fold serial dilution, followed by dissociation. The sensor surface was regenerated with 25 mM NaOH. Kinetic parameters were determined by processing and fitting the data to a 1:1 binding model using BIACORE (registered trademark) T200 Evaluation software, version 2.0 (GE Healthcare). The sensorgrams are shown in Figure 5.
Association rate (ka), dissociation rate (kd), and binding affinity (KD) are listed in Table 3.
The kinetic parameters of M5T1032-Glm toward human, cyno, and mouse latent myostatin were comparable.
[Table 3]
ka, kd, and KD of anti-latent myostatin antibody MST1032-Glm ka (m-ls-1) kd (s-1) KD (M) Human latent myostatin 1.13E+06 8.65E-05 7.66E-11 Mouse latent myostatin 1.41E+06 8.16E-05 5.79E-11 Cyno latent myostatin 1.09E+06 7.79E-05 7.17E-11 Example 8 [0514] In vivo efficacy of anti-latent myostatin antibody on muscle mass and fat mass The in vivo efficacy of mAb M5T1032-Glm was evaluated in mice. Anti-mature myostatin antibody 41C1E4 (as described in US Patent No. 7,632,499) was used as positive control in this study. To avoid potential immunomodulation due to a mouse anti-human antibody response, in vivo studies were performed in immune-deficient Severe Combined Immunodeficient (SCID) mice. Five-week-old SCID (C.B-17 SCID) mice (Charles River Laboratories Japan, Inc.(Kanagawa, JAPAN)) were treated with monoclonal antibodies at various doses or vehicle (PBS) given intravenously once per week for two weeks. On day 0, 7 and 14, full body lean mass and fat mass of the mice were assessed by nuclear magnetic resonance (NMR) (the minispec LF-50, Bruker Bio Spin, (Kanagawa, JAPAN)). The animals were euthanized on day 14, and the gas-trocnemius and quadriceps muscles were dissected and weighed.
[0515] Statistical significance was determined by ANOVA, a Student's t-test and a Dunnett's test with JMP 9 software (SAS, Inc.). A p value of less than 0.05 was considered sig-nificant.
[0516] The results of this experiment are shown in Figures 6A-C. Both antibodies (M5T1032-Glm and 41C1E4) increased lean body mass dose-dependently, and M5T1032-Glm, when administered at 40 mg/kg, significantly decreased fat mass compared with the vehicle (PBS) group on day 14.
[0517] After two-week treatment, the antibodies administrated at 10 mg/kg and 40 mg/kg resulted in significant increment of quadriceps and gastrocnemius wet weight relative to the vehicle group.
Example 9 [0518] Comparison of in vivo efficacy of various myostatin related antibodies All experimental settings for the comparison were the same as in Example 8.
Various myostatin related antibodies, 41C1E4, REGN, OGD, and MY0-029 were tested in five-week-old SCID mice for two weeks. REGN, OGD, and MY0-029 are anti-mature myostatin antibodies described in WO 2011/150008 as H4H1657N2, WO
2013/186719 as OGD1Ø0., and WO 2004/037861 as MY0-029, respectively. These antibodies were administrated intravenously once per week. Lean body mass and fat mass were examined by full-body NMR scanning on day 0 and 14. Grip strength was measured with a grip strength test meter (e.g., GPM-100B, MELQUEST Ltd., (Toyama, JAPAN)) on day 14. As shown in the results presented in Figures 7A-C, the lean body mass was increased by these antibodies except for MY0-029 (P>0.05).
Grip strength of the M5T1032-Glm-administered group was significantly increased relative to the vehicle (PBS) group, at 10 mg/kg (P<0.05). 41C1E4 and REGN also sig-nificantly increased grip strength compared with the vehicle (PBS). Full body fat mass tended to be reduced by 10 mg/kg of M5T1032-Glm. Fat mass reduction efficacy of M5T1032-Glm was stronger than that of other anti-mature myostatin antibodies.

Example 10 [0519] Humanization of anti-latent myostatin antibody Amino acid residues of an antibody variable region are numbered according to Kabat (Kabat et al., Sequence of proteins of immunological interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)).
[0520] Variable regions of the heavy and light chains for humanized MST1032 antibodies were designed. Some of the humanized MST1032 antibodies contain back mutation in framework region. The polynucleotides of the designed heavy and light chain variable regions were synthesised by GenScript Inc., and were cloned into expression vectors containing the heavy chain constant region SG1 sequence (SEQ ID NO: 52 (the amino acid sequence is shown in SEQ ID NO: 9)) and the light chain constant region sequence (SEQ ID NO: 54 (the amino acid sequence is shown in SEQ ID NO: 10)), re-spectively. Humanized antibodies were transiently expressed in F5293-F cells, and HEK Blue Assay and BIACORE (registered trademark) analysis were carried out as described above. As shown in Figure 8 and Table 4, and compared to Figures 1 and 2, humanized antibody (M51032L000-SG1) showed comparable inhibition activity and affinity to chimeric antibody (MST1032-G1m).
[Table 4]
Kinetic parameters of humanized anti-latent myostatin antibody ka (N/1-1-s-1) kd (s-1) KD (M) MST1032-G1m 1.13E+06 8.65E-05 7.66E-11 MS1032L000-SG1 1.27E+06 9.52E-05 7.50E-11 Example 11 [0521] Generation of pH-dependent anti-latent myostatin antibody To generate pH-dependent anti-latent myostatin antibodies, histidine scanning mu-tagenesis was conducted for all CDRs of mAb M51032L000-SG1. Each amino acid in the CDRs was individually mutated to histidine using In-Fusion HD Cloning Kit (Clontech Inc. or Takara Bio company) according to the manufacturer's instructions.
After confirming through sequencing that each variant was mutated correctly, variants were transiently expressed and purified by the method described above. All histidine-substituted variants were evaluated by a modified BIACORE (registered trademark) assay as compared to that described above. Briefly, an additional dissociation phase at pH5.8 was integrated into the BIACORE (registered trademark) assay immediately after the dissociation phase at pH7.4. This is to assess the pH-dependent dissociation between antibody (Ab) and antigen (Ag) from the complexes formed at pH7.4 as opposed to the corresponding dissociation at pH5.8. The dissociation rate at pH5.8 buffer was determined by processing and fitting data using the Scrubber 2.0 (BioLogic Software) curve fitting software.
[05221 As shown in Figure 9, the parental antibody (Ab001) showed no reduction in binding response at pH5.8 compared to the pH7.4 dissociation phase. Several of the single-histidine substitutions resulted in a moderate to strong reduction in binding response at pH5.8 compared to the pH7.4 dissociation phase. The dissociation rate at pH5.8 for each of the single-histidine substitution variants are shown in Table 5. As shown in Table 5, Ab002 showed the fastest Ab/Ag complex dissociation rate at pH5.8, more than 200 fold faster than parental antibody (Ab001). Antibodies with a combination of these mutations in the CDRs were then generated. The CDR sequences of the an-tibodies containing these various mutations are shown in Table 6.
[Table 51 pH5.8 dissociation rate for single histidine substituted anti-latent myostatin antibodies Name of variable region Abbreviation kd (1/s) Heavy chain Light chain M103205H000 M103202L000 Ab001 2.92E-05 M103205H001 M103202L000 Ab002 6.09E-03 M103205H009 M103202L000 Ab003 8.80E-04 M103205H026 M103202L000 Ab004 1.86E-04 M103205H028 M103202L000 Ab005 7.63E-05 M103205H000 M103202L008 Ab006 8.58E-05 M103205H028 M103202L008 Ab007 4.15E-04 [Table 6]

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1 & 22i2222212 22 ,2 212 = :1 5ii' Example 12 [0523] Affinity improvement of pH-dependent anti-latent myostatin antibody To identify mutations which improve affinity at pH7.4 and/or in vitro inhibition activity, more than 500 variants were generated for heavy and light chain respectively, using at least one variant generated in Example 11 as a template. These variants had each amino acid in the CDRs substituted with 18 other amino acids, excluding the original amino acid and Cysteine. The binding ability of variants to human latent myostatin was assessed at 37 degrees C under pH7.4 using BIACORE (registered trademark) 4000 instrument (GE Healthcare). Culture supernatant containing variants expressed in FS293-F cells was prepared with ACES pH7.4 buffer containing 10mg/m1 BSA and 0.1mg/m1 carboxymethyl dextran (CMD). Each antibody was captured onto the flow cells until the capture level reached around 200 RU. Then 25 nM human latent myostatin was injected over the flow cells. An additional dissociation phase at pH5.8 was integrated into the BIACORE (registered trademark) assay immediately after the dissociation phase at pH7.4. This additional dissociation phase assesses the pH-dependent dissociation between antibody (Ab) and antigen (Ag) from the complexes formed at pH7.4. The flow cell surface was regenerated with 25 mM NaOH.
Kinetic parameters were determined using BIACORE (registered trademark) 4000 Evaluation software, version 2.0 (GE Healthcare). Analysis of additional dissociation at pH5.8 was performed using Scrubber2 (BioLogic Software).
[0524] Variants with improved affinity at pH7.4 and/or in vitro inhibition activity were selected, and combined with mutations improving pH dependency identified in Example 11. After combination of these mutations, four variants (MS1032L001, 02, 03, and 04) were selected, and transiently expressed with SG1 or F760 (SEQ ID
NO:
11 and 51) for BIACORE (registered trademark) binding kinetic analysis, in vitro and/
or in vivo assays. Both SG1 and F760 are human heavy chain constant regions.
The binding affinities of SG1 against human Fc gamma Rs are comparable to that for the natural IgG1 constant region, but that of F760 is abolished by Fc modification (also described herein as silent Fc). Amino acid and nucleotide sequences of the four anti-myostatin variants are shown in Table 2a.
Example 13 [0525] Characterization of pH-dependent MS1032 variants (HEK Blue Assay (BMP1 and Spontaneous activation)) All experimental settings were the same in Examples 3 and 4. As shown in Figure 10, all variants showed comparable inhibition activity against human latent myostatin to M51032L000-SG1.
Example 14 [0526]
Characterization of pH-dependent MS1032 variants (BIACORE (registered trademark)) All experimental settings were the same as in Example 7, except that measurements were also performed in ACES pH5.8 buffer in addition to ACES pH7.4 condition.
Some antibodies were only measured against human latent myostatin. Antibody capture level was aimed at 185 RU and 18.5 RU, for avidity and affinity assay, re-spectively. Kinetic parameters were determined using 1:1 Binding fitting using BIACORE (registered trademark) T200 Evaluation software, version 2.0 (GE
Healthcare). The sensorgrams of all antibodies against human latent myostatin with avidity condition are shown in Figure 11, and ka, kd, and KD calculated from different conditions are listed in Tables 7-10. Under the avidity condition, all antibodies except MS1032L000-SG I showed a faster dissociation rate under acidic pH than neutral pH.
Under the affinity condition, all antibodies except M51032L000-SG I showed a weak interaction with latent myostatin under acidic pH and therefore, kinetic parameters were not determined.
[Table 7]
ka, kd, and KD of avidity condition assay under neutral pH
Human latent myostatin Mouse latent myostatin Cyno latent myostatin 102 kd (s-1) KO (M) ka (Res') kd (54) KD (M) ka kd (s-i) KB (M) MS10321_000-SG1 1.27E4-06 9.52E-05 7.50E-11 1.52E+06 7,18E-05 4.71E41 1.21E+06 8.08E-05 6,66E-11 MS1032L001-SG1 1.27E+06 2.16E-04 1.70E-10 1.45E+06 2.17E-04 1.49E-10 1.19E+06 1.97E-04 1.65E-10 MS10321002-SG1 836E1-05 2.49E-04 2.98E-10 N/T N/T N/T N/T N/T
N/T
MS1032L003-5G1 9.81E+05 1.92E-04 1.96E-10 N/T N/T N/T N/T N/T
N/T
M510321004-SG1 1.33E+06 2.17E-04 1.63E-10 N/T NIT N/T NIT N/T
NIT
NIT: not tested [Table 8]
ka, kd, and KD of avidity condition assay under acidic pH
Homan latent myostatin Mouse latent myostatin Cyno latent rnyostatIr ka kd (s) KD (M) ka (M is 1) kd (s4) KD (M) ka (Ms) kd (s KD (M) MS10321_000-561 1,60E+06 1.28E-04 7.97E-11 232E+06 2,02E-05 8.70E-12 1.79E+06 1.58E-05 8.83E-12 MS1032L001-SG1 1.22E+06 2.42E-02 1.99E-08 1.02E+06 6.25E-03 6.15E-09 7.64E+05 6.80E-03 &90E-09 A/610321..002 -SG/ 1.70E+06 4.14E-02 2.43E-08 N/T N/T N/T NIT
N/T N/T
MS103a003-SG1 5.43E+05 5.24E-03 9.66E-09 N/T N/T N/T N/T N/T
N/T
M51032L004-5G1 7.81E+05 7.13E-03 9.13E-09 N/T N/T N/T NIT N/T
N/T
N/T: not tested [Table 9]
ka, kd, and KD of affinity condition assay under neutral pH
Human latent myostatin Mouse latent myostatin Cyno latent myostatin ka (M1s-1) kd (S-1) KO (M) ka (M-1s-1) kd (s-1) KO (M) ka (M-1s-1) kd (s-I) KO (M) M51032L000-SG1 1,07E+06 2.45E04 2.30E-10 1.31E+06 1.60E-04 1.22E-10 1,02E+06 2.13E-04 109E-10 MS1032L001-SG1 1.64E+06 1.29E-03 7.88E-3.0 1.50E+06 1.07E-03 7.13E-10 1.15E+06 1.10E-03 9.54E-10 MS1032L002-SG1 1.09E+06 1.79E-03 1.64E-09 N/T N/T N/T N/T N/T
N/T
MS1032L003-SG1 1.08E+06 1.17E-03 1.08E-09 N/T N/T N/T N/T N/T
N/T
14.451032L004-5G1 1.54E+06 1.25E-03 8.12E-10 N/T N/T N/T N/T
N/T N/T
N/T: not tested [Table 10]
ka, kd, and KD of affinity condition assay under acidic pH
Human latent myostatin Mouse latent myostattn Cyno latent myostatin ko (Nr1s-1) kd (s-1) KD (M) ka (M4s4) kd (s4) KD ;M) ko (M40 kd (i1) KD (M) ivIS10321.000-SG1 1.09E+06 2.35E-04 2.15E-10 1.62E+06 708E05 4.38E-11 1.41E+06 1.96E-04 1.39E-10 MS10321_001-561 n.d. n.d. n.d. n.d. n.d. n.d. n.d.
n.d. n.d.
MS10321_002-561 n.d. n.d. n.d. N/T N/T N/T N/T N/T
NTT
MS10321003-561 n.d. n.d. n.d. N/T N/T N/T N/T N/T
NIT
N1510321.004-561 n.d. n.d. n.d. N/T N/T N/T N/T NIT
N/T
n.d.: not determined, N/T: not tested Example 15 [0527] Comparison of plasma total myostatin concentration between antibodies with Fc gamma R binding and an abolished Fc gamma R binding in mice In vivo test using C.B-17 SCID mice The accumulation of endogenous myostatin was assessed in vivo upon administration of anti-latent myostatin antibody in C.B-17 SCID mice (In Vivos, Singapore).
An anti-latent myostatin antibody (3mg/m1) was administered at a single dose of 10 ml/kg into the caudal vein. Blood was collected 5 minutes, 7 hours, 1 day, 2 days, 3 days, 7 days, 14 days, 21 days, and 28 days after administration. The collected blood was cen-trifuged immediately at 14,000 rpm in 4 degrees C for 10 minutes to separate the plasma. The separated plasma was stored at or below -80 degrees C until measurement.
The anti-latent myostatin antibodies used were M51032L000-SG1 and MS1032L000-F760.
Measurement of total myostatin concentration in plasma by electrochemilumi-nescence (ECL) [0528] The concentration of total myostatin in mouse plasma was measured by ECL. Anti-mature myostatin-immobilized plates were prepared by dispensing anti-mature myostatin antibody RK35 (as described in WO 2009/058346) onto a MULTI-ARRAY
96-well plate (Meso Scale Discovery) and incubated overnight at 4 degrees C.
Mature myostatin calibration curve samples and mouse plasma samples diluted 40-fold or more were prepared. The samples were mixed in an acidic solution (0.2 M
Glycine-HC1, pH2.5) to dissociate mature myostatin from its binding protein (such as propeptide). Subsequently, the samples were added onto an anti-mature myostatin-immobilized plate, and allowed to bind for 1 hour at room temperature before washing.
Next, SULFO TAG labelled anti-mature myostatin antibody RK22 (as described in WO 2009/058346) was added and the plate was incubated for 1 hour at room tem-perature before washing. Read Buffer T (x4) (Meso Scale Discovery) was immediately added to the plate and signal was detected by SECTOR Imager 2400 (Meso Scale Discovery). The mature myostatin concentration was calculated based on the response of the calibration curve using the analytical software SOFTmax PRO (Molecular Devices). The time course of total myostatin concentration in plasma after intravenous administration of anti-latent myostatin antibody measured by this method is shown in Figure 12.
Effect of Fc gamma R binding on myostatin accumulation in vivo [0529] After administration of antibody MS1032L000-F760, the plasma total myostatin concentration at day 28 accumulated 248 fold compared to plasma total myostatin con-centration at 5 minutes. In contrast, after administration of M51032L000-SG1, plasma total myostatin concentration at day 28 accumulated 37 fold compared to plasma total myostatin concentration at 5 minutes. An approximately 7 fold difference of plasma total myostatin concentration at day28 was observed between M51032L000-F760 (silent Fc) and M51032L000-SG1 due to Fc gamma R binding. In human soluble IL-6R (hsIL-6R), no significant difference in plasma hsIL-6R concentration was observed between anti-hsIL-6R antibody-F760 (silent Fc) and -SG1 as described in WO
2013/125667. Since hsIL-6R is a monomeric antigen, antibody-hsIL-6R complex contains only 1 Fc. Therefore, the result in WO 2013/125667 suggested that an antibody-antigen complex with 1 Fc has no significant binding to Fc gamma R in vivo to accelerate the uptake of immune complex by cells. On the other hand, with a multimeric antigen (such as myostatin), antibody can make a large immune complex and the antibody-antigen complex contains more than 2 Fc. Therefore, a significant difference in plasma antigen concentration can be observed between F760 and due to strong avidity binding against Fc gamma R. The result suggests that can form a large immune complex which contains more than 2 antibodies with myostatin.
Example 16 [0530] Comparison of plasma total myostatin concentration between non-pH
dependent anti-latent myostatin antibody and pH-dependent anti-latent myostatin antibody in mice In vivo test using C.B-17 SCID mice The in vivo accumulation of endogenous mouse myostatin was assessed after admin-istering anti-latent myostatin antibody in C.B-17 SCID mice (In Vivos, Singapore) as described in Example 15. The anti-latent myostatin antibodies used were MS1032L001-SG1 and MS1032L001-F760.
Measurement of total myostatin concentration in plasma by ECL
[0531] The concentration of total myostatin in mouse plasma was measured by ECL as described in Example 15. The time course of plasma total myostatin concentration after intravenous administration of anti-latent myostatin antibody as measured by this method is shown in Figure 13.

Effect of pH-dependent myostatin binding on myostatin accumulation in vivo [0532] The pH-dependent anti-latent myostatin antibodies (MS1032L001-SG1 and MS1032L001-F760) were tested in vivo and comparison of plasma total myostatin concentration was made. The total myostatin concentration measurement results after administration of M51032L000-SG1 and M51032L000-F760 described in Example 15 are also shown in Figure 13. M51032L000 is non pH-dependent antibody and M51032L001 is pH-dependent antibody. As shown in Figure 13, total myostatin con-centration after administration of M51032L001-F760 was reduced compared to MS1032L000-F760 due to pH-dependent binding. Moreover, total myostatin con-centration after administration of MS1032L001-SG1 was dramatically reduced compared to that of MS1032L000-SG1 due to pH-dependent binding and increased cellular uptake by Fc gamma R binding. It is expected that MS1032L001-SG1 has a superior property of enhancing myostatin clearance from plasma as a "sweeping antibody".
Example 17 [0533] In vivo efficacy of pH-dependent anti-latent myostatin antibody All experimental settings were the same as in Example 8. As shown in Figure 14, when administrated intravenously to SCID mice at doses of 0.2, 1, and 5 mg/kg, MS1032L000-SG1 (non-sweeping antibody) and MS1032L001-SG1 (sweeping antibody) increased skeletal muscle mass dose-dependently after 2 weeks.
M51032L001-SG1 significantly increased quadriceps wet weight and grip strength at 1 and 5 mg/kg, and significantly increased lean body mass and gastrocnemius wet weight at 5 mg/kg relative to the vehicle group (PBS). M51032L001-SG1 also sig-nificantly decreased fat mass at 1 and 5 mg/kg relative to the vehicle group (PBS).
M51032L000-SG1 showed no increase in lean body mass at 0.2 mg/kg. On the other hand, M51032L001-SG1 drastically increased lean body mass, quadriceps wet weight, gastrocnemius wet weight and grip strength at 0.2 mg/kg. Hence, a pH-dependent binding antibody shows greater muscle mass growth and muscle power improvement relative to a non pH-dependent binding antibody.
Example 18 [0534] Expression and purification of human and mouse latent GDF11 Human GDF11 with Flag-tag on N-terminus (also described herein as Flag-hGDF11, human latent GDF11, hGDF11, or human GDF11, SEQ ID NO: 85) were expressed transiently using FreeStyle293-F cells (Thermo Fisher). Conditioned media expressing Flag-hGDF11 was applied to a column packed with anti-Flag M2 affinity resin (Sigma) and eluted with Flag peptide (Sigma). Fractions containing Flag-hGDF11 were collected and subsequently subjected to a Superdex 200 gel filtration column (GE

healthcare) equilibrated with lx PBS. Fractions containing the Flag-hGDF11 were then pooled and stored at -80 degrees C.
Example 19 [0535] Characterization of anti-latent myostatin antibody (ELISA) Uncoated ELISA plates (NUNC-IMMUNO plate MAXISORP surface, Nalge Nunc International) were coated with 20 micro L of 50nM streptavidin (GenScript) for 2 hour at room temperature. Plates were then washed with PBST three times and blocked with 50 micro L 20% Blocking One (Nacalai Tesque) for overnight. On the next day, each well of the plates was incubated with biotinylated human latent myostatin at 2nM/wel1/20 micro L or biotinylated human latent GDF11 at 20nM/wel1/20 micro L

for 2 hours. After washing, 20 micro L of antibody sample was added to the wells and the plates were left for 1 hour. The plates were washed and 20 micro L of anti-human IgG-horseradish peroxidase (HRP) (Abcam) diluted in HEPES-buffered saline was added, and the plates were left for another hour. The wells were washed again, and then 50 micro L ABTS (KPL) was added to each well, and the plates were incubated for 1 hour. The signal was detected at 405 nm in a colorimetric plate reader.
The results of the binding experiment are shown in Figure 15. M5T1032-Glm bound to latent myostatin (i.e., non-covalent complex of mature myostatin and propeptides), but didn't bind to hGDF11. These results show that M5T1032-Glm specifically binds to myostatin, not to hGDF11.
Example 20 [0536] Characterization of anti-latent myostatin antibody (HEK Blue Assay (BMP1 and spontaneous activation)) A reporter gene assay was used to assess the biological activity of active GDF11 in vitro. HEK-B1ueTM TGF-beta cells (Invivogen) that express a Smad3/4-binding element (SBE)-inducible SEAP reporter gene, allow the detection of bioactive by monitoring the activation of the activin type 1 and type 2 receptors.
Active GDF11 stimulates the production and secretion of SEAP into the cell supernatant. The quantity of SEAP secreted is then assessed using QUANTIBlueTm (Invivogen).
[0537] HEK-B1ueTM TGF-beta cells were maintained in DMEM medium (Gibco) sup-plemented with 10% fetal bovine serum, 50 micro g/mL streptomycin, 50U/mL
penicillin, 100 micro g/mL NormocinTM, 30 micro g/mL of Blasticidin, 200 micro g/
mL of HygroGoldTM and 100 micro g/mL of ZeocinTM. During the functional assay, cells were changed to assay medium (DMEM with 0.1% bovine serum albumin, streptomycin, penicillin and NormocinTM) and seeded to a 96-well plate. Human GDF11 was incubated with or without recombinant human BMP1 (Calbiochem) and anti-latent antibody (M5T1032-G1m) at 37 degrees C overnight. The sample mixtures were transferred to cells. After 24-hour incubation, the cell supernatant was mixed with QUANTIBlueTm and the optical density at 620 nm was measured in a colorimetric plate reader. As shown as Figure 16, MST1032-Glm did not prevent protease-mediated or spontaneous activation of human GDF11 and thus failed to inhibit the secretion of SEAP.
Example 21 [0538] Further optimization of MS1032 variants to enhace the sweeping effect As the pH dependent antibody, M51032L001-SG1, showed superior efficacy in mouse, further optimization was conducted to increase pH dependency, to enhance up-take into cells, to increase stability, and so on by introducing mutations into the antibody CDRs or by changing framework regions. More than a thousand variants were assessed in a BIACORE (registered trademark) and/or HEK Blue Assay as descrived above, and MS1032L006-SG1, MS1032L007-SG1, MS1032L010-SG1, MS1032L011-SG1, M51032L012-SG1, M51032L018-SG1, M51032L019-SG1, MS1032L021-SG1, MS1032L023-SG1, MS1032L024-SG1, MS1032L025-SG1, and M51032L026-SG1 were generated. Their amino acid and nucleotide sequences of these generate antibodies are shown in Table 11.

t,..) MS1032 variants and their DNA and amino acid sequences (shown as SEQ ID NOs) ,- =

Variable region Constant region --.1 i¨
oe Heavy Light Heavy Light P
Antibody name Abbreviation DNA PROTEIN DNA
PROTEIN DNA PROTEIN DNA PROTEIN
MS_M 103205H795-SG 1/M 103202L889-SK1 MS1032 L006-SG1 100 MS_M103205H1004-SG1/M103202L889-SK1 MS1032 L007-SG1 101 MS_M103205 H1046-SG 1/M103202L889-SK1 MS1032L010-SG1 102 MS_M103205H1047-SG1/M103202L889-SK1 MS1032L011-SG1 103 MS_M103205 H1057-SG 1/M103202L889-SK1 MS1032L012-SG1 104 MS_M103205H1186-SG 1/M103202L889-SK1 MS1032L018-SG1 105 MS_M103240H795-SG1/M103202L1045-SK1 MS1032L019-SG1 106 MS_M103245H795-SG1/M103202L1045-SK1 MS1032L021-SG1 107 L.

MS_M103245H1233-SG1/M103202L1045-SK1 MS1032L023-SG1 108 MS_M103205H795-SG1/M103202L1060-SK1 MS1032L024-SG1 100 MS_M103240H1246-SG1/M103202L1045-SK1 M51032L025-SG1 109 ' MS_M103240H795-SG1/M103209L1045-SK1 MS1032L026-SG1 106 i ...]

n ,-i --t..--, =
cA
7:-:--, oe --.1 4=.
oe --.1 C
t..) =
I-IVR amino acid sequences of MS1032 variants (shown as SEQ ID NOs) Hyper Variable region (HVR) Antibody name Abbreviation H1 H2 MS_M103205H795-SG1/M103202L889-SK1 MS1032L006-SG1 114 58 MS_M103205H1004-SG1/M103202L889-SK1 MS1032L007-SG1 114 116 MS_M103205H1046-SG1/M103202L889-SK1 MS1032L010-SG1 57 117 MS_M103205H1047-SG1/M103202L889-SK1 MS1032L011-SG1 57 118 MS_M103205H1057-SG1/M103202L889-SK1 MS1032L012-SG1 57 119 MS_M103205H1186-SG1/M103202L889-SK1 MS1032L018-SG1 114 118 MS_M103240H795-SG1/M103202L1045-SK1 MS1032L019-SG1 114 58 MS_M103245H795-SG1/M103202L1045-SK1 MS1032L021-SG1 114 58 MS_M103245H1233-SG1/M103202L1045-SK1 MS1032L023-SG1 115 58 63 123 71 74 .
MS_M103205H795-SG1/M103202L1060-SK1 MS1032L024-SG1 114 58 MS_M103240H1246-SG1/M103202L1045-SK1 MS1032L025-SG1 115 120 , , MS_M103240H795-SG1/M103209L1045-SK1 MS1032L026-SG1 114 58 63 123 71 74 , , Iv n ,-i k....-) c7, -a oe .6.
oe [0539] The affinity of MS1032 variant binding to human, cynomolgus monkey (cyno), or mouse latent myostatin at pH7.4 and pH5.8 was determined at 37 degrees C using BIACORE (registered trademark) T200 instrument (GE Healthcare) to assess the effect of pH on antigen binding. ProA/G (Pierce) was immobilized onto all flow cells of a CM4 chip using an amine coupling kit (GE Healthcare). All antibodies and analytes were prepared in ACES pH7.4 or pH5.8 buffer containing 20 mM ACES, 150 mM
NaC1, 1.2 mM CaC12, 0.05% Tween 20, 0.005% NaN3. Each antibody was captured onto the sensor surface by proA/G. Antibody capture levels were typically 130 to 240 resonance units (RU). Human, cyno, and mouse latent myostatin was injected at 3.125 to 50 nM prepared by two-fold serial dilution, followed by dissociation. The sensor surface was regenerated each cycle with 10 mM Glycine HC1 pH1.5. Binding affinity was determined by processing and fitting the data to a 1:1 binding model using BIACORE (registered trademark) T200 Evaluation software, version 2.0 (GE
Healthcare).
[0540] The affinity (KD) of MS1032 variants binding to human, cynomolgus monkey (cyno), and mouse latent myostatin at pH7.4 and pH5.8 are shown in Table 12.
All variants showed a KD ratio ((KD at pH5.8)/(KD at pH7.4)) of over 5, indicating pH
dependent binding to latent myostatin.

k.>
Kinetic parameters of MS1032 variants _______________________________________________________________________________ ______________________________ AD -4 CP
ii Name of variable region Human latent myostatin Cyno latent myostatin Mouse latent myostatin Fr 4.

KD pH 7.4 KO pH 5.8 ratio of KD at KD pH 7.4 KD pH 5.8 ratio of KO at KD pH
7.4 KD pH 5.8 ratio of KD at t,17; at Ab name Heavy chain Light chaint4 (M) (M) pH 5.8/pH 7.4 (M) (M) pH 5.8/pH 7.4 (M) (M) pH 5.8/pH 7.4 MS1032L006-SG1 M103205H795 M103202L889 2.85E-10 3.40E-08 119 3.43E-10 2.59E-08 76 2.99E-10 2.26E-08 76 MS10321007-SG1 M103205H1004 M1032021889 2.75E-10 4.62E-08 168 3.39E-10 3.47E-08 102 3.19E-10 3.08E-08 97 MS1032L010-SG1 M103205H1046 M103202L889 3.51E-10 4.38E-08 125 4.19E-10 1.61E-08 38 3.14E-10 1.09E-08 35 MS10321.011-5G1 M103205H1047 M103202L889 4.19E-10 143E-07 341 5.02E-10 4.35E-08 87 3.62E-10 2.92E-08 81 .
MS1032L012-SG1 M103205H1057 M103202L889 4.07E-10 8.49E-08 209 4.92E-10 3.58E-08 73 4.13E-10 2.48E-08 60 w .
ps, A
ps, MS10321018-SG1 M103205H1186 M1032021889 3.29E-10 n.d. n.d.
3.54E-10 8.09E-08 229 2.72E-10 n.d. n.d. ps, ,,,F, MS1032L019-SG1 M103240H795 M103202L1045 2.73E-10 4.58E-08 168 3.26E-10 3.56E-08 109 2.71E-10 2.87E-08 106 .

A
, , MS1032L021-SG1 M103245H795 M10320211045 2.57E-10 5.23E-08 204 3.09E-10 3.10E-08 100 2.61E-10 2.94E-08 113 , M51032L023-5G1 M103245H1233 M103202L1045 2.60E-10 1.61E-08 62 2.99E-10 7.71E-09 26 2.66E-10 6.40E-09 24 tv1S1032L024-SG1 M103205H795 M103202L1060 1.64E-10 1.52E-09 9 1.89E-10 9.29E-10 5 1.76E-10 1.02E-09 6 M51032L025-5G1 M103240H1246 M1032021_1045 2.76E-10 2.55E-08 92 3.40E-10 1.41E-08 41 2.59E-10 9.88E-09 38 n.d.: not determined v n ,-3 v w c, , Example 22 [0541] Evaluation of the neutralization activity of further optimized variants in HEK Blue Assay We evaluated the neutralization activity of MS1032L006-SG1, MS1032L007-SG1, MS1032L010-SG1, MS1032L011-SG1, MS1032L012-SG1, MS1032L018-SG1, MS1032L019-SG1, MS1032L021-SG1, MS1032L023-SG1 and MS1032L025-SG1 against human latent myostatin as described in Example 3. As shown in Fig. 17, all variants showed comparable activity to M51032L001-SG1.
Example 23 [0542] Comparison of plasma total myostatin concentration between non-pH
dependent anti-latent myostatin antibody and different pH-dependent anti-latent myostatin an-tibodies in mice In vivo test using C.B-17 SCID mice The accumulation of endogenous myostatin was assessed in vivo upon administration of anti-latent myostatin antibody in C.B-17 SCID mice (In Vivos, Singapore).
An anti-latent myostatin antibody (3mg/m1) was administered at a single dose of 10 ml/kg into the caudal vein. Blood was collected 5 minutes, 7 hours, 1 day, 2 days, 3 days, 7 days, 14 days, 21 days, and 28 days after administration. The collected blood was cen-trifuged immediately at 14,000 rpm in 4 degrees C for 10 minutes to separate the plasma. The separated plasma was stored at or below -80 degrees C until measurement.
The anti-latent myostatin antibodies tested were M51032L000-SG1, M51032L001-SG1, M51032L006-SG1, MS1032L011-SG1, M51032L018-SG1, MS1032L019-SG1, MS1032L021-SG1 and MS1032L025-SG1.
Measurement of total myostatin concentration in plasma by electrochemilumi-nescence (ECL) [0543] The concentration of total myostatin in mouse plasma was measured by ECL. Anti-mature myostatin-immobilized plates were prepared by dispensing biotinylated anti-mature myostatin antibody RK35 (as described in WO 2009/058346) onto a MULTI-ARRAY 96-well streptavidin plate (Meso Scale Discovery) and incubated in blocking buffer for 2 hours at room temperature. Mature myostatin calibration curve samples and mouse plasma samples diluted 40-fold or more were prepared. The samples were mixed in an acidic solution (0.2 M Glycine-HC1, pH2.5) to dissociate mature myostatin from its binding protein (such as propeptide). Subsequently, the samples were added onto an anti-mature myostatin-immobilized plate, and allowed to bind for 1 hour at room temperature before washing. Next, SULFO TAG labelled anti-mature myostatin antibody RK22 (as described in WO 2009/058346) was added and the plate was incubated for 1 hour at room temperature before washing. Read Buffer T (x4) (Meso Scale Discovery) was immediately added to the plate and signal was detected by SECTOR Imager 2400 (Meso Scale Discovery). The mature myostatin concentration was calculated based on the response of the calibration curve using the analytical software SOFTmax PRO (Molecular Devices). The time course of total myostatin con-centration in plasma after intravenous administration of anti-latent myostatin antibody measured by this method is shown in Figure 18.
Effect of pH-dependent myostatin binding on myostatin accumulation in mice in vivo study [0544] The effect of pH-dependency on myostatin accumulation in mice was compared using non pH-dependent anti-latent myostatin antibody (M51032L000-SG1) and different pH-dependent anti-latent myostatin antibodies (MS1032L001-SG1, M51032L006-SG1, MS1032L011-SG1, M51032L018-SG1, M51032L019-SG1, MS1032L021-SG1 and MS1032L025-SG1). Introduction of pH-dependency sig-nificantly accelerates myostatin clearance from SCID mouse plasma. As shown in Figure 18, pH-dependent anti-latent myostatin antibodies (M51032L001-SG1, M51032L006-SG1, MS1032L011-SG1, M51032L018-SG1, M51032L019-SG1, MS1032L021-SG1 and MS1032L025-SG1) could reduce myostatin accumulation compared to non pH-dependent anti-latent myostatin antibody (M51032L000-SG1) at day 28.
Example 24 [0545]
Comparison of plasma total myostatin concentration between non-pH dependent anti-latent myostatin antibody and anti-latent myostatin antibodies with both pH- and Fc engineering in cynomologous monkey In vivo test using cynomolgus monkey The accumulation of endogenous myostatin was assessed in vivo upon administration of anti-latent myostatin antibody in 2-4 year old Macaca fascicularis (cynomolgus monkey) from Cambodia (Shin Nippon Biomedical Laboratories Ltd., Japan). A
dose level of 30 mg/kg was injected into the cephalic vein of the forearm using a disposable syringe, extension tube, indwelling needle, and infusion pump. The dosing speed was 30 minutes per body. Blood was collected before the start of dosing and either minutes, 7 hours and 1, 2, 3, 7, 14, 21, 28, 35, 42, 49 and 56 days after the end of dosing, or 5 minutes and 2, 4, and 7 hours and 1, 2, 3, 7, 14, 21, 28, 35, 42, 49 and 56 days after the end of dosing. Blood was drawn from the femoral vein with a syringe containing heparin sodium. The blood was immediately cooled on ice, and plasma was obtained by centrifugation at 4 degrees C, 1700 x g for 10 minutes. The plasma samples were stored in a deep freezer (acceptable range: -70 degrees C or below) until measurement. The anti-latent myostatin antibodies used were M51032L000-SG1, MS1032L006-SG1012, MS1032L006-SG1016, MS1032L006-SG1029, MS1032L006-SG1031, MS1032L006-SG1033, and MS1032L006-SG1034 (herein, 5G1012, 5G1016, 5G1029, 5G1031, 5G1033, and 5G1034 are the heavy chain constant regions constructed based on SG1 as described below).
[0546] A dose level of 2mg/kg was administered into the cephalic vein of the forearm or saphenous vein using a disposable syringe, and indwelling needle for the anti-latent myostatin antibodies MS1032L019-SG1079, MS1032L019-SG1071, MS1032L019-SG1080, MS1032L019-SG1074, MS1032L019-5G1081, and M51032L019-5G1077 (herein, 5G1079, 5G1071, 5G1080, 5G1074, 5G1081, and 5G1077 are the heavy chain constant regions constructed based on SG1 as described below). Blood was collected before the start of dosing and 5 minutes and 2, 4, and 7 hours and 1, 2, 3, 7, 14 days after the end of dosing. The blood was processed as described above. Plasma samples were stored in a deep freezer (acceptable range: -70 degrees C or below) until measurement.
Measurement of total myostatin concentration in plasma by electrochemilumi-nescence (ECL) [0547] The concentration of total myostatin in monkey plasma was measured by ECL as described in Example 23. The time course of plasma total myostatin concentration after intravenous administration of anti-latent myostatin antibody as measured by this method is shown in Figure 19.
Measurement of ADA in monkey plasma using electrochemiluminescence (ECL) [0548] Biotinylated drug was coated onto a MULTI-ARRAY 96-well streptavidin plate (Meso Scale Discovery) and incubated in low cross buffer (Candor) for 2 hours at room temperature. Monkey plasma samples were diluted 20 fold in low cross buffer before addition to the plate. Samples were incubated overnight at 4 C. On the next day, the plate was washed three times with wash buffer before addition of SULFO TAG

labelled anti monkey IgG secondary antibody (Thermo Fisher Scientific). After in-cubating for an hour at room temperature, the plate was washed three times with wash buffer. Read Buffer T (x4) (Meso Scale Discovery) was immediately added to the plate and signal was detected using a SECTOR Imager 2400 (Meso Scale Discovery).
Effect of pH-dependent and Fc engineering on myostatin accumulation in monkey in vivo [0549] In cynomolgus monkey, administration of non pH-dependent antibody (M51032L000-SG1) resulted in at least 60 fold increase in myostatin concentration from baseline at day 28. At day 28, pH-dependent anti-latent myostatin antibodies M51032L006-5G1012 and M51032L006-5G1033 resulted in 3 fold and 8 fold increase from baseline respectively. The strong sweeping was mainly contributed by the increase in affinity to cynomolgus monkey Fc gamma RIIb. At day 28, pH-dependent anti-latent myostatin antibodies MS1032L006-SG1029, M51032L006-5G1031 and M51032L006-5G1034 could sweep antigen to below baseline. The reason for strong sweeping of M51032L006-5G1029, M51032L006-5G1031 and M51032L006-5G1034 are an increase in non-specific uptake in the cell due to increase in positive charge cluster of the antibody and an increase in Fc gamma R-mediated cellular uptake due to enhanced binding to Fc gamma R.
[0550] Administration of pH-dependent anti-latent myostatin antibodies MS1032L019-SG1079, MS1032L019-5G1071, MS1032L019-SG1080, MS1032L019-SG1074, MS1032L019-5G1081 and MS1032L019-SG1077 reduced myostatin concentration to below the detection limit (<0.25ng/mL) from day 1 in cynomolgus monkey. On day 14, the concentration of myostatin increased above the detection limit for M51032L019-5G1079 and M51032L019-5G1071 while the con-centration of myostatin remained below the detection limit for M51032L019-5G1080, MS1032L019-SG1074, MS1032L019-5G1081 and MS1032L019-SG1077. The weaker suppression of MS1032L019-SG1079 and MS1032L019-SG1071 could be due to difference in pI mutations.
[0551] The data suggests that strong sweeping of myostatin from the plasma could be achieved by mutations that increase binding to Fc gamma RIM or combining mutations that increase positive charge of the antibody and increase binding to Fc gamma RIM. It is expected that strong sweeping of myostatin could be achieved in human by combining mutations that increase positive charge of the antibody and increase binding to Fc gamma R.
Example 25 [0552] In vivo efficacy of further optimized variants in mouse As described in Example 8, the in vivo efficacy was evaluated in Scid mouse using M51032L006-SG1, MS1032L011-SG1, M51032L018-SG1, M51032L019-SG1, and M51032L025-SG1. Three independent studies were conducted and M51032L001 was used as the control.
[0553] The results of these experiments are shown in Figures 20A-20I. All antibodies (MS1032L006-SG1, MS1032L011-SG1, M51032L018-SG1, M51032L019-SG1, and M51032L025-SG1) showed a dose-dependent increase in lean body mass (LBM) as well as appendicular grip strength. The antibodies also showed a decrease in the body fat mass dose-dependently.
Example 26 [0554] Epitope mapping of anti-latent myostatin antibodies Additional anti-human latent myostatin antibodies were generated for mapping of their corresponding binding epitopes. Two NZW rabbits were immunized and harvested as described in Example 2. 1760 antibody producing B-cell lines identified were further screened for their ability to block BMP1 mediated activation of human latent myostatin. Briefly, B-cell supernatant containing secreted antibodies were incubated with human latent myostatin in the presence of recombinant human (R&D Systems) at 37 degrees C overnight. 50 micro L of the reaction mix were then transferred to Meso Scale Discovery MULTI-ARRAY 96-well plate coated with anti-mature myostain antibody RK35 (as described in WO 2009/058346). After 1 hour in-cubation at room temperature with shaking, the plates were incubated with biotinylated anti-mature myostatin antibody RK22 (as described in WO 2009/058346) followed by SULFO-tagged streptavidin. Read Buffer T (x4) (Meso Scale Discovery) was then added to the plate and ECL signal was detected by SECTOR Imager 2400 (Meso Scale Discovery). 94 lines showing different levels of neutralizing activities were selected for downstream analysis (M5T1495-M5T1588). The variable regions of these selected lines were cloned as described in Example 2, except an expression vector with the heavy chain constant region F1332m sequence (SEQ ID NO: 193) was used.
[0555] The inhibitory activity on human latent myostatin activation was further evaluated for a panel of 7 anti-latent myostatin antibodies (M5T1032, M5T1504, M5T1538, M5T1551, M5T1558, M5T1572, and M5T1573; the sequence identifiers for amino acid sequences of these antibodies are shown in Table 13). As shown in Figure 21, all of the antibodies were able to inhibit the BMP1 mediated activation of human latent myostatin in a dose-dependent manner.
[Table 13]
Amino acid sequences of anti-latent myostatin antibodies SEQ ID NO:
Antibody VH VL HVR-H1 HVR-H2 HVR-113 HVR-L1 [0556] 4 antibodies that worked well in Western blotting were selected for epitope mapping.
Fragments of the N-terminal propeptide coding region of human latent myostatin were cloned into a pGEX4.1 vector so as to produce GST tagged propeptide fragments of 100 amino acid each, with 80 amino acid overlap (Figure 22A). Protein expression was induced in transformed BL21 competent cells with Overnight Express Autoinduction System (Merck Millipore) and protein was extracted using the BugBuster protein ex-traction reagent (Novagen). Expression of the desired protein fragments at about 37kDa were verified by Western blotting analysis as described in Example 6 (anti-GST

antibody (Abcam)) (Figure 22B). When tested with anti-human latent myostatin an-tibodies, as shown in Figure 22C, although all 4 antibodies could inhibit latent myostatin activation, they recognized different epitopes on human latent myostatin.
MST1032 antibody could detect the first five fragments, no bands were detected after the removal of amino acid 81-100 (SEQ ID NO:78). Both MST1538 and M5T1572 an-tibodies bind to the first three fragments only, no bands were detected in the absence of amino acid 41-60 of SEQ ID NO:78. Antibody M5T1573 only bound strongly to the first two fragments, suggesting that its epitope lies within amino acid 21-40 of SEQ ID
NO:78 (Figure 22D).
Example 27 [0557] Development of novel Fc gamma RIM-enhanced Fc variants In this example, Fc engineering to enhance myostatin clearance is illustrated.
[0558] It has been demonstrated in WO 2013/125667 that clearance of a soluble antigen can be enhanced by its administration of antigen-binding molecules comprising an Fc domain displaying an increased affinity for Fc gamma RM. Furthermore, Fc variants that can show enhanced binding to human Fc gamma RHb have been illustrated in WO
2012/115241 and WO 2014/030728. It has been also illustrated that these Fc variants can show selectively enhanced binding to human Fc gamma RIIb and decreased binding to other active Fc gamma Rs. This selective enhancement of Fc gamma RIIb binding can be favorable not only for clearance of soluble antigen but also for de-creasing the risk of undesired effector functions and immune response.
[0559] For development of an antibody drug, efficacy, pharmacokinetics, and safety should be evaluated in non-human animals in which the drug is pharmacologically active. If it is active only in human, alternative approaches such as the use of a surrogate antibody must be considered (Int. J. Tox. 28: 230-253 (2009)). However it would not be easy to precisely predict the effects of the interaction between the Fc region and Fc gamma Rs in human using a surrogate antibody, because the expression patterns and/or functions of Fc gamma Rs in non-human animals are not always the same as in human. It would be preferable that the Fc regions of antibody drugs should have cross-reactivity to non-human animals, especially to cynomolgus monkey which has close expression patterns and functions of Fc gamma Rs to human, so that the results obtained in non-human animals could be extrapolated into human.
[0560] Therefore Fc variants that display cross-reactivity to human and cynoFc gamma Rs were developed in this study.
Affinity measurement of an existing Fc gamma RIIb-enhanced Fc variant against human and monkey Fc gamma Rs [0561] The heavy chain gene of the human Fc gamma RIIb enhanced variant (herein, "Fc gamma Ruth enhanced" means "with enhanced Fc gamma Ruth-binding activity") disclosed in WO 2012/115241 was generated by substituting Pro at position 238 in EU
numbering with Asp in the heavy chain of MS1032L006-SG1 which is named M103205H795-SG1 (VH, SEQ ID NO: 86; CH, SEQ ID NO: 9). The resultant heavy chain is referred to as M103205H795-MY009 (VH, SEQ ID NO: 86; CH, SEQ ID NO:
252). As the antibody light chain, M103202L889-SK1 (VL, SEQ ID NO: 96; CL, SEQ

ID NO: 10) was used. The recombinant antibody was expressed according to the method shown in Example 34.
[0562] The extracellular domains of Fc gamma Rs were prepared in the following manner.
The synthesis of the genes for the extracellular domain of human Fc gamma Rs were carried out in methods known to those skilled in the art based on the information registered in the NCBI. Specifically, Fc gamma RIIa was based on the sequence of NCBI accession # NM 001136219.1, Fc gamma Ruth was on NM 004001.3, Fc gamma RIIIa was on NM 001127593.1, respectively. Allotypes of Fc gamma RIIa and Fc gamma RIIIa were prepared according to the reports about polymorphism for Fc gamma RIIa (J. Exp. Med. 172:19-25 (1990)) and for Fc gamma RIIIa (J. Clin.
Invest.
100(5):1059-1070 (1997)), respectively. The synthesis of the gene for the extracellular domain of cynomolgus monkey Fc gamma Rs were constructed by cloning cDNA of each Fc gamma Rs from cynomolgus monkeys using the methods known to those skilled in the art. The amino acid sequences of the constructed extracellular domains of Fc gamma Rs were shown in the sequence list: (SEQ ID NO 210 for human Fc gamma RIIaR, SEQ ID NO 211 for human Fc gamma RIIaH, SEQ ID NO 214 for human Fc gamma Ruth, SEQ ID NO 217 for human Fc gamma RIIIaF, SEQ ID NO 218 for human Fc gamma RIIIaV, SEQ ID NO 220 for cyno Fc gamma RIIal, SEQ ID NO
221 for cyno Fc gamma RIIa2, SEQ ID NO 222 for cyno Fc gamma RIIa3, SEQ ID
NO 223 for cyno Fc gamma Ruth, SEQ ID NO 224 for cyno Fc gamma RIIIaS). Then His-tag was added to their C-terminus and the obtained each gene was inserted into ex-pression vector designed for mammalian cell expression. The expression vector was in-troduced into the human embryonic kidney cell-derived FreeStyle293 cells (Invitrogen) to express the target protein. After culturing, the resulting culture su-pernatant was filtered and purified by the following four steps in principle.
The cation exchange chromatography using SP Sepharose FF was performed as the first step, an affinity chromatography against the His-tag (HisTrap HP) as the second step, a gel filtration column chromatography (Superdex200) as the third step, and sterile filtration as the fourth step. The absorbance at 280 nm of the purified proteins were measured using a spectrophotometer and the concentration of the purified protein was de-termined using an extinction coefficient calculated by the method of PACE
(Protein Science 4:2411-2423 (1995)).

[0563] The kinetic analysis of interactions between these antibodies and Fc gamma R was carried out using BIACORE (registered trademark) T200 or BIACORE (registered trademark) 4000 (GE Healthcare). HBS-EP+ (GE Healthcare) was used as the running buffer, and the measurement temperature was set to 25 degrees C. A chip produced by immobilizing Protein A, Protein A/G or mouse anti-human IgG kappa light chain (BD
Biosciences) onto a Series S Sensor Chip CM4 or CM5 (GE Healthcare) by the amine-coupling method was used. An antibody of interest was captured onto this chip to interact with each Fc gamma R that had been diluted with the running buffer, and binding to the antibody was measured. After the measurement, the antibody captured on the chip was washed off by allowing reaction with 10 mM glycine-HC1, pH1.5 and 25 mM NaOH so that the chip was regenerated and used repeatedly. The sensorgrams obtained as measurement results were analyzed by the 1:1 Langmuir binding model using the BIACORE (registered trademark) Evaluation Software to calculate the binding rate constant ka (L/mol/s) and dissociation rate constant kd (1/s), and the dis-sociation constant KD (mol/L) was calculated from these values. Since the binding of MS1032L006-MY009 to human Fc gamma RIIaH, human Fc gamma RIIIaV, and cynoFc gamma RIIIaS was weak, kinetic parameters such as KD could not be calculated from the above-mentioned analytical method. Regarding such interactions, KD values were calculated using the following 1:1 binding model described in BIACORE (registered trademark) T100 Software Handbook BR1006-48 Edition AE.
[0564] The behavior of interacting molecules according to the 1:1 binding model on BIACORE (registered trademark) can be described by Equation 1: Req =C x R max/

(KD+C)+RI, wherein, Req is a plot of steady-state binding levels to analyte con-centration, C is concentration, RI is bulk refractive index contribution in the sample, and Rmax is analyte binding capacity of the surface. When this equation is rearranged, KD can be expressed as Equation 2: KD=C x Rmax/(Req-RI)-C. KD can be calculated by substituting the values of Rmax, RI, and C into Equation 1 or Equation 2.
From the current measurement conditions, RI=0, C=2 micro mol/L can be used.
Furthermore, the Rmax value obtained when globally fitting the sensorgram obtained as a result of analyzing the interaction of each Fc gamma R with IgG1 using the 1:1 Langmuir binding model was divided by the amount of SG1 captured, this was multiplied by the amount of MY009 captured, and the resulting value was used as Rmax. This cal-culation is based on the hypothesis that the limit quantity of each Fc gamma R
that can be bound by SG1 remains unchanged for all variants produced by introducing mutations into SG1, and the Rmax at the time of measurement is proportional to the amount of antibody bound on the chip at the time of measurement. Req was defined as the amount of binding of each Fc gamma R to each variant on the sensor chip observed at the time of measurement.

[0565] Table 14 shows the result of kinetic analysis of SG1 and MY009 against human and cynoFc gamma Rs. The KD values in cells filled in gray were calculated using [Equation 21 since their affinity was too weak to be correctly determined by kinetic analysis. Values of KD fold were calculated by dividing the KD value of SG1 by the KD value of the variant for each Fc gamma R.
[0566] As shown in Table 14, human Fc gamma Ruth-enhanced variant MY009 does not show enhanced binding to cynoFc gamma Ruth but shows enhanced binding to human Fc gamma RHb. Its affinity to cynoFc gamma Ruth was rather decreased by 0.4-fold compared to SG1, indicating that human Fc gamma Ruth-enhanced variant MY009 does not have cross-reactivity to cynoFc gamma Rs.
Development of novel Fc variant that shows enhanced binding to both human and cynoFc gamma Rub [0567] Ideally, novel Fc variant should have enhanced binding to both human and cynoFc gamma Ruth selectively and a decreased binding to other active Fc gamma Rs.
However, because the putative Fc binding residues in cynoFc gamma Ruth are completely the same as either allotype of cynoFc gamma RIIa (Figure 23), it is theo-retically impossible to achieve selective enhancement of cynoFc gamma Ruth binding over cynoFc gamma RIIa. Therefore a novel Fc variant should have selectively enhanced binding to human Fc gamma Ruth, cynoFc gamma Ruth and cynoFc gamma RIIa.
[0568] In order to obtain a novel Fc variant that shows selectively enhanced binding to both human and cyno Fc gamma Ruth, the results of comprehensive mutagenesis study performed in WO 2012/115241 were used. In the comprehensive mutagenesis study, comprehensive mutations were introduced into all the positions in Fc gamma R
binding regions in IgG1 antibodies and binding to each Fc gamma R was analyzed comprehensively as shown in the following procedures.
[0569]

Cross reactivity of a human FcyRlIb-enhanced variant to cynoFcyRs KD(M) for cynoFc KD (A) for humanFcgRs KD fold for cynoFceRs (SDI =1) KD fold for humanFceRs(SG1 =1 ) oe Heavy chain name Subatitutbn FcgRilai Fc2R.1182 Fc2RIla3 FcgRIlb FcgRIllaS FcgRIMH rcellaR
FG2RUO FcgP:i1-,/ FcgRllal Fc11;11a2 Fc2RlIa3 FcgRIlb FceRMaS FcaRllaH
FcaRlIaR FogRIlb FcaRIllaV t=J
MI 03205H795-SG1 - 42E-06 52E-06 15E-05 1.7E-06 42E-07 93E-07 1 4E-06 4.7E-06 ' -06 1.0 1.0 1.0 10 1000 1.00 1.0 10 1.000 0.41 032C5H795-MYCCO P2380 25E-C6 2.6E-05 31 E-C6 43E-06 ,r1.0&04, 75E-05 2 0E-C6 1.4E-C$5 5E-4 02 02 05 0.4 0.004 0.01 0.1 33 0002 La ,4 oe [0570] The variable region of a glypican 3 antibody comprising the CDR of GpH7 which is an anti-glypican 3 antibody with improved plasma kinetics disclosed in WO
2009/041062 was used as the antibody heavy chain variable region (GpH7: SEQ ID

NO: 225). Similarly, for the antibody light chain, GpL16-k0 (SEQ ID NO: 226) of the glypican 3 antibody with improved plasma kinetics disclosed in WO 2009/041062 was used. Furthermore, B3 (SEQ ID NO: 228) in which a K439E mutation has been in-troduced into Gld (SEQ ID NO: 227) produced by removing the C terminal Gly and Lys of IgG1 was used as the antibody H chain constant region. This heavy chain, which has been made by fusing GpH7 and B3, is referred to as GpH7-B3 (VH, SEQ
ID
NO: 225; CH, SEQ ID NO: 228).
[0571] With respect to GpH7-B3, the amino acid residues that are considered to be involved in Fc gamma R binding and the surrounding amino acid residues (positions 234 to 239, 265 to 271, 295, 296, 298, 300, and 324 to 337, according to EU numbering) were sub-stituted respectively with 18 amino acid residues excluding the original amino acid residue and Cys. These Fc variants are referred to as B3 variants. B3 variants were expressed and the binding of protein-A purified antibodies to each Fc gamma R
(Fc gamma RIIa type H, Fc gamma RIIa type R, Fc gamma Ruth, and Fc gamma RIIIaF) was comprehensively evaluated as follows. Analysis of interaction between each altered antibody and the Fc gamma receptor prepared as mentioned above was carried out using BIACORE (registered trademark) T100 (GE Healthcare), BIACORE
(registered trademark) T200 (GE Healthcare), BIACORE (registered trademark) A100, or BIACORE (registered trademark) 4000. HBS-EP+ (GE Healthcare) was used as the running buffer, and the measurement temperature was set to 25 degrees C. Chips produced by immobilizing Protein A (Thermo Scientific), Protein A/G (Thermo Scientific), or Protein L (ACTIGEN or BioVision) by the amine coupling method to a Series S sensor Chip CM5 or CM4 (GE Healthcare) were used. After capturing of an-tibodies of interest onto these sensor chips, an Fc gamma receptor diluted with the running buffer was allowed to interact, the amount bound to an antibody was measured. However, since the amount of Fc gamma receptor bound depends on the amount of the captured antibodies, the amount of Fc gamma receptor bound was divided by the amount of each antibody captured to obtain corrected values, and these values were compared. Furthermore, antibodies captured onto the chips were washed by 10 mM glycine-HC1, pH1.5, and the chips were regenerated and used repeatedly.
The value of the amount of Fc gamma R binding of each B3 variant was divided by the value of the amount of Fc gamma R binding of the parental B3 antibody (an antibody having the sequence of a naturally-occurring human IgG1 at positions 234 to 239, 265 to 271, 295, 296, 298, 300, and 324 to 337, according to EU numbering). The value obtained by multiplying this value by 100 was used as an indicator of the relative Fc gamma R-binding activity of each variant.
[05721 Table 15 shows the binding profile of promising substitutions selected based on the following criteria (more than 40% to human Fc gamma Ruth, lower than 100% to human Fc gamma RIIaR and Fc gamma RIIaH, less than 10 % to human Fc gamma RIIIaF, compared to those of parental B3 antibody).
[Table 151 Binding profile of selected substitutions against human FcyRs ______________ Binding to human Fog.Rs (parent B3 =100) Mutati FegRITaii FecfRITalt -b = = FegRIII) FegiRTIMV
on G236N 54 ____________________ 81 ________ 75 ______ 8 (11937Y 77 3 ______________________ 0 __________ G23717) 74 _________________ 0 95 ________ 0 ___________ P238E 21 ___________________ 2 _________ 97 _____ P238Y 93 ___________________ 9 _________ 111 P238.N1 8(3 9 97 9 ___________ P238Q 19 __________________ 4 ________ 13 ______ -1 _________ S239P 49 4 (31 : -1 V266F 91 8 88 ________ 2 __________ S267H 27 0 ' 1 S2671_. f63 :3 112 N3251, f9;3 13 116 0 N325F.: 19 13 40 4 I A3271, 37 12 46 8 [05731 In the next step, cross-reactivity of these substitutions to cynoFc gamma Rs was evaluated. These 16 mutations were introduced into M103205H795-SG1. The variants were expressed using M103202L889-SK1 as light chain and their affinity to cynoFc gamma RIIal, IIa2, IIa3, Tub, IIIaS were analyzed.
[05741 Table 16 shows the binding profile of these 16 variants to cynoFc gamma Rs. The value of the amount of Fc gamma R binding was obtained by dividing the amount of Fc gamma R bound by the amount of each antibody captured. This value was further normalized using the value for SG1 as 100.

[Table 16]
Binding profile of selected substitutions to cynoFcyRs __________________________________________________________________________ , ' Binding to eynoFcgRs (SG1 ¨ 100) CH Name Substitution FegRlIal FegRIIa2 .FegRIIa3 FegRIIb FealliaS
SG.1 100 , 100 100 100 100 , MY001 (4236N 83 86 . 62 85 15 50165 . 0237Y 18 17 . 7,0 30 7 SG166 (3237D 10 10 17 8 5 ______ SG167 . P238Y _______ 18 ______ 1,1 ___ 38 99 ____ 1-\...._ .___..,._.
SO169 P238N1 17 16 ,..,... 29 SG1.70 , P238Q 16, 15 . 21 18 __ 12 SG171. ' S239P 17 90 1.8 90 ____ 8 SG1.72 V266F 1.1 17 9 -.' - ; 16 31.
St..-173 S267L 13 14 ' 21 12 __ 94 SG174 ' S267H 13 16 20 11 15 SG1.75 . N3251,1 13 13 17 14 8 ______ SG 17(3 N:3251 14 15 19 _ 16 20 _ .
SG177 N39.51, 15 ¨ 15 25 _____ 18 , SG178 N325E 29 21 29 96 3(3 [0575] Among selected 16 Fc variants, only MY001 maintained binding to cynoFc gamma Ruth by 85% compared to SG1. Additionally MY001 showed reduced binding to cynoFc gamma RIIIaS and its binding to cynoFc gamma RIIal, IIa2, and IIa3 is maintained. As a result, G236N mutation is the unique substitution which shows similar binding profile to both human and cynoFc gamma Rs.
[0576] Although G236N substitution shows cross-reactivity to both human and cyno Fc gamma Rs, its affinity to Fc gamma Ruth is lower than SG1 (75% for human Fc gamma Ruth and 85% for cynoFc gamma RHb, respectively). In order to increase its affinity to Fc gamma Ruth, additional substitutions were evaluated.
Specifically, sub-stitutions that showed increased binding to human Fc gamma RHb, reduced binding to human Fc gamma RIIIa and increased selectivity to human Fc gamma RHb over human Fc gamma RIIa were selected based on the result of comprehensive mu-tagenesis study (Table 17).

[Table 17]
Binding profile of additionally selected substitutions to human FcyRs Binding to human FcgRs (parent B3 =100) Mutation FcgRIIaH FcgRlIaR FcgRIlb FcgRIIIaV

[0577] Among these substitutions, L234W and L234Y were selected for increasing se-lectivity to human Fc gamma Ruth over human Fc gamma RIIa. And G236A, G236S, A330R, A330K and P33 lE were selected for decreasing binding to human Fc gamma RIIIa. All other substitutions were selected for increasing binding to human Fc gamma RIM. The cross-reactivity of the selected substitutions to cynoFc gamma Rs were evaluated. In addition, three substitutions, P396M, V264I, and E233D were also evaluated. The substitutions were introduced into M103205H795-SG1 and the variants were expressed using M103202L889-SK1 as light chain.
[0578] Table 18 shows kinetic analysis of selected substitutions including G236N against both human and cynoFc gamma Rs. Specifically, substitutions were introduced into M103205H795-SG1. The variants were expressed using M103202L889-SK1 as light chain and their affinity to cynoFc gamma RIIal, IIa2, IIa3, lib, IIIaS were analyzed.
The KD values in cells filled in gray were calculated using [Equation 21 since their affinity was too weak to be correctly determined by kinetic analysis. Values of KD
fold were calculated by dividing the KD value of SG1 by the KD value of the variant for each Fc gamma R.
[0579] G236N showed comparable binding affinity to cynoFc gamma RIIal, cynoFc gamma RIIa2, cynoFc gamma RIIa3, and cynoFc gamma RHb to SG1. Although the affinity to human Fc gamma Ruth is reduced to 0.6-fold, affinities to human Fc gamma RIIaH
and human Fc gamma RIIaR are reduced to 0.2-fold and 0.1-fold, respectively, indicating that this substitution has high selectivity to human Fc gamma Ruth over human Fc gamma RIIa. Furthermore, its affinities to cynoFc gamma RIIIaS and human Fc gamma RIIIaV are 0.03-fold and 0.04-fold, respectively, which is favorable for eliminating ADCC activity. Based on this result, G236N showed almost ideal cross-reactivity to human and cynoFc gamma Rs although its affinity to both human and cynoFc gamma Ruth should be enhanced.
[0580] Among additional substitutions evaluated, S298L, G236A, P331E, E233D, K334Y, K334M, L235W, S239V, K334I, L234W, K328T, Q295L, K334V, K326T, P396M, I332D, H268E, P271G, S267A and H268D showed increased binding to both human and cynoFc gamma RIM. Specifically, H268D showed the largest effect (7-fold for human Fc gamma RIM and 5.3-fold for cynoFc gamma RHb). With respect to the effect of reduction in Fc gamma RIIIa binding, G2365, A330K, and G236D showed less than 0.5-fold affinity compared to SG1.
[0581] In order to enhance the affinity to both human and cynoFc gamma RIM of MY001, combinations of substitutions listed in Table 18 were evaluated. Specifically, sub-stitutions were introduced into M103205H795-SG1. The variants were expressed using M103202L889-SK1 as the light chain and their affinity was analyzed. Table 19 shows the result of kinetic analysis against human and cynoFc gamma Rs. The KD
values in cells filled in gray were calculated using [Equation 21 since their affinity was too weak to be correctly determined by kinetic analysis. Values of KD fold were calculated by dividing the KD value of SG1 by the KD value of the variant for each Fc gamma R.

k.>
Kinetic analysis of variants against human and cynoFcyRs o -a KO (M) for cynoFcgRs KO (M) for humanFces KO fold forcynoFcgRs (501 = 1) 1(0 fold for humanFcgRs (5G1 = 1) Heavy chain name Substitution FcgRlIal FcgRita2 0cgRlIa3 FcgRIlb FcgRIllaS FcgRIlaH FcgRlIaR FcgRIlb fcgRIllaV fcgRIlal Fc.gRlIa2 FcgRlIa3 FcgRilb 'FcgRIllaS FcgRIlaH FcgRIlaR FcgRIlb FcgRIllaV
4103205H795-SG1 - 3.2E-06 5.1E-06 1.7E-05 1.8E-06 3.6E-07 6.9E-07 1.1E-06 4.6E-06 8.1E-07 1.0 1.0 1.0 1.0 1.00 1.0 1.0 1.0 1.00 1-, -a ce M103205H795-MY016 p330 2.9E-06 3.4E-06 3.2E-05 1.7E-06 2.3E-07 8.5E-07 9.4E-07 2.9E-06 3.7E-07 1.1 1.5 5.3 1.2 1.57 0.8\ 1.2 1.6 2.19 2.0E-06' 1.9E-06 3.6E-06 9.2E-07 3.2E-07 1.3E-06 1.8E-06 2.3E-06 7.3E-07 1.6 2.7 4.7 2.0 1.13 0.5 0.6 2.Q 1.11 4.5E-06 4.7E-06 5.2E-05 2.0E-06 3.3E-07 2.6E-06 5.1E-06, 3.7E-06 4.3E-07 0.7 1.1 0.9 0.9 1.09 0.3 0.2 1.2 1.9E

2.3E-06 2.8E-05 4.7E-06 1.2E-06 4.9E-07 5.0E-07 7.4E-07 2.6E-06 7.0E-07 1.4 1.8 3.6 L5 0.73 1.4 1.5 14 1.16 M103205H795-MY001 p236N
2.9E-06 3.6E-05 1.5E-05 1.8E-06' 1.2E-05 3.2E-06\ 8.2E-06 7.7E-06 L86-O5 1.1 14 1.1, 1.Q 0.05 0.2 OA \ 0.6 0.04 6.5E-06 6.8E-06 1.8E-05 4.2E-06 3.4E-04 1.4E-06 1.3E-06 2.5E-06 3.0E-06 0.5 0.8 0.9 0.4 0.11 0.5 0.8 1.8 0.27 5.0E-06 3.7E-06 9.2E-06 2.5E-06 2.8E-07 4.0E-07 4.5E-07 3.1E-06 5.1E-07 0.6 1.4 1.8 0.7 1.29 1.7 2.4 1.5 1.59 2.0E-06 2.3E-06 6.2E-06 1.1E-06 2.7E-07 9.2E-05 2.1E-07 3.6E-06, 5.6E-07 1.5 2.2 2.7 1.6 1.33 7.5 5.2 1.3 1.45 2.6E-06 3.1E-06 1.2E-05 2.2E-06 1.3E-06 1.4E-07 5.2E-07 9.2E-06 5.2E-06 1.2 1.6 1.4 0.8 0.28 4.9 2.1 0.5 0.16 M103205H795-MY008 ,9239V
4.2E-06 3.7E-06 2.4E-05 1.7E-06 3.7E-07 1.6E-06 1.8E-06 2.6E-06 6.3E-07 0.8 1.4 0.7 1.1 0.97 0.4 0.6 1.8 1.29 M103205H795-MY037 S23924 , 3.8E-06 4.5E-06 9.6E-06 2.4E-06 2.0E-07 1.10-06 9.1E-07 2.6E-06 3.7E-07 0.8 1.1 1.01 0.8 1.80 0.6 1.2 1.2 2.19 M103205H795-MY038 $2391 , 3.9E-06 4.5E-06 1.1E-05 1.9E-06 3.1E-07 1.2E-06 1.5E-06 2.6E-06 5.5E-07 0.8 1.1 1.5 0.9 1.16 0.6 0.7 1.8 1.47 0 o 3.5E-06 5.0E-06 1.6E-05 2.0E-06 2.2E-07 1.2E-06 1.8E-06 5.1E-06 2.2E-07 0.9 1.0 1.1 0.5 1.64 0.6 0.6 0.5 3.68 ..., o 2.3E-06 2.8E-06 9.9E-06 1.0E-06 1.5E-07 4.9E-07 1.7E-07 6.6E-07 3.4E-07 1.4 1.8 1.7 1.6 2.40 1.4 6.5 7.0 2.38 o rv M103205H795-MY017 ?4268E
2.7E-06 2.9E-06 5.5E-05 6.8E-07 9.8E-05 4.5E-07 2.8E-07 9.4E-07 1.4E-07 1.2 1.8 3.1 2.6õ 3.67 1.5 3.9 4.9 5.79 h) rv M103205H795-MY063 _______________________________________________ 112680 1.4E-06 1.5E-06 2.0E-06 3.4E-07 6.7E-08 2.7E-07 2.1E-07 6.6E-07 L6E-07 2.3 3.4 8.5 5.3 5.37 2.6 5.2 7.CI 5.06 rv;;
M103205H795-FAY012 p271G
2.1E-06 2.3E-06 7.9E-06 1.1E-06 2.0E-07 4.4E-07 2.3E-07 9.0E-07 4.8E-07 1.5 2.2 2.2 1.6 1.80 1.6 4.8 5.1 1.69 1241, co 53E-06 1.1E-05 4.4E-06 3.5E-06 3.7E-07 1.8E-06 1.2E-06 3.0E-Of 6.1E-01 0.6 0.5 3.9 0.; 0.97 0.4 0.9 1.5 1.33 0 o 4.0E-06 4.1E-06 7.4E-06' 3.5E-0d 2.6E-07 1.8E-06 1.2E-06 3.1E-06 4.8E-07 0.8 1.2 2.i 0.5 1.38 0.4 0.9 1.5 1.69 I
1-..

1.2E-06 1.3E-06 5.1E-06 6.7E-07 1.7E-07 2.4E-07 5.0E-07 2.0E-06 3.4E-07 2.7 3.9 3.3 2.7- 2.12 2.9 2.2 2.3 2.38 ...1 5.1E-07 3.1E-06 8.0E-06 1.6E-06 4.4E-07 1.0E-06 9.8E-07 4.0E-06 5.3E-07 6.3 1.6 2.1 1.1 0.82 0.7 1.3., 1.2 1.53 1.60-06 1.6E-06 6.5E-06 7.3E-07 8.6E-08 4.4E-07 3.9E-07 1.6E-06 2.4E-07 2.0 3.2 2.6 2.5 4.19 1.6 2.8 2.9 3.38 5.8E-06 6.1E-05 1.8E-05 44E-06 8.4E-07 2.9E-06 1.5E-06 3.0E-06 1.2E-06 0.6 0.1 0.9 0.4 0.43 0.2 0.7 14 0.68 2.1E-06 2.7E-06 5.7E-06 1.0E-06 5.8E-07 3.7E-07 5.2E-07 2.1E-0f 1.4E-06 1.5 1.9 3.0 1.8 0.62 1.9 2.1 2.2 0.58 2.5E-06 2.5E-06 1.2E-05 1.50-06 4.0E-07 5.2E-07 9.6E-071 4.80-06 6.9E-07 1.3 2.0 1.4 1.2 0.90 1.3 1.1 1.0 1.17 2.5E-06 3.0E-06 2.3E-05 2.40-06 6.0E-07 4.8E-07 1.3E-06 6.20-06 2.2E-06 1.3 1.7 0.7 0.8 0.60 1.4 0.8 0.7 0.37 M103205H795-MY044 _______________________________________________ P331E
3.2E-06 3.5E-06 5.2E-09 1.6E-06 4.7E-07 1.2E-06 9.2E-07 3.3E-06 9.0E-07 1.0 1.5 0.3 1.1 0.77 0.6 1.2 1.4 0.90 1.0E-06 1.2E-06 2.6E-06 4.8E-07 5.3E-08 2.5E-07 4.8E-07 1.5E-06 1.2E-07 3.2 4.3 6.5 3.8 6.79 2.8 2.3 33 6.75 M103205H795-MY045 1(3340 5.7E-06 4.0E-06 6.8E-06 2.2E-06 1.4E-07 1.60-06 1.6E-06 2.9E-0E 2.4E-07 0.6 1.3 2.5 0.8 2.57 0.4 0.7 1.6 3.381 '19 M10320514795-MY064 1(3341 2.4E-06 2.6E-06 6.9E-06 1.10-116 1.2E-07 5.0E-07 6.8E-07 2.46-06 2.2E-07 1.3 2.0 2.5 1.5 3.00 1.4 1.6 1.9 3.66. n M103205H795-MY065 ,K334V
2.3E-06 2.8E-06 4.2E-06 1.3E-04 1.1E-07 5.2E-07 5.1E-07 1.90-06 2.3E-07 1.4 1.8 4.0 1.4 3.27 1.3 2.2 2.4 3.52, M103205H795-MY066 1(3341' 2.1E-06 2.6E-06 6.7E-06 1.40-06 1.6E-07 6.4E-07 9.7E-07 2.8E-06 2.9E-07 1.5 2.0 23 1.3 2.25 1.1 1.1 1.6 2.79 C--,-'19 2.1E-06 2.30-136 6.7E-06 1.10-02 1.0E-07 5.3E-07 7.6E-07 2.8E-06 2.0E-07 1.5 2.2 2.5 1.6 3.60 1.3 1.4 1.fi 4.05 C=J

1.3E-04 1.5E-06 4.7E-06 6.0E-07 1.0E-07 3.3E-07 3.2E-07 1.6E-06 2.4E-07 2.5 3.4 3.6 3.0 3.60 2.3. 3.4 2.9 3.38 .11 c, oe t=J
Kinetic analysis of variants which are composed of C236N and additional substitutions Z
KC:kW for cynoFcgRs KD1M) for humanF9Rs K Cs fold for cyn oFcgRs (561 1) KO fold for human FcgRs (SG1,-,1) Heavy chain name Substitutions FcgRlialice0(a7fcgRiia.TcgRitbfcgINNagfcgRilatfcg40ailFcgRubrcgRIatekcgRiialFcg RIia2FcgRIta3FcgRiltfcelilaSEcgrillal-fcgfUtagFcgRillfcgRflial, 4.=

3.26-065.16-06 1.7E-061.8606 3.6E-07 6.9E-07 1.1E-064.6E-06 8.1E-07 1.0 1.0 1.0 1.0 7..00 1.0 16 10, LOG --a oe 2.9E-06 3.6E-06 1.5E-051.8E-06 15/.2E-0 3.2E-06 8.2E-0E7.7E-06 .1.8605 1.1 1.4 1.1 1.0 0.03 0.2 0.2 0.6 0.04 7) t=J

4.82-053.46-06 2.4E-052.66-06 7.62-062.82-06 3.82-063.7E-067.7E-06 0.7 13 0.7 0.7 0.05 0.2 0.3 1.2 0.11 1.4E-06 1.7E-06 7.0E-0691E-07 5.4E-06 1.16-06 3.1E063.3E-06 1.2E-05 23 3.0 2.4 2.0 0.07 0.6 0.4 1.4 0.07 2.76-062.36-0(6.66-06.8.56-07 4.6E-06 1.8E-06 7.8E-061.5E-06 7.8E-06 1.2 2.2 2.6 2.I 0.08, 0.4 0.k 3.1 0.1c1 4.36-064.22-06 1.9E-051.4E-06 1.7E-06 2.1E-06 1.5E-0617.56-07 3.3E-06 0.7 1.; 0.9 1.3 0.21, 0.3 0.7 6.1 0.25 4.6E-06 4.8E06 1.8E-051.9E-06' 2.1E-06 2.2E-06 2.8E-062.0E-06,7A2-06 0.7 1.1 0.9 0.9 0.17 0.3 0.4 2.3 0.11 M1032051-$795-MY052 G236N/H268E/P396N1 1.4E-06 1.5E-06 3.960646E-07 1.5E-06 83E-07 1A60672E-07 A.66-06 2.3 34 4.4\ 3.8 0.24 0.8 OA 6.4 0.18 pA103205H795-MY101 G236N/H268E/4330K/P396M
1.1E-06 1.26-06 3.6E-00.5E-07 3.2E-06 4.9E-07(8.7E-074,9E-07 1 1E-06 2.9 4.3 4.7 5.1, 0.11 1.4 1.3 9.4/ 0.07 M103205H7954Rf103 G236N/H2686(4330R/P396M
1.4E-06 1.3E-06 3.6E-06.8E-07 2.66-06,4.12-07 1.2E-067.2E-07 '43E-06 2.6, 3.9 4.7 4.7 0.14, 1.7 0.9 6.4, 0.18 1.2E-06 9.0E-07 1.3E-06 3.96-07/ 1.3E-06 1.5E-0611.4E-065.7E-07 596-06 2.7 5.7 13.1 46 0.28 0.5 0.8 8.1 0.11 M1032054$795-MY107 6236N/S239V/H268E/A330R/P396M
9.06-077.36-07 1.22-063.06-07 2.26-06 9.5E-07 1.4E-065.0E-07 5.8E-06 3.6 7.0 14.2 6.0 0.16 0.7 0.8 9.2 0.14 M103205H795-MY109 236N/S239v/H268E/A3308/9396M
8.7E-07 7.1E-07 1.2E-062.8E-07 20E-06 9.4E-07 1.3E-065 5E-07 4.2E-06 3.1 71 14.21 6.4 0.18 0.7 0.8 8.4 0.19 M1032091795-W111 1.234W/0236N/H268E/4330OUP396M
1.7E-06 1.86-06 4.2E-065.5E-07 2.3E-06 4.7E-07 2.0E-061.1E-06 3.1E-06 1.9 2.8 4.0 3.3 0.16 1.5 0.6 4.2 0.26 M103205H795-W113 t.234W/G236N/H268E/A330R/P396M
1.6E-06 1.7E-06 406-06_ .0E-07 2.0E-06 1.3E-06 1.8E-069.8E-07 3 7E-06 2.0 3.0 4.3 3.6 0.18 0.5 0.6 4.7 0.22 o M103205H795-MY115 0236N/32674,1H268E/P39EM
2.36-062.76-06 7.6E-067.8E-071 8.5E-07 9.6E-07 6.4E-073.2E-07 3.6E-06 1.4 1.9 2.2 2.3 0.42 0.7 13 14.4 0.22 ...
o 2.6E-06 3.2E-069.9E-01.0E-08 1.0E-06 2.2E-06 1.0E-066.0E-07 7.96-06 1.2 1.6 1.7 1.8 0.36 0.3 1.1 7.7 0.10 o iu i4103209-1795-W141 G236N/5267A/P396M
2.2E-06 2.8E-06 2.0E-051.8E-06 4.7E-06 1.2E-06 1.66-061.16-06, 1.2E-06 1.5 1.8 0.6 1.0 0.08 OA 0.7 4.2 0.07 h) 8.9E-07 8.9E-07 3.8E-06r4.4E-07 2.3E-06 6.5E-07 1.3E-067.4E-07 7.7E-06 3.6 5.7 4.6 4.1 0.16 1.1 0,8 6.2 0.11 iu 1.9E-06 2.4E-06 1.0E-059.8E-07 3.6E-06 1.8E-06 2.3E-061.2E-06 9.1606 1.7 2.1 1.7 1.8 0.10 0.4 0.5 3.8 0.09 o 1-.t-11 1.7E-07 1.8E-06 4.86-068.5E-07) 3.1E-06 2.2E-06 2.1E-0689E-07 8.8E-06 18.8 2.8 3.5 2.1 0.12 0.3 0.5 5.2 0.09 0 =
M103209-1795-MY147 ,L235W/G236N/H268E/P396M
2.2E-06 2.2E-06 6.6E-069.6E-07 5.5E-06 1.2E-06 1.1E-068.3E-07 1.1E-05 1.5 2.3 2.6 1.9 0.07 0.6 1.0 5.5 0.07 0 5.86-07 5.6E-07 1.4E-061.7E-07 2.2E-06 3.6E-07 7.1E-0739E-07 1.0E-05 5.5 9.1 12.; 10.6 0.17 1.9 1.5 11.8 0.08 =
1-.
M103205H795-W198 G236h1/32390-12680/43306/P396M
4.42-073.32-07 1.4E-062.4607 2.7E-05 53E-07,,8.0E-07-4.2E-07 6.9E-06 7.3 15.5 12.1 7.5 0.13 1.2 1.4 11.0 0.12 ...) M103205H795-M(199 0236N/H2686/4.3308 1.92-062.06-06 1.2E-051.1E-06 4.1E-06 1.1E-06 2.8E-0619E-06 1.3E-06 1.7 2.6 1.4 1.6, 0.09\ 0.6 0.4 2.4 0.06 103205i-1795-MY200 0236N/5239V/112686/4330K
1.7E-06 1.5E-065.06061.1E-06 4.4E-06 1.76-062.72-061,46-06' 1.5E-05 1.8 3.4 3.4 1.6 0.06 0.4 0.4 3.3, 0.05 103209-1795-W201 6236N/H2680/A33C4( 1,46-061.46-06 3.9E-064.3E-07/ 5.7E-06 9.1607 1.9E-061.1E-06 2.4E-05 2.3 3.6 4.4, 4.2 0.06 0.8 0.6 4.2 0.03 8.6607 6.4E-07 2.2E-064.6E-07 4.0E-061'1.2E-06 1.2E-067.1E-07 9.4E-06 3.7 8.0 7 7 3.9 0.09 0.6 0.5 6.5 0.09 M103205H795-W204 1G236N/S236N/H263E/Q2951/A3301( 1.3E-06 1.4E-06 5.2E-068.1E-07. 3.5E-06 1.3E-06 2.8E0613E-06 1.1E-05 2.5 3.6 3.3 2.2 0.10 0.5 0.4, 3.5 0.07 M103205H795-MY206 G236N/H2680/02951/4330( 9.0E-07 1.1E-06 3.0E-062.5E-0/ 6,0E-0614.7E-07 1.36-065.66-01 1.3E-051 3.6 4.6 5.7 7.2 0.06 1.5 0.0 8.2 0.06 M103205H795-MY206 6236N/S239#H2680/0295t/A330K
8.1E-07J.92-07'3.0E-064.5E-07 3.3E-06 1.0E-06 1.46069.0E-07 8.2E-06 4.0 6.5, 5.71 4.0 0.11 0.7 0.E 5.1 0.10 5E-06 1 4E-06 61E-0681E-07 4.12-06 1.3E-06 2.86-0g1.6E-061 8.06-06/ 2.1 36 2.5 2.2 0.09 OS OA 2.9 0.10 M103205H795-W208 ,G236N/H2680/02951 1.2E-06,1.5E-06 6.76-06,46-07"28E-O6 8.3E-07 2.1E-061.1E-06 1.0E-05 2.7 3.4 2.5 2.8 0.13 0.8 0.5 4.2 0.08 M1032.05H795-W209 G236N/H268)/0299./K3267/4330K
6.3E-07 6.9E-07 1.9E 19E-07 2.2E-06 5.7E-07 8.8E-075.0E-07 1.4E-05 5.1 7.4 8.9 9.6 0.16 1.2 1.3 9.2 0.06 't:J
M1032058795-W210 0236N/H2680/1026T/A33C4( 9.5E-07 8.4E-07 2.3E-02.8E-07 2.4E-06 8.8E-07 1.4E-0039.8E-07 3.1E-05 3.4 6.1 7.4i 6.4 0.15 0.6 0.6 4.7 0.03 n M103205H795-MY211 G236N/S239V/H2680/83267/A3301( 7.5E-07 5.8E-07 2.7E-004.7E-07 3.0E-06 1.1E-06 1.3E-068.2E-07' 4.8E436 4.3 8.8, 6.3 3.8 0.12 0.d 0.8 5.6 0.17 14103205H735-MY212 0236NA239V/H2680/422951./k3267/A330K
.5.6E-07 5.3E-07 2.92-063.7E-07/ 2.66-068.06-07 1.3606636-07.6.56-06 5.7 9.6 5.9 4.9 0.14 0.9 0.8 7.3 0.12 4/1103209-1795-W288 iG236N/Q2951.
1.5E-06 2.0E-06 1.1E-051.2E-06 1.3E-05 1.4E-068.5E-0Z 5.1E-06 6.86-05, 2.1 2.6 1.5 1.6 0.03 0.5 0.1 0.9 0.01 V
t=J

1.3E-06 1.5E-06 86E-067.4E-07 5.2E-06 8.3E-07 3.5E-062.9E-06 7.7E-06 2.5 3.4 2.0 2.4 0.07 0.8 0.3 1.6 0.11 .=
M1032091795-W350 1.235W/G236N/H268E/A3303(1P396M
2.1E-062.2E-06 4.760961E-07 6.6E-06 9.6E-07 9.0E-076.7E-07 1.2E-OS 1.5 2.3 36 3.0 0.05 0.7 1.2 6.9 0.07 ..=7N
-.....

2.9E-06 2.4E-06 6.4E067.4E-07 1.16-05 1.6E-06 1.9E-061.4E-06 1.6E-06 1.1 2.1 2.7 2.4 0.03, 0.4 0.6, 3.3 0.05 M1032091795-MY440r 235W/G236N/H2690/0296./A3301( 1.6E-06 1.8E-06 4SE-064.6607 9.3E-06/ 9.1E-07 1.1E-098.0E-07 2.7E-05' 2.0 2.8 3.5 3.9 0.04 0.8 1.0 5.8 0.03 --a A103205H795-MY518 ,-235W/G236N/H2680/0295L/K3267/433CK
8.9E-07 9.0E-07 2.4E-062.6E-07 4.2E-06 8.5E-07 6.7E-074,8E-07 2.3E-03 3.6 5.7 7.1 6.9 0.09 0.8 1.6 9.6 0.04 4-oe --a [0582] All the variants suppressed affinities to human Fc gamma RIIIaV by less than 0.26-fold and to cynoFc gamma RIIIaS by less than 0.42-fold compared to SG1.
Fur-thermore, all the variants except for MY047, MY051, and MY141 successfully showed enhanced binding to both human and cynoFc gamma RIth compared to MY001 and their human Fc gamma RIIa binding were maintained to less than 2-fold compared to SG1. Among them, MY201, MY210, MY206, MY144, MY103, MY212, MY105, MY205, MY109, MY107, MY209, MY101, MY518, MY198 and MY197 showed enhanced binding both to human and cynoFc gamma RIth by more than 4-fold compared to SG1. Above all, MY205, MY209, MY198, and MY197 showed enhanced binding to both human and cynoFc gamma Ruth binding by more than 7-fold.
[0583] It was illustrated in W02014030728 that substitutions at position 396 in CH3 domain enhanced affinity to human Fc gamma RIIb. Comprehensive mutagenesis was in-troduced into position 396 of M103205H795-MY052, which contains G236N/H268E/P396M substitutions. The resultant variants were expressed using M103202L889-SK1 as light chain and their affinity to human and cynoFc gamma Rs were evaluated (Table 20). Values of KD fold were calculated by dividing the KD
value of SG1 by the KD value of the variant for each Fc gamma R.
[0584] Among substitutions tested, P396I, P396K, and P396L maintained human Fc gamma Ruth binding by more than 5-fold compared to SG1 and only P396L substitution enhanced affinity to human Fc gamma RIM compared to MY052. With respect to the binding to cynoFc gamma RIM, parent MY052 showed the highest affinity which is a 3.9-fold increase from SG1.
[0585] Since G236N showed ideal cross-reactivity to human and cynoFc gamma Rs, other substitutions on position 236 which were not evaluated in the preceding examples were tested. Specifically, Asn in M103205H795-MY201 were substituted with Met, His, Val, Gln, Leu, Thr, and Ile. The resultant variants were expressed using M103202L889-SK1 as light chain and their affinity to human and cynoFc gamma Rs were evaluated (Table 21). The KD values in cells filled in gray were calculated using [Equation 21 since their affinity was too weak to be correctly determined by kinetic analysis. Values of KD fold were calculated by dividing the KD value of SG1 by the KD value of the variant for each Fc gamma R.

k..>
Kinetic analysis of P396 variants derived from MA1052 o _ pa -a KD IM) for cynoFcgRs KO (M) for humankgfts 1(0 fold for cynoFcg,Rs (SG1 = 1 KD fold for humanFcgRs (SG1 = 1) Heavy chain name Substitutions FcgRIla1 Fc8RlIa2 FcgRlIa3 FcgRilb FcgR111aS
FcgR11aH FcgRIlaR FcgRIlb FcgRillaV FcgRlial FcgRlia2 Fc4Ri1a3 Fogiblb .cgRIllaS FcgRIlaH .cgRliaR Fc8111113 FcgRillaV iT 0 .1.
M10320514795-SG1 3.2E-06 5.1E-06 1.7E-05 1.8E-06 3.6E-07 6.9E-07 1.1E-06 4.66-06 8.1E-07 1.0 1.0 1.0 1.0 1.00 1.0 1.0 1.0 1.00 " --1 co M103205H795-MY052 G236N/H268E/P396M 1.4E-06 1.5E-06 3.9E-06 4.6E-07 1.5E-06 8.3E-07 1.4E-06 7.2E-07 1.5E-06 2.3 3.4 4.4 3.9 0.24 0.8 0.8 6.4 0.54 0 tio/
,---, M103205H795-MY168 p236N/H268E/P396A 2.2E-06 2.5E-06 1.0E-05 1.2E-06 4.5E-06 1.6E-06 2.8E-06 1.7E-06 8.1E-06 1.5 2.0 1.7 1.5 0.08 0.4 0.4 2.7 0.10 M103205H795-MY169 G236N/14268E/P3960 2.3E-06 2.3E-06 9.9E-06 1.0E-06 3.5E-06 1.2E-06 2.5E-06 1.3E-0E 6.2E-06 1.4 2.2 1.7 1.8 0.10 0.6 0.4 3.5 0.13 M1032051-1795-MY170 G236N/H268E/P396E 2.3E-06 2.4E-06 1.2E-05 1.2E-06 2.9E-06 1.7E-06 2.5E-06 1.6E-06 6.3E-06 1.4 2.1 1.4 1.5 0.12 0.4 0.4 2.9 0.13 M103205H795-MY171 3236N/H268E/P396F 2.3E-06 2.3E-06 9.2E-06 9.8E-07 3.3E-06 1.2E-06 2.1E-06 1.2E-06 5.9E-06 1.4 2.2 1.8 1.8 0.11 0.6 0.5 3.8 0.14 M1032051-1795-MY172 G236N/H268E/P3966 2.7E-06 2.8E-06 1.1E-05. 1.2E-06 3.3E-06 1.7E-06 3.1E-06 1.8E-06 6.5E-06 1.2 1.8 1.5 1.5 0.11 0.4 0.4 2.6 0.12 6/1103205H795-MY173 3236N/14268f/P396H 1.7E-06 1.8E-06 8.0E 7.8E-07 3.1E-06 1.0E-06 1.9E-06 1.0E-06 7.0E-06 1.9 2.8 2.1 2.3 0.12 0.7 0.6 4.6 0.17 M103205147954.4Y174 G236N/14268E/P3961 1.5E-06 1.6E-06 6.4E- 6.5E-07 2.5E-06 8.1E-07 1.4E-06 8.5E-07 6.8E-06 2.1 3.2 2.7 2.8 0.14 0.9 0.8 5.4 0.12 M103205H795-MY175 3236N/H268E/P396K 1.4E-06 1.5E-06 7.5E 7.1E-07 2.6E-06 8.6E-07 1.5E-06 8.3E-07 7.6E-06 2.3 3.4 2.3 2.5 0.14 0.8 0.7 5.5 0.11 M1032051-1795-NW176 3236N/142613E/P3961. 1.3E-06 1.4E-06 6.0E 6.1E-07 2.2E-06 7.0E-07 1.3E-06 7.0E-07 6.2E-06 2.5 3.6 2.µ 3.0j 0.16 1.0 0.8 6.6 0.13 M103205H795-MY177 0236N/H268E/P396N 1.9E-06 1.8E-06 8.1E 8.3E-07 2.9E-06 1.0E-06 2.0E-06 1.16-0e 6.2E-06 1.7 2.8 2.1 2.2 0.12 0.7 0.6, 4.2 0.13 M103205H795-MY178 3236N/H268E/P396Q 2.0E-06 2.1E-06 8.7E 9.5E-07 3.0E-06 1.2E-06 2.3E-06 1.3E-0E 4.5E-06 1.6 2.4 2.0 1.9 0.12 0.6 0.5 35 0.18 M1032051-1795-MY179 3236N/H268E/P396R 1.9E-06 2.0E-06 7.9E 9.8E-07 2.7E-06 1.1E-06 2.1E-06 1.2E-0E 5.9E-06 1.7 2.6 2.2 1.8 0.13 0.6 0.5 3.8 0.14 0 M103205H795-MY180 5236N/H268E/P3965 2.6E-06 2.7E-06 1.2E-0 1.3E-06 3.1E-06 1.7E-06 3.0E-06 1.9E-OE 5.9E-06 1.2 1.9 1.4 1.4 0.12 0.4 0.4 2.4 0.14 o Wo11032-66i795--NliI81 5236N/H268E/P396T.._, 23E-06 2.3E-06 9.2E-06 1.0E-06 4.1E-06 1.3E-06 2.5E-06 1.41-0E 73E-06 1.4 2.2 1.8 1.8 0.09 0.5 0.4 3.3 0.11 w o M103205H795-MY182 3236N/H268E/P396V 2.1E-06 2.2E-06 7.8E-06 9.6E-07 3.6E-06 1.3E-06 2.1E-06 1.2E-0E 7.0E-06 15 2.3 2.2 1.9 0.10 0.5 0.5, 3.8 0.12 ro ar.
64103205H795-MY183 3236N/14268E/P396W 2.1E-06 2.0E-06 8.4E-06 9.7E-07 3.5E-06 1.2E-06 2.1E-06 1.1E-06 7.2E-06 1.5 2.6 2.0 1.9 0.10 0.6 0.5 4.2 0.11 ^) ro M1032051-1795-MY184 3236N/H268E/P396Y 1.9E-06 2.0E-06 8.4E-06 9.0E-07 3.3E-06 1.1E-06 2.0E-06 1.1E-06 6.8E-06 1.7 2.6 2.0 2.0 0.11, 0.E 0.6 4.2 0.12 ro c7re o 3' ar.
I
I.
.4 V
n i:
v t=-) =
c, -.
ri --.1 oe --.) Kinetic analysis of G236 aria nts derived from MY201 KO (A) for cvnacgits 1(0(M) for humanfctIts KO fold for cynoFcglis (S61 1' KO fold for humanFcgits (561 1) la'.
Heavy Main name Substitutions Fcgfilla1 FcgRila2 FcgMla3 FcgRith FcgRIllaS FcgRilaH FcgRIlaR FcgRUb FcgRillaV FcgRlIal FcgRlIa2 Fcglilla3 FcgRilb EcgRIllaS FcgRliaH FcgRilaR FcgRIlb FcgRMaV
M103205H795-SG1 3.2E-06 5.1E-06 1.7E-05 1.8E-06 3.6E-07 6.9E-07 1.3.E-06 4.6E-06 8.1E-07 1.0 1.0 1.0 10 1.(30 1.0 1.0 1.0 1.00 C.) oe M103205H795-0701 6236N/H2680/A330K 1.4E-06 1.4E-06 3.9E-06 4.3E-07 5.7E-06 9.1E-07 1.9E-06 1.1E-06 2.4E45 23 3.6 4.4 4.2 0.06 0.8 0.6 4.2 0.03 I-, t=J
M103205H795-MY260 G236M/H268D/A330K 4.5E-06 3.76-06 1.4E-05 1.66-06 6.5E-07 9.1E-07 1.8E-06 6.1E-06 3.0E-0E 07 1.4 1.2 1.1 0.55 0.8 0.6 0.8, 0.27 M1032051-1795-MY261 3236H/H26813/A3306 3.7E-06, 4.0E-06 5.5E-06 1.5E-06 2.4E-06 2.4E-07 1.1E-06 5.3E-06 1.1E-05 0.6 1.3 3.1 1.2 0.15 2.9 1.0 0.9 0.07 M103205H795-MY262 3236WH268131/A3301C 1.9E-06 1.7E-06 8.0E46 5.6E-07 2.86-06 1.4E-07 7.1E47 6.0E-06 5.0E4E 1.7 3.0 2.1 32 0.13 4.9 1.5 0.8 0.16 M103205H795-MY263 3236Q/H2680/A33CK 3.2E-06 3.0E46 7.3E46 1.0E-06 7.8E-07 3.2E47 7.1E-07 3.5E-06 2.36-06 1.0 1.7 2.3 1.8 0.46, 2.7 15 1.3 0.35 M1032051-1795-MY264 32361./H268D/A330K 5.6E-06 5.5E46 1.6E-05 2.6E-015, 3.7E-06 1.5E-06 2.3E46 1.2E-050CM3-0 0.6 0.9 1.1 0.7 0.10 0.5 05 0.4 0.01 2.2E46 1.9E46 4.8E-06/ 5.9E-07 2.0E-06 2.1E-07 4.4E-07 2.2E-06 5.4E-0E, 15 2.7 3.5 3.1 0.18 33 2.5 2.1 0.15 M103205H795-MY267 ,32361/H2680/A330K
2.4E46 2.0E-06 7.3E-06 8.4E47 2.7E-06 2.0E47 7.1E-07 5.7E-06 1.56-0E, 1.Z
2.6 2.3 2.1 0.13 3.5 1.5 0.8 0.05 1-.00 Z
CN

oe w Kinetic analysis of variants derived from 1V11'265 .....
m --I
50(M) for cynoFcgRs 80(M) for human6c8Rs KD fo Id for cynoFcgRs(501.1 KO fold for humanFqiRs (501=1) .c.õ, 4eavy cha in name Substttutions FcgRife1 Fcg6tia2 FcgRli 83 FcgRi lb rcgRi 1146 Fcgfiliall FcgROaR FcgR0b FcgRinaV FcgRiiel FcgRita2 FcgRila3 FcgRI ill FcgRII faS FcgRItail 'cgROaR FcgRitb r.cgfti I iaV CD .I...
--I

3.25-0E 5.1E-06 1.7E-05 1.8E-06 3.6E-07 6.9E-07 1.1E-06 4.6E-06 8.1E-07 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 t") X
t=-) Co4 2.2E-OE 1.9E-06 4.8E-06 5.9E-07, 2.0E-06 2.1E-07 4.4E-07 2.2E-06 5.4E-06 1.5 2.7 3.5 3.1 0.2 3.3 2.5 2.1, 0.2 ,--, 5.0E-OE 4.75-06 7.5E-06, 1.1E-06 3.2E-06 3.9E-07 5.6E-07 2.5E-06 8.7E-06 0.6 1.1, 2.8 1.E 0.1 1.8 2.0 1.8 0.1 3.1E-05 3.1E-06 5.5E-06 7.9E-07 2.75-06k 2 6E-07 5.55-07 2.4E-0E 6.7E-06 1.O 1.6 3.1, 21 0.1 2.7 2.0 1.9 0.1 .

1.85406 1.8E-06 3.5E-06 4.7E-07 2.1E-06 1.82-07/ 3.4E-07 1.7E-06 5.4E-06 11 2.8 41 3.8 0.2 31 3.2 2.7 0.2 1.9E-06 1.6E-06 4.2E-06 4.7E-07 1.8E-06 2.4E-07 4.9E-07 1.9E-06 5.4E-06 1.7 3.2 4.0 3.8 0.2 21 22 2.4 0.2 2.1E-06 1.85-06 4.4E-06 5.8E-07/ 1.62-06 2.2E-07 5.1E-07 2.35-01 4.8E-06 1.5 2.8 3.5 3.1 0.2 3.1 2.2, 2.0 0.2 M1032091795-M2464 A231i/G236T/H2680/8330K
1.95-04 1õ55-06 3.92-06 5.0E-07/ 1.7E-06 1.7E-07 2.6E-071 1.6E-06 4.4E-06 1.7 3.4 4.4 3.E 0.2 4.1 4.2 2.8 0.2 M1032091795-M1465 .4231K/G236T/H26813/4330K
2.0E-06f 2.8E-06 5.2E-06 6.3E-07 1.95-06 2.5E-0 6.0E-07 2.8E-06 5.5E-06 1.6 1.6 3.3 2.2 0.2 2.8 1.8 1.6 0.1 1.9E-06 1.8E-08, 4.4E-06 5.1E-07 1.8E-06 1.7E-07 2.6E-07 1.4E-06 4.3E-06 1.7 2.8 3.5 3.5. 0.2 4.1 4.2õ 3.3 0.2 1.95-06 1.9E-06 4.7E-06 5.1E-07 1.7E-06 1.8E-07( 3.6E-07 2.0E-06 4.8E-06 1.7( 2.7 3.6 3.! 0.2 3.8 3.1 2.3 0.2 , 2.8E-06 2.5E-06 5.2E-06 6.5E-07 2.35-0 2.8E-07 4.7E-01 2.6E-0E 6.8E-06 1.1 2.0 3.; 2.8 0.2 2.5 2.3 1.8 0.1 M1032051-1795-M1469 A231P/G236r/H2680/43305 2.2E-06 2.1E-06, 4.9E-06 6.1E-07 2.2E-06 2.0E-07 4.3E-07 2.4E-06 59E-06 1.5 24 3.? 3.e 0.2 3.5 2.0, 1.9 0.1 M10320511795-W470 A2314162361/H2680/A333( 2.2E-06 1.85-06 4.9E-06 5.5E-07 1.7E-06 2.15-07/ 4.7E-07 2.3E-04 5.1E-06 1.5 2.8 3.5 3.3 0.2 3.3 2.3 2.0 01 0 '01032091795-W471 A2318/G2361/1426543/A3305 2.6E-0E 2.1E-06 4.5E-06 7.0E-07 1.8E-06 2.3E-07 4.7E-07 2.8E-06 5.3E-06 1.2 2.4. 3.8 2.E 0.2 3.0 2.3 16 0.2 o w o 2.4E-06 2.1E-06 5.1E-08 6.6E-07 2.3E-06 2.5E-07 498-07 2.5E-06 622-06 1.1 2.4 3.8 2.7 0.2 2.8 2.2 1.8 0.1 o r.

1.6E-06 2.6E-04 5.5E-06 5.85-07 1.7E-06 2.2E-07 4.9E-07 2.2E-06 5.2E-06 2.0 2.0 3.1 3.1 0.2 3.1 2.2 2.1., 0.2 4.
r.

2.0E-06 1.8E-06 4.8E-06 5.0E-07 1.6E-06 1.7E-07 3.4E-07 2.0E-06 4.6E-06 1.6 2.2 3.5 3.E 0.2 4.1 3.2 2.3 0.2 r.
.__.
M1032091795-M1475 A231W/G2361/1-12680/A.33CE
1.0E-06 1.0E-OE 2.3E-06 ,.9 2.75-07 1.7E-06 9.0E-08 2.1E-07 1.1E-06 5.0E-06 3.2 5.1, 7.4 6.7 0.2 7.7 5.2 4.2 0.2 M10320521795-MY476 A231Y/G2361;412680/A330K
1.4E-06 1.3E-06 2.6E-06, 3.9E-07 2.0E-06 1.4E-07 2.5E-0/ 1.2E-06 5.5E-06 2.3 3.9 6.5 4.6 0.2 4.9 4.4 36 0.1 co M1032091795-M1441 R232A/G236qH2680/4330K
3.9E-05 3.3E-06 8.02-06, 8.8E-07, 2.6E-06 3.6E-07 60E-07 2.8E-06 8.1E-06, 0.8 1. 2.1 2.0 0.2 1.9 1.8 1.6 0.1 o1 4.
M10320511795-M2442 P23213/0 2361:412680/A330K
4.4E-06 4.2E-06 6.8E-06 1.1E-06 2.1E-06 4.6E-07 4.2E-07 1.8E-06 4.7E-06 0.7 12 2.5 1.E 02 1.5 2.6 2.6 0.2 1 1-=

508-06 835-08 1 7E-06 21E-06 538-07 6.4E-0/ 235-06E 5 9E-06 0.7/ 10 1.9 1 5 01 13 1.7 1.6 0.1 ...J
M103205H795-MY444 P232F/G2369/112680/.43301( 1.2E-06 1.1E-06 1.9E-06 3.0E-07 1.6E-0E, 1.1E-07 1.4E-07 7.4E-07 5.2E-06 2.7 4.6' 8.9 6.0 0.2 6.3 71 6.21 0.2 M10320511795-M1445 p232G/G236T/H2680/A330( 5.6E-06 5.55-0E 1.4E-05 1.4E-06 3.52-0E, 56E-07 1.15-06 4.25-06 9.5E-06 0.6 0.9 1.2, 1.3 0.1 1.2 1.0 1.1 0.1 M103205H795-MY446 P232H/023617142680/A33C1( 2.55-01 2.2E-06 4.7E-06 6.3E-07 2.92-08 2.6E-07 4.8E-07 2.45-06 7.1E-06 1.3 2.3 3.6' 2.5, 0.1 2.7 2.3 1.9 0.1 M103205H795-7,4Y447 P232 VG236T/H2680/8330K
9.9E-07 8.8E-07 1.5E-06 2.3E-07 3.15-CE 9.7E-08 9.8E-08 4.4E-07 4.0E-06 3.2 5.8 11.0 78 0.3 7.1 11.2 10.5 0.2 M10320511795-M8448 P232K/62.362/H2680/83301( 9.6E-06 7.5E-00 2.1E-05 2.35-06 6.5E-06 9.3E-07 2.1E-06 8.45-06 1.3E-05 0.3 0.? 0.8 0.2 0.1 0.7 0.5 0.5 0.1 M103205H795-MY449 P2321/0236r/H2680/8330K
8.7E-07 9.4E-07 1.5E-06 2.3E-07 1.0E-06 9.85-08 7.4E-08 2.9E-07 3.7E-06 3.7 5.4 11.3 7.5 0.4 7.0 14.8 15.8 0.2 M10320S1-t795-M1450 P232M/G236T/1-t268D/A3308 1.3E-06 1.1E-06 2.2E-06, 2.8E-07 1.3E-06 1.3E-07 1.2E-07 4.7E-07 4.0E-06 2.5 4.6 7.7 6.4 0.3 5.3 9.2 9.8 0.2 3.6E-06 4.2E-06 6.7E-06 1.0E-06 2.7E-06 4.1E07 6.45-01 3.05-06 7.1E-06 0.9 1.2 2.5 1.2 0.1 1.7 1.7 1.5 0.1 M1032091795-M1452 P2320./G236T/3-4268Q/833C8( 3.4E-06 3.1E-06 6.5E-06 9.2E-07 2.8E-06 3.6E-07 6.2E-07 2.9E-06 6.1E-06 0.9 1.6 2.2 2.C., 0.1 1.9 1.6 1.6 0.1 M103205H795-Mf453 P2328/62367/112680/43308 6.2E-06 9.9E-06 2.1E-05 2.1E-06 6.5E-06 7.5E-07 1.6E-06 6.5E-06 1.4E-05 0.5 0.5 as 0.9 0.1 0.9 0.7 0.7 0.1 V
M1032091795-M2454 p2325/0236T/H2680/43306 3.85-06 3.1E-06 7.6E-06, 1.0E-05 2.8E-06 4.1E-07 7.6E-07 3.1E06 5.9E-06 01 1.6 2.2 1.E 0.1 1.74 1.4 1.5 0.1 n 3.1E-06 2.92-02 5.4E-06 9.2E-07 2.8E-06 3.1E-07 4.6E-07 2.2E-00 6.6E-06 1.0 1.8 3.1 2.0 0.1 2.2., 2.4' 2.1 0.1 c..z., 1.9E-06 1.7E-06 3.4E-06 5.2E-07 2.0E-06 1.7E-07 2.9E-07 1.1E-0E 5.35-06 1.7 3.8 5.0 3.; 0.2 4.1 3.8 4.; 0.2 V

3.1E-07 8.4E-07 1.2E-06 2.0E-07 1.1E-06 1.2E-07 7.3E-08 3.3E-07 3.6E-06 4.0 6.1 14.2 9.0 0.3 5.1 15.1, 13.9 0.2 1J
r.11032091795-MY458 P232Y/G2367/1-12680/43301( 1.8E-0E 1.65-02 3.0E-06 4.3E-07 1.9E-06 1.6E-07 1.7E-07 9.9E-07 5.3E-06 1.1 3.2 5.7 4.2 0.2 4.3 6.5 4.6 0.2 ,==', CN
.......
6-.0 --I
.4---I

[0586] Although all the variants showed decreased affinity to both human and cynoFc gamma RIM compared to the parent MY201, MY265, which is produced by sub-stituting Asn with Thr at position 236 of MY201, maintained enhanced binding by 2.1-fold for human Fc gamma RIM and 3.1-fold for cynoFc gamma RIM, respectively compared to SG1. MY265 also maintained reduced binding to both human and cynoFc gamma RIIIa. Although affinity to human Fc gamma RIIaH and Fc gamma RIIaR was enhanced by more than 2.5-fold compared to SG1, G236T is the second favorable sub-stitution at position 236.
[0587] In order to improve the selectivity and affinity of MY265 to human Fc gamma RIM, comprehensive mutagenesis study into position 231 and 232 was performed.
Specifically position 231 and 232 of M103205H795-MY265 were substituted with amino acid residues excluding the original amino acid and Cys. The resultant variants were expressed using M103202L889-SK1 as light chain and their affinity to human and cynoFc gamma Rs were evaluated (Table 22). Values of KD fold were calculated by dividing the KD value of SG1 by the KD value of the variant for each Fc gamma R.
[0588] With respect to affinity to Fc gamma RIM, the addition of A23 1T, A231M, A231V, A231G, A231F, A231I, A231L, A231, A231W, P232V, P232Y, P232F, P232M, P232I, P232W, P232L increased binding to both human and cynoFc gamma RIM
compared to parent MY265. Above all, addition of A231T and A231G reduced human Fc gamma RIIaR binding compared to MY265.
[0589] The combination of the substitutions which was not evaluated in the preceding examples were evaluated. The substitutions tested and revealed to be promising were introduced into M103205H795-SG1 in combination. The resultant variants were expressed using M103202L889-SK1 as light chain and their affinity to human and cynoFc gamma Rs were evaluated. Table 23 shows the result and the KD values in cells filled in gray were calculated using [Equation 21 since their affinity was too weak to be correctly determined by kinetic analysis. Values of KD fold were calculated by dividing the KD value of SG1 by the KD value of the variant for each Fc gamma R.
The values for MY209 which was selected as a promising variants in Table 19 were shown again for comparison.
[0590] Among the variants tested, only MY213 showed higher binding affinity to both human and cynoFc gamma RIlb compared to MY209. However, the affinities of MY213 to human Fc gamma RIIaH and human Fc gamma RIIaR are higher than those of MY209.

Na Kinetic analysis of other combinations Z
1(0(M) for cynoFcgRs KD JM) for humanFcgfts ICD fold for cyno . = (561 = 1)07 IC0 fold for humanFcgRs 561= 1) .-.
Heavy thainname Substitutions FcgRI181 Fcgly.132 FcgRilafcgRilb EggRillaSF RilaH EgiRliaR Fccdtlib Fcg1:11118VFcgRila1 cop/0182 f cgRlia3 - lib FcgR11185FcgRilaH Fq3R118R FcgRilb FcgitillaV
4..
MI03205H795-561 3.2E-06 5.1E-06 1.7E-05 1.8E-06 3.6E-07 6.9E-07 1.1E-06 4.6E-06 8.1E-07 1.0 1.0 1. 1.0 1.00 1.0 _ 1.0 1.0 1.00 oe S236N/H2680/Q2951./K326T/A330K 6.3E-07 6.9E-07 1.9E-0; 1.9E-07 2.2E-05 5.7E-07 8.8E-07 5.0E-07 1.4E-05 5.1 7.4 8. 9.5 0.16 1.2 1.3 9.2 0.06 3236E/H268E/P396M 4.1E-00 4.9E-06 2.5E-05 1.8E-06 1.41-07 1.9E-07 1.7E-07 9.5E-07 5.8E-07 0.8 1.0 0. 1.0 2.57 3.6 6.5 4.8 1.40 >

3236.5/H268E/P396M 1.1E-06 1.3E-06 2.9E-06 4,6E-07 2.8E-07 3.7E-0; 8.3E-08, 6.8E-07 1.6E-06 2.9 3.9 5.= 3.9 1.29 18.6 13.3 6.8 0.51 8236M/H2680/A33003968 2.3E-06 2.6E-06 6.4E-06 6.5E-07 2.2E-07 1.5E-07 7.2E-07 2.1E-06 1.1E-06 1.4 2.0 2. 2.8 1.64 2.8 1.5 2.2 0.74 0236H/H268D/A330K/P396K = 1.8E-06 2.1E-06 2.6E-0; 5.3E-07 1.1E-06 7.3E-08 3.7E-07 2.6E-06 3.7E-06 1.8 2.4 6 ' 3.4 0.3; 9.5 3.;
1.8 0.22 3236V/H268D/A330K/P396K 7.6E-07 7.0E-07 3.1E-06 1.9E-07 8.7E-07 4.4E-08 2.3E-07 2.1E-06 2.3E-06 4.2 7.3 5. 9.5 0.41 15.7 4.8 2.2 0.35 32360/H2680/A330K/P3961( 1.7E-06 1.9E-06 6.2E-06 4.3E-07 2.9E-07 9.2E-08 2.5E-07 1.2E-06 8.7E-07 1.; 2.7 2. 4.2 1.24 7.5 4.4 3.8 0.93 32361/H2680/A330K/P3961( 3.7E-06 3.5E-06 1.1E-05 1.2E-06 1.7E-06 5.1E-07 1.0E-06 5.0E-06 9.7E-06 0.9 1.5 1. 1.5 0.21 1.4 1.1 0.9 0.08 p2361/H2680/A330K/P396K 8.8E-07 8.0E-07 2.0E-06 2.3E-07 6.2E-07 8.1E-08 1.7E-07 9.5E-07 2.5E-06 3.6 6.4 8. 7.8 0.58 8.5 6.5 4.8 0.32 02361/H2680/A330K/P396K 1.1E-06 1.1E-06 4.5E-06, 3.1E-07 1.2E-06 5.3E-08 2.51-07/ 2.4E-06 5.7E-06 2.9 4.6 3.: 5.8 0.30 13.0 4.4 1.9 0.14 3236M/H268D/Q29511A3301( 3.0E-06 3.6E-06 1.0E-0 1.1E-06 7.4E-07 5.3E-07 1.5E-06 4.4E-06 3.4E-06 1.1 1.4 1. 1.6 0.49 1.3 0.7 1.0 0.24 M103205H795-MY279 G236H/H2680/Q2951/43301( 2.8E-06 3.3E-06 4.7E- 9.1E-07 2.4E-0; 1.4E-07 7.2E-07, 4.6E-06 7.01..06 1.1 1.5 3.. 2.0 0.15 4.; 1.5 1.0 0.12 0236V/H2680/Q2951/A3301( 1.0E-06 1.2E-06 4.61-Oq 3.3E-07 1.8E-06 8.1E-08 4.5E-071 2.9E-06 4.4E-06 3.2 4.3 3. 5.; 0.20 8.5 2.4 1.6 0.18 0 o 52360/H2680/Q2951/A330K 1.9E-06 2.5E-06 9.1E-0 6.2E-07 7.4E-07 1.3E-07 3.9E-07 1.8E-06 2.1E-06 1.7 2.0 1. 2.9 0.49 5.3 2.8 2.6 0.39 ..., o M103205H795.MY285 12361/H2680/Q2951./A330K 1.6E-06 1.7E-06 6.8E- 5.8E-07 2.9E-0; 1.1E-07 5.6E-07 3.9E-06 1.6E-05 2.0 3.0 2. 3.1 0.12 6.3 2.0 1.2 0.05 0 ro M103205H795-MY213 G236N/H2680/A330K/P396K 5.3E-07 5.2E-07 1.3E- 1.6E-07 1.9E-06 3.5E-07 6.9E-07 3.6E-07 1.1E-05 6.0 9.8 13. 11.3 0.19 2.0 1.6 12.8 0.07 lib ro M1032051-1795-MY242 0238i/H2680/A330K/P3968 4.5E-06 4.2E-06 5.9E- 1.1E-06 5.3E-07 8.2E-07 6.3E-07 1.9E-06 3.2E-06 0.7 1.2 2.= 1.6 0.68 0.8 1.7 2.4 0.25 ro fv1103205H795-MY219 5237Y/H2680/A330K 3.5E-06 4.0E-06 531 1.3E-06 6.2E-06 2.8E-06 1.6E-07 3.4E-07- 9.1E-05 0.9 1.3 3. 1.4 0.06 0.2 6.9 13.5 0.01 1-=

3237Y/H2680/A330K/P396K 1.9E-06 2.5E-06 2.5E-0e 5.1E-07 3.9E-06 1.3E-06 6.2E-08 1.3E-07 1.8E-05 1.7 2.0 6.: 3.5 0.09 0.5 17.7 35.4 0.04 co =

3237Y/H2680/Q2.951/A330K 3.0E-06 3.1E-06 3.61-06, 1.1E-0; 9.7E-06 2.2E-06 1.6E-07 3.01-01 1.4E44 1.1 1.6 4. 1.6 0.04 0.3 6.9 15.3 0.01 o lib =

.3237Y/H268D/0295L/A330K/P396K 1.6E-06 2.2E-06 1.9E-06 4.7E-07 4.2E-06 9.8E-07 8.1E-08 1.5E-07 3.7E45 2.0 2.3 8. 3.8 0.09 0.7 13.6 30.7 0.02 1-=
...1 5237Y/H2680/Q2951./A330K/P396M 1.51-0; 2.6E-06 2.2E-06 4.5E-07 4.0E-06 9.5E-07 7.6E-08. 1.4E-07 2.8E-0; 2.1 2.0 7. 4.0 0.09 0.7 14.;
32.9 0.03 M103205H795.mv293 3237Y/H268E/Q2951/A330K/P396K 2.8E-06 3.3E-06 3.91-0; 8.6E-07 4.2E-06 1.3E-06 7.3E-08 1.5E-07 3.9E-05 1.1 1.5 4. 2.1 0.09 0.5 15.1 30.7 0.02 3236T/1-l268E/A330K 3.3E-06 4.6E-06 1.1E-05 1.5E-06 2.3E-06 4.5E-07 8.6E-07 4.1E-06 8.8E-06 1.0 1.1 1. 12 0.16 1.5 1.3 1.1 0.09 M103205H795-MY309 3236T/14268E/A3308/P396m 2.5E-06 2.5E-06 7.0E 7.1E-07 1.2E-06 1.4E-07 2.6E-07 1.6E-06 4.3E-06, 1.3 2.0 2. 2.5 0.30 4.9 4.2 2.9 0.19 M103205H795-MY310 32361114268E/K3261/A330K 3.0E-06 2.9E-06 7.2E 9.5E-07 1.81-06 4.0E-07 5.5E-07' 2.9E-06 6.91.061 1.1 1.8 2. 1.9 0.20 1.7 2.0 1.6 0.12 M10320514795-Mv311 5236T/m2688,10.2951IA3308 2.3E-06 3.4E-06 8.6E-01 7.8E-07 2.4E-06 2.0E-07 4.5E-071 2.1E-06 7.1E.06j 1.4 1.; 2.4 2.3 0.15 3.5 2.4 2.2 0.11 m103205H795-MY312 32361/H268E/K3261/A330K/P3961v1 1.41-06 1.58.06 3.7E0; 4.0E-07 6.5E-07 1.1E-07 1.7E-071 1.1106 2.81.04 2.3 3.4 4.= 4.5 0.55,. 6.3 6.5 4.2 0.29 M103205H795-MY316 32361./H268E/Q2951./A330K/P396K
5.11-068.61-06 2.2E-05 2.0E-06 2.4E-06 4.4E-07 1.1E-06 4.0E-06 1.7E- 0.6 0.6 0.8 0.9 0.15 1.6 1.0 1.2 0.05 3236L/H268E/K326T/A330K/P396M 7.9E-06 7.7E-0; 3.2E-05 2.2E-06 1.5E-0; 1.11-06. 1.3E-06 4.8E-06 8.7E-0 0.4 0.7 0.5 0.8 0.24 0.6 0.8 1.0 0.09 M103205H795-mv321 3236H/H2680/A33010P396M 2.3E-06 2.2E-06 2.91-06 6.4E-07 1.1E-06 7.1E-08 3.8E-07 2.6E-06 341-06 1.4 2.3 5.9 2.8 0.33 9.7 2.9 1.8 0.24 'CI
M103205=4795-MY325 32360/H2680/A3301( 4.0E-06 4.4E-06 58E-06 9.8E-07 4.0E-06 5.1E-07 3.0E-07 2.6E-07 8.1E-06 0.8 1.2. 2.9 1.8 0.09 1.4 3.7 17.7 0.10 n 32360/H268E/02951./A3301( 3.4E-06 4.8E-0; 5.3E-0; 1.0E-06 4.5E-06 3.5E-07 2.0E-07 1.7E-07 9.9E-06 0.9 1.1 3.2 1.8 0.08 2.0 5.5 27.1 0.08 fv1103205H795-MY329 ,3236D/H2680/0295L/A3301( 1.9E-06 3.1E-06 3.7E-06, 5.9E-07 2.3E-06 2.7E-07 2.1E-07 1.6E-01 6.4E-06 1.7 1.6 4.6 3.1 0.16 2.6 5.21 28.8 0.13 c.....;,..
V

.11 µ7N
........

--I
4..
oe Na Kinetic analysis of other combinations -_______________________________________________________________________________ ______________________________ AD -4 Ko (A) for cynoFcgRs KO JM) for humanFcgRs KD fold for cynoFcls ($61 =1) 1(17 fold for humanFcgRs (G1= 1) Heavychafn name Substitutions Fc4Rli31 fcgRfla2 FcgRlIa3 FcgRilb 'icellfaSIFcgRIIaH FcgRilaR FcgRIlb FcgRIllaWcgRlIa1 FcgRlIa2 FcgRlIa3 cgRfib 7gRilla5FcgliflaH FcgRlIaR FcgRilb FcgRIllaNi fv1103205H795-9G1. 3.2E-06 5.1E-06 1.7E-05 1.8E-06 3.6E-07 6.9E-07 1.1E-06 4.6E-06 8.1E-07 1.0 1.0 1.0 1.0 1.00 10 1.0 1.0 1.00 oe 6236N/H2680/02951/1(326T/A330K 6.3E-07 6.9E-07 1.9E-06 1.9E-07 2.2E-06 5.7E-07 8.8E-07 5.0E-07 1.4E-05 5.1 7.4 8.9 9.5 0.16 1.2k 1.3 9.2 0.06 t.)..2 f=J

G2360/H2680/Q2951./A3300/P3961C 4.3E-06 4.9E-06 2.3E-05 1.1E-06 2.3E-06 3.1E-07 8.3E-07 3.3E-06 1.7E-05 0.7 1.0 0.7 1.6 0.16 2.1 1.3 1.4 0.05 ed ,-, 6236L/H26130/02951/K3267/A330K/0396K 2.6E-06 2.9E-06 8.8E-06 6.0E-07 9.4E-07 2.6E-07 4.2E-07 1.8E-06 7.0E-06 1.1 1.8 1.9 3.0 0.38 2.7 2.6 2.6, 0.12 G2361./H2680/0295t/A3300396M 5.1E-06 8.5E-06 5.1E-05 1.3E-06 2.6E-06 3.7E-0i 1.0E-06 3.9E-06 2.5E-05 0.6 0.0 0.3 1.4 0.14 1.9 1.1 1.2 0.03 G236T/H2680/029511A330K 1.4E-06 1.6E-06 4.4E-06 4.2E-07 2.2E-06 1.4E-07 3.5E-07 1.80-0E 5.7E-06 2.3 3.2 3.9 4.3 0.16 4.9 3.1 2.6 0.14 M103205H795-MY339 G2364/H2680/K326T/A3300396K 3.4E-06 3.0E-06 9.8E-06 7.5E-07 7.6E-07 4.1E-07 6.3E-07 2.8E- 5.6E-06 0.9 1.7 1.7 2.4 0.47 1.7 1.7 1.6 0.14 6236N/H2.68E/4330K/P396K 9.3E-07 1.0E-06 3.3E-06 2.9E-07( 2.8E-06 4.5E-07 8.5E-07 4.8E-0 1.0E-05 3.4 51 5.2 6.2 0.13 1.5 1.3 9.6 0.00 1Y11032C15H795-MY386 G2361/H2680/02951./K32617A330K 5.2E-06 5.6E-06 1.6E-05 1.6E-06 2.5E-06 8.1E-07 1.7E-06 4.6E 2.4E-05 0.6 0.9 1.2 1.1 0.14 0.9 0.6 1.0 0.03 f4103205H795-MY3138 62367152391/H2680/A33471( 2.2E-06 1.7E-06 1.3E-06 7.6E-07 1.6E-06 3.3E-07, 2.3E-07 1.5E- 4.3E-06 1.!
3.0 13.; 2.4 0.23 2.1 4.8 3.1 0.19 G2367/92391/442680/A3301( . 2.0E-06 1.60.04 1.4E-06 7.0E-07 1.5E-06 2.7E-07 1.9E-07 L1E-0 3.5E-06 LE 3.2 12.1 2.6 0.24 2.6 5.4 4.2 0.23 6236T/H2680/A3308/P3321 1.8E-06 2.2E-06 3.8E-06 6.3E-07 2.4E-06 4.2E-07 7.4E-07 2.5E-Oe' 7.80-06 1.8 2.3 41 2.9 0.15 1.6 1.5 1.8 0.10 6236111-12680/52981./A330K 3.0E-07 2.3E-045 3.9E-03 7.0E-07 4.0E-06 4.3E-07 4.1E-07 1.7E-06 9.3E-06 10.7 2.2 4.4 2.6 0.09 1.6 2.7 2.7 0.09 G2367/5239V/H2680/A330K 2.3E-06 1.9E-06 1.4E-06 8.3E-07 2.5E-06 2.4E-07 2.0E-07 1.6E-06 4.7E-06 14 2_7 12.1 2.2 0.14 2.9 51 2.9 0.17 0 G236115239V/H2680/5298L/A3301( 2-6E-07 1.6E-06 1.5E-06 7.6E-07 4.4E-06 3.5E-07, 1.6E-07 6.9E-07 8.8047c 12.3 3.2 11.3 2.4 0.08 2.0 6.9 6.7 0.09 0 6236r/H2680/02951../s298L/A3308 1.4E-07 1.6E-06 2.9E-06 3.9E-07 3.8E-06 2.2E-07 2.6E-07, 1.2E 06 9.0E-06 22.9 3.2 5.9 4.6 0.09 3.1 4.2 3.8 0.09 1..i G2367/5239V/H2680/Q2951/52981./A330K 11E-07 1.3E-06 7.9E-07 4.5E-07 3.0E-06 2.2E-07 8.5E-08 5.1E-07 6.7E-06 21.3 3.9 211 4.0 0.12 3.1 12.9 9.0 0.12 0 i.
0.

P2320/G2367/5239V/H268D/C12951./A330K 2.5E-06 23E-06 2.1E-06 9.6E-07 2.0E-06 4.9E-07 3.4E-0Y 1.4E-0E 310-06 1.3 2.2 8.1 1.9 0.18 1.4 3.2 3.3 0.23 r.
r.

P2320/G236T/H2680/Q2951/A3301( 2.5E-06 3.3E-06 4.40-06 8.0E-07 2.2E-06 2.9E-07 3.0E-07 1.5E-06 5.10-0E 1.3 1.5 3.9 2.3 0.16 2.4 3.7 3.1 0.16 -A2326/0236T/5239WH2680/A330k 1.7E-06 1.2E-06 9.3E-07 5.3E-07 1.9E-06 2.4E-07 1.7E-07 1.10-06 5.70-06 1.9, 4.3 18.3 34 0.19 2.9 6.5 4.2 034 ,... t...) A2316/0236119239v/H2680/Q2951./A330K 1.4E-06 1.3E-06 1.10-06 4.8E-07 2.1E-06 1.9E-07 2.1E-07 2.1E-06 5.5E-06 2.3 3.9 15.5 3.8 0.17 IC 5.2 4.2 0.15 co A2316/6236T/H2680/C12951/A330K 1.0E-06 1.1E-06 2.7E-06 3.1E-07 2.0E-06 1.1E-07 2.8E-07 1.0E-06 4.5E-06 3.2 4.6 6.3 51 0.18 6.3 3.9 4.6 0.11 0.

A231T/G2361/5239WH2680/A3301( 1.6E-06 1.1E-06 1.1E-06 5.1E-07 1.4E-06 2.1E-07 1.60-07 1.2E-06 4.30-06 2.0 4.6 15.5 3.5 0.26 3.3 6.9 31 0.19 1-=
...]

A2311102367/9239v/H268D/Q2951IA3301( 1.3E-06 2.2E-06 1.3E-0e 4.6E-07 1.6E-06 1.7E-07 2.1E-07 1.20-0e 4.3E-0e 2.5 4.3 13.3 3.9 0.23 4.1 5.2 3.8 0.19 A.410320514795-mY527 A2311102367/H268D/Q295LIA330K 1.1E-06 1.30-06 3.4E-OÃ 3.4E-02 1.5E-06 1.1E-07 2.9E-07 1.4E-06 4.5E-06 2.3 3.9 5.0 5.3 0.24 6.3 3.9 3.3 0.18 1.234w/02367/H2660/A3308 1.4E-06 11E-06 3.2E-0E 4.1E-07 2.0E-06 3.5E-07 5.6E-07 2.2E-06 4.2E-06 2.3 34 5.3 4.4 0.18 2.0 2.0 2.1 0.19 1.234W/G2367/142680/Q2951./A330K 1.0E-06 1.4E-06 3.2E-0E 3.0E-07 2.2E-06 2.3E-07, 5.3E-02 1.4E-06 4.0E-06 3.2 3.6 5.3 6.0 0.16 3.cµ
2.1 3.3 0.20 M10320541795-8.47530 G236T/H2680/1C3261/A3301( 1.30-06 1.10-062.5E-06 3.2E-07 8.6E-07 1.7E-07 27E47 1.6E-06 3.6E-06 2.5 4.6 6.a 5.6 0.42 4.2 4.2 2.9 0.23 1.234W/G2361/H2680/K326T/A330K 1.1E-06 9.2E-07 2.2E-0C 2.7E-07 1.0E-06 2.9E-07 4.1E-07 1-5E-Of 2.9E-0e 2.9 5.5 7.7 6.7 0.36 2.4 2.7 3.1 0.28 1234W/G2367/5239v/H268D/A330K 7.7E-07 6.0E-07 7.4E-07 2.3E-07. 8.6E-07 2.1E-07 1.9E-01 8.3E-07 1.7E-0e 4.2 8.5 234 71 0.42 3.3 5.8 5.1 0.48 1.234w/G2367/5239v/1-42680/42951/A330K 8.0E-07 7.3E-07 1.20-0E 2.5E-07 1.1E-00 2.1E-07 2.0E-07 91E-01 1.80-06 4.0 7.0 14.1 7.2 0.33 3.3 5.5 44 0.45 62367/9239V/H2680/02951JA330K 1.3E-06 1.3E-06 1.2E-06 4.26-0/ 1.7E-06 1.7E-07 1.9E-07 1.28-06 4.3E-06 2.5 3.9 14.2 4.3 0.21 4.1 5.8 3.8 0.19 A2316/L234w/6236T/H2680/A330K 13E-06 1.4E-06 3.00-06 4.1E-07 2.1E-06 3.9E-07 6.4E-07 1.9E-06 5.2E-06 2.1 3.6 5.7 4.4 0.17 1.8 1.7 2.4 0.16 A2310,4234w/G236115239v/H7.68o/A330K 1.2E-06 1.1E-06 9.8E-07 4.0E-07 1.6E-06 2.9E-07 2.2E-07 9.9E-07 2.7E-0e 2.7 4.6 17.3 4.% 0.23 2.4 5.0 4.4 0.30 'V

A231O/L234w/G236119239v/H2680/0295t./A3301( 1.2E-06 1.1E-06 1.40-06 3.2E-07 1.5E-0tµ 2.8E-07 3.7E-07 1.0E-06 3.3E-0e 2.1 4.6 121 5.6 0.24 2.5 3.0 4.6 0.25 n A2310.234W/13236T/H26817/02951./A330K 9.6E-07 1.1E-06 3.0E-0E, 2.8E-07 2.4E-06 21E-07 6.2E-07 1.3E-06 5.7E-0e, 3.3 4.0 5.7 64 0.15 2.8 LEI 3.5 0.14 c...;,..
'V
Na ill µ7N
-., --I

oe Binding profile of the variants tested in all human FcyR transgenic mice _______________________________________________________________________________ ________________________________ Cr' KO (M.) forcynoFcgRs KRIM) for humanaces KD fold for cynoFtgais (5G1 o 1) KO fold for humanf cgRs (S.G1= 11_ IF' Heavy chain name pi 5E010 NO Substitutions FoKR1141 FotRila2 '0.011143 Fcitlillb FoKRIINSFcgRilaf)cKRIHR acKRIlb Fc6.RIllaVac9R1141 FatRiia2 ceRlIa3pcaRIlb cgRillaSscaffilati FcactIlaR
Fcatillb km61110 ts..) M10320514795-501 ,S01 9 = 3.2E-00 5.1E-06 1.7E-05 1.8E-06 3.6E-07 6.9E-07 1.1E-06 4.6E-00 8.1E-o, 1.0 1.0 1.0 1.0 1.00 1.0 1.o, 1.0 1.00 oc (44 62366/14268E/43300/P396M 1.1E-06 1.2E-06 3.6E-06 3.5E-07 3.2E-06 4.99-07!
8.7E-07 4.9E-07 1.1E-05 2.9 4.3 4.7 5.1 0.11 1.4 1.3' 9.1 0.07 0236N/H268E/n3301(/P39661/54006/0413K 1.39-06 1.3E-06 3.8E-06 3.88=0? 3.9E-06 5.7E-07 1.1E-06 5.9E-07 1.3E-05 2.5 3.9 4.5 4.7 0.09 12 1.0 7.0 0.06 M103205H795-MY201 -MY201 231 p236N/H268D/A330K 1.4E-06 1.4E-06 3.9E-06 4.3E-07 5.78- 9.1E-07 1.9E-06 1.10-00 2.4E-05 2.3 3.6 4.4 4.2 0.06! 0.8 0.0 4.21 0.03 p236N/H2680/Q311R/A330100413K 9.2E-07 7.6E-07 1.9E-06 3.5E-07 5.2E- 1.0E-06 1.6E-06 1.2E-00 1.4E-05 3.5 6.7 8.9 5.1 0.07 0.7 0.?
3.6 0.06 G236N/H268olc295tiA3300 9.0E-01 1.1E-06 3.0E-06 2.5E-07 6.0E- 4.7E-07 1.3E-06 5.6E-07 1.3E-05 3.6 4.6 5.7 7.2 0.06 1.5 0.8 8.2f 0.06 234 A2366/112680/0.295L/Q311R/A330A/0413K 8.9E-07 1.1E-06, 2.2E-06 3.1E-07 4.9E-0A 6.7E-07 1.29-06, 7.2E-07 1.9E-05 3.6 4.6 7.7 5.8 0.07 1.0 0.9 6.4 0.04 f6l10320514795-MY335 MY335 235 G2361/H26817/Q311R/A3300/D413K 2.00-06 1.7E-06 2.7E-06 6.5E-07 1.59-00 2.07 2.9E-07 2.38-129. 5.0E-06 1.6 3.0 6.3 2.8 0.24 2.4 3.E, 2.0 0.16 I.
6.6 Example 28 [0591] Evaluation of clearance of myostatin using Fc gamma Ruth-enhanced Fc variants in all human Fc gamma R transgenic mice The effect of clearance of myostatin by Fc gamma Ruth-enhanced Fc variant con-structed in Example 27 was evaluated in a mouse in which all murine Fc gamma Rs have been deleted and human Fc gamma Rs, encoded as transgenes, have been inserted into the mouse genome (Proc. Natl. Acad. Sci., 2012, 109, 6181). In this mice all mouse Fc gamma Rs are substituted with human ones so that the effect of affinity en-hancement to human Fc gamma Ruth on clearance of soluble antigen can be evaluated in mouse. Furthermore, p1-increasing substitutions were evaluated in combination with Fc gamma RHb-enhanced Fc variants constructed in Example 27.
Preparation and profile of tested variants [0592] The tested 8 antibodies and their binding profiles are summarized in Table 24. The heavy chain, MS103205H795-PK2, was prepared by introducing p1-increasing sub-stitutions (S400R/D413K) into MS103205H795-MY101. MS103205H795-MY351, MS103205H795-MY344, MS103205H795-MY335 were prepared by introducing another p1-increasing substitutions (Q311R/D413K) into MS103205H795-MY201, MS103205H795-MY205, MS103205H795-MY265, respectively. All the M51032L006 variants were expressed with M103202L889-SK1 as light chain according to the method shown in Example 27 and their affinities to human and cynoFc gamma Rs were evaluated using the method in Example 27.
[0593] Based on the SPR analysis summarized in Table 24, it was confirmed that the p1-increasing substitutions do not affect the Fc gamma R binding of Fc gamma Ruth-enhanced Fc variants. MY101 and PK2, which was prepared by introducing 5400R/D413K into MY101, showed 9-fold and 8-fold enhanced human Fc gamma RHb binding, respectively. The affinities of MY101 and PK2 to human Fc gamma RIIa were remained comparable to SG1. With respect to other human and cynoFc gamma Rs, they showed almost the same binding profile. Similarly, MY351 showed similar binding profile with that of parent MY201, MY344 with that of parent MY205, and MY335 with that of parent MY265 (Table 21), respectively. These results indicate that either pair of p1-increasing substitutions, 5400R/D413K or Q311R/D413K, does not affect the affinities to human and cynoFc gamma Rs.
PK study in all human Fc gamma R transgenic mice In vivo test using all human Fc gamma R transgenic mice [0594] The elimination of myostatin and anti-latent myostatin antibody were assessed in vivo upon co-administration of anti-latent myostatin antibody and human latent myostatin in all human Fc gamma R transgenic mice (Figures 24 and 25). An anti-latent myostatin antibody (0.3 mg/ml) and latent myostatin (0.05 mg/ml) were ad-ministered at a single dose of 10 ml/kg into the caudal vein. Anti-CD4 antibody (1 mg/
ml) was administered three times (every 10days) at a dose of 10 ml/kg into the caudal vein to suppress anti-drug antibody. Blood was collected at 5 minutes, 15 minutes, 1 hour, 4 hours, 7 hours, 1 day, 2 days, 7 days, 14 days, 21 days, and 28 days after ad-ministration. The collected blood was centrifuged immediately at 15,000 rpm in degrees C for 5 minutes to separate the plasma. The separated plasma was stored at or below -20 degrees C until measurement. The anti-latent myostatin antibodies used were MS1032L006-SG1, MS1032L006-MY101, MS1032L006-PK2, MS1032L006-MY201, MS1032L006-MY351, MS1032L006-MY205, MS1032L006-MY344 and MS1032L006-MY335.
Measurement of total myostatin concentration in plasma by electrochemiluminescence (ECL) [0595] The concentration of total myostatin in mouse plasma was measured by ECL. Anti-mature myostatin antibody-immobilized plates were prepared by dispensing anti-mature myostatin antibody RK35 (WO 2009058346) onto a MULTI-ARRAY 96-well plate (Meso Scale Discovery) and incubated overnight at 4 degrees C. Mature myostatin calibration curve samples and mouse plasma samples diluted 4-fold or more were prepared. The samples were mixed in an acidic solution (0.2 M Glycine-HC1, pH2.5) to dissociate mature myostatin from its binding protein (such as propeptide).
Subsequently, the samples were added onto an anti-mature myostatin antibody-im-mobilized plate, and allowed to bind for 1 hour at room temperature before washing.
Next, BIOTIN TAG labelled anti-mature myostatin antibody RK22 (WO
2009/058346) was added and the plate was incubated for 1 hour at room temperature before washing. Next, SULFO TAG labelled streptavidin (Meso Scale Discovery) was added and the plate was incubated for 1 hour at room temperature before washing.
Read Buffer T (x4) (Meso Scale Discovery) was immediately added to the plate and signal was detected by SECTOR Imager 2400 (Meso Scale Discovery). The mature myostatin concentration was calculated based on the response of the calibration curve using the analytical software SOFTmax PRO (Molecular Devices). The time course of total myostatin concentration in plasma after intravenous administration of anti-latent myostatin antibody and latent myostatin measured by this method is shown in Figure 24.
Measurement of anti-latent myostatin antibody concentration in plasma by high-performance liquid chromatography-electrospray tandem mass spectrometry (LC/ESI-MS/MS) [0596] Anti-latent myostatin antibody concentration in mouse plasma was measured by LC/
ESI-MS/MS. The concentrations of calibration standards were 1.56 25, 3.125, 6.25, 12.5, 25, 50, 100, and 200 micro g/mL in mice plasma. Three micro L of the cal-ibration standards and plasma samples were added to 50 micro L of Ab-Capture Mag (ProteNova), and allowed to incubate for 2 hours at room temperature.
Afterward, the magnetic beads were recovered from samples and washed twice with 0.2 mL of 10 mmol/L PBS with 0.05% Tween 20. Subsequently, the magnetic beads were washed with 10 mmol/L PBS to ensure the removal of Tween 20. After washing, the magnetic beads were suspended in 25 micro L of 7.5 mol/L Urea, 8 mmol/L dithiothreitol and 1 micro g/mL lysozyme (chicken egg white) in 50 mmol/L ammonium bicarbonate and the suspended samples were incubated for 45 minutes at 56 degrees C. Then, 2 micro L
of 500 mmol/L iodoacetoamide was added and the samples were incubated for 30 minutes at 37 degrees C in the dark. Next, Lysyl Endopeptidase digestion was carried out by adding 150 micro L of 0.67 micro g/mL Lysyl Endopeptidase for Biochemistry (Wako) in 50 mmol/L ammonium bicarbonate and the samples were incubated for 3 hr at 37 degrees C. Subsequently, tryptic digestion was carried out by adding 10 micro L
of 10 micro g/mL sequencing grade modified trypsin (Promega) in 50 mmol/L
ammonium bicarbonate. Samples were allowed to digest under mixing overnight at degrees C, and quenched by adding 5 micro L of 10% trifluoroacetic acid. Fifty micro L of digestion samples were subjected to analysis by LC/ESI-MS/MS. LC/ESI-MS/MS
was performed using Xevo TQ-S triple quadrupole instrument (Waters) equipped with 2D I-class UPLC (Waters). Anti-latent myostatin antibody specific peptide YAFGQGTK and Lysozyme specific peptide GTDVQAWIR as an internal standard were monitored by the selected reaction monitoring (SRM). SRM transition was [M+2f112+ (m/z 436.2) to y8 ion (m/z 637.3) for anti-latent myostatin antibody, and [M+2f112+ (m/z 523.3) to y8 ion (m/z 545.3) for lysozyme. Internal calibration curve was constructed by the weighted (1/x or 1/x2) linear regression using the peak area plotted against the concentrations. The concentration in mouse plasma was calculated from the calibration curve using the analytical software Masslynx Ver.4.1 (Waters).
The time course of antibody concentration in plasma after intravenous administration of anti-latent myostatin antibody and latent myostatin measured by this method is shown in Figure 25.
Effect of pI and Fc gamma R binding on myostatin concentration in vivo [0597] After administration of M51032L006-SG1, plasma total myostatin concentration at 7 hours decreased 5 fold compared to plasma total myostatin concentration at 5 minutes. In contrast, after administration of M51032L006-MY101, M51032L006-MY201 and M51032L006-MY205, plasma total myostatin con-centration at 7 hours decreased 28-200 fold compared to plasma total myostatin con-centration at 5 minutes. Furthermore, after administration of MS1032L006-PK2, MS1032L006-MY351, MS1032L006-MY344 and MS1032L006-MY335, plasma total myostatin concentration at 7 hours decreased 361-419 fold compared to plasma total myostatin concentration at 5 minutes. On the other hands, the difference in each antibody's concentration at each sampling point compared to MS1032L006-SG1's was approximately within 2 fold, and pI variants did not increase antibody elimination from plasma. Both human Fc gamma Ruth binding enhanced antibodies and p1-increasing substitutions increased elimination of myostatin, but not antibodies.
High pI variants have more positive charge in plasma. Since this positive charge interacts with cell surface of negative charge, antigen-antibody immune complex of high pI variants get closer to cell surface, resulted in increased cellular uptake of antigen-antibody immune complex of high pI variants.
Example 29 [0598] Evaluation of clearance of myostatin using Fc gamma Ruth-enhanced Fc variants in monkey The effect of enhanced binding of Fc variants developed in Example 27 to cynoFc gamma Ruth on myostatin sweeping was evaluated in cynomolgus monkey. And the combination effect with p1-increasing substitutions were also evaluated.
Preparation and profile of tested variants [0599] The tested 14 antibodies and their binding profiles are summarized in Table 25. It has been reported that Fc variant with enhanced binding to FcRn at acidic pH
improve the antibody half-life in vivo (J. Biol. Chem. 2006 281:23514-23524 (2006); Nat.
Biotechnol. 28:157-159 (2010), Clin Pharm. & Thera. 89(2):283-290 (2011)). To improve the antibody half-life without binding to rheumatoid factor we combined these substitutions with Fc gamma RHb enhanced and p1-increased Fc variants.
[0600] The heavy chain, M5103205H795-5G1012, M5103205H795-5G1029, MS103205 H795-SG1031, MS103205H795-SG1033, MS103205H795-SG1034 were prepared by introducing a N434A substitution into M5103205H795-MY101, MS103205 H795-MY344, MS103205H795-MY351, MS103205H795-MY201, M5103205H795-MY335, respectively. M5103205H795-5G1016 was prepared by in-troducing p1-increasing substitutions (Q311R/D399K) into M5103205H795-5G1012.
M5103240H795-5G1071 and M5103240H795-5G1079 were prepared by introducing M428L/N434A/Y436T/ Q438R/5440E substitutions and N434A/Q438R/5440E sub-stitutions into MS103240 H795-MY344 respectively (M5103240H795: VH, SEQ ID
NO; 92).
[0601] M5103240H795-5G1074 and M5103240H795-5G1077 were prepared by in-troducing p1-increasing substitutions Q311R/P343R and M428L/N434A/Y436T/Q438R/5440E substitutions into MS103240H795-MY209 and MS103240H795-MY518, respectively. MS103240H795-SG1080 and MS103240H795-SG1081 were prepared by introducing p1-increasing substitutions Q311R/P343R and N434A/Q438R/S440E substitutions into MS103240H795-MY209 and MS103240H795-MY518, respectively. MS103240H795-SG1071 was prepared by introducing p1-increasing substitutions Q311R/D413K and M428L/N434A/Y436T/Q438R/5440E substitutions into MS103240H795-MY205.
MS103240H795-SG1079 was prepared by introducing p1-increasing substitutions Q311R/D413K and N434A/Q438R/S440E substitutions into MS103240H795-MY205.
These M51032L006 variants and M51032L019 variants were expressed with M103202L889-SK1 and M103202L1045-SK1 respectively (M103202L1045: VL, SEQ ID NO: 97) as light chain according to the method shown in Example 34 and their affinities to human and cynoFc gamma Rs were evaluated using the method in Example 27. In this example, the value of KD fold for each Fc variant was calculated by dividing the KD value of the parent SG1 by the KD value of the variant for each Fc gamma R. For example, the values of KD fold for M51032L006-5G1012 and M51032L019-5G1071 were calculated by divinding the KD values of M51032L006-SG1 and M51032L019-SG1 by the KD values of MS1032L006-SG1012 and MS1032L019-SG1071 respectively.

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[0602] The SPR analysis results are summarized in Table 25. Among these variants, SG1012, SG1016, SG1074 and SG1080 show the strongest affinity to human Fc gamma RIM, which has 10 fold enhanced affinity to human Fc gamma RIM compared to SG1.
29.2. PK study in monkey In vivo test using cynomolgus monkey [0603] The accumulation of endogenous myostatin was assessed in vivo upon administration of anti-latent myostatin antibody in 2-4 year old Macaca fascicularis (cynomolgus monkey) from Cambodia (Shin Nippon Biomedical Laboratories Ltd., Japan). A
dose level of 30 mg/kg was injected into the cephalic vein of the forearm using a disposable syringe, extension tube, indwelling needle, and infusion pump. The dosing speed was 30 minutes per body. Blood was collected before the start of dosing and either minutes, 7 hours and 1, 2, 3, 7, 14, 21, 28, 35, 42, 49 and 56 days after the end of dosing, or 5 minutes and 2, 4, and 7 hours and 1, 2, 3, 7, 14, 21, 28, 35, 42, 49 and 56 days after the end of dosing. Blood was drawn from the femoral vein with a syringe containing heparin sodium. The blood was immediately cooled on ice, and plasma was obtained by centrifugation at 4 degrees C, 1700 x g for 10 minutes. The plasma samples were stored in a deep freezer (acceptable range: -70 degrees C or below) until measurement. The anti-latent myostatin antibodies used were M51032L000-SG1, MS1032L006-SG1012, MS1032L006-SG1016, MS1032L006-SG1029, MS1032L006-SG1031, MS1032L006-SG1033, and MS1032L006-SG1034 (herein, 5G1012, 5G1016, 5G1029, 5G1031, 5G1033, and 5G1034 are the heavy chain constant regions constructed based on SG1 as described below).
[0604] A dose level of 2mg/kg was administered into the cephalic vein of the forearm or saphenous vein using a disposable syringe, and indwelling needle for the anti-latent myostatin antibodies MS1032L019-SG1079, MS1032L019-SG1071, MS1032L019-SG1080, MS1032L019-SG1074, MS1032L019-5G1081, and M51032L019-5G1077 (herein, 5G1079, 5G1071, 5G1080, 5G1074, 5G1081, and 5G1077 are the heavy chain constant regions constructed based on SG1 as described below). Blood was collected before the start of dosing and 5 minutes and 2, 4, and 7 hours and 1, 2, 3, 7, 14 days after the end of dosing. The blood was processed as described above. Plasma samples were stored in a deep freezer (acceptable range: -70 degrees C or below) until measurement.
Measurement of total myostatin concentration in plasma by electrochemilumi-nescence (ECL) [0605] The concentration of total myostatin in monkey plasma was measured by ECL as described in Example 23. The time course of plasma total myostatin concentration after intravenous administration of anti-latent myostatin antibody as measured by this method is shown in Figure 26.
Measurement of ADA in monkey plasma using electrochemiluminescence (ECL) [0606] Biotinylated drug was coated onto a MULTI-ARRAY 96-well streptavidin plate (Meso Scale Discovery) and incubated in low cross buffer (Candor) for 2 hours at room temperature. Monkey plasma samples were diluted 20 fold in low cross buffer before addition to the plate. Samples were incubated overnight at 4 C. On the next day, the plate was washed three times with wash buffer before addition of SULFO TAG

labelled anti monkey IgG secondary antibody (Thermo Fisher Scientific). After in-cubating for an hour at room temperature, the plate was washed three times with wash buffer. Read Buffer T (x4) (Meso Scale Discovery) was immediately added to the plate and signal was detected using a SECTOR Imager 2400 (Meso Scale Discovery).
Effect of pH-dependent and Fc engineering on myostatin accumulation in monkey in vivo [0607] In cynomolgus monkey, administration of non pH-dependent antibody (M51032L000-SG1) resulted in at least 60 fold increase in myostatin concentration from baseline at day 28. At day 28, pH-dependent anti-latent myostatin antibodies M51032L006-5G1012 and M51032L006-5G1033 resulted in 3 fold and 8 fold increase from baseline respectively. The strong sweeping was mainly contributed by the increase in affinity to cynomolgus monkey Fc gamma RIIb. At day 28, pH-dependent anti-latent myostatin antibodies MS1032L006-SG1029, M51032L006-5G1031 and M51032L006-5G1034 could sweep antigen to below baseline. The reason for strong sweeping of M51032L006-5G1029, M51032L006-5G1031 and M51032L006-5G1034 are an increase in non-specific uptake in the cell due to increase in positive charge cluster of the antibody and an increase in Fc gamma R-mediated cellular uptake due to enhanced binding to Fc gamma R.
[0608] Administration of pH-dependent anti-latent myostatin antibodies MS1032L019-SG1079, MS1032L019-5G1071, MS1032L019-SG1080, MS1032L019-SG1074, MS1032L019-5G1081 and MS1032L019-SG1077 reduced myostatin concentration to below the detection limit (<0.25ng/mL) from day 1 in cynomolgus monkey. On day 14, the concentration of myostatin increased above the detection limit for M51032L019-5G1079 and M51032L019-5G1071 while the con-centration of myostatin remained below the detection limit for M51032L019-5G1080, MS1032L019-SG1074, MS1032L019-5G1081 and MS1032L019-SG1077. The weaker suppression of MS1032L019-SG1079 and MS1032L019-SG1071 could be due to difference in pI mutations.
[0609] The data suggests that strong sweeping of myostatin from the plasma could be achieved by mutations that increase binding to Fc gamma RIIb or combining mutations that increase positive charge of the antibody and increase binding to Fc gamma RIM. It is expected that strong sweeping of myostatin could be achieved in human by combining mutations that increase positive charge of the antibody and increase binding to Fc gamma R.
Example 30 [0610] Screening of p1-increased substitutions to enhance clearance of myostatin.
To enhance the clearance of myostatin, pI increased substitutions in Fc portion of antibody were evaluated in this example. The method of adding amino acid sub-stitutions to the antibody constant region to increase pI is not particularly limited, but for example, it can be performed by the method described in WO 2014/145159. As in the case with the variable region, amino acid substitutions introduced into the constant region are preferably those that decrease the number of negatively charged amino acids (such as aspartic acid and glutamic acid) while increasing the positively charged amino acids (such as arginine and lysine). Furthermore, amino acid substitutions may be in-troduced at any position in the antibody constant region, and may be a single amino acid substitution or a combination of multiple amino acid substitutions.
Without particular limitation, the sites for introducing amino acid substitutions are preferably positions where amino acid side chains may be exposed on the antibody molecule surface. Particularly preferable examples include the method of introducing a com-bination of multiple amino acid substitutions at such positions that may be exposed on the antibody molecule surface. Alternatively, the multiple amino acid substitutions in-troduced here are preferably positioned so that they are structurally close to each other.
Furthermore, without particular limitation, the multiple amino acid substitutions in-troduced herein are preferably substitutions to positively charged amino acids, so that preferably they result in a state where multiple positive charges are present at structurally proximal positions.
Preparation and profile of tested variants [0611] The tested antibodies are summarized in Table 26. The heavy chain, MS103205H795-SG141 was prepared by introducing p1-increasing substitutions Q311R/D399R into M5103205H795-SG1. Other heavy chain variants were also prepared by introducing respective substitutions represented in Table 26 into M5103205H795-SG1. All the M51032L006 variants were expressed with M103202L889-SK1 as light chain according to the method shown in Example 34.
Mouse Fc gamma RI-binding assay of p1-increased Fc variants using BIACORE
(registered trademark) [0612] Regarding the produced Fc region variant-containing antibodies, binding assays between soluble mouse Fc gamma Rh and antigen-antibody complexes were performed using BIACORE (registered trademark) T200 (GE Healthcare). Soluble mouse Fc gamma RII was produced in the form of a His-tagged molecule by a method known to those skilled in the art. An appropriate amount of an anti-His antibody was fixed onto Sensor chip CM5 (GE Healthcare) by the amine coupling method using a His capture kit (GE Healthcare) to capture mouse Fc gamma RII. Next, an antibody-antigen complex and a running buffer (as a reference solution) were injected, and in-teraction was allowed to take place with the mouse Fc gamma Rh captured onto the sensor chip. 20 mM N-(2-Acetamido)-2-aminoethanesulfonic acid, 150 mM NaC1, 1.2 mM CaC12, and 0.05% (w/v) Tween 20 at pH7.4 was used as the running buffer, and the respective buffer was also used to dilute the soluble mouse Fc gamma RII.
To re-generate the sensor chip, 10 mM glycine-HC1 at pH1.5 was used. All measurements were carried out at 25 degrees C. Analyses were performed based on binding (RU) calculated from sensorgrams obtained by the measurements, and relative values when the binding amount of SG1 was defined as 1.00 are shown. To calculate the pa-rameters, the BIACORE (registered trademark) T100 Evaluation Software (GE
Healthcare) was used [0613] The SPR analysis results are summarized in Table 26. A few Fc variants were shown to have enhanced affinity toward mouse Fc gamma Rh fixed on the BIACORE
(registered trademark) sensor chip.
[0614] While not being restricted to a particular theory, this result can be explained as follows. The BIACORE (registered trademark) sensor chip is known to be negatively charged, and this charged state can be considered to resemble the cell membrane surface. More specifically, the affinity of an antigen-antibody complex for mouse Fc gamma Rh fixed onto the negatively charged BIACORE (registered trademark) sensor chip is surmised to resemble the manner in which the antigen-antibody complex binds to mouse Fc gamma Rh present on a similarly negatively charged cell membrane surface.

[Table 26]
Summary of pi-increased Fc variants lie-asy chain name Sub atimtione cellular uptake biacore IC binding M10320511795-601 1.00 1.00 M10320511795-30141 ' Q3118/13399E 4.42 1.64 11103205E19541375m Q311111110413K 3.43 1.21 M1032051179541378= 3,. 400R/1)413K 3.86 1.23 11103205H79541383m Q311R34001D413K . 5.68 1.41 11103205H795-P1484m Q3111143312R 0.00 ' 1.33 111032051179541485m Q31111`N31 all 1.88 1.28 M1032051179541486m Q31111/N315K 1.52 1.20 M103205R79541487ro ' Q311E4.1384.11 1.23 1.46 11103205r179541488zu ' Q311E.V384X 1.45 1.43 M1032051179541489= Q311E.S3 ISE 1.68 1.24 1110320511795-P1490m Q311E:VI8K 1.33 1.28 111032061179541491zu Q311R/33311 1.37 Los 11103200795-P1492m Q31111(E333K 1.13 0.38 /II 032051479541493m Q3111T335E 0.09 1.10 M103205H795-P1494m Q311/1/11351t 1.12 1.14 M103205/179541495m Q31114,8337E 1.76 1.30 111032051179541496m Q31111163371 1.69 1.28 111032051179541497m Q311E/Q342R 1.38 1.49 11103200795-P1498m Q311KQ342K ' 1.56 1.38 , 111032051179541499m Q3111141343E 3.61 1.98 M1032051179541 DOO= Q31117343K 1.82 '1.32 M1032051179541501m ' Q31111/1)413R 2.40 1.82 11103205117954152.21n Q31111/E285E 2.13 1.24 111032051E1795-P1323m Q3112.111285K 1.91 1.19 111032051179541524m Q3 1 moa4n. 3.66 1.40 1110320511795-P1525m Q3111110341K 3.56 1.31 , , 111032051179541526m Q31111fG385R 1.20 1.04 1110320M1954 I 627m o 3 I litiG385K 0.92 0.98 1/1032051179541528m Q3111VE38SE 3.02 1.12 1110320611795-P 1529m Q311R1E3881 2.86 1.08 111032051179541530m Q311RiN39OR 3.12 1.09 11103205H79541531m Q311V1390K 2.35 1.01 11103205H79541532m Q311111:401R 4.16 1.23 M103205B79541533re Q31111D401K 3,52 1.18 1110320511795-P1534m Q311R/G402E 3.12 1.12 1110320511795415352n Q311/tiG46.1.1C 2.48 1.11 1110320311795418367n ' Q31111,042OR 2.05 1.16 .
1110320511795-P I a37= Q311R7422E 177 1.32 M103205/179541338m ' Q311/t/V422K 1.84 1.11 111032051179541639mQ311.11.1A431R 3.20 0.90 , 1 111032051179641540m 320401110413K 7.64 1.73 111032051179541541m 11401.SD4 1 3X 7.17 1.57 M1032051179541 o42m G402EiD413K 3.32 ' 1.21 M1032.051179541543= 6402K/1)413K 3.70 1.19 M103205E179541544= Q3111D4013/04131 7.66 2.04 1.11032031179541345ra Q31111/10401K/5413K 7.73 1.50 /1103205H79541546m Q3111W402E.0413K 5.10 1.41 11103205117954 I 547m Q,511EiG402X.04131 4.85 1.33 11103205B79541548m Q3111110365R13401R 2.30 1.37 11103205H795415492n Q311/1/0385EiG40211 2.03 1.22 11103205117954 I 550m Q311E/EaRAR 10401E 6.43 1.63 111032051179541551m Q3111.13881VG402E 2.91 1.33 , 11103205H79541552zu 0385R04011111)413K 4.44 2.19 141032051479541553m 0385G402E)D413K 2.66 1.47 111032051179541555m E3881VG402111D413K 3.71 2.01 Here, the antibodies produced by introducing p1-increasing modifications into the Fc region are antibodies in which the charge of the Fc region is more towards the positive side when compared with before introduction of the modifications. Therefore, the Coulombic interaction between the Fc region (positive charge) and the sensor chip surface (negative charge) can be considered to have been strengthened by the p1-increasing amino acid modifications. Furthermore, such effects are expected to take place similarly on the same negatively charged cell membrane surface;
therefore, they are also expected to show an effect of accelerating the speed of uptake into cells in vivo.
[0616] Among the pI increased Fc variants with two amino acid substitution from SG1, the antigen-antibody complex made by SG141, P1499m, P1501m and P1540m shows highest binding to human Fc gamma RIM. The amino acid substitutions on Q311R/D399R, Q311R/P343R, Q311R/D413R and D401R/D413K are supposed to have strong charge effect on binding to human Fc gamma Ruth on the sensor chip.
Cellular uptake of p1-increased Fc variants [0617] To evaluate the rate of intracellular uptake into a human Fc gamma Ruth-expressing cell line, following assay was performed. An MDCK (Madin-Darby canine kidney) cell line that constitutively expresses human Fc gamma Ruth was produced by known methods. Using these cells, intracellular uptake of antigen-antibody complexes was evaluated.
[0618] Specifically, pHrodoRed (Life Technlogies) was used to label human latent Myostatin (antigen) according to an established protocol, and antigen-antibody complexes were formed in a culture solution with the antibody concentration being 10 mg/mL and the antigen concentration being 2.5 mg/mL. The culture solution containing the antigen-antibody complexes was added to culture plates of the above-mentioned MDCK cells which constitutively express human Fc gamma RHb and incubated for one hour, and then the fluorescence intensity of the antigen taken up into the cells was quantified using InCell Analyzer 6000 (GE healthcare). The amount of antigen taken up was presented as relative values to the SG1 value which is taken as 1.00.
[0619] The quantification results of cellular uptake were summarized in Table 26. Strong fluorescence derived from the antigen in the cells was observed in several Fc variants.
[0620] While not being restricted to a particular theory, this result can be explained as follows.
[0621] The antigen and antibodies added to the cell culture solution form antigen-antibody complexes in the culture solution. The antigen-antibody complexes bind to human Fc gamma RIM expressed on the cell membrane via the antibody Fc region, and are taken up into the cells in a receptor-dependent manner. Antibodies used in this experiment binds to antigen in a pH-dependent manner; therefore, the antibody can dissociate from DEMANDE OU BREVET VOLUMINEUX
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Claims (15)

Claims
1. [Claim 1] An isolated antibody that binds to latent myostatin, wherein the antibody comprises:
   (a) (i) a HVR-H3 comprising the amino acid sequence GVPAX1SX2 GGDX3, wherein X1 is Y or H, X2 is T or H, X3 is L or K (SEQ ID NO:
   128), (ii) a HVR-L3 comprising the amino acid sequence AGGYGGGX1YA, wherein X1 is L or R (SEQ ID NO: 131), and (iii) a HVR-H2 comprising the amino acid sequence IISX1AGX2X3YX4X5X6 WAKX7, 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) a HVR-H1 comprising the amino acid sequence X1X2DIS, wherein X1 is S or H, X2 is Y, T, D or E (SEQ ID NO: 126), (ii) a HVR-H2 comprising the amino acid sequence IISX1AGX2X3YX4X5X6 WAKX7, 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), and (iii) a HVR-H3 comprising the amino acid sequence GVPAX1SX2 GGDX3, wherein X1 is Y or H, X2 is T or H, X3 is L or K (SEQ ID NO:
   128);
   (c) (i) a HVR-H1 comprising the amino acid sequence X1X2DIS, wherein X1 is S or H, X2 is Y, T, D or E (SEQ ID NO: 126), (ii) a HVR-H2 comprising the amino acid sequence IISX1AGX2X3YX4X5X6 WAKX7, 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), (iii) a HVR-H3 comprising the amino acid sequence GVPAX1SX2 GGDX3, wherein X1 is Y or H, X2 is T or H, X3 is L or K (SEQ ID NO:
   128), (iv) a HVR-L1 comprising the amino acid sequence X1X2SQX3 VX4X5X6NWLS, wherein X1 is Q or T, X2 is S or T, X3 is S or E, X4 is Y or F, X5 is D or H, X6 is N, D, A or E (SEQ ID NO: 129); (v) a HVR-L2 comprising the amino acid sequence WAX1TLAX2, wherein X1 is S
   or E, X2 is S, Y, F or W (SEQ ID NO: 130); and (vi) a HVR-L3 comprising the amino acid sequence AGGYGGGX1YA, wherein X1 is L or R (SEQ ID NO: 131); or (d) (i) a HVR-L1 comprising the amino acid sequence X1X2SQX3VX4X
   5X6NWLS, wherein X1 is Q or T, X2 is S or T, X3 is S or E, X4 is Y or F, X5 is D or H, X6 is N, D, A or E (SEQ ID NO: 129); (ii) a HVR-L2 comprising the amino acid sequence WAX1TLAX2, wherein X1 is S or E, X2 is S, Y, F or W (SEQ ID NO: 130); and (iii) a HVR-L3 comprising the amino acid sequence AGGYGGGX1YA, wherein X1 is L or R (SEQ ID NO: 131).
2. [Claim 2] The antibody of claim 1 (b), further comprising a heavy chain variable domain framework FR1 comprising the amino acid sequence of any one of SEQ ID NOs: 132-134; FR2 comprising the amino acid sequence of any one of SEQ ID NOs: 135-136; FR3 comprising the amino acid sequence of SEQ ID NO: 137; and FR4 comprising the amino acid sequence of SEQ ID NO: 138.
3. [Claim 3] The antibody of claim 1 (d), further comprising a light chain variable domain framework FR1 comprising the amino acid sequence of SEQ
   ID NO: 139; FR2 comprising the amino acid sequence of any one of SEQ ID NOs: 140-141; FR3 comprising the amino acid sequence of any one of SEQ ID NOs: 142-143; and FR4 comprising the amino acid sequence of SEQ ID NO: 144.
4. [Claim 4] An isolated antibody that binds to latent myostatin, comprising (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 13, 16-30, 32-34, and 86-95; (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 15, 31, 35-38, and 96-99; or (c) 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.
5. [Claim 5] A polypeptide comprising a variant Fc region comprising at least one amino acid alteration in a parent Fc region, wherein the ratio of [KD
   value of the parent Fc region for monkey Fc gamma RIIb]/[KD value of the variant Fc region for monkey Fc gamma RIlb] is 2.0 or greater, and the ratio of [KD value of the parent Fc region for monkey Fc gamma RIIIa]/[KD value of the variant Fc region for monkey Fc gamma RIIIa] is 0.5 or smaller.
6. [Claim 6] The polypeptide of claim 5, wherein the KD value of the variant Fc region for monkey Fc gamma RIIb is 1.0x10-6 M or smaller, and the KD value of the variant Fc region for monkey Fc gamma RIIIa is 5.0x10 7 M or greater.
7. [Claim 7] The polypeptide of claim 5 or 6, wherein the variant Fc region comprises at least one amino acid alteration of 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.
8. [Claim 8] The polypeptide of claim 7, wherein the variant Fc region comprises at least two amino acid alterations comprising:
   (a) one amino acid alteration at position 236, and (b) at least one amino acid alteration of at least one position selected from the group consisting of:
   (i) position 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) position: 231, 232, 235, 239, 268, 295, 298, 326, 330, and 396; or (iii) position 268, 295, 326, and 330;
   according to EU numbering.
9. [Claim 9] The polypeptide according to claim 7 or 8, wherein 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, (1) 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.
10. [Claim 10] The polypeptide according to claim 9, wherein 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;
    according to EU numbering.
11. [Claim 11] A polypeptide comprising a variant Fc region comprising at least two amino acid alterations in a parent Fc region, wherein each of the amino acid alterations increases the isoelectric point (pI) of the variant Fc region compared with that of the parent Fc region.
12. [Claim 12] The polypeptide of claim 11, wherein the variant Fc region comprises at least two amino acid alterations of 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.
13. [Claim 13] The polypeptide of claim 12, wherein the variant Fc region comprises Arg or Lys at each of the positions selected.
14. [Claim 14] A polypeptide comprising a variant Fc region comprising amino acid alterations described in Tables 14-30.
15. [Claim 15] A polypeptide comprising the amino acid sequence of any one of SEQ
    ID NOs: 229-381.