AU2016372934B2

AU2016372934B2 - Anti-myostatin antibodies, polypeptides containing variant Fc regions, and methods of use

Info

Publication number: AU2016372934B2
Application number: AU2016372934A
Authority: AU
Inventors: Yuji Hori; Tomoyuki Igawa; Hitoshi KATADA; Taichi Kuramochi
Original assignee: Chugai Pharmaceutical Co Ltd
Current assignee: Chugai Pharmaceutical Co Ltd
Priority date: 2015-12-18
Filing date: 2016-12-16
Publication date: 2023-10-05
Anticipated expiration: 2036-12-16
Also published as: KR101820637B1; SG11201610812WA; CN108473562B; JP2024023427A; CA3002422A1; KR20230027321A; HK1254755A1; TW202231662A; PH12018501280A1; JP2026001164A; KR102709693B1; KR20170085028A; TW202446795A; JP2017112997A; EP3390443A4; TWI605057B; AU2016372934A1; SG10201707267RA; MY189425A; TWI868415B

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

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 IgGI 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 RIa (CD32A), Fc gamma RIb (CD32B), Fc gamma RIIIa (CD16A), and Fc gamma RII (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 RIb 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 RIb 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 RIb, the immunoreceptor tyrosine-based inhibitory motif (ITIM) contained in the intracellular domain of Fc gamma RIb 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 RIb 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 RIb is also expressed on dendritic cells, macrophages, activated neu trophils, mast cells, and basophils. Fc gamma RIb 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 RIb has been elucidated so far through studies using Fc gamma RIb knockout mice. There are reports that in Fc gamma RIb 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 RIb 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 RIIb, 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 RIb 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] IgGI, mainly used as a commercially available therapeutic antibody, is known to bind not only to Fc gamma RIIb, 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 IgGI, by utilizing an Fc region with enhanced Fc gamma RIIb binding, or improved Fc gamma RIb-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 RIb 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 RIb on mast cells using molecules, in which the Fc portion of an IgG with enhanced Fc gamma RIb binding is fused to the Fc portion of IgE that binds to an IgE receptor Fc epsilon RI, causes phosphorylation of Fc gamma RIb, 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 RIb-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 RIb binding enhances the anti-tumor effect of anti-CD40 antibodies. Accordingly, antibodies with enhanced Fc gamma RIb 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 RIb 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 RIb binding may enhance inhibitory effects on cells expressing RTK having constitutively activated mutations.

[0022] Antibodies having an Fc with improved Fc gamma RIub-binding activity have been reported (see, e.g., NPL 40). In this Literature, Fc gamma RIb-binding activity was improved by adding alterations such as S267E/L328F, G236D/S267E, and S239D/S267E to an antibody Fc region. Among them, the antibody introduced with the S267E/L328F mutation most strongly binds to Fc gamma RIb, and maintains the same level of binding to Fc gamma RIa and Fc gamma RIa type H in which a residue at position 131 of Fc gamma RIa is His as that of a naturally-occurring IgG1. 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 RIb binding, which means the Fc gamma RIlb-binding selectivity is not improved in comparison with type-R Fc gamma RIa (see, e.g., PTL 15).

[0023] Only the effect of enhancing Fc gamma RIa binding and not the enhancement of Fc gamma RIb binding is considered to have influence on cells such as platelets which express Fc gamma RIa but do not express Fc gamma RIb (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 RIa 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 RIa 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 RIb 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 RIa is also expressed on dendritic cells, when antibodies having an Fc with enhanced selective binding to Fc gamma RIb are used as pharmaceuticals, antigens are not readily presented by dendritic cells and such due to enhanced selective binding to Fc gamma RIb, 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 RIla 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 RIb binding shows significantly enhanced type-R Fc gamma RIla binding compared with that of a naturally-occurring IgG1. Therefore, its value as a pharmaceutical for patients carrying type-R Fc gamma RIla is considerably reduced. Types H and R of Fc gamma RIla 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 RIb or dendritic cells in which the interaction between Fc gamma RIb and the antibody Fc portion is inhibited by an anti-Fc gamma RIb antibody, dendritic cells have been reported to mature (see, e.g., NPL 63 and NPL 64). This report suggests that Fc gamma RIb 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 RIla is expressed on the dendritic cell surface in addition to Fc gamma RIub; therefore, even if binding to in hibitory Fc gamma RIIb is enhanced and if binding to activating Fc gamma R such as Fc gamma RIla 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 RIla-binding activity is considered to be important in providing antibodies with an immunosup- pressive action.

[0028] Therefore, when considering the generation of a pharmaceutical that utilizes the Fc gamma RIb binding-mediated immunosuppressive action, there is a need for an Fc variant that not only has enhanced Fc gamma RIlb-binding activity, but also has binding to both Fc gamma RIa 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 IgG1.

[0029] Meanwhile, cases where amino acid alterations were introduced into the Fc region to increase the Fc gamma RIlb-binding selectivity have been reported so far (see, e.g., NPL 65). However, all variants said to have improved Fc gamma RIb selectivity as reported in this literature showed decreased Fc gamma RIb binding compared with that of a naturally-occurring IgG1. Therefore, it is considered to be difficult for these variants to actually induce an Fc gamma RIlb-mediated immunosuppressive reaction more strongly than IgG1.

[0030] Furthermore, since Fc gamma RIb 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 RIa 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 RIub-binding activity.

[0031] From these results, in producing therapeutic antibodies to be used for treating au toimmune diseases and cancer utilizing Fc gamma RIb, it is important that compared with those of a naturally-occurring IgG, the activities of binding to both Fc gamma RJla allotypes are maintained or decreased, and Fc gamma RIb binding is enhanced. However, Fc gamma RIb shares 93% sequence identity in the extracellular region with that of Fc gamma RJla 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 RIb binding as compared to each allotype of Fc gamma RIIa, which involves distin guishing highly homologous sequences between Fc gamma RJla and Fc gamma RIb. In spite of those difficulties, several Fc region variants have been identified so far, which has selective binding activity to Fc gamma RIb 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 RIb 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 RIb in relation to monkey Fe gamma R. Owing to the absence of such an Fc variant, the effects of the Fc variant selectively binding to Fc gamma RIb 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 CHI 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

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

PTL 21: WO 2007/114319 PTL 22: WO 2009/041643 PTL 23: WO 2014/145159 PTL 24: WO 2012/016227 Non-Patent Literature

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Summary of Invention Technical Problem

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

[0037a] The present invention provides an isolated antibody that binds to latent myostatin, wherein the antibody comprises: (1) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 114, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 114, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 116, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (iii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 117, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and

12a

HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (v) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 119, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (vi) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 114, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (vii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 114, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 123, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (viii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 115, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 123, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (ix) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 114, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 124, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 125, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (x) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 115,

12b

HVR-H2 comprising the amino acid sequence of SEQ ID NO: 120, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 123, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74.

[0037b] Any reference to any prior art in this specification is not, and should not be taken as an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge.

[0037c] The term "comprise" and variants of the term such as "comprises" or ''comprising" are used herein to denote the inclusion of a stated integer or stated integers but not to exclude any other integer or any other integers, unless in the context or usage an exclusive interpretation of the term is required.

[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 embodiments, 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 l la,

12c

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, l la, 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, Ila, 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 11a 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 11a 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 11a 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 11a. 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 11a. 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 GVPAX1 SX 2GGDX 3, wherein X 1 is Y or H, X 2 is T or H, X 3 is L or K (SEQ ID NO: 128), (ii) a HVR-L3 comprising the amino acid sequence AGGYGGGXiYA, wherein X 1is L or R (SEQ ID NO: 131), and (iii) a HVR-H2 comprising the amino acid sequence IISXAGX 2 X3YX4X5X6 WAKX 7, wherein X 1 is Yor H, X2 is S or K, X 3 is T, Mor K, X 4 is Yor 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 X 1X 2 DIS, wherein X 1 is S or H, X 2 is Y, T, D or E (SEQ ID NO: 126), (ii) a HVR-H2 comprising the amino acid sequence IISX 1AGX 2X 3 YX 4 X X5 WAKX7, 6 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 GVPAXSX 2GGDX 3 ,

wherein X 1is Y or H, X 2 is T or H, X 3 is L or K (SEQ ID NO: 128); (c) (i) a HVR-H1 comprising the amino acid sequence X 1X 2 DIS, wherein X 1 is S or H, X 2 is Y, T, D or E (SEQ ID NO: 126), (ii) a HVR-H2 comprising the amino acid sequence IISX 1AGX 2X 3 YX 4 X X5 WAKX7, 6 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, X 7 is G or K (SEQ ID NO: 127), (iii) a HVR-H3 comprising the amino acid sequence GVPAX1 SX 2GGDX 3, wherein X1 is Y or H, X 2 is T or H, X 3 is L or K (SEQ ID NO: 128), (iv) a HVR-L comprising the amino acid sequence X 1X 2 SQX 3 VX4XsXNWLS, wherein X1 is Q or T, X 2 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 WAX 1TLAX 2, wherein X 1is S or E, X2 is S, Y, F or W (SEQ ID NO: 130); and (vi) a HVR-L3 comprising the amino acid sequence AGGYGGGXiYA, wherein X 1is L or R (SEQ ID NO: 131); (d) (i) a HVR-L1 comprising the amino acid sequence XX 2 SQX 3VX 4 X5 X6 NWLS, wherein X 1is Q or T, X 2 is S or T, X 3 is S or E, X 4 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 TLAX 2 , wherein X 1is S or E, X 2 is S, Y, F or W (SEQ ID NO: 130); and (iii) a HVR L3 comprising the amino acid sequence AGGYGGGXiYA, wherein X 1is L or R (SEQ ID NO: 131). In some embodiments the antibody of (b) further comprises a heavy chain variable domain framework FRI 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 FRI 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 GVPAXSX 2GGDX 3

, wherein X 1is Y or H, X 2 is T or H, X 3 is L or K (SEQ ID NO: 128), (b) a HVR-L3 comprising the amino acid sequence AGGYGGGXiYA, wherein X 1is L or R (SEQ ID NO: 131), and (c) a HVR-H2 comprising the amino acid sequence IISXAGX 2 XYX 3 4X

5X 6WAKX 7, wherein X1 is Yor H, X 2 is S or K, X 3 is T, Mor K, X4 is Yor 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 X 1X 2 DIS, wherein X1 is S or H, X 2 is Y, T, D or E (SEQ ID NO: 126), (b) a HVR-H2 comprising the amino acid sequence IISX1 AGX 2 X3 YX4X5XWAKX 7, wherein X1 is Y or H, X2 is S or K, X 3 is T, M or K, X 4 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 GVPAX1 SX 2GGDX 3 ,

wherein X 1is Y or H, X 2 is T or H, X 3 is L or K (SEQ ID NO: 128). In further em bodiments, the antibody comprises a heavy chain variable domain framework FRI 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-L1 comprising the amino acid sequence X 1 X 2 SQX 3 VX 4

X 5X NWLS, 6 wherein X1 is Qor T,X 2 is S or T, X 3 is S or E, X4 is Yor F, X5 is Dor H, X 6 is N, D, A or E (SEQ ID NO: 129); (b) a HVR-L2 comprising the amino acid sequence WAXTLAX 2, 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 AGGYGGGXiYA, 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 X 1 X 2 SQX 3 VX4X5X6

NWLS, wherein X is 1 Q or T, X 2 is S or T, X 3 is S or E, X 4 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 WAXTLAX 2, 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 AGGYGGGXiYA, wherein X 1is L or R (SEQ ID NO: 131). In a further embodiment, the antibody further comprises a light chain variable domain framework FRI 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 FRI 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 RIIB-binding polypeptides comprising variant Fc regions and methods of using the same. In some embodiments, a variant Fc region with enhanced Fc gamma RIlb-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 RIlb]/[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 RIIla]/[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 RIlb]/[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 RIIla]/[KD 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 RIa (type H)]/[KD value of the variant Fc region for human Fc gamma RIa (type H)] is 5.0 or smaller. In further embodiments, the ratio of

[KD value of the parent Fc region for human Fc gamma RIa (type R)]/[KD value of the variant Fc region for human Fc gamma RIa (type R)] is 5.0 or smaller. In another embodiment, the KD value of the variant Fc region for monkey Fc gamma RIb is 1.Ox10-6 M or smaller. In another embodiment, the KD value of the variant Fc region for monkey Fc gamma RIIIa is 5.0x10-7 M or greater. In another embodiment, the KD value of the variant Fc region for human Fc gamma RIb is 2.0x10 6 M or smaller. In another embodiment, the KD value of the variant Fc region for human Fc gamma RIIIa is 1.Ox106 M or greater. In another embodiment, the KD value of the variant Fc region for human Fe gamma RIa (type H) is 1.Ox10-7 M or greater. In another embodiment, the KD value of the variant Fc region for human Fe gamma RIa (type R) is 2.0x10-7 M or greater.

[0059] In some embodiments, a variant Fc region with enhanced Fe gamma RIlb-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 Fe gamma RIlb-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 Fe gamma RIlb-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,and396, according to EU numbering.

[0062] In further embodiments, the variant Fc region with enhanced Fe gamma RIlb-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 Fe gamma RIlb-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 Fe gamma RIIb-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, 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.

[0065] In further embodiments, the variant Fc region with enhanced Fc gamma RIlb-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 (pI)-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 IgG1. 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.]Figure 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.2]Figure 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.3]Figure 3 illustrates binding of anti-latent myostatin antibody to propeptide domain, as described in Example 5.

[fig.4]Figure 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.5]Figures 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.6A]Figure 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.6B]Figure 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.6C]Figure 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.7A]Figure 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 MYO-029) was administered to SCID mice, and full body lean mass was measured.

[fig.7B]Figure 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 MYO-029) was administered to SCID mice, and grip strength was measured.

[fig.7C]Figure 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 MYO-029) was administered to SCID mice, and change of full body fat mass from Day 0 to Day 14 was measured.

[fig.8]Figure 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.9]Figure 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: AbOO (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.10]Figure 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 BMP1 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 MS1032LO01-SG1, MS1032LO02-SG1, MS1032LO03-SG1, and MS1032LO04-SG1 are described respectively as MSLO-O1, MSLO-02, MSLO-03, and MSLO-04 in the Figure. Comparable inhibition of proteolytic and spontaneous ac tivation of latent myostatin to MS1032LOOO-SG1 was achieved by MS1032LO01-SG1, MS1032LO02-SG1, MS1032LO03-SG1, and MS1032LO04-SG1.

[fig.11]Figures 11A-IIF illustrate BIACORE (registered trademark) sensorgrams of pH-dependent anti-latent myostatin antibody, as described in Example 14. Kinetic pa rameters of MST1032-SG1 (A), MS1032LOOO-SG1 (B), MS1032LOO1-SG1 (C), MS1032LO02-SG1 (D), MS1032LO03-SG1 (E), and MS1032LO04-SG1 (F), were measured at neutral pH and acidic pH.

[fig.12]Figure 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 (MS1032L00-SG1) and anti-myostatin an tibodies with abolished Fc gamma R binding (MS1032L00-F760).

[fig.13]Figure 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 (MS1032LO01-SG1 or MS1032LO01-F760) and non pH-dependent anti myostatin antibody (MS1032LO00-SG1 or MS1032L00-F760).

[fig.14]Figures 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 (MS1032LO01-SG1; described as MSLO1 in the Figure) or non pH-dependent anti-latent myostatin antibody (MS1032L00-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.15]Figure 15 illustrates binding activity of anti-latent myostatin antibody MST1032 to latent myostatin and GDF11, as described in Example 19.

[fig.16]Figure 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 GDFi1 released by BMPi protease (proteolytic) or by 37 degrees C incubation without BMPi (spontaneous) was measured in the presence of anti-latent myostatin antibody, using HEK Blue Assay.

[fig.17]Figure 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.18]Figure 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 (MS1032LO00-SG1) and different pH-dependent anti-latent myostatin antibodies (MS1032LO01-SG1, MS1032LO06-SG1, MS1032LO11-SG1, MS1032LO18-SG1, MS1032LO19-SG1, MS1032LO21-SG1 and MS1032LO25-SG1).

[fig.19]Figures 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 (MS1032LO00-SG1) and pH-dependent anti latent myostatin antibodies with Fc engineering (MS1032LO06-SG1012, MS1032LO06-SG1016, MS1032LO06-SG1029, MS1032LO06-SG1031, MS1032LO06-SG1033, MS1032LO06-SG1034). (B) The effect of Fc engineering on myostatin clearance in vivo was assessed by comparing anti-latent myostatin an tibodies (MS1032LO19-SG1079, MS1032LO19-SG1071, MS1032LO19-SG1080, MS1032LO19-SG1074, MS1032LO19-SG1081, and MS1032LO19-SG1077).

[fig.20A]Figure 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. MS1032LO06-SG1, MS1032LO11-SG1, and MS1032LO18-SG1 were administered to Scid mice, and lean body mass (A)was measured.

[fig.20B]Figure 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. MS1032LO06-SG1, MS1032LO19-SG1, and MS1032LO25-SG1 were administered to Scid mice, and lean body mass (B) was measured.

[fig.20C]Figure 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. MS1032LO01-SG, MS1032LO06-SG1, and MS1032LO11-SG1 were administered to Scid mice, and lean body mass (C) was measured.

[fig.20D]Figure 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. MS1032LO06-SG1, MS1032LO11-SG1, and MS1032LO18-SG1 were administered to Scid mice, and grip strength (D) was measured.

[fig.20E]Figure 20E, together with Figures 20A-D, 20F-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. MS1032LO06-SG1, MS1032LO19-SG1, and MS1032LO25-SG1 were administered to Scid mice, and grip strength (E) was measured.

[fig.20F]Figure 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. MS1032LO01-SG, MS1032LO06-SG1, and MS1032LO11-SG1 were administered to Scid mice, and grip strength (F) was measured.

[fig.20G]Figure 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. MS1032LO06-SG1, MS1032LO11-SG1, and MS1032LO18-SG1 were administered to Scid mice, and body fat mass (G) was measured.

[fig.20H]Figure 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. MS1032LO06-SG1, MS1032LO19-SG1, and MS1032LO25-SG1 were administered to Scid mice, and body fat mass (H) was measured.

[fig.20]Figure 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. MS1032LO1-SG, MS1032LO06-SG1, and MS1032LO11-SG1 were administered to Scid mice, and body fat mass (I) was measured.

[fig.21]Figure 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 (MST1032, MST1504, MST1538, MST1551, MST1558, MST1572, and MST1573).

[fig.22A]Figure 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.22B]Figure 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.22C]Figure 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 (100ng).

[fig.22D]Figure 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.23]Figure 23 illustrates an alignment of amino acid sequences of cynomolgus (cyno) Fc gamma RIal, Fc gamma RIla2, Fc gamma RIla3, Fc gamma RIb, human Fc gamma RIaH, Fc gamma RIaR, and Fc gamma RIb. Squared regions indicate putative residues which interact with Fc domain.

[fig.24]Figure 24 illustrates the time course of total myostatin concentration in plasma after intravenous administration of an anti-myostatin antibody with an Fc gamma RIhb enhanced Fc variant in all human Fc gamma R transgenic mice, as described in Example 28. The effects of Fc gamma RIlb-enhanced Fc variants on antigen elimination through human Fc gamma RIb were evaluated.

[fig.25]Figure 25 illustrates the time course of antibody concentration in plasma after intravenous administration of an anti-myostatin antibody with an Fc gamma RIlb enhanced Fc variant in all human Fc gamma R transgenic mice, as described in Example 28. The effects of Fc gamma RIlb-enhanced Fc variants on antibody pharma cokinetics were evaluated.

[fig.26]Figures 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 (MS1032LO00-SG1) and pH-dependent anti latent myostatin antibodies with Fc engineering (MS1032LO06-SG1012, MS1032LO06-SG1016, MS1032LO06-SG1029, MS1032LO06-SG1031, MS1032LO06-SG1033, MS1032LO06-SG1034). (B) The effect of Fc engineering on myostatin clearance in vivo was assessed by comparing anti-latent myostatin an tibodies (MS1032LO19-SG1079, MS1032LO19-SG1071, MS1032LO19-SG1080,

MS1032LO19-SG1074, MS1032LO19-SG1081, and MS1032LO19-SG1077).

[fig.27A]Figure 27A illustrates the time course of total myostatin concentration in plasma after intravenous administration of an anti-myostatin antibody with a pI increased Fc variant in human FcRn transgenic mice, as described in Example 30. The effects of pI-increased Fc variants on antigen elimination were evaluated.

[fig.27B]Figure 27B illustrates the time course of antibody concentration in plasma after intravenous administration of an anti-myostatin antibody with a pI-increased Fc variant in human FcRn transgenic mice, as described in Example 30. The effects of pI increased Fc variants on antibody pharmacokinetics were evaluated.

[fig.28A]Figure 28A illustrates the time course of total myostatin concentration in plasma after intravenous administration of an anti-myostatin antibody with a pI increased Fc variant in human FcRn transgenic mice, as described in Example 30. The effects of pI-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.28B]Figure 28B illustrates the time course of antibody concentration in plasma after intravenous administration of an anti-myostatin antibody with a pI-increased Fc variant in human FcRn transgenic mice, as described in Example 30. The effects of pI 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.29]Figure 29 illustrates the time course of total myostatin concentration in plasma after intravenous administration of an anti-myostatin antibody with an Fc gamma RIIb enhanced Fc variant in human Fc gamma RIb transgenic mice, as described in Example 31. The effects of Fc gamma RIlb-enhanced Fc variants on antigen elimination through human Fc gamma RIb 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 RIlb enhanced Fc variant in human Fc gamma RIb transgenic mice, as described in Example 31. The effects of Fc gamma RIlb-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 RIb-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 RIlb 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. Calos, 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., 10-1 M or less, e.g., from 10-1 M to 10- 13 M, e.g., from 10 9 M to 10-13M). 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(ab') 2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0082] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks 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., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1, and IgA 2. 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., At 2 1 1 , 1131, 112 5, y9o, Re8 6 , Re88 , Sm 15 3 , Bi 2 12 , 32, Pb2 12 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: FRI, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FRI-HI(L)-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 Cl 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 (HI, H2, H3), and three in the VL (L, L2, L3). Exemplary HVRs herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (LI), 50-52 (L2), 91-96 (L3), 26-32 (HI), 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 (LI), 50-56 (L2), 89-97 (L3), 31-35b (HI), 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 (LI), 46-55 (L2), 89-96 (L3), 30-35b (HI), 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 (HI), 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 (CHI, 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 IgGI 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 21-100 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 GDF11is 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 Fe gamma RIlb-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-i (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 11a or 13. In some embodiments, the anti-myostatin antibody competes for binding latent myostatin with an antibody described in Table 11a 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 11a 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-i (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, 10- 8M, 10-9 M, 10-10 M, 10-" M, 10 12 M, or less. In another embodiment, the KD value of the antibody at acidic pH can be

10-9 M, 10-8 M, 10-7 M, 10-6 M, or 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, 10-8 M, 10-9 M, 10-10 M, 10-1 M, 10-12 M, or less. In another embodiment, the KD value of the antibody at pH5.8 can be 10-9 M, 10-8 M, 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., sect-). 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 least2,3,5,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100, 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- I/s, 10-6 1/s, or less. In another embodiment, the kd value of the antibody at acidic pH can be 10-3 I/s, 10-2 1/s, 10-1 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, 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, 10-1 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, 10-1 M, 10-9 M, 10-10 M, 10-11 M, 10-12 M, or less. In another embodiment, the KD value of the antibody at acidic pH can be 10-9 M, 10-8 M, 10-7 M, 10-6 M, or 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 canbe 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, 10-1 M, 10-9 M, 10-10 M, 10-11 M, 1012 M, or less. In another embodiment, the KD value of the antibody at pH5.8 can be 10-9 M, 10-1 M, 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, 10-5I/s, 10-6 1/s, or less. In another embodiment, the kd value of the antibody at acidic pH can be 10-3 1/s, 10-2 1/s, 10-1 1/s, or greater. 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, 10-1 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 10.0. 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 10.0. 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-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.

[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-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. 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-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.

[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 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: 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-H1 (SEQ ID NO: 55), at positions 1, and 2; (b) in HVR-H2 (SEQ ID NO: 58), at positions

4, 7, 8, 10, 11, 12, and 16; (c) in HVR-H3 (SEQ ID NO: 61), at positions 5, 7, and 11; (d) in HVR-L1 (SEQ ID NO: 65), at positions 1, 2, 5, 7, 8, and 9; (e) in HVR-L2 (SEQ ID NO:70), at positions 3, and 7; and (f) in HVR-L3 (SEQ ID NO:73), at position 8.

[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), SlH; Y2T, D, or E; (b) in HVR-H2 (SEQ ID NO: 58), Y4H; S7K; T8M or K; Y10K; AlIM or E; S12E; G16K; (c) in HVR-H3 (SEQ ID NO: 61), Y5H; T7H; LI1K; (d) in HVR-L1 (SEQ ID NO: 65), QIT; S2T; S5E; Y7F; D8H; N9D or A or E; (e) in HVR-L2 (SEQ ID NO: 70), S3E; S7Y, F or W; and (f) in HVR-L3 (SEQ ID NO: 73), L8R.

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

[0164] In any of the above 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 FRI 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, FRI 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 LI 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 10 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-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. 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 10 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-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. 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-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. 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 32-34 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-H1 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 10 amino acids have been substituted, inserted and/or deleted in any one of SEQ ID NOs: 14, 151-156. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., 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 11a 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(ab') 2 fragment. In another em bodiment, the antibody is a full length IgG antibody, e.g., an intact IgG Ior 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., 10-1 M or less, e.g., from 10-1 M to 10-13 M, e.g., from 10 9 M to 10-13 M).

[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 ( 125 I)-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 1[ 2 1]-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-20TM; Packard) is added, and the plates are counted onaTOPCOUNT 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-dimethylaminopropyl)-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/ml (-0.2 micro M) before injection at a flow rate of 5 micro /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-20 TM) surfactant (PBST) at 25 degrees C at a flow rate of approximately 25 micro1/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 kff/k 0 See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 106 M- 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(ab') 2, Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, 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(ab') 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 XENOMOUSE TM 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; lie Val Arg (R) Lys; Gin; Asn Lys Asn (N) Gin; His; Asp, Lys; Arg Gin Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gin (Q) Asn; Glu Asn Glu (E) Asp; Gin Asp Gly (G) Ala Ala His (H) Asn; Gin; Lys; Arg Arg Ile (T) Leu; Val; Met; Ala; Phe; Norleucine Leu Len (L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gin; 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 (GlcNAc), 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 IgGI, 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, ACT1TM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96 (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 Cl qand hence lacks CDC activity. See, e.g., Cl qand 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) Cl 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 US2005/0014934A1 (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; Al18 (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 RIlb-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 RIb compared to the parent Fc region. In particular embodiments, the monkey Fc gamma RIb is cynomolgus monkey Fc gamma RIb (SEQ ID NO: 223).

[0230] In certain embodiments, the ratio of [KD value of a parent Fc region for monkey Fc gamma RIlb]/[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 RIIIa]/[KD value of a variant Fc region for monkey Fc gamma RIIa] 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 RIb 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 RI. In certain embodiments, the ratio of [KD value of a parent Fc region for human Fc gamma RIb]/[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 RIIIa]/[KD 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 Fe gamma RIb has the sequence of SEQ ID NOS:212, 213, or 214. In certain embodiments, the human Fe 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 Fe gamma RIa (type H) than for human Fe gamma RI. In certain em bodiments, the ratio of [KD value of a parent Fc region for human Fe gamma RIa (type H)]/[KD value of a variant Fc region for human Fe gamma RIa (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 Fe gamma RIa (type R) than for human Fe gamma RI. In certain embodiments, the ratio of [KD value of a parent Fc region for human Fe gamma RIa (type R)]/[KD value of a variant Fc region for human Fe gamma RIa (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 Fe gamma RIa (type H) has the sequence of SEQ ID NO:211. In certain embodiments, the human Fe 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 Fe gamma RIIa]/[KD value of a variant Fc region for monkey Fe 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 Fe gamma RIIa is selected from monkey Fe gamma RIIal (e.g., cynomolgus Fe gamma RIIal (SEQ ID NO:220)), monkey Fe gamma RIIa2 (e.g., cynomolgus Fe gamma RIIa2 (SEQ ID NO:221)), and monkey Fe gamma RIIa3 (e.g., cynomolgus Fe gamma RIIa3 (SEQ ID NO:222).

[0234] In another embodiment, the KD value of the variant Fc region for monkey Fe gamma RIIb can be 1.Ox1O-6 M or smaller, 9.0x10-7 M or smaller, 8.0x10-7 M or smaller, 7.0x10-7 M or smaller, 6.0x10-7 M or smaller, 5.0x10-7 M or smaller, 4.0x10-7 M or smaller, 3.0x10-7 M or smaller, 2.0x10-7 M or smaller, or 1.0x10 7 M or smaller. In another embodiment, the KD value of the variant Fc region for monkey Fe gamma RIIIa can be 5.Ox10-7 M or greater, 6.0x10-7 M or greater, 7.0x10-7 M or greater, 8.Ox10 7 M or greater, 9.Ox1O7 M or greater, 1.0x10 6 M or greater, 2.0x10-6 M or greater,

3.OxlO-6 M or greater, 4.OxlO-6 M or greater, 5.OxlO-6 M or greater, 6.OxlO- 6 M or greater, 7.OxlO-6 M or greater, 8.Ox1O-6 M or greater, 9.x1O- 6 M or greater, or1.xO- 5 M or greater. In another embodiment, the KD value of the variant Fc region for human Fe gamma RIb can be 2.0x10-6 M or smaller, 1.0x10-6 M or smaller, 9.0x10-7 M or smaller, 8.Ox1O-7 M or smaller, 7.Ox10- 7 M or smaller, 6.OxlO-7M or smaller, 5.Ox10- 7 M or smaller, 4.OxlO- 7 M or smaller, 3.Ox10- 7 M or smaller, 2.Ox10- 7 M or smaller, or 1.Ox1-7 M or smaller. In another embodiment, the KD value of the variant Fc region for human Fe gamma RIIIa can be 1.0x10-6 M or greater, 2.0x10-6 M or greater, 3.Ox1O 6M or greater, 4.0x10-6 M or greater, 5.0x10-6 M or greater, 6.OxlO-6 M or greater, 7.0x10-6 M or greater, 8.0x10-6 M or greater, 9.0x10-6 M or greater, 1.0x10-5 M or 5 greater, 2.0x10-5 M or greater, 3.OxlO-5 M or greater, 4.OxlO-5 M or greater, or 5.0x10 M or greater. In another embodiment, the KD value of the variant Fc region for human Fe gamma RIa (type H) can be 1.0x10-7 M or greater, 2.0x10-7 M or greater, 3.0x10-7 M or greater, 4.0x10-7 M or greater, 5.0x10-7 M or greater, 6.0x10-7 M or greater, 7.0x10 7 M or greater, 8.0x10 7 M or greater, 9.Ox1O7 M or greater, 1.0x10 6 M or 6 greater, 2.0x10 6 M or greater, 3.OxlO6 M or greater, 4.0x10 6 M or greater, or 5.0x10 M or greater. In another embodiment, the KD value of the variant Fc region for human 7 Fe gamma RIa (type R) can be 2.0x10 7 M or greater, 3.0x10 7 M or greater, 4.0x10 M or greater, 5.0x10 7 M or greater, 6.OxlO7 M or greater, 7.OxlO7 M or greater, 8.0x10 7 M or greater, 9.0x10 7 M or greater, 1.0x10 6 M or greater, 2.0x10 6 M or greater, 3.0x10-6 M or greater, 4.0x10-6 M or greater, or 5.0x10-6 M or greater.

[0235] In another embodiment, the KD value of the variant Fc region for monkey Fe gamma RIa can be 1.0x10-6 M or smaller, 9.0x10-7 M or smaller, 8.0x10-7 M or smaller, 7.0x10-7 M or smaller, 6.OxlO-7 M or smaller, 5.OxlO-7 M or smaller, 4.0x10-7 M or smaller, 3.0x10-7 M or smaller, 2.OxlO-7 M or smaller, or 1.0x10-7 M or smaller. In certain embodiments, monkey Fe gamma RIa can be selected from any one of monkey Fe gamma RIlal, monkey Fe gamma RIIa2, and monkey Fe 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 Fe gamma receptors, Fe gamma R or FcgR) refers to receptors that may bind to the Fc region of IgGI, IgG2, IgG3, and IgG4 monoclonal antibodies, and practically means any member of the family of proteins encoded by the Fe gamma receptor genes. In humans, this family includes Fe gamma RI (CD64) including isoforms Fe gamma RIa, Fe gamma Rb, and Fe gamma RIc; Fe gamma RII (CD32) including isoforms Fe gamma RIla (including allotypes

H131 (type H) and R131 (type R)), Fe gamma RIb (including Fe gamma RIIb-1 and Fe gamma RIIb-2), and Fe gamma RIIc; and Fe gamma RIII (CD16) including isoforms Fe gamma RIIIa (including allotypes V158 and F158), and Fe gamma RIIIb (including allotypes Fe gamma RIIIb-NA1 and Fe gamma RIIIb-NA2), and any human Fe gamma Rs, Fe gamma R isoforms or allotypes yet to be discovered, but is not limited thereto. Fe gamma RIlb Iand Fe gamma RIIb2 have been reported as splicing variants of human Fe gamma RIlb. In addition, a splicing variant named Fe gamma RIIb3 has been reported (J Exp Med, 1989, 170: 1369-1385). In addition to these splicing variants, human Fe gamma RIb 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 Fe gamma RIb includes every previously-reported genetic polymorphism, as well as Fe gamma RIb (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 Fe gamma RIIa, there are two allotypes, one where the amino acid at position 131 of Fe gamma RIa 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 Fe gamma R includes human, mouse, rat, rabbit, and monkey-derived Fe gamma Rs but is not limited thereto, and may be derived from any organism. Mouse Fe gamma Rs include Fe gamma RI (CD64), Fe gamma RII (CD32), Fe gamma RIII (CD16), and Fe gamma RIII-2 (CD16-2), and any mouse Fe gamma Rs, or Fe gamma R isoforms, but are not limited thereto. Unless otherwise specified, the term "monkey Fe gamma R" or variation thereof, refers to cynomolgus Fe gamma RIlal (SEQ ID NO:220), Fe gamma RIIa2 (SEQ ID NO:221), Fe gamma RIIa3 (SEQ ID NO:222), Fe gamma RIb (SEQ ID NO:223), or Fe gamma RIIIaS (SEQ ID NO:224).

[0240] The polynucleotide sequence of human Fe gamma RI is set forth in SEQ ID NO: 199 (NM_000566.3); the polynucleotide sequence of human Fe gamma RIla is set forth in SEQ ID NO: 200 (BC020823.1) or SEQ ID NO:201 (NM_001136219.1); the polynu cleotide sequence of human Fe gamma RIb is set forth in SEQ ID NO: 202 (BC146678.1) or SEQ ID NO:203 (NM_004001.3); the polynucleotide sequence of human Fe 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 Fe gamma RIIIb is set forth in SEQ ID NO: 206 (BC128562.1).

[0241] The amino acid sequence of human Fe gamma RI is set forth in SEQ ID NO: 207 (NP000557.1); the amino acid sequence of human Fe gamma RIla 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 RIb is set forth in SEQ ID NO: 212 (AA146679.1), 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 RII is set forth in SEQ ID NO: 219 (AA28563.1).

[0242] The amino acid sequence of cynomolgus monkey Fc gamma RIa 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 RIb 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 RIlb-binding activity compared to a corresponding reference Fc gamma RIlb-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 RIb has the sequence of cynomolgus monkey Fc gamma RIb (SEQ ID NO:223). In certain embodiments, the Fc gamma RIb has the sequence of human Fc gamma RIb (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 RIlb-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 RIb has the sequence of cynomolgus monkey Fc gamma RIb (SEQ ID NO:223). In certain embodiments, the Fc gamma RIb has the sequence of human Fc gamma RIb (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 RIlb-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 RIlb-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 RIb has the sequence of cynomolgus monkey Fc gamma RIb (SEQ ID NO:223). In certain embodiments, the Fc gamma RIb has the sequence of human Fc gamma RIb (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 RIlb-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 RIb has the sequence of cynomolgus monkey Fc gamma RIb (SEQ ID NO:223). In certain embodiments, the Fc gamma RIb has the sequence of human Fc gamma RIb (e.g., SEQ ID NOS:212, 213, or 214).

[0248] In a further embodiment, the variant Fc region with enhanced Fc gamma RIlb 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; (in) 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 RIb has the sequence of cynomolgus monkey Fc gamma RIIb (SEQ ID NO:223). In certain em bodiments, the Fc gamma RIb has the sequence of human Fc gamma RIIb (e.g., SEQ ID NOS:212, 213, or 214).

[0249] In a further embodiment, the variant Fc region with enhanced Fc gamma RIIb 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 RIlb-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 RIIb-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 RIIb-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 RIlb-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 RIlb-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 RIIb-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 Fe gamma RIlb-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 RIlb-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 RIb has the sequence of cynomolgus monkey Fc gamma RIb (SEQ ID NO:223). In certain embodiments, the Fc gamma RIb has the sequence of human Fc gamma RIb (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 RIlb-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 RIb has the sequence of cynomolgus monkey Fc gamma RIb (SEQ ID NO:223). In certain em bodiments, the Fc gamma RIb has the sequence of human Fc gamma RIb (e.g., SEQ ID NOS:212, 213, or 214).

[0253] In a further embodiment, the variant Fc region with enhanced Fc gamma RIlb 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 RIb-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 RIlb-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 RIub-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 RIlb-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 RIlb-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 RIlb-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 RIlb-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 RIlb-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 RIlb-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 RIlb-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 RIb has the sequence of cynomolgus monkey Fc gamma RIb (SEQ ID NO:223). In certain embodiments, the Fc gamma RIb has the sequence of human Fc gamma RIb (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 pH6.0. 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 pH7.0. 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 (US2009/0035836), m-N02-Tyr (pKa 7.45), 3,5-Br2-Tyr (pKa 7.21), and 3,5-12-Tyr (pKa 7.38) (Heyl 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 (NH 3+ 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 RIlb-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 RIlb-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 RIlb-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 RIb-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 RIb has the sequence of cynomolgus monkey Fc gamma RIb (SEQ ID NO:223). In certain embodiments, the Fc gamma RIb has the sequence of human Fc gamma RIb (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 RIb-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 RIb has the sequence of cynomolgus monkey Fc gamma RIb (SEQ ID NO:223). In certain embodiments, the Fc gamma RIb has the sequence of human Fc gamma RIb (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 RIb-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 (IgGI, 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 IgGI, 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 IgG I(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):157-159 (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 EP1752471 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. Immunol.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 JP2015-021371and 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 (IgG1, 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 RIb and Fc gamma RIa. Preferably, an alteration is in troduced in the loop structure of the CH2 domain of one of the Fc regions that enhances Fc gamma RIb-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 CH3 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 CH Iand 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 82:488-492 (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 orless, 12 orless, 14 orless, 16 orless, 18 orless, or20 orless.

[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 IgGIconstant 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 RIlb-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 - 4 6 (1988) Elsevier Science Publishers BV; Patthy (Cell 61(1):13-14 (1990)); Ullrich (Cell 61(2):203-212 (1990)); Massague (Cell 69(6):1067-1070 (1992)); Miyajima et al. (Annu. Rev. Immunol. 10:295-331 (1992)); Taga et al. (FASEB J. 6:3387-3396 (1992)); Fantl et al. (Annu. Rev. Biochem. 62:453-481 (1993)); Smith et al. (Cell 76(6):959-962 (1994)); and Flower (Biochim. Biophys. Acta 1422(3): 207-234 (1999)).

[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, CD1O, 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-IBB, 4Dc, 6-keto-PGFIa, 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, ADAM1O, ADAM12, ADAM15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha 1-antitrypsin, alpha-V/beta-i 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, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4, BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neu rotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, CI, 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, CDIIa, CDIib, CDIIc, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CLi, CX3CRi, CXCL, CXCLi, 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(i-3)-IGF-I (brain IGF-

1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2/EphB4, EPO, ERCC, E-selectin, ET-1, factor Ila, factor VII, factor VIIIc, factor IX, fibroblast ac tivation protein (FAP), Fas, FcRi, 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, FZDI, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZDIO, 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 Ilb/Illa (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, HMI.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-IR, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-iR, 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 factor, integrin alpha2, integrin alpha3, integrin alpha4, integrin alpha4/betai, integrin alpha4/beta7, integrin alpha5 (alpha V), integrin alpha5/betai, integrin alpha5/beta3, integrin alpha6, integrin betal, 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-i bp, 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, MMACi, 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 (Muci), 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, OGGI, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, serum albumin, sFRP 3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T-cell receptor (for example, T-cell receptor alpha/beta), TdT, TECK, TEMI, TEM5, TEM7, TEM8, TERT, testis PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific, TGF-betaRl (ALK-5), TGF betaRII, TGF-betaRIlb, 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 Ri Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF1lB (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), TNFRSFlA (TNF RI CD120a, p55-60), TNFRSFlB (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL Ri 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, TALL, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A/VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSFlA (TNF-a Conectin, DIF, TNFSF2), TNFSFlB (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGMi, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT Iligand), TNFSF7 (CD27 ligand

CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-RI, 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- I(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, WNTiOA, WNTiOB, WNT1, WNT16, XCLi, XCL2, XCRi, XCRi, XEDAR, XIAP, XPD, HMGBi, IgA, A beta, CD81, CD97, CD98, DDRi, DKKi, EREG, Hsp90, IL-17/IL-17R, IL-20/IL-20R, oxidized LDL, PCSK9, prekallikrein, RON, TMEM16F, SODI, Chromogranin A, Chro mogranin B, tau, VAPi, high molecular weight kininogen, IL-31, IL-31R, Navi.i, Navi.2, Navi.3, Navi.4, Navi.5, Navi.6, Navi.7, Navi.8, Navi.9, EPCR, Ci, Clq, Clr, Cs, 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 SIP; 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 PLANTIBODIES TM 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 SV40 (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 (CVi); 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 FS4 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, SOCl 2 , 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 IgGI, 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 RIlb-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 RIlb-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 RIlb 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 RIJIa-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 RIlb-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 RIlb 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 RIa (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 RIa (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 RIa (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 RIa (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 RIub-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 RIb has the sequence of cynomolgus monkey Fc gamma RIb (SEQ ID NO:223). In certain embodiments, the Fc gamma RIb has the sequence of human Fc gamma RIb (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 RIIb-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 RIlb-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,and396, according to EU numbering. In certain embodiments, the Fc gamma RIIb has the sequence of cynomolgus monkey Fc gamma RIIb (SEQ ID NO:223). In certain em bodiments, the Fc gamma RIb has the sequence of human Fc gamma RIIb (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 RIlb-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 RIb has the sequence of cynomolgus monkey Fc gamma RIb (SEQ ID NO:223). In certain em bodiments, the Fc gamma RIb has the sequence of human Fc gamma RIb (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 RIlb-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 RIb has the sequence of cynomolgus monkey Fc gamma RIb (SEQ ID NO:223). In certain embodiments, the Fc gamma RIb has the sequence of human Fc gamma RIb (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 RIlb-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 RIb has the sequence of cynomolgus monkey Fc gamma RIb (SEQ ID NO:223). In certain embodiments, the Fc gamma RIb has the sequence of human Fc gamma RIb (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 RIlb-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 RIb has the sequence of cynomolgus monkey Fc gamma RIb (SEQ ID NO:223). In certain embodiments, the Fc gamma RIb has the sequence of human Fc gamma RIb (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 RIub-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 RIb has the sequence of cynomolgus monkey Fc gamma RIb (SEQ ID NO:223). In certain embodiments, the Fc gamma RIb has the sequence of human Fc gamma RIb (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 RIub-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 RIb-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 RIlb-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 RIlb-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 RIlb-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 RIlb-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 RIlb-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 RIb has the sequence of cynomolgus monkey Fc gamma RIb (SEQ ID NO:223). In certain embodiments, the Fc gamma RIb has the sequence of human Fc gamma RIb (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 RIlb-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 RIlb-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 RIb has the sequence of cynomolgus monkey Fc gamma RIb (SEQ ID NO:223). In certain embodiments, the Fc gamma RIb has the sequence of human Fc gamma RIb (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 Fe gamma RIlb-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 RIlb-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 RIlb-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 RIlb-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 RIlb-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 RIlb-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 RIlb-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 RIlb-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 RIlb-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 RIlb-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 RIlb-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 RIlb-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 RIb has the sequence of cynomolgus monkey Fc gamma RIb (SEQ ID NO:223). In certain embodiments, the Fc gamma RIb has the sequence of human Fc gamma RIb (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 pl'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,and431, 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):4005-4010 (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 RIlb-binding activity preferably means, for example, that the ratio of [KD value of a parent Fc region for Fc gamma RIIb]/[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.OxlO-6 M or smaller, 1.0x10-6 M or smaller, 9.Ox1O-7 M or smaller, 8.0x10-7 M or smaller, 7.0x10-7 M or smaller, 6.0x10-7 M or smaller, 5.0x10-7 M or smaller, 4.0x10-7 M or smaller, 3.Ox10-7 M or smaller, 2.0x10-7 M or smaller, 1.0x10 7 M or smaller, 9.0x10 8 M or smaller, 8.Ox1O M or smaller, 7.0x10 8 M or smaller, 6.Ox1O8 M or smaller, 5.Ox1O M or smaller.

[0390] Furthermore, a variant Fc region with enhanced binding selectivity to Fc gamma RIb compared to Fc gamma RIIa refers to an Fc region where: (a) Fc gamma RIlb binding activity is enhanced, and Fc gamma RIa-binding activity is maintained or decreased; (b) Fc gamma RIub-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 RIb-binding activity; or (c) Fc gamma RIub-binding activity is decreased and Fc gamma RIIa binding activity is also decreased, but the degree of decrease of Fc gamma RIub binding activity is less than the degree of decrease of Fc gamma RIla-binding activity. Whether or not a variant Fc region has improved binding selectivity for Fc gamma RIb 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 RIb of the variant Fc region (the ratio of [KD value of the variant Fc region for Fc gamma RJla]/[KD value of the variant Fc region for Fc gamma RIb]), with the ratio of the KD value for Fc gamma RIIa to the KD value for Fc gamma RIb of the parent Fc region (the ratio of [KD value of the parent Fc region for Fc gamma Rla]/[KD value of the parent Fc region for Fc gamma RIIb]), 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 RIb rather than for Fc gamma RIIa in comparison with the parent Fc region. Especially in human, the Fc gamma RIub-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 RIb 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 RIb compared to human Fc gamma RIIa (type R) is important for enhancing binding selectivity to Fc gamma RIb compared to Fc gamma RIIa in humans.

[0391] When a variant Fc region of the present invention has higher binding activity against Fc gamma RIb and lower binding activity against Fc gamma RIII (such as Fc gamma RIIa or Fc gamma RIIIb) compared to those of a parent Fc region, the variant Fc region can be said to be Fc gamma RIlb-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):6591-6604 (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 0 425 235 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. 45:4336-4343 (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 At 2 1 1, 1131, 112 5, y9o, Re' 6, Re 188, Sm 15 3 , Bi2 12 , 21 32,Pb 2 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-I11, 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-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), 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-diazoniumbenzoyl)-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-isothiocyanatobenzyl-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, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(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, 1251, 3H, and 131I, 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 10 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., 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 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 RIIb 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 RIb 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 RIlb 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 RIb 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 RIb 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 RIb 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 RIb 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 crosslinking between Fc gamma RIIb 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 Fe gamma RIb 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 (DENVI, 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 RIa-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 RIla-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 RIa-dependent binding and suppress monocyte adhesion by Fe gamma RIlb-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 RIhb 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, TNFRSF1GA, TNFRSF1GB, TNFRSF1OC, TNFRSF1GD, 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 RIb. 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 RIb, 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 RIb 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 p-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 (FS293-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 NaCl 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 1x 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/CH 3CN gradient. Fractions containing human mature myostatin were pooled, dried and stored at -80 degrees C. To reconstitute, human mature myostatin was dissolved in 4mM HCl.

[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. EL4 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 hour, 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 MST0255-287, 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 ZR-96 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 Gim 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 k0MC 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 FS293-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 NaCl, 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-Blue T M 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-Blue T MTGF-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 Normocin T M, 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 BMP1 (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 QUANTIBlueT and the optical density at 620 nm was measured in a col orimetric plate reader. As shown as Figure 1, mAb MST1032-GIm (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. MST1032-Glm showed almost comparable inhibition activity against human, cynomolgus, and mouse latent myostatin.

[Table 2a] 2 S0000 0 00 0 0 00 0 o o o-4 o- - - -- 0 o o o o o o o o o o o o 0 0. ..c

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-I - KTLn Ln -I -I Ln O w Ln k.0O m m m m m m m m 0 o D D Q Q Q D o C 0 0 a 2°

C < ~I N 0o w ON0 Co CD 000 D C D DC DC 000 0 0 O~ z zz LU (r C,4 m m rn o m (1 r o m (U 00CD 0CC ~mommomomm CDCCDCDD 000 000 > c: C. > a

< <m m oD o m - - n mm e nm Sz m m q q t t t IZ e Z KT KT 0)0 *C0

o ~ 00 00000000a m

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2 0 o H N rHH 0 0 HFH F0

-m mu u 0 n, L nLU Lh ,LflfLr 000 00 ) DC C D00 00 H H) (D (NJ CD rD r) mm H H1 l0 2 1 -1 rlrf0 0 0 nf o o (I 0 Lf Ln HHHHHHHH

[Table 2b]

0 ooC) 0 00C0 0C)000

tv o0 0 )0 0 )0 00)0 0

000 00m000mm0)00m

m . L-4-- m wwMmmmWmwm

Ln n Un LA L

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CLl)flf~ 0 C) 0 0 0 lflfL) Lfl flV)Ln LfLLfLfLfL U LL00000000 10 0C

>r n0 0000 0~ 6 o N o I-I *~~w~-0 6 C) 0 TwIODQ O '

-V, I- I 0 M O ro 00 OM r-M c6 co

0 r O rn m m m coc

E~~ 0 0 0 0 0 0 mLn Li L-Li L- J

I* Cu 0 0 ~0 0 0 0 0 0 Nc.c~: IT TINN T T JTI q

2EU' 2i 2 :22 2 : i E IE

Example 4

[0510] 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 HEK-BlueTMTGF-beta cells. After 24-hour incubation, cell supernatant was mixed with QUANTIBlueTMand 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 MST1032-Glm, myostatin activation was inhibited and thus a lower SEAP level was detected in the cell supernatant. MST1032-Glm inhibited spontaneous activation of latent myostatin, and showed almost comparable inhibition activity against human, cynomolgus, and mouse latent myostatin. Example 5

[0511] 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/welI20 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 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 3. MST1032-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 MST1032-Glm specifically binds to the myostatin propeptide (e.g., the prppeptide portion), but not to active myostatin (e.g., mature region). Example 6

[0512] Characterization of anti-latent myostatin antibody (Western Blot) Mouse latent myostatin was incubated with recombinant human BMP1 (R&D Systems), with or without MST1032-Glm at 37 degrees C overnight. The samples were then mixed with 4x reducing SDS-PAGE sample buffer (Wako) and heated at 95 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 GDF8 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 MST1032-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 NaCl, 1.2 mM CaC 2 , 0.05% Tween 20, 0.005% NaN 3). 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 MST1032-GIm toward human, cyno, and mouse latent myostatin were comparable.

[Table 3] ka, kd, and KD of anti-latent myostatin antibody MST1032-Glm

ka (M 's) kd (s1 ) 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 MST1032-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 (MST1032-GIm and 41C1E4) increased lean body mass dose-dependently, and MST1032-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 MYO-029 were tested in five-week-old SCID mice for two weeks. REGN, OGD, and MYO-029 are anti-mature myostatin antibodies described in WO 2011/150008 as H4H1657N2, WO 2013/186719 as OGD1.0.0., and WO 2004/037861 as MYO-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 MYO-029 (P>0.05). Grip strength of the MST1032-Gm-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 MST1032-GIm. Fat mass reduction efficacy of MST1032-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 SKI sequence (SEQ ID NO: 54 (the amino acid sequence is shown in SEQ ID NO: 10)), re spectively. Humanized antibodies were transiently expressed in FS293-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 (MS1032LO00-SG1) showed comparable inhibition activity and affinity to chimeric antibody (MST1032-Glm).

[Table 4] Kinetic parameters of humanized anti-latent myostatin antibody

ka(M's') kd(s') KD(M) MST1032-Glm 1.13E+06 8.65E-05 7.66E-11 MS1032LOOO-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 MS1032LOOO-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.

[0522] As shown in Figure 9, the parental antibody (AbOO) 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 (AbO01). 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 5] pH5. 8 dissociation rate for single histidine substituted anti-latent myostatin antibodies

Name of variable region Abbreviation kd(1s) Heavy chain Light chain M103205H000 M103202L000 AbO01 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]

o r-o a0o o a n o o a

ee e oe ee o oe oeo

n 0

r r r r

~J*J ~ y

o o a

En CO(f C -1f E.n

n n t'n'E in EI'n 4i En En~t E n~ 17 L

D En nE D (n LD nK ot

- n NEn t n EnEn- n EnEnEn E

c -<

r- 00 0 '00,0' ro'o no00r0 hENi oo

.c a o in in ' n EN'EN m E nN n in > r

EnEo EEE n:E Eno NE oEN 0 o x o

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/ml BSA and 0.1mg/ml 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 IgG Iconstant 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 MS1032L00-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 MS1032LO00-SG1 showed a faster dissociation rate under acidic pH than neutral pH. Under the affinity condition, all antibodies except MS1032L00-SG1 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 myostati n Cyno latent myostatin 1 ka (M s 1) kd (s- ) KD (M) ka {M s ) kd (s-1) KD (M) ka (M- s') kd (s-') KD (M) MS1032LOOO-SG1 1.27E+06 9.52E-05 7.50&11 1.S2E+06 7.18E-05 4.71E-11 1.21E+06 808E-05 666-11 MS1032LO01-SG1 1.27E+06 2.16E-04 170E-10 145E+06 217E-04 1.49E-10 1,19E+06 197E-04 1.65E-10 MS1032LO02-SG1 8.36E+OS 249E-04 2.98E-10 N/T N/T N/T N/T N/T N/T MS1032LO03-SG1 9.81E+05 1.92E-04 196E10 N/T N/T N/T N/T N/T N/T MS1032LO04-SG1 1,33E+06 217E-04 163E-10 N/T N/T N/T N/T N/T N/T N/T: not tested

[Table 8] ka, kd, and KD of avidity condition assay under acidic pH Human latent myostatin Mouse latent myostatin Cyno latent myostatin 4 ka (Ms } 4 kd (s ) KD (M) ka (M s) kd (s ) KD {M) ka (M's4 ) kd (si} KD (M) MS1032LOOO-SG1 .GOE+06 1.28E-04 7.97E-11 232E+06 2.02E-05 8,70E-12 179E+06 1.58E-05 883E-12 MS1032LOO1-SG1 122E+06 2.42E-02 199E-08 1.02E+06 6.25E-03 6.1SE-09 7.64E+05 6.0E-03 890E-09 MS1032LO02-SG1 1.70E+06 4144E02 243E-08 N/T N/T N/T N/T N/T NIT MS1032LOO3-SG1 5,43E+05 5.24&03 9.66&09 N/T N/T N/T N/T N/T N/T MS1032L004-SG1 7.81E+05 7.13E03 9.13E-09 N/T N/T N/T N/T 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 1 ka (M s ) kd fs ) KD (M) ka(M's) kd(s } KD(M) ka(M-s') kd (s-) KD(M) MS1032LO00-SG1 107E+06 245E-04 2,30E-10 131E+06 L60E04 122E-101L02E+06 2,13E-04 2.09E-10 MS1032LO01-SG1 1.64E+06 129E-03 7.88E-10 150E+06 107E-03 713E-10 115E+06 1OE-03 954E-10 MS1032LOO2-SG1 LO9E+06 L79E&03 164E-09 N/T N/T N/T N/T N/T N/T MS1032LO03-SG1 LO8E+06 117E-03 1O8E-09 N/T N/T N/T N/T N/T N/T MS1032LOO4-SG1 154E+06 125E-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 myostatin Cyno latent myostatin 4 4 ko (M's') kd (s-) KD (M) ka (Mis-) kd (s ) KD (M) a (M's ) kd (s-) KD (M) MS1032LOOO-SG1 1.09E+06 2.35E-04 2.15E-10 1.62E+06 7.O8E-05 4.38E-11 141E+06 1.96E-04 1.39E-10 MS1032LOO1-SG1 n.d. nd. n.d. nd nd. nd. n.d. n.d. n.d. MS1032LO02-SGI n.d. n.d. n.d. N/T N/T N/T N/T N/T N/T MS1032LO03-SG1 n.d. n.d. n.d. N/T N/T N/T N/T N/T N/T MS1032LOO4-SG1 n.d. n.d. n.d, N/T N/T N/T N/T N/T N/T n.d.: not determined, N/T: not tested Example 15

[0527] Comparison of plasma total myostatin concentration between antibodies with Fe gamma R binding and an abolished Fe 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/ml) 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 MS1032L00-SG1 and MS1032L00-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 HCl, 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 MS1032L00-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 MS1032L00-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 MS1032L00-F760 (silent Fc) and MS1032LO00-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 SG1 due to strong avidity binding against Fc gamma R. The result suggests that MST1032 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 MS1032LO01-SG1 and MS1032LO01-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 (MS1032LO01-SG1 and MS1032LO01-F760) were tested in vivo and comparison of plasma total myostatin concentration was made. The total myostatin concentration measurement results after administration of MS1032LOOO-SG1 and MS1032LOOO-F760 described in Example 15 are also shown in Figure 13. MS1032LOOO is non pH-dependent antibody and MS1032LOO1is pH-dependent antibody. As shown in Figure 13, total myostatin con centration after administration of MS1032LOO1-F760 was reduced compared to MS1032LO00-F760 due to pH-dependent binding. Moreover, total myostatin con centration after administration of MS1032LO01-SG1 was dramatically reduced compared to that of MS1032LOOO-SG1 due to pH-dependent binding and increased cellular uptake by Fc gamma R binding. It is expected that MS1032LOO1-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, MS1032LO00-SG1 (non-sweeping antibody) and MS1032LO01-SG1 (sweeping antibody) increased skeletal muscle mass dose-dependently after 2 weeks. MS1032LOO1-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). MS1032LOO1-SG1 also sig nificantly decreased fat mass at 1 and 5 mg/kg relative to the vehicle group (PBS). MS1032LOOO-SG1showed no increase in lean body mass at 0.2 mg/kg. On the other hand, MS1032LOO1-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 1x 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/well/20 micro L or biotinylated human latent GDF11 at 20nM/well/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. MST1032-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 MST1032-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-Blue T M TGF-beta cells (Invivogen) that express a Smad3/4-binding element (SBE)-inducible SEAP reporter gene, allow the detection of bioactive GDF11 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-Blue T MTGF-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 Normocin T M, 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 (MST1032-Glm) at 37 degrees C overnight. The sample mixtures were transferred to cells. After 24-hour incubation, the cell supernatant was mixed with QUANTIBlueTMand 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, MS1032LO01-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 MS1032LO06-SG1, MS1032LO07-SG1, MS1032LO10-SG1, MS1032LO11-SG1, MS1032LO12-SG1, MS1032LO18-SG1, MS1032LO19-SG1, MS1032LO21-SG1, MS1032LO23-SG1, MS1032LO24-SG1, MS1032LO25-SG1, and MS1032LO26-SG1 were generated. Their amino acid and nucleotide sequences of these generate antibodies are shown in Table 11.

[Table 11a]

0 0) 0 0 0) 0) 0) 0)a .- Z C- tn0c tn00 0 Ln 0 0) Ln 0 Ln 0 Ln 0 Ln 0 Ln 0 Ln 0 Ln 0 Ln 0 Ln 0

z c W o oo oo oo oo oo o zHof o o o

z

Z

ric Oo Cu 0 00 0C 00 00 0

H o o1 o4m ot N N N OOm

rt C L

0 c NN N0N NN N N N N NN

oM M mro oco mo r

C - , (/ Lq O E n co Ln C4 m Ln oV

'H H 'H 'H 'Hi 'H'H H 'H H 'H H

O O O O 0 4 CO4 CH MN

L Ln LnL L In I- I0000nC ILn in Xi cIn Idn i Ii, mI,U i

cn Nq N N N -4 r Cq )M M M- 4 C r- (N M r-1

' H 'H I 'H H H

in ILnIL I I LI I I n I oiL

[Table 1lb]

rN H H tH H t t t t z t z

-N N N N N N N rN N rN N rN

-J 'n Nn Nn N N N N NN N LN >

Ga C

T ammmmmmmmmmmm

> 0 00 00 0 0 0 0 ~0 V - Ln V) in inV V n in Vn V)nV

mN N N N NN N N N C

L L mma)MLnLn I In In In n In I

~00 00 000NN 0 0 NI N N N 0 0 N 0N 0 eqrrnCfM m mN rrn Nrn N

1r H Hr 0 0 0 0 E

0 ID N N I L In In In

Z~ In oin in in 0 n in n 0 N 0 0D 0 0 01 M 0 4M 0

m N N Nq N N Nq N N N N N 0 00- 0,10o0

O I n I V I I V iu I VI UI f ) )

In2n2n2n2n2nuj2 2 2 2 2

[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 NaCl, 1.2 mM CaCl 2 , 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 HC 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.

[Table 12]

0 "o) fN Lfl c00 r m 00 D tD

E 0 00 00 00 0 00 a: 00 O ~ O 4- ~0 0 0 0 N 0 0 0 04 0

00 N

o00 X 0 00 0 0 00 00 0n 0 X0 17 ¶ -44 ¶4) cc ¶ 4 ¶4 ¶ 4 ¶

W L UJ LU U L LU LU LU wJ L CL r" N rv (N -41 a' c

19~~ a CL 0)0 L .

00 N (N 0 C~ O 0N 1- NN'

QO 0 00 00 00 00 CC, 00 m~ 00 0 999CU¶ c9 0 L ULU LW wU LwLU

(N (V* N NT LOm

r- 0

C 00 00 00 co 00 c (Nc 0

LO(N ' 0 00 0 C,

00 00 0 N 00 00 00C0,0 9 0 Cf 0 0 0q (N00 m 0 H - -4 '1e N

'~r, NC ko mC M IT LO 00 ul L m4 v, (

00 0 0 0> 0- 0N 0 0 0a

u) LU LU Li U LULnU L -~ ~r 1-14 00N(0 N (N . ' D D N 4N enm m N N ( -4 N

00 00 00 00) 00 00 c-, 0 4) ~ 0 0 0 22 22222

0 0l N~~ ( - 4NN - F '

'D~~~~ A '

Example 22

[0541] Evaluation of the neutralization activity of further optimized variants in HEK Blue Assay We evaluated the neutralization activity of MS1032LO06-SG1, MS1032LO07-SG1, MS1032LO10-SG1, MS1032LO11-SG1, MS1032LO12-SG1, MS1032LO18-SG1, MS1032LO19-SG1, MS1032LO21-SG1, MS1032LO23-SG1 and MS1032LO25-SG1 against human latent myostatin as described in Example 3. As shown in Fig. 17, all variants showed comparable activity to MS1032LOO1-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/ml) 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 MS1032LOOO-SG1, MS1032LOO1-SG1, MS1032LO06-SG1, MS1032LO11-SG1, MS1032LO18-SG1, MS1032LO19-SG1, MS1032LO21-SG1 and MS1032LO25-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-HCl, 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 (MS1032L00-SG1) and different pH-dependent anti-latent myostatin antibodies (MS1032LO01-SG1, MS1032LO06-SG1, MS1032LO11-SG1, MS1032LO18-SG1, MS1032LO19-SG1, MS1032LO21-SG1 and MS1032LO25-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 (MS1032LO01-SG1, MS1032LO06-SG1, MS1032LO11-SG1, MS1032LO18-SG1, MS1032LO19-SG1, MS1032LO21-SG1 and MS1032LO25-SG1) could reduce myostatin accumulation compared to non pH-dependent anti-latent myostatin antibody (MS1032L00-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 5 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 MS1032L00-SG1,

MS1032LO06-SG1012, MS1032LO06-SG1016, MS1032LO06-SG1029, MS1032LO06-SG1031, MS1032LO06-SG1033, and MS1032LO06-SG1034 (herein, SG1012, SG1016, SG1029, SG1031, SG1033, and SG1034 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 MS1032LO19-SG1079, MS1032LO19-SG1071, MS1032LO19-SG1080, MS1032LO19-SG1074, MS1032LO19-SG1081, and MS1032LO19-SG1077 (herein, SG1079, SG1071, SG1080, SG1074, SG1081, and SG1077 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 4oC. 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 (MS1032LO00-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 MS1032LO06-SG1012 and MS1032LO06-SG1033 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 MS1032LO06-SG1029, MS1032LO06-SG1031 and MS1032LO06-SG1034 could sweep antigen to below baseline. The reason for strong sweeping of MS1032LO06-SG1029, MS1032LO06-SG1031 and MS1032LO06-SG1034 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 MS1032LO19-SG1079, MS1032LO19-SG1071, MS1032LO19-SG1080, MS1032LO19-SG1074, MS1032LO19-SG1081 and MS1032LO19-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 MS1032LO19-SG1079 and MS1032LO19-SG1071 while the con centration of myostatin remained below the detection limit for MS1032LO19-SG1080, MS1032LO19-SG1074, MS1032LO19-SG1081 and MS1032LO19-SG1077. The weaker suppression of MS1032LO19-SG1079 and MS1032LO19-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 RIb or combining mutations that increase positive charge of the antibody and increase binding to Fc gamma RI. 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 MS1032LO06-SG1, MS1032LO11-SG1, MS1032LO18-SG1, MS1032LO19-SG1, and MS1032LO25-SG1. Three independent studies were conducted and MS1032LO01 was used as the control.

[0553] The results of these experiments are shown in Figures 20A-201. All antibodies (MS1032LO06-SG1, MS1032LO11-SG1, MS1032LO18-SG1, MS1032LO19-SG1, and MS1032LO25-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 BMP1 (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 (MST1495-MST1588). 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 (MST1032, MST1504, MST1538, MST1551, MST1558, MST1572, and MST1573; 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-H3 HVR-L1 HVR-L2 HVR-L3 MST1032 12 14 55 58 61 65 70 73 MST1504 145 151 157 163 169 175 181 187 MST1538 146 152 158 164 170 176 182 188 MST1551 147 153 159 165 171 177 183 189 MST1558 148 154 160 166 172 178 184 190 MST1572 149 155 161 167 173 179 185 191 MST1573 150 156 162 168 174 180 186 192

[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 (Abeam)) (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 MST1572 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 MST1573 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 RIlb-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 RIIb. Furthermore, Fc variants that can show enhanced binding to human Fc gamma RIb 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 RIb and decreased binding to other active Fc gamma Rs. This selective enhancement of Fc gamma RIb 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 RIlb-enhanced Fc variant against human and monkey Fc gamma Rs

[0561] The heavy chain gene of the human Fc gamma RIb enhanced variant (herein, "Fc gamma RIb enhanced" means "with enhanced Fe gamma RIlb-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 MS1032LO06-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 RIa was based on the sequence of NCBI accession # NM_001136219.1, Fc gamma RIb was on NM_004001.3, Fc gamma RIIIa was on NM_001127593.1, respectively. Allotypes of Fc gamma RIa and Fc gamma RIIIa were prepared according to the reports about polymorphism for Fc gamma RIa (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 RIaR, SEQ ID NO 211 for human Fc gamma RIIaH, SEQ ID NO 214 for human Fc gamma RIIb, 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 RIlal, 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 RIIb, SEQ ID NO 224 for cyno Fc gamma RIIaS). 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-HCl, 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 MS1032LO06-MY009 to human Fc gamma RIIaH, human Fc gamma RIIIaV, and cynoFc gamma RIIaS 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=, 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 2] 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 RIlb-enhanced variant MY009 does not show enhanced binding to cynoFc gamma RIb but shows enhanced binding to human Fc gamma RIb. Its affinity to cynoFc gamma RIb was rather decreased by 0.4-fold compared to SG1, indicating that human Fc gamma RIlb-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 RIb

[0567] Ideally, novel Fc variant should have enhanced binding to both human and cynoFc gamma RIb selectively and a decreased binding to other active Fc gamma Rs. However, because the putative Fc binding residues in cynoFc gamma RIb are completely the same as either allotype of cynoFc gamma RIa (Figure 23), it is theo retically impossible to achieve selective enhancement of cynoFc gamma RIb binding over cynoFc gamma RIa. Therefore a novel Fc variant should have selectively enhanced binding to human Fc gamma RIb, cynoFc gamma RIb and cynoFc gamma RIa.

[0568] In order to obtain a novel Fc variant that shows selectively enhanced binding to both human and cyno Fc gamma RIb, 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 IgG Iantibodies and binding to each Fc gamma R was analyzed comprehensively as shown in the following procedures.

[0569]

WO2017/104783 PCT1JP20161087487

[Table14] 3,, (Hc(3:

Hi -~ 0 Hi

~ (HC

O~

88

0 ~ Hf~ ~as Hi Hi~ 0 H

H (H 0 H~Hi H as ~iH

qH

as

E 0

0 Hi ~i) NO

5

H ~'

~H4HJ o -H I

H~ (HWHJ

(H Hi HI Hi N Hi Hi Hi N

a

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(H3~

[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 IgGIwas 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 RIa type H, Fc gamma RIa type R, Fc gamma RIb, and Fc gamma RIIaF) 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-HCl, 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 IgG Iat 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.

[0572] Table 15 shows the binding profile of promising substitutions selected based on the following criteria (more than 40% to human Fc gamma RIIb, lower than 100% to human Fc gamma RIaR and Fc gamma RIaH, less than 10 % to human Fc gamma RIIIaF, compared to those of parental B3 antibody).

[Table 15] Binding profile of selected substitutions against human Fc7Rs

Bindin___ to human Fe-R s( B3 =10 Mutati FegRIIaH FegRIaR FcgRIIb FcgRIIjaV on G236N 54 81 75 8 G237Y 77 4 85 0 G237D 74 0 95 0 P238E 21 2 97 1 P238Y 93 9 11 3 P238M 86 9 97 2 S19 4 43 -1____ 4 -1 V266F 91 88 2 S267H 27 3 421 S267L 63 3 112 2 N325L 93 13 116 0 N825E 49 13 40 4 N325I 76 8 97 1 N325F 65 6 72 -1 A327I 37 12 46 8

[0573] 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, Ilb, IIlaS were analyzed.

[0574] 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 cynoFcaRs(SG1 = 100) CH Name ubstitutio Fe Ra1 Be-5 2 Fe!aR2I1a3 FRIb FgIIaS SG1 1 0100 100 100 100 MY001 G236N 83 86 62 85 15 SG165 G237Y 18 17 30 30 7 SG166 G27D10 10 17 8 5 S067 P238Y 18 14 38 22 14 SG18231E2116 30 28 13 SG169 P28M17 16 32 22 22 SG170 m 16 15 21 18 1 SG171 S P17 20 18 20 8 SG172 F14 1 27 16 1 SG173 S267L 13 14 21 12 24 SG174 P267H 13 16 20 11 1 SG175 N325F 1 13 17 14 8 SG176 14 1 19 16 20 SG177 N325L 15 15 25 1 21 SG8 N325E 22 21 22 36 G 17 A3271 19 17 2 19 48

[0575] Among selected 16 Fc variants, only MYO Imaintained binding to cynoFc gamma RIb by 85% compared to SG1. Additionally MY001 showed reduced binding to cynoFc gamma RIIIaS and its binding to cynoFc gamma RIal, 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 RIb is lower than SG1 (75% for human Fc gamma RIb and 85% for cynoFc gamma RIb, respectively). In order to increase its affinity to Fc gamma RIb, additional substitutions were evaluated. Specifically, sub stitutions that showed increased binding to human Fc gamma RIb, reduced binding to human Fc gamma RIIIa and increased selectivity to human Fc gamma RIb over human Fc gamma RIla were selected based on the result of comprehensive mu tagenesis study (Table 17).

[Table 17] Binding profile of additionally selected substitutions to human FeyRs Binding to human FcgRs (parent B3 =100) Mutation FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV L234W 64 63 90 36 L234Y 38 50 55 63 L235W 136 128 130 40 G236D 109 91 212 25 G236A 165 168 94 65 G236E 142 131 117 33 G236S 147 161 87 33 S239N 99 80 124 111 S2391 82 75 108 47 S239V 79 71 107 48 S267A 176 125 283 145 H268D 173 142 307 220 H268E 169 132 283 205 P271G 152 130 252 93 P271D 94 55 110 85 P271E 90 51 105 87 Q295L 128 148 156 135 S298L 99 71 114 51 K326T 138 127 195 164 A327N 95 48 123 32 L328T 156 135 179 53 A330R 116 123 92 73 A330K 121 127 96 86 P331E 96 67 100 46 1332D 129 139 198 235 K3341 169 137 174 187 K334V 172 132 184 176 K334Y 157 131 160 188 K334M 151 127 148 183 K334D 97 72 108 155

[0577] Among these substitutions, L234W and L234Y were selected for increasing se lectivity to human Fc gamma RIb over human Fc gamma RIIa. And G236A, G236S, A330R, A330K and P331E were selected for decreasing binding to human Fc gamma RIIIa. All other substitutions were selected for increasing binding to human Fc gamma RIb. 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 RI~al, IIa2, IIa3, Ilb, IIaS were analyzed. The KD values in cells filled in gray were calculated using [Equation 2] 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 RIlal, cynoFc gamma RIIa2, cynoFc gamma RIIa3, and cynoFc gamma RIb to SG1. Although the affinity to human Fc gamma RIb is reduced to 0.6-fold, affinities to human Fc gamma RIIaH and human Fc gamma RIaR are reduced to 0.2-fold and 0.1-fold, respectively, indicating that this substitution has high selectivity to human Fc gamma RIb over human Fc gamma RIIa. Furthermore, its affinities to cynoFc gamma RIIaS 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 RIb should be enhanced.

[0580] Among additional substitutions evaluated, S298L, G236A, P331E, E233D, K334Y, K334M, L235W, S239V, K3341, L234W, K328T, Q295L, K334V, K326T, P396M, 1332D, H268E, P271G, S267A and H268D showed increased binding to both human and cynoFc gamma RIb. Specifically, H268D showed the largest effect (7-fold for human Fc gamma RIb and 5.3-fold for cynoFc gamma RIb). With respect to the effect of reduction in Fc gamma RIIIa binding, G236S, 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 RIb of MYO01, 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 2] 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.

[Taible 18]

- c jo - r r- - 6A-jt -6 -J1 L

r - - - Un - ~ aw

-- a

( - -i - ! 1U

LU- - -J~ 1I-

- - r -LfU~L

- f.-~ - U

- r,I - r---:K -6 - ULU 4)L -UL - . ULaU- UL UL ~ 1U44

rO4 44 0 LUL u s - a~ u u- - - - --- -a~ -00 - . i

- ---------4 -------

~~2 ~l LU LUL LU - L UL LUULULav L§L .U -U U-. -UL -LUW U -- - - - - *1 U

- -1 1- 1- - r - ? -- r - -- - -- r LU-U 164 U., U4L -ANL

- L..e.. e .. - -U - L U

[Table 19] ~ -N.7 Ii . . - - 10- ~ ~ t 0 -00 0 ~0 ~ 2 - 00t

- Oj r000 ~o -- 00 0 -j~ 0- 0 0 j

1 10

0 - 0 0 - t - : - 1 I

r

- •- - 0 - - - -00 00 - --- 0- - --0 -? -00 0 0~ -~ - ~ - -99 u -- n999 00000 000r

n - - 900n

1 0 0 0L L 0 0 L I 4 0000 1. L t .. 0 0

V 000 00-t n0~ 0 ~ < 00 0 0 0 0 00 0 0 0 0

-I1- t - 6 I L.j oflOLa S 4 0

- -_ It

- 0 00 6-~~i- .. !~ L$

t • j J - 0- - 10

[0582] All the variants suppressed affinities to human Fe 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 RIb compared to MYO Iand 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, MY1O, MY518, MY198 and MY197 showed enhanced binding both to human and cynoFc gamma RIb by more than 4-fold compared to SG1. Above all, MY205, MY209, MY198, and MY197 showed enhanced binding to both human and cynoFc gamma RIb 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, P3961, P396K, and P396L maintained human Fc gamma RIb binding by more than 5-fold compared to SG1 and only P396L substitution enhanced affinity to human Fc gamma RIb compared to MY052. With respect to the binding to cynoFc gamma RIb, parent MY052 showed the highest affinity which is a 3.9-fold increase from SGl.

[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

[Table20] 1 3 c.: a,no -aaa -a

, -n0 0 00 6© & 616 uj 6 6

666y H C Ooo et

6 62ar to

Ooo 4?''4© 4

1ea -666

oeo 6 6 !r

1 4 e 4e * 14

' -a

I -' --- '( - - - r -

(4N a e~

(N -N (N( N( N , (N :1;: o

(2 a'2

t.

444 N 900 99 9 n 4 4 44,,a na(4

1 09c99n 9999r9r

2 . 44.. . .4'44

- -- a (4

a ca a aa aato aa- a ala

a c a a a a o a aa a a 4,44o()(~( 4 44 44

. 2. 2 2 2 2 24 2. 2 Z4Z Z 2+ 74to

y- afa ac a aa

1 1 2N 2

[Table 21]

L11 -g~Na L

cI I u1g~

- --a Ic I e 2u

~itI~Ke ei5 I

NII 3 Nf r

-~9 99

-- C - OC 9

H 9

e ->- N

[Table 22] - - T N -NT c T 7C]

LCC . A ' o

' u 4 c -i ic -ic io c ioc~o-icc c ic ic ico ic t ic

-A~ - 1A - -N-A NNCCA 1 -cC-C-1 - c 0 -ACl CC , - CC ~f ~A ~C Cl t N -CC~NC C C

1 - N

1 1~CN~NCCCCl, N CAN NNN ~CN NN NN N N N-N N N0 CC CCAN N N!C

1~ NNN tN NC-C -CC -I _ -C C CC._C C J

aN ; - - A N NN-N-N1C NC.'N1N - -NL! N } N N- C NtCCN CACCc t - r JC1] + - CC CC -- C - C C C C ~~ "~

~AciANNArNNNN ~ ~c00N0Cic~i0 C-N

• • - (- NCNNN NNNCCCCC N0

NN N C C cCCF

4 -CNNTCCC CCC-.Q C N N N

-- - NN

4 - - {

) - i I 4 - L CC-CN5 - - N G 1 .. t CC L L C .C LI C LNCT .I . E A C I. A. C

r a s CCC -- A 1NN N N C NN - N N -- ) N AA C N C C CC N ~ N -CC C ' C|* ") CCC N-NC CCU-CCCCC 4 CN N AC N N C

T- F N r - -. .. r- - T T- E -T r... T

~ NCC CCCC -D

CCC~CC- - NCCC•C CN~ - 2 C .N CC-C CC-CA CCC-4 C 1

f s - .

- 1 -

CCCC LN

-1 ~C sC LL NNNLt|LLN

C, -C - - NNC CNN.,. LiCO N L -CC -CCC C.C. y -.. ~SI CC N

T- NN A' C 4 C-- -F - - - C - CC CC; C C4 CC C - - --- NT- T -r-- C - ;CC T -CCCCCC' CCCCCCCCCCCC' - -FN-- ~ N - N ---- CC--T T- - - --N-C- - r - -T - C - -N C

NANNN N NC - - ) 6

-CCAC| N NN C

C NN N NNNLNN A CC C N

~C~CN 1 C~NCNNONCCNNN -C-C-C-C-C-C -C -CCC CC 1CNNN[ NNN FN

C[ C'- C A - NNN-NC-N -W N- NNN•OCCArC-CCCN-N1

ts - C .. -. . OONCLLA CLL'LL NL 1 t L u INNN

- t~- CC- *

CCCe o u e

'4N CCN NN - NN NN N NNN NNNeo C N NN NN NNN CA CA AN CN1 CA - - A N -C - C - -u r s ONLNNNLu ulN- 1NNN- - -CLA. 0 0 r L0 t L i 0- 0 0 s C C-00 .NN

[0586] Although all the variants showed decreased affinity to both human and cynoFc gamma RIb 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 RIb and 3.1-fold for cynoFc gamma RIIb, respectively compared to SG1. MY265 also maintained reduced binding to both human and cynoFc gamma RIIIa. Although affinity to human Fc gamma RIaH and Fc gamma RI~aR 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 RIIb, comprehensive mutagenesis study into position 231 and 232 was performed. Specifically position 231 and 232 of M103205H795-MY265 were substituted with 18 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 RIIb, the addition of A23IT, A231M, A231V, A231G, A231F, A231I, A231L, A231, A231W, P232V, P232Y, P232F, P232M, P2321, P232W, P232L increased binding to both human and cynoFc gamma RIb compared to parent MY265. Above all, addition of A231T and A231G reduced human Fc gamma RIaR 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 2] 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 RIb compared to MY209. However, the affinities of MY213 to human Fc gamma RIaH and human Fc gamma RIaR are higher than those of MY209.

WO2017/104783 PCT1JP20161087487

[Table23A] 6!66666o6~6~6A~ ALA a A.2L L~ 6~666 6~666666 6j6W~6~~ 6 fAA 6 -A LA' 6 ~ ~A-Th '~6 a ~ ~a ~A 6 a 6 a ~, a a ~ - c~- - 6

~ A6ja ajl6 AL 66666 aj66A'$ 1 66 66 vA~aa66faa

LLAL~

aZ~ 6 f6aaa ~Z666a~~6j66aaI~;4a J a ' a - aH ~U ~ AL 666

~-i~-ilv-~ ~ '4

6 ala

666alaaa~ajAA6a~6

~UAaf6~66v-iIaL66a6~a66lAAla6 ~ + a I 1 666v-LL6i6l6aaaa U-AL U-K

A-AL6fI66AALtA-46ALAAVLALA aa66j4~6

A-A'AA-A-A'~AA'2A-6U66A66~A-AAAAA'~A-AA'A o66~a~v-~66j6v-ia a~6

Wa WWWAALALAAALALLAAALA~LL WALL LLA ALL ALA LALLALL aALLAAa

ALLL~i1A j } 9 9 ALA AAA-Z

91 6ALAAU-ah~46AALA~66~ALALaU

aaaaf6&6

~ a a

LALUaWWLALAL aLAs WALL ALA LLLAULLL

~ t 66 a aLLAhALLLaaa I A~a AL LAL U qL a AALAa ALL a LA. ALA a U-aaALAAA'-LLAAALLAav-AaAAAAA-~Lv}A'La U9 a ~AAWAAL9a~ 66 v-La ALA 6~v4 - A' A' a a aa 66 A' 9 A AL A A LAALALAU99 a~aA-~ a a a a

ALL ALA ALL U a ALA ALL AALa ALL ALL A6 AL a a A'LA-A~UALAA-ALAA-tLALALAUAL WIALL 'AjA'Av-AA~A'A 66 aLa a a LA

O ~ 7

a AL a A'L ALL a a LAI ALL ALL ALL N- a A'- A~. A'AL A'A~aLAS A'L -~ a a v-ALA'L ~ 6 LAIATh A4Av-A LA. A" A a 6a a A'L AL' A'- A~-A ALA LL4 LALIAAL LLA aA 'A a'ALa a ALA ULA'L

ALL AALa ALL ALL a AAL ALL LAA~U U U A-A a A- LA U A-AL

aa aa A' aa UaUaU~ UUUa A-A A-A ~ v-A U v-A A-A ALAa a~vA U a a A-A a a alU ALA ALL a A'- AL<A-' a - a U a a

LAAL~AiL¶~<L+ aaaaa~aaaaaaa UAALLAAALLAUU A~ ~ ~ a AL 99Aa ALALLAL ~ '~V?9 LAS a1 a9LAAL ~A~a rAAAIAI AL LA UL' ALL a a ALL AL ALL Lfl ALL AAA ALA 4U ALA ALL U ALLAALAI ALL ALL ALA ALA ALL ALLA-' A'A aLa AL ALLALL U AXiaLa AAA~1 'A A'AL¶ v-A - a LA a aLL) a aa ALAa a A-~ U~U~a ULALA U AAU A -~ a~AL-A aaajv-A A' ULLA a a v-v v--A U a AL LA v-A~iLLA a a~%~A-4 a /A a A'~ a a

9 *~ aala ~ AAlALL~I'~ tA~A'9LaI A

~-I~v- LLy~ALL U ~AALALU6A'AAALLN'A-AAAUIvAA a a aLa a 166. Ala a 6 ALLALA

ALLAL WAU ALL U LABALA LLA ALL U ALA ALL aLL ALA ALA aU ALA ALL LALLALL ALL ALL ALL a ALLLALL a ALLLALL U ALL AALLAAL ~ A-~L aa a alala A-' Alaa a UIALAOA U AAA a a U ALU ALA a a a a~a ALL A-ALa A'Aa U a - j-'--- 4 4 L - ALA a LAL~AAL a 6616

~ A' ~faLL'aaaaAAL aaUUUU: a ~ ~ a aALLLAAA CAL

a U ALAALA fv-AIAL U a a~ c~> a a a aI QAA~UALA~ a ALA-AAL a ~LAj~> AL) ALA LA~ ALA ~ ALA a ALL1 A ASAA-~LLL~ U LA~ 44 'A - LALAA-A v-LA U ALL ALA ~ALL AL aA 'A 4 4 AL(j-ALAILA'- 414 2LA UAAA~'Aj~ ~ a A-AA~ a ~faV~ 0I~3 c AL a a a ALALLLL~a ~ "-' a a a a LLLI a a ALL4a LZL ALL ~A9~A ~ ULA i?2~l~ ~ a~ -A~ ~ v-A a AL ~ 'AALLAjLA a a a v-na a a - LA a ~-a a a a v-A ~. V~ ~lii~A~H AL1L ~ ~AALZ ~LLL Aji -'LLLL ~ -~ '~LLAL AL~ AA$A LA-LA' A' A lUAU U UlaLU U LALAALAIU U U ULU U AU a A--LA' AA'~ ~ U A->U U U U U U ALLALL AaLa AL-A aAaaa U aAUUALLLUAUUUIU aLa ULULA UUUUUUALLAAUU A CAL aLa A-A a aAa aLa aaa a v-A a A'LLaLAa a a a AaAaaaAala a a aAa - ~A A'A' AL a -A~jA A A AL

- I _ laaalaa - ULAULA AL6A f~ AA>VLLALA ALAALLAAA-A ~ U A-' ALL ALA v-A ALA ~AAL a ~ alan AALA? UAALA A a a v-A 'Ala " aAa a vAL aLA' A-LA LAAA-AU

LAAZLALILLLLLI LA - ZLALLALLLLI LXLIALL LLL £ ZLALALLSLItLLLL XLLLLL A' A' L.-jA' A' LA- A' AL aAa 11~ ALA a alA/AL/A ALA ALALLA-A-ALA a a AL I a ALA a a a ALL U U a ~ AALLAALAIa ALA a ALA AL-A AALA ~ ~AA' ALA 112 ~A A--ia A' A'- IA-ALL ALA N-va ~z a ALA ALA ALA A/AU AA ALA ALA U~ ALA ALA ALA An A ALA AL-A LA-lU ULA/A U ALA a a U a U a LA- a a~ LAAALALA a a AAL ALA U A/A 112 A/A ALa aLALAAL a a a a a a a " WLALL) LA a a a a a a AL a a a a a a a a a l~ l~ a LLALALaaALA)AALaaaaA'ALaaALaaaAL a a v-A -A 1~il~ ~ A-Ajg~a ALA-LA a aLa a a a a a A-A a a aa ALlaaaaaa I A-A AlA-A-ALA ALAALA-AALAU U ~ I ~AA~12L ALA-A ~'A-A -A 'LA-A U v-A A-A aaAvALaaaa A--A ~AAA-ALAA v-LA-A aav-AaA-LAaaaa-ALa

WO2017/104783 PCT1JP20161087487

[Table23B]

= dj Li i~ a a a aaaa aa a a aa a a ti da uia ~Iaa a a a Wa a 2~fNo~Nao~,aapr~aa ,a.~aaaar9oaIaIaaa~aN ~ C .LLiuLLW&Jaaaaaaaaaaaaaa aaWa ~Caaaaaaaaaa~aaaaaaaaCaa.~a ai.1 ~ ~

a a~aaaaaaa WaWa sJaaaI aa a WaWaW Waa aa aaa a o~ ~

~ ~

4trja~~aaaa?~aaaaat4acp'taaar~aaaN'aaar-aaaa

o L~oc,~aoC 0 OW 00

oi t5 dNa ri a ,,eO~iri ~#r,~dc'i asraa~~i,.iaa aa N ~ci 000 00 aaaoar'lmnoiLOiOorobaOQ,.naoaaAA 'aariaorNa

~ ~a0 oooo aooooo 00 Woo 0 0 000 0

~ ~ ~~aaaaao Ca aa.aaa Wammania N

~L ~ ~a0 a~aja a a ~ a 0 ~aaa oLfo~ 0 ~ 0 ~a

C -.C a~ ~ 0 0 a ~~a ~ ~O4a.flJarN ~ ~~ ~ a ~ N a aN a~~ CI 2 ~ ~I-I~rIr~~aaa ~aaIa~~~ ' ~ a ~ ~ ania ri aaaaaa a niar-Inia CIa CC

aoaaaa aNanra,~aaNa

E ~ ~ C C ~ LAa~a ~i ~, ~ a a a aaaa aa a LA AiALALA LAL ii LA A AlA 1 ALA A A LA C ~

~~a~oa~o0o W0~0O0 00O00000a~a00~ ja a a a C000~W00,000000I00CWWl000000O0lO0000OW00 C C' CI~Cl a~4 IC CI a CI a a a a C-I W Cl Ci NCi[C CI CI C-i aNH a a CI a Ci

WO2017/104783 PCT1JP20161087487

[Table24] ~I0 0~00~

OTO 0

H 000 00~0~ 000000~00~

0~TT~1~

~

L

~ o~o aI~I~'~ us o~o us us WLOW

10 1~ 0

E 0 ~

K ~ osu~ 00 0~0 0~W~

ousoojo ~H 0 ~ sL~o s~si 2 -1 ~ 0b '10 ~ 00555 5550 0 5555

OKI

K vs I-o Ii L HI ~[~ ~ ojo

50 '~1' 50~5050dc~c1j ~j ~ 0

:051 xIx:0 '5 'Hi rJ

555 500 I 0~ ~I I 0 050

010010

I ~ u.TIXii ~0~00i000O00

__ n,000000 0~0~ ~1

Example 28

[0591] Evaluation of clearance of myostatin using Fe gamma RIb-enhanced Fc variants in all human Fe gamma R transgenic mice The effect of clearance of myostatin by Fe gamma RIlb-enhanced Fc variant con structed in Example 27 was evaluated in a mouse in which all murine Fe gamma Rs have been deleted and human Fe gamma Rs, encoded as transgenes, have been inserted into the mouse genome (Proc. Natl. Acad. Sci., 2012, 109, 6181). In this mice all mouse Fe gamma Rs are substituted with human ones so that the effect of affinity en hancement to human Fe gamma RIb on clearance of soluble antigen can be evaluated in mouse. Furthermore, pI-increasing substitutions were evaluated in combination with Fe gamma RIlb-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 pI-increasing sub stitutions (S400R/D413K) into MS103205H795-MY101. MS103205H795-MY351, MS103205H795-MY344, MS103205H795-MY335 were prepared by introducing another pI-increasing substitutions (Q311R/D413K) into MS103205H795-MY201, MS103205H795-MY205, MS103205H795-MY265, respectively. All the MS1032LO06 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 pI increasing substitutions do not affect the Fe gamma R binding of Fe gamma RIIb enhanced Fc variants. MY101 and PK2, which was prepared by introducing S400R/D413K into MY1O, showed 9-fold and 8-fold enhanced human Fe gamma RIb binding, respectively. The affinities of MY1O and PK2 to human Fe 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 pI-increasing substitutions, S400R/D413K or Q311R/D413K, does not affect the affinities to human and cynoFc gamma Rs. PK study in all human Fe gamma R transgenic mice In vivo test using all human Fe 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 Fe 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 4 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 MS1032LO06-SG1, MS1032LO06-MY101, MS1032LO06-PK2, MS1032LO06-MY201, MS1032LO06-MY351, MS1032LO06-MY205, MS1032LO06-MY344 and MS1032LO06-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-HCl, 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 37 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+2H]2+ (m/z 436.2) to y8 ion (m/z 637.3) for anti-latent myostatin antibody, and

[M+2H]2+ (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 p1 and Fc gamma R binding on myostatin concentration in vivo

[0597] After administration of MS1032LO06-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 MS1032LO06-MY101, MS1032LO06-MY201 and MS1032LO06-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 MS1032LO06-PK2, MS1032LO06-MY351, MS1032LO06-MY344 and MS1032LO06-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 MS1032LO06-SG1's was approximately within 2 fold, and pI variants did not increase antibody elimination from plasma. Both human Fc gamma RIb binding enhanced antibodies and pI-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 RIb-enhanced Fc variants in monkey The effect of enhanced binding of Fc variants developed in Example 27 to cynoFc gamma RIb on myostatin sweeping was evaluated in cynomolgus monkey. And the combination effect with pI-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 RIb enhanced and pI-increased Fc variants.

[0600] The heavy chain, MS103205H795-SG1012, MS103205H795-SG1029, MS103205 H795-SG1031, MS103205H795-SG1033, MS103205H795-SG1034 were prepared by introducing a N434A substitution into MS103205H795-MY1O1, MS103205 H795-MY344, MS103205H795-MY351, MS103205H795-MY201, MS103205H795-MY335, respectively. MS103205H795-SG1016 was prepared by in troducing pI-increasing substitutions (Q311R/D399K) into MS103205H795-SG1012. MS103240H795-SG1071 and MS103240H795-SG1079 were prepared by introducing M428L/N434A/Y436T/ Q438R/S440E substitutions and N434A/Q438R/S440E sub stitutions into MS103240 H795-MY344 respectively (MS103240H795: VH, SEQ ID NO; 92).

[0601] MS103240H795-SG1074 and MS103240H795-SG1077 were prepared by in troducing pI-increasing substitutions Q311R/P343R and M428L/N434A/Y436T/Q438R/S440E substitutions into MS103240H795-MY209 and MS103240H795-MY518, respectively. MS103240H795-SG1080 and

MS103240H795-SG1081 were prepared by introducing pI-increasing substitutions Q311R/P343R and N434A/Q438R/S440E substitutions into MS103240H795-MY209 and MS103240H795-MY518, respectively. MS103240H795-SG1071 was prepared by introducing pI-increasing substitutions Q311R/D413K and M428L/N434A/Y436T/Q438R/S440E substitutions into MS103240H795-MY205. MS103240H795-SG1079 was prepared by introducing pI-increasing substitutions Q311R/D413K and N434A/Q438R/S440E substitutions into MS103240H795-MY205. These MS1032LO06 variants and MS1032LO19 variants were expressed with M103202L889-SK1 and M103202L1045-SKI 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 MS1032LO06-SG1012 and MS1032LO19-SG1071 were calculated by divinding the KD values of MS1032LO06-SG1 and MS1032LO19-SG1 by the KD values of MS1032LO06-SG1012 and MS1032LO19-SG1071 respectively.

WO2017/104783 PCT1JP20161087487

[Table25] 000000 000000

3 300 f 00 0

~ .0300 0000030j0030000 j 3,3 0

~99~9 0Nw~ooo0303~o03No 9~r 03 30 30 003&303 10300030303

9 :9999 03 '~ V ~9~?9999 ~ ~ 99999999~99 0 099 ~9o3033'o~9' W iN 0000 Nj ~003,03 *999~I9~999 03 03333000 N 003

0? _ 9999 999 0303003030303 N33O~030303030303

00 999099o:999999 ~0oo~ ~

V~O~ 0 9 0 ???'#i? "1000333000303000003300 03 0~ 3' 9 00 0 N 3030 00 000 "'3ONN03NN03 3OINONN0ON

O~0 0303030 O~ <0303 ON 0303 ,,~,030 ~3N00N4003N0030

~' 9, ,99' 3 3$T~9 99990~9,~9

03000000303000

~03

903

03

9

~I033 30

N NNNNN N ~NNN 30 *tXr0z~ 3 0 .03 O 030300303o N 000003 N 3S~N 000 '3 NNNNNN 4N o N~03 ~0~NN 0

030300303 10030o03~0 00000 000000 0000003'300000 030 OO 00

~

"N N '0 0

[0602] The SPR analysis results are summarized in Table 25. Among these variants, SG1012, SG1016, SG1074 and SG1080 show the strongest affinity to human Fe gamma RIb, which has 10 fold enhanced affinity to human Fc gamma RIb compared to SGl. 29.2. PK study in monkey In vivo test using cynomolaus 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 5 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 MS1032L00-SG1, MS1032LO06-SG1012, MS1032LO06-SG1016, MS1032LO06-SG1029, MS1032LO06-SG1031, MS1032LO06-SG1033, and MS1032LO06-SG1034 (herein, SG1012, SG1016, SG1029, SG1031, SG1033, and SG1034 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 MS1032LO19-SG1079, MS1032LO19-SG1071, MS1032LO19-SG1080, MS1032LO19-SG1074, MS1032LO19-SG1081, and MS1032LO19-SG1077 (herein, SG1079, SG1071, SG1080, SG1074, SG1081, and SG1077 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 4oC. 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 (MS1032LO00-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 MS1032LO06-SG1012 and MS1032LO06-SG1033 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 MS1032LO06-SG1029, MS1032LO06-SG1031 and MS1032LO06-SG1034 could sweep antigen to below baseline. The reason for strong sweeping of MS1032LO06-SG1029, MS1032LO06-SG1031 and MS1032LO06-SG1034 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 MS1032LO19-SG1079, MS1032LO19-SG1071, MS1032LO19-SG1080, MS1032LO19-SG1074, MS1032LO19-SG1081 and MS1032LO19-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 MS1032LO19-SG1079 and MS1032LO19-SG1071 while the con centration of myostatin remained below the detection limit for MS1032LO19-SG1080, MS1032LO19-SG1074, MS1032LO19-SG1081 and MS1032LO19-SG1077. The weaker suppression of MS1032LO19-SG1079 and MS1032LO19-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 RI. 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 pI-increasing substitutions Q311R/D399R into MS103205H795-SG1. Other heavy chain variants were also prepared by introducing respective substitutions represented in Table 26 into MS103205H795-SG1. All the MS1032LO06 variants were expressed with M103202L889-SK1 as light chain according to the method shown in Example 34. Mouse Fc gamma RII-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 RII and antigen-antibody complexes were performed using BIACORE (registered trademark) T200 (GE Healthcare). Soluble mouse Fe 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 RII captured onto the sensor chip. 20 mM N-(2-Acetamido)-2-aminoethanesulfonic acid, 150 mM NaCl, 1.2 mM CaCl2, 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-HCl at pHl.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 RII 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 RII 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 RII present on a similarly negatively charged cell membrane surface.

WO2017/104783 PCT1JP20161087487

[Table26] ___________________ Summaryofp1-increasedFcvariants

Mi~ ~ L00 Ml0320~5SGl41 Q311Rt~399P ~ 64 102011 1 4 - :1 ~.1030 ?1 I 020 P1333 1~ 0P~D4 ~ 1A M120. Q1~ 0~0 1020 P1 1 ~N1~ 1. 14032 1 ~&1SIC t L~0 1410&0 P 4 7 1 ~334 1A 10 9-1 ~4 M032051i~ 4 ~ ~24 0~0 '~P1 1 ___________

1 ~141 102 14 ____________________

1f ____________

03 P144 140 _____________________ __________ ___________

~4m ______

1. 14 1410~051I~ P4 1410JH P4 Q1~ M120~ ~1 ~1 43 _______

0320~If P 1 1RD41 _____________ _____________

I41030~1i -.1 1 *R 1 1~4 1403 P 1410320 G41 14103 ~P1 3m ~311RIG34 _____________

10 ~05 1 Em ______________________________ ______________

G335~ ____________ _____________

L33 10320 - 1 m ~ _______________ LOS 0~01~ 1 ~1 - L09 14100 ~P1 1 1 ~S~0 ~ QI 1R ____________

141020 9P1 11Rt040 I ____________________________ O4~ 11 ~42R ~42 -p43 ______

1410320 1~ 0 1 41 7~4 1032 Hi P1 1 ~D4 1410320~ 9 1 2m G40- 17 A 141020117 1544 Q~ D 1 1 __________ ___________

1410303I~ P1 ~ Q1 ~D41 ~10 ________________

141 20 7 ~ 1410 _________________

14030li7 Pf _________________

10& 7~1 1 0401.

0320 1? 0401 041 _______________

14103~0IP Pf33 ~G4 041 ___________________ 33 40 041 3~71 ~2~01

[0615] Heretheantibodiesproducedbyintroducingp1-increasingmodificationsintotheFe 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 pI 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 RIb. 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 RIlb on the sensor chip. Cellular uptake of p1-increased Fc variants

[0617] To evaluate the rate of intracellular uptake into a human Fc gamma RIlb-expressing cell line, following assay was performed. An MDCK (Madin-Darby canine kidney) cell line that constitutively expresses human Fc gamma RIlb 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 RIlb 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 RIb 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 the antigen in the endosomes (acidic pH conditions) inside the cells. Since the dis sociated antigen is labeled with pHrodoRed as described earlier, it fluoresces in the endosomes. Thus, a strong fluorescence intensity inside the cell is thought to indicate that the uptake of the antigen-antibody complexes into the cells is taking place more quickly or in larger amounts.

[0622] Among the pI increased Fc variants with two amino acid substitution from SG1, the antigen-antibody complex made by SG141, P1375m, P1378m, P1499m, P1524m, P1525m, P1532m, P1533m, P1540m, P1541m and P1543m shows stronger antigen uptake into the cells. The amino acid substitutions on Q311R/D399R, Q311R/D413K, S400R/D413K, Q311R/P343R, Q311R/G341R, Q311R/G341K, Q311R/D401R, Q311R/D401K, D401R/D413K, D401K/D413K and G402K/D413K are supposed to have strong charge effect on antigen antibody complex uptake into the cells. PK study in human FcRn transgenic mouse In vivo test using human FcRn transgenic mice

[0623] The elimination of myostatin and anti-latent myostatin antibody were assessed in vivo upon co-administration of anti-latent myostatin antibody and latent myostatin in human FcRn transgenic mice in which mouse FcRn was substituted with human FcRn. An anti-latent myostatin antibody (0.1 mg/ml) and mouse latent myostatin (0.05 mg/ ml) were administered at a single dose of 10 ml/kg into the caudal vein in the ex periment PK-2 (described in Figure 27). An anti-latent myostatin antibody (0.3 mg/ ml), human latent myostatin (0.05 mg/ml) and human normal immunoglobulin (CSL Behring AG) (100 mg/ml) were administered at a single dose of 10 ml/kg into the caudal vein in the experiment PK-4 (described in Figure 28). Blood was collected at 5 minutes, 15min, 1 hour, 4 hours, 7 hours, 1 day, 2days, 7 days, 14 days, 21 days, and 28 days after administration in the experiment PK-2. Blood was collected at 5 minutes, 1 hour, 4 hours, 7 hours, 1 day, 7 days, 14 days, 21 days, and 30 days after admin istration in the experiment PK-4. The collected blood was centrifuged immediately at 15,000 rpm in 4 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 an tibodies used were MS1032LO06-SG1, MS1032LO06-P1375m, MS1032LO06-P1378m, MS1032LO06-P1383m in the experiment PK-2, and MS1032LO06-P1375m, MS1032LO06-P1499m in the experiment PK-4. Measurement of total myostatin concentration in plasma by electrochemilumi nescence (ECL)

[0624] The concentration of total myostatin in mouse plasma was measured by ECL as described in Example 28 (Ravetch PK). The time course of total myostatin con centration in plasma after intravenous administration of anti-latent myostatin antibody and latent myostatin measured by this method is shown in Figures 27A and 28A.

Measurement of anti-latent myostatin antibody concentration in plasma by enzyme linked immunosorbent assay (ELISA)

[0625] The concentration of anti-latent myostatin antibody in mouse plasma was measured by ELISA in the experiment PK-2. Anti-human IgG (gamma-chain specific) F(ab')2 antibody fragment (Sigma) was dispensed onto a Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and allowed to stand overnight at 4 degrees C to prepare anti human IgG-immobilized plates. Calibration curve samples having plasma concen trations of 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, and 0.039 micro g/ml, and mouse plasma samples diluted 100-fold or more were prepared. Then, the samples were dispensed onto the anti-human IgG-immobilized plates, and allowed to stand for 1 hour at room temperature. Subsequently, Goat Anti-Human IgG (gamma-chain specific) Biotin conjugate (Southern Biotech) was added to react for 1 hour at room temperature. Then, Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was added to react for one hour at room temperature, and chromogenic reaction was carried out using TMB One Component HRP Microwell Substrate (BioFX Labo ratories) as a substrate. After stopping the reaction with 1 N sulfuric acid (Showa Chemical), the absorbance at 450 nm was measured by a microplate reader. The con centration in mouse plasma was calculated from the absorbance of the calibration curve using the analytical software SOFTmax PRO (Molecular Devices). 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 27B.

[0626] The concentration of anti-latent myostatin antibody in mouse plasma was measured by a Gyrolab Bioaffy TM CD (Gyros) in the experiment PK-4. Biotinylated anti-human IgG Fc antibody was flowed over the streptavidin bead column within the mi crostructure of the CDs. Calibration curve samples having plasma concentrations of 0.5, 1, 2, 4, 8, 16, and 32 micro g/ml spiking plasma concentrations of 5 mg/mL of human normal immunoglobulin (CSL Behring AG) and 200 micro g/ml of mouse latent myostatin, and mouse plasma samples diluted 25-fold or more spiking plasma concentrations of 200 micro g/ml of mice latent myostatin were prepared. Then the samples were added into CDs and flowed over the bead column. Subsequently, Alexa labeled Goat anti-human IgG polyclonal antibody (BETHYL) was added into CDs and flowed over the bead column. The concentration in mouse plasma was calculated from the response of the calibration curve using the Gyrolab Evaluator Program. 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 28B. Effect of p1 on myostatin concentration in vivo

[0627] After administration of MS1032LO06-SG1, plasma total myostatin concentration at 7 hours decreased 12 fold compared to plasma total myostatin concentration at 5 minutes in the experiment PK-2. In contrast, after administration of MS1032LO06-P1375m, MS1032LO06-P1378m and MS1032LO06-P1383m, plasma total myostatin concentration at 7 hours decreased 26-67 fold compared to plasma total myostatin concentration at 5 minutes in the experiment PK-2. Antibody concentration of MS1032LO06-P1378m and MS1032LO06-P1383m decreased more than 3 fold compared to that of MS1032LO06-SG1, although the difference in MS1032LO06-P1375m's concentration at each sampling point compared to MS1032LO06-SG1's was within 2 fold in the experiment PK-2. pI variants including the amino acid substitutions on Q311R/D413K showed enhancement of myostatin elimination from plasma without enhancement of antibody elimination from plasma.

[0628] Furthermore, total myostatin concentration and antibody concentration after admin istration of MS1032LO06-P1499m were evaluated under co-administration of human normal immunoglobulin to mimic human plasma. After administration of MS1032LO06-P1375m and MS1032LO06-P1499m, plasma total myostatin con centration at 7 hours decreased 3.4 and 5.3 fold compared to plasma total myostatin concentration at 5 minutes, and total myostatin concentration and antibody con centration at each sampling point in MS1032LO06-P1499m was within 1.5 fold compared to that in MS1032LO06-P1375m in the experiment PK-4. pI variants including the amino acid substitutions on Q311R/P343R also showed enhancement of myostatin elimination from plasma without enhancement of antibody elimination from plasma.

[0629] 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 31

[0630] PI-increased substitutions in combination with Fc gamma RIlb-enhanced Fc variant The effect of clearance of myostatin by Fc gamma RIb-enhanced Fc variant in com bination with other pI-increasing substitutions were evaluated in human Fc gamma RIb transgenic mice. Human Fc gamma RIb transgenic mice were generated by mi croinjection of BAC (bacterial artificial chromosome) vector containing all the exons of human FCGR2B gene into the pronucleus of fertilized eggs of C57BL/6N by standard techniques (see, e.g., J. Immunol., 2015 Oct 1; 195(7): 3198-205). Mouse Fc gamma RIb deficient mice were generated using zinc finger nucleases (ZFNs) which was designed to target exon 1. The Humanized Fc gamma RIb mice were established by crossing human Fe gamma RIlb transgenic mice with mouse Fe gamma RIlb KO mice. Using this mouse, the combination effect of affinity enhancement to human Fc gamma RIb and pI increase on clearance of soluble antigen can be evaluated. Preparation and profile of tested variants

[0631] The tested 7 antibodies and their binding profiles are summarized in Table 27. The heavy chain, MS103205H795-MY352, MS103205H795-PK55, MS103205H795-PK56, MS103205H795-PK57, were prepared by introducing pI increasing substitutions Q311R/D413R, Q311R/P343R, Q311R/P343R/D413R, Q311R/N384R/D413R, respectively into MS103205H795-MY201. All the MS1032LO06 variants were expressed with M103202L889-SK1 as light chain according to the method shown in Example 30 and their affinities to human and cynoFc gamma Rs were evaluated using the method in Example 27.

WO2017/104783 PCT1JP20161087487

[Table27] 6 66 666

~6666 666

~ - 66666

~ ~~66 666

SI C) II) W C) U)

!C1:1:1, C) ~ N,~ I

E ~66r~66

~ ~466~5d

IOU)

[0632] Based on the SPR analysis summarized in Table 27, it was confirmed that the pI increasing substitutions do not affect the Fe gamma R binding of Fe gamma RIIb enhanced Fc variants. MY201 and MY351, which was prepared by introducing Q311R/D413K into MY201, show 6-fold and 5-fold enhanced human Fe gamma RIb binding, respectively. With respect to other human and cynoFc gamma Rs, they show almost the same binding profile. Similarly, MY352, PK55, PK56 and PK57 showed similar binding profile with parent MY201 (Table 27). These results indicates that either pair of p-increasing substitutions does not affect the affinities against human and cynoFc gamma Rs. PK study in human Fe gamma RIlb transgenic mice In vivo test using human Fe gamma RIb transgenic mice

[0633] 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 human Fe gamma RIb transgenic mice in which mouse Fe gamma RII was substituted with human Fe gamma RI. An anti-latent myostatin antibody (0.3 mg/ml) and latent myostatin (0.05 mg/ml) were administered at a single dose of 10 ml/ kg into the caudal vein. Blood was collected at 5 minutes, 1 hour, 4 hours, 7 hours, 1 day, 7 days, 14 days, 21 days, and 28 days after administration. The collected blood was centrifuged immediately at 15,000 rpm in 4 degrees C for 5 minutes to separate the plasma. The separated plasma was stored at or below -20 degrees C until mea surement. The anti-latent myostatin antibodies used were MS1032LO06-SG1, MS1032LO06-MY201, MS1032LO06-MY351, MS1032LO06-MY352, MS1032LO06-PK55, MS1032LO06-PK56 and MS1032LO06-PK57. Measurement of total myostatin concentration in plasma by electrochemilumi nescence

[0634] The concentration of total myostatin in mouse plasma was measured by electro chemiluminescence (ECL) as described in Example 28. 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 29. Measurement of anti-latent myostatin antibody concentration in plasma by enzyme linked immunosorbent assay (ELISA)

[0635] The concentration of anti-latent myostatin antibody in mouse plasma was measured by ELISA. Anti-human IgG (gamma-chain specific) F(ab')2 antibody fragment (Sigma) was dispensed onto a Nunc-ImmunoPlate MaxiSorp (Nalge Nunc Inter national) and allowed to stand overnight at 4 degrees C to prepare anti-human IgG immobilized plates. Calibration curve samples having plasma concentrations of 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, and 0.039 micro g/ml, and mouse plasma samples diluted 100-fold or more were prepared. Then, the samples were dispensed onto the anti-human IgG-immobilized plates, and allowed to stand for 1 hour at room tem perature. Subsequently, Goat Anti-Human IgG (gamma-chain specific) Biotin conjugate (Southern Biotech) was added to react for 1 hour at room temperature. Then, Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was added to react for one hour at room temperature, and chromogenic reaction was carried out using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as a substrate. After stopping the reaction with 1 N sulfuric acid (Showa Chemical), the absorbance at 450 nm was measured by a microplate reader. The concentration in mouse plasma was calculated from the absorbance of the calibration curve using the analytical software SOFTmax PRO (Molecular Devices). 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 30. Effect of p1 and Fc gamma R binding on myostatin concentration in vivo

[0636] After administration of MS1032LO06-MY201, plasma total myostatin concentration at 7 hours decreased 8 fold compared to plasma total myostatin concentration at 5 minutes. In contrast, after administration of MS1032LO06-PK57, plasma total myostatin concentration at 7 hours decreased 376 fold compared to plasma total myostatin concentration at 5 minutes. Other high pI variants showed similar en hancement of myostatin elimination from plasma. Antibody concentration of high pI variants at 28 days decreased 2-3 fold compared to antibody concentration of MS1032LO06-SG1, and pI variants showed slight enhancement of antibody elimination from plasma.

[0637] 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 32

[0638] Development of human Fc gamma RIlb-enhanced Fc variants Human Fc gamma RIlb-enhanced Fc variants with anti-myostatin variable region were constructed and their affinity for human Fc gamma Rs was evaluated. Specifically, human Fc gamma RIlb-enhanced Fc variants were constructed by in troducing substitutions into MS103240H795-SG1 (VH, SEQ ID NO: 92; CH, SEQ ID NO: 9) in combination with T250V/T307P substitutions. In addition, substitutions (Q311R/D413K or Q311R/P343R) were also introduced. The variants were expressed using M103202L1045-SK1 as light chain and their affinity to human Fc gamma Rs was analyzed according to the method shown in Example 34. Table 28 shows the result of kinetic analysis against human and cynoFc gamma Rs. The KD values in cells filled in gray were calculated using [Equation 2] since their affinity was too weak to be correctly determined by kinetic analysis.

[0639] All the variants evaluated showed enhanced affinity to human Fc gamma RIb and reduced binding to human Fc gamma RIaR, Fc gamma RIaH, Fc gamma RIIIaV compared to SG1. The affinity to human Fc gamma RIb varies between 4.1-fold and 30.5-fold increase compared to SG1. The affinity to human Fc gamma RIaR was between 0.02-fold and 0.22-fold. The affinities to human Fc gamma RIaH and human Fc gamma RIIIaV were less than 0.04-fold and 0.012-fold, respectively. It was revealed that the substitutions (T250V/T307P) does not affect the binding profile to human Fc gamma Rs by comparing the affinities of MY009 (P238D) and MY214 (P238D/T250V/T307P). On the other hand, both pair of p-increasing substitutions (Q311R/D413K and Q311R/P343R) decreased the binding affinity about 0.5-fold compared to the variants which does not contain the p-increasing substitutions. For example, although TT14 showed 30.5-fold higher affinity to human Fc gamma RIIb, it showed only 16.1-fold and 14.5-fold increase in combination with Q311R/D413K (TT33) and Q311R/P343R (TT32), respectively.

[0640] Furthermore, the substitutions used in Example 29 for improving antibody half-life and reducing binding to rheumatoid factor were introduced into human Fc gamma RIub-enhanced Fc variants and their affinity against human Fc gamma Rs were analyzed (Table 29). In this SPR analysis, all the data were obtained using mouse anti human IgG kappa light chain for capturing the antibodies of interest. Table 30 shows the result and the KD values in cells filled in gray were calculated using [Equation 2] since their affinity was too weak to be correctly determined by kinetic analysis.

[Table28]

-- - - - -~ - -- C 8of888 ,

D'A r A n h

j1j-- .t-h 99a 9 , 9

31 - LWGW L L U LW WL U W LUiL u

- - --

s K e H

E Ie n .'---

a e

N n N-NN-N N

1 . - -N- .. -- - ..

C n

N m r' r -s NN -NA '

10n- n -- N N N N - Nn - -A

LnK n

[Table 29] Amino acid sequences of heavy chain constant regions comprising human FeyRIIb enhanced Fc variants (shown as SEQ ID NOs)

+M4428 +N434A +N434A +Y436T +N434A CH - +Q438R +Y436T +438R +0438R +S440E +438R +S440E +5440E +S440E MY009 252 278 304 330 356 MY214 253 279 305 331 357 08 254 280 306 332 358 0T9 255 281 307 333 359 TT0 256 282 308 334 360

[11 257 283 309 335 361 T12 258 284 310 336 362 T13 259 285 311 337 363 T14 260 286 312 338 364 T15 261 287 313 339 365 20 262 288 314 340 366 T21 263 289 315 341 367 T22 264 290 316 342 368 T23 265 291 317 343 369

T25 267 293 319 345 371 T26 268 294 320 346 372 T27 269 295 321 347 373 T28 270 296 322 348 374 T29 271 297 323 349 375

T31 273 299 325 351 377 T32 274 300 326 352 378 T33 275 301 327 353 379 34 276 302 328 354 380 5 277 303 329 355 381

[Table 30]

1 r

C - O C~

0 ccicc

C1 -. 1 -0

n0

;4 T - - - 0 0 -0 s .

- -c 1

[0641] SG1 and TT33 were evaluated again in this measurement in which capturing method was different from that in Table 28. As a result, "KD fold" values of TT33 were consistent with those obtained with Table 28 (16.1-fold in Table 28 and 15.7-fold in Table 30 against human Fe gamma RIb). TT92 and TT93, which were constructed by introducing N434A/Q438R/S440E and M428L/N434A/Y436T/Q438R/S440E into TT33, showed comparable binding profile to TT33. Similarly, TT90 and TT91 derived from TT32, TT72 and TT73 derived from TT31, TT70 and TT71 derived from TT30, TT68 and TT69 derived from TT21, and TT66 and TT67 derived from TT20 show comparable binding profile to those of parental antibodies shown in Table 28. These results indicate that substitutions for improving antibody half-life and reducing binding to rheumatoid factor do not affect binding profile of human Fc gamma RIlb-enhanced variants against human Fc gamma Rs. Example 33

[0642] Cell imaging analysis of Fc gamma RIlb-enhanced Fc variants To evaluate the intracellular uptake of antigen-antibody complex formed by the antibody described in Example 32, cell imaging assay described in Example 30 was performed. To avoid the signal saturation, the antigen-antibody complexes were formed in a culture solution with the antibody concentration being 1.25 mg/mL and the antigen concentration being 0.34 mg/mL in this example.

[0643] Assay results are shown in Figure 31. By increasing the binding affinity to human Fc gamma RIIb, cellular uptake of antigen-antibody complex were increased. In case of TT14, which has 30 fold enhanced binding affinity to human Fc gamma RIlb compared with SG1, the antigen florescent intensity inside cells was 7.5 fold increased as that of SG1.

[0644] Although pI-increasing substitutions (Q311R/D413K and Q311R/P343R) decreased the binding affinity to human Fc gamma RIb about 0.5-fold compared to the variants which does not contain the pI-increasing substitutions, the cellular uptake of antigen antibody complex of Fc variants with pI increased substitutions was increased compared to Fc variants without pI increased substitutions. TT33 shows 36 fold increase in antigen antibody uptake into the cells compared to SG1, while its parent TT14 shows 7.5 fold increase in cellular uptake. Furthermore, TT33 shows comparable cellular uptake of antigen-antibody complex into the cells compared to SG1071, SG1074, SG1077, SG1079, SG1O80 and SG1O81 which shows strong sweeping in Cynomolugus monkey. These results suggest that combining of pI increased sub stitutions with Fc gamma RIb enhanced Fc variants is the powerful tool for antigen sweeping. Example 34

[0645] Construction of antibody expression vectors: and expression and purification of an tibodies Synthesis of full-length genes encoding the nucleotide sequences of the H chain and L chain of the antibody variable regions was carried out by production methods known to those skilled in the art using Assemble PCR and such. Introduction of amino acid substitutions was carried out by methods known to those skilled in the art using PCR or such. The obtained plasmid fragment was inserted into an animal cell expression vector, and the H-chain expression vector and L-chain expression vector were produced. The nucleotide sequence of the obtained expression vector was determined by methods known to those skilled in the art. The produced plasmids were introduced transiently into the HEK293H cell line derived from human embryonic kidney cancer cells (Invitrogen) or into FreeStyle293 cells (Invitrogen) for antibody expression. The obtained culture supernatant was collected, and then passed through a 0.22 micro m MILLEX(R)-GV filter (Millipore), or through a 0.45 micro m MILLEX(R)-GV filter (Millipore) to obtain the culture supernatant. Antibodies were purified from the obtained culture supernatant by methods known to those skilled in the art using rProtein A Sepharose Fast Flow (GE Healthcare) or Protein G Sepharose 4 Fast Flow (GE Healthcare). For the concentration of the purified antibodies, their absorbance at 280 nm was measured using a spectrophotometer. From the obtained value, the ex tinction coefficient calculated by the methods such as PACE was used to calculate the antibody concentration (Protein Sci. 4:2411-2423 (1995)).

[0646] Although the foregoing invention has been described in some detail by way of il lustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The dis closures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.

SEQUENCE LISTING SEQUENCE LISTING

<110> CHUGAI SEIYAKU <110> CHUGAI SEIYAKU KABUSHIKI KABUSHIKI KAISHA KAISHA

<120> ANTI-MYOSTATIN ANTIBODIES, <120> ANTI-MYOSTATIN ANTIBODIES, POLYPEPTIDES POLYPEPTIDES CONTAINING CONTAINING VARIANT Fc VARIANT FC REGIONS, AND REGIONS, ANDMETHODS METHODSOF OF USEUSE <130> <130> C1-A1518P C1-A1518P

<150> <150> JP 2015-247070 JP 2015-247070 <151> <151> 2015-12-18 2015-12-18

<160> <160> 381 381

<170> <170> PatentIn version PatentIn version3.5 3.5

<210> <210> 1 1 <211> <211> 375 375 <212> <212> PRT PRT <213> <213> Homo sapiens Homo sapiens

<400> <400> 1 1

Met Gln Met Gln Lys Lys Leu Leu Gln Gln Leu Leu Cys Cys Val Val Tyr Tyr Ile Ile Tyr Tyr Leu Leu Phe Phe Met Met Leu Leu Ile Ile 1 1 5 5 10 10 15 15

Val Ala Val Ala Gly Gly Pro Pro Val Val Asp Asp Leu Leu Asn Asn Glu Glu Asn Asn Ser Ser Glu Glu Gln Gln Lys Lys Glu Glu Asn Asn 20 20 25 25 30 30

Val Glu Val Glu Lys Lys Glu Glu Gly Gly Leu Leu Cys Cys Asn Asn Ala Ala Cys Cys Thr Thr Trp Trp Arg Arg Gln Gln Asn Asn Thr Thr 35 35 40 40 45 45

Lys Ser Lys Ser Ser SerArg ArgIle Ile Glu Glu AlaAla IleIle Lys Lys Ile Ile Gln Leu Gln Ile Ile Ser LeuLys Ser Lys Leu Leu 50 50 55 55 60 60

Arg Leu Arg Leu Glu Glu Thr Thr Ala Ala Pro Pro Asn Asn Ile Ile Ser Ser Lys Lys Asp Asp Val Val Ile Ile Arg Arg Gln Gln Leu Leu

70 70 75 75 80 80

Leu Pro Leu Pro Lys Lys Ala Ala Pro Pro Pro Pro Leu Leu Arg Arg Glu Glu Leu Leu Ile Ile Asp Asp Gln Gln Tyr Tyr Asp Asp Val Val 85 85 90 90 95 95

Gln Arg Gln Arg Asp AspAsp AspSer Ser Ser Ser AspAsp GlyGly Ser Ser Leu Leu Glu Asp Glu Asp Asp Asp AspTyr Asp Tyr His His

1

100 105 105 110 110

Ala Thr Ala Thr Thr ThrGlu GluThr Thr Ile Ile IleIle ThrThr Met Met Pro Pro Thr Ser Thr Glu Glu Asp SerPhe Asp Phe Leu Leu 115 115 120 120 125 125

Met Gln Met Gln Val ValAsp AspGly Gly Lys Lys ProPro LysLys Cys Cys Cys Cys Phe Lys Phe Phe Phe Phe LysSer Phe Ser Ser Ser 130 130 135 135 140 140

Lys Ile Lys Ile Gln GlnTyr TyrAsn Asn Lys Lys ValVal ValVal Lys Lys Ala Ala Gln Trp Gln Leu Leu Ile TrpTyr Ile Tyr Leu Leu 145 145 150 150 155 155 160 160

Arg Pro Arg Pro Val Val Glu Glu Thr Thr Pro Pro Thr Thr Thr Thr Val Val Phe Phe Val Val Gln Gln Ile Ile Leu Leu Arg Arg Leu Leu 165 165 170 170 175 175

Ile Lys Pro Ile Lys ProMet MetLys LysAsp Asp GlyGly ThrThr Arg Arg Tyr Tyr Thr Thr Gly Arg Gly Ile IleSer Arg Ser Leu Leu 180 180 185 185 190 190

Lys Leu Lys Leu Asp Asp Met Met Asn Asn Pro Pro Gly Gly Thr Thr Gly Gly Ile Ile Trp Trp Gln Gln Ser Ser Ile Ile Asp Asp Val Val 195 195 200 200 205 205

Lys Thr Lys Thr Val Val Leu Leu Gln Gln Asn Asn Trp Trp Leu Leu Lys Lys Gln Gln Pro Pro Glu Glu Ser Ser Asn Asn Leu Leu Gly Gly 210 210 215 215 220 220

Ile Glu Ile Ile Glu IleLys LysAla AlaLeu Leu AspAsp GluGlu Asn Asn Gly Gly His His Asp Ala Asp Leu LeuVal Ala Val Thr Thr 225 225 230 230 235 235 240 240

Phe Pro Phe Pro Gly GlyPro ProGly GlyGlu Glu AspAsp GlyGly Leu Leu Asn Asn Pro Pro Phe Glu Phe Leu LeuVal Glu Val Lys Lys 245 245 250 250 255 255

Val Thr Val Thr Asp Asp Thr Thr Pro Pro Lys Lys Arg Arg Ser Ser Arg Arg Arg Arg Asp Asp Phe Phe Gly Gly Leu Leu Asp Asp Cys Cys 260 260 265 265 270 270

Asp Glu Asp Glu His His Ser Ser Thr Thr Glu Glu Ser Ser Arg Arg Cys Cys Cys Cys Arg Arg Tyr Tyr Pro Pro Leu Leu Thr Thr

2

Val Val 275 275 280 280 285 285

Asp Phe Asp Phe Glu Glu Ala Ala Phe Phe Gly Gly Trp Trp Asp Asp Trp Trp Ile Ile Ile Ile Ala Ala Pro Pro Lys Lys Arg Arg Tyr Tyr 290 290 295 295 300 300

Lys Ala Lys Ala Asn AsnTyr TyrCys Cys Ser Ser GlyGly GluGlu Cys Cys Glu Glu Phe Phe Phe Val Val Leu PheGln Leu Gln Lys Lys 305 305 310 310 315 315 320 320

Tyr Pro Tyr Pro His HisThr ThrHis His Leu Leu ValVal HisHis Gln Gln Ala Ala Asn Arg Asn Pro Pro Gly ArgSer Gly Ser Ala Ala 325 325 330 330 335 335

Gly Pro Gly Pro Cys Cys Cys Cys Thr Thr Pro Pro Thr Thr Lys Lys Met Met Ser Ser Pro Pro Ile Ile Asn Asn Met Met Leu Leu Tyr Tyr 340 340 345 345 350 350

Phe Asn Gly Phe Asn GlyLys LysGlu GluGln Gln IleIle IleIle Tyr Tyr Gly Gly Lys Lys Ile Ala Ile Pro ProMet Ala Met Val Val 355 355 360 360 365 365

Val Asp Val Asp Arg ArgCys CysGly Gly Cys Cys SerSer 370 370 375 375

<210> <210> 2 2 <211> <211> 109 109 <212> <212> PRT PRT <213> <213> Homo sapiens Homo sapiens

<400> <400> 2 2

Asp Phe Asp Phe Gly GlyLeu LeuAsp Asp Cys Cys AspAsp GluGlu His His Ser Ser Thr Ser Thr Glu Glu Arg SerCys Arg Cys Cys Cys 1 1 5 5 10 10 15 15

Arg Tyr Arg Tyr Pro Pro Leu Leu Thr Thr Val Val Asp Asp Phe Phe Glu Glu Ala Ala Phe Phe Gly Gly Trp Trp Asp Asp Trp Trp Ile Ile 20 20 25 25 30 30

Ile Ala Pro Ile Ala ProLys LysArg ArgTyr Tyr LysLys AlaAla Asn Asn Tyr Tyr Cys Cys Ser Glu Ser Gly GlyCys Glu Cys Glu Glu 35 35 40 40 45 45

3

Phe Val Phe Val Phe PheLeu LeuGln GlnLys Lys TyrTyr ProPro His His Thr Thr His His Leu His Leu Val ValGln His Gln Ala Ala 50 50 55 55 60 60

Asn Pro Asn Pro Arg ArgGly GlySer Ser Ala Ala GlyGly ProPro Cys Cys Cys Cys Thr Thr Thr Pro Pro Lys ThrMet Lys Met Ser Ser

70 70 75 75 80 80

Pro Ile Asn Pro Ile AsnMet MetLeu LeuTyr Tyr PhePhe AsnAsn Gly Gly Lys Lys Glu Glu Gln Ile Gln Ile IleTyr Ile Tyr Gly Gly 85 85 90 90 95 95

Lys Ile Lys Ile Pro ProAla AlaMet Met Val Val ValVal AspAsp Arg Arg Cys Cys Gly Ser Gly Cys Cys Ser 100 100 105 105

<210> <210> 3 3 <211> <211> 375 375 <212> <212> PRT PRT <213> <213> Macaca fascicularis Macaca fascicularis

<400> <400> 3 3

Met Gln Met Gln Lys LysLeu LeuGln Gln Leu Leu CysCys ValVal Tyr Tyr Ile Ile Tyr Phe Tyr Leu Leu Met PheLeu Met Leu Ile Ile 1 1 5 5 10 10 15 15

Arg Leu Arg Leu Glu Glu Thr Thr Ala Ala Pro Pro Asn Asn Ile Ile Ser Ser Lys Lys Asp Asp Ala Ala Ile Ile Arg Arg Gln Gln Leu Leu

70 70 75 75 80 80

4

Gln Arg Gln Arg Asp AspAsp AspSer Ser Ser Ser AspAsp GlyGly Ser Ser Leu Leu Glu Asp Glu Asp Asp Asp AspTyr Asp Tyr His His 100 100 105 105 110 110

Phe Pro Phe Pro Gly GlyPro ProGly Gly Glu Glu AspAsp GlyGly Leu Leu Asn Asn Pro Leu Pro Phe Phe Glu LeuVal Glu Val Lys Lys 245 245 250 250 255 255

Val Thr Val Thr Asp AspThr ThrPro Pro Lys Lys ArgArg SerSer Arg Arg Arg Arg Asp Gly Asp Phe Phe Leu GlyAsp Leu Asp Cys Cys 260 260 265 265 270 270

5

Asp Glu Asp Glu His HisSer SerThr Thr Glu Glu SerSer ArgArg Cys Cys Cys Cys Arg Pro Arg Tyr Tyr Leu ProThr Leu Thr Val Val 275 275 280 280 285 285

Lys Ala Lys Ala Asn Asn Tyr Tyr Cys Cys Ser Ser Gly Gly Glu Glu Cys Cys Glu Glu Phe Phe Val Val Phe Phe Leu Leu Gln Gln Lys Lys 305 305 310 310 315 315 320 320

Val Asp Val Asp Arg ArgCys CysGly Gly Cys Cys SerSer 370 370 375 375

<210> <210> 4 4 <211> <211> 109 109 <212> <212> PRT PRT <213> <213> Macaca fascicularis Macaca fascicularis

<400> <400> 4 4

6

Phe Val Phe Val Phe PheLeu LeuGln GlnLys Lys TyrTyr ProPro His His Thr Thr His Val His Leu Leu His ValGln His Gln Ala Ala 50 50 55 55 60 60

70 70 75 75 80 80

Pro Ile Pro Ile Asn AsnMet MetLeu LeuTyr Tyr PhePhe AsnAsn Gly Gly Lys Lys Glu Ile Glu Gln Gln Ile IleTyr Ile Tyr Gly Gly 85 85 90 90 95 95

Lys Ile Lys Ile Pro Pro Ala Ala Met Met Val Val Val Val Asp Asp Arg Arg Cys Cys Gly Gly Cys Cys Ser Ser 100 100 105 105

<210> <210> 5 5 <211> <211> 376 376 <212> <212> PRT PRT <213> <213> Mus musculus Mus musculus

<400> <400> 5 5

Met Met Met Met Gln GlnLys LysLeu Leu Gln Gln MetMet TyrTyr Val Val Tyr Tyr Ile Leu Ile Tyr Tyr Phe LeuMet Phe Met Leu Leu 1 1 5 5 10 10 15 15

Ile Ala Ala Ile Ala AlaGly GlyPro ProVal Val AspAsp LeuLeu Asn Asn Glu Glu Gly Gly Ser Arg Ser Glu GluGlu Arg Glu Glu Glu 20 20 25 25 30 30

Asn Val Asn Val Glu Glu Lys Lys Glu Glu Gly Gly Leu Leu Cys Cys Asn Asn Ala Ala Cys Cys Ala Ala Trp Trp Arg Arg Gln Gln Asn Asn 35 35 40 40 45 45

Thr Arg Thr Arg Tyr TyrSer SerArg Arg Ile Ile GluGlu AlaAla Ile Ile Lys Lys Ile Ile Ile Gln Gln Leu IleSer Leu Ser Lys Lys 50 50 55 55 60 60

Leu Arg Leu Arg Leu LeuGlu GluThr Thr Ala Ala ProPro AsnAsn Ile Ile Ser Ser Lys Ala Lys Asp Asp Ile AlaArg Ile Arg Gln Gln

70 70 75 75 80 80

Leu Leu Leu Leu Pro ProArg ArgAla Ala Pro Pro ProPro LeuLeu Arg Arg Glu Glu Leu Asp Leu Ile Ile Gln AspTyr Gln Tyr Asp Asp 85 85 90 90 95 95

7

Val Gln Val Gln Arg Arg Asp Asp Asp Asp Ser Ser Ser Ser Asp Asp Gly Gly Ser Ser Leu Leu Glu Glu Asp Asp Asp Asp Asp Asp Tyr Tyr 100 100 105 105 110 110

His Ala His Ala Thr ThrThr ThrGlu Glu Thr Thr IleIle IleIle Thr Thr Met Met Pro Glu Pro Thr Thr Ser GluAsp Ser Asp Phe Phe 115 115 120 120 125 125

Leu Met Leu Met Gln GlnAla AlaAsp Asp Gly Gly LysLys ProPro Lys Lys Cys Cys Cys Phe Cys Phe Phe Lys PhePhe Lys Phe Ser Ser 130 130 135 135 140 140

Ser Lys Ile Ser Lys IleGln GlnTyr TyrAsn Asn LysLys ValVal Val Val Lys Lys Ala Ala Gln Trp Gln Leu LeuIle Trp Ile Tyr Tyr 145 145 150 150 155 155 160 160

Leu Arg Leu Arg Pro ProVal ValLys Lys Thr Thr ProPro ThrThr Thr Thr Val Val Phe Gln Phe Val Val Ile GlnLeu Ile Leu Arg Arg 165 165 170 170 175 175

Leu Ile Leu Ile Lys LysPro ProMet Met Lys Lys AspAsp GlyGly Thr Thr Arg Arg Tyr Gly Tyr Thr Thr Ile GlyArg Ile Arg Ser Ser 180 180 185 185 190 190

Leu Lys Leu Lys Leu LeuAsp AspMet Met Ser Ser ProPro GlyGly Thr Thr Gly Gly Ile Gln Ile Trp Trp Ser GlnIle Ser Ile Asp Asp 195 195 200 200 205 205

Val Lys Val Lys Thr Thr Val Val Leu Leu Gln Gln Asn Asn Trp Trp Leu Leu Lys Lys Gln Gln Pro Pro Glu Glu Ser Ser Asn Asn Leu Leu 210 210 215 215 220 220

Gly Ile Gly Ile Glu GluIle IleLys Lys Ala Ala LeuLeu AspAsp Glu Glu Asn Asn Gly Asp Gly His His Leu AspAla Leu Ala Val Val 225 225 230 230 235 235 240 240

Thr Phe Thr Phe Pro ProGly GlyPro Pro Gly Gly GluGlu AspAsp Gly Gly Leu Leu Asn Phe Asn Pro Pro Leu PheGlu Leu Glu Val Val 245 245 250 250 255 255

Lys Val Lys Val Thr Thr Asp Asp Thr Thr Pro Pro Lys Lys Arg Arg Ser Ser Arg Arg Arg Arg Asp Asp Phe Phe Gly Gly Leu Leu Asp Asp

8

260 265 265 270 270

Cys Asp Cys Asp Glu Glu His His Ser Ser Thr Thr Glu Glu Ser Ser Arg Arg Cys Cys Cys Cys Arg Arg Tyr Tyr Pro Pro Leu Leu Thr Thr 275 275 280 280 285 285

Val Asp Val Asp Phe PheGlu GluAla Ala Phe Phe GlyGly TrpTrp Asp Asp Trp Trp Ile Ala Ile Ile Ile Pro AlaLys Pro Lys Arg Arg 290 290 295 295 300 300

Tyr Lys Tyr Lys Ala Ala Asn Asn Tyr Tyr Cys Cys Ser Ser Gly Gly Glu Glu Cys Cys Glu Glu Phe Phe Val Val Phe Phe Leu Leu Gln Gln 305 305 310 310 315 315 320 320

Lys Tyr Lys Tyr Pro ProHis HisThr Thr His His LeuLeu ValVal His His Gln Gln Ala Pro Ala Asn Asn Arg ProGly Arg Gly Ser Ser 325 325 330 330 335 335

Ala Gly Ala Gly Pro ProCys CysCys Cys Thr Thr ProPro ThrThr Lys Lys Met Met Ser Ile Ser Pro Pro Asn IleMet Asn Met Leu Leu 340 340 345 345 350 350

Tyr Phe Tyr Phe Asn AsnGly GlyLys Lys Glu Glu GlnGln IleIle Ile Ile Tyr Tyr Gly Ile Gly Lys Lys Pro IleAla Pro Ala Met Met 355 355 360 360 365 365

Val Val Val Val Asp AspArg ArgCys Cys Gly Gly CysCys SerSer 370 370 375 375

<210> <210> 6 6 <211> <211> 109 109 <212> <212> PRT PRT <213> <213> Mus musculus Mus musculus

<400> <400> 6 6

Ile Ala Pro Ile Ala ProLys LysArg ArgTyr Tyr LysLys AlaAla Asn Asn Tyr Tyr Cys Cys Ser Glu Ser Gly GlyCys Glu Cys

9

Glu Glu 35 35 40 40 45 45

Phe Val Phe Phe Val PheLeu LeuGln GlnLys Lys TyrTyr ProPro His His Thr Thr His His Leu His Leu Val ValGln His Gln Ala Ala 50 50 55 55 60 60

70 70 75 75 80 80

<210> <210> 7 7 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<220> <220> <223> An artificially <223> An artificially synthesized synthesized sequence sequence

<400> <400: > 7 7

Ala Ser Ala Ser Thr ThrLys LysGly Gly Pro Pro SerSer ValVal Phe Phe Pro Pro Leu Pro Leu Ala Ala Ser ProSer Ser Ser Lys Lys 1 1 5 5 10 10 15 15

Ser Thr Ser Ser Thr SerGly GlyGly GlyThr Thr AlaAla AlaAla Leu Leu Gly Gly Cys Cys Leu Lys Leu Val ValAsp Lys Asp Tyr Tyr 20 20 25 25 30 30

Phe Pro Glu Phe Pro GluPro ProVal ValThr Thr ValVal SerSer Trp Trp Asn Asn Ser Ser Gly Leu Gly Ala AlaThr Leu Thr Ser Ser 35 35 40 40 45 45

Gly Val Gly Val His HisThr ThrPhe Phe Pro Pro AlaAla ValVal Leu Leu Gln Gln Ser Gly Ser Ser Ser Leu GlyTyr Leu Tyr Ser Ser 50 50 55 55 60 60

Leu Ser Leu Ser Ser SerVal ValVal Val Thr Thr ValVal ProPro Ser Ser Ser Ser Ser Gly Ser Leu Leu Thr GlyGln Thr Gln Thr Thr

70 70 75 75 80 80

10

Tyr Ile Tyr Ile Cys Cys Asn Asn Val Val Asn Asn His His Lys Lys Pro Pro Ser Ser Asn Asn Thr Thr Lys Lys Val Val Asp Asp Lys Lys 85 85 90 90 95 95

Lys Val Lys Val Glu GluPro ProLys Lys Ser Ser CysCys AspAsp Lys Lys Thr Thr His Cys His Thr Thr Pro CysPro Pro Pro Cys Cys 100 100 105 105 110 110

Pro Ala Pro Ala Pro ProGlu GluLeu LeuLeu Leu GlyGly GlyGly Pro Pro Ser Ser Val Val Phe Phe Phe Leu LeuPro Phe Pro Pro Pro 115 115 120 120 125 125

Lys Pro Lys Pro Lys LysAsp AspThr Thr Leu Leu MetMet IleIle Ser Ser Arg Arg Thr Glu Thr Pro Pro Val GluThr Val Thr Cys Cys 130 130 135 135 140 140

Val Val Val Val Val Val Asp Asp Val Val Ser Ser His His Glu Glu Asp Asp Pro Pro Glu Glu Val Val Lys Lys Phe Phe Asn Asn Trp Trp 145 145 150 150 155 155 160 160

Tyr Val Tyr Val Asp Asp Gly Gly Val Val Glu Glu Val Val His His Asn Asn Ala Ala Lys Lys Thr Thr Lys Lys Pro Pro Arg Arg Glu Glu 165 165 170 170 175 175

Glu Gln Glu Gln Tyr TyrAsn AsnSer Ser Thr Thr TyrTyr ArgArg Val Val Val Val Ser Leu Ser Val Val Thr LeuVal Thr Val Leu Leu 180 180 185 185 190 190

His Gln His Gln Asp AspTrp TrpLeu Leu Asn Asn GlyGly LysLys Glu Glu Tyr Tyr Lys Lys Lys Cys Cys Val LysSer Val Ser Asn Asn 195 195 200 200 205 205

Lys Ala Lys Ala Leu LeuPro ProAla Ala Pro Pro IleIle GluGlu Lys Lys Thr Thr Ile Lys Ile Ser Ser Ala LysLys Ala Lys Gly Gly 210 210 215 215 220 220

Gln Pro Gln Pro Arg Arg Glu Glu Pro Pro Gln Gln Val Val Tyr Tyr Thr Thr Leu Leu Pro Pro Pro Pro Ser Ser Arg Arg Glu Glu Glu Glu 225 225 230 230 235 235 240 240

Met Thr Met Thr Lys Lys Asn Asn Gln Gln Val Val Ser Ser Leu Leu Thr Thr Cys Cys Leu Leu Val Val Lys Lys Gly Gly Phe Phe Tyr Tyr

11

245 250 250 255 255

Pro Ser Pro Ser Asp AspIle IleAla AlaVal Val GluGlu TrpTrp Glu Glu Ser Ser Asn Gln Asn Gly Gly Pro GlnGlu Pro Glu Asn Asn 260 260 265 265 270 270

Asn Tyr Asn Tyr Lys LysThr ThrThr Thr Pro Pro ProPro ValVal Leu Leu Asp Asp Ser Gly Ser Asp Asp Ser GlyPhe Ser Phe Phe Phe 275 275 280 280 285 285

Leu Tyr Leu Tyr Ser SerLys LysLeu Leu Thr Thr ValVal AspAsp Lys Lys Ser Ser Arg Gln Arg Trp Trp Gln GlnGly Gln Gly Asn Asn 290 290 295 295 300 300

Val Phe Val Phe Ser Ser Cys Cys Ser Ser Val Val Met Met His His Glu Glu Ala Ala Leu Leu His His Asn Asn His His Tyr Tyr Thr Thr 305 305 310 310 315 315 320 320

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 8 8 <211> <211> 107 107 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 8 8

Arg Thr Arg Thr Val Val Ala Ala Ala Ala Pro Pro Ser Ser Val Val Phe Phe Ile Ile Phe Phe Pro Pro Pro Pro Ser Ser Asp Asp Glu Glu 1 1 5 5 10 10 15 15

Gln Leu Gln Leu Lys LysSer SerGly Gly Thr Thr AlaAla SerSer Val Val Val Val Cys Leu Cys Leu Leu Asn LeuAsn Asn Asn Phe Phe 20 20 25 25 30 30

Tyr Pro Tyr Pro Arg ArgGlu GluAla Ala Lys Lys ValVal GlnGln Trp Trp Lys Lys Val Asn Val Asp Asp Ala AsnLeu Ala Leu Gln Gln 35 35 40 40 45 45

Ser Gly Ser Gly Asn AsnSer SerGln GlnGlu Glu SerSer ValVal Thr Thr Glu Glu Gln Ser Gln Asp Asp Lys SerAsp Lys Asp Cys Cys 50 50 55 55 60 60

12

Thr Tyr Thr Tyr Ser SerLeu LeuSer Ser Ser Ser ThrThr LeuLeu Thr Thr Leu Leu Ser Ala Ser Lys Lys Asp AlaTyr Asp Tyr Glu Glu

70 70 75 75 80 80

Lys His Lys His Lys LysVal ValTyr Tyr Ala Ala CysCys GluGlu Val Val Thr Thr His Gly His Gln Gln Leu GlySer Leu Ser Ser Ser 85 85 90 90 95 95

Pro Pro Val Val Thr Thr Lys Lys Ser Ser Phe Phe Asn Asn Arg Arg Gly Gly Glu Glu Cys Cys 100 100 105 105

<210> <210> 9 9 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 9 9

Ser Thr Ser Thr Ser SerGly GlyGly Gly Thr Thr AlaAla AlaAla Leu Leu Gly Gly Cys Val Cys Leu Leu Lys ValAsp Lys Asp Tyr Tyr 20 20 25 25 30 30

Phe Pro Phe Pro Glu GluPro ProVal Val Thr Thr ValVal SerSer Trp Trp Asn Asn Ser Ala Ser Gly Gly Leu AlaThr Leu Thr Ser Ser 35 35 40 40 45 45

70 70 75 75 80 80

13

Met Thr Met Thr Lys Lys Asn Asn Gln Gln Val Val Ser Ser Leu Leu Thr Thr Cys Cys Leu Leu Val Val Lys Lys Gly Gly Phe Phe Tyr Tyr 245 245 250 250 255 255

Pro Ser Pro Ser Asp Asp Ile Ile Ala Ala Val Val Glu Glu Trp Trp Glu Glu Ser Ser Asn Asn Gly Gly Gln Gln Pro Pro Glu Glu Asn Asn 260 260 265 265 270 270

14

Asn Tyr Asn Tyr Lys Lys Thr Thr Thr Thr Pro Pro Pro Pro Val Val Leu Leu Asp Asp Ser Ser Asp Asp Gly Gly Ser Ser Phe Phe Phe Phe 275 275 280 280 285 285

Leu Tyr Leu Tyr Ser Ser Lys Lys Leu Leu Thr Thr Val Val Asp Asp Lys Lys Ser Ser Arg Arg Trp Trp Gln Gln Gln Gln Gly Gly Asn Asn 290 290 295 295 300 300

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 10 10 <211> <211> 107 107 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 10 10

Gln Leu Gln Leu Lys Lys Ser Ser Gly Gly Thr Thr Ala Ala Ser Ser Val Val Val Val Cys Cys Leu Leu Leu Leu Asn Asn Asn Asn Phe Phe 20 20 25 25 30 30

Tyr Pro Tyr Pro Arg Arg Glu Glu Ala Ala Lys Lys Val Val Gln Gln Trp Trp Lys Lys Val Val Asp Asp Asn Asn Ala Ala Leu Leu Gln Gln 35 35 40 40 45 45

Ser Gly Asn Ser Gly AsnSer SerGln GlnGlu Glu SerSer ValVal Thr Thr Glu Glu Gln Gln Asp Lys Asp Ser SerAsp Lys Asp Ser Ser 50 50 55 55 60 60

70 70 75 75 80 80

15

Pro Val Pro Val Thr ThrLys LysSer SerPhe Phe AsnAsn ArgArg Gly Gly Glu Glu Cys Cys 100 100 105 105

<210> <210> 11 11 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 11 11

Ser Thr Ser Thr Ser SerGly GlyGly GlyThr Thr AlaAla AlaAla Leu Leu Gly Gly Cys Val Cys Leu Leu Lys ValAsp Lys Asp Tyr Tyr 20 20 25 25 30 30

70 70 75 75 80 80

Tyr Ile Tyr Ile Cys CysAsn AsnVal Val Asn Asn HisHis LysLys Pro Pro Ser Ser Asn Lys Asn Thr Thr Val LysAsp Val Asp Lys Lys 85 85 90 90 95 95

Pro Ala Pro Ala Pro ProGlu GluLeu LeuArg Arg GlyGly GlyGly Pro Pro Lys Lys Val Leu Val Phe Phe Phe LeuPro Phe Pro Pro Pro

16

115 120 120 125 125

Val Val Val Val Val ValAsp AspVal Val Ser Ser HisHis GluGlu Asp Asp Pro Pro Glu Lys Glu Val Val Phe LysAsn Phe Asn Trp Trp 145 145 150 150 155 155 160 160

Gln Pro Gln Pro Arg ArgGlu GluPro Pro Gln Gln ValVal TyrTyr Thr Thr Leu Leu Pro Ser Pro Pro Pro Arg SerAsp Arg Asp Glu Glu 225 225 230 230 235 235 240 240

Leu Thr Leu Thr Lys LysAsn AsnGln Gln Val Val SerSer LeuLeu Thr Thr Cys Cys Leu Lys Leu Val Val Gly LysPhe Gly Phe Tyr Tyr 245 245 250 250 255 255

Pro Ser Pro Ser Asp AspIle IleAla Ala Val Val GluGlu TrpTrp Glu Glu Ser Ser Asn Gln Asn Gly Gly Pro GlnGlu Pro Glu Asn Asn 260 260 265 265 270 270

Leu Tyr Leu Tyr Ser Ser Lys Lys Leu Leu Thr Thr Val Val Asp Asp Lys Lys Ser Ser Arg Arg Trp Trp Gln Gln Gln Gln Gly Gly

17

Asn Asn 290 290 295 295 300 300

Val Phe Val Phe Ser SerCys CysSer Ser Val Val MetMet HisHis Glu Glu Ala Ala Leu Asn Leu His His His AsnTyr His Tyr Thr Thr 305 305 310 310 315 315 320 320

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 12 12 <211> <211> 116 116 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 12 12

Gln Ser Gln Ser Leu LeuGlu GluGlu Glu Ser Ser GlyGly GlyGly Arg Arg Leu Leu Val Pro Val Thr Thr Gly ProGly Gly Gly Ser Ser 1 1 5 5 10 10 15 15

Leu Thr Leu Thr Val ValThr ThrCys Cys Arg Arg ValVal SerSer Gly Gly Ile Ile Asp Ser Asp Leu Leu Ser SerTyr Ser Tyr Asp Asp 20 20 25 25 30 30

Ile Ser Trp Ile Ser TrpVal ValArg ArgGln Gln AlaAla ProPro Gly Gly Lys Lys Gly Gly Leu Trp Leu Glu GluIle Trp Ile Gly Gly 35 35 40 40 45 45

Ile Ile Ser Ile Ile SerTyr TyrAla AlaGly Gly SerSer ThrThr Tyr Tyr Tyr Tyr Ala Ala Ser Ala Ser Trp TrpLys Ala Lys Gly Gly 50 50 55 55 60 60

Arg Phe Arg Phe Thr ThrIle IleSer Ser Lys Lys ThrThr SerSer Thr Thr Thr Thr Val Leu Val Asp Asp Lys LeuMet Lys Met Thr Thr

70 70 75 75 80 80

Ser Leu Thr Ser Leu ThrThr ThrGlu GluAsp Asp ThrThr AlaAla Thr Thr Tyr Tyr Phe Phe Cys Arg Cys Ala AlaGly Arg Gly Val Val 85 85 90 90 95 95

Pro Ala Pro Ala Tyr TyrSer SerThr Thr Gly Gly GlyGly AspAsp Leu Leu Trp Trp Gly Gly Gly Pro Pro Thr GlyLeu Thr Leu Val Val

18

100 105 105 110 110

Thr Val Thr Val Ser SerSer Ser 115 115

<210> <210> 13 13 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 13 13

Gln Val Gln Val Thr ThrLeu LeuLys Lys Glu Glu SerSer GlyGly Pro Pro Val Val Leu Lys Leu Val Val Pro LysThr Pro Thr Glu Glu 1 1 5 5 10 10 15 15

Thr Leu Thr Leu Thr ThrLeu LeuThr Thr Cys Cys ThrThr ValVal Ser Ser Gly Gly Ile Leu Ile Asp Asp Ser LeuSer Ser Ser Tyr Tyr 20 20 25 25 30 30

Asp Ile Asp Ile Ser Ser Trp Trp Val Val Arg Arg Gln Gln Ala Ala Pro Pro Gly Gly Lys Lys Gly Gly Leu Leu Glu Glu Trp Trp Val Val 35 35 40 40 45 45

Gly Ile Gly Ile Ile IleSer SerTyr Tyr Ala Ala GlyGly SerSer Thr Thr Tyr Tyr Tyr Ser Tyr Ala Ala Trp SerAla Trp Ala Lys Lys 50 50 55 55 60 60

Gly Arg Gly Arg Leu LeuThr ThrIle Ile Ser Ser LysLys AspAsp Thr Thr Ser Ser Lys Gln Lys Asn Asn Val GlnVal Val Val Leu Leu

70 70 75 75 80 80

Thr Met Thr Met Thr ThrAsn AsnMet Met Asp Asp ProPro ValVal Asp Asp Thr Thr Ala Tyr Ala Thr Thr Tyr TyrCys Tyr Cys Ala Ala 85 85 90 90 95 95

Arg Gly Arg Gly Val ValPro ProAla Ala Tyr Tyr SerSer ThrThr Gly Gly Gly Gly Asp Trp Asp Leu Leu Gly TrpGln Gly Gln Gly Gly 100 100 105 105 110 110

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

19

<210> <210> 14 14 <211> <211> 110 110 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<220> <220> <223> <223> An artificially An artificiallysynthesized synthesized sequence sequence

<400> <400> 14 14

Ala Gln Ala Gln Val ValLeu LeuThr Thr Gln Gln ThrThr AlaAla Ser Ser Ser Ser Val Ala Val Ser Ser Ala AlaVal Ala Val Gly Gly 1 1 5 5 10 10 15 15

Gly Thr Gly Thr Val ValThr ThrIle Ile Asn Asn CysCys GlnGln Ser Ser Ser Ser Gln Val Gln Ser Ser Tyr ValAsp Tyr Asp Asn Asn 20 20 25 25 30 30

Asn Trp Asn Trp Leu LeuSer SerTrp Trp Phe Phe GlnGln GlnGln Lys Lys Pro Pro Gly Pro Gly Gln Gln Pro ProLys Pro Lys Leu Leu 35 35 40 40 45 45

Leu Ile Leu Ile Tyr TyrTrp TrpAla Ala Ser Ser ThrThr LeuLeu Ala Ala Ser Ser Gly Pro Gly Val Val Ser ProArg Ser Arg Phe Phe 50 50 55 55 60 60

Lys Gly Lys Gly Ser SerGly GlySer Ser Gly Gly ThrThr GlnGln Phe Phe Thr Thr Leu Ile Leu Thr Thr Ser IleAsp Ser Asp Leu Leu

70 70 75 75 80 80

Glu Cys Glu Cys Asp AspAsp AspAla Ala Ala Ala ThrThr TyrTyr Tyr Tyr Cys Cys Ala Gly Ala Gly Gly Tyr GlyGly Tyr Gly Gly Gly 85 85 90 90 95 95

Gly Leu Gly Leu Tyr TyrAla AlaPhe Phe Gly Gly GlyGly GlyGly Thr Thr Lys Lys Val Ile Val Glu Glu Lys Ile Lys 100 100 105 105 110 110

<210> <210> 15 15 <211> <211> 110 110 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 15 15

Asp Ile Asp Ile Val ValMet MetThr Thr Gln Gln SerSer ProPro Ala Ala Thr Thr Leu Leu Leu Ser Ser Ser LeuPro Ser Pro Gly Gly

20

1 5 5 10 10 15 15

Glu Arg Glu Arg Ala AlaThr ThrLeu Leu Ser Ser CysCys GlnGln Ser Ser Ser Ser Gln Val Gln Ser Ser Tyr ValAsp Tyr Asp Asn Asn 20 20 25 25 30 30

Ser Gly Ser Ser Gly SerGly GlySer SerGly GlyThrThr AspAsp Phe Phe Thr Thr Leu Leu Thr Ser Thr Ile IleSer Ser Ser Leu Leu

70 70 75 75 80 80

Gln Pro Gln Pro Glu GluAsp AspAla Ala Ala Ala ThrThr TyrTyr Tyr Tyr Cys Cys Ala Gly Ala Gly Gly Tyr GlyGly Tyr Gly Gly Gly 85 85 90 90 95 95

Gly Leu Gly Leu Tyr TyrAla AlaPhe Phe Gly Gly GlnGln GlyGly Thr Thr Lys Lys Val Ile Val Glu Glu Lys Ile Lys 100 100 105 105 110 110

<210> <210> 16 16 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 16 16

Thr Leu Thr Leu Thr ThrLeu LeuThr Thr Cys Cys ThrThr ValVal Ser Ser Gly Gly Ile Leu Ile Asp Asp Ser LeuHis Ser His Tyr Tyr 20 20 25 25 30 30

21

Gly Ile Gly Ile Ile Ile Ser Ser Tyr Tyr Ala Ala Gly Gly Ser Ser Thr Thr Tyr Tyr Tyr Tyr Ala Ala Ser Ser Trp Trp Ala Ala Lys Lys 50 50 55 55 60 60

70 70 75 75 80 80

Arg Gly Arg Gly Val Val Pro Pro Ala Ala Tyr Tyr Ser Ser Thr Thr Gly Gly Gly Gly Asp Asp Leu Leu Trp Trp Gly Gly Gln Gln Gly Gly 100 100 105 105 110 110

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

<210> <210> 17 17 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 17 17

Gly Ile Gly Ile Ile Ile Ser Ser His His Ala Ala Gly Gly Ser Ser Thr Thr Tyr Tyr Tyr Tyr Ala Ala Ser Ser Trp Trp Ala Ala Lys Lys 50 50 55 55 60 60

Gly Arg Gly Arg Leu LeuThr ThrIle Ile Ser Ser LysLys AspAsp Thr Thr Ser Ser Lys Gln Lys Asn Asn Val GlnVal Val Val

22

Leu Leu

70 70 75 75 80 80

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

<210> <210> 18 18 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 18 18

Asp Ile Asp Ile Ser SerTrp TrpVal Val Arg Arg GlnGln AlaAla Pro Pro Gly Gly Lys Leu Lys Gly Gly Glu LeuTrp Glu Trp Val Val 35 35 40 40 45 45

70 70 75 75 80 80

23

Arg Gly Arg Gly Val Val Pro Pro Ala Ala His His Ser Ser Thr Thr Gly Gly Gly Gly Asp Asp Leu Leu Trp Trp Gly Gly Gln Gln Gly Gly 100 100 105 105 110 110

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

<210> <210> 19 19 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 19 19

70 70 75 75 80 80

Arg Gly Arg Gly Val Val Pro Pro Ala Ala Tyr Tyr Ser Ser His His Gly Gly Gly Gly Asp Asp Leu Leu Trp Trp Gly Gly Gln Gln Gly Gly 100 100 105 105 110 110

24

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

<210> <210> 20 20 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 20 20

Gly Ile Gly Ile Ile IleSer SerHis His Ala Ala GlyGly SerSer Thr Thr Tyr Tyr Tyr Ser Tyr Ala Ala Trp SerAla Trp Ala Lys Lys 50 50 55 55 60 60

70 70 75 75 80 80

Thr Met Thr Met Thr ThrAsn AsnMet MetAsp Asp ProPro ValVal Asp Asp Thr Thr Ala Tyr Ala Thr Thr Tyr TyrCys Tyr Cys Ala Ala 85 85 90 90 95 95

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

<210> <210> 21 21 <211> <211> 119 119 <212> <212> PRT PRT

25

<213> Artificialsequence <213> Artificial sequence

<400> <400> 21 21

70 70 75 75 80 80

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

<210> <210> 22 22 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 22 22

26

70 70 75 75 80 80

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

<210> <210> 23 23 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 23 23

Thr Leu Thr Leu Thr ThrLeu LeuThr Thr Cys Cys ThrThr ValVal Ser Ser Gly Gly Ile Leu Ile Asp Asp Ser LeuHis Ser His Tyr Tyr

27

20 25 25 30 30

70 70 75 75 80 80

Arg Gly Arg Gly Val ValPro ProAla Ala His His SerSer ThrThr Gly Gly Gly Gly Asp Trp Asp Leu Leu Gly TrpGln Gly Gln Gly Gly 100 100 105 105 110 110

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

<210> < 210> 24 24 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 24 24

28

70 70 75 75 80 80

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

<210> <210> 25 25 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 25 25

29

Leu Leu

70 70 75 75 80 80

Arg Gly Arg Gly Val ValPro ProAla Ala Tyr Tyr SerSer HisHis Gly Gly Gly Gly Asp Trp Asp Leu Leu Gly TrpGln Gly Gln Gly Gly 100 100 105 105 110 110

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

<210> <210> 26 26 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 26 26

70 70 75 75 80 80

30

Arg Gly Arg Gly Val Val Pro Pro Ala Ala His His Ser Ser His His Gly Gly Gly Gly Asp Asp Leu Leu Trp Trp Gly Gly Gln Gln Gly Gly 100 100 105 105 110 110

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

<210> <210> 27 27 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 27 27

70 70 75 75 80 80

31

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

<210> <210> 28 28 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 28 28

70 70 75 75 80 80

Arg Gly Arg Gly Val ValPro ProAla Ala His His SerSer HisHis Gly Gly Gly Gly Asp Trp Asp Leu Leu Gly TrpGln Gly Gln Gly Gly 100 100 105 105 110 110

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

<210> <210> 29 29 <211> <211> 119 119 <212> <212> PRT PRT

32

<213> Artificialsequence <213> Artificial sequence

<400> <400> 29 29

Thr Leu Thr Leu Thr ThrLeu LeuThr ThrCys Cys ThrThr ValVal Ser Ser Gly Gly Ile Leu Ile Asp Asp Ser LeuHis Ser His Tyr Tyr 20 20 25 25 30 30

70 70 75 75 80 80

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

<210> <210> 30 30 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 30 30

33

70 70 75 75 80 80

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

<210> < 210> 31 31 <211> <211> 110 110 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 31 31

Asp Ile Asp Ile Val ValMet MetThr Thr Gln Gln SerSer ProPro Ala Ala Thr Thr Leu Leu Leu Ser Ser Ser LeuPro Ser Pro Gly Gly 1 1 5 5 10 10 15 15

Glu Arg Glu Arg Ala AlaThr ThrLeu Leu Ser Ser CysCys GlnGln Ser Ser Ser Ser Gln Val Gln Ser Ser Tyr ValHis Tyr His Asn Asn

34

20 25 25 30 30

Leu Ile Leu Ile Tyr TyrTrp TrpAla AlaSer Ser ThrThr LeuLeu Ala Ala Ser Ser Gly Pro Gly Val Val Ser ProArg Ser Arg Phe Phe 50 50 55 55 60 60

Ser Gly Ser Gly Ser SerGly GlySer SerGly Gly ThrThr AspAsp Phe Phe Thr Thr Leu Ile Leu Thr Thr Ser IleSer Ser Ser Leu Leu

70 70 75 75 80 80

Gln Pro Gln Pro Glu Glu Asp Asp Ala Ala Ala Ala Thr Thr Tyr Tyr Tyr Tyr Cys Cys Ala Ala Gly Gly Gly Gly Tyr Tyr Gly Gly Gly Gly 85 85 90 90 95 95

<210> <210> 32 32 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 32 32

Thr Leu Thr Leu Thr ThrLeu LeuThr Thr Cys Cys ThrThr ValVal Ser Ser Gly Gly Ile Leu Ile Asp Asp Ser LeuHis Ser His Thr Thr 20 20 25 25 30 30

35

70 70 75 75 80 80

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

<210> <210> 33 33 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 33 33

Gly Ile Gly Ile Ile IleSer SerTyr Tyr Ala Ala GlyGly SerSer Thr Thr Tyr Tyr Tyr Ser Tyr Met Met Trp SerAla Trp Ala Lys Lys 50 50 55 55 60 60

70 70 75 75 80 80

Thr Met Thr Met Thr ThrAsn AsnMet Met Asp Asp ProPro ValVal Asp Asp Thr Thr Ala Tyr Ala Thr Thr Tyr TyrCys Tyr Cys

36

Ala Ala 85 85 90 90 95 95

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

<210> <210> 34 34 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 34 34

70 70 75 75 80 80

37

Thr Leu Thr Leu Val ValThr ThrVal ValSer Ser SerSer 115 115

<210> <210> 35 35 <211> <211> 110 110 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 35 35

Glu Arg Glu Arg Ala AlaThr ThrLeu Leu Ser Ser CysCys ThrThr Ser Ser Ser Ser Gln Val Gln Ser Ser Tyr ValHis Tyr His Asn Asn 20 20 25 25 30 30

Leu Ile Leu Ile Tyr TyrTrp TrpAla Ala Ser Ser ThrThr LeuLeu Ala Ala Tyr Tyr Gly Pro Gly Val Val Ser ProArg Ser Arg Phe Phe 50 50 55 55 60 60

Ser Gly Ser Ser Gly SerGly GlySer SerGly Gly ThrThr AspAsp Phe Phe Thr Thr Leu Leu Thr Ser Thr Ile IleSer Ser Ser Leu Leu

70 70 75 75 80 80

Gly Arg Gly Arg Tyr TyrAla AlaPhe Phe Gly Gly GlnGln GlyGly Thr Thr Lys Lys Val Ile Val Glu Glu Lys Ile Lys 100 100 105 105 110 110

<210> <210> 36 36 <211> <211> 110 110 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<220> <220>

38

<223> An artificially <223> An artificially synthesized synthesized sequence sequence

<400> <400> 36 36

Glu Arg Glu Arg Ala AlaThr ThrLeu Leu Ser Ser CysCys GlnGln Ser Ser Ser Ser Gln Val Gln Ser Ser Tyr ValHis Tyr His Asn Asn 20 20 25 25 30 30

Leu Ile Leu Ile Tyr TyrTrp TrpAla Ala Ser Ser ThrThr LeuLeu Ala Ala Phe Phe Gly Pro Gly Val Val Ser ProArg Ser Arg Phe Phe 50 50 55 55 60 60

70 70 75 75 80 80

<210> <210> 37 37 <211> <211> 110 110 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<220> <220> <223> Anartificially <223> An artificially synthesized synthesized sequence sequence

<400> <400> 37 37

Glu Arg Glu Arg Ala AlaThr ThrLeu Leu Ser Ser CysCys GlnGln Thr Thr Ser Ser Gln Val Gln Ser Ser Tyr ValHis Tyr His Asp Asp 20 20 25 25 30 30

39

70 70 75 75 80 80

<210> <210> 38 38 <211> <211> 110 110 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 38 38

Glu Arg Glu Arg Ala AlaThr ThrLeu Leu Ser Ser CysCys ThrThr Ser Ser Ser Ser Gln Val Gln Ser Ser Tyr ValHis Tyr His Asp Asp 20 20 25 25 30 30

40

70 70 75 75 80 80

<210> <210> 39 39 <211> <211> 348 348 <212> <212> DNA DNA <213> <213> Artificial sequence Artificial sequence

<220> <220> <223> <223> An artificially An artificially synthesized synthesized sequence sequence

<400> <400> 39 39 cagtcgctgg aggagtccgg cagtcgctgg aggagtccgg gggtcgcctg gggtcgcctg gtaacgcctg gtaacgcctg gaggatccct gaggatccct gacagtcacc gacagtcacc 60 60

tgcagagtct ctggaatcga tgcagagtct ctggaatcga cctcagtagt cctcagtagt tacgacatca tacgacatca gctgggtccg gctgggtccg ccaggctcca ccaggctcca 120 120

gggaaggggctggagtggat gggaaggggc tggagtggat cggaatcatt cggaatcatt agttatgctg agttatgctg gtagcacata gtagcacata ctacgcgagc ctacgcgage 180 180

tgggcaaaag gccgattcac tgggcaaaag gccgattcac catctccaaa catctccaaa acctcgacca acctcgacca cggtggatct cggtggatct gaaaatgacc gaaaatgacc 240 240

agtctgacaaccgaggacac agtctgacaa ccgaggacac ggccacctat ggccacctat ttctgtgcca ttctgtgcca gaggtgtgcc gaggtgtgcc tgcttatagt tgcttatagt 300 300

actggtggtgacttgtgggg actggtggtg acttgtgggg cccaggcacc cccaggcace ctggtcaccg ctggtcaccg tctcctcc tctcctcc 348 348

<210> <210> 40 40 <211> <211> 357 357 <212> <212> DNA DNA <213> <213> Artificialsequence Artificial sequence

<400> <400> 40 40 caggtgacac tgaaagaaag caggtgacac tgaaagaaag cggccccgtg cggccccgtg ctggtgaagc ctggtgaage caaccgagac caaccgagac actgactctg actgactctg 60 60

acctgcacagtgagtgggat acctgcacag tgagtgggat cgacctgagc cgacctgagc agctacgaca agctacgaca tctcctgggt tctcctgggt gcggcaggca gcggcaggca 120 120

41 cccggaaagg gcctggaatg cccggaaagg gcctggaatg ggtcggcatc ggtcggcatc attagctacg attagctacg ccgggagcac ccgggagcac atactatgcc atactatgcc 180 180 agctgggctaaaggaagact agctgggcta aaggaagact gactatctcc gactatctcc aaggacacct aaggacacct ctaaaaacca ctaaaaacca ggtggtcctg ggtggtcctg 240 240 actatgaccaatatggaccc actatgacca atatggaccc cgtggataca cgtggataca gcaacttact gcaacttact attgtgccag attgtgccag gggcgtccct gggcgtccct 300 300 gcttactctaccggagggga gcttactcta ccggagggga tctgtgggga tctgtgggga cagggaaccc cagggaaccc tggtgacagt tggtgacagt ctctagt ctctagt 357 357

<210> <210> 41 41 <211> <211> 330 330 <212> <212> DNA DNA <213> <213> Artificial sequence Artificial sequence

<400> <400> 41 41 gcgcaagtgc tgacccagac gcgcaagtgc tgacccagac tgcatcatcc tgcatcatcc gtgtctgcag gtgtctgcag ctgtgggagg ctgtgggagg cacagtcacc cacagtcace 60 60

atcaattgccagtccagtca atcaattgcc agtccagtca gagtgtttat gagtgtttat gataacaact gataacaact ggttatcctg ggttatcctg gtttcagcag gtttcagcag 120 120

aaaccagggcagcctcccaa aaaccagggc agcctcccaa gctcctgatc gctcctgatc tattgggcat tattgggcat ccactctggc ccactctggc atctggggtc atctggggtd 180 180

ccatcgcggttcaaaggcag ccatcgcggt tcaaaggcag tggatctggg tggatctggg acacagttca acacagttca ctctcaccat ctctcaccat cagcgacctg cagcgacctg 240 240

gagtgtgacgatgctgccac gagtgtgacg atgctgccac ttactactgt ttactactgt gcaggcggtt gcaggcggtt atggtggtgg atggtggtgg tctgtatgct tctgtatgct 300 300

ttcggcggag ggaccaaggt ttcggcggag ggaccaaggt ggagatcaaa ggagatcaaa 330 330

<210> <210> 42 42 <211> <211> 330 330 <212> <212> DNA DNA <213> <213> Artificialsequence Artificial sequence

<400> <400> 4242 gacattgtga tgactcagtc tcctgccact gacattgtga tgactcagta tcctgccact ctgtcactga ctgtcactga gccctggcga gccctggcga gcgagcaacc gcgagcaacc 60 60

ctgtcttgccagagcagcca ctgtcttgcc agagcagcca gtccgtgtac gtccgtgtac gacaacaatt gacaacaatt ggctgagctg ggctgagctg gttccagcag gttccagcag 120 120

42 aagccagggc agccccctaa aagccagggc agccccctaa actgctgatc actgctgatc tattgggcat tattgggcat caacactggc caacactggc cagcggcgtc 180 cagcggcgtc 180 ccctccagattctccggatc ccctccagat tctccggatc tggcagtggg tggcagtggg actgacttta actgacttta ccctgaccat ccctgaccat cagcagcctc cagcagectc 240 240 cagcccgaggatgccgctac cagcccgagg atgccgctac ctactattgt ctactattgt gctggcgggt gctggcgggt acggaggcgg acggaggcgg gctgtatgca gctgtatgca 300 300 tttggacagg gcacaaaggt tttggacagg gcacaaaggt ggaaatcaaa ggaaatcaaa 330 330

<210> <210> 43 43 <211> <211> 357 357 <212> <212> DNA DNA <213> <213> Artificial sequence Artificial sequence

<400> <400> 43 43 caggtgacac tgaaagaaag caggtgacac tgaaagaaag cggccccgtg cggccccgtg ctggtgaagc ctggtgaage caaccgagac caaccgagac actgactctg actgactctg 60 60

acctgcacagtgagtgggat acctgcacag tgagtgggat cgacctgagc cgacctgage cacaccgaca cacaccgaca tctcctgggt tctcctgggt gcggcaggca gcggcaggca 120 120

cccggaaagg gcctggaatg cccggaaagg gcctggaatg ggtcggcatc ggtcggcatc attagctacg attagctacg ccgggagcac ccgggagcad atactatgcc atactatgcc 180 180

agctgggctaaaggaagact agctgggcta aaggaagact gactatctcc gactatctcc aaggacacct aaggacacct ctaaaaacca ctaaaaacca ggtggtcctg ggtggtcctg 240 240

actatgaccaatatggaccc actatgacca atatggaccc cgtggataca cgtggataca gcaacttact gcaacttact attgtgccag attgtgccag gggcgtccct gggcgtccct 300 300

gcttactctcacggagggga gcttactctc acggagggga tctgtgggga tctgtgggga cagggaaccc cagggaaccc tggtgacagt tggtgacagt ctctagt ctctagt 357 357

<210> <210> 44 44 <211> <211> 357 357 <212> <212> DNA DNA <213> <213> Artificialsequence Artificial sequence

<400> <400> 4444 caggtgacac tgaaagaaag caggtgacac tgaaagaaag cggccccgtg cggccccgtg ctggtgaagc ctggtgaage caaccgagac caaccgagac actgactctg actgactctg 60 60

acctgcacagtgagtgggat acctgcacag tgagtgggat cgacctgagc cgacctgagc cacaccgaca cacaccgaca tctcctgggt tctcctgggt gcggcaggca gcggcaggca 120 120

43 cccggaaagg gcctggaatg cccggaaagg gcctggaatg ggtcggcatc ggtcggcatc attagctacg attagctacg ccgggagcac ccgggagcad atactatatg atactatatg 180 180 agctgggctaaaggaagact agctgggcta aaggaagact gactatctcc gactatctcc aaggacacct aaggacacct ctaaaaacca ctaaaaacca ggtggtcctg ggtggtcctg 240 240 actatgaccaatatggaccc actatgacca atatggaccc cgtggataca cgtggataca gcaacttact gcaacttact attgtgccag attgtgccag gggcgtccct gggcgtccct 300 300 gcttactctcacggagggga gcttactctc acggagggga tctgtgggga tctgtgggga cagggaaccc cagggaaccc tggtgacagt tggtgacagt ctctagt ctctagt 357 357

<210> <210> 45 45 <211> <211> 357 357 <212> <212> DNA DNA <213> <213> Artificial sequence Artificial sequence

<400> <400> 45 45 caggtgacac tgaaagaaag caggtgacac tgaaagaaag cggccccgtg cggccccgtg ctggtgaagc ctggtgaage caaccgagac caaccgagac actgactctg actgactctg 60 60

acctgcacagtgagtgggat acctgcacag tgagtgggat cgacctgagc cgacctgage cactacgaca cactacgaca tctcctgggt tctcctgggt gcggcaggca gcggcaggca 120 120

<210> <210> 46 46 <211> <211> 330 330 <212> <212> DNA DNA <213> <213> Artificial sequence Artificial sequence

<400> <400> 46 46 gacattgtga tgactcagtc tcctgccact gacattgtga tgactcagta tcctgccact ctgtcactga ctgtcactga gccctggcga gccctggcga gcgagcaacc gcgagcaacc 60 60

ctgtcttgcaccagcagcca ctgtcttgca ccagcagcca gtccgtgtac gtccgtgtac cacaacaatt cacaacaatt ggctgagctg ggctgagctg gttccagcag gttccagcag 120 120

44 aagccagggc agccccctaa aagccagggc agccccctaa actgctgatc actgctgatc tattgggcat tattgggcat caacactggc caacactggc ctacggcgtc 180 ctacggcgtc 180 ccctccagattctccggatc ccctccagat tctccggatc tggcagtggg tggcagtggg actgacttta actgacttta ccctgaccat ccctgaccat cagcagcctc cagcageetc 240 240 cagcccgaggatgccgctac cagcccgagg atgccgctac ctactattgt ctactattgt gctggcgggt gctggcgggt acggaggcgg acggaggcgg gcggtatgca gcggtatgca 300 300 tttggacagg gcacaaaggt tttggacagg gcacaaaggt ggaaatcaaa ggaaatcaaa 330 330

<210> <210> 47 47 <211> <211> 330 330 <212> <212> DNA DNA <213> <213> Artificialsequence Artificial sequence

<400> <400> 47 47 gacattgtga tgactcagtc tcctgccact gacattgtga tgactcagtc tcctgccact ctgtcactga ctgtcactga gccctggcga gccctggcga gcgagcaacc gcgagcaacc 60 60

ctgtcttgccagagcagcca ctgtcttgcc agagcagcca gtccgtgtac gtccgtgtac cacaacaatt cacaacaatt ggctgagctg ggctgagctg gttccagcag gttccagcag 120 120

aagccagggcagccccctaa aagccagggc agccccctaa actgctgatc actgctgatc tattgggcat tattgggcat caacactggc caacactggc cttcggcgtc cttcggcgtc 180 180

ccctccagattctccggatc ccctccagat tctccggatc tggcagtggg tggcagtggg actgacttta actgacttta ccctgaccat ccctgaccat cagcagcctc cagcagectc 240 240

cagcccgagg atgccgctac cagcccgagg atgccgctac ctactattgt ctactattgt gctggcgggt gctggcgggt acggaggcgg acggaggcgg gctgtatgca gctgtatgca 300 300

tttggacagg gcacaaaggt tttggacagg gcacaaaggt ggaaatcaaa ggaaatcaaa 330 330

<210> <210> 48 48 <211> <211> 330 330 <212> <212> DNA DNA <213> <213> Artificial sequence Artificial sequence

<400> <400> 48 48 gacattgtga tgactcagtc gacattgtga tgactcagtc tcctgccact tcctgccact ctgtcactga ctgtcactga gccctggcga gccctggcga gcgagcaacc gcgagcaacc 60 60

ctgtcttgccagaccagcca ctgtcttgcc agaccagcca gtccgtgtac gtccgtgtac cacgacaatt cacgacaatt ggctgagctg ggctgagctg gttccagcag gttccagcag 120 120

45 aagccagggc agccccctaa aagccaggga agccccctaa actgctgatc actgctgatc tattgggcat tattgggcat caacactggc caacactgga cagcggcgtc 180 cagcggcgtc 180 ccctccagattctccggatc ccctccagat tctccggatc tggcagtggg tggcagtggg actgacttta actgacttta ccctgaccat ccctgaccat cagcagcctc cagcagectc 240 240 cagcccgaggatgccgctac cagcccgagg atgccgctac ctactattgt ctactattgt gctggcgggt gctggcgggt acggaggcgg acggaggcgg gcggtatgca gcggtatgca 300 300 tttggacagg gcacaaaggt tttggacagg gcacaaaggt ggaaatcaaa ggaaatcaaa 330 330

<210> <210> 49 49 <211> <211> 330 330 <212> <212> DNA DNA <213> <213> Artificial sequence Artificial sequence

<400> <400> 49 49 gacattgtga tgactcagtc gacattgtga tgactcagtc tcctgccact tcctgccact ctgtcactga ctgtcactga gccctggcga gccctggcga gcgagcaacc gcgagcaacc 60 60

ctgtcttgcaccagcagcca ctgtcttgca ccagcagcca gtccgtgtac gtccgtgtac cacgacaatt cacgacaatt ggctgagctg ggctgagctg gttccagcag gttccagcag 120 120

aagccagggcagccccctaa aagccagggc agccccctaa actgctgatc actgctgatc tattgggcat tattgggcat caacactggc caacactggc ctacggcgtc ctacggcgtc 180 180

tttggacagg gcacaaaggt tttggacagg gcacaaaggt ggaaatcaaa ggaaatcaaa 330 330

<210> <210> 50 50 <211> <211> 987 987 <212> <212> DNA DNA <213> <213> Artificialsequence Artificial sequence

<400> <400> 5050 gcttccacca agggcccatc gcttccacca agggcccatc ggtcttcccc ggtcttcccc ctggcaccct ctggcaccct cctccaagtc cctccaagtc cacctctggg cacctctggg 60 60

ggcacagcggccctgggctg ggcacagcgg ccctgggctg cctggtcaag cctggtcaag gactacttcc gactacttcc ccgaaccggt ccgaaccggt gacggtgtcg gacggtgtcg 120 120

46 tggaactcag gcgccctgac tggaactcag gcgccctgac ctccggcgtg ctccggcgtg cacaccttcc cacaccttcc cggctgtcct cggctgtcct acagtcctca 180 acagtcctca 180 ggactctactccctctcctc ggactctact ccctctcctc cgtggtgacc cgtggtgacc gtgccctcct gtgccctcct cgtccttggg cgtccttggg cacccagacc 240 cacccagacc 240 tacatctgca acgtgaatca tacatctgca acgtgaatca caagccctcc caagccctcc aacaccaagg aacaccaagg tggacaagaa tggacaagaa agttgagccc agttgagccc 300 300 aaatcttgtg acaaaactca aaatcttgtg acaaaactca cacatgccca cacatgccca ccgtgcccag ccgtgcccag cacctgaact cacctgaact cctgggggga cctgggggga 360 360 ccgtcagtct tcctcttccc ccgtcagtct tcctcttccc cccaaaaca cccaaaacccaaggacaccc aaggacaccctcatgatctc tcatgatctc ccggacccct ccggacccct 420 420 gaggtcacatgcgtggtggt gaggtcacat gcgtggtggt ggacgtgtcc ggacgtgtcc cacgaagacc cacgaagacc ctgaggtcaa ctgaggtcaa gttcaactgg gttcaactgg 480 480 tacgtggacg gcgtggaggt tacgtggacg gcgtggaggt gcataatgcc gcataatgcc aagacaaagc aagacaaage cgcgggagga cgcgggagga gcagtacaac gcagtacaac 540 540 tccacgtacc gtgtggtctc tccacgtacc gtgtggtctc cgtcctcacc cgtcctcacc gtcctgcacc gtcctgcace aggactggct aggactggct gaatggcaag gaatggcaag 600 600 gagtacaagtgcaaggtctc gagtacaagt gcaaggtctc caacaaagcc caacaaagcc ctcccagccc ctcccagccc ccatcgagaa ccatcgagaa aaccatctcc aaccatctcc 660 660 aaagccaaag ggcagccccg aaagccaaag ggcagccccg agaaccacag agaaccacag gtgtacaccc gtgtacaccc tgcccccatc tgcccccatc ccgggaggaa ccgggaggaa 720 720 atgaccaagaaccaggtctc atgaccaaga accaggtctc cctgacctgc cctgacctgc ctggtcaaag ctggtcaaag gcttctatcc gcttctatco ctccgacatc 780 ctccgacatc 780 gccgtggagtgggagtccaa gccgtggagt gggagtccaa tgggcagccg tgggcagccg gagaacaact gagaacaact acaagaccac acaagaccac gcctcccgtg gcctcccgtg 840 840 ctggactccg acggctcctt ctggactccg acggctcctt cttcctctac cttcctctac tccaagctca tccaagctca ccgtggacaa ccgtggacaa gtccaggtgg 900 gtccaggtgg 900 cagcagggga acgtcttctc cagcagggga acgtcttctc atgctccgtg atgctccgtg atgcatgagg atgcatgagg ctctgcacaa ctctgcacaa ccactacacg 960 ccactacacg 960 cagaagtcgc tctccctgtc cagaagtcgc tctccctgtc tccgtag tccgtag 987 987

<210> <210> 51 51 <211> <211> 984 984 <212> <212> DNA DNA <213> <213> Artificialsequence Artificial sequence

<400> <400> 51 51

47 gctagcaccaagggcccatc gctagcacca agggcccatc ggtcttcccc ggtcttcccc ctggcaccct ctggcaccct cctccaagag cctccaagag cacctctggg cacctctggg 60 60 ggcacagcggccctgggctg ggcacagcgg ccctgggctg cctggtcaag cctggtcaag gactacttcc gactacttcc ccgaaccggt ccgaaccggt gacggtgtcg gacggtgtcg 120 120 tggaactcag gcgccctgac tggaactcag gcgccctgac cagcggcgtg cagcggcgtg cacaccttcc cacaccttcc cggctgtcct cggctgtcct acagtcctca acagtcctca 180 180 ggactctactccctcagcag ggactctact ccctcagcag cgtggtgacc cgtggtgacc gtgccctcca gtgccctcca gcagcttggg gcagcttggg cacccagacc cacccagacc 240 240 tacatctgca acgtgaatca tacatctgca acgtgaatca caagcccagc caagcccage aacaccaagg aacaccaagg tggacaagaa tggacaagaa agttgagccc agttgagccc 300 300 aaatcttgtg acaaaactca aaatcttgtg acaaaactca cacatgccca cacatgccca ccgtgcccag ccgtgcccag cacctgaact cacctgaact ccggggggga ccggggggga 360 360 ccgaaagtct ccgaaagtct tcctcttccc tcctcttccc cccaaaaccc aaggacaccc cccaaaac aaggacaccc tcatgatctc tcatgatctc ccggacccct ccggacccct 420 420 gaggtcacatgcgtggtggt gaggtcacat gcgtggtggt ggacgtgagc ggacgtgage cacgaagacc cacgaagacc ctgaggtcaa ctgaggtcaa gttcaactgg gttcaactgg 480 480 tacgtggacg gcgtggaggt tacgtggacg gcgtggaggt gcataatgcc gcataatgcc aagacaaagc aagacaaage cgcgggagga cgcgggagga gcagtacaac 540 gcagtacaac 540 agcacgtaccgtgtggtcag agcacgtacc gtgtggtcag cgtcctcacc cgtcctcacc gtcctgcacc gtcctgcace aggactggct aggactggct gaatggcaag 600 gaatggcaag 600 gagtacaagtgcaaggtctc gagtacaagt gcaaggtctc caacaaagcc caacaaagcc ctcccagccc ctcccagccc ccatcgagaa ccatcgagaa aaccatctcc aaccatctcc 660 660 aaagccaaagggcagccccg aaagccaaag ggcagccccg agaaccacag agaaccacag gtgtacaccc gtgtacaccc tgcccccatc tgcccccatc ccgggatgag ccgggatgag 720 720 ctgaccaagaaccaggtcag ctgaccaaga accaggtcag cctgacctgc cctgacctgc ctggtcaaag ctggtcaaag gcttctatcc gcttctatcc cagcgacatc cagcgacatc 780 780 gccgtggagtgggagagcaa gccgtggagt gggagagcaa tgggcagccg tgggcagccg gagaacaact gagaacaact acaagaccac acaagaccac gcctcccgtg gcctcccgtg 840 840 ctggactccg acggctcctt ctggactccg acggctcctt cttcctctac cttcctctac agcaagctca agcaagctca ccgtggacaa ccgtggacaa gagcaggtgg gagcaggtgg 900 900 cagcagggga acgtcttctc cagcagggga acgtcttctc atgctccgtg atgctccgtg atgcatgagg atgcatgagg ctctgcacaa ctctgcacaa ccactacacg 960 ccactacacg 960 cagaagagcc tctccctgtc cagaagagcc tctccctgtc tccg tccg 984 984

<210> <210> 52 52 <211> <211> 987 987 <212> <212> DNA DNA <213> <213> Artificialsequence Artificial sequence

48

<400> <400> 52 52 gcttccacca agggcccatc gcttccacca agggcccatc ggtcttcccc ggtcttcccc ctggcaccct ctggcaccct cctccaagtc cctccaagtc gacctctggg gacctctggg 60 60

ggcacagcggccctgggctg ggcacagcgg ccctgggctg cctggtcaag cctggtcaag gactacttcc gactacttcc ccgaaccggt ccgaaccggt gacggtgtcg 120 gacggtgtcg 120 tggaactcag gcgccctgac tggaactcag gcgccctgac cagtggcgtg cagtggcgtg cacaccttcc cacaccttcc cggctgtcct cggctgtcct acagtcctca 180 acagtcctca 180 ggactctact ccctcagtag ggactctact ccctcagtag tgtggtgacc tgtggtgacc gtgccctcca gtgccctcca gtagtttggg gtagtttggg cacccagacc 240 cacccagacc 240 tacatctgca acgtgaatca tacatctgca acgtgaatca caagcccagt caagcccagt aacaccaagg aacaccaagg tggacaagaa tggacaagaa agttgagccc agttgagccc 300 300

aaatcttgtgacaaaactca aaatcttgtg acaaaactca cacatgccca cacatgccca ccgtgcccag ccgtgcccag cacctgagct cacctgagct cctgggggga 360 cctgggggga 360 ccgtcagtct tcctcttccc ccgtcagtct tcctcttccc cccaaaacca cccaaaaccc aaggacaccc aaggacaccc tcatgatctc tcatgatctc ccggacccct ccggacccct 420 420

gaggtcacatgcgtggtggt gaggtcacat gcgtggtggt ggacgtgagt ggacgtgagt cacgaagacc cacgaagacc ctgaggtcaa ctgaggtcaa gttcaactgg gttcaactgg 480 480

tacgtggacg gcgtggaggt tacgtggacg gcgtggaggt gcataatgcc gcataatgcc aagacaaagc aagacaaage cgcgggagga cgcgggagga gcagtacaac gcagtacaac 540 540

agtacgtaccgtgtggtcag agtacgtacc gtgtggtcag tgtcctcacc tgtcctcacc gtcctgcacc gtcctgcace aggactggct aggactggct gaatggcaag gaatggcaag 600 600

gagtacaagtgcaaggtctc gagtacaagt gcaaggtctc caacaaagcc caacaaagcc ctcccagccc ctcccagccc ccatcgagaa ccatcgagaa aaccatctcc aaccatctcc 660 660

aaagccaaagggcagccccg aaagccaaag ggcagccccg agaaccacag agaaccacag gtgtacaccc gtgtacaccc tgcccccatc tgcccccatc ccgggaggag 720 ccgggaggag 720 atgaccaagaaccaggtcag atgaccaaga accaggtcag tctgacctgc tctgacctgc ctggtcaaag ctggtcaaag gcttctatcc gcttctatcc cagtgacatc 780 cagtgacatc 780 gccgtggagtgggagagtaa gccgtggagt gggagagtaa tgggcagccg tgggcagccg gagaacaact gagaacaact acaagaccac acaagaccac gcctcccgtg gcctcccgtg 840 840

ctggactccg acggctcctt ctggactccg acggctcctt cttcctctac cttcctctac agtaagctca agtaagctca ccgtggacaa ccgtggacaa gagtaggtgg gagtaggtgg 900 900

cagcagggga acgtcttctc cagcagggga acgtcttctc atgctccgtg atgctccgtg atgcatgagg atgcatgagg ctctgcacaa ctctgcacaa ccactacacg 960 ccactacacg 960 cagaagagtc tctccctgtc cagaagagtc tctccctgtc tccgtga tccgtga 987 987

49

<210> <210> 53 53 <211> <211> 324 324 <212> <212> DNA DNA <213> <213> Artificial sequence Artificial sequence

<400> <400> 53 53 cgtacggtgg ctgcaccatc cgtacggtgg ctgcaccatc tgtcttcatc tgtcttcatc ttcccgccat ttcccgccat ctgatgagca ctgatgagca gttgaaatct gttgaaatct 60 60

ggaactgcctctgttgtgtg ggaactgcct ctgttgtgtg cctgctgaat cctgctgaat aacttctatc aacttctatc ccagagaggc ccagagaggc caaagtacag caaagtacag 120 120

tggaaggtgg ataacgccct tggaaggtgg ataacgccct ccaatcgggt ccaatcgggt aactcccagg aactcccagg agagtgtcac agagtgtcac agagcaggac agagcaggac 180 180

tccaaggact gcacctactc tccaaggact gcacctactc cctctcctcc cctctcctcc accctgacgc accctgacgc tgtccaaagc tgtccaaage agactacgag agactacgag 240 240

aaacacaaagtctacgcctg aaacacaaag tctacgcctg cgaagtcacc cgaagtcacc catcagggcc catcagggcc tgtcctcgcc tgtcctcgcc cgtcacaaag 300 cgtcacaaag 300 tccttcaaca ggggagagtg tccttcaaca ggggagagtg ttga ttga 324 324

<210> <210> 54 54 <211> <211> 324 324 <212> <212> DNA DNA <213> <213> Artificialsequence Artificial sequence

<400> <400> 54 54 cgtacggtgg ctgcaccatc cgtacggtgg ctgcaccate tgtcttcatc tgtcttcatc ttcccgccat ttcccgccat ctgatgagca ctgatgagca gttgaaatct gttgaaatct 60 60

ggtaccgcctctgttgtgtg ggtaccgcct ctgttgtgtg cctgctgaat cctgctgaat aacttctatc aacttctatc ccagagaggc ccagagaggc caaagtacag caaagtacag 120 120

agtaaggaca gtacctacag agtaaggaca gtacctacag tctcagtagt tctcagtagt accctgacgc accctgacgc tgtccaaagc tgtccaaagc agactacgag 240 agactacgag 240 aaacacaaag tctacgcctg aaacacaaag tctacgcctg cgaagtcacc cgaagtcacc catcagggcc catcagggcc tgtcctcgcc tgtcctcgcc cgtcacaaag 300 cgtcacaaag 300 tccttcaaca ggggagagtg tccttcaaca ggggagagtg ttga ttga 324 324

50

<210> <210> 55 55 <211> <211> 5 5 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 55 55

Ser Tyr Asp Ser Tyr AspIle IleSer Ser 1 1 5 5

<210> <210> 56 56 <211> <211> 5 5 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 56 56

His Tyr His Tyr Asp AspIle IleSer Ser 1 1 5 5

<210> <210> 57 57 <211> <211> 5 5 <212> <212> PRT PRT <213> <213> ArtificialSequence Artificial Sequence

<400> <400> 57 57

His Thr His Thr Asp AspIle IleSer Ser 1 1 5 5

<210> <210> 58 58 <211> <211> 16 16 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 58 58

Ile Ile Ser Ile Ile SerTyr TyrAla AlaGly Gly SerSer ThrThr Tyr Tyr Tyr Tyr Ala Ala Ser Ala Ser Trp TrpLys Ala Lys Gly Gly 1 1 5 5 10 10 15 15

51

<210> <210> 59 59 <211> <211> 16 16 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 59 59

Ile Ile Ser Ile Ile SerHis HisAla AlaGly Gly SerSer ThrThr Tyr Tyr Tyr Tyr Ala Ala Ser Ala Ser Trp TrpLys Ala Lys Gly Gly 1 1 5 5 10 10 15 15

<210> <210> 60 60 <211> <211> 16 16 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 60 60

Ile Ile Ser Ile Ile SerTyr TyrAla AlaGly Gly SerSer ThrThr Tyr Tyr Tyr Tyr Met Met Ser Ala Ser Trp TrpLys Ala Lys Gly Gly 1 1 5 5 10 10 15 15

<210> <210> 61 61 <211> <211> 11 11 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 61 61

Gly Val Gly Val Pro Pro Ala Ala Tyr Tyr Ser Ser Thr Thr Gly Gly Gly Gly Asp Asp Leu Leu 1 1 5 5 10 10

<210> <210> 62 62 <211> <211> 11 11 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 62 62

52

Gly Val Gly Val Pro Pro Ala Ala His His Ser Ser Thr Thr Gly Gly Gly Gly Asp Asp Leu Leu 1 1 5 5 10 10

<210> <210> 63 63 <211> <211> 11 11 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 63 63

Gly Val Gly Val Pro Pro Ala Ala Tyr Tyr Ser Ser His His Gly Gly Gly Gly Asp Asp Leu Leu 1 1 5 5 10 10

<210> <210> 64 64 <211> <211> 11 11 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 64 64

Gly Val Gly Val Pro Pro Ala Ala His His Ser Ser His His Gly Gly Gly Gly Asp Asp Leu Leu 1 1 5 5 10 10

<210> <210> 65 65 <211> <211> 13 13 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 65 65

Gln Ser Gln Ser Ser SerGln GlnSer Ser Val Val TyrTyr AspAsp Asn Asn Asn Asn Trp Ser Trp Leu Leu Ser 1 1 5 5 10 10

<210> <210> 66 66 <211> <211> 13 13 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 66 66

53

Gln Ser Gln Ser Ser SerGln GlnSer Ser Val Val TyrTyr HisHis Asn Asn Asn Asn Trp Ser Trp Leu Leu Ser 1 1 5 5 10 10

<210> <210> 67 67 <211> <211> 13 13 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 67 67

Thr Ser Thr Ser Ser SerGln GlnSer Ser Val Val TyrTyr HisHis Asn Asn Asn Asn Trp Ser Trp Leu Leu Ser 1 1 5 5 10 10

<210> <210> 68 68 <211> <211> 13 13 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 68 68

Gln Thr Gln Thr Ser SerGln GlnSer Ser Val Val TyrTyr HisHis Asp Asp Asn Asn Trp Ser Trp Leu Leu Ser 1 1 5 5 10 10

<210> <210> 69 69 <211> <211> 13 13 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 69 69

Thr Ser Thr Ser Ser SerGln GlnSer SerVal Val TyrTyr HisHis Asp Asp Asn Asn Trp Ser Trp Leu Leu Ser 1 1 5 5 10 10

<210> <210> 70 70 <211> <211> 7 7 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 70 70

54

Trp Ala Trp Ala Ser SerThr ThrLeu Leu Ala Ala SerSer 1 1 5 5

<210> <210> 71 71 <211> <211> 7 7 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 71 71

Trp Ala Trp Ala Ser SerThr ThrLeu Leu Ala Ala TyrTyr 1 1 5 5

<210> <210> 72 72 <211> <211> 7 7 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 72 72

Trp Ala Trp Ala Ser SerThr ThrLeu Leu Ala Ala PhePhe 1 1 5 5

<210> <210> 73 73 <211> <211> 10 10 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 73 73

Ala Gly Ala Gly Gly GlyTyr TyrGly Gly Gly Gly GlyGly LeuLeu Tyr Tyr Ala Ala 1 1 5 5 10 10

<210> <210> 74 74 <211> <211> 10 10 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 74 74

55

Ala Gly Ala Gly Gly Gly Tyr Tyr Gly Gly Gly Gly Gly Gly Arg Arg Tyr Tyr Ala Ala 1 1 5 5 10 10

<210> <210> 75 75 <211> <211> 242 242 <212> <212> PRT PRT <213> <213> Homo sapiens Homo sapiens

<400> <400> 75 75

Glu Asn Glu Asn Ser SerGlu GluGln Gln Lys Lys GluGlu AsnAsn Val Val Glu Glu Lys Gly Lys Glu Glu Leu GlyCys Leu Cys Asn Asn 1 1 5 5 10 10 15 15

Ala Cys Ala Cys Thr ThrTrp TrpArg Arg Gln Gln AsnAsn ThrThr Lys Lys Ser Ser Ser Ile Ser Arg Arg Glu IleAla Glu Ala Ile Ile 20 20 25 25 30 30

Lys Ile Lys Ile Gln Gln Ile Ile Leu Leu Ser Ser Lys Lys Leu Leu Arg Arg Leu Leu Glu Glu Thr Thr Ala Ala Pro Pro Asn Asn Ile Ile 35 35 40 40 45 45

Ser Lys Asp Ser Lys AspVal ValIle IleArg Arg GlnGln LeuLeu Leu Leu Pro Pro Lys Lys Ala Pro Ala Pro ProLeu Pro Leu Arg Arg 50 50 55 55 60 60

Glu Leu Glu Leu Ile IleAsp AspGln Gln Tyr Tyr AspAsp ValVal Gln Gln Arg Arg Asp Ser Asp Asp Asp Ser SerAsp Ser Asp Gly Gly

70 70 75 75 80 80

Ser Leu Ser Leu Glu GluAsp AspAsp AspAsp Asp TyrTyr HisHis Ala Ala Thr Thr Thr Thr Thr Glu Glu Ile ThrIle Ile Ile Thr Thr 85 85 90 90 95 95

Met Pro Met Pro Thr ThrGlu GluSer Ser Asp Asp PhePhe LeuLeu Met Met Gln Gln Val Gly Val Asp Asp Lys GlyPro Lys Pro Lys Lys 100 100 105 105 110 110

Cys Cys Cys Cys Phe PhePhe PheLys Lys Phe Phe SerSer SerSer Lys Lys Ile Ile Gln Asn Gln Tyr Tyr Lys AsnVal Lys Val Val Val 115 115 120 120 125 125

Lys Ala Lys Ala Gln GlnLeu LeuTrp Trp Ile Ile TyrTyr LeuLeu Arg Arg Pro Pro Val Thr Val Glu Glu Pro ThrThr Pro Thr Thr Thr 130 130 135 135 140 140

56

Val Phe Val Phe Val Val Gln Gln Ile Ile Leu Leu Arg Arg Leu Leu Ile Ile Lys Lys Pro Pro Met Met Lys Lys Asp Asp Gly Gly Thr Thr 145 145 150 150 155 155 160 160

Arg Tyr Arg Tyr Thr Thr Gly Gly Ile Ile Arg Arg Ser Ser Leu Leu Lys Lys Leu Leu Asp Asp Met Met Asn Asn Pro Pro Gly Gly Thr Thr 165 165 170 170 175 175

Gly Ile Gly Ile Trp Trp Gln Gln Ser Ser Ile Ile Asp Asp Val Val Lys Lys Thr Thr Val Val Leu Leu Gln Gln Asn Asn Trp Trp Leu Leu 180 180 185 185 190 190

Lys Gln Lys Gln Pro Pro Glu Glu Ser Ser Asn Asn Leu Leu Gly Gly Ile Ile Glu Glu Ile Ile Lys Lys Ala Ala Leu Leu Asp Asp Glu Glu 195 195 200 200 205 205

Asn Gly Asn Gly His His Asp Asp Leu Leu Ala Ala Val Val Thr Thr Phe Phe Pro Pro Gly Gly Pro Pro Gly Gly Glu Glu Asp Asp Gly Gly 210 210 215 215 220 220

Leu Asn Leu Asn Pro Pro Phe Phe Leu Leu Glu Glu Val Val Lys Lys Val Val Thr Thr Asp Asp Thr Thr Pro Pro Lys Lys Arg Arg Ser Ser 225 225 230 230 235 235 240 240

Arg Arg Arg Arg

<210> <210> 76 76 <211> <211> 242 242 <212> <212> PRT PRT <213> <213> Macaca fascicularis Macaca fascicularis

<400> <400> 76 76

Ala Cys Ala Cys Thr Thr Trp Trp Arg Arg Gln Gln Asn Asn Thr Thr Lys Lys Ser Ser Ser Ser Arg Arg Ile Ile Glu Glu Ala Ala Ile Ile 20 20 25 25 30 30

57

Ser Lys Ser Lys Asp AspAla AlaIle IleArg Arg GlnGln LeuLeu Leu Leu Pro Pro Lys Lys Ala Pro Ala Pro ProLeu Pro Leu Arg Arg 50 50 55 55 60 60

70 70 75 75 80 80

Ser Leu Ser Leu Glu GluAsp AspAsp AspAsp Asp TyrTyr HisHis Ala Ala Thr Thr Thr Thr Glu Ile Glu Thr ThrIle Ile Ile Thr Thr 85 85 90 90 95 95

58

Leu Asn Leu Asn Pro ProPhe PheLeu Leu Glu Glu ValVal LysLys Val Val Thr Thr Asp Pro Asp Thr Thr Lys ProArg Lys Arg Ser Ser 225 225 230 230 235 235 240 240

Arg Arg Arg Arg

<210> <210> 77 77 <211> <211> 242 242 <212> <212> PRT PRT <213> <213> Mus musculus Mus musculus

<400> <400> 77 77

Glu Gly Glu Gly Ser SerGlu GluArg Arg Glu Glu GluGlu AsnAsn Val Val Glu Glu Lys Gly Lys Glu Glu Leu GlyCys Leu Cys Asn Asn 1 1 5 5 10 10 15 15

Ala Cys Ala Cys Ala AlaTrp TrpArg Arg Gln Gln AsnAsn ThrThr Arg Arg Tyr Tyr Ser Ile Ser Arg Arg Glu IleAla Glu Ala Ile Ile 20 20 25 25 30 30

Lys Ile Lys Ile Gln GlnIle IleLeu Leu Ser Ser LysLys LeuLeu Arg Arg Leu Leu Glu Ala Glu Thr Thr Pro AlaAsn Pro Asn Ile Ile 35 35 40 40 45 45

Ser Lys Asp Ser Lys AspAla AlaIle IleArg Arg GlnGln LeuLeu Leu Leu Pro Pro Arg Arg Ala Pro Ala Pro ProLeu Pro Leu Arg Arg 50 50 55 55 60 60

70 70 75 75 80 80

Met Pro Met Pro Thr ThrGlu GluSer Ser Asp Asp PhePhe LeuLeu Met Met Gln Gln Ala Gly Ala Asp Asp Lys GlyPro Lys Pro Lys Lys 100 100 105 105 110 110

Cys Cys Cys Cys Phe PhePhe PheLys Lys Phe Phe SerSer SerSer Lys Lys Ile Ile Gln Asn Gln Tyr Tyr Lys AsnVal Lys Val Val Val

59

115 120 120 125 125

Lys Ala Lys Ala Gln GlnLeu LeuTrp Trp Ile Ile TyrTyr LeuLeu Arg Arg Pro Pro Val Thr Val Lys Lys Pro ThrThr Pro Thr Thr Thr 130 130 135 135 140 140

Arg Tyr Arg Tyr Thr Thr Gly Gly Ile Ile Arg Arg Ser Ser Leu Leu Lys Lys Leu Leu Asp Asp Met Met Ser Ser Pro Pro Gly Gly Thr Thr 165 165 170 170 175 175

Gly Ile Gly Ile Trp TrpGln GlnSer Ser Ile Ile AspAsp ValVal Lys Lys Thr Thr Val Gln Val Leu Leu Asn GlnTrp Asn Trp Leu Leu 180 180 185 185 190 190

Lys Gln Lys Gln Pro ProGlu GluSer Ser Asn Asn LeuLeu GlyGly Ile Ile Glu Glu Ile Ala Ile Lys Lys Leu AlaAsp Leu Asp Glu Glu 195 195 200 200 205 205

Arg Arg Arg Arg

<210> <210> 78 78 <211> <211> 266 266 <212> <212> PRT PRT <213> <213> Homo sapiens Homo sapiens

<400> <400> 78 78

60

70 70 75 75 80 80

61

Val Thr Val Thr Asp Asp Thr Thr Pro Pro Lys Lys Arg Arg Ser Ser Arg Arg Arg Arg 260 260 265 265

<210> <210> 79 79 <211> <211> 266 266 <212> <212> PRT PRT <213> <213> Macaca fascicularis Macaca fascicularis

<400> <400> 79 79

70 70 75 75 80 80

62

63

<210> < 210> 80 80 <211> <211> 267 267 <212> <212> PRT PRT <213> <213> Mus musculus Mus musculus

<400> <400> 80 80

Asn Val Asn Val Glu GluLys LysGlu Glu Gly Gly LeuLeu CysCys Asn Asn Ala Ala Cys Trp Cys Ala Ala Arg TrpGln Arg Gln Asn Asn 35 35 40 40 45 45

70 70 75 75 80 80

Leu Leu Leu Leu Pro ProArg ArgAla AlaPro Pro ProPro LeuLeu Arg Arg Glu Glu Leu Asp Leu Ile Ile Gln AspTyr Gln Tyr Asp Asp 85 85 90 90 95 95

64

Leu Arg Leu Arg Pro Pro Val Val Lys Lys Thr Thr Pro Pro Thr Thr Thr Thr Val Val Phe Phe Val Val Gln Gln Ile Ile Leu Leu Arg Arg 165 165 170 170 175 175

Gly Ile Gly Ile Glu Glu Ile Ile Lys Lys Ala Ala Leu Leu Asp Asp Glu Glu Asn Asn Gly Gly His His Asp Asp Leu Leu Ala Ala Val Val 225 225 230 230 235 235 240 240

Thr Phe Thr Phe Pro Pro Gly Gly Pro Pro Gly Gly Glu Glu Asp Asp Gly Gly Leu Leu Asn Asn Pro Pro Phe Phe Leu Leu Glu Glu Val Val 245 245 250 250 255 255

Lys Val Lys Val Thr Thr Asp Asp Thr Thr Pro Pro Lys Lys Arg Arg Ser Ser Arg Arg Arg Arg 260 260 265 265

<210> <210> 81 81 <211> <211> 407 407 <212> <212> PRT PRT <213> <213> Homo sapiens Homo sapiens

<400> <400> 81 81

Met Val Met Val Leu LeuAla AlaAla Ala Pro Pro LeuLeu LeuLeu Leu Leu Gly Gly Phe Leu Phe Leu Leu Leu LeuAla Leu Ala Leu Leu 1 1 5 5 10 10 15 15

Glu Leu Glu Leu Arg ArgPro ProArg Arg Gly Gly GluGlu AlaAla Ala Ala Glu Glu Gly Ala Gly Pro Pro Ala AlaAla Ala Ala

65

Ala Ala 20 20 25 25 30 30

Ala Ala Ala Ala Ala AlaAla AlaAla Ala Ala Ala AlaAla AlaAla Ala Ala Gly Gly Val Gly Val Gly Gly Glu GlyArg Glu Arg Ser Ser 35 35 40 40 45 45

Ser Arg Ser Arg Pro ProAla AlaPro Pro Ser Ser ValVal AlaAla Pro Pro Glu Glu Pro Gly Pro Asp Asp Cys GlyPro Cys Pro Val Val 50 50 55 55 60 60

Cys Val Cys Val Trp TrpArg ArgGln Gln His His SerSer ArgArg Glu Glu Leu Leu Arg Glu Arg Leu Leu Ser GluIle Ser Ile Lys Lys

70 70 75 75 80 80

Ser Gln Ile Ser Gln IleLeu LeuSer SerLys Lys LeuLeu ArgArg Leu Leu Lys Lys Glu Glu Ala Asn Ala Pro ProIle Asn Ile Ser Ser 85 85 90 90 95 95

Arg Glu Arg Glu Val Val Val Val Lys Lys Gln Gln Leu Leu Leu Leu Pro Pro Lys Lys Ala Ala Pro Pro Pro Pro Leu Leu Gln Gln Gln Gln 100 100 105 105 110 110

Ile Leu Asp Ile Leu AspLeu LeuHis HisAsp Asp PhePhe GlnGln Gly Gly Asp Asp Ala Ala Leu Pro Leu Gln GlnGlu Pro Glu Asp Asp 115 115 120 120 125 125

Phe Leu Phe Leu Glu GluGlu GluAsp AspGlu Glu TyrTyr HisHis Ala Ala Thr Thr Thr Thr Glu Val Glu Thr ThrIle Val Ile Ser Ser 130 130 135 135 140 140

Met Ala Met Ala Gln GlnGlu GluThr Thr Asp Asp ProPro AlaAla Val Val Gln Gln Thr Gly Thr Asp Asp Ser GlyPro Ser Pro Leu Leu 145 145 150 150 155 155 160 160

Cys Cys Cys Cys His HisPhe PheHis His Phe Phe SerSer ProPro Lys Lys Val Val Met Thr Met Phe Phe Lys ThrVal Lys Val Leu Leu 165 165 170 170 175 175

Lys Ala Lys Ala Gln GlnLeu LeuTrp Trp Val Val TyrTyr LeuLeu Arg Arg Pro Pro Val Arg Val Pro Pro Pro ArgAla Pro Ala Thr Thr 180 180 185 185 190 190

Val Tyr Val Tyr Leu Leu Gln Gln Ile Ile Leu Leu Arg Arg Leu Leu Lys Lys Pro Pro Leu Leu Thr Thr Gly Gly Glu Glu Gly Gly

66

Thr Thr 195 195 200 200 205 205

Ala Gly Ala Gly Gly GlyGly GlyGly Gly Gly Gly GlyGly ArgArg Arg Arg His His Ile Ile Ile Arg Arg Arg IleSer Arg Ser Leu Leu 210 210 215 215 220 220

Lys Ile Lys Ile Glu GluLeu LeuHis His Ser Ser ArgArg SerSer Gly Gly His His Trp Ser Trp Gln Gln Ile SerAsp Ile Asp Phe Phe 225 225 230 230 235 235 240 240

Lys Gln Lys Gln Val ValLeu LeuHis His Ser Ser TrpTrp PhePhe Arg Arg Gln Gln Pro Ser Pro Gln Gln Asn SerTrp Asn Trp Gly Gly 245 245 250 250 255 255

Ile Glu Ile Ile Glu IleAsn AsnAla AlaPhe Phe AspAsp ProPro Ser Ser Gly Gly Thr Thr Asp Ala Asp Leu LeuVal Ala Val Thr Thr 260 260 265 265 270 270

Ser Leu Gly Ser Leu GlyPro ProGly GlyAla Ala GluGlu GlyGly Leu Leu His His Pro Pro Phe Glu Phe Met MetLeu Glu Leu Arg Arg 275 275 280 280 285 285

Val Leu Val Leu Glu Glu Asn Asn Thr Thr Lys Lys Arg Arg Ser Ser Arg Arg Arg Arg Asn Asn Leu Leu Gly Gly Leu Leu Asp Asp Cys Cys 290 290 295 295 300 300

Asp Glu Asp Glu His HisSer SerSer Ser Glu Glu SerSer ArgArg Cys Cys Cys Cys Arg Pro Arg Tyr Tyr Leu ProThr Leu Thr Val Val 305 305 310 310 315 315 320 320

Asp Phe Asp Phe Glu GluAla AlaPhe Phe Gly Gly TrpTrp AspAsp Trp Trp Ile Ile Ile Pro Ile Ala Ala Lys ProArg Lys Arg Tyr Tyr 325 325 330 330 335 335

Lys Ala Lys Ala Asn Asn Tyr Tyr Cys Cys Ser Ser Gly Gly Gln Gln Cys Cys Glu Glu Tyr Tyr Met Met Phe Phe Met Met Gln Gln Lys Lys 340 340 345 345 350 350

Tyr Pro Tyr Pro His HisThr ThrHis His Leu Leu ValVal GlnGln Gln Gln Ala Ala Asn Arg Asn Pro Pro Gly ArgSer Gly Ser Ala Ala 355 355 360 360 365 365

67

Gly Pro Gly Pro Cys CysCys CysThr Thr Pro Pro ThrThr LysLys Met Met Ser Ser Pro Asn Pro Ile Ile Met AsnLeu Met Leu Tyr Tyr 370 370 375 375 380 380

Phe Asn Asp Phe Asn AspLys LysGln GlnGln Gln IleIle IleIle Tyr Tyr Gly Gly Lys Lys Ile Gly Ile Pro ProMet Gly Met Val Val 385 385 390 390 395 395 400 400

Val Asp Val Asp Arg ArgCys CysGly Gly Cys Cys SerSer 405 405

<210> <210> 82 82 <211> <211> 109 109 <212> <212> PRT PRT <213> <213> Homo sapiens Homo sapiens

<400> <400> 82 82

Asn Leu Asn Leu Gly GlyLeu LeuAsp Asp Cys Cys AspAsp GluGlu His His Ser Ser Ser Ser Ser Glu Glu Arg SerCys Arg Cys Cys Cys 1 1 5 5 10 10 15 15

Arg Tyr Arg Tyr Pro ProLeu LeuThr Thr Val Val AspAsp PhePhe Glu Glu Ala Ala Phe Trp Phe Gly Gly Asp TrpTrp Asp Trp Ile Ile 20 20 25 25 30 30

Ile Ala Pro Ile Ala ProLys LysArg ArgTyr Tyr LysLys AlaAla Asn Asn Tyr Tyr Cys Cys Ser Gln Ser Gly GlyCys Gln Cys Glu Glu 35 35 40 40 45 45

Tyr Met Tyr Met Phe PheMet MetGln Gln Lys Lys TyrTyr ProPro His His Thr Thr His Val His Leu Leu Gln ValGln Gln Gln Ala Ala 50 50 55 55 60 60

70 70 75 75 80 80

Pro Ile Pro Ile Asn AsnMet MetLeu Leu Tyr Tyr PhePhe AsnAsn Asp Asp Lys Lys Gln Ile Gln Gln Gln Ile IleTyr Ile Tyr Gly Gly 85 85 90 90 95 95

Lys Ile Lys Ile Pro ProGly GlyMet Met Val Val ValVal AspAsp Arg Arg Cys Cys Gly Ser Gly Cys Cys Ser 100 100 105 105

68

<210> <210> 83 83 <211> <211> 274 274 <212> <212> PRT PRT <213> <213> Homo sapiens Homo sapiens

<400> <400> 83 83

Ala Glu Ala Glu Gly GlyPro ProAla Ala Ala Ala AlaAla AlaAla Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala AlaAla Ala Ala Ala Ala 1 1 5 5 10 10 15 15

Ala Gly Ala Gly Val ValGly GlyGly Gly Glu Glu ArgArg SerSer Ser Ser Arg Arg Pro Pro Pro Ala Ala Ser ProVal Ser Val Ala Ala 20 20 25 25 30 30

Pro Glu Pro Glu Pro ProAsp AspGly GlyCys Cys ProPro ValVal Cys Cys Val Val Trp Gln Trp Arg Arg His GlnSer His Ser Arg Arg 35 35 40 40 45 45

Glu Leu Glu Leu Arg Arg Leu Leu Glu Glu Ser Ser Ile Ile Lys Lys Ser Ser Gln Gln Ile Ile Leu Leu Ser Ser Lys Lys Leu Leu Arg Arg 50 50 55 55 60 60

Leu Lys Leu Lys Glu GluAla AlaPro Pro Asn Asn IleIle SerSer Arg Arg Glu Glu Val Lys Val Val Val Gln LysLeu Gln Leu Leu Leu

70 70 75 75 80 80

Pro Lys Ala Pro Lys AlaPro ProPro ProLeu Leu GlnGln GlnGln Ile Ile Leu Leu Asp Asp Leu Asp Leu His HisPhe Asp Phe Gln Gln 85 85 90 90 95 95

Gly Asp Gly Asp Ala AlaLeu LeuGln Gln Pro Pro GluGlu AspAsp Phe Phe Leu Leu Glu Asp Glu Glu Glu Glu AspTyr Glu Tyr His His 100 100 105 105 110 110

Ala Thr Ala Thr Thr ThrGlu GluThr Thr Val Val IleIle SerSer Met Met Ala Ala Gln Thr Gln Glu Glu Asp ThrPro Asp Pro Ala Ala 115 115 120 120 125 125

Val Gln Val Gln Thr ThrAsp AspGly Gly Ser Ser ProPro LeuLeu Cys Cys Cys Cys His His His Phe Phe Phe HisSer Phe Ser Pro Pro 130 130 135 135 140 140

Lys Val Lys Val Met MetPhe PheThr Thr Lys Lys ValVal LeuLeu Lys Lys Ala Ala Gln Trp Gln Leu Leu Val TrpTyr Val Tyr Leu Leu 145 145 150 150 155 155 160 160

69

Arg Pro Arg Pro Val Val Pro Pro Arg Arg Pro Pro Ala Ala Thr Thr Val Val Tyr Tyr Leu Leu Gln Gln Ile Ile Leu Leu Arg Arg Leu Leu 165 165 170 170 175 175

Lys Pro Lys Pro Leu LeuThr ThrGly Gly Glu Glu GlyGly ThrThr Ala Ala Gly Gly Gly Gly Gly Gly Gly Gly GlyGly Gly Gly Arg Arg 180 180 185 185 190 190

Arg His Arg His Ile Ile Arg Arg Ile Ile Arg Arg Ser Ser Leu Leu Lys Lys Ile Ile Glu Glu Leu Leu His His Ser Ser Arg Arg Ser Ser 195 195 200 200 205 205

Gly His Gly His Trp Trp Gln Gln Ser Ser Ile Ile Asp Asp Phe Phe Lys Lys Gln Gln Val Val Leu Leu His His Ser Ser Trp Trp Phe Phe 210 210 215 215 220 220

Arg Gln Arg Gln Pro Pro Gln Gln Ser Ser Asn Asn Trp Trp Gly Gly Ile Ile Glu Glu Ile Ile Asn Asn Ala Ala Phe Phe Asp Asp Pro Pro 225 225 230 230 235 235 240 240

Ser Gly Thr Ser Gly ThrAsp AspLeu LeuAla Ala ValVal ThrThr Ser Ser Leu Leu Gly Gly Pro Ala Pro Gly GlyGlu Ala Glu Gly Gly 245 245 250 250 255 255

Leu His Leu His Pro Pro Phe Phe Met Met Glu Glu Leu Leu Arg Arg Val Val Leu Leu Glu Glu Asn Asn Thr Thr Lys Lys Arg Arg Ser Ser 260 260 265 265 270 270

Arg Arg Arg Arg

<210> <210> 84 84 <211> <211> 298 298 <212> <212> PRT PRT <213> <213> Homo sapiens Homo sapiens

<400> <400> 84 84

Glu Leu Glu Leu Arg ArgPro ProArg Arg Gly Gly GluGlu AlaAla Ala Ala Glu Glu Gly Ala Gly Pro Pro Ala AlaAla Ala Ala Ala Ala

70

20 25 25 30 30

Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Val Val Gly Gly Gly Gly Glu Glu Arg Arg Ser Ser 35 35 40 40 45 45

Ser Arg Ser Arg Pro ProAla AlaPro ProSer Ser ValVal AlaAla Pro Pro Glu Glu Pro Pro Asp Cys Asp Gly GlyPro Cys Pro Val Val 50 50 55 55 60 60

70 70 75 75 80 80

Ser Gln Ser Gln Ile IleLeu LeuSer SerLys Lys LeuLeu ArgArg Leu Leu Lys Lys Glu Glu Ala Asn Ala Pro ProIle Asn Ile Ser Ser 85 85 90 90 95 95

Phe Leu Glu Phe Leu GluGlu GluAsp AspGlu Glu TyrTyr HisHis Ala Ala Thr Thr Thr Thr Glu Val Glu Thr ThrIle Val Ile Ser Ser 130 130 135 135 140 140

Val Tyr Val Tyr Leu Leu Gln Gln Ile Ile Leu Leu Arg Arg Leu Leu Lys Lys Pro Pro Leu Leu Thr Thr Gly Gly Glu Glu Gly Gly Thr Thr

71

195 200 200 205 205

Val Leu Val Leu Glu Glu Asn Asn Thr Thr Lys Lys Arg Arg Ser Ser Arg Arg Arg Arg 290 290 295 295

<210> <210> 85 85 <211> <211> 409 409 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 85 85

Met Lys Met Lys Trp TrpVal ValThr Thr Phe Phe LeuLeu LeuLeu Leu Leu Leu Leu Phe Ser Phe Ile Ile Gly SerSer Gly Ser Ala Ala 1 1 5 5 10 10 15 15

Phe Ser Phe Ser Asp AspTyr TyrLys LysAsp Asp AspAsp AspAsp Asp Asp Lys Lys Ala Ala Glu Pro Glu Gly GlyAla Pro Ala Ala Ala 20 20 25 25 30 30

Ala Ala Ala Ala Ala AlaAla AlaAla Ala Ala Ala AlaAla AlaAla Ala Ala Ala Ala Ala Val Ala Gly Gly Gly ValGly Gly Gly Glu Glu 35 35 40 40 45 45

72

Arg Ser Arg Ser Ser Ser Arg Arg Pro Pro Ala Ala Pro Pro Ser Ser Val Val Ala Ala Pro Pro Glu Glu Pro Pro Asp Asp Gly Gly Cys Cys 50 50 55 55 60 60

Pro Val Pro Val Cys Cys Val Val Trp Trp Arg Arg Gln Gln His His Ser Ser Arg Arg Glu Glu Leu Leu Arg Arg Leu Leu Glu Glu Ser Ser

70 70 75 75 80 80

Ile Lys Ser Ile Lys SerGln GlnIle IleLeu Leu SerSer LysLys Leu Leu Arg Arg Leu Leu Lys Ala Lys Glu GluPro Ala Pro Asn Asn 85 85 90 90 95 95

Ile Ser Arg Ile Ser ArgGlu GluVal ValVal Val LysLys GlnGln Leu Leu Leu Leu Pro Pro Lys Pro Lys Ala AlaPro Pro Pro Leu Leu 100 100 105 105 110 110

Gln Gln Gln Gln Ile Ile Leu Leu Asp Asp Leu Leu His His Asp Asp Phe Phe Gln Gln Gly Gly Asp Asp Ala Ala Leu Leu Gln Gln Pro Pro 115 115 120 120 125 125

Glu Asp Glu Asp Phe PheLeu LeuGlu Glu Glu Glu AspAsp GluGlu Tyr Tyr His His Ala Thr Ala Thr Thr Glu ThrThr Glu Thr Val Val 130 130 135 135 140 140

Ile Ser Met Ile Ser MetAla AlaGln GlnGlu Glu ThrThr AspAsp Pro Pro Ala Ala Val Val Gln Asp Gln Thr ThrGly Asp Gly Ser Ser 145 145 150 150 155 155 160 160

Pro Leu Pro Leu Cys CysCys CysHis HisPhe Phe HisHis PhePhe Ser Ser Pro Pro Lys Met Lys Val Val Phe MetThr Phe Thr Lys Lys 165 165 170 170 175 175

Val Leu Val Leu Lys Lys Ala Ala Gln Gln Leu Leu Trp Trp Val Val Tyr Tyr Leu Leu Arg Arg Pro Pro Val Val Pro Pro Arg Arg Pro Pro 180 180 185 185 190 190

Ala Thr Ala Thr Val Val Tyr Tyr Leu Leu Gln Gln Ile Ile Leu Leu Arg Arg Leu Leu Lys Lys Pro Pro Leu Leu Thr Thr Gly Gly Glu Glu 195 195 200 200 205 205

Gly Thr Gly Thr Ala AlaGly GlyGly Gly Gly Gly GlyGly GlyGly Gly Gly Arg Arg Arg Ile Arg His His Arg IleIle Arg Ile Arg Arg 210 210 215 215 220 220

73

Ser Leu Lys Ser Leu LysIle IleGlu GluLeu Leu HisHis SerSer Arg Arg Ser Ser Gly Gly His Gln His Trp TrpSer Gln Ser Ile Ile 225 225 230 230 235 235 240 240

Asp Phe Asp Phe Lys LysGln GlnVal Val Leu Leu HisHis SerSer Trp Trp Phe Phe Arg Pro Arg Gln Gln Gln ProSer Gln Ser Asn Asn 245 245 250 250 255 255

Trp Gly Trp Gly Ile IleGlu GluIle Ile Asn Asn AlaAla PhePhe Asp Asp Pro Pro Ser Thr Ser Gly Gly Asp ThrLeu Asp Leu Ala Ala 260 260 265 265 270 270

Val Thr Val Thr Ser SerLeu LeuGly Gly Pro Pro GlyGly AlaAla Glu Glu Gly Gly Leu Pro Leu His His Phe ProMet Phe Met Glu Glu 275 275 280 280 285 285

Leu Arg Leu Arg Val ValLeu LeuGlu Glu Asn Asn ThrThr LysLys Arg Arg Ser Ser Arg Asn Arg Arg Arg Leu AsnGly Leu Gly Leu Leu 290 290 295 295 300 300

Asp Cys Asp Cys Asp AspGlu GluHis His Ser Ser SerSer GluGlu Ser Ser Arg Arg Cys Arg Cys Cys Cys Tyr ArgPro Tyr Pro Leu Leu 305 305 310 310 315 315 320 320

Thr Val Thr Val Asp AspPhe PheGlu Glu Ala Ala PhePhe GlyGly Trp Trp Asp Asp Trp Ile Trp Ile Ile Ala IlePro Ala Pro Lys Lys 325 325 330 330 335 335

Arg Tyr Arg Tyr Lys LysAla AlaAsn Asn Tyr Tyr CysCys SerSer Gly Gly Gln Gln Cys Tyr Cys Glu Glu Met TyrPhe Met Phe Met Met 340 340 345 345 350 350

Gln Lys Gln Lys Tyr TyrPro ProHis His Thr Thr HisHis LeuLeu Val Val Gln Gln Gln Asn Gln Ala Ala Pro AsnArg Pro Arg Gly Gly 355 355 360 360 365 365

Ser Ala Ser Ala Gly GlyPro ProCys Cys Cys Cys ThrThr ProPro Thr Thr Lys Lys Met Pro Met Ser Ser Ile ProAsn Ile Asn Met Met 370 370 375 375 380 380

Leu Tyr Leu Tyr Phe PheAsn AsnAsp Asp Lys Lys GlnGln GlnGln Ile Ile Ile Ile Tyr Lys Tyr Gly Gly Ile LysPro Ile Pro Gly Gly

74

385 390 390 395 395 400 400

Met Val Met Val Val ValAsp AspArg Arg Cys Cys GlyGly CysCys Ser Ser 405 405

<210> <210> 86 86 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 86 86

Gln Val Gln Val Thr ThrLeu LeuLys LysGlu Glu SerSer GlyGly Pro Pro Val Val Leu Lys Leu Val Val Pro LysThr Pro Thr Glu Glu 1 1 5 5 10 10 15 15

Thr Leu Thr Leu Thr ThrLeu LeuThr ThrCys Cys ThrThr ValVal Ser Ser Gly Gly Ile Leu Ile Asp Asp Ser LeuHis Ser His Asp Asp 20 20 25 25 30 30

70 70 75 75 80 80

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

75

<210> <210> 87 87 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 87 87

Thr Leu Thr Leu Thr ThrLeu LeuThr Thr Cys Cys ThrThr ValVal Ser Ser Gly Gly Ile Leu Ile Asp Asp Ser LeuHis Ser His Asp Asp 20 20 25 25 30 30

Gly Ile Gly Ile Ile Ile Ser Ser Tyr Tyr Ala Ala Gly Gly Ser Ser Met Met Tyr Tyr Tyr Tyr Ala Ala Ser Ser Trp Trp Ala Ala Lys Lys 50 50 55 55 60 60

70 70 75 75 80 80

Arg Gly Arg Gly Val Val Pro Pro Ala Ala Tyr Tyr Ser Ser His His Gly Gly Gly Gly Asp Asp Lys Lys Trp Trp Gly Gly Gln Gln Gly Gly 100 100 105 105 110 110

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

<210> <210> 88 88 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<220> <220>

76

<400> <400> 88 88

Gly Ile Gly Ile Ile Ile Ser Ser Tyr Tyr Ala Ala Gly Gly Lys Lys Thr Thr Tyr Tyr Tyr Tyr Ala Ala Ser Ser Trp Trp Ala Ala Lys Lys 50 50 55 55 60 60

Lys Arg Lys Arg Leu LeuThr ThrIle Ile Ser Ser LysLys AspAsp Thr Thr Ser Ser Lys Gln Lys Asn Asn Val GlnVal Val Val Leu Leu

70 70 75 75 80 80

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

<210> <210> 89 89 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 89 89

77

Gly Ile Gly Ile Ile Ile Ser Ser Tyr Tyr Ala Ala Gly Gly Lys Lys Lys Lys Tyr Tyr Tyr Tyr Ala Ala Ser Ser Trp Trp Ala Ala Lys Lys 50 50 55 55 60 60

70 70 75 75 80 80

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

<210> <210> 90 90 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 90 90

78

Gly Ile Gly Ile Ile IleSer SerTyr Tyr Ala Ala GlyGly SerSer Lys Lys Tyr Tyr Lys Ser Lys Ala Ala Trp SerAla Trp Ala Lys Lys 50 50 55 55 60 60

70 70 75 75 80 80

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

<210> <210> 91 91 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 91 91

Gly Ile Gly Ile Ile IleSer SerTyr Tyr Ala Ala GlyGly LysLys Lys Lys Tyr Tyr Tyr Ser Tyr Ala Ala Trp SerAla Trp Ala Lys Lys

79

50 55 55 60 60

70 70 75 75 80 80

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

<210> <210> 92 92 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 92 92

Gln Val Gln Val Gln GlnLeu LeuVal Val Glu Glu SerSer GlyGly Gly Gly Gly Gly Leu Gln Leu Val Val Pro GlnGly Pro Gly Gly Gly 1 1 5 5 10 10 15 15

Ser Leu Arg Ser Leu ArgLeu LeuSer SerCys Cys AlaAla ValVal Ser Ser Gly Gly Ile Ile Asp Ser Asp Leu LeuHis Ser His Asp Asp 20 20 25 25 30 30

Ser Ile Ile Ser Ile IleSer SerTyr TyrAla Ala GlyGly SerSer Thr Thr Tyr Tyr Tyr Tyr Ala Trp Ala Ser SerAla Trp Ala Lys Lys 50 50 55 55 60 60

70 70 75 75 80 80

80

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

<210> <210> 93 93 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 93 93

Thr Leu Thr Leu Thr ThrLeu LeuThr Thr Cys Cys LysLys ValVal Ser Ser Gly Gly Ile Leu Ile Asp Asp Ser LeuHis Ser His Asp Asp 20 20 25 25 30 30

Ser Ile Ser Ile Ile IleSer SerTyr Tyr Ala Ala GlyGly SerSer Thr Thr Tyr Tyr Tyr Ser Tyr Ala Ala Trp SerAla Trp Ala Lys Lys 50 50 55 55 60 60

70 70 75 75 80 80

Arg Gly Arg Gly Val Val Pro Pro Ala Ala Tyr Tyr Ser Ser His His Gly Gly Gly Gly Asp Asp Leu Leu Trp Trp Gly Gly Gln Gln

81

Gly Gly 100 100 105 105 110 110

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

<210> <210> 94 94 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 94 94

Thr Leu Thr Leu Thr ThrLeu LeuThr Thr Cys Cys LysLys ValVal Ser Ser Gly Gly Ile Leu Ile Asp Asp Ser LeuHis Ser His Glu Glu 20 20 25 25 30 30

70 70 75 75 80 80

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

82

<210> <210> 95 95 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 95 95

Gln Val Gln Val Gln Gln Leu Leu Val Val Glu Glu Ser Ser Gly Gly Gly Gly Gly Gly Leu Leu Val Val Gln Gln Pro Pro Gly Gly Gly Gly 1 1 5 5 10 10 15 15

Ser Leu Arg Ser Leu ArgLeu LeuSer SerCys Cys AlaAla ValVal Ser Ser Gly Gly Ile Ile Asp Ser Asp Leu LeuHis Ser His Glu Glu 20 20 25 25 30 30

Ser Ile Ile Ser Ile IleSer SerTyr TyrAla Ala GlyGly SerSer Lys Lys Tyr Tyr Tyr Tyr Ala Trp Ala Ser SerAla Trp Ala Lys Lys 50 50 55 55 60 60

70 70 75 75 80 80

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

<210> <210> 96 96 <211> <211> 110 110 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<220> <220>

83

<400> <400> 96 96

Glu Arg Glu Arg Ala AlaThr ThrLeu Leu Ser Ser CysCys ThrThr Ser Ser Ser Ser Gln Val Gln Ser Ser Tyr ValHis Tyr His Ala Ala 20 20 25 25 30 30

Leu Ile Leu Ile Tyr Tyr Trp Trp Ala Ala Ser Ser Thr Thr Leu Leu Ala Ala Tyr Tyr Gly Gly Val Val Pro Pro Ser Ser Arg Arg Phe Phe 50 50 55 55 60 60

70 70 75 75 80 80

<210> <210> 97 97 <211> <211> 110 110 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 97 97

Glu Arg Glu Arg Ala AlaThr ThrLeu Leu Ser Ser CysCys ThrThr Thr Thr Ser Ser Gln Val Gln Ser Ser Tyr ValHis Tyr His Glu Glu 20 20 25 25 30 30

84

70 70 75 75 80 80

<210> <210> 98 98 <211> <211> 110 110 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 98 98

Glu Arg Glu Arg Ala AlaThr ThrLeu Leu Ser Ser CysCys ThrThr Ser Ser Ser Ser Gln Val Gln Ser Ser Phe ValHis Phe His Glu Glu 20 20 25 25 30 30

Leu Ile Leu Ile Tyr TyrTrp TrpAla Ala Ser Ser ThrThr LeuLeu Ala Ala Trp Trp Gly Pro Gly Val Val Ser ProArg Ser Arg Phe Phe 50 50 55 55 60 60

85

70 70 75 75 80 80

<210> <210> 99 99 <211> <211> 110 110 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 99 99

Asn Trp Asn Trp Leu LeuSer SerTrp Trp Tyr Tyr GlnGln GlnGln Lys Lys Pro Pro Gly Pro Gly Gln Gln Pro ProLys Pro Lys Leu Leu 35 35 40 40 45 45

70 70 75 75 80 80

Gln Pro Gln Pro Glu GluAsp AspVal Val Ala Ala ThrThr TyrTyr Tyr Tyr Cys Cys Ala Gly Ala Gly Gly Tyr GlyGly Tyr Gly Gly Gly 85 85 90 90 95 95

86

<210> <210> 100 100 <211> <211> 357 357 <212> <212> DNA DNA <213> <213> Artificialsequence Artificial sequence

<400> <400> 100 100 caggtgacac tgaaagaaag caggtgacac tgaaagaaag cggccccgtg cggccccgtg ctggtgaagc ctggtgaage caaccgagac caaccgagac actgactctg actgactctg 60 60

acctgcacagtgagtgggat acctgcacag tgagtgggat cgacctgagc cgacctgage cacgacgaca cacgacgaca tctcctgggt tctcctgggt gcggcaggca 120 gcggcaggca 120 cccggaaagg gcctggaatg cccggaaagg gcctggaatg ggtcggcatc ggtcggcatc attagctacg attagctacg ccgggagcac ccgggagcad atactatgcc atactatgcc 180 180

<210> <210> 101 101 <211> <211> 357 357 <212> <212> DNA DNA <213> <213> Artificialsequence Artificial sequence

<400> <400> 101 101 caggtgacac tgaaagaaag caggtgacac tgaaagaaag cggccccgtg cggccccgtg ctggtgaagc ctggtgaage caaccgagac caaccgagac actgactctg actgactctg 60 60

acctgcacagtgagtgggat acctgcacag tgagtgggat cgacctgagc cgacctgage cacgacgaca cacgacgaca tctcctgggt tctcctgggt gcggcaggca gcggcaggca 120 120

cccggaaagg gcctggaatg cccggaaagg gcctggaatg ggtcggcatc ggtcggcatc attagctacg attagctacg ccgggagcat ccgggagcat gtactatgcc gtactatgcc 180 180 agctgggctaaaggaagact agctgggcta aaggaagact gactatctcc gactatctcc aaggacacct aaggacacct ctaaaaacca ctaaaaacca ggtggtcctg ggtggtcctg 240 240

gcttactctcacggagggga gcttactctc acggagggga taagtgggga taagtgggga cagggaaccc cagggaaccc tggtgacagt tggtgacagt ctctagt ctctagt 357 357

87

<210> <210> 102 102 <211> <211> 357 357 <212> <212> DNA DNA <213> <213> Artificial sequence Artificial sequence

<400> <400> 102 102 caggtgacac tgaaagaaag caggtgacac tgaaagaaag cggccccgtg cggccccgtg ctggtgaagc ctggtgaage caaccgagac caaccgagac actgactctg actgactctg 60 60

acctgcacagtgagtgggat acctgcacag tgagtgggat cgacctgagc cgacctgage cacaccgaca cacaccgaca tctcctgggt tctcctgggt gcggcaggca 120 gcggcaggca 120 cccggaaagg gcctggaatg cccggaaagg gcctggaatg ggtcggcatc ggtcggcatc attagctacg attagctacg ccgggaagac ccgggaagac atactatgcc atactatgcc 180 180

agctgggctaaaaaaagact agctgggcta aaaaaagact gactatctcc gactatctcc aaggacacct aaggacacct ctaaaaacca ctaaaaacca ggtggtcctg ggtggtcctg 240 240

actatgaccaatatggacco actatgacca atatggaccc cgtggataca cgtggataca gcaacttact gcaacttact attgtgccag attgtgccag gggcgtccct gggcgtccct 300 300

<210> <210> 103 103 <211> <211> 357 357 <212> <212> DNA DNA <213> <213> Artificial sequence Artificial sequence

<400> <400> 103 103 caggtgacac tgaaagaaag caggtgacac tgaaagaaag cggccccgtg cggccccgtg ctggtgaagc ctggtgaage caaccgagac caaccgagac actgactctg actgactctg 60 60

cccggaaagg gcctggaatg cccggaaagg gcctggaatg ggtcggcatc ggtcggcatc attagctacg attagctacg ccgggaagaa ccgggaagaa atactatgcc atactatgcc 180 180

88

<210> <210> 104 104 <211> <211> 357 357 <212> <212> DNA DNA <213> <213> Artificialsequence Artificial sequence

<400> <400> 104 104 caggtgacac tgaaagaaag caggtgacac tgaaagaaag cggccccgtg cggccccgtg ctggtgaagc ctggtgaage caaccgagac caaccgagac actgactctg actgactctg 60 60

acctgcacagtgagtgggat acctgcacag tgagtgggat cgacctgagc cgacctgage cacaccgaca cacaccgaca tctcctgggt tctcctgggt gcggcaggca 120 gcggcaggca 120 cccggaaagg gcctggaatg cccggaaagg gcctggaatg ggtcggcatc ggtcggcatc attagctacg attagctacg ccgggagcaa ccgggagcaa atacaaggcc atacaaggcc 180 180

<210> <210> 105 105 <211> <211> 357 357 <212> <212> DNA DNA <213> <213> Artificialsequence Artificial sequence

<400> <400> 105 105 caggtgacac tgaaagaaag caggtgacac tgaaagaaag cggccccgtg cggccccgtg ctggtgaagc ctggtgaage caaccgagac caaccgagac actgactctg actgactctg 60 60

cccggaaagg gcctggaatg cccggaaagg gcctggaatg ggtcggcatc ggtcggcatc attagctacg attagctacg ccgggaagaa ccgggaagaa atactatgcc atactatgcc 180 180 agctgggctaaaggaagact agctgggcta aaggaagact gactatctcc gactatctcc aaggacacct aaggacacct ctaaaaacca ctaaaaacca ggtggtcctg ggtggtcctg 240 240

89

<210> <210> 106 106 <211> <211> 357 357 <212> <212> DNA DNA <213> <213> Artificialsequence Artificial sequence

<400> <400> 106 106 caggtgcagc tggtcgaatc caggtgcage tggtcgaatc tggaggagga tggaggagga ctggtgcagc ctggtgcagc caggagggtc caggagggtc actgcgactg actgcgactg 60 60

agctgtgccgtgtccgggat agctgtgccg tgtccgggat cgacctgtca cgacctgtca cacgacgata cacgacgata ttagctgggt ttagctgggt gagacaggca 120 gagacaggca 120 cccgggaagg gactggaatg cccgggaagg gactggaatg ggtctctatc ggtctctatc attagttacg attagttacg ccggcagcac ccggcagcac ctactatgcc ctactatgcc 180 180

tcttgggcta aaggcaggct tcttgggcta aaggcaggct gaccatttct gaccatttct aaggacacaa aaggacacaa gtaaaaacca gtaaaaacca ggtggtcctg ggtggtcctg 240 240

accatgacaaatatggacco accatgacaa atatggaccc cgtggatact cgtggatact gcaacctact gcaacctact attgcgcccg attgcgcccg gggcgtccct gggcgtccct 300 300

gcttatagccacggcggcga gcttatagcc acggcggcga tctgtgggga tctgtgggga caggggacac caggggacac tggtgactgt tggtgactgt cagctcc cagctcc 357 357

<210> <210> 107 107 <211> <211> 357 357 <212> <212> DNA DNA <213> <213> Artificialsequence Artificial sequence

<400> <400> 107 107 caggtgacac tgaaagaaag caggtgacac tgaaagaaag cggccccgtg cggccccgtg ctggtgaagc ctggtgaage caaccgagac caaccgagac actgactctg actgactctg 60 60

acctgcaaagtgagtgggat acctgcaaag tgagtgggat cgacctgagc cgacctgage cacgacgaca cacgacgaca tctcctgggt tctcctgggt gcggcaggca gcggcaggca 120 120

cccggaaagg gcctggaatg cccggaaagg gcctggaatg ggtctccatc ggtctccatc attagctacg attagctacg ccgggagcac ccgggagcad atactatgcc atactatgcc 180 180 agctgggctaaaggaagact agctgggcta aaggaagact gactatctcc gactatctcc aaggacacct aaggacacct ctaaaaacca ctaaaaacca ggtggtcctg ggtggtcctg 240 240

90

<210> <210> 108 108 <211> <211> 357 357 <212> <212> DNA DNA <213> <213> Artificialsequence Artificial sequence

<400> <400> 108 108 caggtgacac tgaaagaaag caggtgacac tgaaagaaag cggccccgtg cggccccgtg ctggtgaagc ctggtgaage caaccgagac caaccgagac actgactctg actgactctg 60 60

acctgcaaagtgagtgggat acctgcaaag tgagtgggat cgacctgagc cgacctgage cacgaggaca cacgaggaca tctcctgggt tctcctgggt gcggcaggca 120 gcggcaggca 120 cccggaaagg gcctggaatg cccggaaagg gcctggaatg ggtctccatc ggtctccatc attagctacg attagctacg ccgggagcac ccgggagcad atactatgcc atactatgcc 180 180

<210> <210> 109 109 <211> <211> 357 357 <212> <212> DNA DNA <213> <213> Artificialsequence Artificial sequence

<400> <400> 109 109 caggtgcagc tggtcgaatc caggtgcage tggtcgaatc tggaggagga tggaggagga ctggtgcagc ctggtgcagc caggagggtc caggagggtc actgcgactg actgcgactg 60 60

agctgtgccgtgtccgggat agctgtgccg tgtccgggat cgacctgtca cgacctgtca cacgaggata cacgaggata ttagctgggt ttagctgggt gagacaggca gagacaggca 120 120

cccgggaagg gactggaatg cccgggaagg gactggaatg ggtctctatc ggtctctatc attagttacg attagttacg ccggcagcaa ccggcagcaa gtactatgcc gtactatgcc 180 180

accatgacaaatatggaccc accatgacaa atatggaccc cgtggatact cgtggatact gcaacctact gcaacctact attgcgcccg attgcgcccg gggcgtccct gggcgtccct 300 300

91

<210> <210> 110 110 <211> <211> 330 330 <212> <212> DNA DNA <213> <213> Artificial sequence Artificial sequence

<400> <400> 110 110 gacattgtga tgactcagtc gacattgtga tgactcagtc tcctgccact tcctgccact ctgtcactga ctgtcactga gccctggcga gccctggcga gcgagcaacc gcgagcaacc 60 60

ctgtcttgca ccagcagcca ctgtcttgca ccagcagcca gtccgtgtac gtccgtgtac cacgccaatt cacgccaatt ggctgagctg ggctgagctg gttccagcag 120 gttccagcag 120 aagccagggcagccccctaa aagccagggc agccccctaa actgctgatc actgctgatc tattgggcat tattgggcat caacactggc caacactggc ctacggcgtc ctacggcgtc 180 180

ccctccagat tctccggatc ccctccagat tctccggatc tggcagtggg tggcagtggg actgacttta actgacttta ccctgaccat ccctgaccat cagcagcctc cagcagectc 240 240

cagcccgaggatgccgctac cagcccgagg atgccgctac ctactattgt ctactattgt gctggcgggt gctggcgggt acggaggcgg acggaggcgg gcggtatgca gcggtatgca 300 300

tttggacagg gcacaaaggt tttggacagg gcacaaaggt ggaaatcaaa ggaaatcaaa 330 330

<210> <210> 111 111 <211> <211> 330 330 <212> <212> DNA DNA <213> <213> Artificial sequence Artificial sequence

<400> <400> 111 111 gacattgtga tgactcagtc gacattgtga tgactcagtc tcctgccact tcctgccact ctgtcactga ctgtcactga gccctggcga gccctggcga gcgagcaacc gcgagcaacc 60 60

ctgtcttgca ccaccagcca ctgtcttgca ccaccagcca gtccgtgtac gtccgtgtac cacgagaatt cacgagaatt ggctgagctg ggctgagctg gttccagcag gttccagcag 120 120

tttggacagg gcacaaaggt tttggacagg gcacaaaggt ggaaatcaaa ggaaatcaaa 330 330

92

<210> <210> 112 112 <211> <211> 330 330 <212> <212> DNA DNA <213> <213> Artificial sequence Artificial sequence

<400> <400> 112 112 gacattgtga tgactcagtc gacattgtga tgactcagtc tcctgccact tcctgccact ctgtcactga ctgtcactga gccctggcga gccctggcga gcgagcaacc gcgagcaacc 60 60

ctgtcttgca ccagcagcca ctgtcttgca ccagcagcca gtccgtgttc gtccgtgttc cacgagaatt cacgagaatt ggctgagctg ggctgagctg gttccagcag 120 gttccagcag 120 aagccagggcagccccctaa aagccagggc agccccctaa actgctgatc actgctgatc tattgggcat tattgggcat caacactggc caacactggc ctggggcgtc ctggggcgtc 180 180

tttggacagg gcacaaaggt tttggacagg gcacaaaggt ggaaatcaaa ggaaatcaaa 330 330

<210> <210> 113 113 <211> <211> 330 330 <212> <212> DNA DNA <213> <213> Artificial sequence Artificial sequence

<400> <400> 113 113 gacattgtga tgactcagtc gacattgtga tgactcagtc tcctgccact tcctgccact ctgtcactga ctgtcactga gccctggcga gccctggcga gcgagcaacc gcgagcaacc 60 60

ctgtcttgca ccaccagcca ctgtcttgca ccaccagcca gtccgtgtac gtccgtgtac cacgagaatt cacgagaatt ggctgagctg ggctgagctg gtaccagcag gtaccagcag 120 120

cagcccgaggatgtcgctac cagcccgagg atgtcgctac ctactattgt ctactattgt gctggcgggt gctggcgggt acggaggcgg acggaggcgg gcggtatgca gcggtatgca 300 300

tttggacagg gcacaaaggt tttggacagg gcacaaaggt ggaaatcaaa ggaaatcaaa 330 330

93

<210> <210> 114 114 <211> <211> 5 5 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 114 114

His Asp His Asp Asp AspIle IleSer Ser 1 1 5 5

<210> <210> 115 115 <211> <211> 5 5 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 115 115

His Glu His Glu Asp Asp Ile Ile Ser Ser 1 1 5 5

<210> <210> 116 116 <211> <211> 16 16 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 116 116

Ile Ile Ser Ile Ile SerTyr TyrAla AlaGly Gly SerSer MetMet Tyr Tyr Tyr Tyr Ala Ala Ser Ala Ser Trp TrpLys Ala Lys Gly Gly 1 1 5 5 10 10 15 15

<210> <210> 117 117 <211> <211> 16 16 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 117 117

Ile Ile Ser Ile Ile SerTyr TyrAla AlaGly Gly LysLys ThrThr Tyr Tyr Tyr Tyr Ala Ala Ser Ala Ser Trp TrpLys Ala Lys

94

Lys Lys 1 1 5 5 10 10 15 15

<210> <210> 118 118 <211> <211> 16 16 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 118 118

Ile Ile Ser Ile Ile SerTyr TyrAla AlaGly Gly LysLys LysLys Tyr Tyr Tyr Tyr Ala Ala Ser Ala Ser Trp TrpLys Ala Lys Gly Gly 1 1 5 5 10 10 15 15

<210> <210> 119 119 <211> <211> 16 16 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 119 119

Ile Ile Ser Ile Ile SerTyr TyrAla AlaGly Gly SerSer LysLys Tyr Tyr Lys Lys Ala Ala Ser Ala Ser Trp TrpLys Ala Lys Gly Gly 1 1 5 5 10 10 15 15

<210> <210> 120 120 <211> <211> 16 16 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 120 120

Ile Ile Ser Ile Ile SerTyr TyrAla AlaGly Gly SerSer LysLys Tyr Tyr Tyr Tyr Ala Ala Ser Ala Ser Trp TrpLys Ala Lys Gly Gly 1 1 5 5 10 10 15 15

<210> <210> 121 121 <211> <211> 11 11 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

95

<400> < 400> 121 121

Gly Val Gly Val Pro ProAla AlaTyr Tyr Ser Ser HisHis GlyGly Gly Gly Asp Asp Lys Lys 1 1 5 5 10 10

<210> <210> 122 122 <211> <211> 13 13 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 122 122

Thr Ser Thr Ser Ser SerGln GlnSer SerVal Val TyrTyr HisHis Ala Ala Asn Asn Trp Trp Leu Ser Leu Ser 1 1 5 5 10 10

<210> <210> 123 123 <211> <211> 13 13 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 123 123

Thr Thr Thr Thr Ser SerGln GlnSer Ser Val Val TyrTyr HisHis Glu Glu Asn Asn Trp Ser Trp Leu Leu Ser 1 1 5 5 10 10

<210> <210> 124 124 <211> <211> 13 13 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 124 124

Thr Ser Ser Thr Ser SerGln GlnSer SerVal Val PhePhe HisHis Glu Glu Asn Asn Trp Trp Leu Ser Leu Ser 1 1 5 5 10 10

<210> <210> 125 125 <211> <211> 7 7 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

96

<400> <400> 125 125

Trp Ala Trp Ala Ser Ser Thr Thr Leu Leu Ala Ala Trp Trp 1 1 5 5

<210> <210> 126 126 <211> <211> 5 5 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<220> <220> <221> <221> misc_feature misc_feature <222> <222> (1)..(1) (1) . (1) <223> <223> Xaa is Xaa is Ser SerororHis His

<220> <220> <221> <221> misc_feature misc_feature <222> <222> (2)..(2) (2) (2) <223> <223> Xaa is Tyr, Xaa is Tyr,Thr, Thr,Asp Asp or or GluGlu

<400> <400> 126 126

Xaa Xaa Xaa Xaa Asp AspIle IleSer Ser 1 1 5 5

<210> <210> 127 127 <211> <211> 16 16 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<220> <220> <221> <221> misc_feature misc_feature <222> <222> (4)..(4) (4) .(4) <223> <223> Xaa is Tyr Xaa is TyrororHis His

<220> <220> <221> <221> misc_feature misc_feature <222> <222> (7) -(7) (7)..(7) <223> <223> Xaa is Ser Xaa is SerororLys Lys

<220> <220> <221> <221> misc_feature misc_feature <222> <222> (8) -(8) (8)..(8) <223> <223> Xaa is Xaa is Thr, Thr,Met MetororLys Lys

97

<220> <220> <221> <221> misc_feature misc_feature <222> <222> (10)- (10) (10)..(10) <223> <223> Xaa is Tyr Xaa is TyrororLys Lys

<220> <220> <221> <221> misc_feature misc_feature <222> <222> (11)- (11) (11)..(11) <223> <223> Xaa is Xaa is Ala, Ala,Met MetororGlu Glu

<220> <220> <221> <221> misc_feature misc_feature <222> <222> (12)..(12) (12) )..(12) <223> <223> Xaa is Xaa is Ser SerororGlu Glu

<220> <220> <221> <221> misc_feature misc_feature <222> <222> - (16)..(16) (16) (16) <223> <223> Xaa is Xaa is Gly GlyororLys Lys

<400> <400> 127 127

Ile Ile Ser Ile Ile SerXaa XaaAla AlaGly Gly XaaXaa XaaXaa Tyr Tyr Xaa Xaa Xaa Xaa Xaa Ala Xaa Trp TrpLys Ala Lys Xaa Xaa 1 1 5 5 10 10 15 15

<210> <210> 128 128 <211> <211> 11 11 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<220> <220> <221> <221> misc_feature misc_feature <222> <222> - (5)..(5) (5) )..(5) <223> <223> Xaa is Tyr Xaa is Tyror orHis His

<220> <220> <221> <221> misc_feature misc_feature <222> <222> (7)..(7) (7) (7) <223> <223> Xaa is Thr Xaa is Thror orHis His

<220> <220> <221> <221> misc_feature misc_feature <222> <222> (11)..(11) (11)..(11) <223> <223> Xaa is Xaa is Leu LeuororLys Lys

<400> <400> 128 128

Gly Val Gly Val Pro ProAla AlaXaa Xaa Ser Ser XaaXaa GlyGly Gly Gly Asp Asp Xaa Xaa 1 1 5 5 10 10

98

<210> <210> 129 129 <211> <211> 13 13 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<220> <220> <221> <221> misc_feature misc feature <222> <222> (1)..(1) (1) . (1) <223> <223> Xaa is Gln Xaa is Glnor orThr Thr

<220> <220> <221> <221> misc_feature misc feature <222> <222> - (2)..(2) (2) (2) <223> <223> Xaa is Ser Xaa is SerororThr Thr

<220> <220> <221> <221> misc_feature misc feature <222> <222> - (5)..(5) (5) (5) <223> <223> Xaa is Xaa is Ser SerororGlu Glu

<220> <220> <221> <221> misc_feature misc feature <222> <222> (7)..(7) (7) (7) <223> <223> Xaa is Tyr Xaa is Tyror orPhe Phe

<220> <220> <221> <221> misc_feature miso feature <222> <222> (8) -(8) (8)..(8) <223> <223> Xaa is Asp Xaa is AspororHis His

<220> <220> <221> <221> misc_feature misc _feature <222> <222> (9)..(9) (9) -(9) <223> <223> Xaa is Asn, Xaa is Asn,Asp, Asp,Ala Ala or or GluGlu

<400> <400> 129 129

Xaa Xaa Xaa Xaa Ser SerGln GlnXaa Xaa Val Val XaaXaa XaaXaa Xaa Xaa Asn Asn Trp Ser Trp Leu Leu Ser 1 1 5 5 10 10

<210> <210> 130 130 <211> <211> 7 7 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<220> <220>

99

<221> <221> misc_feature misc feature <222> <222> (3) -(3) (3)..(3) <223> <223> Xaa is Ser Xaa is Seror orGlu Glu

<220> <220> <221> <221> misc_feature misc_feature <222> <222> (7)..(7) (7) -(7) <223> <223> Xaa is Ser, Xaa is Ser,Tyr, Tyr,Phe Phe or or TrpTrp

<400> <400> 130 130

Trp Ala Trp Ala Xaa XaaThr ThrLeu Leu Ala Ala XaaXaa 1 1 5 5

<210> <210> 131 131 <211> <211> 10 10 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<220> <220> <221> <221> misc_feature misc_feature <222> <222> (8)..(8) (8) (8) <223> <223> Xaa is Xaa is Leu LeuororArg Arg

<400> <400> 131 131

Ala Gly Ala Gly Gly Gly Tyr Tyr Gly Gly Gly Gly Gly Gly Xaa Xaa Tyr Tyr Ala Ala 1 1 5 5 10 10

<210> <210> 132 132 <211> <211> 30 30 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 132 132

Thr Leu Thr Leu Thr ThrLeu LeuThr Thr Cys Cys ThrThr ValVal Ser Ser Gly Gly Ile Leu Ile Asp Asp Ser Leu Ser 20 20 25 25 30 30

<210> <210> 133 133 <211> <211> 30 30

100

<212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 133 133

Ser Leu Ser Leu Arg ArgLeu LeuSer SerCys Cys AlaAla ValVal Ser Ser Gly Gly Ile Ile Asp Ser Asp Leu Leu Ser 20 20 25 25 30 30

<210> <210> 134 134 <211> <211> 30 30 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 134 134

Thr Leu Thr Leu Thr ThrLeu LeuThr Thr Cys Cys LysLys ValVal Ser Ser Gly Gly Ile Leu Ile Asp Asp Ser Leu Ser 20 20 25 25 30 30

<210> <210> 135 135 <211> <211> 14 14 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 135 135

Trp Val Trp Val Arg Arg Gln Gln Ala Ala Pro Pro Gly Gly Lys Lys Gly Gly Leu Leu Glu Glu Trp Trp Val Val Gly Gly 1 1 5 5 10 10

<210> <210> 136 136 <211> <211> 14 14 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<220> <220>

101

<223> <223> An artificially An artificially synthesized synthesized sequence sequence

<400> <400> 136 136

Trp Val Trp Val Arg ArgGln GlnAla Ala Pro Pro GlyGly LysLys Gly Gly Leu Leu Glu Val Glu Trp Trp Ser Val Ser 1 1 5 5 10 10

<210> <210> 137 137 <211> <211> 32 32 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 137 137

Arg Leu Arg Leu Thr Thr Ile Ile Ser Ser Lys Lys Asp Asp Thr Thr Ser Ser Lys Lys Asn Asn Gln Gln Val Val Val Val Leu Leu Thr Thr 1 1 5 5 10 10 15 15

Met Thr Met Thr Asn Asn Met Met Asp Asp Pro Pro Val Val Asp Asp Thr Thr Ala Ala Thr Thr Tyr Tyr Tyr Tyr Cys Cys Ala Ala Arg Arg 20 20 25 25 30 30

<210> <210> 138 138 <211> <211> 11 11 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 138 138

Trp Gly Trp Gly Gln GlnGly GlyThr Thr Leu Leu ValVal ThrThr Val Val Ser Ser Ser Ser 1 1 5 5 10 10

<210> <210> 139 139 <211> <211> 23 23 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 139 139

102

Glu Arg Glu Arg Ala AlaThr ThrLeu Leu Ser Ser CysCys 20 20

<210> <210> 140 140 <211> <211> 15 15 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 140 140

Trp Phe Trp Phe Gln GlnGln GlnLys Lys Pro Pro GlyGly GlnGln Pro Pro Pro Pro Lys Leu Lys Leu Leu Ile LeuTyr Ile Tyr 1 1 5 5 10 10 15 15

<210> <210> 141 141 <211> <211> 15 15 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 141 141

Trp Tyr Trp Tyr Gln GlnGln GlnLys Lys Pro Pro GlyGly GlnGln Pro Pro Pro Pro Lys Leu Lys Leu Leu Ile LeuTyr Ile Tyr 1 1 5 5 10 10 15 15

<210> <210> 142 142 <211> <211> 32 32 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 142 142

Gly Val Gly Val Pro ProSer SerArg Arg Phe Phe SerSer GlyGly Ser Ser Gly Gly Ser Thr Ser Gly Gly Asp ThrPhe Asp Phe Thr Thr 1 1 5 5 10 10 15 15

Leu Thr Leu Thr Ile IleSer SerSer Ser Leu Leu GlnGln ProPro Glu Glu Asp Asp Ala Thr Ala Ala Ala Tyr ThrTyr Tyr Tyr Cys Cys 20 20 25 25 30 30

<210> <210> 143 143 <211> <211> 32 32 <212> <212> PRT PRT

103

<213> Artificialsequence <213> Artificial sequence

<400> <400> 143 143

Leu Thr Leu Thr Ile IleSer SerSer Ser Leu Leu GlnGln ProPro Glu Glu Asp Asp Val Thr Val Ala Ala Tyr ThrTyr Tyr Tyr Cys Cys 20 20 25 25 30 30

<210> <210> 144 144 <211> <211> 10 10 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 144 144

Phe Gly Gln Phe Gly GlnGly GlyThr ThrLys Lys ValVal GluGlu Ile Ile Lys Lys 1 1 5 5 10 10

<210> <210> 145 145 <211> <211> 121 121 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 145 145

Gln Ser Gln Ser Val ValGlu GluGlu Glu Ser Ser GlyGly GlyGly Arg Arg Leu Leu Val Pro Val Thr Thr Gly ProThr Gly Thr Pro Pro 1 1 5 5 10 10 15 15

Leu Thr Leu Thr Leu LeuThr ThrCys Cys Thr Thr ValVal SerSer Gly Gly Phe Phe Ser Ser Ser Leu Leu Arg SerTyr Arg Tyr Tyr Tyr 20 20 25 25 30 30

Val Ser Val Ser Trp Trp Val Val Arg Arg Gln Gln Ala Ala Pro Pro Gly Gly Lys Lys Gly Gly Leu Leu Glu Glu Trp Trp Ile Ile Gly Gly 35 35 40 40 45 45

104

Ile Ile Tyr Ile Ile TyrThr ThrGly GlyGly Gly AsnAsn ThrThr Asn Asn Tyr Tyr Ala Ala Ser Ala Ser Trp TrpLys Ala Lys Gly Gly 50 50 55 55 60 60

Arg Phe Arg Phe Thr ThrIle IleSer Ser Lys Lys AlaAla SerSer Thr Thr Thr Thr Val Leu Val Asp Asp Lys LeuMet Lys Met Thr Thr

70 70 75 75 80 80

Gly Pro Gly Pro Thr ThrThr ThrGlu Glu Asp Asp ThrThr AlaAla Thr Thr Tyr Tyr Phe Ala Phe Cys Cys Gly AlaPhe Gly Phe Leu Leu 85 85 90 90 95 95

Asp Tyr Asp Tyr Ser Ser His His Tyr Tyr Thr Thr Thr Thr Gly Gly Gly Gly Tyr Tyr Tyr Tyr Leu Leu Asp Asp Leu Leu Trp Trp Gly Gly 100 100 105 105 110 110

Pro Gly Pro Gly Thr ThrLeu LeuVal ValThr Thr ValVal SerSer Ser Ser 115 115 120 120

<210> <210> 146 146 <211> <211> 119 119 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 146 146

Gln Ser Gln Ser Leu LeuGlu GluGlu Glu Ser Ser GlyGly GlyGly Gly Gly Leu Leu Val Pro Val Lys Lys Gly ProGly Gly Gly Thr Thr 1 1 5 5 10 10 15 15

Leu Thr Leu Thr Leu LeuThr ThrCys Cys Lys Lys AlaAla SerSer Gly Gly Ile Ile Asp Ser Asp Phe Phe Ser SerTyr Ser Tyr Tyr Tyr 20 20 25 25 30 30

Tyr Met Tyr Met Cys CysTrp TrpVal Val Arg Arg GlnGln AlaAla Pro Pro Gly Gly Lys Leu Lys Gly Gly Glu LeuTrp Glu Trp Ile Ile 35 35 40 40 45 45

Ala Cys Ala Cys Ile IleTyr TyrAla Ala Gly Gly IleIle SerSer Gly Gly Ser Ser Thr Tyr Thr Tyr Tyr Ala TyrSer Ala Ser Trp Trp 50 50 55 55 60 60

Ala Lys Ala Lys Gly GlyArg ArgPhe Phe Thr Thr IleIle SerSer Lys Lys Thr Thr Ser Thr Ser Ser Ser Thr ThrVal Thr Val Thr Thr

105

70 75 75 80 80

Leu Gln Leu Gln Met MetThr ThrSer Ser Leu Leu ThrThr AlaAla Ala Ala Asp Asp Thr Thr Thr Ala Ala Tyr ThrPhe Tyr Phe Cys Cys 85 85 90 90 95 95

Ala Arg Ala Arg Asp AspPro ProTyr Tyr His His PhePhe AspAsp Tyr Tyr Gly Gly Ser Trp Ser Leu Leu Gly TrpPro Gly Pro Gly Gly 100 100 105 105 110 110

Thr Leu Thr Leu Val ValThr ThrVal Val Ser Ser SerSer 115 115

<210> <210> 147 147 <211> <211> 116 116 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 147 147

Gln Ser Gln Ser Leu LeuGlu GluGlu Glu Ser Ser GlyGly GlyGly Arg Arg Leu Leu Val Pro Val Thr Thr Gly ProThr Gly Thr Pro Pro 1 1 5 5 10 10 15 15

Leu Thr Leu Thr Leu LeuThr ThrCys Cys Thr Thr ValVal SerSer Gly Gly Ile Ile Asp Ser Asp Leu Leu Arg SerTyr Arg Tyr Trp Trp 20 20 25 25 30 30

Met Ser Met Ser Trp TrpVal ValArg Arg Gln Gln AlaAla ProPro Gly Gly Lys Lys Gly Glu Gly Leu Leu Trp GluIle Trp Ile Gly Gly 35 35 40 40 45 45

Ile Ile Ser Ile Ile SerSer SerGly GlyGly Gly ThrThr ThrThr Tyr Tyr Tyr Tyr Ala Ala Ser Ala Ser Trp TrpLys Ala Lys Gly Gly 50 50 55 55 60 60

Arg Phe Arg Phe Thr ThrIle IleSer Ser Lys Lys ThrThr SerSer Thr Thr Thr Thr Val Leu Val Asp Asp Lys LeuIle Lys Ile Thr Thr

70 70 75 75 80 80

Ser Pro Thr Ser Pro ThrThr ThrGlu GluAsp Asp ThrThr AlaAla Thr Thr Tyr Tyr Phe Phe Cys Arg Cys Ala AlaAsp Arg Asp Ser Ser 85 85 90 90 95 95

106

Arg Tyr Arg Tyr Gly Gly Ala Ala Ala Ala Arg Arg Leu Leu Asp Asp Leu Leu Trp Trp Gly Gly Gln Gln Gly Gly Thr Thr Leu Leu Val Val 100 100 105 105 110 110

Thr Val Thr Val Ser SerSer Ser 115 115

<210> <210> 148 148 <211> <211> 109 109 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 148 148

Leu Thr Leu Thr Leu LeuThr ThrCys Cys Thr Thr ValVal SerSer Gly Gly Phe Phe Ser Ser Ser Leu Leu Ser SerTyr Ser Tyr Ile Ile 20 20 25 25 30 30

Met Gly Met Gly Trp TrpVal ValArg Arg Gln Gln AlaAla ProPro Gly Gly Lys Lys Gly Glu Gly Leu Leu Trp GluIle Trp Ile Gly Gly 35 35 40 40 45 45

Phe Ile Phe Ile Tyr TyrVal ValSer SerGly Gly SerSer ThrThr Tyr Tyr Tyr Tyr Ala Trp Ala Ser Ser Ala TrpLys Ala Lys Gly Gly 50 50 55 55 60 60

Arg Phe Arg Phe Thr ThrIle IleSer Ser Arg Arg ThrThr SerSer Thr Thr Thr Thr Val Leu Val Asp Asp Arg LeuIle Arg Ile Thr Thr

70 70 75 75 80 80

Ser Pro Ser Pro Thr ThrThr ThrGlu GluAsp Asp ThrThr AlaAla Thr Thr Tyr Tyr Phe Val Phe Cys Cys Arg ValThr Arg Thr Trp Trp 85 85 90 90 95 95

Asp Leu Asp Leu Trp Trp Gly Gly Pro Pro Gly Gly Thr Thr Leu Leu Val Val Thr Thr Val Val Ser Ser Ser Ser 100 100 105 105

<210> <210> 149 149 <211> <211> 115 115

107

<212> PRT <212> PRT <213> Artificial sequence <213> Artificial sequence

<400> <400> 149 149

Leu Thr Leu Thr Leu LeuThr ThrCys Cys Thr Thr ValVal SerSer Gly Gly Phe Phe Ser Ser Ser Leu Leu Ser SerTyr Ser Tyr Ala Ala 20 20 25 25 30 30

Ile Ile Tyr Ile Ile TyrGly GlyVal ValGly Gly SerSer ThrThr Tyr Tyr Tyr Tyr Ala Ala Ser Ala Ser Trp TrpLys Ala Lys Gly Gly 50 50 55 55 60 60

Arg Phe Arg Phe Thr ThrIle IleSer Ser Lys Lys ThrThr SerSer Thr Thr Thr Thr Val Leu Val Asp Asp Lys LeuIle Lys Ile Ala Ala

70 70 75 75 80 80

Ser Pro Thr Ser Pro ThrThr ThrGlu GluAsp Asp ThrThr AlaAla Thr Thr Tyr Tyr Phe Phe Cys Arg Cys Ala AlaAsp Arg Asp Asp Asp 85 85 90 90 95 95

Tyr Gly Tyr Gly Trp TrpGly GlyTyr Tyr Thr Thr AsnAsn LeuLeu Trp Trp Gly Gly Pro Thr Pro Gly Gly Leu ThrVal Leu Val Thr Thr 100 100 105 105 110 110

Val Ser Val Ser Ser Ser 115 115

<210> <210> 150 150 <211> <211> 117 117 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 150 150

108

Leu Thr Leu Thr Leu LeuThr ThrCys Cys Thr Thr ValVal SerSer Gly Gly Ile Ile Asp Ser Asp Leu Leu Ser SerSer Ser Ser Ser Ser 20 20 25 25 30 30

Met Ile Met Ile Trp TrpVal ValArg Arg Gln Gln AlaAla ProPro Gly Gly Glu Glu Gly Lys Gly Leu Leu Tyr LysIle Tyr Ile Gly Gly 35 35 40 40 45 45

Phe Ile Phe Ile Ser SerLys LysSer SerGly Gly TyrTyr ThrThr Tyr Tyr Tyr Tyr Ala Ala Ser Ala Ser Trp TrpLys Ala Lys Gly Gly 50 50 55 55 60 60

70 70 75 75 80 80

Ser Pro Thr Ser Pro ThrThr ThrGlu GluAsp Asp ThrThr AlaAla Thr Thr Tyr Tyr Phe Phe Cys Arg Cys Ala AlaGly Arg Gly Gly Gly 85 85 90 90 95 95

Tyr Ser Tyr Ser Gly GlyAsp AspTrp Trp Asp Asp TyrTyr PhePhe Thr Thr Leu Leu Trp Pro Trp Gly Gly Gly ProThr Gly Thr Leu Leu 100 100 105 105 110 110

Val Thr Val Thr Val Val Ser Ser Ser Ser 115 115

<210> <210> 151 151 <211> <211> 112 112 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 151 151

Gln Val Gln Val Leu Leu Thr Thr Gln Gln Thr Thr Pro Pro Ser Ser Pro Pro Val Val Ser Ser Ala Ala Ala Ala Val Val Gly Gly Gly Gly 1 1 5 5 10 10 15 15

Thr Val Thr Val Thr Thr Ile Ile Asn Asn Cys Cys Gln Gln Ala Ala Ser Ser Gln Gln Ser Ser Val Val Tyr Tyr Asn Asn Asn Asn

109

Lys Lys 20 20 25 25 30 30

Asn Leu Asn Leu Ala Ala Trp Trp Tyr Tyr Gln Gln Gln Gln Lys Lys Pro Pro Gly Gly Gln Gln Pro Pro Pro Pro Lys Lys Leu Leu Leu Leu 35 35 40 40 45 45

Ile Tyr Tyr Ile Tyr TyrAla AlaSer SerThr Thr LeuLeu ProPro Ser Ser Gly Gly Val Val Ser Arg Ser Ser SerPhe Arg Phe Lys Lys 50 50 55 55 60 60

Gly Ser Gly Ser Gly GlySer SerGly Gly Thr Thr GlnGln PhePhe Thr Thr Leu Leu Thr Ser Thr Ile Ile Gly SerVal Gly Val Gln Gln

70 70 75 75 80 80

Cys Asp Cys Asp Asp AspAla AlaAla Ala Thr Thr TyrTyr TyrTyr Cys Cys Gln Gln Gly Phe Gly Glu Glu Ser PheCys Ser Cys Gly Gly 85 85 90 90 95 95

Ser Ala Asp Ser Ala AspCys CysThr ThrAla Ala PhePhe GlyGly Gly Gly Gly Gly Thr Thr Lys Glu Lys Val ValIle Glu Ile Lys Lys 100 100 105 105 110 110

<210> <210> 152 152 <211> <211> 112 112 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 152 152

Ala Leu Ala Leu Val ValMet MetThr Thr Gln Gln ThrThr ProPro Ala Ala Ser Ser Val Ala Val Ser Ser Ala AlaVal Ala Val Gly Gly 1 1 5 5 10 10 15 15

Gly Thr Gly Thr Val ValThr ThrIle Ile Asn Asn CysCys GlnGln Ala Ala Ser Ser Gln Val Gln Ser Ser Tyr ValGly Tyr Gly Gly Gly 20 20 25 25 30 30

Asn Glu Asn Glu Leu LeuSer SerTrp Trp Tyr Tyr GlnGln GlnGln Glu Glu Pro Pro Gly Pro Gly Gln Gln Pro ProLys Pro Lys Leu Leu 35 35 40 40 45 45

Leu Ile Leu Ile Tyr TyrSer SerAla Ala Ser Ser LysLys LeuLeu Ala Ala Ser Ser Gly Pro Gly Val Val Ser ProArg Ser Arg Phe Phe

110

50 55 55 60 60

Lys Gly Lys Gly Ser Ser Gly Gly Ser Ser Gly Gly Thr Thr Gln Gln Phe Phe Thr Thr Leu Leu Thr Thr Ile Ile Ser Ser Gly Gly Val Val

70 70 75 75 80 80

Gln Cys Gln Cys Asp AspAsp AspAla Ala Ala Ala AlaAla TyrTyr Tyr Tyr Cys Cys Ala Tyr Ala Gly Gly Arg TyrThr Arg Thr Tyr Tyr 85 85 90 90 95 95

Ser Asp Ser Asp Asp AspAsp AspAsn AsnAla Ala PhePhe GlyGly Gly Gly Gly Gly Thr Val Thr Lys Lys Glu ValIle Glu Ile Lys Lys 100 100 105 105 110 110

<210> <210> 153 153 <211> <211> 112 112 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 153 153

Gln Val Gln Val Leu Leu Thr Thr Gln Gln Thr Thr Pro Pro Ala Ala Ser Ser Val Val Ser Ser Ala Ala Ala Ala Val Val Gly Gly Gly Gly 1 1 5 5 10 10 15 15

Thr Val Thr Val Thr Thr Ile Ile Asn Asn Cys Cys Gln Gln Ser Ser Ser Ser Gln Gln Ser Ser Val Val Ala Ala Gly Gly Asn Asn Asp Asp 20 20 25 25 30 30

Ile Tyr Tyr Ile Tyr TyrAla AlaSer SerAsn Asn LeuLeu AlaAla Ser Ser Gly Gly Val Val Ser Arg Ser Ser SerPhe Arg Phe Lys Lys 50 50 55 55 60 60

Gly Ser Gly Ser Gly GlySer SerGly Gly Thr Thr ArgArg PhePhe Thr Thr Leu Leu Thr Ser Thr Ile Ile Asp SerVal Asp Val Gln Gln

70 70 75 75 80 80

Cys Asp Cys Asp Asp AspAla AlaAla Ala Thr Thr TyrTyr TyrTyr Cys Cys Leu Leu Gly Tyr Gly Asn Asn Asp TyrCys Asp Cys Ser Ser 85 85 90 90 95 95

111

Ser Ala Asp Ser Ala AspCys CysAsp AspAla Ala PhePhe GlyGly Gly Gly Gly Gly Thr Thr Lys Glu Lys Val ValIle Glu Ile Lys Lys 100 100 105 105 110 110

<210> <210> 154 154 <211> <211> 109 109 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 154 154

Gln Val Gln Val Leu LeuThr ThrGln Gln Thr Thr ProPro SerSer Ser Ser Thr Thr Ser Ala Ser Ala Ala Val AlaGly Val Gly Gly Gly 1 1 5 5 10 10 15 15

Thr Val Thr Val Thr ThrIle IleAsn Asn Cys Cys GlnGln SerSer Ser Ser Gln Gln Ser Tyr Ser Val Val Asp TyrAsn Asp Asn Asn Asn 20 20 25 25 30 30

Trp Leu Trp Leu Ala AlaTrp TrpTyr Tyr Gln Gln GlnGln LysLys Pro Pro Gly Gly Gln Pro Gln Pro Pro Lys ProArg Lys Arg Leu Leu 35 35 40 40 45 45

Ile Tyr Gly Ile Tyr GlyAla AlaSer SerThr Thr LeuLeu AspAsp Ser Ser Gly Gly Val Val Pro Arg Pro Ser SerPhe Arg Phe Lys Lys 50 50 55 55 60 60

Gly Ser Gly Ser Gly GlySer SerGly Gly Thr Thr GluGlu PhePhe Thr Thr Leu Leu Thr Ser Thr Ile Ile Asp SerLeu Asp Leu Glu Glu

70 70 75 75 80 80

Cys Asp Cys Asp Asp AspAla AlaAla Ala Thr Thr TyrTyr TyrTyr Cys Cys Gln Gln Gly Tyr Gly Gly Gly Ser TyrGly Ser Gly Asn Asn 85 85 90 90 95 95

Ile Tyr Pro Ile Tyr ProPhe PheGly GlyGly Gly GlyGly ThrThr Lys Lys Val Val Glu Glu Ile Lys Ile Lys 100 100 105 105

<210> <210> 155 155 <211> <211> 111 111 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

112

<400> <400> 155 155

Gln Val Gln Val Leu LeuThr ThrGln Gln Thr Thr ProPro SerSer Ser Ser Val Val Ser Ala Ser Ala Ala Val AlaGly Val Gly Gly Gly 1 1 5 5 10 10 15 15

Thr Val Thr Val Thr ThrIle IleAsn AsnCys Cys GlnGln SerSer Ser Ser Gln Gln Ser Tyr Ser Val Val Asn TyrAsn Asn Asn Asn Asn 20 20 25 25 30 30

Tyr Leu Tyr Leu Ala Ala Trp Trp Phe Phe Gln Gln Gln Gln Lys Lys Pro Pro Gly Gly Gln Gln Pro Pro Pro Pro Lys Lys Leu Leu Leu Leu 35 35 40 40 45 45

Ile Tyr Asp Ile Tyr AspAla AlaSer SerLys Lys LeuLeu AlaAla Ser Ser Gly Gly Val Val Pro Arg Pro Ser SerPhe Arg Phe Lys Lys 50 50 55 55 60 60

70 70 75 75 80 80

Cys Asp Cys Asp Asp AspAla AlaAla Ala Thr Thr TyrTyr TyrTyr Cys Cys Gln Gln Gly Tyr Gly Ser Ser Tyr TyrSer Tyr Ser Ser Ser 85 85 90 90 95 95

Gly Trp Gly Trp Tyr TyrArg ArgAla Ala Phe Phe GlyGly GlyGly Gly Gly Thr Thr Lys Glu Lys Val Val Ile GluLys Ile Lys 100 100 105 105 110 110

<210> <210> 156 156 <211> <211> 114 114 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 156 156

Ala Asp Ala Asp Ile IleVal ValMet Met Thr Thr GlnGln ThrThr Pro Pro Ala Ala Ser Glu Ser Val Val Ala GluAla Ala Ala Val Val 1 1 5 5 10 10 15 15

Gly Gly Gly Gly Thr Thr Val Val Thr Thr Ile Ile Lys Lys Cys Cys Gln Gln Ala Ala Ser Ser Glu Glu Ser Ser Ile Ile Gly Gly Asn Asn

113

20 25 25 30 30

Ala Leu Ala Leu Ala Ala Trp Trp Tyr Tyr Gln Gln Gln Gln Lys Lys Pro Pro Gly Gly Gln Gln Pro Pro Pro Pro Lys Lys Leu Leu Leu Leu 35 35 40 40 45 45

Ile Tyr Gly Ile Tyr GlyAla AlaPhe PheAsn Asn LeuLeu GluGlu Ser Ser Gly Gly Val Val Pro Arg Pro Ser SerPhe Arg Phe Arg Arg 50 50 55 55 60 60

Gly Ser Gly Ser Gly Gly Ser Ser Gly Gly Thr Thr Glu Glu Tyr Tyr Thr Thr Leu Leu Thr Thr Ile Ile Ser Ser Asp Asp Leu Leu Glu Glu

70 70 75 75 80 80

Cys Asp Cys Asp Asp AspAla AlaAla Ala Thr Thr TyrTyr TyrTyr Cys Cys Gln Gln Gly Phe Gly Tyr Tyr Trp PheSer Trp Ser Ser Ser 85 85 90 90 95 95

Ser Thr Ile Ser Thr IleTyr TyrAla AlaPhe Phe TrpTrp AlaAla Phe Phe Gly Gly Gly Gly Gly Lys Gly Thr ThrVal Lys Val Glu Glu 100 100 105 105 110 110

Ile Lys Ile Lys

<210> <210> 157 157 <211> <211> 5 5 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 157 157

Arg Tyr Arg Tyr Tyr TyrVal ValSer Ser 1 1 5 5

<210> <210> 158 158 <211> <211> 6 6 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 158 158

114

Ser Tyr Tyr Ser Tyr TyrTyr TyrMet MetCys Cys 1 1 5 5

<210> <210> 159 159 <211> <211> 5 5 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 159 159

Arg Tyr Arg Tyr Trp TrpMet MetSer Ser 1 1 5 5

<210> <210> 160 160 <211> <211> 5 5 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 160 160

Ser Tyr Ile Ser Tyr IleMet MetGly Gly 1 1 5 5

<210> <210> 161 161 <211> <211> 5 5 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 161 161

Ser Tyr Ser Tyr Ala AlaMet MetSer Ser 1 1 5 5

<210> <210> 162 162 <211> <211> 5 5 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 162 162

115

Ser Ser Ser Ser Ser SerMet MetIle Ile 1 1 5 5

<210> <210> 163 163 <211> <211> 16 16 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 163 163

Ile Ile Tyr Ile Ile TyrThr ThrGly GlyGly Gly AsnAsn ThrThr Asn Asn Tyr Tyr Ala Ala Ser Ala Ser Trp TrpLys Ala Lys Gly Gly 1 1 5 5 10 10 15 15

<210> <210> 164 164 <211> <211> 18 18 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 164 164

Cys Ile Cys Ile Tyr TyrAla AlaGly Gly Ile Ile SerSer GlyGly Ser Ser Thr Thr Tyr Ala Tyr Tyr Tyr Ser AlaTrp Ser Trp Ala Ala 1 1 5 5 10 10 15 15

Lys Gly Lys Gly

<210> <210> 165 165 <211> <211> 16 16 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 165 165

Ile Ile Ser Ile Ile SerSer SerGly GlyGly Gly ThrThr ThrThr Tyr Tyr Tyr Tyr Ala Ala Ser Ala Ser Trp TrpLys Ala Lys Gly Gly 1 1 5 5 10 10 15 15

<210> <210> 166 166 <211> <211> 16 16 <212> <212> PRT PRT

116

<213> Artificialsequence <213> Artificial sequence

<400> <400> 166 166

Phe Ile Tyr Phe Ile TyrVal ValSer SerGly Gly SerSer ThrThr Tyr Tyr Tyr Tyr Ala Ala Ser Ala Ser Trp TrpLys Ala Lys Gly Gly 1 1 5 5 10 10 15 15

<210> <210> 167 167 <211> <211> 16 16 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 167 167

Ile Ile Tyr Ile Ile TyrGly GlyVal ValGly Gly SerSer ThrThr Tyr Tyr Tyr Tyr Ala Ala Ser Ala Ser Trp TrpLys Ala Lys Gly Gly 1 1 5 5 10 10 15 15

<210> <210> 168 168 <211> <211> 16 16 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 168 168

Phe Ile Ser Phe Ile SerLys LysSer SerGly Gly TyrTyr ThrThr Tyr Tyr Tyr Tyr Ala Ala Ser Ala Ser Trp TrpLys Ala Lys Gly Gly 1 1 5 5 10 10 15 15

<210> <210> 169 169 <211> <211> 16 16 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 169 169

Phe Leu Phe Leu Asp AspTyr TyrSer SerHis His TyrTyr ThrThr Thr Thr Gly Gly Gly Gly Tyr Leu Tyr Tyr TyrAsp Leu Asp Leu Leu 1 1 5 5 10 10 15 15

117

<210> <210> 170 170 <211> <211> 10 10 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 170 170

Asp Pro Asp Pro Tyr TyrHis HisPhe Phe Asp Asp TyrTyr GlyGly Ser Ser Leu Leu 1 1 5 5 10 10

<210> <210> 171 171 <211> <211> 11 11 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 171 171

Asp Ser Asp Ser Arg ArgTyr TyrGly Gly Ala Ala AlaAla ArgArg Leu Leu Asp Asp Leu Leu 1 1 5 5 10 10

<210> <210> 172 172 <211> <211> 4 4 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 172 172

Thr Trp Thr Trp Asp AspLeu Leu 1 1

<210> <210> 173 173 <211> <211> 10 10 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 173 173

Asp Asp Asp Asp Tyr TyrGly GlyTrp Trp Gly Gly TyrTyr ThrThr Asn Asn Leu Leu 1 1 5 5 10 10

118

<210> <210> 174 174 <211> <211> 12 12 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 174 174

Gly Gly Gly Gly Tyr Tyr Ser Ser Gly Gly Asp Asp Trp Trp Asp Asp Tyr Tyr Phe Phe Thr Thr Leu Leu 1 1 5 5 10 10

<210> <210> 175 175 <211> <211> 13 13 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 175 175

Gln Ala Gln Ala Ser SerGln GlnSer Ser Val Val TyrTyr AsnAsn Asn Asn Lys Lys Asn Ala Asn Leu Leu Ala 1 1 5 5 10 10

<210> <210> 176 176 <211> <211> 13 13 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 176 176

Gln Ala Gln Ala Ser SerGln GlnSer Ser Val Val TyrTyr GlyGly Gly Gly Asn Asn Glu Ser Glu Leu Leu Ser 1 1 5 5 10 10

<210> <210> 177 177 <211> <211> 13 13 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 177 177

Gln Ser Gln Ser Ser Ser Gln Gln Ser Ser Val Val Ala Ala Gly Gly Asn Asn Asp Asp Asn Asn Leu Leu Ala Ala 1 1 5 5 10 10

119

<210> <210> 178 178 <211> <211> 13 13 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 178 178

Gln Ser Gln Ser Ser SerGln GlnSer Ser Val Val TyrTyr AspAsp Asn Asn Asn Asn Trp Ala Trp Leu Leu Ala 1 1 5 5 10 10

<210> <210> 179 179 <211> <211> 13 13 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 179 179

Gln Ser Gln Ser Ser SerGln GlnSer Ser Val Val TyrTyr AsnAsn Asn Asn Asn Asn Tyr Ala Tyr Leu Leu Ala 1 1 5 5 10 10

<210> <210> 180 180 <211> <211> 11 11 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 180 180

Gln Ala Gln Ala Ser SerGlu GluSer Ser Ile Ile GlyGly AsnAsn Ala Ala Leu Leu Ala Ala 1 1 5 5 10 10

<210> <210> 181 181 <211> <211> 7 7 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 181 181

Tyr Ala Tyr Ala Ser SerThr ThrLeu Leu Pro Pro SerSer 1 1 5 5

120

<210> <210> 182 182 <211> <211> 7 7 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 182 182

Ser Ala Ser Ser Ala SerLys LysLeu LeuAla Ala SerSer 1 1 5 5

<210> <210> 183 183 <211> <211> 7 7 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 183 183

Tyr Ala Tyr Ala Ser SerAsn AsnLeu Leu Ala Ala SerSer 1 1 5 5

<210> <210> 184 184 <211> <211> 7 7 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 184 184

Gly Ala Gly Ala Ser SerThr ThrLeu Leu Asp Asp SerSer 1 1 5 5

<210> <210> 185 185 <211> <211> 7 7 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 185 185

Asp Ala Asp Ala Ser SerLys LysLeu Leu Ala Ala SerSer 1 1 5 5

121

<210> <210> 186 186 <211> <211> 7 7 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 186 186

Gly Ala Gly Ala Phe PheAsn AsnLeu Leu Glu Glu SerSer 1 1 5 5

<210> <210> 187 187 <211> <211> 13 13 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 187 187

Gln Gly Gln Gly Glu GluPhe PheSer Ser Cys Cys GlyGly SerSer Ala Ala Asp Asp Cys Ala Cys Thr Thr Ala 1 1 5 5 10 10

<210> <210> 188 188 <211> <211> 12 12 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 188 188

Ala Gly Ala Gly Tyr TyrArg ArgThr Thr Tyr Tyr SerSer AspAsp Asp Asp Asp Asp Asn Ala Asn Ala 1 1 5 5 10 10

<210> <210> 189 189 <211> <211> 13 13 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 189 189

Leu Gly Leu Gly Asn AsnTyr TyrAsp Asp Cys Cys SerSer SerSer Ala Ala Asp Asp Cys Ala Cys Asp Asp Ala 1 1 5 5 10 10

122

<210> <210> 190 190 <211> <211> 10 10 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 190 190

Gln Gly Gln Gly Gly GlyTyr TyrSer Ser Gly Gly AsnAsn IleIle Tyr Tyr Pro Pro 1 1 5 5 10 10

<210> <210> 191 191 <211> <211> 12 12 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 191 191

Gln Gly Gln Gly Ser SerTyr TyrTyr Tyr Ser Ser SerSer GlyGly Trp Trp Tyr Tyr Arg Ala Arg Ala 1 1 5 5 10 10

<210> <210> 192 192 <211> <211> 15 15 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 192 192

Gln Gly Gln Gly Tyr TyrPhe PheTrp Trp Ser Ser SerSer SerSer Thr Thr Ile Ile Tyr Phe Tyr Ala Ala Trp PheAla Trp Ala 1 1 5 5 10 10 15 15

<210> <210> 193 193 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 193 193

123

Leu Ser Leu Ser Ser Ser Val Val Val Val Thr Thr Val Val Pro Pro Ser Ser Ser Ser Ser Ser Leu Leu Gly Gly Thr Thr Gln Gln Thr Thr

70 70 75 75 80 80

Pro Ala Pro Ala Pro ProGlu GluLeu LeuArg Arg ArgArg GlyGly Pro Pro Lys Lys Val Leu Val Phe Phe Phe LeuPro Phe Pro Pro Pro 115 115 120 120 125 125

Tyr Val Tyr Val Asp AspGly GlyVal Val Glu Glu ValVal HisHis Asn Asn Ala Ala Lys Lys Lys Thr Thr Pro LysArg Pro Arg Glu Glu 165 165 170 170 175 175

124

Met Thr Met Thr Lys LysAsn AsnGln Gln Val Val SerSer LeuLeu Thr Thr Cys Cys Leu Lys Leu Val Val Gly LysPhe Gly Phe Tyr Tyr 245 245 250 250 255 255

Pro Ser Pro Ser Asp AspIle IleAla AlaVal Val GluGlu TrpTrp Glu Glu Ser Ser Asn Asn Gly Pro Gly Gln GlnGlu Pro Glu Asn Asn 260 260 265 265 270 270

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 194 194 <211> <211> 324 324 <212> <212> DNA DNA <213> <213> Artificial sequence Artificial sequence

125

<400> <400> 194 194 cgtacggtgg ctgcaccatc cgtacggtgg ctgcaccatc tgtcttcatc tgtcttcatc ttcccgccat ttcccgccat ctgatgagca ctgatgagca gttgaaatct gttgaaatct 60 60

ggaactgcctctgttgtgtg ggaactgcct ctgttgtgtg cctgctgaat cctgctgaat aacttctatc aacttctatc ccagagaggc ccagagagga caaagtacag caaagtacag 120 120

agtaaggactgcacctacag agtaaggact gcacctacag tctcagtagt tctcagtagt accctgacgc accctgacgc tgtccaaagc tgtccaaagc agactacgag agactacgag 240 240

aaacacaaagtctacgcctg aaacacaaag tctacgcctg cgaagtcacc cgaagtcacc catcagggcc catcagggcc tgtcctcgcc tgtcctcgcc cgtcacaaag cgtcacaaag 300 300

tccttcaacaggggagagtg tccttcaaca ggggagagtg ttga ttga 324 324

<210> <210> 195 195 <211> <211> 330 330 <212> <212> PRT PRT <213> <213> Homo sapiens Homo sapiens

<400> <400> 195 195

70 70 75 75 80 80

126

Pro Ala Pro Ala Pro ProGlu GluLeu LeuLeu Leu GlyGly GlyGly Pro Pro Ser Ser Val Leu Val Phe Phe Phe LeuPro Phe Pro Pro Pro 115 115 120 120 125 125

Gln Pro Gln Pro Arg Arg Glu Glu Pro Pro Gln Gln Val Val Tyr Tyr Thr Thr Leu Leu Pro Pro Pro Pro Ser Ser Arg Arg Asp Asp Glu Glu 225 225 230 230 235 235 240 240

127

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro Gly Gly Lys Lys 325 325 330 330

<210> <210> 196 196 <211> <211> 326 326 <212> <212> PRT PRT <213> <213> Homo sapiens Homo sapiens

<400> <400> 196 196

Ala Ser Ala Ser Thr ThrLys LysGly Gly Pro Pro SerSer ValVal Phe Phe Pro Pro Leu Pro Leu Ala Ala Cys ProSer Cys Ser Arg Arg 1 1 5 5 10 10 15 15

Ser Thr Ser Thr Ser SerGlu GluSer SerThr Thr AlaAla AlaAla Leu Leu Gly Gly Cys Cys Leu Lys Leu Val ValAsp Lys Asp Tyr Tyr 20 20 25 25 30 30

Phe Pro Phe Pro Glu GluPro ProVal ValThr Thr ValVal SerSer Trp Trp Asn Asn Ser Ser Gly Leu Gly Ala AlaThr Leu Thr Ser Ser 35 35 40 40 45 45

Leu Ser Leu Ser Ser SerVal ValVal Val Thr Thr ValVal ProPro Ser Ser Ser Ser Asn Gly Asn Phe Phe Thr GlyGln Thr Gln Thr Thr

70 70 75 75 80 80

Tyr Thr Tyr Thr Cys CysAsn AsnVal Val Asp Asp HisHis LysLys Pro Pro Ser Ser Asn Lys Asn Thr Thr Val LysAsp Val Asp Lys Lys

128

85 90 90 95 95

Thr Val Thr Val Glu GluArg ArgLys Lys Cys Cys CysCys ValVal Glu Glu Cys Cys Pro Cys Pro Pro Pro Pro CysAla Pro Ala Pro Pro 100 100 105 105 110 110

Pro Val Pro Val Ala AlaGly GlyPro ProSer Ser ValVal PhePhe Leu Leu Phe Phe Pro Lys Pro Pro Pro Pro LysLys Pro Lys Asp Asp 115 115 120 120 125 125

Thr Leu Thr Leu Met MetIle IleSer Ser Arg Arg ThrThr ProPro Glu Glu Val Val Thr Val Thr Cys Cys Val ValVal Val Val Asp Asp 130 130 135 135 140 140

Val Ser Val Ser His His Glu Glu Asp Asp Pro Pro Glu Glu Val Val Gln Gln Phe Phe Asn Asn Trp Trp Tyr Tyr Val Val Asp Asp Gly Gly 145 145 150 150 155 155 160 160

Val Glu Val Glu Val ValHis HisAsn Asn Ala Ala LysLys ThrThr Lys Lys Pro Pro Arg Glu Arg Glu Glu Gln GluPhe Gln Phe Asn Asn 165 165 170 170 175 175

Ser Thr Phe Ser Thr PheArg ArgVal ValVal Val SerSer ValVal Leu Leu Thr Thr Val Val Val Gln Val His HisAsp Gln Asp Trp Trp 180 180 185 185 190 190

Leu Asn Leu Asn Gly GlyLys LysGlu Glu Tyr Tyr LysLys CysCys Lys Lys Val Val Ser Lys Ser Asn Asn Gly LysLeu Gly Leu Pro Pro 195 195 200 200 205 205

Ala Pro Ala Pro Ile IleGlu GluLys Lys Thr Thr IleIle SerSer Lys Lys Thr Thr Lys Gln Lys Gly Gly Pro GlnArg Pro Arg Glu Glu 210 210 215 215 220 220

Pro Gln Pro Gln Val ValTyr TyrThr ThrLeu Leu ProPro ProPro Ser Ser Arg Arg Glu Met Glu Glu Glu Thr MetLys Thr Lys Asn Asn 225 225 230 230 235 235 240 240

Gln Val Gln Val Ser SerLeu LeuThr Thr Cys Cys LeuLeu ValVal Lys Lys Gly Gly Phe Pro Phe Tyr Tyr Ser ProAsp Ser Asp Ile Ile 245 245 250 250 255 255

Ala Val Ala Val Glu GluTrp TrpGlu Glu Ser Ser AsnAsn GlyGly Gln Gln Pro Pro Glu Asn Glu Asn Asn Tyr AsnLys Tyr Lys

129

Thr Thr 260 260 265 265 270 270

Thr Pro Thr Pro Pro ProMet MetLeu Leu Asp Asp SerSer AspAsp Gly Gly Ser Ser Phe Leu Phe Phe Phe Tyr LeuSer Tyr Ser Lys Lys 275 275 280 280 285 285

Leu Thr Val Leu Thr ValAsp AspLys LysSer Ser ArgArg TrpTrp Gln Gln Gln Gln Gly Gly Asn Phe Asn Val ValSer Phe Ser Cys Cys 290 290 295 295 300 300

Ser Val Met Ser Val MetHis HisGlu GluAla Ala LeuLeu HisHis Asn Asn His His Tyr Tyr Thr Lys Thr Gln GlnSer Lys Ser Leu Leu 305 305 310 310 315 315 320 320

Ser Leu Ser Ser Leu SerPro ProGly GlyLys Lys 325 325

<210> <210> 197 197 <211> <211> 377 377 <212> <212> PRT PRT <213> <213> Homo sapiens Homo sapiens

<400> <400> 197 197

70 70 75 75 80 80

130

Tyr Thr Tyr Thr Cys Cys Asn Asn Val Val Asn Asn His His Lys Lys Pro Pro Ser Ser Asn Asn Thr Thr Lys Lys Val Val Asp Asp Lys Lys 85 85 90 90 95 95

Arg Val Arg Val Glu GluLeu LeuLys Lys Thr Thr ProPro LeuLeu Gly Gly Asp Asp Thr His Thr Thr Thr Thr HisCys Thr Cys Pro Pro 100 100 105 105 110 110

Arg Cys Arg Cys Pro ProGlu GluPro Pro Lys Lys SerSer CysCys Asp Asp Thr Thr Pro Pro Pro Pro Pro Cys ProPro Cys Pro Arg Arg 115 115 120 120 125 125

Cys Pro Cys Pro Glu Glu Pro Pro Lys Lys Ser Ser Cys Cys Asp Asp Thr Thr Pro Pro Pro Pro Pro Pro Cys Cys Pro Pro Arg Arg Cys Cys 130 130 135 135 140 140

Pro Glu Pro Glu Pro ProLys LysSer SerCys Cys AspAsp ThrThr Pro Pro Pro Pro Pro Pro Pro Cys Cys Arg ProCys Arg Cys Pro Pro 145 145 150 150 155 155 160 160

Ala Pro Ala Pro Glu GluLeu LeuLeu Leu Gly Gly GlyGly ProPro Ser Ser Val Val Phe Phe Phe Leu Leu Pro PhePro Pro Pro Lys Lys 165 165 170 170 175 175

Pro Lys Pro Lys Asp AspThr ThrLeu Leu Met Met IleIle SerSer Arg Arg Thr Thr Pro Val Pro Glu Glu Thr ValCys Thr Cys Val Val 180 180 185 185 190 190

Val Val Val Val Asp Asp Val Val Ser Ser His His Glu Glu Asp Asp Pro Pro Glu Glu Val Val Gln Gln Phe Phe Lys Lys Trp Trp Tyr Tyr 195 195 200 200 205 205

Val Asp Val Asp Gly Gly Val Val Glu Glu Val Val His His Asn Asn Ala Ala Lys Lys Thr Thr Lys Lys Pro Pro Arg Arg Glu Glu Glu Glu 210 210 215 215 220 220

Gln Tyr Gln Tyr Asn Asn Ser Ser Thr Thr Phe Phe Arg Arg Val Val Val Val Ser Ser Val Val Leu Leu Thr Thr Val Val Leu Leu His His 225 225 230 230 235 235 240 240

Gln Asp Gln Asp Trp Trp Leu Leu Asn Asn Gly Gly Lys Lys Glu Glu Tyr Tyr Lys Lys Cys Cys Lys Lys Val Val Ser Ser Asn Asn Lys Lys 245 245 250 250 255 255

131

Ala Leu Ala Leu Pro Pro Ala Ala Pro Pro Ile Ile Glu Glu Lys Lys Thr Thr Ile Ile Ser Ser Lys Lys Thr Thr Lys Lys Gly Gly Gln Gln 260 260 265 265 270 270

Pro Arg Pro Arg Glu GluPro ProGln GlnVal Val TyrTyr ThrThr Leu Leu Pro Pro Pro Arg Pro Ser Ser Glu ArgGlu Glu Glu Met Met 275 275 280 280 285 285

Thr Lys Thr Lys Asn AsnGln GlnVal Val Ser Ser LeuLeu ThrThr Cys Cys Leu Leu Val Gly Val Lys Lys Phe GlyTyr Phe Tyr Pro Pro 290 290 295 295 300 300

Ser Asp Ile Ser Asp IleAla AlaVal ValGlu Glu TrpTrp GluGlu Ser Ser Ser Ser Gly Gly Gln Glu Gln Pro ProAsn Glu Asn Asn Asn 305 305 310 310 315 315 320 320

Tyr Asn Tyr Asn Thr ThrThr ThrPro Pro Pro Pro MetMet LeuLeu Asp Asp Ser Ser Asp Ser Asp Gly Gly Phe SerPhe Phe Phe Leu Leu 325 325 330 330 335 335

Tyr Ser Tyr Ser Lys Lys Leu Leu Thr Thr Val Val Asp Asp Lys Lys Ser Ser Arg Arg Trp Trp Gln Gln Gln Gln Gly Gly Asn Asn Ile Ile 340 340 345 345 350 350

Phe Ser Cys Phe Ser CysSer SerVal ValMet Met HisHis GluGlu Ala Ala Leu Leu His His Asn Phe Asn Arg ArgThr Phe Thr Gln Gln 355 355 360 360 365 365

Lys Ser Lys Ser Leu LeuSer SerLeu Leu Ser Ser ProPro GlyGly Lys Lys 370 370 375 375

<210> <210> 198 198 <211> <211> 327 327 <212> <212> PRT PRT <213> <213> Homo sapiens Homo sapiens

<400> <400> 198 198

Ser Thr Ser Ser Thr SerGlu GluSer SerThr Thr AlaAla AlaAla Leu Leu Gly Gly Cys Cys Leu Lys Leu Val ValAsp Lys Asp Tyr Tyr 20 20 25 25 30 30

132

Phe Pro Phe Pro Glu GluPro ProVal ValThr Thr ValVal SerSer Trp Trp Asn Asn Ser Ala Ser Gly Gly Leu AlaThr Leu Thr Ser Ser 35 35 40 40 45 45

Leu Ser Leu Ser Ser SerVal ValVal Val Thr Thr ValVal ProPro Ser Ser Ser Ser Ser Gly Ser Leu Leu Thr GlyLys Thr Lys Thr Thr

70 70 75 75 80 80

Tyr Thr Tyr Thr Cys CysAsn AsnVal Val Asp Asp HisHis LysLys Pro Pro Ser Ser Asn Lys Asn Thr Thr Val LysAsp Val Asp Lys Lys 85 85 90 90 95 95

Arg Val Arg Val Glu GluSer SerLys Lys Tyr Tyr GlyGly ProPro Pro Pro Cys Cys Pro Cys Pro Ser Ser Pro CysAla Pro Ala Pro Pro 100 100 105 105 110 110

Glu Phe Glu Phe Leu LeuGly GlyGly Gly Pro Pro SerSer ValVal Phe Phe Leu Leu Phe Pro Phe Pro Pro Lys ProPro Lys Pro Lys Lys 115 115 120 120 125 125

Asp Thr Asp Thr Leu LeuMet MetIle Ile Ser Ser ArgArg ThrThr Pro Pro Glu Glu Val Cys Val Thr Thr Val CysVal Val Val Val Val 130 130 135 135 140 140

Asp Val Asp Val Ser SerGln GlnGlu Glu Asp Asp ProPro GluGlu Val Val Gln Gln Phe Trp Phe Asn Asn Tyr TrpVal Tyr Val Asp Asp 145 145 150 150 155 155 160 160

Gly Val Gly Val Glu Glu Val Val His His Asn Asn Ala Ala Lys Lys Thr Thr Lys Lys Pro Pro Arg Arg Glu Glu Glu Glu Gln Gln Phe Phe 165 165 170 170 175 175

Asn Ser Asn Ser Thr ThrTyr TyrArg Arg Val Val ValVal SerSer Val Val Leu Leu Thr Leu Thr Val Val His LeuGln His Gln Asp Asp 180 180 185 185 190 190

Trp Leu Trp Leu Asn Asn Gly Gly Lys Lys Glu Glu Tyr Tyr Lys Lys Cys Cys Lys Lys Val Val Ser Ser Asn Asn Lys Lys Gly Gly Leu Leu 195 195 200 200 205 205

133

Pro Ser Pro Ser Ser SerIle IleGlu GluLys Lys ThrThr IleIle Ser Ser Lys Lys Ala Ala Lys Gln Lys Gly GlyPro Gln Pro Arg Arg 210 210 215 215 220 220

Glu Pro Glu Pro Gln GlnVal ValTyr Tyr Thr Thr LeuLeu ProPro Pro Pro Ser Ser Gln Glu Gln Glu Glu Met GluThr Met Thr Lys Lys 225 225 230 230 235 235 240 240

Asn Gln Asn Gln Val ValSer SerLeu Leu Thr Thr CysCys LeuLeu Val Val Lys Lys Gly Tyr Gly Phe Phe Pro TyrSer Pro Ser Asp Asp 245 245 250 250 255 255

Ile Ala Val Ile Ala ValGlu GluTrp TrpGlu Glu SerSer AsnAsn Gly Gly Gln Gln Pro Pro Glu Asn Glu Asn AsnTyr Asn Tyr Lys Lys 260 260 265 265 270 270

Thr Thr Thr Thr Pro ProPro ProVal Val Leu Leu AspAsp SerSer Asp Asp Gly Gly Ser Phe Ser Phe Phe Leu PheTyr Leu Tyr Ser Ser 275 275 280 280 285 285

Arg Leu Arg Leu Thr ThrVal ValAsp Asp Lys Lys SerSer ArgArg Trp Trp Gln Gln Glu Asn Glu Gly Gly Val AsnPhe Val Phe Ser Ser 290 290 295 295 300 300

Cys Ser Cys Ser Val ValMet MetHis His Glu Glu AlaAla LeuLeu His His Asn Asn His Thr His Tyr Tyr Gln ThrLys Gln Lys Ser Ser 305 305 310 310 315 315 320 320

Leu Ser Leu Ser Leu LeuSer SerLeu Leu Gly Gly LysLys 325 325

<210> <210> 199 199 <211> <211> 1125 1125 <212> <212> DNA DNA <213> <213> Homo sapiens Homo sapiens

<400> <400> 199 199 atgtggttct tgacaactct atgtggttct tgacaactct gctcctttgg gctcctttgg gttccagttg gttccagttg atgggcaagt atgggcaagt ggacaccaca ggacaccaca 60 60

aaggcagtgatcactttgca aaggcagtga tcactttgca gcctccatgg gcctccatgg gtcagcgtgt gtcagcgtgt tccaagagga tccaagagga aaccgtaacc aaccgtaacc 120 120

ttgcactgtg aggtgctcca ttgcactgtg aggtgctcca tctgcctggg tctgcctggg agcagctcta agcagctcta cacagtggtt cacagtggtt

134 tctcaatggc tctcaattgc 180 180 acagccactcagacctcgac acagccactc agacctcgac ccccagctac ccccagctac agaatcacct agaatcacct ctgccagtgt ctgccagtgt caatgacagt caatgacagt 240 240 ggtgaatacaggtgccagag ggtgaataca ggtgccagag aggtctctca aggtctctca gggcgaagtg gggcgaagtg accccataca accccataca gctggaaatc gctggaaatc 300 300 cacagaggct ggctactact cacagaggct ggctactact gcaggtctcc gcaggtctcc agcagagtct agcagagtct tcacggaagg tcacggaagg agaacctctg agaacctctg 360 360 gccttgaggtgtcatgcgtg gccttgaggt gtcatgcgtg gaaggataag gaaggataag ctggtgtaca ctggtgtaca atgtgcttta atgtgcttta ctatcgaaat 420 ctatcgaaat 420 ggcaaagcctttaagttttt ggcaaagcct ttaagttttt ccactggaat ccactggaat tctaacctca tctaacctca ccattctgaa ccattctgaa aaccaacata aaccaacata 480 480 agtcacaatggcacctacca agtcacaatg gcacctacca ttgctcaggc ttgctcaggc atgggaaagc atgggaaaga atcgctacac atcgctacac atcagcagga atcagcagga 540 540 atatctgtcactgtgaaaga atatctgtca ctgtgaaaga gctatttcca gctatttcca gctccagtgc gctccagtgc tgaatgcatc tgaatgcatc tgtgacatcc 600 tgtgacatcc 600 ccactcctggaggggaatct ccactcctgg aggggaatct ggtcaccctg ggtcaccctg agctgtgaaa agctgtgaaa caaagttgct caaagttgct cttgcagagg cttgcagagg 660 660 cctggtttgcagctttactt cctggtttgc agctttactt ctccttctac ctccttctac atgggcagca atgggcagca agaccctgcg agaccctgcg aggcaggaac aggcaggaac 720 720 acatcctctgaataccaaat acatcctctg aataccaaat actaactgct actaactgct agaagagaag agaagagaag actctgggtt actctgggtt atactggtgc atactggtgc 780 780 gaggctgcca cagaggatgg gaggctgcca cagaggatgg aaatgtcctt aaatgtcctt aagcgcagcc aagcgcagcc ctgagttgga ctgagttgga gcttcaagtg gcttcaagtg 840 840 cttggcctccagttaccaac cttggcctcc agttaccaac tcctgtctgg tcctgtctgg tttcatgtcc tttcatgtcc ttttctatct ttttctatct ggcagtggga ggcagtggga 900 900 ataatgtttttagtgaacac ataatgtttt tagtgaacac tgttctctgg tgttctctgg gtgacaatac gtgacaatac gtaaagaact gtaaagaact gaaaagaaag gaaaagaaag 960 960 aaaaagtgggatttagaaat aaaaagtggg atttagaaat ctctttggat ctctttggat tctggtcatg tctggtcatg agaagaaggt agaagaaggt aatttccagc 1020 aatttccage 1020 cttcaagaagacagacattt cttcaagaag acagacattt agaagaagag agaagaagag ctgaaatgtc ctgaaatgtc aggaacaaaa aggaacaaaa agaagaacag 1080 agaagaacag 1080 ctgcaggaag gggtgcaccg ctgcaggaag gggtgcaccg gaaggagccc gaaggageec cagggggcca cagggggcca cgtagcgtag 1125 1125

<210> <210> 200 200 <211> <211> 951 951 <212> <212> DNA DNA <213> <213> Homo sapiens Homo sapiens

135

<400> <400> 200 200 atgactatggagacccaaat atgactatgg agacccaaat gtctcagaat gtctcagaat gtatgtccca gtatgtccca gaaacctgtg gaaacctgtg gctgcttcaa 60 gctgcttcaa 60 ccattgacag ttttgctgct ccattgacag ttttgctgct gctggcttct gctggcttct gcagacagtc gcagacagtc aagctgctcc aagctgctcc cccaaaggct cccaaaggct 120 120

gtgctgaaacttgagccccc gtgctgaaac ttgagccccc gtggatcaac gtggatcaac gtgctccagg gtgctccagg aggactctgt aggactctgt gactctgaca gactctgaca 180 180

tgccagggggctcgcageee tgccaggggg ctcgcagccc tgagagcgac tgagagcgac tccattcagt tccattcagt ggttccacaa ggttccacaa tgggaatctc tgggaatctc 240 240

attcccaccc acacgcagcc attcccaccc acacgcagcc cagctacagg cagctacagg ttcaaggcca ttcaaggcca acaacaatga acaacaatga cagcggggag cagcggggag 300 300

tacacgtgcc agactggcca tacacgtgcc agactggcca gaccagcctc gaccagectc agcgaccctg agcgaccctg tgcatctgac tgcatctgac tgtgctttcc tgtgctttcc 360 360

gaatggctggtgctccagac gaatggctgg tgctccagac ccctcacctg ccctcacctg gagttccagg gagttccagg agggagaaac agggagaaac catcatgctg catcatgctg 420 420

aggtgccacagctggaagga aggtgccaca gctggaagga caagcctctg caagcctctg gtcaaggtca gtcaaggtca cattcttcca cattcttcca gaatggaaaa gaatggaaaa 480 480

tcccagaaat tctcccattt tcccagaaat tctcccattt ggatcccacc ggatcccacc ttctccatcc ttctccatcc cacaagcaaa cacaagcaaa ccacagtcac ccacagtcac 540 540

agtggtgattaccactgcac agtggtgatt accactgcac aggaaacata aggaaacata ggctacacgc ggctacacgc tgttctcatc tgttctcatc caagcctgtg caagcctgtg 600 600 accatcactgtccaaggtgcc accatcactg tccaagtgcccagcatgggc cagcatgggc agctcttcac agctcttcac caatgggggt caatgggggt cattgtggct cattgtggct 660 660

gtggtcattgcgactgctgt gtggtcattg cgactgctgt agcagccatt agcagccatt gttgctgctg gttgctgctg tagtggcctt tagtggcctt gatctactgc 720 gatctactgc 720 aggaaaaagcggatttcage aggaaaaaga ggatttcagc caattccact caattccact gatcctgtga gatcctgtga aggctgccca aggctgccca atttgagcca 780 atttgagcca 780 cctggacgtc aaatgattgc cctggacgtc aaatgattgc catcagaaag catcagaaag agacaacttg agacaacttg aagaaaccaa aagaaaccaa caatgactat caatgactat 840 840

gaaacagctgacggcggcta gaaacagctg acggcggcta catgactctg catgactctg aaccccaggg aaccccaggg cacctactga cacctactga cgatgataaa 900 cgatgataaa 900 aacatctacctgactcttcc aacatctacc tgactcttcc tcccaacgac tcccaacgac catgtcaaca catgtcaaca gtaataacta gtaataacta a a 951 951

<210> <210> 201 201 <211> <211> 954 954 <212> <212> DNA DNA <213> <213> Homo sapiens Homo sapiens

<400> <400> 201 201

136 atgactatggagacccaaat atgactatgg agacccaaat gtctcagaat gtctcagaat gtatgtccca gtatgtccca gaaacctgtg gaaacctgtg gctgcttcaa gctgcttcaa 60 60 ccattgacagttttgctgct ccattgacag ttttgctgct gctggcttct gctggcttct gcagacagtc gcagacagte aagctgcagc aagctgcage tcccccaaag tcccccaaag 120 120 gctgtgctgaaacttgagcc gctgtgctga aacttgagcc cccgtggatc cccgtggatc aacgtgctcc aacgtgctcc aggaggactc aggaggacto tgtgactctg tgtgactctg 180 180 acatgccagggggctcgcag acatgccagg gggctcgcag ccctgagagc ccctgagago gactccattc gactccatto agtggttcca agtggttcca caatgggaat caatgggaat 240 240 ctcattccca cccacacgca ctcattccca cccacacgca gcccagctac gcccagctac aggttcaagg aggttcaagg ccaacaacaa ccaacaacaa tgacagcggg tgacagcggg 300 300 gagtacacgtgccagactgg gagtacacgt gccagactgg ccagaccagc ccagaccago ctcagcgacc ctcagcgace ctgtgcatct ctgtgcatct gactgtgctt gactgtgctt 360 360 tccgaatggctggtgctcca tccgaatggc tggtgctcca gacccctcac gacccctcac ctggagttcc ctggagttcc aggagggaga aggagggaga aaccatcatg aaccatcatg 420 420 ctgaggtgcc acagctggaa ctgaggtgcc acagctggaa ggacaagcct ggacaagcct ctggtcaagg ctggtcaagg tcacattctt tcacattctt ccagaatgga 480 ccagaatgga 480 aaatcccagaaattctccca aaatcccaga aattctccca tttggatccc tttggatccc accttctcca accttctcca tcccacaagc tcccacaage aaaccacagt 540 aaaccacagt 540 cacagtggtg attaccactg cacagtggtg attaccactg cacaggaaac cacaggaaac ataggctaca ataggctaca cgctgttctc cgctgttctc atccaagcct 600 atccaagcct 600 gtgaccatcactgtccaagt gtgaccatca ctgtccaagt gcccagcatg gcccagcatg ggcagctctt ggcagctctt caccaatggg caccaatggg gatcattgtg gatcattgtg 660 660 gctgtggtcattgcgactgc gctgtggtca ttgcgactgc tgtagcagcc tgtagcagcc attgttgctg attgttgctg ctgtagtggc ctgtagtggc cttgatctac cttgatctac 720 720 tgcaggaaaaagcggatttc tgcaggaaaa agcggatttc agccaattcc agccaattcc actgatcctg actgatcctg tgaaggctgc tgaaggctgc ccaatttgag ccaatttgag 780 780 ccacctggacgtcaaatgat ccacctggac gtcaaatgat tgccatcaga tgccatcaga aagagacaac aagagacaac ttgaagaaac ttgaagaaac caacaatgac caacaatgac 840 840 tatgaaacag ctgacggcgg tatgaaacag ctgacggcgg ctacatgact ctacatgact ctgaacccca ctgaacccca gggcacctac gggcacctac tgacgatgat tgacgatgat 900 900 aaaaacatctacctgactct aaaaacatct acctgactct tcctcccaac tcctcccaac gaccatgtca gaccatgtca acagtaataa acagtaataa ctaa ctaa 954 954

<210> <210> 202 202 <211> <211> 876 876 <212> <212> DNA DNA <213> <213> Homo sapiens Homo sapiens

<400> <400> 202 202 atgggaatcctgtcattctt atgggaatcc tgtcattctt acctgtcctt acctgtcctt gccactgaga gccactgaga gtgactgggc gtgactggga

137 tgactgcaag tgactgcaag 60 60 tccccccagc cttggggtca tccccccagc cttggggtca tatgcttctg tatgcttctg tggacagctg tggacagctg tgctattcct tgctattect ggctcctgtt ggctcctgtt 120 120 gctgggacacctgcagctcc gctgggacac ctgcagctcc cccaaaggct cccaaaggct gtgctgaaac gtgctgaaac tcgagcccca tcgagcccca gtggatcaac gtggatcaac 180 180 gtgctccaggaggactctgt gtgctccagg aggactctgt gactctgaca gactctgaca tgccggggga tgccggggga ctcacagccc ctcacageee tgagagcgac tgagagcgac 240 240 tccattcagt ggttccacaa tccattcagt ggttccacaa tgggaatctc tgggaatctc attcccaccc attcccaccc acacgcagcc acacgcagcc cagctacagg cagctacagg 300 300 ttcaaggcca acaacaatga ttcaaggcca acaacaatga cagcggggag cagcggggag tacacgtgcc tacacgtgcc agactggcca agactggcca gaccagcctc 360 gaccagectc 360 agcgaccctgtgcatctgac agcgaccctg tgcatctgac tgtgctttct tgtgctttct gagtggctgg gagtggctgg tgctccagac tgctccagac ccctcacctg ccctcacctg 420 420 gagttccaggagggagaaac gagttccagg agggagaaac catcgtgctg catcgtgctg aggtgccaca aggtgccaca gctggaagga gctggaagga caagcctctg caagcctctg 480 480 gtcaaggtcacattcttcca gtcaaggtca cattcttcca gaatggaaaa gaatggaaaa tccaagaaat tccaagaaat tttcccgttc tttcccgttc ggatcccaac ggatcccaac 540 540 ttctccatcc cacaagcaaa ttctccatcc cacaagcaaa ccacagtcac ccacagtcac agtggtgatt agtggtgatt accactgcac accactgcac aggaaacata aggaaacata 600 600 ggctacacgctgtactcatc ggctacacgc tgtactcatc caagcctgtg caagcctgtg accatcactg accatcactg tccaagctcc tccaagctcc cagctcttca cagctcttca 660 660 ccgatggggatcattgtggc ccgatgggga tcattgtggc tgtggtcact tgtggtcact gggattgctg gggattgctg tagcggccat tagcggccat tgttgctgct tgttgctgct 720 720 gtagtggccttgatctactg gtagtggcct tgatctactg caggaaaaag caggaaaaag cggatttcag cggatttcag ccaatcccac ccaatcccac taatcctgat taatcctgat 780 780 gaggctgacaaagttgggga gaggctgaca aagttggggc tgagaacaca tgagaacaca atcacctatt atcacctatt cacttctcat cacttctcat gcacccggat gcacccggat 840 840 gctctggaag agcctgatga gctctggaag agcctgatga ccagaaccgt ccagaaccgt atttag atttag 876 876

<210> <210> 203 203 <211> <211> 933 933 <212> <212> DNA DNA <213> <213> Homo sapiens Homo sapiens

<400> <400> 203 203 atgggaatcc tgtcattctt atgggaatcc tgtcattctt acctgtcctt acctgtcctt gccactgaga gccactgaga gtgactgggc gtgactgggc tgactgcaag tgactgcaag 60 60

tccccccagc cttggggtca tccccccage cttggggtca tatgcttctg tatgcttctg tggacagctg tggacagctg tgctattcct tgctattcct ggctcctgtt 120 ggctcctgtt 120

138 gctgggacacctgcagctcc gctgggacac ctgcagctcc cccaaaggct cccaaaggct gtgctgaaac gtgctgaaac tcgagcccca tcgagcccca gtggatcaac gtggatcaac 180 180 gtgctccaggaggactctgt gtgctccagg aggactctgt gactctgaca gactctgaca tgccggggga tgccggggga ctcacagccc ctcacageee tgagagcgac 240 tgagagcgac 240 tccattcagt ggttccacaa tccattcagt ggttccacaa tgggaatctc tgggaatctc attcccaccc attcccaccc acacgcagcc acacgcagcc cagctacagg 300 cagctacagg 300 ttcaaggcca acaacaatga ttcaaggcca acaacaatga cagcggggag cagcggggag tacacgtgcc tacacgtgcc agactggcca agactggcca gaccagcctc gaccagectc 360 360 agcgaccctgtgcatctgac agcgaccctg tgcatctgac tgtgctttct tgtgctttct gagtggctgg gagtggctgg tgctccagac tgctccagac ccctcacctg ccctcacctg 420 420 gagttccaggagggagaaac gagttccagg agggagaaac catcgtgctg catcgtgctg aggtgccaca aggtgccaca gctggaagga gctggaagga caagcctctg caagcctctg 480 480 gtcaaggtcacattcttcca gtcaaggtca cattcttcca gaatggaaaa gaatggaaaa tccaagaaat tccaagaaat tttcccgttc tttcccgttc ggatcccaac ggatcccaac 540 540 ttctccatcc cacaagcaaa ttctccatcc cacaagcaaa ccacagtcac ccacagtcac agtggtgatt agtggtgatt accactgcac accactgcac aggaaacata aggaaacata 600 600 ggctacacgctgtactcatc ggctacacgc tgtactcatc caagcctgtg caagcctgtg accatcactg accatcactg tccaagctcc tccaagctcc cagctcttca cagctcttca 660 660 ccgatggggatcattgtggc ccgatgggga tcattgtggc tgtggtcact tgtggtcact gggattgctg gggattgctg tagcggccat tagcggccat tgttgctgct tgttgctgct 720 720 gtagtggccttgatctactg gtagtggcct tgatctactg caggaaaaag caggaaaaag cggatttcag cggatttcag ctctcccagg ctctcccagg ataccctgag ataccctgag 780 780 tgcagggaaa tgggagagac tgcagggaaa tgggagagac cctccctgag cctccctgag aaaccagcca aaaccagcca atcccactaa atcccactaa tcctgatgag tcctgatgag 840 840 gctgacaaagttggggctga gctgacaaag ttggggctga gaacacaatc gaacacaatc acctattcac acctattcac ttctcatgca ttctcatgca cccggatgct 900 cccggatgct 900 ctggaagagc ctgatgacca ctggaagage ctgatgacca gaaccgtatt gaaccgtatt tag tag 933 933

<210> <210> 204 204 <211> <211> 765 765 <212> <212> DNA DNA <213> <213> Homo sapiens Homo sapiens

<400> <400> 204 204 atgtggcagc tgctcctccc atgtggcage tgctcctccc aactgctctg aactgctctg ctacttctag ctacttctag tttcagctgg tttcagctgg catgcggact catgcggact 60 60

gaagatctcccaaaggctgt gaagatctcc caaaggctgt ggtgttcctg ggtgttcctg gagcctcaat gagcctcaat ggtacagggt ggtacagggt gctcgagaag gctcgagaag 120 120

139 gacagtgtgactctgaagtg gacagtgtga ctctgaagtg ccagggagcc ccagggagec tactcccctg tactcccctg aggacaattc aggacaattc cacacagtgg cacacagtgg 180 180 tttcacaatg agagcctcat tttcacaatg agagcctcat ctcaagccag ctcaagccag gcctcgagct gcctcgagct acttcattga acttcattga cgctgccaca cgctgccaca 240 240 gttgacgaca gtggagagta gttgacgaca gtggagagta caggtgccag caggtgccag acaaacctct acaaacctct ccaccctcag ccaccctcag tgacccggtg tgacccggtg 300 300 cagctagaagtccatatcgg cagctagaag tccatatcgg ctggctgttg ctggctgttg ctccaggccc ctccaggccc ctcggtgggt ctcggtgggt gttcaaggag gttcaaggag 360 360 gaagaccctattcacctgag gaagacccta ttcacctgag gtgtcacagc gtgtcacaga tggaagaaca tggaagaaca ctgctctgca ctgctctgca taaggtcaca taaggtcaca 420 420 tatttacaga atggcaaagg tatttacaga atggcaaagg caggaagtat caggaagtat tttcatcata tttcatcata attctgactt attctgactt ctacattcca ctacattcca 480 480 aaagccacactcaaagacag aaagccacac tcaaagacag cggctcctac cggctcctac ttctgcaggg ttctgcaggg ggcttgttgg ggcttgttgg gagtaaaaat gagtaaaaat 540 540 gtgtcttcagagactgtgaa gtgtcttcag agactgtgaa catcaccatc catcaccatc actcaaggtt actcaaggtt tgtcagtgtc tgtcagtgtc aaccatctca aaccatctca 600 600 tcattctttc cacctgggta tcattctttc cacctgggta ccaagtctct ccaagtctct ttctgcttgg ttctgcttgg tgatggtact tgatggtact cctttttgca cctttttgca 660 660 gtggacacaggactatattt gtggacacag gactatattt ctctgtgaag ctctgtgaag acaaacattc acaaacattc gaagctcaac gaagctcaac aagagactgg aagagactgg 720 720 aaggaccataaatttaaatg aaggaccata aatttaaatg gagaaaggac gagaaaggac cctcaagaca cctcaagaca aatgaaatga 765 765

<210> <210> 205 205 <211> <211> 765 765 <212> <212> DNA DNA <213> <213> Homo sapiens Homo sapiens

<400> <400> 205 205 atgtggcagc tgctcctccc atgtggcage tgctcctccc aactgctctg aactgctctg ctacttctag ctacttctag tttcagctgg tttcagctgg catgcggact catgcggact 60 60

gaagatctcccaaaggctgt gaagatctcc caaaggctgt ggtgttcctg ggtgttcctg gagcctcaat gagcctcaat ggtacagggt ggtacagggt gctcgagaag 120 gctcgagaag 120 gacagtgtgactctgaagtg gacagtgtga ctctgaagtg ccagggagcc ccagggagec tactcccctg tactcccctg aggacaattc aggacaattc cacacagtgg cacacagtgg 180 180

tttcacaatg agagcctcat tttcacaatg agagcctcat ctcaagccag ctcaagccag gcctcgagct gcctcgagct acttcattga acttcattga cgctgccaca cgctgccaca 240 240

gtcgacgacagtggagagta gtcgacgaca gtggagagta caggtgccag caggtgccag acaaacctct acaaacctct ccaccctcag ccaccctcag tgacccggtg tgacccggtg 300 300

cagctagaagtccatatcgg cagctagaag tccatatcgg ctggctgttg ctggctgttg ctccaggccc ctccaggccc ctcggtgggt ctcggtgggt

140 gttcaaggag gttcaaggag 360 360 gaagaccctattcacctgag gaagacccta ttcacctgag gtgtcacagc gtgtcacagc tggaagaaca tggaagaaca ctgctctgca ctgctctgca taaggtcaca taaggtcaca 420 420 tatttacaga atggcaaagg tatttacaga atggcaaagg caggaagtat caggaagtat tttcatcata tttcatcata attctgactt attctgactt ctacattcca ctacattcca 480 480 aaagccacac tcaaagacag aaagccacac tcaaagacag cggctcctac cggctcctac ttctgcaggg ttctgcaggg ggctttttgg ggctttttgg gagtaaaaat 540 gagtaaaaat 540 gtgtcttcagagactgtgaa gtgtcttcag agactgtgaa catcaccatc catcaccatc actcaaggtt actcaaggtt tggcagtgtc tggcagtgtc aaccatctca aaccatctca 600 600 tcattctttc cacctgggta tcattctttc cacctgggta ccaagtctct ccaagtctct ttctgcttgg ttctgcttgg tgatggtact tgatggtact cctttttgca cctttttgca 660 660 gtggacacag gactatattt gtggacacag gactatattt ctctgtgaag ctctgtgaag acaaacattc acaaacattc gaagctcaac gaagctcaac aagagactgg aagagactgg 720 720 aaggaccataaatttaaatg aaggaccata aatttaaatg gagaaaggac gagaaaggac cctcaagaca cctcaagaca aatgaaatga 765 765

<210> <210> 206 206 <211> <211> 702 702 <212> <212> DNA DNA <213> <213> Homo sapiens Homo sapiens

<400> <400> 206 206 atgtggcagc tgctcctccc atgtggcagc tgctcctccc aactgctctg aactgctctg ctacttctag ctacttctag tttcagctgg tttcagctgg catgcggact catgcggact 60 60

gaagatctcccaaaggctgt gaagatctcc caaaggctgt ggtgttcctg ggtgttcctg gagcctcaat gagcctcaat ggtacagcgt ggtacagcgt gcttgagaag gcttgagaag 120 120

gacagtgtgactctgaagtg gacagtgtga ctctgaagtg ccagggagcc ccagggagec tactcccctg tactcccctg aggacaattc aggacaattc cacacagtgg cacacagtgg 180 180

gtcaacgacagtggagagta gtcaacgaca gtggagagta caggtgccag caggtgccag acaaacctct acaaacctct ccaccctcag ccaccctcag tgacccggtg tgacccggtg 300 300

cagctagaagtccatatcgg cagctagaag tccatatcgg ctggctgttg ctggctgttg ctccaggccc ctccaggccc ctcggtgggt ctcggtgggt gttcaaggag 360 gttcaaggag 360 gaagaccctattcacctgag gaagacccta ttcacctgag gtgtcacagc gtgtcacagc tggaagaaca tggaagaaca ctgctctgca ctgctctgca taaggtcaca taaggtcaca 420 420

tatttacaga atggcaaaga tatttacaga atggcaaaga caggaagtat caggaagtat tttcatcata tttcatcata attctgactt attctgactt ccacattcca ccacattcca 480 480

aaagccacactcaaagatag aaagccacac tcaaagatag cggctcctac cggctcctac ttctgcaggg ttctgcaggg ggcttgttgg ggcttgttgg gagtaaaaat gagtaaaaat 540 540

141 gtgtcttcagagactgtgaa gtgtcttcag agactgtgaa catcaccatc catcaccatc actcaaggtt actcaaggtt tggcagtgtc tggcagtgtc aaccatctca aaccatctca 600 600 tcattctctc cacctgggta tcattctctc cacctgggta ccaagtctct ccaagtctct ttctgcttgg ttctgcttgg tgatggtact tgatggtact cctttttgca cctttttgca 660 660 gtggacacag gactatattt gtggacacag gactatattt ctctgtgaag ctctgtgaag acaaacattt acaaacattt ga ga 702 702

<210> <210> 207 207 <211> <211> 374 374 <212> <212> PRT PRT <213> <213> Homo sapiens Homo sapiens

<400> <400> 207 207

Met Trp Met Trp Phe Phe Leu Leu Thr Thr Thr Thr Leu Leu Leu Leu Leu Leu Trp Trp Val Val Pro Pro Val Val Asp Asp Gly Gly Gln Gln 1 1 5 5 10 10 15 15

Val Asp Val Asp Thr ThrThr ThrLys Lys Ala Ala ValVal IleIle Thr Thr Leu Leu Gln Pro Gln Pro Pro Trp ProVal Trp Val Ser Ser 20 20 25 25 30 30

Val Phe Val Phe Gln GlnGlu GluGlu Glu Thr Thr ValVal ThrThr Leu Leu His His Cys Val Cys Glu Glu Leu ValHis Leu His Leu Leu 35 35 40 40 45 45

Pro Gly Pro Gly Ser SerSer SerSer SerThr Thr GlnGln TrpTrp Phe Phe Leu Leu Asn Asn Gly Ala Gly Thr ThrThr Ala Thr Gln Gln 50 50 55 55 60 60

Thr Ser Thr Ser Thr Thr Pro Pro Ser Ser Tyr Tyr Arg Arg Ile Ile Thr Thr Ser Ser Ala Ala Ser Ser Val Val Asn Asn Asp Asp Ser Ser

70 70 75 75 80 80

Gly Glu Gly Glu Tyr TyrArg ArgCys Cys Gln Gln ArgArg GlyGly Leu Leu Ser Ser Gly Ser Gly Arg Arg Asp SerPro Asp Pro Ile Ile 85 85 90 90 95 95

Gln Leu Gln Leu Glu GluIle IleHis His Arg Arg GlyGly TrpTrp Leu Leu Leu Leu Leu Val Leu Gln Gln Ser ValSer Ser Ser Arg Arg 100 100 105 105 110 110

Val Phe Val Phe Thr Thr Glu Glu Gly Gly Glu Glu Pro Pro Leu Leu Ala Ala Leu Leu Arg Arg Cys Cys His His Ala Ala Trp Trp Lys Lys 115 115 120 120 125 125

142

Asp Lys Asp Lys Leu LeuVal ValTyr Tyr Asn Asn ValVal LeuLeu Tyr Tyr Tyr Tyr Arg Gly Arg Asn Asn Lys GlyAla Lys Ala Phe Phe 130 130 135 135 140 140

Lys Phe Lys Phe Phe PheHis HisTrp Trp Asn Asn SerSer AsnAsn Leu Leu Thr Thr Ile Lys Ile Leu Leu Thr LysAsn Thr Asn Ile Ile 145 145 150 150 155 155 160 160

Ser His Ser His Asn AsnGly GlyThr Thr Tyr Tyr HisHis CysCys Ser Ser Gly Gly Met Lys Met Gly Gly His LysArg His Arg Tyr Tyr 165 165 170 170 175 175

Thr Ser Thr Ser Ala AlaGly GlyIle Ile Ser Ser ValVal ThrThr Val Val Lys Lys Glu Phe Glu Leu Leu Pro PheAla Pro Ala Pro Pro 180 180 185 185 190 190

Val Leu Val Leu Asn Asn Ala Ala Ser Ser Val Val Thr Thr Ser Ser Pro Pro Leu Leu Leu Leu Glu Glu Gly Gly Asn Asn Leu Leu Val Val 195 195 200 200 205 205

Thr Leu Thr Leu Ser SerCys CysGlu Glu Thr Thr LysLys LeuLeu Leu Leu Leu Leu Gln Pro Gln Arg Arg Gly ProLeu Gly Leu Gln Gln 210 210 215 215 220 220

Leu Tyr Leu Tyr Phe PheSer SerPhe Phe Tyr Tyr MetMet GlyGly Ser Ser Lys Lys Thr Arg Thr Leu Leu Gly ArgArg Gly Arg Asn Asn 225 225 230 230 235 235 240 240

Thr Ser Thr Ser Ser SerGlu GluTyr Tyr Gln Gln IleIle LeuLeu Thr Thr Ala Ala Arg Glu Arg Arg Arg Asp GluSer Asp Ser Gly Gly 245 245 250 250 255 255

Leu Tyr Leu Tyr Trp Trp Cys Cys Glu Glu Ala Ala Ala Ala Thr Thr Glu Glu Asp Asp Gly Gly Asn Asn Val Val Leu Leu Lys Lys Arg Arg 260 260 265 265 270 270

Ser Pro Ser Pro Glu GluLeu LeuGlu Glu Leu Leu GlnGln ValVal Leu Leu Gly Gly Leu Leu Leu Gln Gln Pro LeuThr Pro Thr Pro Pro 275 275 280 280 285 285

Val Trp Val Trp Phe Phe His His Val Val Leu Leu Phe Phe Tyr Tyr Leu Leu Ala Ala Val Val Gly Gly Ile Ile Met Met Phe Phe Leu Leu

143

290 295 295 300 300

Val Asn Val Asn Thr ThrVal ValLeu Leu Trp Trp ValVal ThrThr Ile Ile Arg Arg Lys Leu Lys Glu Glu Lys LeuArg Lys Arg Lys Lys 305 305 310 310 315 315 320 320

Lys Lys Lys Lys Trp TrpAsp AspLeu Leu Glu Glu IleIle SerSer Leu Leu Asp Asp Ser His Ser Gly Gly Glu HisLys Glu Lys Lys Lys 325 325 330 330 335 335

Val Ile Val Ile Ser Ser Ser Ser Leu Leu Gln Gln Glu Glu Asp Asp Arg Arg His His Leu Leu Glu Glu Glu Glu Glu Glu Leu Leu Lys Lys 340 340 345 345 350 350

Cys Gln Cys Gln Glu Glu Gln Gln Lys Lys Glu Glu Glu Glu Gln Gln Leu Leu Gln Gln Glu Glu Gly Gly Val Val His His Arg Arg Lys Lys 355 355 360 360 365 365

Glu Pro Glu Pro Gln GlnGly GlyAla Ala Thr Thr 370 370

<210> <210> 208 208 <211> <211> 316 316 <212> <212> PRT PRT <213> <213> Homo sapiens Homo sapiens

<400> <400> 208 208

Met Thr Met Thr Met Met Glu Glu Thr Thr Gln Gln Met Met Ser Ser Gln Gln Asn Asn Val Val Cys Cys Pro Pro Arg Arg Asn Asn Leu Leu 1 1 5 5 10 10 15 15

Trp Leu Trp Leu Leu LeuGln GlnPro Pro Leu Leu ThrThr ValVal Leu Leu Leu Leu Leu Ala Leu Leu Leu Ser AlaAla Ser Ala Asp Asp 20 20 25 25 30 30

Ser Gln Ser Gln Ala AlaAla AlaPro ProPro Pro LysLys AlaAla Val Val Leu Leu Lys Lys Leu Pro Leu Glu GluPro Pro Pro Trp Trp 35 35 40 40 45 45

Ile Asn Val Ile Asn ValLeu LeuGln GlnGlu Glu AspAsp SerSer Val Val Thr Thr Leu Leu Thr Gln Thr Cys CysGly Gln Gly Ala Ala 50 50 55 55 60 60

Arg Ser Arg Ser Pro Pro Glu Glu Ser Ser Asp Asp Ser Ser Ile Ile Gln Gln Trp Trp Phe Phe His His Asn Asn Gly Gly Asn Asn

144

Leu Leu

70 70 75 75 80 80

Ile Pro Thr Ile Pro ThrHis HisThr ThrGln Gln ProPro SerSer Tyr Tyr Arg Arg Phe Phe Lys Asn Lys Ala AlaAsn Asn Asn Asn Asn 85 85 90 90 95 95

Asp Ser Asp Ser Gly GlyGlu GluTyr Tyr Thr Thr CysCys GlnGln Thr Thr Gly Gly Gln Ser Gln Thr Thr Leu SerSer Leu Ser Asp Asp 100 100 105 105 110 110

Pro Val Pro Val His HisLeu LeuThr ThrVal Val LeuLeu SerSer Glu Glu Trp Trp Leu Leu Leu Val Val Gln LeuThr Gln Thr Pro Pro 115 115 120 120 125 125

His Leu His Leu Glu GluPhe PheGln Gln Glu Glu GlyGly GluGlu Thr Thr Ile Ile Met Arg Met Leu Leu Cys ArgHis Cys His Ser Ser 130 130 135 135 140 140

Trp Lys Trp Lys Asp Asp Lys Lys Pro Pro Leu Leu Val Val Lys Lys Val Val Thr Thr Phe Phe Phe Phe Gln Gln Asn Asn Gly Gly Lys Lys 145 145 150 150 155 155 160 160

Ser Gln Lys Ser Gln LysPhe PheSer SerHis His LeuLeu AspAsp Pro Pro Thr Thr Phe Phe Ser Pro Ser Ile IleGln Pro Gln Ala Ala 165 165 170 170 175 175

Asn His Asn His Ser Ser His His Ser Ser Gly Gly Asp Asp Tyr Tyr His His Cys Cys Thr Thr Gly Gly Asn Asn Ile Ile Gly Gly Tyr Tyr 180 180 185 185 190 190

Thr Leu Thr Leu Phe PheSer SerSer Ser Lys Lys ProPro ValVal Thr Thr Ile Ile Thr Gln Thr Val Val Val GlnPro Val Pro Ser Ser 195 195 200 200 205 205

Met Gly Met Gly Ser SerSer SerSer Ser Pro Pro MetMet GlyGly Val Val Ile Ile Val Val Val Ala Ala Val ValIle Val Ile Ala Ala 210 210 215 215 220 220

Thr Ala Thr Ala Val ValAla AlaAla Ala Ile Ile ValVal AlaAla Ala Ala Val Val Val Leu Val Ala Ala Ile LeuTyr Ile Tyr Cys Cys 225 225 230 230 235 235 240 240

145

Arg Lys Arg Lys Lys LysArg ArgIle Ile Ser Ser AlaAla AsnAsn Ser Ser Thr Thr Asp Val Asp Pro Pro Lys ValAla Lys Ala Ala Ala 245 245 250 250 255 255

Gln Phe Gln Phe Glu GluPro ProPro Pro Gly Gly ArgArg GlnGln Met Met Ile Ile Ala Arg Ala Ile Ile Lys ArgArg Lys Arg Gln Gln 260 260 265 265 270 270

Leu Glu Leu Glu Glu GluThr ThrAsn Asn Asn Asn AspAsp TyrTyr Glu Glu Thr Thr Ala Gly Ala Asp Asp Gly GlyTyr Gly Tyr Met Met 275 275 280 280 285 285

Thr Leu Thr Leu Asn AsnPro ProArg Arg Ala Ala ProPro ThrThr Asp Asp Asp Asp Asp Asn Asp Lys Lys Ile AsnTyr Ile Tyr Leu Leu 290 290 295 295 300 300

Thr Leu Thr Leu Pro Pro Pro Pro Asn Asn Asp Asp His His Val Val Asn Asn Ser Ser Asn Asn Asn Asn 305 305 310 310 315 315

<210> <210> 209 209 <211> <211> 317 317 <212> <212> PRT PRT <213> <213> Homo sapiens Homo sapiens

<400> <400> 209 209

Ser Gln Ser Gln Ala AlaAla AlaAla AlaPro Pro ProPro LysLys Ala Ala Val Val Leu Leu Lys Glu Lys Leu LeuPro Glu Pro Pro Pro 35 35 40 40 45 45

Trp Ile Trp Ile Asn AsnVal ValLeu Leu Gln Gln GluGlu AspAsp Ser Ser Val Val Thr Thr Thr Leu Leu Cys ThrGln Cys Gln Gly Gly 50 50 55 55 60 60

Ala Arg Ala Arg Ser SerPro ProGlu Glu Ser Ser AspAsp SerSer Ile Ile Gln Gln Trp His Trp Phe Phe Asn HisGly Asn Gly Asn Asn

70 70 75 75 80 80

146

Leu Ile Leu Ile Pro ProThr ThrHis His Thr Thr GlnGln ProPro Ser Ser Tyr Tyr Arg Lys Arg Phe Phe Ala LysAsn Ala Asn Asn Asn 85 85 90 90 95 95

Asn Asp Asn Asp Ser SerGly GlyGlu Glu Tyr Tyr ThrThr CysCys Gln Gln Thr Thr Gly Thr Gly Gln Gln Ser ThrLeu Ser Leu Ser Ser 100 100 105 105 110 110

Asp Pro Asp Pro Val Val His His Leu Leu Thr Thr Val Val Leu Leu Ser Ser Glu Glu Trp Trp Leu Leu Val Val Leu Leu Gln Gln Thr Thr 115 115 120 120 125 125

Pro His Pro His Leu LeuGlu GluPhe Phe Gln Gln GluGlu GlyGly Glu Glu Thr Thr Ile Leu Ile Met Met Arg LeuCys Arg Cys His His 130 130 135 135 140 140

Ser Trp Lys Ser Trp LysAsp AspLys LysPro Pro LeuLeu ValVal Lys Lys Val Val Thr Thr Phe Gln Phe Phe PheAsn Gln Asn Gly Gly 145 145 150 150 155 155 160 160

Lys Ser Lys Ser Gln GlnLys LysPhe Phe Ser Ser HisHis LeuLeu Asp Asp Pro Pro Thr Ser Thr Phe Phe Ile SerPro Ile Pro Gln Gln 165 165 170 170 175 175

Ala Asn Ala Asn His HisSer SerHis His Ser Ser GlyGly AspAsp Tyr Tyr His His Cys Gly Cys Thr Thr Asn GlyIle Asn Ile Gly Gly 180 180 185 185 190 190

Tyr Thr Tyr Thr Leu LeuPhe PheSer Ser Ser Ser LysLys ProPro Val Val Thr Thr Ile Val Ile Thr Thr Gln ValVal Gln Val Pro Pro 195 195 200 200 205 205

Ser Met Ser Met Gly GlySer SerSer SerSer Ser ProPro MetMet Gly Gly Ile Ile Ile Ala Ile Val Val Val AlaVal Val Val Ile Ile 210 210 215 215 220 220

Ala Thr Ala Thr Ala AlaVal ValAla Ala Ala Ala IleIle ValVal Ala Ala Ala Ala Val Ala Val Val Val Leu AlaIle Leu Ile Tyr Tyr 225 225 230 230 235 235 240 240

Cys Arg Cys Arg Lys LysLys LysArg Arg Ile Ile SerSer AlaAla Asn Asn Ser Ser Thr Pro Thr Asp Asp Val ProLys Val Lys Ala Ala 245 245 250 250 255 255

147

Ala Gln Ala Gln Phe PheGlu GluPro Pro Pro Pro GlyGly ArgArg Gln Gln Met Met Ile Ile Ile Ala Ala Arg IleLys Arg Lys Arg Arg 260 260 265 265 270 270

Gln Leu Gln Leu Glu Glu Glu Glu Thr Thr Asn Asn Asn Asn Asp Asp Tyr Tyr Glu Glu Thr Thr Ala Ala Asp Asp Gly Gly Gly Gly Tyr Tyr 275 275 280 280 285 285

Met Thr Met Thr Leu LeuAsn AsnPro Pro Arg Arg AlaAla ProPro Thr Thr Asp Asp Asp Lys Asp Asp Asp Asn LysIle Asn Ile Tyr Tyr 290 290 295 295 300 300

Leu Thr Leu Thr Leu Leu Pro Pro Pro Pro Asn Asn Asp Asp His His Val Val Asn Asn Ser Ser Asn Asn Asn Asn 305 305 310 310 315 315

<210> <210> 210 210 <211> <211> 218 218 <212> <212> PRT PRT <213> <213> Homo sapiens Homo sapiens

<400> <400> 210 210

Trp Leu Trp Leu Leu Leu Gln Gln Pro Pro Leu Leu Thr Thr Val Val Leu Leu Leu Leu Leu Leu Leu Leu Ala Ala Ser Ser Ala Ala Asp Asp 20 20 25 25 30 30

Ser Gln Ala Ser Gln AlaAla AlaAla AlaPro Pro ProPro LysLys Ala Ala Val Val Leu Leu Lys Glu Lys Leu LeuPro Glu Pro Pro Pro 35 35 40 40 45 45

Trp Ile Trp Ile Asn Asn Val Val Leu Leu Gln Gln Glu Glu Asp Asp Ser Ser Val Val Thr Thr Leu Leu Thr Thr Cys Cys Gln Gln Gly Gly 50 50 55 55 60 60

Ala Arg Ala Arg Ser Ser Pro Pro Glu Glu Ser Ser Asp Asp Ser Ser Ile Ile Gln Gln Trp Trp Phe Phe His His Asn Asn Gly Gly Asn Asn

70 70 75 75 80 80

Leu Ile Leu Ile Pro Pro Thr Thr His His Thr Thr Gln Gln Pro Pro Ser Ser Tyr Tyr Arg Arg Phe Phe Lys Lys Ala Ala Asn Asn Asn Asn 85 85 90 90 95 95

148

Asn Asp Asn Asp Ser Ser Gly Gly Glu Glu Tyr Tyr Thr Thr Cys Cys Gln Gln Thr Thr Gly Gly Gln Gln Thr Thr Ser Ser Leu Leu Ser Ser 100 100 105 105 110 110

Pro His Leu Pro His LeuGlu GluPhe PheGln Gln GluGlu GlyGly Glu Glu Thr Thr Ile Ile Met Arg Met Leu LeuCys Arg Cys His His 130 130 135 135 140 140

Lys Ser Lys Ser Gln GlnLys LysPhe Phe Ser Ser ArgArg LeuLeu Asp Asp Pro Pro Thr Ser Thr Phe Phe Ile SerPro Ile Pro Gln Gln 165 165 170 170 175 175

Ser Met Gly Ser Met GlySer SerSer SerSer Ser ProPro MetMet Gly Gly Ile Ile 210 210 215 215

<210> <210> 211 211 <211> <211> 218 218 <212> <212> PRT PRT <213> <213> Homo sapiens Homo sapiens

<400> <400> 211 211

Met Thr Met Thr Met MetGlu GluThr Thr Gln Gln MetMet SerSer Gln Gln Asn Asn Val Pro Val Cys Cys Arg ProAsn Arg Asn Leu Leu 1 1 5 5 10 10 15 15

149

70 70 75 75 80 80

150

<210> < 210> 212 212 <211> <211> 291 291 <212> <212> PRT PRT <213> <213> Homo sapiens Homo sapiens

<400> <400> 212 212

Met Gly Met Gly Ile Ile Leu Leu Ser Ser Phe Phe Leu Leu Pro Pro Val Val Leu Leu Ala Ala Thr Thr Glu Glu Ser Ser Asp Asp Trp Trp 1 1 5 5 10 10 15 15

Ala Asp Ala Asp Cys Cys Lys Lys Ser Ser Pro Pro Gln Gln Pro Pro Trp Trp Gly Gly His His Met Met Leu Leu Leu Leu Trp Trp Thr Thr 20 20 25 25 30 30

Ala Val Ala Val Leu LeuPhe PheLeu Leu Ala Ala ProPro ValVal Ala Ala Gly Gly Thr Ala Thr Pro Pro Ala AlaPro Ala Pro Pro Pro 35 35 40 40 45 45

Lys Ala Lys Ala Val Val Leu Leu Lys Lys Leu Leu Glu Glu Pro Pro Gln Gln Trp Trp Ile Ile Asn Asn Val Val Leu Leu Gln Gln Glu Glu 50 50 55 55 60 60

Asp Ser Asp Ser Val ValThr ThrLeu Leu Thr Thr CysCys ArgArg Gly Gly Thr Thr His Pro His Ser Ser Glu ProSer Glu Ser Asp Asp

70 70 75 75 80 80

Ser Ile Gln Ser Ile GlnTrp TrpPhe PheHis His AsnAsn GlyGly Asn Asn Leu Leu Ile Ile Pro His Pro Thr ThrThr His Thr Gln Gln 85 85 90 90 95 95

Pro Ser Tyr Pro Ser TyrArg ArgPhe PheLys Lys AlaAla AsnAsn Asn Asn Asn Asn Asp Asp Ser Glu Ser Gly GlyTyr Glu Tyr Thr Thr 100 100 105 105 110 110

Cys Gln Cys Gln Thr ThrGly GlyGln Gln Thr Thr SerSer LeuLeu Ser Ser Asp Asp Pro His Pro Val Val Leu HisThr Leu Thr Val Val 115 115 120 120 125 125

Leu Ser Leu Ser Glu GluTrp TrpLeu Leu Val Val LeuLeu GlnGln Thr Thr Pro Pro His Glu His Leu Leu Phe GluGln Phe Gln Glu Glu 130 130 135 135 140 140

151

Gly Glu Gly Glu Thr ThrIle IleVal Val Leu Leu ArgArg CysCys His His Ser Ser Trp Asp Trp Lys Lys Lys AspPro Lys Pro Leu Leu 145 145 150 150 155 155 160 160

Val Lys Val Lys Val Val Thr Thr Phe Phe Phe Phe Gln Gln Asn Asn Gly Gly Lys Lys Ser Ser Lys Lys Lys Lys Phe Phe Ser Ser Arg Arg 165 165 170 170 175 175

Ser Asp Ser Asp Pro ProAsn AsnPhe Phe Ser Ser IleIle ProPro Gln Gln Ala Ala Asn Ser Asn His His His SerSer His Ser Gly Gly 180 180 185 185 190 190

Asp Tyr Asp Tyr His HisCys CysThr Thr Gly Gly AsnAsn IleIle Gly Gly Tyr Tyr Thr Tyr Thr Leu Leu Ser TyrSer Ser Ser Lys Lys 195 195 200 200 205 205

Pro Val Pro Val Thr ThrIle IleThr ThrVal Val GlnGln AlaAla Pro Pro Ser Ser Ser Ser Ser Met Ser Pro ProGly Met Gly Ile Ile 210 210 215 215 220 220

Ile Val Ala Ile Val AlaVal ValVal ValThr Thr GlyGly IleIle Ala Ala Val Val Ala Ala Ala Val Ala Ile IleAla Val Ala Ala Ala 225 225 230 230 235 235 240 240

Val Val Val Val Ala Ala Leu Leu Ile Ile Tyr Tyr Cys Cys Arg Arg Lys Lys Lys Lys Arg Arg Ile Ile Ser Ser Ala Ala Asn Asn Pro Pro 245 245 250 250 255 255

Thr Asn Thr Asn Pro ProAsp AspGlu Glu Ala Ala AspAsp LysLys Val Val Gly Gly Ala Asn Ala Glu Glu Thr AsnIle Thr Ile Thr Thr 260 260 265 265 270 270

Tyr Ser Tyr Ser Leu Leu Leu Leu Met Met His His Pro Pro Asp Asp Ala Ala Leu Leu Glu Glu Glu Glu Pro Pro Asp Asp Asp Asp Gln Gln 275 275 280 280 285 285

Asn Arg Asn Arg Ile Ile 290 290

<210> <210> 213 213 <211> <211> 310 310 <212> <212> PRT PRT

152

<213> <213> Homo sapiens Homo sapiens

<400> <400> 213 213

70 70 75 75 80 80

Pro Ser Pro Ser Tyr TyrArg ArgPhe Phe Lys Lys AlaAla AsnAsn Asn Asn Asn Asn Asp Gly Asp Ser Ser Glu GlyTyr Glu Tyr Thr Thr 100 100 105 105 110 110

Leu Ser Leu Ser Glu Glu Trp Trp Leu Leu Val Val Leu Leu Gln Gln Thr Thr Pro Pro His His Leu Leu Glu Glu Phe Phe Gln Gln Glu Glu 130 130 135 135 140 140

Val Lys Val Lys Val Val Thr Thr Phe Phe Phe Phe Gln Gln Asn Asn Gly Gly Lys Lys Ser Ser Lys Lys Lys Lys Phe Phe Ser Ser

153

Arg Arg 165 165 170 170 175 175

Ser Asp Ser Asp Pro ProAsn AsnPhe PheSer Ser IleIle ProPro Gln Gln Ala Ala Asn Ser Asn His His His SerSer His Ser Gly Gly 180 180 185 185 190 190

Pro Val Thr Pro Val ThrIle IleThr ThrVal Val GlnGln AlaAla Pro Pro Ser Ser Ser Ser Ser Met Ser Pro ProGly Met Gly Ile Ile 210 210 215 215 220 220

Val Val Val Val Ala Ala Leu Leu Ile Ile Tyr Tyr Cys Cys Arg Arg Lys Lys Lys Lys Arg Arg Ile Ile Ser Ser Ala Ala Leu Leu Pro Pro 245 245 250 250 255 255

Gly Tyr Gly Tyr Pro ProGlu GluCys Cys Arg Arg GluGlu MetMet Gly Gly Glu Glu Thr Pro Thr Leu Leu Glu ProLys Glu Lys Pro Pro 260 260 265 265 270 270

Ala Asn Ala Asn Pro ProThr ThrAsn Asn Pro Pro AspAsp GluGlu Ala Ala Asp Asp Lys Gly Lys Val Val Ala GlyGlu Ala Glu Asn Asn 275 275 280 280 285 285

Thr Ile Thr Ile Thr ThrTyr TyrSer Ser Leu Leu LeuLeu MetMet His His Pro Pro Asp Leu Asp Ala Ala Glu LeuGlu Glu Glu Pro Pro 290 290 295 295 300 300

Asp Asp Asp Asp Gln GlnAsn AsnArg Arg Ile Ile 305 305 310 310

<210> <210> 214 214 <211> <211> 217 217 <212> <212> PRT PRT <213> <213> Homo sapiens Homo sapiens

<400> <400> 214 214

154

70 70 75 75 80 80

Ser Ile Ser Ile Gln GlnTrp TrpPhe PheHis His AsnAsn GlyGly Asn Asn Leu Leu Ile Thr Ile Pro Pro His ThrThr His Thr Gln Gln 85 85 90 90 95 95

Val Lys Val Lys Val ValThr ThrPhe Phe Phe Phe GlnGln AsnAsn Gly Gly Lys Lys Ser Lys Ser Lys Lys Phe LysSer Phe Ser Arg Arg 165 165 170 170 175 175

155

Ser Asp Pro Ser Asp ProAsn AsnPhe PheSer Ser IleIle ProPro Gln Gln Ala Ala Asn Asn His His His Ser SerSer His Ser Gly Gly 180 180 185 185 190 190

Pro Val Pro Val Thr ThrIle IleThr ThrVal Val GlnGln AlaAla Pro Pro 210 210 215 215

<210> <210> 215 215 <211> <211> 254 254 <212> <212> PRT PRT <213> <213> Homo sapiens Homo sapiens

<400> <400> 215 215

Met Trp Met Trp Gln GlnLeu LeuLeu Leu Leu Leu ProPro ThrThr Ala Ala Leu Leu Leu Leu Leu Leu Leu Val LeuSer Val Ser Ala Ala 1 1 5 5 10 10 15 15

Gly Met Gly Met Arg Arg Thr Thr Glu Glu Asp Asp Leu Leu Pro Pro Lys Lys Ala Ala Val Val Val Val Phe Phe Leu Leu Glu Glu Pro Pro 20 20 25 25 30 30

Gln Trp Gln Trp Tyr TyrArg ArgVal Val Leu Leu GluGlu LysLys Asp Asp Ser Ser Val Leu Val Thr Thr Lys LeuCys Lys Cys Gln Gln 35 35 40 40 45 45

Gly Ala Gly Ala Tyr Tyr Ser Ser Pro Pro Glu Glu Asp Asp Asn Asn Ser Ser Thr Thr Gln Gln Trp Trp Phe Phe His His Asn Asn Glu Glu 50 50 55 55 60 60

Ser Leu Ile Ser Leu IleSer SerSer SerGln Gln AlaAla SerSer Ser Ser Tyr Tyr Phe Phe Ile Ala Ile Asp AspAla Ala Ala Thr Thr

70 70 75 75 80 80

Val Asp Val Asp Asp AspSer SerGly Gly Glu Glu TyrTyr ArgArg Cys Cys Gln Gln Thr Leu Thr Asn Asn Ser LeuThr Ser Thr Leu Leu 85 85 90 90 95 95

Ser Asp Pro Ser Asp ProVal ValGln GlnLeu Leu GluGlu ValVal His His Ile Ile Gly Gly Trp Leu Trp Leu LeuLeu Leu Leu Gln Gln 100 100 105 105 110 110

156

Ala Pro Ala Pro Arg Arg Trp Trp Val Val Phe Phe Lys Lys Glu Glu Glu Glu Asp Asp Pro Pro Ile Ile His His Leu Leu Arg Arg Cys Cys 115 115 120 120 125 125

His Ser His Ser Trp Trp Lys Lys Asn Asn Thr Thr Ala Ala Leu Leu His His Lys Lys Val Val Thr Thr Tyr Tyr Leu Leu Gln Gln Asn Asn 130 130 135 135 140 140

Gly Lys Gly Lys Gly GlyArg ArgLys Lys Tyr Tyr PhePhe HisHis His His Asn Asn Ser Phe Ser Asp Asp Tyr PheIle Tyr Ile Pro Pro 145 145 150 150 155 155 160 160

Lys Ala Lys Ala Thr Thr Leu Leu Lys Lys Asp Asp Ser Ser Gly Gly Ser Ser Tyr Tyr Phe Phe Cys Cys Arg Arg Gly Gly Leu Leu Val Val 165 165 170 170 175 175

Gly Ser Gly Ser Lys Lys Asn Asn Val Val Ser Ser Ser Ser Glu Glu Thr Thr Val Val Asn Asn Ile Ile Thr Thr Ile Ile Thr Thr Gln Gln 180 180 185 185 190 190

Gly Leu Gly Leu Ser Ser Val Val Ser Ser Thr Thr Ile Ile Ser Ser Ser Ser Phe Phe Phe Phe Pro Pro Pro Pro Gly Gly Tyr Tyr Gln Gln 195 195 200 200 205 205

Val Ser Val Ser Phe Phe Cys Cys Leu Leu Val Val Met Met Val Val Leu Leu Leu Leu Phe Phe Ala Ala Val Val Asp Asp Thr Thr Gly Gly 210 210 215 215 220 220

Leu Tyr Leu Tyr Phe Phe Ser Ser Val Val Lys Lys Thr Thr Asn Asn Ile Ile Arg Arg Ser Ser Ser Ser Thr Thr Arg Arg Asp Asp Trp Trp 225 225 230 230 235 235 240 240

Lys Asp Lys Asp His HisLys LysPhe Phe Lys Lys TrpTrp ArgArg Lys Lys Asp Asp Pro Asp Pro Gln Gln Lys Asp Lys 245 245 250 250

<210> <210> 216 216 <211> <211> 254 254 <212> <212> PRT PRT <213> <213> Homo sapiens Homo sapiens

<400> <400> 216 216

157

Ser Leu Ile Ser Leu IleSer SerSer SerGln GlnAlaAla SerSer Ser Ser Tyr Tyr Phe Phe Ile Ala Ile Asp AspAla Ala Ala Thr Thr

70 70 75 75 80 80

Ser Asp Ser Asp Pro ProVal ValGln GlnLeu Leu GluGlu ValVal His His Ile Ile Gly Leu Gly Trp Trp Leu LeuLeu Leu Leu Gln Gln 100 100 105 105 110 110

Ala Pro Ala Pro Arg ArgTrp TrpVal Val Phe Phe LysLys GluGlu Glu Glu Asp Asp Pro His Pro Ile Ile Leu HisArg Leu Arg Cys Cys 115 115 120 120 125 125

His Ser His Ser Trp TrpLys LysAsn Asn Thr Thr AlaAla LeuLeu His His Lys Lys Val Tyr Val Thr Thr Leu TyrGln Leu Gln Asn Asn 130 130 135 135 140 140

Lys Ala Lys Ala Thr Thr Leu Leu Lys Lys Asp Asp Ser Ser Gly Gly Ser Ser Tyr Tyr Phe Phe Cys Cys Arg Arg Gly Gly Leu Leu Phe Phe 165 165 170 170 175 175

Gly Ser Gly Ser Lys LysAsn AsnVal Val Ser Ser SerSer GluGlu Thr Thr Val Val Asn Thr Asn Ile Ile Ile ThrThr Ile Thr Gln Gln 180 180 185 185 190 190

158

Gly Leu Gly Leu Ala AlaVal ValSer Ser Thr Thr IleIle SerSer Ser Ser Phe Phe Phe Pro Phe Pro Pro Gly ProTyr Gly Tyr Gln Gln 195 195 200 200 205 205

Val Ser Val Ser Phe PheCys CysLeu Leu Val Val MetMet ValVal Leu Leu Leu Leu Phe Val Phe Ala Ala Asp ValThr Asp Thr Gly Gly 210 210 215 215 220 220

Leu Tyr Leu Tyr Phe PheSer SerVal Val Lys Lys ThrThr AsnAsn Ile Ile Arg Arg Ser Thr Ser Ser Ser Arg ThrAsp Arg Asp Trp Trp 225 225 230 230 235 235 240 240

<210> <210> 217 217 <211> <211> 208 208 <212> <212> PRT PRT <213> <213> Homo sapiens Homo sapiens

<400> <400> 217 217

Gly Met Gly Met Arg ArgThr ThrGlu Glu Asp Asp LeuLeu ProPro Lys Lys Ala Ala Val Phe Val Val Val Leu PheGlu Leu Glu Pro Pro 20 20 25 25 30 30

Gly Ala Gly Ala Tyr TyrSer SerPro Pro Glu Glu AspAsp AsnAsn Ser Ser Thr Thr Gln Phe Gln Trp Trp His PheAsn His Asn Glu Glu 50 50 55 55 60 60

Ser Leu Ser Leu Ile IleSer SerSer Ser Gln Gln AlaAla SerSer Ser Ser Tyr Tyr Phe Asp Phe Ile Ile Ala AspAla Ala Ala Thr Thr

70 70 75 75 80 80

Val Asp Val Asp Asp AspSer SerGly Gly Glu Glu TyrTyr ArgArg Cys Cys Gln Gln Thr Leu Thr Asn Asn Ser LeuThr Ser Thr Leu Leu

159

85 90 90 95 95

Lys Ala Lys Ala Thr ThrLeu LeuLys Lys Asp Asp SerSer GlyGly Ser Ser Tyr Tyr Phe Arg Phe Cys Cys Gly ArgLeu Gly Leu Phe Phe 165 165 170 170 175 175

<210> <210> 218 218 <211> <211> 208 208 <212> <212> PRT PRT <213> <213> Homo sapiens Homo sapiens

<400> <400> 218 218

160

70 70 75 75 80 80

Lys Ala Lys Ala Thr ThrLeu LeuLys Lys Asp Asp SerSer GlyGly Ser Ser Tyr Tyr Phe Arg Phe Cys Cys Gly ArgLeu Gly Leu Val Val 165 165 170 170 175 175

161

<210> <210> 219 219 <211> <211> 233 233 <212> <212> PRT PRT <213> <213> Homo sapiens Homo sapiens

<400> <400> 219 219

Gln Trp Gln Trp Tyr TyrSer SerVal Val Leu Leu GluGlu LysLys Asp Asp Ser Ser Val Leu Val Thr Thr Lys LeuCys Lys Cys Gln Gln 35 35 40 40 45 45

Ser Leu Ser Leu Ile IleSer SerSer SerGln Gln AlaAla SerSer Ser Ser Tyr Tyr Phe Asp Phe Ile Ile Ala AspAla Ala Ala Thr Thr

70 70 75 75 80 80

Val Asn Val Asn Asp AspSer SerGly Gly Glu Glu TyrTyr ArgArg Cys Cys Gln Gln Thr Leu Thr Asn Asn Ser LeuThr Ser Thr Leu Leu 85 85 90 90 95 95

Gly Lys Gly Lys Asp AspArg ArgLys Lys Tyr Tyr PhePhe HisHis His His Asn Asn Ser Phe Ser Asp Asp His PheIle His Ile Pro Pro 145 145 150 150 155 155 160 160

162

Gly Leu Gly Leu Ala AlaVal ValSer Ser Thr Thr IleIle SerSer Ser Ser Phe Phe Ser Pro Ser Pro Pro Gly ProTyr Gly Tyr Gln Gln 195 195 200 200 205 205

Leu Tyr Leu Tyr Phe PheSer SerVal Val Lys Lys ThrThr AsnAsn Ile Ile 225 225 230 230

<210> <210> 220 220 <211> <211> 211 211 <212> <212> PRT PRT <213> <213> Macaca fascicularis Macaca fascicularis

<400> <400> 220 220

Met Ser Met Ser Gln GlnAsn AsnVal Val Cys Cys ProPro GlyGly Asn Asn Leu Leu Trp Leu Trp Leu Leu Gln LeuPro Gln Pro Leu Leu 1 1 5 5 10 10 15 15

Thr Val Thr Val Leu LeuLeu LeuLeu Leu Leu Leu AlaAla SerSer Ala Ala Asp Asp Ser Thr Ser Gln Gln Ala ThrPro Ala Pro Pro Pro 20 20 25 25 30 30

Lys Ala Lys Ala Val ValLeu LeuLys Lys Leu Leu GluGlu ProPro Pro Pro Trp Trp Ile Val Ile Asn Asn Leu ValArg Leu Arg Glu Glu 35 35 40 40 45 45

Asp Ser Asp Ser Val ValThr ThrLeu Leu Thr Thr CysCys GlyGly Gly Gly Ala Ala His Pro His Ser Ser Asp ProSer Asp Ser Asp Asp 50 50 55 55 60 60

Ser Thr Ser Thr Gln GlnTrp TrpPhe PheHis His AsnAsn GlyGly Asn Asn Leu Leu Ile Ile Pro His Pro Thr ThrThr His Thr Gln Gln

70 70 75 75 80 80

163

Pro Ser Pro Ser Tyr TyrArg ArgPhe Phe Lys Lys AlaAla AsnAsn Asn Asn Asn Asn Asp Gly Asp Ser Ser Glu GlyTyr Glu Tyr Arg Arg 85 85 90 90 95 95

Cys Gln Cys Gln Thr ThrGly GlyArg Arg Thr Thr SerSer LeuLeu Ser Ser Asp Asp Pro His Pro Val Val Leu HisThr Leu Thr Val Val 100 100 105 105 110 110

Leu Ser Leu Ser Glu GluTrp TrpLeu Leu Ala Ala LeuLeu GlnGln Thr Thr Pro Pro His Glu His Leu Leu Phe GluArg Phe Arg Glu Glu 115 115 120 120 125 125

Gly Glu Gly Glu Thr ThrIle IleMet Met Leu Leu ArgArg CysCys His His Ser Ser Trp Asp Trp Lys Lys Lys AspPro Lys Pro Leu Leu 130 130 135 135 140 140

Ile Lys Val Ile Lys ValThr ThrPhe PhePhe Phe GlnGln AsnAsn Gly Gly Ile Ile Ala Ala Lys Phe Lys Lys LysSer Phe Ser His His 145 145 150 150 155 155 160 160

Met Asp Met Asp Pro ProAsn AsnPhe Phe Ser Ser IleIle ProPro Arg Arg Ala Ala Asn Ser Asn His His His SerSer His Ser Gly Gly 165 165 170 170 175 175

Asp Tyr Asp Tyr His HisCys CysThr Thr Gly Gly AsnAsn IleIle Gly Gly Tyr Tyr Thr Tyr Thr Pro Pro Ser TyrSer Ser Ser Lys Lys 180 180 185 185 190 190

Pro Val Pro Val Thr ThrIle IleThr ThrVal Val GlnGln ValVal Pro Pro Ser Ser Val Ser Val Gly Gly Ser SerSer Ser Ser Pro Pro 195 195 200 200 205 205

Met Gly Met Gly Ile Ile 210 210

<210> <210> 221 221 <211> <211> 211 211 <212> <212> PRT PRT <213> <213> Macaca fascicularis Macaca fascicularis

<400> <400> 221 221

Met Ser Met Ser Gln GlnAsn AsnVal Val Cys Cys ProPro GlyGly Asn Asn Leu Leu Trp Leu Trp Leu Leu Gln LeuPro Gln Pro Leu Leu

164

1 5 5 10 10 15 15

Ser Thr Gln Ser Thr GlnTrp TrpPhe PheHis HisAsnAsn GlyGly Asn Asn Leu Leu Ile Ile Pro His Pro Thr ThrThr His Thr Gln Gln

70 70 75 75 80 80

Leu Ser Leu Ser Glu GluTrp TrpLeu Leu Ala Ala LeuLeu GlnGln Thr Thr Thr Thr His Glu His Leu Leu Phe GluArg Phe Arg Glu Glu 115 115 120 120 125 125

Ile Lys Val Ile Lys ValAla AlaPhe PhePhe Phe GlnGln AsnAsn Gly Gly Lys Lys Ser Ser Lys Phe Lys Asn AsnSer Phe Ser His His 145 145 150 150 155 155 160 160

Met Asn Met Asn Pro ProAsn AsnPhe Phe Ser Ser IleIle ProPro Gln Gln Ala Ala Asn Ser Asn His His His SerSer His Ser Gly Gly 165 165 170 170 175 175

Asp Tyr Asp Tyr His HisCys CysThr Thr Gly Gly AsnAsn IleIle Gly Gly Tyr Tyr Thr Tyr Thr Pro Pro Ser TyrSer Ser Ser Lys Lys

165

180 185 185 190 190

Pro Val Thr Pro Val ThrIle IleThr ThrVal Val GlnGln ValVal Pro Pro Ser Ser Val Val Gly Ser Gly Ser SerSer Ser Ser Pro Pro 195 195 200 200 205 205

Met Gly Met Gly Ile Ile 210 210

<210> <210> 222 222 <211> <211> 211 211 <212> <212> PRT PRT <213> <213> Macaca fascicularis Macaca fascicularis

<400> <400> 222 222

Ser Thr Gln Ser Thr GlnTrp TrpPhe PheHis His AsnAsn GlyGly Asn Asn Leu Leu Ile Ile Pro His Pro Thr ThrThr His Thr Gln Gln

70 70 75 75 80 80

Leu Ser Leu Ser Glu GluTrp TrpLeu Leu Ala Ala LeuLeu GlnGln Thr Thr Thr Thr His Glu His Leu Leu Phe GluArg Phe Arg Glu Glu

166

115 120 120 125 125

Ile Lys Val Ile Lys ValAla AlaPhe PhePhe Phe GlnGln AsnAsn Gly Gly Ile Ile Ser Ser Lys Phe Lys Lys LysSer Phe Ser Pro Pro 145 145 150 150 155 155 160 160

Met Asn Met Asn Pro ProAsn AsnPhe Phe Ser Ser IleIle ProPro Arg Arg Ala Ala Asn Ser Asn His His His SerSer His Ser Gly Gly 165 165 170 170 175 175

Met Gly Met Gly Ile Ile 210 210

<210> <210> 223 223 <211> <211> 217 217 <212> <212> PRT PRT <213> <213> Macaca fascicularis Macaca fascicularis

<400> <400> 223 223

Ala Asp Ala Asp Cys Cys Lys Lys Ser Ser Ser Ser Gln Gln Pro Pro Trp Trp Gly Gly His His Met Met Leu Leu Leu Leu Trp Trp Thr Thr 20 20 25 25 30 30

Lys Ala Lys Ala Val Val Leu Leu Lys Lys Leu Leu Glu Glu Pro Pro Pro Pro Trp Trp Ile Ile Asn Asn Val Val Leu Leu Arg Arg

167

Glu Glu 50 50 55 55 60 60

Asp Ser Asp Ser Val ValThr ThrLeu Leu Thr Thr CysCys GlyGly Gly Gly Ala Ala His Pro His Ser Ser Asp ProSer Asp Ser Asp Asp

70 70 75 75 80 80

Ser Thr Gln Ser Thr GlnTrp TrpPhe PheHis His AsnAsn GlyGly Asn Asn Leu Leu Ile Ile Pro His Pro Thr ThrThr His Thr Gln Gln 85 85 90 90 95 95

Pro Ser Tyr Pro Ser TyrArg ArgPhe PheLys Lys AlaAla AsnAsn Asn Asn Asn Asn Asp Asp Ser Glu Ser Gly GlyTyr Glu Tyr Arg Arg 100 100 105 105 110 110

Cys Gln Cys Gln Thr ThrGly GlyArg Arg Thr Thr SerSer LeuLeu Ser Ser Asp Asp Pro His Pro Val Val Leu HisThr Leu Thr Val Val 115 115 120 120 125 125

Leu Ser Leu Ser Glu GluTrp TrpLeu Leu Ala Ala LeuLeu GlnGln Thr Thr Pro Pro His Glu His Leu Leu Phe GluArg Phe Arg Glu Glu 130 130 135 135 140 140

Gly Glu Gly Glu Thr ThrIle IleMet Met Leu Leu ArgArg CysCys His His Ser Ser Trp Asp Trp Lys Lys Lys AspPro Lys Pro Leu Leu 145 145 150 150 155 155 160 160

Ile Lys Val Ile Lys ValThr ThrPhe PhePhe Phe GlnGln AsnAsn Gly Gly Ile Ile Ser Ser Lys Phe Lys Lys LysSer Phe Ser His His 165 165 170 170 175 175

Met Asn Met Asn Pro ProAsn AsnPhe Phe Ser Ser IleIle ProPro Gln Gln Ala Ala Asn Ser Asn His His His SerSer His Ser Gly Gly 180 180 185 185 190 190

Asp Tyr Asp Tyr His HisCys CysThr Thr Gly Gly AsnAsn IleIle Gly Gly Tyr Tyr Thr Tyr Thr Pro Pro Ser TyrSer Ser Ser Lys Lys 195 195 200 200 205 205

Pro Val Thr Pro Val ThrIle IleThr ThrVal Val GlnGln ValVal Pro Pro 210 210 215 215

<210> <210> 224 224 <211> <211> 208 208

168

<212> <212> PRT PRT <213> <213> Macaca fascicularis Macaca fascicularis

<400> <400> 224 224

Gly Met Gly Met Arg Arg Ala Ala Glu Glu Asp Asp Leu Leu Pro Pro Lys Lys Ala Ala Val Val Val Val Phe Phe Leu Leu Glu Glu Pro Pro 20 20 25 25 30 30

Gly Ala Gly Ala Tyr Tyr Ser Ser Pro Pro Glu Glu Asp Asp Asn Asn Ser Ser Thr Thr Arg Arg Trp Trp Phe Phe His His Asn Asn Glu Glu 50 50 55 55 60 60

Ser Leu Ile Ser Leu IleSer SerSer SerGln Gln ThrThr SerSer Ser Ser Tyr Tyr Phe Phe Ile Ala Ile Ala AlaAla Ala Ala Arg Arg

70 70 75 75 80 80

Val Asn Val Asn Asn AsnSer SerGly Gly Glu Glu TyrTyr ArgArg Cys Cys Gln Gln Thr Leu Thr Ser Ser Ser LeuThr Ser Thr Leu Leu 85 85 90 90 95 95

Ala Pro Ala Pro Arg Arg Trp Trp Val Val Phe Phe Lys Lys Glu Glu Glu Glu Glu Glu Ser Ser Ile Ile His His Leu Leu Arg Arg Cys Cys 115 115 120 120 125 125

His Ser His Ser Trp TrpLys LysAsn Asn Thr Thr LeuLeu LeuLeu His His Lys Lys Val Tyr Val Thr Thr Leu TyrGln Leu Gln Asn Asn 130 130 135 135 140 140

Gly Lys Gly Lys Gly GlyArg ArgLys Lys Tyr Tyr PhePhe HisHis Gln Gln Asn Asn Ser Phe Ser Asp Asp Tyr PheIle Tyr Ile Pro Pro 145 145 150 150 155 155 160 160

169

Lys Ala Lys Ala Thr Thr Leu Leu Lys Lys Asp Asp Ser Ser Gly Gly Ser Ser Tyr Tyr Phe Phe Cys Cys Arg Arg Gly Gly Leu Leu Ile Ile 165 165 170 170 175 175

Asp Leu Asp Leu Ala AlaVal ValSer Ser Ser Ser IleIle SerSer Ser Ser Phe Phe Phe Pro Phe Pro Pro Gly ProTyr Gly Tyr Gln Gln 195 195 200 200 205 205

<210> <210> 225 225 <211> <211> 115 115 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 225 225

Gln Val Gln Val Gln GlnLeu LeuVal Val Gln Gln SerSer GlyGly Ala Ala Glu Glu Val Lys Val Lys Lys Pro LysGly Pro Gly Ala Ala 1 1 5 5 10 10 15 15

Ser Val Ser Val Thr ThrVal ValSer SerCys Cys LysLys AlaAla Ser Ser Gly Gly Tyr Tyr Thr Thr Thr Phe PheAsp Thr Asp Tyr Tyr 20 20 25 25 30 30

Glu Met Glu Met His His Trp Trp Ile Ile Arg Arg Gln Gln Pro Pro Pro Pro Gly Gly Glu Glu Gly Gly Leu Leu Glu Glu Trp Trp Ile Ile 35 35 40 40 45 45

Gly Ala Gly Ala Ile IleAsp AspPro Pro Lys Lys ThrThr GlyGly Asp Asp Thr Thr Ala Ser Ala Tyr Tyr Glu SerSer Glu Ser Phe Phe 50 50 55 55 60 60

Gln Asp Gln Asp Arg ArgVal ValThr Thr Leu Leu ThrThr AlaAla Asp Asp Lys Lys Ser Ser Ser Thr Thr Thr SerAla Thr Ala Tyr Tyr

70 70 75 75 80 80

Met Glu Met Glu Leu LeuSer SerSer Ser Leu Leu ThrThr SerSer Glu Glu Asp Asp Thr Val Thr Ala Ala Tyr ValTyr Tyr Tyr Cys Cys 85 85 90 90 95 95

Thr Arg Thr Arg Phe PheTyr TyrSer Ser Tyr Tyr ThrThr TyrTyr Trp Trp Gly Gly Gln Thr Gln Gly Gly Leu ThrVal Leu Val

170

Thr Thr 100 100 105 105 110 110

Val Ser Val Ser Ser Ser 115 115

<210> <210> 226 226 <211> <211> 219 219 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 226 226

Asp Ile Asp Ile Val ValMet MetThr Thr Gln Gln SerSer ProPro Leu Leu Ser Ser Leu Val Leu Pro Pro Thr ValPro Thr Pro Gly Gly 1 1 5 5 10 10 15 15

Glu Pro Glu Pro Ala AlaSer SerIle Ile Ser Ser CysCys GlnGln Ala Ala Ser Ser Glu Leu Glu Ser Ser Val LeuHis Val His Ser Ser 20 20 25 25 30 30

Asn Arg Asn Arg Asn Asn Thr Thr Tyr Tyr Leu Leu His His Trp Trp Tyr Tyr Leu Leu Gln Gln Lys Lys Pro Pro Gly Gly Gln Gln Ser Ser 35 35 40 40 45 45

Pro Gln Leu Pro Gln LeuLeu LeuIle IleTyr Tyr LysLys ValVal Ser Ser Asn Asn Arg Arg Phe Gly Phe Ser SerVal Gly Val Pro Pro 50 50 55 55 60 60

Asp Arg Asp Arg Phe PheSer SerGly Gly Ser Ser GlyGly SerSer Gly Gly Thr Thr Asp Thr Asp Phe Phe Leu ThrLys Leu Lys Ile Ile

70 70 75 75 80 80

Ser Arg Val Ser Arg ValGlu GluAla AlaGlu Glu AspAsp ValVal Gly Gly Val Val Tyr Tyr Tyr Ser Tyr Cys CysGln Ser Gln Asn Asn 85 85 90 90 95 95

Thr His Thr His Val ValPro ProPro Pro Thr Thr PhePhe GlyGly Gln Gln Gly Gly Thr Val Thr Lys Lys Glu ValIle Glu Ile Glu Glu 100 100 105 105 110 110

Arg Thr Arg Thr Val Val Ala Ala Ala Ala Pro Pro Ser Ser Val Val Phe Phe Ile Ile Phe Phe Pro Pro Pro Pro Ser Ser Asp Asp Glu Glu 115 115 120 120 125 125

171

Gln Leu Gln Leu Lys LysSer SerGly Gly Thr Thr AlaAla SerSer Val Val Val Val Cys Leu Cys Leu Leu Asn LeuAsn Asn Asn Phe Phe 130 130 135 135 140 140

Tyr Pro Tyr Pro Arg Arg Glu Glu Ala Ala Lys Lys Val Val Gln Gln Trp Trp Lys Lys Val Val Asp Asp Asn Asn Ala Ala Leu Leu Gln Gln 145 145 150 150 155 155 160 160

Ser Gly Ser Gly Asn AsnSer SerGln GlnGlu Glu SerSer ValVal Thr Thr Glu Glu Gln Gln Asp Lys Asp Ser SerAsp Lys Asp Ser Ser 165 165 170 170 175 175

Thr Tyr Thr Tyr Ser SerLeu LeuSer Ser Ser Ser ThrThr LeuLeu Thr Thr Leu Leu Ser Ala Ser Lys Lys Asp AlaTyr Asp Tyr Glu Glu 180 180 185 185 190 190

Lys His Lys His Lys LysVal ValTyr Tyr Ala Ala CysCys GluGlu Val Val Thr Thr His Gly His Gln Gln Leu GlySer Leu Ser Ser Ser 195 195 200 200 205 205

Pro Val Pro Val Thr ThrLys LysSer SerPhe Phe AsnAsn ArgArg Gly Gly Glu Glu Cys Cys 210 210 215 215

<210> <210> 227 227 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 227 227

Ser Thr Ser Thr Ser SerGly GlyGly GlyThr Thr AlaAla AlaAla Leu Leu Gly Gly Cys Cys Leu Lys Leu Val ValAsp Lys Asp Tyr Tyr 20 20 25 25 30 30

172

70 70 75 75 80 80

Pro Ala Pro Ala Pro ProGlu GluLeu Leu Leu Leu GlyGly GlyGly Pro Pro Ser Ser Val Leu Val Phe Phe Phe LeuPro Phe Pro Pro Pro 115 115 120 120 125 125

173

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 228 228 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 228 228

174

70 70 75 75 80 80

175

Gln Glu Gln Glu Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 229 229 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 229 229

176

70 70 75 75 80 80

Pro Ala Pro Ala Pro ProGlu GluLeu Leu Leu Leu AsnAsn GlyGly Pro Pro Ser Ser Val Leu Val Phe Phe Phe LeuPro Phe Pro Pro Pro 115 115 120 120 125 125

Val Val Val Val Val Val Asp Asp Val Val Ser Ser Glu Glu Glu Glu Asp Asp Pro Pro Glu Glu Val Val Lys Lys Phe Phe Asn Asn Trp Trp 145 145 150 150 155 155 160 160

177

Lys Ala Lys Ala Leu LeuPro ProLys Lys Pro Pro IleIle GluGlu Lys Lys Thr Thr Ile Lys Ile Ser Ser Ala LysLys Ala Lys Gly Gly 210 210 215 215 220 220

Gln Pro Gln Pro Arg ArgGlu GluPro Pro Gln Gln ValVal TyrTyr Thr Thr Leu Leu Pro Ser Pro Pro Pro Arg SerGlu Arg Glu Glu Glu 225 225 230 230 235 235 240 240

Asn Tyr Asn Tyr Lys LysThr ThrThr Thr Pro Pro MetMet ValVal Leu Leu Asp Asp Ser Gly Ser Asp Asp Ser GlyPhe Ser Phe Phe Phe 275 275 280 280 285 285

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 230 230 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 230 230

178

70 70 75 75 80 80

Pro Ala Pro Ala Pro ProGlu GluLeu LeuLeu Leu AsnAsn GlyGly Pro Pro Ser Ser Val Leu Val Phe Phe Phe LeuPro Phe Pro Pro Pro 115 115 120 120 125 125

179

Asn Tyr Asn Tyr Lys LysThr ThrThr Thr Pro Pro MetMet ValVal Leu Leu Asp Asp Arg Gly Arg Asp Asp Ser GlyPhe Ser Phe Phe Phe 275 275 280 280 285 285

Leu Tyr Leu Tyr Ser Ser Lys Lys Leu Leu Thr Thr Val Val Lys Lys Lys Lys Ser Ser Arg Arg Trp Trp Gln Gln Gln Gln Gly Gly Asn Asn 290 290 295 295 300 300

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 231 231 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

180

<400> <400> 231 231

70 70 75 75 80 80

Val Val Val Val Val Val Asp Asp Val Val Ser Ser Asp Asp Glu Glu Asp Asp Pro Pro Glu Glu Val Val Lys Lys Phe Phe Asn Asn Trp Trp 145 145 150 150 155 155 160 160

181

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

182

<210> <210> 232 232 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 232 232

70 70 75 75 80 80

183

Glu Gln Glu Gln Tyr Tyr Asn Asn Ser Ser Thr Thr Tyr Tyr Arg Arg Val Val Val Val Ser Ser Val Val Leu Leu Thr Thr Val Val Leu Leu 180 180 185 185 190 190

His Arg His Arg Asp AspTrp TrpLeu Leu Asn Asn GlyGly LysLys Glu Glu Tyr Tyr Lys Lys Lys Cys Cys Val LysSer Val Ser Asn Asn 195 195 200 200 205 205

184

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 233 233 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 233 233

70 70 75 75 80 80

Pro Ala Pro Ala Pro ProGlu GluLeu LeuLeu Leu AsnAsn GlyGly Pro Pro Ser Ser Val Val Phe Phe Phe Leu LeuPro Phe Pro Pro Pro 115 115 120 120 125 125

185

Glu Leu Glu Leu Tyr TyrAsn AsnSer Ser Thr Thr TyrTyr ArgArg Val Val Val Val Ser Leu Ser Val Val Thr LeuVal Thr Val Leu Leu 180 180 185 185 190 190

186

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 234 234 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 234 234

70 70 75 75 80 80

187

Val Val Val Val Val ValAsp AspVal Val Ser Ser AspAsp GluGlu Asp Asp Pro Pro Glu Lys Glu Val Val Phe LysAsn Phe Asn Trp Trp 145 145 150 150 155 155 160 160

188

Leu Tyr Leu Tyr Ser SerLys LysLeu Leu Thr Thr ValVal LysLys Lys Lys Ser Ser Arg Gln Arg Trp Trp Gln GlnGly Gln Gly Asn Asn 290 290 295 295 300 300

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 235 235 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 235 235

70 70 75 75 80 80

189

Pro Ala Pro Ala Pro ProGlu GluLeu LeuLeu Leu ThrThr GlyGly Pro Pro Ser Ser Val Leu Val Phe Phe Phe LeuPro Phe Pro Pro Pro 115 115 120 120 125 125

190

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 236 236 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 236 236

70 70 75 75 80 80

191

192

Val Phe Val Phe Ser SerCys CysSer Ser Val Val MetMet HisHis Glu Glu Ala Ala Leu Ala Leu His His His AlaTyr His Tyr Thr Thr 305 305 310 310 315 315 320 320

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 237 237 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 237 237

193

70 70 75 75 80 80

194

Asn Tyr Asn Tyr Lys LysThr ThrThr Thr Pro Pro MetMet ValVal Leu Leu Arg Arg Ser Gly Ser Asp Asp Ser GlyPhe Ser Phe Phe Phe 275 275 280 280 285 285

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 238 238 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 238 238

195

70 70 75 75 80 80

196

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 239 239 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 239 239

197

Gly Val Gly Val His His Thr Thr Phe Phe Pro Pro Ala Ala Val Val Leu Leu Gln Gln Ser Ser Ser Ser Gly Gly Leu Leu Tyr Tyr Ser Ser 50 50 55 55 60 60

70 70 75 75 80 80

198

Val Phe Val Phe Ser Ser Cys Cys Ser Ser Val Val Met Met His His Glu Glu Ala Ala Leu Leu His His Ala Ala His His Tyr Tyr Thr Thr 305 305 310 310 315 315 320 320

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 240 240 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 240 240

199

70 70 75 75 80 80

200

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 241 241 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 241 241

201

70 70 75 75 80 80

202

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 242 242 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

203

<400> <400> 242 242

70 70 75 75 80 80

204

Val Phe Val Phe Ser SerCys CysSer Ser Val Val LeuLeu HisHis Glu Glu Ala Ala Leu Ala Leu His His His AlaThr His Thr Thr Thr 305 305 310 310 315 315 320 320

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

205

<210> <210> 243 243 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 243 243

70 70 75 75 80 80

Val Val Val Val Val ValAsp AspVal Val Ser Ser AspAsp GluGlu Asp Asp Pro Pro Glu Lys Glu Val Val Phe LysAsn Phe Asn

206

Trp Trp 145 145 150 150 155 155 160 160

Thr Ala Thr Ala Leu LeuPro ProLys Lys Pro Pro IleIle GluGlu Lys Lys Thr Thr Ile Lys Ile Ser Ser Ala LysLys Ala Lys Gly Gly 210 210 215 215 220 220

Gln Arg Gln Arg Arg Arg Glu Glu Pro Pro Gln Gln Val Val Tyr Tyr Thr Thr Leu Leu Pro Pro Pro Pro Ser Ser Arg Arg Glu Glu Glu Glu 225 225 230 230 235 235 240 240

207

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 244 244 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 244 244

70 70 75 75 80 80

Pro Ala Pro Ala Pro ProGlu GluLeu LeuTrp Trp AsnAsn GlyGly Pro Pro Ser Ser Val Val Phe Phe Phe Leu LeuPro Phe Pro Pro Pro 115 115 120 120 125 125

Lys Pro Lys Pro Lys LysAsp AspThr Thr Leu Leu MetMet IleIle Ser Ser Arg Arg Thr Glu Thr Pro Pro Val GluThr Val Thr

208

Cys Cys 130 130 135 135 140 140

209

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 245 245 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 245 245

70 70 75 75 80 80

Pro Ala Pro Ala Pro ProGlu GluLeu Leu Leu Leu AsnAsn GlyGly Pro Pro Ser Ser Val Leu Val Phe Phe Phe LeuPro Phe Pro

210

Pro Pro 115 115 120 120 125 125

211

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 246 246 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 246 246

70 70 75 75 80 80

Lys Val Lys Val Glu GluPro ProLys Lys Ser Ser CysCys AspAsp Lys Lys Thr Thr His Cys His Thr Thr Pro CysPro Pro Pro

212

Cys Cys 100 100 105 105 110 110

Gln Arg Gln Arg Arg ArgGlu GluPro Pro Gln Gln ValVal TyrTyr Thr Thr Leu Leu Pro Ser Pro Pro Pro Arg SerGlu Arg Glu Glu Glu 225 225 230 230 235 235 240 240

213

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 247 247 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 247 247

70 70 75 75 80 80

Tyr Ile Tyr Ile Cys Cys Asn Asn Val Val Asn Asn His His Lys Lys Pro Pro Ser Ser Asn Asn Thr Thr Lys Lys Val Val Asp Asp

214

Lys Lys 85 85 90 90 95 95

215

Leu Tyr Leu Tyr Ser SerLys LysLeu LeuThr Thr ValVal AspAsp Lys Lys Ser Ser Arg Gln Arg Trp Trp Gln GlnGly Gln Gly Asn Asn 290 290 295 295 300 300

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 248 248 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 248 248

Leu Ser Leu Ser Ser SerVal ValVal Val Thr Thr ValVal ProPro Ser Ser Ser Ser Ser Gly Ser Leu Leu Thr GlyGln Thr Gln

216

Thr Thr

70 70 75 75 80 80

217

Leu Tyr Leu Tyr Ser SerLys LysLeu Leu Thr Thr ValVal ArgArg Lys Lys Ser Ser Arg Gln Arg Trp Trp Gln GlnGly Gln Gly Asn Asn 290 290 295 295 300 300

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 249 249 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 249 249

Gly Val Gly Val His HisThr ThrPhe Phe Pro Pro AlaAla ValVal Leu Leu Gln Gln Ser Gly Ser Ser Ser Leu GlyTyr Leu Tyr

218

Ser Ser 50 50 55 55 60 60

70 70 75 75 80 80

Gln Arg Gln Arg Arg ArgGlu GluPro Pro Gln Gln ValVal TyrTyr Thr Thr Leu Leu Pro Ser Pro Pro Pro Arg SerGlu Arg Glu

219

Glu Glu 225 225 230 230 235 235 240 240

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 250 250 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 250 250

Phe Pro Phe Pro Glu GluPro ProVal Val Thr Thr ValVal SerSer Trp Trp Asn Asn Ser Ala Ser Gly Gly Leu AlaThr Leu Thr

220

Ser Ser 35 35 40 40 45 45

Leu Ser Leu Ser Ser SerVal ValVal ValThr Thr ValVal ProPro Ser Ser Ser Ser Ser Gly Ser Leu Leu Thr GlyGln Thr Gln Thr Thr

70 70 75 75 80 80

Lys Ala Lys Ala Leu LeuPro ProLys Lys Pro Pro IleIle GluGlu Lys Lys Thr Thr Ile Lys Ile Ser Ser Ala LysLys Ala Lys

221

Gly Gly 210 210 215 215 220 220

Pro Ser Asp Pro Ser AspIle IleAla AlaVal Val GluGlu TrpTrp Glu Glu Ser Ser Asn Asn Gly Pro Gly Gln GlnGlu Pro Glu Asn Asn 260 260 265 265 270 270

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 251 251 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 251 251

Ser Thr Ser Thr Ser SerGly GlyGly GlyThr Thr AlaAla AlaAla Leu Leu Gly Gly Cys Val Cys Leu Leu Lys ValAsp Lys Asp

222

Tyr Tyr 20 20 25 25 30 30

70 70 75 75 80 80

Pro Ala Pro Pro Ala ProGlu GluLeu LeuLeu Leu AsnAsn GlyGly Pro Pro Ser Ser Val Val Phe Phe Phe Leu LeuPro Phe Pro Pro Pro 115 115 120 120 125 125

His Arg His Arg Asp AspTrp TrpLeu Leu Asn Asn GlyGly LysLys Glu Glu Tyr Tyr Lys Lys Lys Cys Cys Val LysSer Val Ser

223

Asn Asn 195 195 200 200 205 205

Lys Ala Lys Ala Leu LeuPro ProLys LysPro Pro IleIle GluGlu Lys Lys Thr Thr Ile Lys Ile Ser Ser Ala LysLys Ala Lys Gly Gly 210 210 215 215 220 220

Pro Ser Asp Pro Ser AspIle IleAla AlaVal Val GluGlu TrpTrp Glu Glu Ser Ser Arg Arg Gly Pro Gly Gln GlnGlu Pro Glu Asn Asn 260 260 265 265 270 270

Leu Tyr Leu Tyr Ser SerLys LysLeu LeuThr Thr ValVal ArgArg Lys Lys Ser Ser Arg Gln Arg Trp Trp Gln GlnGly Gln Gly Asn Asn 290 290 295 295 300 300

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 252 252 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 252 252

Ala Ser Ala Ser Thr ThrLys LysGly Gly Pro Pro SerSer ValVal Phe Phe Pro Pro Leu Pro Leu Ala Ala Ser ProSer Ser Ser

224

Lys Lys 1 1 5 5 10 10 15 15

70 70 75 75 80 80

Pro Ala Pro Ala Pro ProGlu GluLeu Leu Leu Leu GlyGly GlyGly Asp Asp Ser Ser Val Leu Val Phe Phe Phe LeuPro Phe Pro Pro Pro 115 115 120 120 125 125

Glu Gln Glu Gln Tyr TyrAsn AsnSer Ser Thr Thr TyrTyr ArgArg Val Val Val Val Ser Leu Ser Val Val Thr LeuVal Thr Val

225

Leu Leu 180 180 185 185 190 190

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 253 253 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<220> <220>

226

<223> Anartificially <223> An artificiallysynthesized synthesizedsequence sequence

<400> <400> 253 253

70 70 75 75 80 80

Pro Ala Pro Ala Pro ProGlu GluLeu LeuLeu Leu GlyGly GlyGly Asp Asp Ser Ser Val Leu Val Phe Phe Phe LeuPro Phe Pro Pro Pro 115 115 120 120 125 125

Lys Pro Lys Pro Lys LysAsp AspVal Val Leu Leu MetMet IleIle Ser Ser Arg Arg Thr Glu Thr Pro Pro Val GluThr Val Thr Cys Cys 130 130 135 135 140 140

Tyr Val Tyr Val Asp Asp Gly Gly Val Val Glu Glu Val Val His His Asn Asn Ala Ala Lys Lys Thr Thr Lys Lys Pro Pro Arg Arg

227

Glu Glu 165 165 170 170 175 175

Glu Gln Glu Gln Tyr TyrAsn AsnSer Ser Thr Thr TyrTyr ArgArg Val Val Val Val Ser Leu Ser Val Val Pro LeuVal Pro Val Leu Leu 180 180 185 185 190 190

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> < 220> 254 254

228

<211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 254 254

70 70 75 75 80 80

Val Val Val Val Val Val Asp Asp Val Val Ser Ser His His Glu Glu Asp Asp Pro Pro Glu Glu Val Val Lys Lys Phe Phe Asn Asn Trp Trp

229

145 150 150 155 155 160 160

230

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 255 255 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 255 255

70 70 75 75 80 80

Lys Pro Lys Pro Lys LysAsp AspVal Val Leu Leu MetMet IleIle Ser Ser Arg Arg Thr Glu Thr Pro Pro Val GluThr Val Thr Cys Cys

231

130 135 135 140 140

Val Val Val Val Ile Ile Asp Asp Val Val Ser Ser His His Glu Glu Asp Asp Pro Pro Glu Glu Val Val Lys Lys Phe Phe Asn Asn Trp Trp 145 145 150 150 155 155 160 160

Val Phe Val Phe Ser SerCys CysSer Ser Val Val MetMet HisHis Glu Glu Ala Ala Leu Asn Leu His His His AsnTyr His Tyr

232

Thr Thr 305 305 310 310 315 315 320 320

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 256 256 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 256 256

70 70 75 75 80 80

Pro Ala Pro Ala Pro ProGlu GluLeu LeuLeu Leu GlyGly GlyGly Asp Asp Ser Ser Val Leu Val Phe Phe Phe LeuPro Phe Pro Pro Pro

233

115 120 120 125 125

Val Val Val Val Val ValAsp AspVal Val Ala Ala HisHis GluGlu Asp Asp Pro Pro Glu Lys Glu Val Val Phe LysAsn Phe Asn Trp Trp 145 145 150 150 155 155 160 160

Leu Tyr Leu Tyr Ser SerLys LysLeu Leu Thr Thr ValVal AspAsp Lys Lys Ser Ser Arg Gln Arg Trp Trp Gln GlnGly Gln Gly

234

Asn Asn 290 290 295 295 300 300

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 257 257 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 257 257

70 70 75 75 80 80

Lys Val Lys Val Glu GluPro ProLys Lys Ser Ser CysCys AspAsp Lys Lys Thr Thr His Cys His Thr Thr Pro CysPro Pro Pro Cys Cys

235

100 105 105 110 110

Pro Ala Pro Ala Pro ProGlu GluTyr TyrLeu Leu GlyGly GlyGly Asp Asp Ser Ser Val Val Phe Phe Phe Leu LeuPro Phe Pro Pro Pro 115 115 120 120 125 125

Asn Tyr Asn Tyr Lys LysThr ThrThr Thr Pro Pro ProPro ValVal Leu Leu Asp Asp Ser Gly Ser Asp Asp Ser GlyPhe Ser Phe

236

Phe Phe 275 275 280 280 285 285

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 258 258 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 258 258

70 70 75 75 80 80

Tyr Ile Tyr Ile Cys Cys Asn Asn Val Val Asn Asn His His Lys Lys Pro Pro Ser Ser Asn Asn Thr Thr Lys Lys Val Val Asp Asp Lys Lys

237

85 90 90 95 95

Pro Ala Pro Ala Pro ProGlu GluTyr Tyr Leu Leu GlyGly GlyGly Asp Asp Ser Ser Val Leu Val Phe Phe Phe LeuPro Phe Pro Pro Pro 115 115 120 120 125 125

Val Val Val Val Val Val Asp Asp Val Val Ala Ala His His Glu Glu Asp Asp Pro Pro Glu Glu Val Val Lys Lys Phe Phe Asn Asn Trp Trp 145 145 150 150 155 155 160 160

Pro Ser Pro Ser Asp AspIle IleAla Ala Val Val GluGlu TrpTrp Glu Glu Ser Ser Asn Gln Asn Gly Gly Pro GlnGlu Pro Glu

238

Asn Asn 260 260 265 265 270 270

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 259 259 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 259 259

239

70 75 75 80 80

Val Val Val Val Ile IleAsp AspVal Val Ala Ala HisHis GluGlu Asp Asp Pro Pro Glu Lys Glu Val Val Phe LysAsn Phe Asn Trp Trp 145 145 150 150 155 155 160 160

Met Thr Met Thr Lys LysAsn AsnGln Gln Val Val SerSer LeuLeu Thr Thr Cys Cys Leu Lys Leu Val Val Gly LysPhe Gly Phe

240

Tyr Tyr 245 245 250 250 255 255

Gln Lys Gln Lys Ser Ser Leu Leu Ser Ser Leu Leu Ser Ser Pro Pro 325 325

<210> <210> 260 260 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 260 260

Gly Val Gly Val His HisThr ThrPhe Phe Pro Pro AlaAla ValVal Leu Leu Gln Gln Ser Gly Ser Ser Ser Leu GlyTyr Leu Tyr Ser Ser

241

50 55 55 60 60

70 70 75 75 80 80

Pro Ala Pro Ala Pro ProGlu GluTyr TyrLeu Leu GlyGly GlyGly Asp Asp Ser Ser Val Leu Val Phe Phe Phe LeuPro Phe Pro Pro Pro 115 115 120 120 125 125

Gln Pro Gln Pro Arg Arg Glu Glu Pro Pro Gln Gln Val Val Tyr Tyr Thr Thr Leu Leu Pro Pro Pro Pro Ser Ser Arg Arg Glu Glu Glu Glu

242

225 230 230 235 235 240 240

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 261 261 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 261 261

Phe Pro Glu Phe Pro GluPro ProVal ValThr Thr ValVal SerSer Trp Trp Asn Asn Ser Ser Gly Leu Gly Ala AlaThr Leu Thr Ser Ser

243

35 40 40 45 45

70 70 75 75 80 80

Val Val Val Val Ile Ile Asp Asp Val Val Ala Ala His His Glu Glu Asp Asp Pro Pro Glu Glu Val Val Lys Lys Phe Phe Asn Asn Trp Trp 145 145 150 150 155 155 160 160

Lys Ala Lys Ala Leu LeuPro ProLys Lys Pro Pro IleIle GluGlu Lys Lys Thr Thr Ile Lys Ile Ser Ser Ala LysLys Ala Lys Gly Gly

244

210 215 215 220 220

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 262 262 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 262 262

Ser Thr Ser Ser Thr SerGly GlyGly GlyThr Thr AlaAla AlaAla Leu Leu Gly Gly Cys Cys Leu Lys Leu Val ValAsp Lys Asp Tyr Tyr

245

20 25 25 30 30

70 70 75 75 80 80

His Arg His Arg Asp AspTrp TrpLeu Leu Asn Asn GlyGly LysLys Glu Glu Tyr Tyr Lys Lys Lys Cys Cys Val LysSer Val Ser Asn Asn

246

195 200 200 205 205

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 263 263 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 263 263

Ala Ser Ala Ser Thr ThrLys LysGly Gly Pro Pro SerSer ValVal Phe Phe Pro Pro Leu Pro Leu Ala Ala Ser ProSer Ser Ser Lys Lys

247

1 5 5 10 10 15 15

70 70 75 75 80 80

Glu Gln Glu Gln Tyr TyrAsn AsnSer Ser Thr Thr TyrTyr ArgArg Val Val Val Val Ser Leu Ser Val Val Pro LeuVal Pro Val Leu Leu

248

180 185 185 190 190

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 264 264 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

249

<400> <400> 264 264

70 70 75 75 80 80

Tyr Val Tyr Val Asp AspGly GlyVal Val Glu Glu ValVal HisHis Asn Asn Ala Ala Lys Lys Lys Thr Thr Pro LysArg Pro Arg Glu Glu

250

165 170 170 175 175

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 265 265 <211> <211> 328 328

251

<212> PRT <212> PRT <213> Artificial sequence <213> Artificial sequence

<400> <400> 265 265

70 70 75 75 80 80

Val Val Val Val Ile Ile Asp Asp Val Val Ser Ser His His Glu Glu Asp Asp Pro Pro Glu Glu Val Val Lys Lys Phe Phe Asn Asn Trp Trp 145 145 150 150 155 155

252

160

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro

253

325

<210> <210> 266 266 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 266 266

70 70 75 75 80 80

Pro Ala Pro Pro Ala ProGlu GluLeu LeuLeu Leu GlyGly GlyGly Asp Asp Ser Ser Val Val Phe Phe Phe Leu LeuPro Phe Pro Pro Pro 115 115 120 120 125 125

254

Val Phe Val Phe Ser SerCys CysSer Ser Val Val MetMet HisHis Glu Glu Ala Ala Leu Asn Leu His His His AsnTyr His Tyr Thr Thr

255

305 310 310 315 315 320 320

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 267 267 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 267 267

70 70 75 75 80 80

256

Leu Tyr Leu Tyr Ser Ser Lys Lys Leu Leu Thr Thr Val Val Lys Lys Lys Lys Ser Ser Arg Arg Trp Trp Gln Gln Gln Gln Gly Gly Asn Asn

257

290 295 295 300 300

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 268 268 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 268 268

70 70 75 75 80 80

258

Asn Tyr Asn Tyr Lys LysThr ThrThr Thr Pro Pro ProPro ValVal Leu Leu Asp Asp Ser Gly Ser Asp Asp Ser GlyPhe Ser Phe Phe Phe

259

275 280 280 285 285

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 269 269 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 269 269

70 70 75 75 80 80

260

Pro Ser Pro Ser Asp AspIle IleAla AlaVal Val GluGlu TrpTrp Glu Glu Ser Ser Asn Gln Asn Gly Gly Pro GlnGlu Pro Glu Asn Asn

261

260 265 265 270 270

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 270 270 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 270 270

70 70 75 75 80 80

262

Pro Ala Pro Pro Ala ProGlu GluTyr TyrLeu Leu GlyGly GlyGly Asp Asp Ser Ser Val Val Phe Phe Phe Leu LeuPro Phe Pro Pro Pro 115 115 120 120 125 125

263

245 250 250 255 255

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 271 271 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 271 271

264

70 70 75 75 80 80

Gln Pro Gln Pro Arg Arg Glu Glu Pro Pro Gln Gln Val Val Tyr Tyr Thr Thr Leu Leu Pro Pro Pro Pro Ser Ser Arg Arg Glu Glu Glu Glu 225 225 230 230 235 235

265

240

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 272 272 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 272 272

266

70 70 75 75 80 80

267

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 273 273 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 273 273

268

70 70 75 75 80 80

His Arg His Arg Asp Asp Trp Trp Leu Leu Asn Asn Gly Gly Lys Lys Glu Glu Tyr Tyr Lys Lys Cys Cys Lys Lys Val Val Ser Ser Asn Asn 195 195 200 200 205 205

269

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 274 274 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 274 274

270

70 70 75 75 80 80

271

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 275 275 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

272

<400> <400> 275 275

70 70 75 75 80 80

273

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 276 276 <211> <211> 328 328 <212> <212> PRT PRT

274

<213> Artificialsequence <213> Artificial sequence

<400> <400> 276 276

70 70 75 75 80 80

275

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

276

<210> <210> 277 277 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 277 277

70 70 75 75 80 80

277

Val Phe Val Phe Ser Ser Cys Cys Ser Ser Val Val Met Met His His Glu Glu Ala Ala Leu Leu His His Asn Asn His His Tyr Tyr Thr Thr 305 305 310 310 315 315

278

320

Gln Lys Gln Lys Ser SerLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 278 278 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 278 278

70 70 75 75 80 80

279

280

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 279 279 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 279 279

70 70 75 75 80 80

281

282

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 280 280 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 280 280

70 70 75 75 80 80

283

284

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 281 281 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 281 281

70 70 75 75 80 80

285

286

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 282 282 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 282 282

287

70 70 75 75 80 80

288

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 283 283 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 283 283

289

70 70 75 75 80 80

290

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 284 284 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 284 284

291

70 70 75 75 80 80

292

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 285 285 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 285 285

293

70 70 75 75 80 80

294

Arg Lys Arg Lys Glu Glu Leu Leu Ser Ser Leu Leu Ser Ser Pro Pro 325 325

<210> <210> 286 286 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 286 286

295

70 70 75 75 80 80

296

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 287 287 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

297

<400> <400> 287 287

70 70 75 75 80 80

298

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

299

<210> <210> 288 288 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 288 288

70 70 75 75 80 80

300

301

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 289 289 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 289 289

70 70 75 75 80 80

Pro Ala Pro Ala Pro ProGlu GluLeu LeuLeu Leu GlyGly GlyGly Asp Asp Ser Ser Val Val Phe Phe Phe Leu LeuPro Phe Pro Pro Pro 115 115 120 120 125 125

302

303

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 290 290 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 290 290

70 70 75 75 80 80

304

305

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 291 291 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 291 291

70 70 75 75 80 80

306

307

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 292 292 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 292 292

70 70 75 75 80 80

308

309

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 293 293 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 293 293

310

70 70 75 75 80 80

Lys Val Lys Val Glu Glu Pro Pro Lys Lys Ser Ser Cys Cys Asp Asp Lys Lys Thr Thr His His Thr Thr Cys Cys Pro Pro Pro Pro Cys Cys 100 100 105 105 110 110

311

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 294 294 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 294 294

312

70 70 75 75 80 80

313

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 295 295 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 295 295

314

70 70 75 75 80 80

315

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 296 296 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 296 296

316

70 70 75 75 80 80

317

Arg Lys Arg Lys Glu Glu Leu Leu Ser Ser Leu Leu Ser Ser Pro Pro 325 325

<210> <210> 297 297 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 297 297

318

70 70 75 75 80 80

319

Arg Lys Arg Lys Glu Glu Leu Leu Ser Ser Leu Leu Ser Ser Pro Pro 325 325

<210> <210> 298 298 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

320

<400> <400> 298 298

70 70 75 75 80 80

321

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

322

<210> <210> 299 299 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 299 299

70 70 75 75 80 80

Val Val Val Val Ile Ile Asp Asp Val Val Ala Ala His His Glu Glu Asp Asp Pro Pro Glu Glu Val Val Lys Lys Phe Phe Asn Asn

323

Trp Trp 145 145 150 150 155 155 160 160

324

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 300 300 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 300 300

70 70 75 75 80 80

Lys Pro Lys Pro Lys LysAsp AspVal Val Leu Leu MetMet IleIle Ser Ser Arg Arg Thr Glu Thr Pro Pro Val GluThr Val Thr

325

Cys Cys 130 130 135 135 140 140

326

Arg Lys Arg Lys Glu Glu Leu Leu Ser Ser Leu Leu Ser Ser Pro Pro 325 325

<210> <210> 301 301 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 301 301

70 70 75 75 80 80

Pro Ala Pro Ala Pro ProGlu GluTyr Tyr Leu Leu GlyGly GlyGly Asp Asp Ser Ser Val Leu Val Phe Phe Phe LeuPro Phe Pro

327

Pro Pro 115 115 120 120 125 125

328

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 302 302 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 302 302

70 70 75 75 80 80

329

Cys Cys 100 100 105 105 110 110

330

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 303 303 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 303 303

70 70 75 75 80 80

331

Lys Lys 85 85 90 90 95 95

332

Leu Tyr Leu Tyr Ser SerLys LysLeu LeuThr Thr ValVal LysLys Lys Lys Ser Ser Arg Gln Arg Trp Trp Gln GlnGly Gln Gly Asn Asn 290 290 295 295 300 300

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 304 304 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 304 304

333

Thr Thr

70 70 75 75 80 80

334

Val Phe Val Phe Ser SerCys CysSer Ser Val Val MetMet HisHis Glu Glu Ala Ala Leu Ala Leu His His His AlaThr His Thr Thr Thr 305 305 310 310 315 315 320 320

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 305 305 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 305 305

335

Ser Ser 50 50 55 55 60 60

70 70 75 75 80 80

Gln Pro Gln Pro Arg Arg Glu Glu Pro Pro Gln Gln Val Val Tyr Tyr Thr Thr Leu Leu Pro Pro Pro Pro Ser Ser Arg Arg Glu Glu

336

Glu Glu 225 225 230 230 235 235 240 240

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 306 306 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 306 306

Phe Pro Phe Pro Glu GluPro ProVal ValThr Thr ValVal SerSer Trp Trp Asn Asn Ser Ala Ser Gly Gly Leu AlaThr Leu Thr

337

Ser Ser 35 35 40 40 45 45

70 70 75 75 80 80

338

Gly Gly 210 210 215 215 220 220

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 307 307 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 307 307

339

Tyr Tyr 20 20 25 25 30 30

70 70 75 75 80 80

His Gln His Gln Asp AspTrp TrpLeu Leu Asn Asn GlyGly LysLys Glu Glu Tyr Tyr Lys Lys Lys Cys Cys Val LysSer Val Ser

340

Asn Asn 195 195 200 200 205 205

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 308 308 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 308 308

341

Lys Lys 1 1 5 5 10 10 15 15

70 70 75 75 80 80

Glu Gln Glu Gln Tyr TyrAsn AsnSer Ser Thr Thr TyrTyr ArgArg Val Val Val Val Ser Leu Ser Val Val Pro LeuVal Pro Val

342

Leu Leu 180 180 185 185 190 190

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 309 309 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<220> <220>

343

<223> Anartificially <223> An artificially synthesized synthesized sequence sequence

<400> <400> 309 309

70 70 75 75 80 80

344

Glu Glu 165 165 170 170 175 175

Arg Lys Arg Lys Glu Glu Leu Leu Ser Ser Leu Leu Ser Ser Pro Pro 325 325

<210> <210> 310 310

345

<400> <400> 310 310

70 70 75 75 80 80

Val Val Val Val Val Val Asp Asp Val Val Ala Ala His His Glu Glu Asp Asp Pro Pro Glu Glu Val Val Lys Lys Phe Phe Asn Asn Trp Trp

346

145 150 150 155 155 160 160

347

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 311 311 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 311 311

70 70 75 75 80 80

348

130 135 135 140 140

Val Phe Val Phe Ser Ser Cys Cys Ser Ser Val Val Met Met His His Glu Glu Ala Ala Leu Leu His His Ala Ala His His Thr Thr

349

Thr Thr 305 305 310 310 315 315 320 320

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 312 312 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 312 312

70 70 75 75 80 80

Pro Ala Pro Ala Pro ProGlu GluTyr TyrLeu Leu GlyGly GlyGly Asp Asp Ser Ser Val Leu Val Phe Phe Phe LeuPro Phe Pro Pro Pro

350

115 120 120 125 125

Val Val Val Val Ile IleAsp AspVal Val Ser Ser HisHis GluGlu Asp Asp Pro Pro Glu Lys Glu Val Val Phe LysAsn Phe Asn Trp Trp 145 145 150 150 155 155 160 160

351

Asn Asn 290 290 295 295 300 300

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 313 313 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 313 313

70 70 75 75 80 80

352

100 105 105 110 110

353

Phe Phe 275 275 280 280 285 285

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 314 314 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 314 314

70 70 75 75 80 80

Tyr Ile Tyr Ile Cys CysAsn AsnVal Val Asn Asn HisHis LysLys Pro Pro Ser Ser Asn Lys Asn Thr Thr Val LysAsp Val Asp Lys Lys

354

85 90 90 95 95

355

Asn Asn 260 260 265 265 270 270

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 315 315 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 315 315

356

70 75 75 80 80

357

Tyr Tyr 245 245 250 250 255 255

Arg Lys Arg Lys Glu Glu Leu Leu Ser Ser Leu Leu Ser Ser Pro Pro 325 325

<210> <210> 316 316 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 316 316

358

50 55 55 60 60

70 70 75 75 80 80

Gln Arg Gln Arg Arg Arg Glu Glu Pro Pro Gln Gln Val Val Tyr Tyr Thr Thr Leu Leu Pro Pro Pro Pro Ser Ser Arg Arg Glu Glu Glu Glu

359

225 230 230 235 235 240 240

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 317 317 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 317 317

360

35 40 40 45 45

70 70 75 75 80 80

361

210 215 215 220 220

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 318 318 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 318 318

362

20 25 25 30 30

70 70 75 75 80 80

363

195 200 200 205 205

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 319 319 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 319 319

364

1 5 5 10 10 15 15

70 70 75 75 80 80

365

180 185 185 190 190

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 320 320 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

366

<400> <400> 320 320

70 70 75 75 80 80

367

165 170 170 175 175

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 321 321 <211> <211> 328 328

368

<212> PRT <212> PRT <213> Artificial sequence <213> Artificial sequence

<400> <400> 321 321

70 70 75 75 80 80

Val Val Val Val Val Val Asp Asp Val Val Ser Ser His His Glu Glu Asp Asp Pro Pro Glu Glu Val Val Lys Lys Phe Phe Asn Asn Trp Trp 145 145 150 150 155 155

369

160

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro

370

325

<210> <210> 322 322 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 322 322

70 70 75 75 80 80

371

Val Phe Val Phe Ser SerCys CysSer Ser Val Val MetMet HisHis Glu Glu Ala Ala Leu Ala Leu His His His AlaThr His Thr Thr Thr

372

305 310 310 315 315 320 320

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 323 323 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 323 323

70 70 75 75 80 80

373

374

290 295 295 300 300

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 324 324 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 324 324

70 70 75 75 80 80

375

Asn Tyr Asn Tyr Lys Lys Thr Thr Thr Thr Pro Pro Pro Pro Val Val Leu Leu Asp Asp Ser Ser Asp Asp Gly Gly Ser Ser Phe Phe Phe Phe

376

275 280 280 285 285

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 325 325 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 325 325

70 70 75 75 80 80

377

Pro Ser Pro Ser Asp AspIle IleAla Ala Val Val GluGlu TrpTrp Glu Glu Ser Ser Asn Gln Asn Gly Gly Pro GlnGlu Pro Glu Asn Asn

378

260 265 265 270 270

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 326 326 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 326 326

70 70 75 75 80 80

379

Met Thr Met Thr Lys LysAsn AsnGln Gln Val Val SerSer LeuLeu Thr Thr Cys Cys Leu Lys Leu Val Val Gly LysPhe Gly Phe Tyr Tyr

380

245 250 250 255 255

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 327 327 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 327 327

381

70 70 75 75 80 80

382

240

Arg Lys Arg Lys Glu Glu Leu Leu Ser Ser Leu Leu Ser Ser Pro Pro 325 325

<210> <210> 328 328 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 328 328

383

70 70 75 75 80 80

384

Leu Tyr Ser Leu Tyr SerLys LysLeu LeuThr Thr ValVal AspAsp Lys Lys Ser Ser Arg Arg Trp Gln Trp Gln GlnGly Gln Gly Asn Asn 290 290 295 295 300 300

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 329 329 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 329 329

385

70 70 75 75 80 80

386

Val Phe Val Phe Ser Ser Cys Cys Ser Ser Val Val Met Met His His Glu Glu Ala Ala Leu Leu His His Ala Ala His His Thr Thr Thr Thr 305 305 310 310 315 315 320 320

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 330 330 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 330 330

387

70 70 75 75 80 80

388

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 331 331 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

389

<400> <400> 331 331

70 70 75 75 80 80

390

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 332 332 <211> <211> 328 328 <212> <212> PRT PRT

391

<213> Artificialsequence <213> Artificial sequence

<400> <400> 332 332

70 70 75 75 80 80

392

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

393

<210> <210> 333 333 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 333 333

70 70 75 75 80 80

394

Val Phe Val Phe Ser SerCys CysSer Ser Val Val LeuLeu HisHis Glu Glu Ala Ala Leu Ala Leu His His His AlaThr His Thr Thr Thr 305 305 310 310 315 315

395

320

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 334 334 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 334 334

70 70 75 75 80 80

396

397

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 335 335 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 335 335

70 70 75 75 80 80

398

399

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 336 336 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 336 336

70 70 75 75 80 80

400

401

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 337 337 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 337 337

70 70 75 75 80 80

402

403

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 338 338 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 338 338

404

70 70 75 75 80 80

405

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 339 339 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 339 339

406

70 70 75 75 80 80

407

Arg Lys Arg Lys Glu Glu Leu Leu Ser Ser Leu Leu Ser Ser Pro Pro 325 325

<210> <210> 340 340 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 340 340

408

70 70 75 75 80 80

409

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 341 341 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 341 341

410

70 70 75 75 80 80

411

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 342 342 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 342 342

412

70 70 75 75 80 80

413

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 343 343 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

414

<400> <400> 343 343

70 70 75 75 80 80

415

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

416

<210> <210> 344 344 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 344 344

70 70 75 75 80 80

417

418

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 345 345 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 345 345

70 70 75 75 80 80

419

420

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 346 346 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 346 346

70 70 75 75 80 80

421

422

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 347 347 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 347 347

70 70 75 75 80 80

423

424

Arg Lys Arg Lys Glu Glu Leu Leu Ser Ser Leu Leu Ser Ser Pro Pro 325 325

<210> <210> 348 348 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 348 348

70 70 75 75 80 80

425

426

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 349 349 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 349 349

427

70 70 75 75 80 80

428

Arg Lys Arg Lys Glu Glu Leu Leu Ser Ser Leu Leu Ser Ser Pro Pro 325 325

<210> <210> 350 350 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 350 350

429

70 70 75 75 80 80

430

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 351 351 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 351 351

431

70 70 75 75 80 80

432

Val Phe Val Phe Ser Ser Cys Cys Ser Ser Val Val Leu Leu His His Glu Glu Ala Ala Leu Leu His His Ala Ala His His Thr Thr Thr Thr 305 305 310 310 315 315 320 320

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 352 352 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 352 352

433

70 70 75 75 80 80

434

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 353 353 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 353 353

435

70 70 75 75 80 80

436

Arg Lys Arg Lys Glu Glu Leu Leu Ser Ser Leu Leu Ser Ser Pro Pro 325 325

<210> <210> 354 354 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

437

<400> <400> 354 354

70 70 75 75 80 80

438

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

439

<210> <210> 355 355 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 355 355

70 70 75 75 80 80

440

Trp Trp 145 145 150 150 155 155 160 160

441

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 356 356 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 356 356

70 70 75 75 80 80

442

Cys Cys 130 130 135 135 140 140

443

Val Phe Val Phe Ser Ser Cys Cys Ser Ser Val Val Leu Leu His His Glu Glu Ala Ala Leu Leu His His Ala Ala His His Tyr Tyr Thr Thr 305 305 310 310 315 315 320 320

Arg Lys Arg Lys Glu Glu Leu Leu Ser Ser Leu Leu Ser Ser Pro Pro 325 325

<210> <210> 357 357 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 357 357

70 70 75 75 80 80

Pro Ala Pro Ala Pro ProGlu GluLeu Leu Leu Leu GlyGly GlyGly Asp Asp Ser Ser Val Leu Val Phe Phe Phe LeuPro Phe Pro

444

Pro Pro 115 115 120 120 125 125

445

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 358 358 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 358 358

70 70 75 75 80 80

446

Cys Cys 100 100 105 105 110 110

447

Val Phe Val Phe Ser SerCys CysSer Ser Val Val LeuLeu HisHis Glu Glu Ala Ala Leu Ala Leu His His His AlaTyr His Tyr Thr Thr 305 305 310 310 315 315 320 320

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 359 359 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 359 359

70 70 75 75 80 80

448

Lys Lys 85 85 90 90 95 95

449

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 360 360 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 360 360

Leu Ser Leu Ser Ser Ser Val Val Val Val Thr Thr Val Val Pro Pro Ser Ser Ser Ser Ser Ser Leu Leu Gly Gly Thr Thr Gln Gln

450

Thr Thr

70 70 75 75 80 80

451

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 361 361 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 361 361

452

Ser Ser 50 50 55 55 60 60

70 70 75 75 80 80

453

Glu Glu 225 225 230 230 235 235 240 240

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 362 362 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 362 362

454

Ser Ser 35 35 40 40 45 45

70 70 75 75 80 80

455

Gly Gly 210 210 215 215 220 220

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 363 363 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 363 363

456

Tyr Tyr 20 20 25 25 30 30

70 70 75 75 80 80

457

Asn Asn 195 195 200 200 205 205

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 364 364 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 364 364

458

Lys Lys 1 1 5 5 10 10 15 15

70 70 75 75 80 80

459

Leu Leu 180 180 185 185 190 190

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 365 365 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<220> <220>

460

<400> <400> 365 365

70 70 75 75 80 80

461

Glu Glu 165 165 170 170 175 175

Arg Lys Arg Lys Glu Glu Leu Leu Ser Ser Leu Leu Ser Ser Pro Pro 325 325

<210> <210> 366 366

462

<400> <400> 366 366

70 70 75 75 80 80

463

145 150 150 155 155 160 160

464

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 367 367 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 367 367

70 70 75 75 80 80

465

130 135 135 140 140

Val Phe Val Phe Ser SerCys CysSer Ser Val Val LeuLeu HisHis Glu Glu Ala Ala Leu Ala Leu His His His AlaTyr His Tyr

466

Thr Thr 305 305 310 310 315 315 320 320

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 368 368 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 368 368

70 70 75 75 80 80

467

115 120 120 125 125

468

Asn Asn 290 290 295 295 300 300

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 369 369 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 369 369

70 70 75 75 80 80

469

100 105 105 110 110

Asn Tyr Asn Tyr Lys Lys Thr Thr Thr Thr Pro Pro Pro Pro Val Val Leu Leu Asp Asp Ser Ser Asp Asp Gly Gly Ser Ser Phe Phe

470

Phe Phe 275 275 280 280 285 285

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 370 370 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 370 370

70 70 75 75 80 80

471

85 90 90 95 95

Pro Ser Pro Ser Asp AspIle IleAla AlaVal Val GluGlu TrpTrp Glu Glu Ser Ser Asn Gln Asn Gly Gly Pro GlnGlu Pro Glu

472

Asn Asn 260 260 265 265 270 270

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 371 371 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 371 371

473

70 75 75 80 80

474

Tyr Tyr 245 245 250 250 255 255

Arg Lys Arg Lys Glu Glu Leu Leu Ser Ser Leu Leu Ser Ser Pro Pro 325 325

<210> <210> 372 372 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 372 372

475

50 55 55 60 60

70 70 75 75 80 80

476

225 230 230 235 235 240 240

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 373 373 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 373 373

Phe Pro Phe Pro Glu GluPro ProVal ValThr Thr ValVal SerSer Trp Trp Asn Asn Ser Ser Gly Leu Gly Ala AlaThr Leu Thr Ser Ser

477

35 40 40 45 45

70 70 75 75 80 80

478

210 215 215 220 220

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 374 374 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 374 374

479

20 25 25 30 30

70 70 75 75 80 80

480

195 200 200 205 205

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 375 375 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 375 375

481

1 5 5 10 10 15 15

70 70 75 75 80 80

482

180 185 185 190 190

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 376 376 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

483

<400> <400> 376 376

70 70 75 75 80 80

484

165 170 170 175 175

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 377 377 <211> <211> 328 328

485

<212> <212> PRT PRT <213> Artificial sequence <213> Artificial sequence

<400> <400> 377 377

70 70 75 75 80 80

Val Val Val Val Ile Ile Asp Asp Val Val Ala Ala His His Glu Glu Asp Asp Pro Pro Glu Glu Val Val Lys Lys Phe Phe Asn Asn Trp Trp 145 145 150 150 155 155

486

160

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro

487

325

<210> <210> 378 378 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificialsequence Artificial sequence

<400> <400> 378 378

70 70 75 75 80 80

488

Val Phe Val Phe Ser SerCys CysSer Ser Val Val LeuLeu HisHis Glu Glu Ala Ala Leu Ala Leu His His His AlaTyr His Tyr Thr Thr

489

305 310 310 315 315 320 320

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 379 379 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 379 379

70 70 75 75 80 80

490

491

290 295 295 300 300

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 380 380 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 380 380

70 70 75 75 80 80

492

493

275 280 280 285 285

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

<210> <210> 381 381 <211> <211> 328 328 <212> <212> PRT PRT <213> <213> Artificial sequence Artificial sequence

<400> <400> 381 381

70 70 75 75 80 80

494

495

260 265 265 270 270

Arg Lys Arg Lys Glu GluLeu LeuSer Ser Leu Leu SerSer ProPro 325 325

496

Claims

[Claim 1] An isolated antibody that binds to latent myostatin, wherein the antibody comprises: (1) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 114, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 114, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 116, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (iii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 117, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (v) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 119, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122,

HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (vi) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 114, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (vii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 114, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 123, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (viii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 115, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 123, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (ix) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 114, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 124, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 125, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; or (x) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 115, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 120, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 123, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74.
[Claim 2] The antibody of claim 1(i) to (vi) and (ix) comprising a heavy chain variable domain framework FRI (H-FR1) comprising the amino acid sequence of SEQ ID NO: 132; a heavy chain variable domain framework FR2 (H-FR2) comprising the amino acid sequence of SEQ ID NO: 135; a heavy chain variable domain framework FR3 (H-FR3) comprising the amino acid sequence of SEQ ID NO: 137; a heavy chain variable domain framework FR4 (H-FR4) comprising the amino acid sequence of SEQ ID NO: 138, a light chain variable domain framework FRI (L-FR1) comprising the amino acid sequence of SEQ ID NO: 139; a light chain variable domain framework FR2 (L-FR2) comprising the amino acid sequence of SEQ ID NO: 140; a light chain variable domain framework FR3 (L-FR3) comprising the amino acid sequence of SEQ ID NO: 142; and a light chain variable domain framework FR4 (L-FR4) comprising the amino acid sequence of SEQ ID NO: 144; or the antibody of claim 1 (vii) or (x), comprising a H-FRi comprising the amino acid sequence of SEQ ID NO: 133; a H-FR2 comprising the amino acid sequence of SEQ ID NO: 136; a H-FR3 comprising the amino acid sequence of SEQ ID NO: 137; a H-FR4 comprising the amino acid sequence of SEQ ID NO: 138; a L-FR1 comprising the amino acid sequence of SEQ ID NO: 139; a L-FR2 comprising the amino acid sequence of SEQ ID NO: 140; a L-FR3 comprising the amino acid sequence of SEQ ID NO: 142; and a L-FR4 comprising the amino acid sequence of SEQ ID NO: 144; or the antibody of claim 1 (vii) or (viii), comprising a H-FRi comprising the amino acid sequence of SEQ ID NO: 134; a H-FR2 comprising the amino acid sequence of SEQ ID NO: 136; a H-FR3 comprising the amino acid sequence of SEQ ID NO: 137; a H-FR4 comprising the amino acid sequence of SEQ ID NO: 138; a L-FR1 comprising the amino acid sequence of SEQ ID NO: 139; a L-FR2 comprising the amino acid sequence of SEQ ID NO: 140; a L-FR3 comprising the amino acid sequence of SEQ ID NO: 142; and a L-FR4 comprising the amino acid sequence of SEQ ID NO: 144; or the antibody of claim 1 (vii), comprising a H-FRI comprising the amino acid sequence of SEQ ID NO: 133; a H-FR2 comprising the amino acid sequence of SEQ ID NO: 136; a H-FR3 comprising the amino acid sequence of SEQ ID NO: 137; a H-FR4 comprising the amino acid sequence of SEQ ID NO: 138; a L-FR1 comprising the amino acid sequence of SEQ ID NO: 139; a L-FR2 comprising the amino acid sequence of SEQ ID NO: 141; a L-FR3 comprising the amino acid sequence of SEQ ID NO: 143; and a L-FR4 comprising the amino acid sequence of SEQ ID NO: 144.
[Claim 3] The antibody of claim 1 or 2, comprising: (i) a VH sequence of SEQ ID NO: 86 and a VL sequence of SEQ ID NO: 96; or (ii) a VH sequence of SEQ ID NO: 86 and a VL sequence of SEQ ID NO: 98; or (iii) a VH sequence of SEQ ID NO: 87 and a VL sequence of SEQ ID NO: 96; or (iv) a VH sequence of SEQ ID NO: 88 and a VL sequence of SEQ ID NO: 96; or (v) a VH sequence of SEQ ID NO: 89 and a VL sequence of SEQ ID NO: 96; or (vi) a VH sequence of SEQ ID NO: 90 and a VL sequence of SEQ ID NO: 96; or (vii) a VH sequence of SEQ ID NO: 91 and a VL sequence of SEQ ID NO: 96; or (viii) a VH sequence of SEQ ID NO: 92 and a VL sequence of SEQ ID NO: 97; or (ix) a VH sequence of SEQ ID NO: 92 and a VL sequence of SEQ ID NO: 99; or (x) a VH sequence of SEQ ID NO: 93 and a VL sequence of SEQ ID NO: 97; or (xi) a VH sequence of SEQ ID NO: 94 and a VL sequence of SEQ ID NO: 97; or (xii) a VH sequence of SEQ ID NO: 95 and a VL sequence of SEQ ID NO: 97.
[Claim 4] The antibody of any one of claims 1 to 3, which comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 352, and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 10.
[Claim 5] The antibody of claim 1 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 RlIb]/[KD value of the variant Fc region for monkey Fc gamma RIIb] 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.
[Claim 6] The antibody of claim 5, wherein the KD value of the variant Fc region for monkey Fc gamma RIlb 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.
[Claim 7] The antibody 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.
[Claim 8] The antibody 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,and396; (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.
[Claim 9] The antibody 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, Gin, Tyr at position 238, (i) Ile, Leu, Asn, Pro, Val at position 239, () 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.
[Claim 10] The antibody 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, () Lys, Arg at position 330, and (k) Lys, Met at position 396; according to EU numbering.
[Claim 11] The antibody of claim 1 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.
[Claim 12] The antibody 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.
[Claim 13] The antibody of claim 12, wherein the variant Fc region comprises Arg or Lys at each of the positions selected.