EA040713B1 - ANTIBODY TO MYOSTATIN AND METHODS FOR ITS PRODUCTION, ISOLATED NUCLEIC ACID ENCODING ANTIBODY, HOST CELL AND PHARMACEUTICAL COMPOSITION FOR INCREASING MYOSTATIN CLEARANCE - Google Patents

Description

The field of technology to which the invention belongs

The present invention relates to antibodies to myostatin and method of their use. The present invention also relates to polypeptides containing variants of the Fc regions, and methods of their use.

Prerequisites for the creation of the invention

Myostatin, also referred to as differentiation factor-8 (GDF8), is a secreted protein and is a member of the growth-transforming factor-beta (TGF-beta) superfamily of proteins. Members of this superfamily have growth regulating and morphogenetic properties (see, for example, NPL1, NPL2 and PTL1). Myostatin is expressed primarily in developing adult skeletal and musculature and functions as a negative regulator of muscle growth. Systemic overexpression of myostatin in adult mice leads to muscle wasting (see, for example, NPL3), while in the opposite case, myostatin-deactivated mice are characterized by hypertrophy and hyperplasia of skeletal muscles, leading to a 2-3-fold increase in muscle mass by compared to their wild-type littermates (see, for example, NPL4).

Like other members of the TGF-beta family, myostatin is synthesized as a large precursor protein that contains an N-terminal propeptide domain and a C-terminal domain, which is an active molecule (see, for example, NPL5; PTL2). The two myostatin precursor molecules are covalently linked via a single disulfide bond present in the C-terminal domain of the growth factor. Active mature myostatin (a disulfide-linked homodimer consisting of the C-terminal domain of the growth factor) is released from cleavage of the peptide bond between the N-terminal propeptide domain and the C-terminal domain of the growth factor, i.e. Arg266-Asp267 using a furin-type proprotein convertase in both strands of the homodimeric precursor. However, the resulting three peptides (two propeptides and one mature myostatin (i.e., a disulfide-linked homodimer consisting of growth factor domains)) remain associated, forming a non-covalent inactive complex, which is referred to as latent myostatin. Mature myostatin can then be released from latent myostatin via propeptide cleavage. Representatives of the family of metalloproteinases, which are bone morphogenetic protein (protein involved in osteogenesis) 1 (BMP1), cleave a single peptide bond in the propeptide, Arg98-Asp99, which is accompanied by the release of mature active myostatin in the form of a homodimer (see, for example, NPL6). In addition, latent myostatin can also be activated in vitro by dissociating the complex with either acid treatment or heat treatment (see, for example, NPL7).

Myostatin exerts its actions through a family of transmembrane heterotetrameric serine/threonine kinase receptors, the activation of which increases receptor transphosphorylation, leading to stimulation of serine/threonine kinase activity. It has been established that the myostatin pathway includes an active myostatin dimer that binds to the activin type IIB receptor (ActRIIB) with high affinity, which further leads to the emergence and activation of transphosphorylation of the low affinity receptor, activin-like kinase 4 (ALK4) or activin-like kinase 5 (ALK5). It was also found that the Smad 2 and Smad 3 proteins are then activated and form a complex with Smad 4, which then translocates into the nucleus to activate the transcription of the target gene. ActRIIB has been shown to be able to mediate the effects of myostatin in vivo, as expression of the dominant negative form of ActRIIB in mice mimics myostatin gene knockout (see, for example, NPL8).

A variety of disorders or conditions are associated with muscle wasting (i.e., loss or functional impairment of muscle tissue), such as muscular dystrophy (MD; including Duchenne muscular dystrophy), amyotrophic lateral sclerosis (ALS), muscle atrophy, organ atrophy, infirmity, congestive obstructive pulmonary disease (COPD), sarcopenia and cachexia resulting from cancer or other disorders, as well as kidney disease, heart failure, or liver disease and disease. Patients may benefit from increased muscle mass and/or muscle strength; however, only limited treatments for these disorders are currently available. Thus, because of its role as a negative regulator of skeletal muscle growth, myostatin becomes an important target for therapeutic or prophylactic intervention in said disorders or conditions, or for monitoring the development of said disorders or conditions. In particular, agents that inhibit the activity of myostatin may be valuable therapeutic agents.

Inhibition of myostatin expression leads to both muscle hypertrophy and hyperplasia (NPL9). Myostatin is a negative regulator of muscle regeneration after injury, and the absence of myostatin in myostatin-null mice results in accelerated muscle regeneration (see, for example, NPL10). Anti-myostatin antibodies (GDF8), which are described, for example, in PTL3, PTL4, PTL5, PTL6 and PTL7, and PTL8, PTL9 and PTL10, have been found to bind to myostatin and inhibit myostatin activity in vitro and in vivo, including myostatin activity, associated with

- 1 040713 negative regulation of skeletal muscle mass. Myostatin-neutralizing antibodies increase body weight, skeletal muscle mass and muscle size, as well as skeletal muscle strength in wild-type mice (see, for example, NPL11) and in mdx mice (muscular dystrophy model) (see, for example, NPL12; NPL13 ). However, the antibodies known in the art are all specific for mature myostatin but not for latent myostatin, and the described strategies for inhibiting myostatin activity have used antibodies that can bind to and neutralize mature myostatin.

Antibodies are attracting attention as pharmaceuticals due to their high blood stability and few side effects (see, for example, NPL14 and NPL15). Nearly all current commercially available therapeutic antibodies are of the human IgG1 subclass. One of the known functions of IgG-class antibodies is antibody-mediated cell-dependent cytotoxicity (hereinafter referred to as ADCC activity in the context of the present description) (see, for example, NPL16). For an antibody to exhibit ADCC activity, the Fc region of an antibody must bind to the Fc-gamma receptor (hereinafter referred to 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.

For humans, the isoforms Fc-gamma RIa (CD64A), Fc-gamma RIIa (CD32A), Fc-gamma RIIb (CD32B), Fc-gamma RIIIa (CD16A) and Fc-gamma RIIIb (CD16B) are described as proteins of the Fc-gamma family. R, in addition, the corresponding allotypes are also described (see, for example, NPL17). Fc-gamma RIa, Fc-gamma RIIa and Fc-gamma RIIIa are called activating Fc-gamma R because they have immunologically active functions, and Fc-gamma RIIb is called inhibitory Fc-gamma R because it has immunosuppressive functions (see, e.g., , NPL18).

Several amino acid residues in the antibody hinge region and the CH2 domain and a sugar chain attached to Asn at position 297 (EU numbering) that is linked to the CH2 domain (see ., for example, NPL19, NPL20 and NPL21). To date, various variants have been obtained that have the ability to bind to Fcgamma R, primarily antibodies with mutations introduced at these sites; and variants of the Fc region having higher binding activities for the Fc-activating R gamma (see, for example, PTL11, PTL12, PTL13 and PTL14).

When the activating Fc-gamma R cross-links with the immune complex, it phosphorylates immunoreceptor tyrosine-based activating motifs (ITAM) within the intracellular domain or common gamma chain of the FcR (interaction partner), activates the SYK signal transducer, and triggers an inflammatory immune response. by initiating activation of the signal cascade (see, for example, NPL22).

Fc-gamma RIIb is the only Fc-gamma R that is expressed on B cells (see, for example, NPL23). It has been established that the interaction of the Fc region of the antibody with the Fc-gamma of RIIb suppresses the primary immune response of B cells (see, for example, NPL24). In addition, it has been described that when Fc-gamma RIIb on B cells and the B cell receptor (BCR) are cross-linked via an immune complex in the blood, B cell activation and antibody production by B cells is suppressed (see, e.g., NPL25). This immunosuppressive signal transduction mediated by BCR and Fc-gamma RIIb requires an immunoreceptor tyrosine-based inhibitory motif (ITIM) within the intracellular domain of Fc-gamma RIIb (see, for example, NPL26 and NPL27). When ITIM is phosphorylated after signal transduction, SH2-containing inositol polyphosphate 5-phosphatase (SHIP) is recruited, transduction of the other Fc-gamma R signaling cascade is inhibited, and the inflammatory immune response is suppressed (see, for example, NPL28). In addition, Fc-gamma RIIb aggregation alone is known to result in transient suppression of 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. NPL29) .

Fc-gamma RIIb is also expressed on dendritic cells, macrophages, activated neutrophils, mast cells, and basophils. Fc-gamma RIIb inhibits activating Fc-gamma R functions such as phagocytosis and release of pro-inflammatory cytokines in these cells and suppresses inflammatory immune responses (see, for example, NPL30).

To date, to study the immunosuppressive functions of Fc-gamma RIIb, studies have been carried out using Fc-gamma RIIb deactivated mice. With their help, it was found that in mice with Fc-gamma RIIb turned off, the humoral immune response is not regulated appropriately (see, for example, NPL31), sensitivity to collagen-induced arthritis (CIA) increases (see, for example, NPL32), there are reminiscent lupus symptoms and symptoms resembling Goodpasture's syndrome (see, for example, NPL33).

In addition, human autoimmune diseases are known to be associated with Fc-gamma RIIb dysregulation. For example, a relationship has been described between genetic polymorphisms in the transmembrane region and promoter region of Fc-gamma RIIb and the incidence of systemic lupus erythematosus (SLE) (see, for example, NPL34, NPL35, NPL36, NPL37 and NPL38) and a decrease in Fc-gamma RIIb expression by surfaces of B cells in SLE patients (see, for example, NPL39 and NPL40).

Based on mouse model data and the clinical results themselves, Fc-gamma RIIb is believed to play a role in the control of autoimmune diseases and inflammatory diseases through recruitment primarily of B cells, and is a promising target molecule for the control of autoimmune diseases. and inflammatory diseases.

It is known that IgG1, which are mainly used as commercially available therapeutic antibodies, bind not only to Fc-gamma RIIb, but also have strong binding to activating Fc-gamma R (see, for example, NPL41). This makes it possible to develop therapeutic antibodies with greater immunosuppressive properties than those of IgG1 by using an Fc region with increased ability to bind to Fc-gamma RIIb or with improved binding selectivity to Fc-gamma RIIb compared to binding to 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 increased ability to bind to Fc-gamma RIIb can inhibit B cell activation (see, for example, NPL42). Cross-linking of Fc-gamma RIIb on B cells and IgE bound to the B cell receptor has been described to suppress B cell differentiation into plasma cells, resulting in suppression of IgE production; and in mice transplanted with human PBMCs, human IgG and IgM concentrations are maintained while human IgE concentrations are reduced (see, for example, NPL43). It has been described that when Fc-gamma RUB and CD79b, which is a constitutive molecule of the B-cell receptor complex, are cross-linked with an antibody, in addition to affecting IgE, B-cell proliferation is suppressed in vitro and arthritis symptoms are alleviated in a collagen-induced arthritis model ( see, for example, NPL44).

In addition to affecting B cells, cross-linking of Fc-epsilon RI and Fcgamma RIIb on mast cells has also been described using molecules in which an IgG Fc region with increased ability to bind to Fc-gamma RIIb is fused to an IgE Fc region that binds to the IgE receptor

Fc-epsilon RI causes phosphorylation of Fc-gamma RIIb, thereby suppressing Fc-epsilon RI-dependent calcium influx. This suggests that inhibition of degranulation via Fc-gamma RIIb stimulation can be achieved by increasing Fc-gamma RIIb binding (see, for example, NPL45).

Thus, an antibody that has an Fc with improved ability to bind to Fc-gamma RIIb is expected to be promising as a therapeutic agent in inflammatory diseases such as autoimmune disease.

In addition, the activation of macrophages and dendritic cells by Toll-like receptor 4 upon stimulation with LPS has been reported to be suppressed in the presence of an antibody-antigen immune complex, and it is suggested that this also represents immune complex actions via Fc-gamma RIIb ( see, for example, NPL46 and NPL47). Thus, it is expected that the use of antibodies with increased ability to bind to Fc-gamma RIIb may enhance TLR-mediated inhibitory effects on signal activation; therefore, it is expected that these antibodies may be promising as therapeutic agents in inflammatory diseases such as autoimmune diseases.

In addition, it is expected that mutants with increased ability to bind to Fc-gamma RIIb may be promising as therapeutic agents in cancer, as well as in inflammatory diseases such as autoimmune diseases. To date, it has been established that the Fc-gamma of RIIb plays an important role in the agonistic activity of agonist antibodies to the TNF receptor superfamily. In particular, it can be assumed that interaction with Fc-gamma RIIb is required for the manifestation of agonistic activity of antibodies to CD40, DR4, DR5, CD30 and CD137, which are members of the TNF receptor family (see, for example, NPL48, NPL49, NPL50, NPL51 , NPL52, NPL53 and NPL54). NPL55 demonstrated that the use of antibodies with increased ability to bind to Fc-gamma RIIb increases the antitumor effect of antibodies to CD40. Thus, it can be assumed that antibodies with increased ability to bind to Fc-gamma RIIb may influence the increase in agonist activity of agonist antibodies, including antibodies to the TNF receptor superfamily.

In addition, cell proliferation has been found to be suppressed by using an antibody that recognizes Kit, a type of receptor tyrosine kinase (RTK), to cross-link Fc-gamma RIIb and Kit on Kit-expressing cells. Similar actions are described even in cases in which said Kit was constitutively activated and had mutations that cause tumorigenesis (see, for example, NPL56). Thus, it can be assumed that the use of antibodies with increased ability to bind to RIIb Fc-gamma may increase the inhibitory effects on cells that express RTK having constitutively activating mutations.

Antibodies having Fc with increased Fc-gamma RIIb-binding activity have been described (see, for example, NPL57). This publication describes the enhancement of Fc-gamma RIIb binding activity by introducing changes such as S267E/L328F, G236D/S267E and S239D/S267E to the Fc region of an antibody. Among them, the antibody with the introduced S267E/L328F mutation most strongly bound to Fc-gamma.

- 3 040713

RIIb, and retained the same level of binding to Fc-gamma Ria and Fc-gamma RIIa type H, in which the residue at position 131 of Fc-gamma RIIa is His, as in naturally occurring IgG1. However, another publication describes that this change increases the binding of Fc-gamma RIIa type R, in which the residue at position 131 Fc-gamma RIIa is Arg, several hundred times relative to the level of the same binding of Fc-gamma RIIb, which means that the selectivity of Fc-gamma RIIb binding is not increased compared to Fc-gamma RIIa type R (see, for example, PTL1).

It is believed that only exposure to increased binding to Fc-gamma RIIa, but not increased binding to Fc-gamma RIIb, should affect cells, such as platelets, that express Fc-gamma RIIa but do not express Fc-gamma RIIb (see, e.g. NPL58). For example, the group of patients who were administered bevacizumab, an anti-VEGF antibody, is known to have an increased risk of thromboembolism (see, for example, NPL59). In addition, similarly, thromboembolism was detected in clinical trials of developed anti-CD40 ligand antibodies, and the clinical trial was discontinued (see, for example, NPL60). Both variants of these antibodies have been studied in more recent studies using animal models and it has been suggested that the administered antibodies caused aggregation by Fc-gamma RIIa binding on platelets and led to blood clot formation (see, for example, NPL61 and NPL62). In systemic lupus erythematosus, which is an autoimmune disease, platelets are activated through an Fc-gamma RIIa dependent mechanism and platelet activation has been described to correlate with the severity of symptoms (see, for example, NPL63). The introduction of an antibody with increased ability to bind to Fc-gamma RIIa to those patients who already had a high risk of developing thromboembolism, the risk of developing thromboembolism may increase, which is extremely dangerous.

In addition, antibodies with increased ability to bind to Fc-gamma RIIa have been described to enhance macrophage-mediated antibody-dependent cellular phagocytosis (ADCP) (see, for example, NPL64). When antigens to be bound by antibodies are phagocytosed by macrophages, it is believed that the antibodies themselves are also simultaneously phagocytosed. When antibodies are administered as pharmaceutical agents, it is expected that peptide fragments from the administered antibodies are also likely to be presented as an antigen, increasing the risk of producing antibodies against therapeutic antibodies (anti-therapeutic antibodies). More specifically, increased ability to bind to Fc-gamma RIIa may increase the risk of developing antibodies against therapeutic antibodies, and this may significantly reduce their value as pharmaceutical agents. In addition, Fc-gamma RIIb on dendritic cells is thought to be involved in peripheral tolerance by inhibiting dendritic cell activation induced by immune complexes formed between antigens and antibodies, or by suppressing antigen presentation to T cells via activation of Fcgamma receptors (see e.g. NPL65). Since Fc-gamma RIIa is also expressed on dendritic cells, when antibodies that have an Fc with increased selectivity to bind to Fc-gamma RIIb were used as pharmaceutical agents, antigens could not be easily presented by dendritic cells and the like. due to increased selectivity to bind to Fc-gamma RIIb, and the risk of developing anti-drug antibodies could be relatively reduced. These antibodies may also be valuable in this regard.

More specifically, value as pharmaceuticals can be substantially reduced with increased ability to bind to Fc-gamma RIIa, resulting in an increased risk of thrombus formation from platelet aggregation and an increased risk of anti-therapeutic antibody production due to increased immunogenicity.

From this point of view, the above Fc variant with increased ability to bind to Fcgamma RIIb is characterized by a significantly increased binding of R-type Fc-gamma RIIa compared to naturally occurring IgG1. Thus, its value as a pharmaceutical for patients who carry R-type Fc-gamma RIIa is greatly reduced. Types H and R Fc-gamma RIIa occur in Caucasians and African Americans with approximately the same frequency (see, for example, NPL66 and NPL67). Thus, when this Fc variant is used for the treatment of autoimmune diseases, the number of patients for whom it can be safely used to achieve its effectiveness as a pharmaceutical agent may be limited.

In addition, maturation of dendritic cells has been found to occur if the dendritic cells are deficient in Fc-gamma RIIb or if the interaction between Fc-gamma RIIb and the Fc region of an antibody to Fc-gamma RIIb is inhibited in dendritic cells (see e.g. NPL68 and NPL69). In this study, it is suggested that RIIb Fc-gamma actively inhibits the maturation of dendritic cells in a stable state in the absence of inflammation or the like. and in the absence of activation. Fc-gamma RIIa is expressed on the surface of dendritic cells in addition to Fc-gamma RIIb; therefore, even if binding to inhibitory Fc-gamma RIIb is increased and if binding to activating Fc-gamma R such as Fc-gamma RIIa is also increased, maturation of dendritic cells may be enhanced as a result. More specifically, increasing not only Fc-gamma RIIb-4 040713 binding activity, but also the ratio of Fc-gamma RIIb-binding activity and Fc-gamma

RIIa-binding activity is considered as an important parameter in the development of antibodies with immunosuppressive action.

Thus, in developing a pharmaceutical that utilizes Fc-gamma RIIb binding-mediated immunosuppressive activity, there is a need for an Fc variant that not only has increased Fc-gamma RIIb binding activity, but also has the ability to bind to both Fc-gamma allotypes. RIIa H and R, which is maintained at a similar level or attenuated to a lower level compared to naturally occurring IgG1.

However, studies have now been published in which amino acid changes have been introduced into the Fc region to increase binding selectivity to Fc-gamma RIIb (see, for example, NPL70). However, all variants reported in this publication that had improved selectivity for Fc-gamma RIIb were found to have reduced ability to bind to Fc-gamma RIIb compared to naturally occurring IgG1. Thus, in fact, it seems difficult for these variants to induce an Fc-gamma RIIb mediated immunosuppressive response that is stronger than IgG1.

In addition, since the Fc-gamma of RIIb plays an important role for the above agonist antibodies, it is expected that an increase in their binding activity will lead to an increase in agonist activity. However, when binding to Fc-gamma RIIa is increased in a similar manner, activities such as ADCC activity and ADCP activity may be inadvertently displayed, and this may lead to side effects. From this point of view, it is preferable to be able to selectively increase the Fc-gamma RIIb-binding activity.

From these results it follows that in the production of therapeutic antibodies intended for the treatment of autoimmune diseases and cancer, in which Fc-gamma RIIb is involved, it is important that, compared to naturally occurring IgG1, the binding activity to both Fc-gamma RIIa allotypes is maintained. or decreased, and binding to Fc-gamma RIIb increased. However, Fc-gamma RIIb shares 93% extracellular sequence identity with Fc-gamma RIIa, which is one of the activating Fc-gamma Rs, and is structurally very similar. Fc-gamma RIIa, H-type and R-type allotypes are known, in which the amino acid at position 131 is His (H-type) or Arg (R-type), and each of them interacts differently with antibodies ( see, for example, NPL71). Thus, it can be a difficult problem to generate an Fc region variant with increased selective binding to Fc-gamma RIIb over each of the Fc-gamma RIIa allotypes, including the ability to distinguish between highly homologous sequences in Fc-gamma RIIa and Fc-gamma RIIb. In light of these difficulties, several Fc variants have now been identified that have selective binding activity for Fc-gamma RIIb over Fc-gamma RIIa by performing a wide-ranging analysis of amino acid modifications in the Fc region (see, for example, PTL16, PTL17, PTL18, PTL19 and PTL20).

To date, a variant Fc region has been described with selective binding to Fcgamma RIIb among human Fc-gamma R, but there is no evidence of a variant Fc region with selective binding to Fc-gamma RIIb among monkey Fc-gamma R. Given the absence of this Fc variant, the effects of the Fc variant selectively binding to Fc-gamma RIIb have not yet been extensively tested in monkeys.

In addition to the above, it has been described that by modifying the charge of amino acid residues that can be displayed on the surface of an antibody, resulting in an increase or decrease in the isoelectric point (pI) of the antibody, the blood half-life of the antibody can be controlled (see, for example, PTL21 and PTL22). These patents demonstrate that it is possible to prolong the plasma half-life of an antibody by lowering the pI of the antibody and vice versa.

In addition, it has been described that incorporation of an antigen into cells can be increased by modifying the charge of specific amino acid residues, primarily in the CH3 domain of an antibody, to increase its pI (see, for example, PTL23). In addition, it is described that by modifying the charge of amino acid residues in the constant region (mainly in the CH1 domain) in order to reduce pI, the plasma half-life of the antibody can be prolonged (see, for example, PTL24).

Link List

Patent Literature.

[PTL1] US Patent No. 5827733.

[PTL2] WO 1994/021681.

[PTL3] US Patent No. 6096506.

[PTL4] US Patent No. 7261893.

[PTL5] US Patent No. 7320789.

[PTL6] US Patent No. 7807159.

[PTL7] US Patent No. 7888486.

[PTL8] WO 2005/094446.

- 5 040713

[PTL9] WO 2007/047112.

[PTL10] WO 2010/070094.

[PTL11] WO 2000/042072.

[PTL12] WO 2006/019447.

[PTL13] WO 2004/099249.

[PTL14] WO 2004/029207.

[PTL15] US Patent Application Publication No. US 2009/0136485.

[PTL16] WO 2012/115241.

[PTL17] WO 2013/047752.

[PTL18] WO 2013/125667.

[PTL19] WO 2014/030728.

[PTL20] WO 2014/163101.

[PTL21] WO 2007/114319.

[PTL22] WO 2009/041643.

[PTL23] WO 2014/145159.

[PTL24] WO 2012/016227.

non-patent literature.

[NPL2] Hoodless et al., Curr. top. microbiol. Immunol. 228, 1998, p. 235-272.

[NPL3] Zimmers et al., Science 296(5572), 2002, p. 1486-1488.

[NPL4] McPherron et al., Nature 387(6628), 1997, p. 83-90.

[NPL5] McPherron and Lee, Proc. Natl. Acad. sci. USA 94(23), 1997, p. 12457-12461.

[NPL6] Szlama et al., FEBS J 280(16), 2013, p. 3822-3839.

[NPL7] Lee, PloS One 3(2), 2008, p. e1628.

[NPL8] Lee, Proc. Natl. Acad. sci. USA 98(16), 2001, p. 9306-9311.

[NPL9] McPherron et al., Nature 387(6628), 1997, p. 83-90.

[NPL10] McCroskery et al., J Cell Sci. 118(15), 2005, p. 3531-3541.

[NPL11] Whittemore et al., Biochem. Biophys. Res. commun. 300(4), 2003, p. 965-971.

[NPL12] Bogdanovich et al., Nature 420(6914), 2002, p. 418-421.

[NPL13] Wagner., Ann. Neurol. 52(6), 2002, p. 832-836.

[NPL14] Reichert et al., Nat. Biotechnol. 23, 2005, p. 1073-1078.

[NPL15] Pavlou et al., Eur. J Pharm. Biopharm. 59, 2005, p. 389-396.

[NPL16] Clark et al., Chem. Immunol. 65, 1997, p. 88-110.

[NPL17] Jefferis et al., Immunol. Lett. 82, 2002, p. 57-65.

[NPL18] Smith et al., Nat. Rev. Immunol. 10, 2010, p. 328-343.

[NPL19] Radaev et al., J. Biol. Chem. 276, 2001, p. 16478-16483.

[NPL20] Greenwood et al., Eur. J. Immunol. 23, 1993, p. 1098-1104.

[NPL21] Morgan et al., Immunology 86, 1995, p. 319-324.

[NPL22] Nimmerjahn et al., Nat. Rev. Immunol. 8, 2008, p. 34-47.

[NPL23] Amigorena et al., Eur. J. Immunol. 19, 1989, p. 1379-1385.

[NPL24] Sinclair, J. Exp. Med. 129, 1969, p. 1183-1201.

[NPL25] Heyman, Immunol. Lett. 88, 2003, p. 157-161.

[NPL26] Amigorena et al., Science 256, 1992, p. 1808-1812.

[NPL27] Muta et al., Nature 368, 1994, p. 70-73.

[NPL28] Ravetch, Science 290, 2000, p. 84-89.

[NPL29] Fournier et al., J. Immunol. 181, 2008, p. 5350-5359.

[NPL30] Smith et al., Nat. Rev. Immunol. 10, 2010, p. 328-343.

[NPL31] J. Immunol. 163, 1999, p. 618-622.

[NPL32] Yuasa et al., J. Exp. Med. 189, 1999, p. 187-194.

[NPL33] Nakamura et al., J. Exp. Med. 191, 2000, p. 899-906.

[NPL34] Blank, Hum. Genet. 117, 2005, p. 220-227.

[NPL35] Olferiev et al., J. Biol. Chem. 282, 2007, p. 1738-1746.

[NPL36] Chen et al., Arthritis Rheum. 54, 2006, p. 3908-3917.

[NPL37] Floto et al., Nat. Med. 11, 2005, p. 1056-1058.

[NPL38] Li et al., J. Immunol. 176, 2006, p. 5321-5328.

[NPL39] Mackay et al., J. Exp. Med. 203, 2006, p. 2157-2164.

[NPL40] Yang et al., J. Immunol. 178, 2007, p. 3272-3280.

[NPL41] Bruhns et al., Blood 113, 2009, p. 3716-3725.

[NPL42] Chu et al., Mol. Immunol. 45, 2008, p. 3926-3933.

[NPL43] Chu et al., J. Allergy Clin. Immunol. 129, 2012, p. 1102-1115.

[NPL44] Veri et al., Arthritis Rheum. 62, 20-10, p. 1933-1943.

[NPL45] Cemerski et al., Immunol. Lett. 143, 2012, p. 34-43.

[NPL46] Wenink et al., J. Immunol. 183, 2009, p. 4509-4520.

- 6 040713

[NPL47] Zhang et al., J. Immunol. 182, 2009, p. 554-562.

[NPL48] Ravetch, Science 333, 2011, p. 1030-1034.

[NPL49] Wilson et al., Cancer Cell 19, 2011, p. 101-113.

[NPL50] Kohrt et al., J. Clin. Invest. 122, 2012, p. 1066-1075.

[NPL51] Xu et al., J. Immunol. 171, 2003, p. 562-568.

[NPL52] Zhang et al., Blood 108, 2006, p. 705-710.

[NPL53] Chuntharapai et al., J. Immunol. 166, 2001, p. 4891-4898.

[NPL54] Ravetch et al., Proc. Natl. Acad. sci. USA 109, 2012, p. 10966-10971.

[NPL55] Ravetch, Science 333, 2011, p. 1030-1034.

[NPL56] Cemerski et al., Immunol. Lett. 143, 2002, p. 28-33.

[NPL57] Chu et al., Mol. Immunol. 45, 2008, p. 3926-3933.

[NPL58] Smith et al., Nat. Rev. Immunol. 10, 2010, p. 328-343.

[NPL59] Scappaticci et al., J. Natl. cancer. Inst. 99, 2007, p. 1232-1239.

[NPL60] Arthritis Rheum. 48, 2003, p. 719-727.

[NPL61] Meyer et al., J. Thromb. haemost. 7, 2008, p. 171-181.

[NPL62] Robles-Carrillo et al., J. Immunol. 185, 2010, p. 1577-1583.

[NPL63] Duffau et al., Sci. Transl. Med. 2:47ra63, 2010.

[NPL64] Richards et al., Mol. Cancer Ther. 7, 2008, p. 2517-2527.

[NPL65] Desai et al., J. Immunol. 178, 2007, p. 6217-6226.

[NPL66] Salmon et al., J. Clin. Invest. 97, 1996, p. 1348-1354.

[NPL67] Manger et al., Arthritis Rheum. 41, 1998, p. 1181-1189.

[NPL68] Boruchov et al., J. Clin. Invest. 115, 2005, p. 2914-2923.

[NPL69] Dhodapkar et al., Proc. Natl. Acad. sci. USA 102, 2005, p. 2910-2915.

[NPL70] Armor et al., Mol. Immunol. 40, 2003, p. 585-593.

[NPL71] Warmerdam et al., J. Exp. Med. 172, 1990, p. 19-25.

Brief summary of the invention

The present invention relates to antibodies to myostatin and methods of their use. The invention also relates to proteins containing a variant Fc region and methods for their use.

In some embodiments, an isolated anti-myostatin antibody of the present invention binds to latent myostatin. In other embodiments, the antibody binds to an epitope within a fragment consisting of amino acids 21-100 of the myostatin propeptide (SEQ ID NO: 78). In some embodiments, the isolated anti-myostatin antibody of the present invention inhibits myostatin activation. In other embodiments, the antibody blocks the release of mature myostatin from latent myostatin. In other embodiments, the antibody blocks the proteolytic release of mature myostatin. In other embodiments, the antibody blocks the spontaneous release of mature myostatin. In other embodiments, the antibody does not bind to mature myostatin. In other embodiments, the antibody binds to the same epitope as the antibody shown in Table 1. 13. In other embodiments, the antibody binds to the same epitope as an antibody that contains the VH and VL pair shown in Table 13. 13. In other embodiments of the invention, the antibody binds to the same epitope as the antibody presented in table. 2a. In other embodiments, the antibody binds to the same epitope as an antibody that contains the VH and VL pair shown in Table 1. 2a. In other embodiments, the antibody binds to the same epitope as the antibody shown in Table 1. 11a. In other embodiments, the antibody binds to the same epitope as an antibody that contains the VH and VL pair shown in Table 1. 11a. In other embodiments, the antibody binds to the same epitope as the antibody shown in Table 1. 2a, 11a or 13. In other embodiments of the invention, the antibody binds to the same epitope as the antibody that contains the pair of VH and VL listed in table. 2a, 11a, or 13. In some embodiments, the isolated anti-myostatin antibody of the present invention binds latent myostatin with higher affinity at neutral pH than at acidic pH. In some embodiments, the anti-myostatin antibody binds latent myostatin with higher affinity at pH 7.4 than at pH 5.8. In some embodiments, an isolated anti-myostatin antibody of the present invention binds to a polypeptide fragment consisting of amino acids 21-100 of the myostatin propeptide (SEQ ID NO: 78) with higher affinity at pH 7.4 than at pH 5. ,8. In some embodiments, the antibody binds to the same myostatin epitope as the antibody shown in Table 1. 13 with higher affinity at neutral pH than at acidic pH. In additional embodiments, the anti-myostatin antibody binds to the same epitope as the antibody shown in Table 1. 13 with higher affinity at pH 7.4 than at pH 5.8. In other embodiments, the antibody binds to the same epitope as an antibody that contains the VH and VL pair specified in

- 7 040713 tab. 13 with higher affinity at pH 7.4 than at pH 5.8. In some embodiments, the antibody binds to the same myostatin epitope as the antibody shown in Table 1. 2a with higher affinity at neutral pH than at acidic pH. In some embodiments, the antibody binds to the same myostatin epitope as the antibody shown in Table 1. 2a with higher affinity at pH 7.4 than at pH 5.8. In other embodiments, the antibody binds to the same epitope as an antibody that contains the VH and VL pair shown in Table 1. 2a with higher affinity at pH 7.4 than at pH 5.8. In additional embodiments, the anti-myostatin antibody binds to the same epitope as the antibody shown in Table 1. 11a, with higher affinity at neutral pH than at acidic pH. In other embodiments, the antibody binds to the same myostatin epitope as the antibody shown in Table 1. 11a at pH 7.4 than at pH 5.8. In other embodiments, the antibody binds to the same epitope as an antibody that contains the VH and VL pair shown in Table 1. 11a, with higher affinity at pH 7.4 than at pH 5.8. In additional embodiments, the anti-myostatin antibody binds to the same epitope as the antibody shown in Table 1. 2a, 11a or 13 with higher affinity at neutral pH than at acidic pH. In other embodiments, the antibody binds to the same myostatin epitope as the antibody shown in Table 1. 2a, 11a or 13 with higher affinity at pH 7.4 than at pH 5.8. In other embodiments, the antibody binds to the same epitope as an antibody that contains the VH and VL pair shown in Table 1. 2a, 11a or 13 with higher affinity at pH 7.4 than at pH 5.8.

In some embodiments, an isolated anti-myostatin antibody of the present invention competes for binding to latent myostatin with an antibody as defined herein. In some embodiments, the isolated anti-myostatin antibody of the present invention competes for binding to latent myostatin with the antibody shown in Table 1. 13. In some embodiments, the isolated anti-myostatin antibody of the present invention competes for binding to latent myostatin, with an antibody that contains the VH and VL pair shown in Table 13. 13. In some embodiments of the invention, the antibody competes for binding to latent myostatin with the antibody shown in table. 2a. In some embodiments, an isolated anti-myostatin antibody of the present invention competes for binding to latent myostatin with an antibody that contains the VH and VL pair shown in Table 1. 2a. In some embodiments of the invention, the antibody competes for binding to latent myostatin with the antibody shown in table. 11a. In some embodiments, an isolated anti-myostatin antibody of the present invention competes for binding to latent myostatin with an antibody that contains the VH and VL pair shown in Table 1. 11a. In additional embodiments, the anti-myostatin antibody competes for binding to latent myostatin with the antibody shown in Table 1. 2a, 11a, or 13. In further embodiments, the anti-myostatin antibody competes for binding to latent myostatin with an antibody that contains the VH and VL pair shown in Table 2. 2a, 11a, or 13. In other embodiments, the anti-myostatin antibody binds latent myostatin with higher affinity at neutral pH than at acidic pH. In other embodiments, the anti-myostatin antibody binds latent myostatin with higher affinity at pH 7.4 than at pH 5.8. In other embodiments, the anti-myostatin antibody binds to a polypeptide fragment consisting of amino acids 21-100 of the myostatin propeptide (SEQ ID NO: 78) with higher affinity at pH 7.4 than at pH 5.8. Methods for assessing the ability of an antibody to compete with a reference antibody for binding to latent myostatin are described herein and are known in the art.

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

In some embodiments, an anti-myostatin antibody of the invention comprises:

- 8 040713 (a) (I) HVR-H3 containing the amino acid sequence GVPAX1SX2GGDX3, in which X1 is Y or H, X2 is T or H, X ₃ is L or K (SEQ ID NO: 128), (II) HVR -L3 containing the amino acid sequence AGGYGGGX ₁ YA, in which X ₁ is L or R (SEQ ID NO: 131), and (III) HVR-H2 containing the amino acid sequence IISX1AGX ₂ X ₃ YX ₄ X ₅ X ₆ WAKX ₇ , in which X1 is Y or H, X ₂ is S or K, X ₃ is T, M or K, X ₄ is Y or K, X ₅ is A, M or E, X ₆ is S or E, X ₇ is G or K (SEQ ID NO: 127);

(b) (I) HVR-H1 containing the amino acid sequence X ₁ X ₂ DIS, in which X ₁ is S or H, X ₂ is Y, T, D or E (SEQ ID NO: 126), (II) HVR -H2 containing the amino acid sequence IISX ₁ AGX ₂ X ₃ YX ₄ X ₅ X ₆ WAKX ₇ in which X1 is Y or H, X ₂ is S or K, X ₃ is T, M or K, X4 is Y or K , X ₅ is A, M or E, X ₆ is S or E, X ₇ is G or K (SEQ ID NO: 127), and (III) HVR-H3 containing the amino acid sequence GVPAX1SX2GGDX3 in which X1 is Y or H, X ₂ is T or H, X ₃ is L or K (SEQ ID NO: 128);

(c) (I) HVR-H1 containing the amino acid sequence X1X2DIS, in which X1 is S or H, X ₂ is Y, T, D or E (SEQ ID NO: 126), (II) HVR-H2 containing the amino acid the sequence IISX1AGX ₂ X ₃ YX ₄ X ₅ X ₆ WAKX ₇ in which X1 is Y or H, X ₂ is S or K, X ₃ is T, M or K, X ₄ is Y or K, X ₅ is A, M or E, X ₆ is S or E, X ₇ is G or K (SEQ ID NO: 127), (III) HVR-H3 containing the amino acid sequence GVPAX1SX2GGDX3, in which X1 is Y or H, X ₂ is T or H, X ₃ is L or K (SEQ ID NO: 128), (IV) HVR-L1 containing the amino acid sequence X ₁ X ₂ SQX ₃ VX ₄ X ₅ X ₆ NWLS where X1 is Q or T, X ₂ is S or T, X ₃ is S or E, X4 is Y or F, X ₅ is D or H, X ₆ is N, D, A or E (SEQ ID NO: 129); (V) HVR-L2 containing the amino acid sequence WAX1TLAX2, in which X1 is S or E, X ₂ is S, Y, F or W (SEQ ID NO: 130); and (VI) HVR-L3 containing the amino acid sequence AGGYGGGX1YA, in which X1 is L or R (SEQ ID NO: 131);

(d) (I) HVR-L1 containing the amino acid sequence X1X ₂ SQX ₃ VX ₄ X ₅ X ₆ NWLS, in which X1 is Q or T, X ₂ is S or T, X ₃ is S or E, X ₄ is Y or F, X ₅ is D or H, X ₆ is N, D, A or E (SEQ ID NO: 129); (II) HVR-L2 containing the amino acid sequence WAX1TLAX2, in which X1 is S or E, X ₂ is S, Y, F or W (SEQ ID NO: 130); and (III) HVR-L3 containing the amino acid sequence AGGYGGGX1YA, in which X1 is L or R (SEQ ID NO: 131). In some embodiments of the invention, the antibody specified in subparagraph (b), further comprises a framework plot of the variable domain of the heavy chain FR1 containing one of the amino acid sequences of SEQ ID NO: 132-134; FR2 containing one of the amino acid sequences of SEQ ID NO: 135-136; FR3 containing 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 subparagraph (d) further comprises an FR1 light chain variable domain framework region comprising the amino acid sequence of SEQ ID NO: 139; FR2 containing one of the amino acid sequences of SEQ ID NO: 140-141; FR3 containing one of the amino acid sequences of SEQ ID NO: 142-143; and FR4 containing the amino acid sequence of SEQ ID NO: 144.

In some embodiments, the isolated anti-myostatin antibody of the present invention comprises (a) an HVR-H3 containing the amino acid sequence GVPAX1SX2GGDX3, wherein X1 is Y or H, _X2 is T or H, _X3 is L or K ( SEQ ID NO: 128), (b) HVR-L3 containing the amino acid sequence AGGYGGGX1YA, in which X1 is L or R (SEQ ID NO: 131), and (c) HVR-H2, containing the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7, in which X1 is Y or H, X ₂ is S or K, X ₃ is T, M or K, X ₄ is Y or K, X ₅ is A, M or E, X ₆ is S or E, X ₇ is G or K (SEQ ID NO: 127).

In some embodiments, the isolated anti-myostatin antibody of the present invention comprises (a) an HVR-H1 comprising the amino acid sequence X1X2DIS, wherein X1 is S or H, X ₂ is Y, T, D, or E (SEQ ID NO : 126), (b) HVR-H2 containing the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7, in which 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, X ₆ is S or E, X ₇ is G or K (SEQ ID NO: 127), and (c) HVR-H3 containing the amino acid sequence GVPAX1SX2GGDX3 wherein X1 is Y or H, X ₂ is T or H , X3 is L or K (SEQ ID NO: 128). In other embodiments, the antibody comprises a FR1 heavy chain variable domain framework region comprising one of the amino acid sequences of SEQ ID NOs: 132-134; FR2 containing one of the amino acid sequences of SEQ ID NO: 135-136; FR3 containing the amino acid sequence of SEQ ID NO: 137; and FR4 containing the amino acid sequence of SEQ ID NO: 138. In others

- 9 040713 embodiments of the invention, the antibody further comprises (a) HVR-L1 containing the amino acid sequence X ₁ X ₂ SQX ₃ VX ₄ X ₅ X ₆ NWLS, in which X ₁ is Q or T, X2 is S or T, X ₃ is S or E, X ₄ is Y or F, X ₅ is D or H, X ₆ is N, D, A or E (SEQ ID NO: 129); (b) HVR-L2 containing the amino acid sequence WAX1TLAX2, in which X ₁ denotes S or E, X ₂ denotes S, Y, F or W (SEQ ID NO: 130); and (c) HVR-L3 containing the amino acid sequence AGGYGGGX ₁ YA, wherein X ₁ is L or R (SEQ ID NO: 131).

In some embodiments, the isolated anti-myostatin antibody of the present invention comprises (a) an HVR-L1 comprising the amino acid sequence X ₁ X ₂ SQX ₃ VX ₄ X ₅ X ₆ NWLS, wherein X ₁ is Q or T, X ₂ is S or T, X ₃ is S or E, X4 is Y or F, X ₅ is D or H, X ₆ is N, D, A or E (SEQ ID NO: 129); (b) HVR-L2 containing the amino acid sequence WAX1TLAX2, in which X1 is S or E, X ₂ is S, Y, F or W (SEQ ID NO: 130); and (c) HVR-L3 containing the amino acid sequence AGGYGGGX ₁ YA, wherein X ₁ is L or R (SEQ ID NO: 131). In a further embodiment, the antibody further comprises an FR1 light chain variable domain framework region comprising the amino acid sequence of SEQ ID NO: 139; FR2 containing one of the amino acid sequences of SEQ ID NO: 140-141; FR3 containing one of the amino acid sequences of SEQ ID NO: 142-143; and FR4 containing the amino acid sequence of SEQ ID NO: 144.

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

In some embodiments, an isolated anti-myostatin antibody of the present invention comprises (a) a VH sequence that is at least 95% identical to one of the amino acid sequences of SEQ ID NOs: 13, 16-30, 32-34, and 86-95 ; (b) a VL sequence at least 95% identical to one of the amino acid sequences of SEQ ID NOs: 15, 31, 35-38 and 96-99; or (c) a VH sequence as specified in subparagraph (a) and a VL sequence as specified in subparagraph (b). In other embodiments, the antibody comprises a VH sequence as set forth in one of SEQ ID NOs: 13, 16-30, 32-34, and 86-95. In other embodiments, the antibody comprises a VL sequence as set forth in one of SEQ ID NOs: 15, 31, 35-38, and 96-99. In some embodiments, the antibody comprises a VH sequence as set forth in one of SEQ ID NOs: 13, 16-30, 32-34, and 86-95. In other embodiments, the antibody comprises the VH sequence shown in one of SEQ ID NOs: 13, 16-30, 32-34, and 86-95; and the VL sequence shown in one of SEQ ID NOs: 15, 31, 35-38 and 96-99.

The invention also relates to isolated nucleic acids encoding an anti-myostatin antibody of the present invention. The invention also relates to host cells that contain the nucleic acid of the present invention. The invention also relates to a method for producing an antibody, which comprises culturing a host cell according to the present invention so as to obtain an antibody.

In some aspects of the invention, a method for producing an antibody to myostatin is provided, which consists in the fact that: (a) cultivating a host cell proposed in the present invention, so as to obtain an antibody; or (b) immunizing the animal with a polypeptide, wherein the polypeptide contains a region corresponding to amino acids at positions 21-100 of the myostatin propeptide (SEQ ID NO: 78).

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

The invention also relates to a pharmaceutical composition which contains an anti-myostatin antibody of the present invention and a pharmaceutically acceptable carrier.

The invention also relates to polypeptides containing variants of the Fc regions, and methods for their preparation and use.

One embodiment of the invention is an Fc-gamma RIIb-binding polypeptide

- 10 040713 types containing variants of the Fc regions, and methods for their use. In some embodiments, a variant Fc region with increased Fc-gamma RIIb binding activity of the present invention contains at least one amino acid change in the parent Fc region. In other embodiments, the ratio [KD value of parental Fc region for simian Fc-gamma RIIb]/[KD value of variant Fc region for simian Fc-gamma RIIb] is 2.0 or greater. In other embodiments, the ratio [KD value of the parent Fc region for simian Fc-gamma RIIIa]/[KD value of the variant Fc region for simian Fc-gamma RIIIa] is 0.5 or less. In other embodiments, the ratio [KD value of parent Fc region for human Fc-gamma RIIb]/[KD value of variant Fc region for human Fc-gamma RIIb] is 2.0 or greater. In other embodiments, the ratio [KD value of the parental Fc region for human Fc-gamma RIIIa]/[KD value of the variant Fc region for human Fc-gamma RIIIa] is 0.5 or less. In other embodiments, the ratio [KD value of parental Fc region for human Fc-gamma RIIa (H-type)]/[KD value of variant Fc region for human Fc-gamma RIIa (H-type)] is 5.0 or less. In other embodiments, the ratio [KD value of parental Fc region for human Fc-gamma RIIa (R) type]/[KD value of variant Fc region for human Fc-gamma RIIa (R-type)] is 5.0 or less . In another embodiment, the Fc region variant KD value for the simian Fc-gamma RIIb is 1.0 x 10 ^-6 M or less. In another embodiment of the invention, the KD value of the Fc region variant for the simian Fc-gamma RIIIa is 5.0 x 10' ⁷ M or greater. In another embodiment of the invention, the KD value of the Fc region variant for human Fc-gamma RIIb is 2.0 x 10 ^-6 M or less. In another embodiment of the invention, the KD value of the Fc region variant for the human Fc-gamma RIIIa is 1.0 x 10 ^-6 M or more. In another embodiment of the invention, the KD value of the Fc region variant for human Fc-gamma RIIa (H-type) is 1.0x10 ^-7 M or more. In another embodiment, the Fc region variant KD value for human Fc-gamma RIIa (R-type) is 2.0 x 10 ^-7 M or greater.

In some embodiments, a variant Fc region with increased Fc-gamma RIIb binding activity of the present invention contains at least one amino acid change at at least one position selected from the group consisting of 231, 232, 233, 234, 235, 236, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334 and 396, according to EU numbering.

In other embodiments, a variant Fc region with increased Fc-gamma RIIb binding activity comprises at least two amino acid changes, comprising (a) one amino acid change at position 236 and (b) at least one amino acid change at at least one position selected from the group consisting of: (I) positions: 231, 232, 233, 234, 235, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334 and 396; (II) provisions: 231, 232, 235, 239, 268, 295, 298, 326, 330 and 396; or (III) provisions 268, 295, 326 and 330; according to EU numbering.

In other embodiments, a variant Fc region with increased Fc-gamma RIIb binding activity comprises at least two amino acid changes, comprising (a) one amino acid change at position 236 and (b) at least one amino acid change at at least one position selected from the group consisting of 231, 232, 233, 234, 235, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332 , 334 and 396, according to the EU numbering.

In other embodiments, a variant Fc region with increased Fc-gamma RIIb binding activity comprises at least two amino acid changes, comprising (a) one amino acid change at position 236 and (b) at least one amino acid change at at least one position , selected from the group consisting of 231, 232, 235, 239, 268, 295, 298, 326, 330 and 396, according to EU numbering.

In other embodiments, a variant Fc region with increased Fc-gamma RIIb binding activity comprises at least two amino acid changes, comprising (a) one amino acid change at position 236 and (b) at least one amino acid change at at least one position , selected from the group consisting of 268, 295, 326 and 330, according to the EU numbering.

In some embodiments, the Fc region variant with increased Fc-gamma RIIb 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; (6) 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; (l)

- 11 040713

Phe at position 266; (m) Ala, His, Leu at position 267; (m) Asp, Glu at position 268; (o) Asp, Glu, Gly at position 271; (p) Leu at position 295; (p) Leu at position 298; (c) Glu, Phe, Ile, Leu at position 325; (r) Thr at position 326; (y) Ile, Asn at position 327; (f) Thr at position 328; (x) Lys, Arg at position 330; (c) Glu at position 331; (h) Asp at position 332; (w) 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 other embodiments, the Fc region variant with increased Fc-gamma RIIb 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; (e) 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.

Another embodiment of the invention is a polypeptide that contains a variant Fc region with an increased isoelectric point (pI) and a method of using it. In some embodiments, a polypeptide that contains an elevated pI variant Fc region includes at least two amino acid changes in the parent Fc region. In other embodiments, each of the amino acid changes increases the isoelectric point (pI) of the variant Fc region relative to the parent Fc region. In other embodiments, the amino acid may be displayed on the surface of a variant Fc region. In other embodiments, the polypeptide comprises a variant Fc region and an antigen binding domain. In other embodiments of the invention, the antigen-binding activity of the antigen-binding domain varies depending on the concentration of ions. In other embodiments, the increased pI variant Fc region of the present invention contains at least two amino acid changes at at least two positions selected from the group consisting of 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422 and 431, according to EU numbering. In other embodiments, the elevated pI variant Fc region contains Arg or Lys at each of the selected positions.

In some embodiments, the Fc region variant of the present invention contains the amino acid changes shown in Table 1. 14-30.

In some embodiments, the polypeptide comprises a variant Fc region of the present invention. In other embodiments, the parental Fc region is derived from human IgG1. In other embodiments, the polypeptide is an antibody. In other embodiments, the polypeptide is an Fc-containing fusion protein.

The invention provides a polypeptide containing one of the amino acid sequences of SEQ ID NO: 229-381.

The invention also relates to an isolated nucleic acid which encodes a polypeptide containing a variant Fc region according to the invention. The invention also relates to a host cell that contains the nucleic acid of the present invention. The invention also relates to a method for producing a polypeptide that contains a variant Fc region, which consists in cultivating the host proposed in the present invention, so as to obtain a polypeptide.

The invention also relates to a pharmaceutical composition that contains a polypeptide containing a variant of the Fc region proposed in the present invention, and a pharmaceutically acceptable carrier.

In particular, the present invention provides the following.

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

[2] The antibody according to [1], where the antibody:

(a) blocks the release of mature myostatin from latent myostatin;

(b) blocks the proteolytic release of mature myostatin;

(c) blocks the spontaneous release of mature myostatin; or (d) does not bind to mature myostatin; or binds to an epitope within a fragment consisting of amino acids 21-100 of the myostatin propeptide (SEQ ID NO: 78).

[3] The antibody according to [1] or item [2], where the antibody competes for binding to latent myostatin with an antibody that contains a pair of VH and VL indicated in tables 2a, 11a or 13, or binds to the same epitope as the specified antibody.

[4] An antibody according to one of [1] to [3], which binds to latent myostatin with higher affinity at neutral pH than at acidic pH, or which binds to latent myostatin with higher affinity at pH 7.4 than at pH 5.8.

[5] An antibody according to one of [1]-[4], which is (a) a monoclonal antibody, (b)

- 12 040713 human, humanized or chimeric antibody; (c) a full-length IgG-type antibody; or (d) an antibody fragment that binds to myostatin.

[6] An antibody according to one of [1]-[5], wherein the antibody comprises:

(a) (I) HVR-H3 containing the amino acid sequence GVPAX1SX2GGDX3, in which X1 is Y or H, X2 is T or H, X ₃ is L or K (SEQ ID NO: 128), (II) HVR-L3, containing the amino acid sequence AGGYGGGX1YA, in which X1 is L or R (SEQ ID NO: 131), and (III) HVR-H2, containing the amino acid sequence nSX1AGX ₂ X ₃ YX ₄ X ₅ X ₆ WAKX ₇ , in which X1 is Y or H, X ₂ is S or K, X ₃ is T, M or K, X ₄ is Y or K, X ₅ is A, M or E, X ₆ is S or E, X ₇ is G or K (SEQ ID NO: 127);

(b) (I) HVR-H1 containing the amino acid sequence X1X2DIS, in which X1 is S or H, X ₂ is Y, T, D or E (SEQ ID NO: 126), (II) HVR-H2 containing the amino acid sequence IISX1AGX2X3YX4X5X6WAKX7 in which X1 is Y or H, X ₂ is S or K, X ₃ is T, M or K, X ₄ is Y or K, X ₅ is A, M or E, X ₆ is S or E, X ₇ is G or K (SEQ ID NO: 127), and (III) HVR-H3 containing the amino acid sequence GVPAX1SX2GGDX3 wherein X1 is Y or H, X ₂ is T or H, X ₃ is L or K (SEQ ID NO: 128);

(c) (I) HVR-H1 containing the amino acid sequence X1X2DIS, in which X1 is S or H, X ₂ is Y, T, D or E (SEQ ID NO: 126), (II) HVR-H2 containing the amino acid the sequence IISX1AGX ₂ X ₃ YX ₄ X ₅ X ₆ WAKX ₇ in which X1 is Y or H, X ₂ is S or K, X ₃ is T, M or K, X ₄ is Y or K, X ₅ is A, M or E, X ₆ is S or E, X ₇ is G or K (SEQ ID NO: 127), (III) HVR-H3 containing the amino acid sequence GVPAX1SX2GGDX3, in which X1 is Y or H, X ₂ is T or H, X ₃ is L or K (SEQ ID NO: 128), (IV) HVR-L1 containing the amino acid sequence X1X2SQX3VX4X5X6NWLS, in which X1 is Q or T, X ₂ is S or T, X ₃ is S or E, X ₄ is Y or F, X ₅ is D or H, X ₆ is N, D, A or E (SEQ ID NO: 129); (V) HVR-L2 containing the amino acid sequence WAX1TLAX2, in which X1 is S or E, X ₂ is S, Y, F or W (SEQ ID NO: 130); and (VI) HVR-L3 containing the amino acid sequence AGGYGGGX1YA, in which X1 is L or R (SEQ ID NO: 131);

(d) (I) HVR-L1 containing the amino acid sequence X1X ₂ SQX ₃ VX ₄ X ₅ X ₆ NWLS, in which X1 is Q or T, X ₂ is S or T, X ₃ is S or E, X ₄ is Y or F, X ₅ is D or H, X6 is N, D, A or E (SEQ ID NO: 129); (II) HVR-L2 containing the amino acid sequence WAX1TLAX2, in which X1 is S or E, X ₂ is S, Y, F or W (SEQ ID NO: 130); and (III) HVR-L3 containing the amino acid sequence AGGYGGGX1YA, in which X1 is L or R (SEQ ID NO: 131).

[7] The antibody according to [6](b), further comprising a frame plot of the variable domain of the heavy chain FR1, which contains one of the amino acid sequences of SEQ ID NO: 132-134; FR2, which contains one of the amino acid sequences of SEQ ID NO: 135-136; FR3, which contains the amino acid sequence of SEQ ID NO: 137; and FR4, which contains the amino acid sequence of SEQ ID NO: 138.

[8] The antibody according to [6](d), further comprising a framework region of the FR1 light chain variable domain, which contains the amino acid sequence of SEQ ID NO: 139; FR2, which contains one of the amino acid sequences of SEQ ID NO: 140-141; FR3, which contains one of the amino acid sequences of SEQ ID NO: 142-143; and FR4, which contains the amino acid sequence of SEQ ID NO: 144.

[9] An antibody according to one of [1]-[5] containing (a) a VH sequence at least 95% identical to one of the amino acid sequences of SEQ ID NOs: 13, 16-30, 32-34 and 8695 ; (b) a VL sequence at least 95% identical to one of the amino acid sequences of SEQ ID NOs: 15, 31, 35-38 and 96-99; or (c) a VH sequence as set forth in one of SEQ ID NOs: 13, 16-30, 32-34, and 86-95 and a VL sequence as set forth in one of SEQ ID NOs: 15, 31, 35-38, and 96 -99.

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

[11] A host cell containing the nucleic acid of [10].

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

(a) culture the host cell according to [11] so as to obtain an antibody; or (b) immunizing the animal with a polypeptide, wherein the polypeptide contains a region corresponding to positions 21-100 of the myostatin propeptide (SEQ ID NO: 78).

[13] A pharmaceutical composition comprising an antibody according to one of [1]-[9] and a pharmaceutically acceptable carrier.

[14] A polypeptide comprising a variant Fc region that contains at least one amino acid change in the parent Fc region for which the ratio of [KD value of the parent Fc region for simian Fc-gamma RIIb]/[KD value of the variant Fc region for simian Fc-

- 13 040713 gamma RIIb] is 2.0 or more, and the ratio [KD value of parental Fc region for simian Fc-gamma RIIIa]/[KD value of variant Fc region for simian Fc-gamma RIIIa] is 0.5 or less .

[15] A polypeptide according to [14], which also has a ratio of [KD value of parental Fc region for human Fcgamma RIIb]/[KD value of variant Fc region for human Fcgamma RIIb] is 2.0 or more, and the ratio of [ the KD value of the parental Fc region for human Fc-gamma RSha]/[the KD value of the variant Fc region for human Fc-gamma RIIIa] is 0.5 or less.

[16] The polypeptide of [15] wherein also the ratio [KD value of parental Fc region for human Fc-gamma RIIa (H-type)]/[KD value of variant Fc region for human Fc-gamma RIIa (H-type) )] is 5.0 or less.

[17] The polypeptide of [16] wherein also the ratio [KD value of parental Fc region for human Fc-gamma RIIa (R-type)]/[KD value of variant Fc region for human Fc-gamma RIIa (R-type) )] is 5.0 or less.

[18] A polypeptide according to [14], wherein the KD value of the Fc region variant for simian Fcgamma RIIb is 1.0x10'6 M or less, and the KD value of the Fc region variant for simian Fcgamma RIIIa is 5.0x10'7 M or more.

[19] A polypeptide according to [15], wherein the KD value of the Fc region variant for human Fcgamma RIIb is 2.0x10'6 M or less, and the KD value of the Fc region variant for human Fcgamma RIIIa is 1.0x10'6 M or more.

[20] A polypeptide according to [16], wherein the Fc region variant KD value for human Fcgamma RIIa (H-type) is 1.0 x 10'7 M or more.

[21] A polypeptide according to [17], wherein the Fc region variant KD value for human Fcgamma RIIa (R-type) is 2.0x10'7 M or more.

[22] A polypeptide according to one of [14]-[21], wherein the Fc region variant contains at least one amino acid change at at least one position selected from the group consisting of 231, 232, 233, 234 , 235, 236, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334 and 396, according to EU numbering.

[23] The polypeptide of [22], wherein the Fc region variant contains at least two amino acid changes, including:

(a) one amino acid change at position 236; and (b) at least one amino acid change at at least one position selected from the group consisting of:

(I) provisions 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) provisions: 231, 232, 235, 239, 268, 295, 298, 326, 330 and 396; or (III) provisions 268, 295, 326 and 330; according to EU numbering.

[24] The polypeptide of [22] or [23] wherein the Fc region variant contains 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, (m ) Ala, His, Leu at position 267, (n) Asp, Glu at position 268, (o) Asp, Glu, Gly at position 271, (p) Leu at position 295, (p) Leu at position 298, (c) ) Glu, Phe, Ile, Leu at position 325, (r) Thr at position 326, (y) Ile, Asn at position 327, (f) Thr at position 328,

- 14 040713 (x) Lys, Arg at position 330, (v) Glu at position 331, (h) Asp at position 332, (w) 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.

[25] The polypeptide of [24], wherein the Fc region variant contains at least one amino acid selected from the group consisting of:

(a) Gly, Thr at position 231, (b) Asp at position 232, (c) Tgr 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.

[26] A polypeptide comprising a variant Fc region that contains at least two amino acid changes in the parent Fc region, where each of the amino acid changes increases the isoelectric point (pI) of the variant Fc region compared to the parent Fc region.

[27] The polypeptide of [26] wherein the amino acid changes are exposed on the surface of the variant Fc region.

[28] The polypeptide of [26] or p. [27] also containing an antigen-binding domain.

[29] The polypeptide of [28], wherein the antigen-binding activity of the antigen-binding domain varies with ion concentration.

[30] A polypeptide according to one of [26]-[29], wherein the Fc region variant contains at least two amino acid changes at at least two positions selected from the group consisting of 285, 311, 312, 315 , 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422 and 431, according to EU numbering.

[31] The polypeptide of [30], wherein the Fc region variant contains Arg or Lys at each of the selected positions.

[32] A polypeptide containing a variant of the Fc region, which contains the amino acid changes listed in table. 14-30.

[33] A polypeptide according to one of [14]-[32], wherein the parental Fc region is derived from human IgG1.

[34] A polypeptide according to one of [14]-[33], wherein the polypeptide is an antibody or an Fc fusion protein.

[35] A polypeptide comprising one of the amino acid sequences of SEQ ID NO: 229-381.

[36] An isolated nucleic acid encoding a polypeptide according to one of [14]-[35].

[37] A host cell containing the nucleic acid of [36].

[38] A method for producing a polypeptide containing an Fc region variant, comprising culturing a host cell according to [37] so as to obtain a polypeptide.

[39] A pharmaceutical composition comprising a polypeptide according to one of [14]-[35] and a pharmaceutically acceptable carrier.

Brief description of the drawings

The drawings show:

in fig. 1 Inhibition of proteolytic activation of latent myostatin by anti-latent myostatin antibody as illustrated in Example 3. Active myostatin activity released from latent myostatin by BMP1 protease was measured in the presence of anti-latent myostatin antibody using the HEK Blue™ assay;

in fig. 2 - Inhibition of spontaneous activation of latent myostatin by anti-latent myostatin antibody as illustrated in Example 4. The activity of active myostatin released from latent myostatin by incubation at 37° C. was measured in the presence of anti-latent myostatin antibody using the HEK Blue™ assay;

in fig. 3 - binding of antibodies to latent myostatin with the propeptide domain, as illustrated in example 5;

in fig. 4 - results of Western blot analysis against myostatin propeptide as illustrated in Example 6. Proteolytic cleavage of myostatin propeptide with

- 15 040713

BMP1 was assessed in the presence and absence of antibodies to latent myostatin;

in fig. 5A-5C are BIACORE™-derived sensograms characterizing the binding of anti-latent myostatin antibody MST1032-Glm to human latent myostatin (A), monkey cynomolgus latent myostatin (B), and mouse latent myostatin (C) as illustrated in Example 7;

in fig. 6 - Inhibition of proteolytic and spontaneous activation of latent myostatin by a humanized antibody to latent myostatin, as illustrated in example 8. The activity of active myostatin released from latent myostatin by the action of BMP1 protease (proteolytic) or incubation at 37°C without BMP1 (spontaneous) was measured in the presence of latent myostatin antibodies using the HEK Blue™ assay;

in fig. 7 - BIACORE™ generated sensorgrams characterizing the binding of variants to a substituted histidine of an anti-latent myostatin antibody as illustrated in Example 9. The antibody/antigen complexes were allowed to dissociate at pH 7.4 followed by further dissociation at pH 5.8 (indicated by an arrow) to study pH-dependent interactions. The following antibodies were tested in this experiment: Ab001 (curve indicated by black solid line), Ab002 (curve indicated by black dotted line), Ab003 (curve indicated by black dotted line), Ab004 (curve indicated by gray dashed line), Ab005 (curve indicated by gray dashed line). , indicated by a gray solid line), Ab006 (curve indicated by gray long strokes) and Ab007 (curve indicated by black long strokes);

in fig. 8 - inhibition of proteolytic and spontaneous activation of latent myostatin by pH-dependent antibodies to latent myostatin, as illustrated in example 11. The activity of active myostatin released from latent myostatin by the action of BMP1 protease (proteolytic), or incubation at 37°C without BMP1 (spontaneous) was measured in presence of anti-latent myostatin antibody using the HEK Blue™ assay. In the drawing, antibodies MS1032LO01-SG1, MS1032LO02-SG1, MS1032LO03-SG1 and MS1032LO04-SG1 are designated as MSLO-01, MSLO-02, MSLO-03 and MSLO-04, respectively. Inhibition of proteolytic and spontaneous activation of latent myostatin, comparable to MS1032LO00-SG1, was achieved using MS1032LO01-SG1, MS1032LO02-SG1, MS1032LO03-SG1 and MS1032LO04-SG1;

in fig. 9A-9E are BIACORE™ generated sensorgrams characterizing the binding of a pH-dependent antibody to latent myostatin as illustrated in Example 12. Kinetic parameters of MST1032-SG1 (A), MS1032LO00-SG1 (B), MS1032LO01-SG1 (C), MS1032LO02-SG1 (D), MS1032LO03-SG1 (D) and MS1032LO04-SG1 (E) were measured at neutral pH and acidic pH;

in fig. 10 is a time course of plasma myostatin concentration following intravenous administration of an anti-myostatin antibody to mice as illustrated in Example 13. The effects of Fc-gamma R-mediated cell uptake of antibody/antigen complexes on myostatin clearance in vivo were assessed by comparing anti-myostatin antibodies having the ability to bind with Fc-gamma R (MS1032LO00-SG1), and anti-myostatin antibodies with eliminated ability to bind to Fc-gamma R (MS1032LO00-F760);

in fig. 11 is a time course of plasma myostatin concentration following intravenous administration of anti-myostatin antibody to mice as illustrated in Example 14. The effects of pH dependent anti-myostatin antibody binding on myostatin clearance in vivo were assessed by comparing pH dependent anti-myostatin antibody (MS1032LO01-SG1 or MS1032LO01- F760) and a pH independent anti-myostatin antibody (MS1032LO00-SG1 or MS1032LO00-F760);

in fig. 12: Binding activity of latent myostatin antibody MST1032 to latent myostatin and GDF11 as illustrated in Example 16;

in fig. 13 - Inhibitory activity of the latent myostatin antibody MST1032 against proteolytic and spontaneous activation of GDF11 as illustrated in Example 17. The activity of active GDF11 released by the action of BMP1 protease (proteolytic) or incubation at 37°C without BMP1 (spontaneous) was measured in the presence of antibody to latent myostatin using the HEK Blue™ assay;

in fig. 14 Inhibition of proteolytic activation of latent myostatin by anti-latent myostatin antibody as illustrated in Example 19. The activity of active myostatin released by BMP1 protease was measured in the presence of anti-latent myostatin antibody using the HEK Blue™ assay;

in fig. 15 is a time course of plasma myostatin concentration following intravenous administration of anti-latent myostatin antibody to mice as illustrated in Example 20. Effects of pH dependence on myostatin clearance in vivo were assessed by comparing pH independent anti-latent myostatin antibody (MS1032LO00-SG1) and various pH-dependent antibodies to latent myostatin (MS1032LO01-SG1, MS1032LO06-SG1, MS1032LO11-SG1, MS1032LO18-SG1, MS1032LO19-SG1, MS1032LO21-SG1 and MS1032LO25-SG1);

in fig. 16A and 16B are time-lapse plasma myostatin concentrations following intravenous administration of anti-latent myostatin antibody to cynomolgus monkeys as illustrated in Example 21. (A) The effect of pH dependence and Fc region construction on myostatin clearance in vivo was assessed by comparison of pH-independent antibody to latent myostatin (MS1032LO00-SG1) and pH-dependent antibody to latent myostatin with engineered Fc (MS1032LO06-SG1012,

MS1032LO06-SG1016, MS1032LO06-SG1029, MS1032LO06-SG1031, MS1032LO06-SG1033,

MS1032LO06-SG1034). (B) The effect of Fc construction on myostatin clearance in vivo was assessed by comparing antibodies to latent myostatin (MS1032LO19-SG1079, MS1032LO19-SG1071, MS1032LO19-SG1080, MS1032LO19-SG1074, MS1032LO19-SG1081 and MS1032LO19-SG1077);

in fig. 17 Inhibitory activity against latent myostatin activation of antibodies to latent myostatin as illustrated in Example 22. Amounts of mature myostatin released from latent myostatin by BMP1 protease were measured in the presence of antibodies to latent myostatin (MST1032, MST1504, MST1538, MST1551, MST1558 , MST1572 and MST1573);

in fig. 18A is a schematic diagram of latent myostatin fragments of 100 amino acids each designed for epitope mapping of antibodies to latent myostatin as illustrated in Example 22;

in fig. 18B shows results of Western blot analysis against GST labeled human latent myostatin (GST-hMSTN) fragments using an anti-GST antibody as illustrated in Example 22. Each lane corresponds to: 1, GST-hMSTN 1-100 aa; 2, GST-hMSTN 21-120 ac; 3, GST-hMSTN 41-140 aa; 4, GST-hMSTN 61-160 ac; 5, GST-hMSTN 81-180 ac; 6, GST-hMSTN 101-200ac; 7, GST-hMSTN 121-220 ac; 8, GST-hMSTN 141-241 aa; 9, GST control;

in fig. 18B - Western blot analysis results against GST-labeled human latent myostatin (GST-hMSTN) fragments using antibodies to latent myostatin (MST1032, MST1538, MST1572 and MST1573) as illustrated in Example 22. Each band corresponds to: 1, GST- hMSTN 1-100 ac; 2, GST-hMSTN 21-120 ac; 3, GST-hMSTN 41-140 aa; 4, GSThMSTN 61-160 ac; 5, GST-hMSTN 81-180 ac; 6, GST-hMSTN 101-200ac; 7, GST-hMSTN 121-220 ac; 8, GST-hMSTN 141-241 aa; 9, GST control; 10, human latent myostatin (100 ng);

in fig. 18D is a summary of Western blot D analysis and predicted epitope positions for latent myostatin antibodies (MST1032, MST1538, MST1572 and MST1573) as illustrated in Example 22;

in fig. 19 - comparative analysis of the primary structure of the amino acid sequences of simian (cynomolgus, (cyno)) Fc-gamma RIIa1, Fc-gamma RIIa2, Fc-gamma RIIa3, Fc-gamma RIIb, human Fc-gamma RIIaH, Fc-gamma RIIaR and Fc-gamma RIIb. The squared regions correspond to putative residues that interact with the Fc domain;

in fig. 20 is a time course of total plasma myostatin concentration following intravenous administration of an anti-myostatin antibody with an Fc variant with increased ability to bind Fc-gamma RIIb to all human Fc-gamma R transgenic mice as illustrated in Example 24. The effects of Fc variants with increased the ability to bind to Fc-gamma RIIb to eliminate antigen by human Fc-gamma RIIb;

in fig. 21 is a time course of plasma antibody concentration following intravenous administration of an anti-myostatin antibody with an Fc variant with increased ability to bind to Fc-gamma RIIb to all human Fc-gamma R transgenic mice, as illustrated in Example 24. The effects of Fc variants with increased ability were assessed. bind to Fc-gamma RIIb on antibody pharmacokinetics;

in fig. 22A and 22B are time-dependent plasma myostatin concentrations following intravenous administration of anti-latent myostatin antibodies to cynomolgus monkey as illustrated in Example 25. latent myostatin (MS1032LO00-SG1) and pH-dependent antibodies to latent myostatin with an engineered Fc region (MS1032LO06-SG1012, MS1032LO06-SG1016, MS1032LO06-SG1029, MS1032LO06-SG1031, MS1032LO06-SG1033,

MS1032LO06-SG1034). (B) The effect of Fc construction on myostatin clearance in vivo was assessed by comparing antibodies to latent myostatin (MS1032LO19-SG1079, MS1032LO19-SG1071, MS1032LO19-SG1080, MS1032LO19-SG1074, MS1032LO19-SG1081, and MS1032LO19-SG1077);

in fig. 23A is a time course of total plasma myostatin concentration following intravenous administration of an anti-myostatin antibody with elevated pI Fc variant to human FcRn transgenic mice as illustrated in Example 26. The effects of elevated pI Fc variants on antigen clearance were assessed;

in fig. 23B is a time course of plasma antibody concentration following intravenous administration of an anti-myostatin antibody with elevated pI Fc variant to human FcRn transgenic mice as illustrated in Example 26. The effects of elevated pI Fc variants on antibody pharmacokinetics were assessed;

in fig. 24A Time-dependence of total plasma myostatin concentration after intravenous administration of an anti-myostatin antibody with an Fc variant with increased pI human transgenic.

FcRn mice as illustrated in Example 26. The effects of elevated pI Fc variants on antigen elimination were assessed. In this assay, human normal immunoglobulin was over-administered together with an anti-myostatin antibody to mimic the situation in human plasma;

in fig. 24B is a time course of plasma antibody concentration following intravenous administration of an anti-myostatin antibody with elevated pI Fc variant to human FcRn transgenic mice as illustrated in Example 26. The effects of elevated pI Fc variants on antibody pharmacokinetics were assessed. In this assay, human normal immunoglobulin was over-administered together with an anti-myostatin antibody to mimic the situation in human plasma;

in fig. 25 is a time course of total plasma myostatin concentration following intravenous administration of an anti-myostatin antibody with an Fc variant with increased ability to bind to Fc-gamma RIIb human Fc-gamma RIIb transgenic mice as illustrated in Example 27. The effects of Fc variants with increased ability were assessed. bind to Fc-gamma RIIb to eliminate the antigen by human Fc-gamma RIIb;

in fig. 26 is a time course of plasma antibody concentration following intravenous administration of an anti-myostatin antibody with enhanced Fc variant binding to Fc-gamma RIIb human Fc-gamma RIIb transgenic mice as illustrated in Example 27. The effects of Fc variants with enhanced binding were assessed. with Fc-gamma RIIb on antibody pharmacokinetics;

in fig. 27 - Cell imaging assay results of anti-myostatin antibodies with an Fc variant with increased ability to bind to Fc-gamma RIIb as illustrated in Example 29. Each antibody was complexed with fluorescently labeled myostatin and intracellular uptake of the antigen-antibody complex was measured in cells expressing human Fc-gamma RIIb.

Description of embodiments of the invention

The techniques and procedures described or referenced herein are generally well known to those skilled in the art and are widely used using conventional methodologies such as those described by Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 2001; Current Protocols in Molecular Biology (ed. F.M. Ausubel et al., 2003); Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (ed. M.J. MacPherson, B.D. Hames and G.R. Taylor, 1995), Antibodies, A Laboratory Manual, ed. Harlow and Lane, 1988 and Animal Cell Culture (ed. R.I. Freshney 1987); Oligonucleotide Synthesis (ed. M.J. Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (ed. J.E. Cellis, 1998) from Academic Press; Animal Cell Culture (ed. R.I. Freshney, 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998), Plenum Press; Cell and Tissue Culture: Laboratory Procedures (ed. A. Doyle, J. B. Griffiths and D. G. Newell, 1993-8), J. Wiley and Sons; Handbook of Experimental Immunology (ed. D.M. Weir and C.C. Blackwell); Gene Transfer Vectors for Mammalian Cells (ed. J. M. Miller and M. P. Calos, 1987); PCR: The Polymerase Chain Reaction, (ed. Mullis et al., 1994); Current Protocols in Immunology (ed. J.E. Coligan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (ed. D. Catty., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (ed. P. Shepherd and C. Dean, Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (ed. M. Zanetti and J. D. Capra, Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., J.B. Lippincott Company, 1993).

I. Definitions.

Unless otherwise indicated, the technical and scientific terms used in the present description have the meanings well known to the ordinary specialist in the field in which the present invention relates. In 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, a general interpretation of many of the concepts used in this application is provided for those skilled in the art. All references cited herein, including patent applications and patent publications, are hereby incorporated by reference in their entirety.

For the purposes of interpreting this specification, the following terms are to be used and, where possible, terms used in the singular also include their use in the plural and vice versa. It should be obvious that the terminology used in the present description is presented only for the purpose of describing specific embodiments of the invention and is not intended to limit its scope. In the event that any of the following definitions conflicts with those in any document incorporated by reference in the specification, the following definition shall be used.

For the purposes of this specification, an acceptor human framework region means a framework region that contains the amino acid sequence of a light chain variable domain (VL) framework region or a heavy chain variable domain (VH) framework region derived from a human immunoglobulin framework region or a human consensus framework region. area indicated below. An acceptor human framework derived from a human immunoglobulin framework or a human consensus framework may have the same amino acid sequence or may contain alterations in the amino acid sequence. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the sequence of the human VL acceptor framework is identical to the sequence of the human immunoglobulin VL framework or the sequence of the human consensus framework.

The term affinity refers to the total strength of all non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). As used herein, unless otherwise indicated, binding affinity refers to the intrinsic binding affinity of a molecule, reflecting a 1:1 interaction between the components of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can generally be characterized by the dissociation constant (Kd).

Affinity can be assessed using conventional methods known in this field, including those presented in the present description. Specific options for measuring binding affinity are provided below by way of illustration.

An affinity matured antibody means an antibody with one or more changes in one or more hypervariable regions (HVRs) compared to a parent antibody that does not contain said changes, said changes leading to an increase in the affinity of the antibody for the antigen.

The terms anti-myostatin antibody and antibody that binds to myostatin refer to an antibody that has the ability to bind to myostatin with sufficient affinity to enable the antibody to be used as a diagnostic and/or therapeutic agent that targets myostatin. In one embodiment, the level 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 by, for example, radioimmunoassay (RIA). In some embodiments, the antibody that binds to myostatin has a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM, or less than or 0.001 nM or less (eg 10 ^-8 M or less, eg 10 ^-8 to 10 ^-13 M, eg 10 ^-9 to 10 ^-13 M). In some embodiments, the anti-myostatin antibody binds to a myostatin epitope that is conserved for myostatin from different species.

In the context of the present description, the term antibody is used in its broadest sense and refers to various antibody structures, including (but not limited to) monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies) and antibody fragments, provided that they have the desired antigen-binding activity.

The term antibody fragment refers to a molecule, other than an intact antibody, that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include (but are not limited to) Fv, Fab, Fab', Fab'-SH, F(ab')2; dimeric antibodies (diabodi); linear antibodies; single chain antibody molecules (eg scFv); and multispecific antibodies formed from antibody fragments.

The term antibody that binds to the same epitope as the reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen when assessed by a competition assay (competitive assay) and/or, conversely, the reference antibody blocks the binding of an antibody to its antigen when assessed by a competitive assay. An example of competitive analysis is presented in the present description.

The term chimeric antibody refers to an antibody in which part of the heavy and/or light chain is from a particular source or species, and the remainder of the heavy and/or light chain is from a different source or species.

The term antibody class refers to the type of constant domain or constant region that/which is included in its heavy chain. Five main classes of antibodies are known: IgA, IgD, IgE, IgG and IgM, and some of them can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains that correspond to the different immunoglobulin classes are referred to as alpha, delta, epsilon, gamma, and mu (α, δ, ε, γ, and μ), respectively.

The concept of a cytotoxic agent in the context of the present description refers to a substance that

- 19 040713 paradise inhibits or interferes with cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (eg, At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and radioactive isotopes Lu) ; chemotherapeutic agents or drugs (eg, 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 low molecular weight toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various anti-tumor or anti-cancer agents listed below.

The concept of effector functions refers to those types of biological activity that are associated with the Fc region of the antibody, which vary depending on the isotype of the antibody. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-mediated cell-dependent cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (eg B cell receptor) and activation of B cells.

The concept of an effective amount of an agent, for example, a pharmaceutical composition, means an amount effective in doses and for a period of time necessary to achieve the desired therapeutic or prophylactic result.

The term epitope includes any determinant that is capable of being bound by an antibody. An epitope is a region of an antigen that binds to an antibody whose target is the antigen and includes specific amino acids that are in direct contact with the antibody. Epitope determinants may include reactive surface groups of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structure characteristics and/or specific charge characteristics. In general, antibodies specific for a particular target antigen should preferentially recognize an epitope on the target antigen in a complex mixture of proteins and/or macromolecules.

Fc receptor or FcR means a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR is a receptor that binds to an IgG-type antibody (gamma receptor) and includes receptors of the subclass Fc-gamma RI, Fc-gamma RII, and Fc-gamma RIII, including allelic variants and resulting alternative splicing forms these receptors. Fc-gamma RII receptors include Fc-gamma RIIA (activating receptor) and Fc-gamma RIIB (inhibiting receptor), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor Fc-gamma RIIA contains immunoreceptor tyrosine-based activating motifs (ITAM) in its cytoplasmic domain. Inhibitory receptor Fc-gamma RIIB contains an immunoreceptor tyrosine receptor (ITIM) inhibitory motif in its cytoplasmic domain (see, for example, Daeron, Annu. Rev. Immunol. 15, 1997, c. 203-234). An overview of FcR information is provided, for example, by Ravetch and Kinet, Annu. Rev. Immunol. 9, 1991, p. 457-492; Capel et al., Immunomethods 4, 1994, p. 25-34 and de Haas et al., J. Lab. Clin. Med. 126, 1995, p. 330-341. Under the concept of FcR, specified in the present description, other FcR fall, including those that will be identified in the future.

The term Fc receptor or FcR also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the embryo (Guyer et al., J. Immunol. 117, 1976, c. 587-593 and Kim et al., J. Immunol. 24 , 1994, pp. 2429-2434) and regulates immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, for example, Ghetie and Ward., Immunol. Today 18(12), 1997, cc.592-598; Ghetie et al., Nature Biotechnology 15(7), 1997, c. 637 -640 Hinton et al., J Biol Chem 279(8), 2004, pp. 6213-6216 WO 2004/92219 (Hinton et al.) In vivo binding to human FcRn and human serum half-life An FcRn having high binding affinity for polypeptides can be evaluated, for example, in transgenic mice or in transfected human cell lines expressing human FcRn or in primates administered polypeptides with a variant Fc region.WO 2000/42072 (Presta) describes antibody variants with increased or decreased ability to bind to FcR (see, for example, also Shields et al., J. Biol. Chem. 9(2), 2001, c. 6591-6604).

The term Fc-region in the context of the present description refers to the C-terminal region of the heavy chain of an immunoglobulin, which contains at least part of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise noted, the numbering of amino acid residues in the Fco region or constant region follows the EU numbering system, which is also referred to as the EU index, as described by Kabat E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

- 20 040713

The term Fc region-containing antibody refers to an antibody that contains an Fc region. The terminal lysine (EU numbering residue 447) of the Fc region can be removed, for example, during purification of the antibody or by recombinant construction of the nucleic acid encoding the antibody. Thus, a composition that contains an antibody having an Fc region of the present invention may contain an antibody with K447, all antibodies that have K447 removed, or a mixture of antibodies with and without a K447 residue.

The term framework region, or FR, refers to variable domain residues other than hypervariable region (HVR) residues. The variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically have the following location in the VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms full antibody, intact antibody, and whole antibody are used interchangeably herein to refer to an antibody having a structure substantially similar to the native antibody structure, or having heavy chains that contain the Fc region described herein.

The functional Fc region has the effector function of the native sequence Fc region. Examples of effector functions include C1q binding; (CDC); Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (eg B cell receptor; BCR), etc. These effector functions generally require the Fc region to be combined with a binding domain (eg, an antibody variable domain) and can be assessed using various assays, such as those provided in the Definitions section of this specification.

The terms host cell, host cell line, and host cell culture are used interchangeably and refer to cells into which an exogenous nucleic acid has been introduced, including progeny of said cells. Host cells include transformants and transformed cells, which include the primary transformed cell and its progeny, regardless of the number of passages. The offspring may not be completely identical in nucleic acid composition to the parent cell, but may contain mutations. Under the scope of the invention falls mutant progeny, which has the same function or biological activity, which is found as a result of screening or selected from the original transformed cell.

A human antibody is an antibody having an amino acid sequence that corresponds to that of an antibody produced in the human body or human cells, or derived from a non-human source that uses a human antibody spectrum or other human antibody-encoding sequences. Specifically excluded from this definition of a human antibody is a humanized antibody containing non-human antigen-binding residues.

The human consensus framework region is a framework region that includes the most frequently occurring amino acid residues in selected human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is made from a subset of variable domain sequences. Typically, a subgroup of sequences is the subset described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., NIH Publication 91-3242, Bethesda MD, vols. 1-3, 1991. In one embodiment, implementation of the invention regarding VL subgroup is a subgroup kappa I according to Kabat and others, above. In one embodiment of the invention regarding VH, the subgroup is subgroup III according to Kabat et al., supra.

The term humanized antibody refers to a chimeric antibody that contains amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, a humanized antibody may 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 regions of the non-human antibody and all or substantially all of the FRs correspond to regions human antibody. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. The term humanized form of an antibody, such as a non-human antibody, refers to an antibody that has been humanized.

The concept of a hypervariable region or HVR in the context of the present description refers to each of the regions of the variable domain of an antibody, the sequences of which are hypervariable (complementarity determining regions or CDRs) and / or form loops of a certain structure (hypervariable loops), and / or contain antigen-contacting residues ( contact with the antigen). Typically, antibodies contain six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). As used herein, examples of HVRs include (a) hypervariable loops comprising amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196, 1987, c. 901-917); (b) CDRs including amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) ( Kabat et al., Sequences of Proteins

- 21 040713 of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991, NIH publication 91-3242); (c) antigen contact regions, including amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2) and 93-101 ( H3) (MacCallum et al., J. Mol. Biol. 262, 1996, pp. 732-745); and (d) combinations of residues specified in subparagraphs (a), (b) and/or (c), including amino acid residues HVR 46-56 (L2), 47-56 (L2), 48-56 (L2), 49 -56 (L2), 26-35 (H1), 26-35b (H1), 4965 (H2), 93-102 (H3) and 94-102 (H3).

Unless specifically stated otherwise, residues in HVR and other residues in the variable domain (eg, residues in FR) are numbered according to Kabat et al., supra.

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

The individual or subject is a mammal. Mammals include, but are not limited to, domesticated animals (for example, cows, sheep, cats, dogs, and horses), primates (for example, humans and non-human primates, such as monkeys), rabbits, and rodents (for example, mice and rats). In some embodiments of the invention, the individual or subject is a human.

An isolated antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic assays (such as, for example, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic assays (such as, for example, as ion exchange chromatography or reverse phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chrom. In 848, 2007, p. 79-87.

An isolated nucleic acid is 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 a cell that normally includes a nucleic acid molecule, but in which the nucleic acid molecule is present outside the chromosome or in which its localization on the chromosome differs from its naturally occurring localization on the chromosome.

The term isolated nucleic acid encoding an anti-myostatin antibody refers to one or more nucleic acid molecules encoding heavy and light chains of an antibody (or fragments thereof), including the specified molecule(s) in one vector or in different vectors, and the specified ( s) nucleic acid(s) molecule(s) present(s) at one or more positions in the host cell.

The term monoclonal antibody in the context of the present description refers to an antibody obtained from a population of substantially homogeneous antibodies, i. individual antibodies in a population are identical and/or bind to the same epitope, with the exception of possible variants of antibodies, for example, containing mutations that occur naturally or arise during the production of a preparation of a monoclonal antibody, these variants are usually present in minor quantities. Unlike polyclonal antibody preparations, which typically include different antibodies to different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single antigen determinant. Thus, the adjective monoclonal defines the specificity of an antibody, characterizing it as derived from a substantially homogeneous population of antibodies, rather than being engineered to meet the requirements for producing an antibody by any particular method. For example, monoclonal antibodies that can be used according to the present invention can be created using various technologies, including (but not limited to) the hybridoma method, recombinant DNA methods, phage display methods, and methods based on the use of transgenic animals, which contain all or a portion of human immunoglobulin loci, these methods and other exemplary methods for producing monoclonal antibodies are provided herein.

The term naked antibody refers to an antibody that has not been conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a radioactive label. The naked antibody may be present in the pharmaceutical composition.

The term native antibodies refers to naturally occurring immunoglobulin molecules with different structures. For example, native IgG antibodies are heterotetrameric glycoproteins with a molecular weight of approximately 150,000 Da, consisting of two identical light chains and two identical heavy chains linked by disulfide bridges. In the N-terminal to C-terminal direction, each heavy chain has a variable region (VH), which is also referred to as a heavy chain variable domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also referred to as a variable light domain or a light chain variable domain, followed by a light chain constant domain (CL). The light chain of an antibody, depending on the amino acid sequence of its constant

- 22 040713 domain can belong to one of two types, which denote kappa (k) and lambda (λ).

Having a native sequence Fc region contains an amino acid sequence identical to the amino acid sequence of the Fc region, which occurs in nature. The native sequence of human Fc regions includes the native sequence of human IgG1 Fc regions (non-A and A allotypes); native sequence of the Fc region of human IgG2; the native sequence of the human IgG3 Fc region and the native sequence of the human IgG4 Fc region, as well as their naturally occurring variants.

The term package insert refers to the instructions that are usually included in the commercial packaging of therapeutic products, containing information about the indications, use, dose, route of administration, combination therapy, contraindications and / or precautions associated with the use of these therapeutic products. .

Percent (%) amino acid sequence identity relative to the reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the polypeptide reference sequence, after sequence alignment and, if necessary, the introduction of gaps to achieve the maximum percent sequence identity, and without considering any conservative substitutions in as a component in the evaluation of sequence identity. Comparison for purposes of determining percent amino acid sequence identity can be performed in a variety of ways known in the art using, for example, publicly available computer programs such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the compared sequences. However, for the purposes of the present invention, % amino acid sequence identity values are obtained using the ALIGN-2 sequence comparison computer program. The sequence comparison computer program ALIGN-2 is copyrighted by Genentech, Inc. and the source code has been provided in the U.S. user documentation. Copyright Office, Washington D.C., 20559, where it is registered as U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, CA. California, or it can be compiled from source code. The ALIGN-2 program can be compiled for use on the UNIX operating system, including Digital UNIX V4.0D. All parameters required for sequence comparison are set by ALIGN-2 and should not be changed. In situations in which ALIGN-2 is used to compare amino acid sequences, the % amino acid sequence identity of a given amino acid sequence A compared to, with, or relative to a given amino acid sequence B (which can alternatively be referred to as a given amino acid sequence A that has or contains a specific % amino acid sequence identity compared to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 x fraction X/Y where X is the number of amino acid residues determined to be identical matches when assessed using the ALIGN-2 sequence comparison software , where A and B were compared using the program, and where Y denotes the total number of amino acid residues in B. It should be obvious that if the length of the amino acid sequence A is not equal to the length of the amino acid sequence B, then % The % Amino Acid Sequence Identity of A to B cannot be equal to % Amino Acid Sequence Identity of B to A. Unless specifically stated otherwise, all % Amino Acid Sequence Identities mentioned herein were obtained using the ALIGN-2 computer program described in the previous paragraph.

The term pharmaceutical composition refers to a preparation that is in such a form that it provides the biological activity of the active substance included in its composition, which should be effective, and which does not contain additional components that have unacceptable toxicity for the individual to whom the composition should be administered.

A pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical composition, other than the active ingredient, that is non-toxic to the individual. Pharmaceutically acceptable carriers include, but are not limited to, a buffer, excipient, stabilizer, or preservative.

The term myostatin as used herein can refer to any native myostatin from any vertebrate animal, including mammals such as primates (eg humans) and rodents (eg mice and rats). Unless specifically stated otherwise, the term myostatin refers to a human myostatin protein having the amino acid sequence shown in SEQ ID NO: 1 and containing the terminal human myostatin propeptide domain shown in SEQ ID NO: 75 or 78. The term refers to a full-length non-processed 23 040713 bath myostatin, as well as to the form of myostatin, which is formed as a result of processing in the cell. The term also includes naturally occurring variants of human myostatin, such as splicing variants or allelic variants. The amino acid sequence of human myostatin (promyostatin) is shown in SEQ ID NO: 1. The amino acid sequence of exemplary human myostatin growth factor C-terminal domain is shown in SEQ ID NO: 2. The amino acid sequence of exemplary human myostatin N-terminal propeptide domain shown in SEQ ID NO: 75 or 78. Active mature myostatin is a disulfide-linked homodimer consisting of two C-terminal growth factor domains. Inactive latent myostatin is a non-covalently associated complex of two propeptides and mature myostatin. As described herein, the antibodies of the invention bind to inactive latent myostatin but do not bind to mature active myostatin homodimer. In some embodiments, the antibodies of the invention bind to an epitope within amino acids 21-100 of the myostatin propeptide (SEQ ID NO: 78) but do not bind to the mature active myostatin homodimer. The amino acid sequence of exemplary cynomolgus monkey myostatin and mouse myostatin (promyostatin) are shown in SEQ ID NOS: 3 and 5, respectively. The amino acid sequence of the exemplary C-terminal domain of monkey cynomolgus myostatin growth factor and mouse myostatin is shown in SEQ ID NOs: 4 and 6, respectively. The amino acid sequence of the exemplary N-terminal propeptide domain of cynomolgus monkey myostatin and mouse myostatin is shown in SEQ ID NOs: 76 or 79 and 77 or 80, respectively. GDF-11 (BMP-11) is a closely related molecule to myostatin, both of which are members of the TGF-beta superfamily. Similar to myostatin, GDF11 is synthesized first as a precursor polypeptide and then cleaved into an N-terminal prodomain and a C-terminal mature GDF11. The amino acid sequence of human GDF11 (precursor) is shown in SEQ ID NO: 81. The amino acid sequence of the C-terminal mature human GDF11 is shown in SEQ ID NO: 82. The amino acid sequence of the N-terminal prodomain of human GDF11 is shown in SEQ ID NO: 83 or 84. sequence SEQ ID NO: 1, 3, 5, 78, 79, 80, 81 and 84 include a signal sequence. It corresponds to amino acids 1-24, and they are removed during processing in the cell.

As used herein, treatment (and its grammatical variations, such as treat or process of treatment) refers to a clinical intervention to alter the natural course of a disease in an individual being treated, and it can be done either for prophylaxis or in the course of developing a clinical pathology. The desired actions of treatment are (but not limited to) prevention of the onset or recurrence of the disease, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastases, reduction in the rate of disease progression, alleviation or temporary amelioration of the disease state, and remission or improvement in prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or slow the progression of a disease.

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

The variant Fc region contains an amino acid sequence that differs from the native sequence of the Fc region due to the presence of at least one amino acid modification (change), preferably one or more amino acid(s) substitution(s). Preferably, the Fc region variant has at least one amino acid substitution relative to the native Fc region sequence of the parent polypeptide, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in the native Fc region sequence. or in the Fc region of the parent polypeptide. In the context of the present description, the Fc region variant should preferably have at least about 80% homology with the native sequence of the Fc region and/or Fc region of the parent polypeptide, and most preferably at least about 90% homology with it, more preferably at at least about 95% homology with it.

The concept of a vector in the context of the present description refers to a nucleic acid molecule,

- 24 040713 which has the ability to propagate another nucleic acid with which it is associated.

The concept includes a vector in the form of a self-replicating nucleic acid structure, as well as a vector included in the genome of the host cell into which it is introduced. Some vectors have the ability to control the expression of the nucleic acids to which they are operably linked. These vectors in the context of the present description are referred to as expression vectors.

II. Compositions and methods.

One of the objects of the invention are, in particular, antibodies to myostatin and their uses. Some embodiments of the invention are antibodies that bind to myostatin. The antibodies of the invention can be used, for example, to diagnose or treat a disease.

One of the objects of the invention are, in particular C5, polypeptides containing variants of the Fco regions, and their uses. One of the embodiments of the invention are polypeptides containing variants of the Fc regions with increased Fc-gamma RIIb-binding activity. Another embodiment of the invention are polypeptides containing variant Fc regions with increased pI. In specific embodiments, the polypeptides of the invention are antibodies. Polypeptides containing a variant Fc region of the invention can be used, for example, to diagnose or treat a disease.

A. Examples of anti-myostatin antibodies and polypeptides containing variant Fc regions.

One of the objects of the invention are isolated antibodies that bind to myostatin. In some embodiments, an anti-myostatin antibody of the present invention binds to latent myostatin. In other embodiments, an anti-myostatin antibody of the present invention binds to a 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 other embodiments, the antibody binds to an epitope in a fragment consisting of amino acids 21-100 of the myostatin propeptide (SEQ ID NO: 78). The propeptide is included in latent myostatin as one of the components, as described above. In some embodiments, an anti-myostatin antibody of the present invention inhibits myostatin activation. In some embodiments, the anti-myostatin antibody blocks the release of mature myostatin from latent myostatin. Mature myostatin is known to be released through proteolytic and non-proteolytic processes from latent myostatin. An anti-myostatin antibody of the present invention can block the proteolytic and/or non-proteolytic release of mature myostatin from latent myostatin. In some embodiments, the anti-myostatin antibody blocks the proteolytic cleavage of latent myostatin. In some embodiments, the anti-myostatin antibody blocks the access of a protease to latent myostatin (primarily the proteolytic cleavage site (Arg98-Asp99) of latent myostatin). In other embodiments, the protease may be a member of the BMP1/TLD metalloprotease family, such as BMP1, TED, tolloid-like protein-1 (TLL-1), or tolloid-like protein-2 (TLL-2). In another embodiment, the anti-myostatin antibody blocks the non-proteolytic release of mature myostatin from latent myostatin. In the context of the present description, non-proteolytic release means the spontaneous release of mature myostatin from latent myostatin, which is not accompanied by proteolytic cleavage of latent myostatin. Non-proteolytic release includes, for example, the release of mature myostatin by incubation of latent myostatin, for example at 37° C., in the absence of a protease that cleaves latent myostatin. In some embodiments, an 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 the antibody shown in Table 1. 2a. In some embodiments, the anti-myostatin antibody competes for binding to latent myostatin with the antibody shown in Table 1. 2a. In additional embodiments, the anti-myostatin antibody competes for binding to latent myostatin, with an antibody that contains the VH and VL pair shown in Table 1. 2a. In some embodiments, the anti-myostatin antibody competes for binding to the amino acid 21-100 fragment of the myostatin propeptide (SEQ ID NO: 78) of the antibody shown in Table 1. 2a. In other embodiments, the anti-myostatin antibody binds to the same epitope as the antibody shown in Table 1. 11a or 13. In some embodiments, the anti-myostatin antibody competes for binding to latent myostatin with the antibody shown in Table 11a. 11a or 13. In some embodiments, the anti-myostatin antibody competes for binding to the amino acid 21-100 fragment of the myostatin propeptide (SEQ ID NO: 78) with the antibody shown in Table 11a. 11a or 13.

In some embodiments, an anti-myostatin antibody of the present invention binds to latent myostatin and inhibits myostatin activation. In other embodiments, the antibody: (a) blocks the release of mature myostatin from latent myostatin; (b) blocks the proteolytic release of mature myostatin; (c) blocks spontaneous release of mature myostatin or (d) does not bind to mature myostatin; or binds to an epitope within a fragment consisting of amino acids 21-100 of the myostatin propeptide (SEQ ID NO: 78). In other embodiments, the antibody competes for binding to latent myostatin or binds to the same epitope as an antibody that contains the VH and VL pair shown in Table 1. 2a, 11a, or 13. In other embodiments, the antibody binds latent myostatin with higher affinity at neutral pH (eg, pH 7.4) than at acidic pH (eg, pH 5.8). In other embodiments, the antibody is (a) a monoclonal antibody, (b) a human, humanized, or chimeric antibody; (c) a full-length IgG-type antibody; or (d) an antibody fragment that binds to latent myostatin or myostatin propeptide.

In another embodiment, the anti-myostatin antibody of the present invention does not bind to GDF11 (growth factor 11). In some embodiments, an anti-myostatin antibody of the present invention does not inhibit GDF11 activation. In some 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 some embodiments, the anti-myostatin antibody does not block proteolytic cleavage of latent GDF11. In some embodiments, the anti-myostatin antibody does not block protease access to latent GDF11 (primarily the proteolytic cleavage site of latent GDF11). In other embodiments, the protease may be a metalloprotease from the BMP1/TLD family, such as BMP1, TED, tolloid-like protein-1 (TLL-1), or tolloid-like protein-2 (TLL-2). Under non-proteolytic release in the context of the present description refers to the spontaneous release of Mature GDF11 from latent GDF11, which is not carried out through proteolytic cleavage of latent GDF11. Non-proteolytic release includes, for example, releasing mature GDF11 by incubating latent GDF11, for example at 37° C., in the absence of a protease that cleaves latent GDF11. Most of the anti-myostatin antibodies known to date are not specific for myostatin. These antibodies have high affinity for other members of the TGF-beta superfamily, such as GDF11, and neutralize their biological activity. GDF11 plays an important role in the process of embryogenesis and is responsible for the homeotic transformation of the axial skeleton. Mice homozygous for the disabled GDF11 died in the perinatal period, mice with one copy of the wild type GDF11 gene were viable, but had skeletal defects. Since GDF11 plays an important role in the process of embryogenesis, an antagonist that inhibits GDF11 theoretically carries safety risks, which can manifest itself as either toxicity to treated patients or reproductive toxicity, for example, to women of childbearing potential. Thus, there is a need for specific inhibition of myostatin activity in the treatment of myostatin-associated disorders, especially in women of childbearing potential.

Another object of the invention are antibodies to myostatin with pH-dependent binding characteristics. In the context of the present description, the term pH-dependent binding means that the antibody has a reduced ability to bind to myostatin at acidic pH compared to its binding at neutral pH (both terms can be used interchangeably for the purposes of this description). 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 some embodiments, the antibodies of the present invention bind to myostatin with an affinity of at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10,000 or more times higher at neutral pH than at acidic pH. In some embodiments, antibodies bind to myostatin (eg, latent myostatin or myostatin propeptide) with higher affinity at pH 7.4 than at pH 5.8. In other embodiments, the antibodies bind to myostatin with an affinity of at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 1θ0, 200, 400, 1000, 10000 or more times higher at pH 7.4 than at pH 5.8.

When the antigen is a soluble protein, binding of the antibody to the antigen may result in an extended plasma half-life of the antigen (i.e., reduced clearance of the antigen from plasma), since the antibody may have a longer plasma half-life than the antigen itself, and can serve as a carrier for an antigen. This is a consequence of the recycling of the antigen-antibody complex by FcRn through the endosomal pathway in the cell (Roopenian, Nat. Rev. Immunol. 7(9), 2007, c. 715-725). However, an antibody with pH-dependent binding characteristics that binds to its antigen in a neutral extracellular environment, thereby releasing the antigen into acidic endosomal compartments upon cell entry, would be expected to have advantageous antigen neutralization and clearance properties relative to its antigen. a copy that binds in a pH-independent manner (Igawa et al., Nature Biotechnol. 28(11), 2010, p.

1203-1207; Devanaboyina et al., mAbs 5(6), 2013, p. 851-859; WO 2009/125825).

For the purposes of this specification, the affinity of an antibody for myostatin is expressed in terms of the KD of the antibody. The KD of an antibody means the dissociation equilibrium constant of the antibody-antigen interaction. The higher the KD value, which characterizes the binding of an antibody to its antigen, the weaker its binding affinity for the specified specific antigen. Thus, in the context of the present description, the concept of higher affinity at neutral pH than at acidic pH (or the equivalent concept of pH-dependent binding) means that the KD characterizing the binding of the antibody to myostatin at acidic pH is higher than the KD characterizing the binding of the antibody with 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 characterizing the binding of the antibody to myostatin at acidic pH is at least 2 times higher than the KD characterizing binding of the antibody to myostatin at neutral pH. Thus, the present invention includes antibodies whose binding to myostatin at acidic pH is characterized by a KD value that is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10,000 or more times the KD of myostatin binding at neutral pH. In another embodiment, the KD value at neutral pH may be 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 , 10 ^-11 , 10 ^-12 M or less. In another embodiment of the invention, the value of KD at acidic pH may be 10 ^-9 M, 10 ^-8 M, 10 ^-7 M, 10 ^-6 M or more.

In other embodiments, an antibody is said to bind to myostatin (e.g., latent myostatin or myostatin propeptide) with higher affinity at neutral pH than at acidic pH if the KD value of antibody binding to myostatin at pH 5.8 is at least 2 times higher than the KD, which characterizes the binding of the antibody to myostatin at pH 7.4. In some embodiments of the invention, the binding of the proposed antibodies to myostatin at pH 5.8 is characterized by a KD value 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 the KD characterizing the binding of the antibody to myostatin at pH 7.4. In another embodiment, the KD value of the antibody at pH 7.4 may be 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 , 10 ^-11 , 10 ^-12 M or less. In another embodiment, the KD value of the antibody at pH 5.8 may be 10 ^-9 , 10 ^-8 , 10 ^-7 , 10 ^-6 M or more.

The binding characteristics of an antibody to a particular antigen can also be expressed as the kd of an antibody. The kd value of an antibody denotes the dissociation rate constant of an antibody relative to a particular antigen and is expressed as reciprocals of a second (ie, s ⁻¹ ). An increased kd value means weaker binding of the antibody to its antigen. Thus, the present invention includes antibodies whose binding to myostatin has a higher kd value at acidic pH than at neutral pH. The present invention includes antibodies whose binding to myostatin at acidic pH is characterized by a kd value 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 the kd characterizing the binding of the antibody to myostatin at neutral pH. In another embodiment, the kd value of the antibody at neutral pH may be 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 1/s or less. In another embodiment, the kd value of the antibody at acidic pH may be 10 ^-3 , 10 ^-2 , 10 ^-1 1/s or more. The invention also includes antibodies whose binding to myostatin (eg, latent myostatin or myostatin propeptide) has a higher kd value at pH 5.8 than at pH 7.4. The invention includes antibodies whose binding to myostatin at pH 5.8 is characterized by a kd value 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 the kd characterizing the binding of the antibody to myostatin at pH 7.4. In another embodiment, the kd value of the antibody at pH 7.4 may be 10 ^-2 , 10 -3 , 10 ^-4 , 10 ^-5 , 10 ^-6 1/s or less. In another embodiment, the kd value of the antibody at pH 5.8 may be 10 ^-3 , 10 ^-2 , 10 ^-1 1/s or more.

In some cases, the reduced ability to bind to myostatin at acidic pH compared to the ability to bind at neutral pH is expressed as the ratio of the KD value, which characterizes the binding of the antibody to myostatin at acidic pH, and the KD value, which characterizes the binding of the antibody to myostatin at neutral pH (or vice versa ). For example, an antibody may be considered to have a reduced ability to bind to myostatin at acidic pH compared to the ability to bind at neutral pH for the purposes of the present invention if the antibody has an acid/neutral pH KD ratio of 2 or more. In some embodiments, the KD ratio at pH 5.8/pH 7.4 for an anti-myostatin antibody of the present invention is 2 or more. In some embodiments, the acid/neutral pH KD ratio for an antibody of the present invention may be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,

- 27 040713

90, 95, 100, 200, 400, 1000, 10000 or more. In another embodiment, the KD value of the antibody at neutral pH may be 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 , 10 ^-11 , 10 ^-12 M or less. In another embodiment, the KD value of the antibody at acidic pH may be 10-9, 10 ^-8 , 10 ^-7 , 10 ^-6 M or more. In other instances, an antibody may be considered to have a reduced ability to bind to myostatin (eg, latent myostatin) at acidic pH relative to its ability to bind at neutral pH if the antibody has a KD ratio at pH 5.8/pH 7.4 of 2 or more. In some exemplary embodiments of the invention, the KD ratio at pH 5.8/pH 7.4 for an antibody may be 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more. In another embodiment, the KD value of the antibody at pH 7.4 may be 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 , 10 ^-11 , 10 ^-12 M or less. In another embodiment, the KD value of the antibody at pH 5.8 may be 10 ^-9 , 10 ^-8 , 10 ^-7 , 10 ^-6 M or more.

In some cases, the reduced ability to bind to myostatin at acidic pH compared to the ability to bind at neutral pH is expressed as the ratio of the kd value characterizing the binding of the antibody to myostatin at acidic pH and the kd value characterizing the binding of the antibody to myostatin at neutral pH (or vice versa). ). For example, an antibody may be considered to have a reduced ability to bind to myostatin at acidic pH compared to the ability to bind at neutral pH for the purposes of the present invention if the acid/neutral pH kd ratio of the antibody is 2 or more. In some embodiments, the kd ratio at pH 5.8/pH 7.4 for an anti-myostatin antibody of the present invention is 2 or more. In some embodiments, the acid/neutral pH kd ratio for an antibody of the present invention may be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more. In another embodiment, the kd value of the antibody at neutral pH may be 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 1/s or less. In another embodiment, the kd value of the antibody at acidic pH may be 10 ^-3 , 10 ^-2 , 10 ^-1 1/s or more. In some exemplary embodiments of the invention, the ratio kd at pH 5.8/pH 7.4 for an antibody may be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 , 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more. In another embodiment, the kd value of the antibody at pH 7.4 may be 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 1/s or less. In another embodiment, the kd value of the antibody at pH 5.8 may be 10 ^-3 , 10 ^-2 , 10 ^-1 1/s or more.

In the context of the present description, the term acidic pH means a pH of 4.0 to 6.5. The concept of acidic pH includes one of the following pH values 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5 .0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2 , 6.3, 6.4 and 6.5. In specific aspects of the invention, the acidic pH is 5.8.

In the context of the present description, the concept of neutral pH means a pH from 6.7 to about 10.0. The concept of neutral pH includes one of the following pH values 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7 .7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 , 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and 10.0. In specific aspects of the invention, neutral pH is 7.4.

The KD and kd values described herein can be determined using a biosensor based surface plasmon resonance method to characterize antibody-antigen interactions (see, for example, Example 7 herein). The KD values and kd values can be determined at 25 or 37°C.

In some embodiments, an anti-myostatin antibody of the present invention binds to more than one species of myostatin. In other embodiments, the anti-myostatin antibody binds to human and non-human myostatin. In other embodiments, the anti-myostatin antibody binds to human, murine, and monkey myostatin (eg, cynomolgus monkeys, rhesus monkeys, marmosets, chimpanzees, or baboons).

In some embodiments, an anti-myostatin antibody of the present invention binds to latent myostatin from more than one species. In other embodiments, the anti-myostatin antibody binds to latent human and non-human myostatin. In other embodiments, the anti-myostatin antibody binds to latent human, mouse, and monkey myostatin.

In some embodiments, an anti-myostatin antibody of the present invention binds to a myostatin propeptide from more than one species. In other embodiments, the anti-myostatin antibody binds to a human and non-human myostatin propeptide. In other embodiments, the anti-myostatin antibody binds to human, mouse, and monkey myostatin propeptide.

Another aspect of the invention is an anti-myostatin antibody that forms an immune complex (ie, an antigen-antibody complex) with myostatin. In some embodiments, the invention

- 28 040713 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 that contains two myostatin molecules and the antibody has two antigen binding sites. An anti-myostatin antibody may bind to the same epitope on the myostatin molecule or may bind to different epitopes on the myostatin molecule, most likely such an antibody is a bispecific antibody. In general, when two or more antibodies form an immune complex with two or more antigens, the resulting immune complex can bind strongly to Fc receptors present on cell surfaces due to avidity effects through the Fc regions of the antibodies in the complex and can then absorbed by cells with high efficiency. Thus, the above anti-myostatin antibody, which has the ability to form an immune complex containing two or more anti-myostatin antibodies and two or more molecules of myostatin, can lead to a rapid clearance of myostatin from plasma in vivo due to strong binding to Fc receptors. due to the effects of avidity.

In addition, it is contemplated that an antibody with pH-dependent binding characteristics may have advantageous antigen-clearance-neutralizing properties over its copy that binds in a pH-independent manner (Igawa et al., Nature Biotech. 28(11), 2010, c 1203-1207; Devanaboyina et al., mAbs 5(6), 2013, pp. 851-859; WO 2009/125825). Thus, an antibody having the above two properties, i. an antibody having pH-dependent binding characteristics and forming an immune complex, which contains two or more antibodies with two or more antigens, should have even more advantageous properties regarding the accelerated elimination of antigens from plasma (WO 2013/081143).

Another object of the invention is an antibody to myostatin, which contains at least one, two, three, four, five or six HVRs selected from (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NO: 55-57, 114 -115, 126; (b) HVR-H2 which contains one of the amino acid sequences of SEQ ID NOs: 58-60, 116-120, 127; (c) HVR-H3 which contains one of the amino acid sequences of SEQ ID NOs: 61-64, 121, 128; (d) HVR-L1 which contains one of the amino acid sequences of SEQ ID NOs: 65-69, 122-124, 129; (e) HVR-L2 which contains one of the amino acid sequences of SEQ ID NOs: 70-72, 125, 130; and (e) HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 73-74, 131.

Another object of the invention is an antibody to myostatin, which contains at least one, two, three, four, five or six HVR selected from (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NO: 55-57; (b) HVR-H2, which contains one of the amino acid sequences of SEQ ID NO: 58-60; (c) HVR-H3, which contains one of the amino acid sequences of SEQ ID NO: 61-64; (d) HVR-L1, which contains one of the amino acid sequences of SEQ ID NO: 65-69; (e) HVR-L2, which contains one of the amino acid sequences of SEQ ID NO: 70-72; and (e) HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 73-74.

One of the objects of the invention is an antibody to myostatin, which contains at least one, two, three, four, five or six hypervariable regions (HVR) selected from (a) HVRH1, which contains one of the amino acid sequences of SEQ ID NO: 114- 115; (b) HVR-H2, which contains one of the amino acid sequences of SEQ ID NO: 116-120; (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 121; (d) HVR-L1, which contains one of the amino acid sequences of SEQ ID NO: 122-124; (e) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 125; and (e) HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 73-74.

Another object of the invention is an antibody to myostatin, which contains at least one, two, three, four, five or six HVR selected from (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 114; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 58; (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 63; (d) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 71; and (e) an HVR-L3 which contains the amino acid sequence of SEQ ID NO: 74. Another aspect of the invention is an anti-myostatin antibody which contains at least one, two, three, four, five, or six HVRs selected from (a) HVRs -H1, which contains the amino acid sequence of SEQ ID NO: 114; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 58; (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 63; (d) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 123; (e) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 71; and (e) HVR-L3 which contains the amino acid sequence of SEQ ID NO: 74.

Another object of the invention is an antibody to myostatin which contains at least one, two, three, four, five or six HVRs selected from (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 126; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 127; (c) HVR-H3 which contains the amino acid sequence of SEQ

ID NO: 128; (d) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 129; (e)

HVR-L2, which contains the amino acid sequence of SEQ ID NO: 130; and (e) HVR-L3 which contains the amino acid sequence of SEQ ID NO: 131.

One of the objects of the invention is an antibody that contains at least one, at least two or all three VH HVR sequences selected from (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NO: 55-57, 114 -115, 126; (b) HVR-H2, which contains one of the amino acid sequences of SEQ ID NO: 58-60, 116-120, 127; (c) HVRH3 which contains one of the amino acid sequences of SEQ ID NOs: 61-64, 121, 128. In one embodiment, the antibody comprises an HVR-H3 which contains one of the amino acid sequences of SEQ ID NOs: 61-64, 121 , 128. In another embodiment, the antibody comprises HVR-H3, which contains one of the amino acid sequences of SEQ ID NOs: 61-64, 121, 128, and HVR-L3, which contains one of the amino acid sequences of SEQ ID NOs: 73-74 , 131. In a further embodiment of the invention, the antibody comprises HVR-H3 which contains one of the amino acid sequences of SEQ ID NOs: 61-64, 121, 128, HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 73-74, 131, and HVR-H2, which contains one of the amino acid sequences of SEQ ID NOs: 58-60, 116-120, 127. In the following embodiment, the antibody contains (a) HVR-H1, which contains one of the amino acid sequences th SEQ ID NO: 55-57, 114-115, 126; (b) HVR-H2, which contains one of the amino acid sequences of SEQ ID NO: 58-60, 116-120, 127; and (c) HVR-H3 which contains one of the amino acid sequences of SEQ ID NOs: 61-64, 121, 128.

One of the objects of the invention is an antibody that contains at least one, at least two or all three VH HVR sequences selected from (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NO: 55-57; (b) HVR-H2, which contains one of the amino acid sequences of SEQ ID NO: 58-60; and (c) HVR-H3 which contains one of the amino acid sequences of SEQ ID NOs: 61-64. In one of the embodiments of the invention, the antibody contains HVR-H3, which contains one of the amino acid sequences of SEQ ID NO: 61-64. In another embodiment, the antibody contains HVR-H3, which contains one of the amino acid sequences of SEQ ID NO: 61-64, and HVR-L3, which contains one of the amino acid sequences of SEQ ID NO: 73-74. In a further embodiment, the antibody comprises HVR-H3, which contains one of the amino acid sequences of SEQ ID NOs: 61-64, HVR-L3, which contains one of the amino acid sequences of SEQ ID NOs: 73-74, and HVR-H2, which contains one of the amino acid sequences of SEQ ID NO: 58-60. In the following embodiment of the invention, the antibody contains (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NO: 55-57; (b) HVR-H2, which contains one of the amino acid sequences of SEQ ID NO: 58-60; and (c) HVR-H3 which contains one of the amino acid sequences of SEQ ID NOs: 61-64.

Another object of the invention is an antibody that contains at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NO: 114-115; (b) HVR-H2, which contains one of the amino acid sequences of SEQ ID NO: 116-120; and (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 121. In one embodiment, the antibody contains HVR-H3, which contains the amino acid sequence of SEQ ID NO: 121. In another embodiment, the antibody contains HVR-H3 , which contains the amino acid sequence of SEQ ID NO: 121, and HVR-L3, which contains one of the amino acid sequences of SEQ ID NO: 73-74. In a further embodiment, the antibody comprises HVR-H3, which contains the amino acid sequence of SEQ ID NO: 121, HVR-L3, which contains one of the amino acid sequences of SEQ ID NO: 73-74, and HVRH2, which contains one of the amino acid sequences of SEQ ID NO: 116-120. In the following embodiment of the invention, the antibody contains (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NO: 114-115; (b) HVR-H2, which contains one of the amino acid sequences of SEQ ID NO: 116-120; and (c) HVR-H3 which contains the amino acid sequence of SEQ ID NO: 121.

Another object of the invention is an antibody that contains at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 114; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 58; and (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 63. In one embodiment, the antibody contains HVRH3, which contains the amino acid sequence of SEQ ID NO: 63. In another embodiment, the antibody contains HVR-H3, which contains the amino acid sequence of SEQ ID NO: 63, and HVR-L3, which contains the amino acid sequence of SEQ ID NO:

- 30 040713

74. In a further embodiment, the antibody comprises HVR-H3 which contains the amino acid sequence of SEQ ID NO: 63, HVR-L3 which contains the amino acid sequence of SEQ ID NO: 74, and HVR-H2 which contains the amino acid sequence of SEQ ID NO: 58. In another embodiment of the invention, the antibody contains (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 114; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 58; and (c) HVR-H3 which contains the amino acid sequence of SEQ ID NO: 63.

Another object of the invention is an antibody containing at least one, at least two or all three HVR VH sequences selected from (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 126; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 127; and (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 128. In one embodiment, the antibody comprises HVR-H3, which contains the amino acid sequence of SEQ ID NO: 128. In another embodiment, the antibody comprises HVR-H3 , which contains the amino acid sequence of SEQ ID NO: 128, and HVR-L3, which contains the amino acid sequence of SEQ ID NO: 131. In the following embodiment, the antibody contains HVR-H3, which contains the amino acid sequence of SEQ ID NO: 128, HVR-L3 , which contains the amino acid sequence of SEQ ID NO: 131, and HVR-H2, which contains the amino acid sequence of SEQ ID NO: 127. In another embodiment, the antibody contains (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 126; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 127; and (c) HVR-H3 which contains the amino acid sequence of SEQ ID NO: 128.

Another object of the invention is an antibody containing at least one, at least two or all three HVR VL sequences selected from (a) HVR-L1, which contains one of the amino acid sequences of SEQ ID NO: 65-69,122-124, 129; (b) HVR-L2, which contains one of the amino acid sequences of SEQ ID NO: 70-72, 125, 130; and (c) HVR-L3, which contains one of the amino acid sequences of SEQ ID NO: 73-74, 131. In one embodiment, the antibody contains (a) HVR-L1, which contains one of the amino acid sequences of SEQ ID NO: 65 -69.122-124, 129; (b) HVR-L2, which contains one of the amino acid sequences of SEQ ID NO: 70-72, 125, 130; and (c) HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 73-74, 131.

Another object of the invention is an antibody containing at least one, at least two or all three HVR VL sequences selected from (a) HVR-L1, which contains one of the amino acid sequences of SEQ ID NO: 65-69; (b) HVR-L2, which contains one of the amino acid sequences of SEQ ID NO: 70-72; and (c) HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 73-74. In one of the embodiments of the invention, the antibody contains (a) HVR-L1, which contains one of the amino acid sequences of SEQ ID NO: 65-69; (b) HVR-L2, which contains one of the amino acid sequences of SEQ ID NO: 70-72; and (c) HVR-L3 which contains one of the amino acid sequences of SEQ ID NO: 7374.

Another object of the invention is an antibody containing at least one, at least two or all three HVR VL sequences selected from (a) HVR-L1, which contains one of the amino acid sequences of SEQ ID NO: 122-124; (b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 125; and (c) HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 73-74. In one of the embodiments of the invention, the antibody contains (a) HVR-L1, which contains one of the amino acid sequences of SEQ ID NO: 122124; (b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 125; and (c) HVRL3 which contains one of the amino acid sequences of SEQ ID NOs: 73-74.

Another object of the invention is an antibody containing at least one, at least two or all three HVR VL sequences selected from (a) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 71; and (c) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 74. In one embodiment, the antibody contains (a) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 71; and (c) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 74. Another object of the invention is an antibody containing at least one, at least two, or all three HVR VL sequences selected from (a) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 123; (b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 71; and (c) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 74. In one embodiment, the antibody contains (a) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 123; (b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 71; and (c) HVR-L3 which contains the amino acid sequence of SEQ ID NO: 74.

Another object of the invention is an antibody containing at least one, at least two or all three HVR VL sequences selected from (a) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 129; (b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 130; and (c) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 131. In one embodiment, the antibody contains (a) HVRL1, which contains the amino acid sequence of SEQ ID NO: 129; (b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 130; and (c) HVR-L3 which contains the amino acid sequence of SEQ ID NO 131.

According to another aspect of the invention, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three HVR VH sequences selected from (I) HVR-H1, which comprises one of the amino acid sequences of SEQ ID NOs: 55-57, 114, 115, 126, (II) HVR-H2 which contains one of the amino acid sequences of SEQ ID NOs: 58-60, 116-120, 127, and (III) HVR-H3 which contains one from the amino acid sequences of SEQ ID NOs: 61-64, 121, 128; and (b) a VL domain comprising at least one, at least two, or all three HVR VL sequences selected from (I) HVR-L1, which comprises one of the amino acid sequences of SEQ ID NOs: 65-69, 122- 124, 129, (II) HVR-L2 which contains one of the amino acid sequences of SEQ ID NOs: 70-72, 125, 130, and (in HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 73-74, 131.

According to another aspect of the invention, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three HVR VH sequences selected from (I) HVR-H1, which comprises one of the amino acid sequences of SEQ ID NOs: 55-57, (II) HVR-H2 which contains one of the amino acid sequences of SEQ ID NOs: 58-60, and (III) HVR-H3 which contains one of the amino acid sequences of SEQ ID NOs: 61-64; and (b) a VL domain containing at least one, at least two, or all three HVR VL sequences selected from (I) HVR-L1, which contains one of the amino acid sequences of SEQ ID NO: 65-69, (II ) HVR-L2, which contains one of the amino acid sequences of SEQ ID NO: 70-72, and (c) HVR-L3, which contains one of the amino acid sequences of SEQ ID NO: 73-74.

According to another aspect of the invention, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three HVR VH sequences selected from (I) HVR-H1, which comprises one of the amino acid sequences of SEQ ID NOs: 114-115, (II) HVR-H2 which contains one of the amino acid sequences of SEQ ID NOs: 116-120, and (III) HVR-H3 which contains one of the amino acid sequences of SEQ ID NOs: 121; and (b) a VL domain containing at least one, at least two, or all three HVR VL sequences selected from (I) HVR-L1, which contains one of the amino acid sequences of SEQ ID NO: 122-124, (II ) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 125, and (c) HVR-L3, which contains one of the amino acid sequences of SEQ ID NO: 73-74.

According to another aspect of the invention, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three HVR VH sequences selected from (I) HVR-H1, which comprises the amino acid sequence of SEQ ID NO: 114, (II) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 58, and (III) HVRH3, which contains the amino acid sequence of SEQ ID NO: 63; and (b) a VL domain comprising at least one, at least two, or all three HVR VL sequences selected from (I) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 122, (II) HVR-L2 , which contains the amino acid sequence of SEQ ID NO: 71, and (c) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 74. According to another object of the invention, the antibody of the invention contains (a) a VH domain containing at least at least one, at least two or all three HVR VH sequences selected from (I) HVR-H1 which contains the amino acid sequence of SEQ ID NO: 114, (II) HVR-H2 which contains the amino acid sequence of SEQ ID NO: 58, and (III) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 63; and (b) a VL domain containing at least one, at least two, or all three HVR VL sequences selected from (I) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 123, (II) HVR-L2 , which contains the amino acid sequence of SEQ ID NO: 71, and (c) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 74.

According to another aspect of the invention, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three HVR VH sequences selected from (I) HVR-H1, which comprises the amino acid sequence of SEQ ID NO: 126, (II) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 127, and (III) HVRH3, which contains the amino acid sequence of SEQ ID NO: 128; and (b) a VL domain comprising at least one, at least two, or all three HVR VL sequences selected from (I) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 129, (II ) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 1301, and (c) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 131.

Another object of the invention is an antibody containing (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NO: 55-57, 114-115, 126; (b) HVR-H2 which contains one of the amino acid sequences of SEQ ID NOs: 58-60, 116-120, 127; (c) HVR-H3 which contains one of the amino acid sequences of SEQ ID NOs: 61-64, 121, 128; (d) HVR-L1 which contains one of the amino acid sequences of SEQ ID NOs: 65-69, 122-124, 129; (e) HVR-L2 which contains one of the amino acid sequences of SEQ ID NOs: 70-72, 125, 130; and (e) HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 73-74, 131.

Another object of the invention is an antibody containing (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NO: 55-57, 114-115; (b) HVR-H2, which contains one of the amino acid sequences of SEQ ID NO: 58-60, 116-120; (c) HVR-H3 which contains one of the amino acid sequences of SEQ ID NOs: 61-64, 121; (d) HVR-L1 which contains one of the amino acid sequences of SEQ ID NOs: 65-69, 122-124; (e) HVR-L2, which contains one of the amino acid sequences of SEQ ID NO: 70-72, 125; and (e) HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 73-74.

Another object of the invention is an antibody containing (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NO: 114-115; (b) HVR-H2, which contains one of the amino acid sequences of SEQ ID NO: 116-120; (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 121; (d) HVR-L1, which contains one of the amino acid sequences of SEQ ID NO: 122-124; (e) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 125; and (e) HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 73-74.

Another object of the invention is an antibody containing (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 114; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 58; (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 63; (d) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 71; and (e) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 74. Another object of the invention is an antibody containing (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 114; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 58; (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 63; (d) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 123; (e) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 71; and (e) HVR-L3 which contains the amino acid sequence of SEQ ID NO: 74.

Another object of the invention is an antibody containing (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 126; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 127; (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 128; (d) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 129; (e) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 130; and (e) HVRL3 which contains the amino acid sequence of SEQ ID NO: 131.

In some embodiments, any one or more amino acids in the anti-myostatin antibody provided herein are substituted at the following HV positions: (a) 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 (e) in HVR-L3 (SEQ ID NO: 73) at position 8.

In some embodiments of the invention, one or more amino acid(s) substitution(s) in the antibody to myostatin is(are) a conservative(s) substitution(s) presented in the present description. In some embodiments, one or more of the following amino acid substitutions may be made in any combination: (a) to HVR-H1 (SEQ ID NO: 55) S1H; Y2T, D or E; (b) in HVR-H2 (SEQ ID NO: 58) Y4H; S7K; T8M or K; Y10K; A11M or E; S12E; G16K; (c) in HVR-H3 (SEQ ID NO: 61) Y5H; T7H; L11K; (d) in HVR-L1 (SEQ ID NO: 65) Q1T; S2T; S5E; Y7F; D8H; N9D or A or E; (e) in HVR-L2 (SEQ ID NO: 70) S3E; S7Y, F or W; and (e) in HVR-L3 (SEQ ID NO: 73) L8R.

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

In any of the above embodiments, the anti-myostatin antibody may be humanized. In one embodiment, the anti-myostatin antibody comprises the HVRs of any of the above embodiments and further comprises an acceptor human framework, eg, a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-myostatin antibody comprises an HVR as defined in any of the above embodiments and further comprises a VH or VL containing the FR sequence. In a further embodiment, the anti-myostatin antibody comprises the following FR sequences of the heavy chain and/or light chain variable domain: in the heavy chain variable domain, FR1 contains one of the amino acid sequences of SEQ ID NO: 132-134, FR2 contains one of the amino acid sequences of SEQ ID NO: : 135-136, FR3 contains one of the amino acid sequences of SEQ ID NO: 137, FR4 contains one of the amino acid sequences of SEQ ID NO: 138. In the light chain variable domain, FR1 contains the amino acid sequence of SEQ ID NO: 139, FR2 contains one of the amino acid sequences SEQ ID NO: 140-141, FR3 contains one of the amino acid sequences of SEQ ID NO: 142-143, FR4 contains the amino acid sequence of SEQ ID NO: 144.

One of the objects of the invention is an antibody to myostatin, containing at least one, two, three, four, five or six HVR selected from (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NO: 157-162; (b) HVR-H2, which contains one of the amino acid sequences of SEQ ID NO: 163-168; (c) HVR-H3, which contains one of the amino acid sequences of SEQ ID NO: 169-174; (d) HVR-L1, which contains one of the amino acid sequences of SEQ ID NO: 175-180; (e) HVR-L2, which contains one of the amino acid sequences of SEQ ID NO: 181-186; and (e) HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 187-192.

One of the objects of the invention is an antibody containing at least one, at least two or all three HVR VH sequences selected from (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NO: 157-162; (b) HVR-H2, which contains one of the amino acid sequences of SEQ ID NO: 163-168; and (c) HVR-H3 which contains one of the amino acid sequences of SEQ ID NOs: 169-174. In one of the embodiments of the invention, the antibody contains HVR-H3, which contains one of the amino acid sequences of SEQ ID NO: 169-174. In another embodiment, the antibody comprises HVR-H3 which contains one of the amino acid sequences of SEQ ID NOs: 169-174 and HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 187-192. In another embodiment, the antibody comprises HVR-H3, which contains one of the amino acid sequences of SEQ ID NO: 169-174, HVR-L3, which contains one of the amino acid sequences of SEQ ID NO: 187-192, and HVR-H2, which contains one of the amino acid sequences of SEQ ID NO: 163-168. In the following embodiment of the invention, the antibody contains (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NO: 157-162; (b) HVR-H2, which contains one of the amino acid sequences of SEQ ID NO: 163-168; and (c) HVR-H3 which contains one of the amino acid sequences of SEQ ID NOs: 169-174.

Another object of the invention is an antibody containing at least one, at least two or all three HVR VL sequences selected from (a) HVR-L1, which contains one of the amino acid sequences of SEQ ID NO: 175-180; (b) HVR-L2, which contains one of the amino acid sequences of SEQ ID NO: 181-186; and (c) HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 187-192. In one of the embodiments of the invention, the antibody contains (a) HVR-L1, which contains one of the amino acid sequences of SEQ ID NO: 175-180; (b) HVR-L2, which contains one of the amino acid sequences of SEQ ID NO: 181-186; and (c) HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 187-192.

Another object of the invention is an antibody according to the invention, which contains (a) a VH domain containing at least one, at least two or all three HVR VH sequences selected from (I) HVR-H1, which contains one of the amino acids sequences of SEQ ID NO: 157-162, (II) HVR-H2, which contains one of the amino acid sequences of SEQ ID NO: 163-168, and (III) HVR-H3, which contains one of the amino acid sequences of SEQ ID NO: 169- 174; and (b) a VL domain containing at least one, at least two, or all three HVR VL sequences selected from (I) HVR-L1, which contains one of the amino acid sequences of SEQ ID NO: 175-180, (II ) HVR-L2, which contains one of the amino acid sequences of SEQ ID NO: 181-186, and (c) HVR-L3, which contains one of the amino acid sequences of SEQ ID NO: 187-192.

Another object of the invention is an antibody containing (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NO: 157-162; (b) HVR-H2, which contains one of the amino acid sequences of SEQ ID NO: 163-168; (c) HVR-H3, which contains one of the amino acid sequences of SEQ ID NO: 169-174; (d) HVR-L1, which contains one of the amino acid sequences of SEQ ID NO: 175-180; (e) HVR-L2 which contains one of the amino acid sequences of SEQ ID NOs: 181-186; and (e) HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 187-192.

In another aspect of the invention, the anti-myostatin antibody comprises a heavy chain variable domain (VH) sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of the amino acid sequences of SEQ ID NOs: 13, 16-30, 32-34 and 86-95. In some embodiments, a VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence. , but an antibody to myostatin containing the specified sequence retains the ability to bind to myostatin. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in one of SEQ ID NOs: 13, 16-30, and 32-34. In some embodiments, the substitutions, insertions, or deletions affect regions outside of the HVR (ie, FR). Optionally, the anti-myostatin antibody contains the VH sequence shown in one of SEQ ID NOs: 13, 16-30, and 32-34, including post-translational modifications of said sequence. In a particular embodiment, the VH contains one, two, or three HVRs selected from: (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NOs: 55-57, (b) HVR-H2, which contains one of the amino acids sequences of SEQ ID NO: 58-60, and (c) HVR-H3, which contains one of the amino acid sequences of SEQ ID NO: 61-64.

In another aspect of the invention, the anti-myostatin antibody comprises a heavy chain variable domain (VH) sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of the amino acid sequences of SEQ ID NOs: 13, 16-30, 32-34 and 86-95. In some embodiments, a VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence. , but an antibody to myostatin containing the specified sequence retains the ability to bind to myostatin. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in one of SEQ ID NOs: 13, 16-30, 32-34, and 86-95. In some embodiments, the substitutions, insertions, or deletions affect regions outside of the HVR (ie, FR). Optionally, the anti-myostatin antibody contains the VH sequence shown in one of SEQ ID NOs: 13, 16-30, 32-34, and 86-95, including post-translational modifications of said sequence. In a specific embodiment, the VH contains one, two, or three HVRs selected from: (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NOs: 55-57, 114-115, 126, (b) HVRH2, which contains one of the amino acid sequences of SEQ ID NOs: 58-60, 116-120, 127, and (c) HVR-H3, which contains one of the amino acid sequences of SEQ ID NOs: 61-64, 121, 128.

In another aspect of the invention, the anti-myostatin antibody comprises a VH sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to one of the amino acid sequences of SEQ ID NOs: 13, 16 -30, 32, 33, and 34. In some embodiments, a VH sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical contains substitutions (e.g., conservative substitutions ), insertions or deletions relative to the reference sequence, but an anti-myostatin antibody containing said sequence retains the ability to bind to myostatin. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in one of SEQ ID NOs: 13, 16-30, 32, 33, and 34. In some embodiments, the substitution, insertion, or deletion affect areas outside the HVR (i.e. FR). Optionally, the anti-myostatin antibody contains the VH sequence shown in one of SEQ ID NOs: 13, 16-30, 32, 33 and 34, including post-translational modifications of said sequence. In a particular embodiment, the VH contains one, two, or three HVRs selected from: (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NOs: 55-57, (b) HVR-H2, which contains one of the amino acids sequences of SEQ ID NO: 58-60, and (c) HVR-H3, which contains one of the amino acid sequences of SEQ ID NO: 61-64.

In another aspect of the invention, an anti-myostatin antibody comprises a VH sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to one of the amino acid sequences of SEQ ID NOs: 86-95 . In some embodiments, a VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence. , but an antibody to myostatin containing the specified sequence retains the ability to bind to myostatin. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in one of SEQ ID NOs: 86-95. In some embodiments, the substitutions, insertions, or deletions affect regions outside of the HVR (ie, FR). Optionally, the anti-myostatin antibody contains the VH sequence shown in one of SEQ ID NOs: 86-95, including post-translational modifications of said sequence. In a particular embodiment, the VH contains one, two, or three HVRs selected from: (a)

HVR-H1, which contains one of the amino acid sequences of SEQ ID NO: 114-115, 126, (b)

HVR-H2 which contains one of the amino acid sequences of SEQ ID NOs: 116-120, 127, and (c) HVR-H3 which contains one of the amino acid sequences of SEQ ID NOs: 121, 128.

In another aspect of the invention, the anti-myostatin antibody comprises a VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 86. In some embodiments, carrying out the invention, a VH sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical contains substitutions (for example, conservative substitutions), insertions or deletions relative to the reference sequence, but the antibody to myostatin containing the specified sequence retains the ability to bind to myostatin. In some embodiments, a total of 1 to 10 amino acids are changed, inserted and/or deleted by deletion in SEQ ID NO: 86.

In some embodiments, the substitutions, insertions, or deletions affect regions outside of the HVR (ie, FR). Optionally, the anti-myostatin antibody contains the VH sequence shown in SEQ ID NO: 86, including post-translational modifications of said sequence. In a specific embodiment, the VH contains one, two, or three HVRs selected from: (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 114, (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 58 , and (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 63. In another aspect of the invention, the anti-myostatin antibody contains a VH sequence that is at least , 98, 99, or 100% identical to the amino acid sequence of SEQ ID NO: 92. In some embodiments, a VH sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% contains substitutions (eg, conservative substitutions), insertions or deletions relative to the reference sequence, but an antibody to myostatin containing the specified sequence retains the ability to bind to myostatin. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in SEQ ID NO: 92. In some embodiments, the substitutions, insertions, or deletions affect regions outside the HVR (i.e., FR). Optionally, the anti-myostatin antibody contains the VH sequence shown in SEQ ID NO: 92, including post-translational modifications of said sequence. In a specific embodiment, the VH contains one, two, or three HVRs selected from: (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 114, (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 58 , and (c) HVR-H3 which contains the amino acid sequence of SEQ ID NO: 63.

In another aspect of the invention, the anti-myostatin antibody contains a VL sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to one of the amino acid sequences of SEQ ID NOs: 15, 31 , 35-38 and 96-99. In some embodiments, a VL sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-myostatin antibody containing said sequence retains the ability to bind to myostatin. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in one of SEQ ID NOs: 15, 31, 3538, and 96-99. In some embodiments, the substitutions, insertions, or deletions affect regions outside of the HVR (ie, FR). Optionally, the anti-myostatin antibody contains the VL sequence shown in one of SEQ ID NOs: 15, 31, 35-38 and 96-99, including post-translational modifications of said sequence. In a particular embodiment, the VL contains one, two or three HVRs selected from: (a) HVR-L1 which contains one of the amino acid sequences of SEQ ID NOs: 65-69, 122-124, 129; (b) HVR-L2, which contains one of the amino acid sequences of SEQ ID NO: 70-72, 125, 130; and (c) HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 73-74, 131.

In another aspect of the invention, the anti-myostatin antibody contains a VL sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to one of the amino acid sequences of SEQ ID NOs: 15, 31 , 35, 36, 37, and 38. In some embodiments, a VL sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical contains substitutions (e.g., conservative substitutions ), insertions or deletions relative to the reference sequence, but an anti-myostatin antibody containing said sequence retains the ability to bind to myostatin. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in one of SEQ ID NOs: 15, 31, 35, 36, 37, and 38. In some embodiments, the substitution, insertion, or deletion affect areas outside the HVR (i.e. FR). Optionally, the anti-myostatin antibody contains the VL sequence shown in one of SEQ ID NOs: 15, 31, 35, 36, 37 and 38, including post-translational modifications of said sequence. In a particular embodiment, the VL contains one, two or three HVRs selected from: (a) HVR-L1 which contains one of the amino acid sequences of SEQ ID NOs: 65-69; (b) HVR-L2, which contains one of the amino acid sequences of SEQ ID NO: 70-72; and (c) HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 73-74.

In another aspect of the invention, the anti-myostatin antibody comprises a VL sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to one of the amino acid sequences of SEQ ID NOs: 96-99 . In some embodiments, a VL sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence , but an antibody to myostatin containing the specified sequence retains the ability to bind to myostatin. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in one of SEQ ID NOs: 96-99. In some embodiments, the substitutions, insertions, or deletions affect regions outside of the HVR (ie, FR). Optionally, the anti-myostatin antibody contains the VL sequence shown in one of SEQ ID NOs: 96-99, including post-translational modifications of said sequence. In a specific embodiment, the VL contains one, two or three HVRs selected from: (a) HVR-L1 which contains one of the amino acid sequences of SEQ ID NOs: 122-124, 129; (b) HVR-L2, which contains one of the amino acid sequences of SEQ ID NO: 125, 130; and (c) HVR-L3 which contains the amino acid sequence of SEQ ID NO: 131.

In another aspect of the invention, the anti-myostatin antibody comprises a VL sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 96. In some embodiments, implementation of the invention, a VL sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical contains substitutions (for example, conservative substitutions), insertions or deletions relative to the reference sequence, but the antibody to myostatin containing the specified sequence retains the ability to bind to myostatin. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in SEQ ID NO: 96. In some embodiments, the substitutions, insertions, or deletions affect regions outside the HVR (i.e., FR). Optionally, the anti-myostatin antibody contains the VL sequence shown in SEQ ID NO: 96, including post-translational modifications of said sequence. In a specific embodiment of the invention, the VL contains one, two or three HVRs selected from: (a) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 71; and (c) HVR-L3 which contains the amino acid sequence of SEQ ID NO: 74.

In another aspect of the invention, the anti-myostatin antibody comprises a VL sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 97. In some embodiments, implementation of the invention, a VL sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical contains substitutions (for example, conservative substitutions), insertions or deletions relative to the reference sequence, but the antibody to myostatin containing the specified sequence retains the ability to bind to myostatin. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in SEQ ID NO: 97. In some embodiments, the substitutions, insertions, or deletions affect regions outside the HVR (i.e., FR). Optionally, the anti-myostatin antibody contains the VL sequence shown in SEQ ID NO: 97, including post-translational modifications of said sequence. In a specific embodiment of the invention, the VL contains one, two or three HVRs selected from: (a) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 123; (b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 71; and (c) HVR-L3 which contains the amino acid sequence of SEQ ID NO: 74.

Another object of the invention is an antibody to myostatin, where the antibody contains VH, presented in one of the above embodiments of the invention, and VL, presented in one of the above embodiments of the invention. In one embodiment, the antibody comprises the VH and VL sequences shown in one of SEQ ID NOs: 13, 16-30, 32-34, and 86-95 and one of SEQ ID NOs: 15, 31, 35-38 and 96-99, respectively, including post-translational modifications of these sequences. In one embodiment, the antibody comprises the VH and VL sequences shown in one of SEQ ID NOs: 13, 16-30, 3234, and 86-95 and in one of SEQ ID NOs: 15, 31, 35-38, and 96- 99, respectively, including post-translational modifications of these sequences. In one of the embodiments of the invention, the antibody contains the VH and VL sequences specified in one of SEQ ID NO: 86-95 and one of SEQ ID NO: 96-99, respectively, including post-translational modifications of these sequences.

Another object of the invention is an antibody to myostatin, where the antibody contains VH, presented in any of the above embodiments of the invention, and VL, presented in any of the above embodiments of the invention. In one of the embodiments

- 37 040713 of the invention, the antibody contains the VH and VL sequences indicated in one of SEQ ID NOs: 13, 1630 and 32-34 and in one of SEQ ID NOs: 15, 31 and 35-38, respectively, including post-translational modifications of these sequences . In one of the embodiments of the invention, the antibody contains the sequences VH and VL specified in one of SEQ ID NO: 13, 16-30 and 32-34 and in one of SEQ

ID NOs: 15, 31 and 35-38, including post-translational modifications of these sequences.

Another object of the invention is an antibody to myostatin, where the antibody contains VH, presented in any of the above embodiments of the invention, and VL, presented in any of the above embodiments of the invention. In one embodiment, the antibody comprises the VH and VL sequences shown in one of SEQ ID NOs: 86-95 and one of SEQ ID NOs: 96-99, respectively, including post-translational modifications of these sequences. In one embodiment, the antibody comprises the VH and VL sequences shown in one of SEQ ID NOs: 86-95 and one of SEQ ID NOs: 96-99, including post-translational modifications of these sequences. In one embodiment, the antibody comprises the VH and VL sequences shown in one of SEQ ID NOs: 86-95 and one of SEQ ID NOs: 96-99, including post-translational modifications of these sequences. Another object of the invention is an antibody to myostatin, where the antibody contains VH, presented in any of the above embodiments of the invention, and VL, presented in any of the above embodiments of the invention. In one embodiment, the antibody contains the VH and VL sequences shown in SEQ ID NO: 86 and SEQ ID NO: 96, respectively, including post-translational modifications of these sequences. In one of the embodiments of the invention, the antibody contains the VH and VL sequences indicated in SEQ ID NO: 92 and SEQ ID NO: 97, respectively, including post-translational modifications of these sequences.

Another object of the invention is an antibody to myostatin containing a VH sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to one of the amino acid sequences of SEQ ID NO: 12, 145 -150. In some embodiments, a VH sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-myostatin antibody containing said sequence retains the ability to bind to myostatin. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in one of SEQ ID NO: 12, 145150. In some embodiments, the substitutions, insertions, or deletions affect regions outside the HVR (i.e., .FR). Optionally, the anti-myostatin antibody contains the VH sequence shown in one of SEQ ID NOs: 12, 145-150, including post-translational modifications of said sequence. In a particular embodiment, the VH contains one, two, or three HVRs selected from: (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NO: 55, 157-162, (b) HVR-H2, which contains one from the amino acid sequences of SEQ ID NO: 58, 163-168, and (c) HVR-H3, which contains one of the amino acid sequences of SEQ ID NO: 61, 169-174.

In another aspect of the invention, the anti-myostatin antibody contains a VL sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to one of the amino acid sequences of SEQ ID NOs: 14, 151 -156. In some embodiments, a VL sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-myostatin antibody containing said sequence retains the ability to bind to myostatin. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in one of SEQ ID NO: 14, 151156. In some embodiments, the substitutions, insertions, or deletions affect regions outside the HVR (i.e., .FR). Optionally, the anti-myostatin antibody contains the VL sequence shown in one of SEQ ID NOs: 14, 151-156, including post-translational modifications of said sequence. In a specific embodiment of the invention, the VL contains one, two or three HVRs selected from: (a) HVR-L1, which contains one of the amino acid sequences of SEQ ID NO: 65, 175-180; (b) HVR-L2, which contains one of the amino acid sequences of SEQ ID NO: 70, 181-186; and (c) HVR-L3 which contains one of the amino acid sequences of SEQ ID NO: 73, 187-192.

Another object of the invention is an antibody to myostatin, where the antibody contains VH, presented in any of the above embodiments of the invention, and VL, presented in any of the above embodiments of the invention. In one embodiment, the antibody comprises the VH and VL sequences shown in one of SEQ ID NO: 12, 145150 and one of SEQ ID NO: 14, 151-156, respectively, including post-translational modifications of these sequences.

In some embodiments, an anti-myostatin antibody of the present invention comprises a VH as shown in any of the above embodiments and a heavy chain constant region comprising one of the amino acid sequences of SEQ ID NOs: 7, 9, 11, 193, 195-198, 227, 228, 229-381. In some embodiments, an anti-myostatin antibody of the present invention comprises a VL as defined in any of the above embodiments and a light chain constant region comprising one of the amino acid sequences of SEQ ID NOS: 8 and 10.

The following object of the invention is an antibody that binds to the same epitope as the antibody to myostatin presented in the present description. The next object of the invention is an antibody that binds to the same epitope as the antibody listed in table. 2a. The next object of the invention is an antibody that binds to the same epitope as the antibody listed in table. 11a or 13. In some embodiments, the invention is an antibody that binds to an epitope within a myostatin propeptide fragment consisting of amino acids 21-100 of SEQ ID NO: 78. Alternatively, the antibody binds to a myostatin propeptide fragment that consists of amino acids 21-80, 41-100, 21-60, 41-80, 61-10o, 21-40, 41-60, 61-80 or 81-100 SEQ ID NO: 78.

In another embodiment, the anti-myostatin antibody of any of the above embodiments is a monoclonal antibody, including a chimeric, humanized, or human antibody. In one embodiment, the anti-myostatin antibody is an antibody fragment, such as an Fv, Fab, Fab', dimeric antibody, or F(ab') ₂ fragment. In another embodiment, the antibody is a full-length antibody, for example, an intact antibody of an IgG1 or IgG4 isotype, or another class or isotype of antibodies described herein.

In a further aspect of the invention, the anti-myostatin antibody referred to in any of the above embodiments of the invention may have any of the features, singly or in combination, as described in sections 1-7 below.

1. Affinity of antibodies.

In some embodiments, an antibody provided herein has a dissociation constant (Kd) value of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0, 01 nM or less, or 0.001 nM or less (eg 10 ^-8 M or less, eg 10 ^-8 to 10 ^-13 M, eg 10 ^-9 to 10 ^-13 M).

In one embodiment, the Kd value is measured using a radiolabeled antigen (RIA) binding assay. In one of the embodiments of the invention, RIA is carried out using the Fab version of the antibody of interest and its antigen. For example, the binding affinity of a Fab to an antigen is measured in solution by equilibrating the Fab with a minimum concentration of ¹²⁵ I-labeled antigen in the presence of titrated dilutions of unlabeled antigen, then capturing the bound antigen on an anti-Fab sensitized plate (see, e.g., Chen et al., J. Mol Biol 293, 1999, pp. 865-881). MICROTITER multi-well plates (Thermo Scientific) are sensitized overnight with anti-Fab capture antibody (Cappel Labs) at 5 µg/mL in 50 mM sodium carbonate (pH 9.6) and then blocked with 2% (w/v) bovine serum albumin in PBS for 2-5 hours at room temperature (about 23°C). In a non-absorbent plate (Nunc, #269620), 100 or 26 pM I labeled antigen is mixed with serial dilutions of the Fab of interest (e.g., anti-VEGF antibody, Fab-12, see Presta et al., Cancer Res. 57, 1997, pp. 4593-4599). The Fab of interest is then incubated overnight; however, the incubation can be carried out for a longer period of time (for example, for 65 hours) in order to ensure that equilibrium is reached. The mixtures are then transferred to a prepared capture plate and incubated at room temperature (eg, for 1 hour). The solution is then removed and the plate washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. After the plates are dry, 150 µl/well of scintillation fluid ((MICROSCINT-20™; Packard) is added to the plates and the plates are read using a TOPCOUNT™ gamma counter (Packard) for 10 minutes. For competitive binding assays choose the concentration of each Fab, providing a level of binding that is less than or equal to 20% of the maximum.

In another embodiment of the invention, Kd is measured by surface plasmon resonance using a BIACORE® device. For example, the assay is performed with a BIACORE®-2000 or BIACORE®-3000 device (GE Healthcare Inc., Piscataway, NJ) at 25° C. using antigen immobilized on CM5 chips with an immobilization level corresponding to ~ 10 response units (RU). In one embodiment, carboxymethylated dextran (CM5, GE Healthcare Inc.) biosensor chips are activated with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) hydrochloride according to the supplier's instructions. . The antigen is diluted with 10 mM sodium acetate, pH 4.8 to a concentration of 5

- 39 040713 μg/ml (~0.2 μM) before injection at a flow rate of 5 μl/min to achieve an immobilization level corresponding to approximately 10 response units (RU) of the cross-linked protein. After injection of the antigen, 1 M ethanolamine is injected to block the unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (from 0.78 to 500 nM) in PBS supplemented with 0.05% polysorbate 20 (TWEEN-20™) as a surfactant (PBS) are injected at 25°C with a flow rate of approximately 25 μl /min The rates of the association reaction (k _on ) and the dissociation reaction (k _off ) are calculated using a simple 1:1 Langmuir binding model (Evaluation software version 3.2, BIACORE®) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium constant of the dissociation reaction (Kd) is calculated as the ratio k _off /k _on (see, for example, Chen et al., J Mol Biol 293, 1999, pp. 865-881). If the association reaction rate exceeds 106 M'^s' ¹ as measured by the surface plasmon resonance method described above, then the association reaction rate can be determined using the fluorescence quenching method, which measures the increase or decrease in fluorescence emission intensity (excitation wavelength 295 nm ; emission wavelength 340 nm, bandwidth 16 nm) at 25°C applied at a concentration of 20 nM anti-antigen antibody (Fab-format) in PBS, pH 7.2, in the presence of increasing concentrations of antigen, measuring with a spectrometer such as as a spectrophotometer equipped with a flow stop unit (Aviv Instruments) or an SLM-AMINCO™ 8000 series spectrophotometer (ThermoSpectronic) while stirring the contents of the cuvette.

2. Fragments of antibodies.

In some embodiments, the antibody of the present invention 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 some antibody fragments, see Hudson et al., Nat. Med. 9, 2003, p. 129-134. For a review of scFv fragments, see, for example, Plueckthun in: The Pharmacology of Monoclonal Antibodies, ed. Rosenburg and Moore, Springer, New York, vol. 113, 1994, p. 269-315; see also WO 93/16185; and US Pat.

Dimeric antibodies are antibody fragments with two antigen-binding sites, which can be divalent or bispecific (see, for example, EP 0404097; WO 1993/01161; Hudson et al., Nat. Med. 9, 2003, c. 129-134; and Holliger et al., Proc Natl Acad Sci USA 90, 1993, pp. 6444-6448). Trimeric and tetrameric antibodies are also described in Hudson et al., Nat. Med. 9, 2003, p. 129-134).

Single domain antibodies are antibody fragments containing all or part of the heavy chain variable domain, or all or part of the light chain variable domain of an antibody. In some embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, for example, US No. 6,248,516).

Antibody fragments can be generated by a variety of techniques including, but not limited to, proteolytic cleavage of an intact antibody, and also generated using recombinant host cells (eg, E. coli or phage) described herein.

3. Chimeric and humanized antibodies.

In some embodiments of the invention, the antibody specified in the present description is a chimeric antibody. Some chimeric antibodies are described, for example, in US No. 4816567; and Morrison et al., Proc. Natl. Acad. sci. USA 81, 1984, p. 6851-6855. In one example, a chimeric antibody comprises a non-human variable region (eg, a variable region from an antibody from mice, rats, hamsters, rabbits, or non-human primates such as monkeys) and a human constant region. In another example, the chimeric antibody is a class-switched antibody whose class or subclass has been changed from the parent antibody. Chimeric antibodies include their antigen-binding fragments.

In some embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody contains one or more variable domains in which the HVRs, eg CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. The humanized antibody may optionally contain at least a portion of a human constant region. In some embodiments, certain FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (eg, an antibody from which HVR residues are derived), for example, to maintain or improve the specificity or affinity of the antibody.

Information about humanized antibodies and methods for their creation is summarized, for example, in Almagro,

- 40 040713

front. biosci. 13, 2008, p. 1619-1633, and are also described in Riechmann et al., Nature 332, 1988, p. 323-329; Queen et al., Proc. Natl. Acad. sci. USA 86, 1989, p. 10029-10033; US No. 5821337, 7527791, 6982321 and 7087409; Kashmiri et al., Methods 36, 2005, p. 25-34 (description of transplantation of SDR (a-CDR)); Padlan, Mol. Immunol. 28, 1991, p. 489-498 (description of applying a new coating); Dall'Acqua et al., Methods 36, 2005, p. 43-60 (description of permutation FR); and Osbourn et al., Methods 36, 2005, p. 61-68 and Klimka et al., Br. J. Cancer 83, 2000, p. 252-260 (describing a directed selection approach for FR permutation).

Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using best fit methods (see, for example, Sims et al., J. Immunol. 151, 1993, c. 2296-2308, framework regions derived from the consensus sequence of a specific subgroup of human antibody light and heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA 89, 1992, pp. 4285-4289 and Presta et al., J. Immunol. 151, 1993, pp. 2623-2632), human mature (with somatic mutations) or human germline frameworks (see, for example, Almagro and Fransson, Front. Biosci. 13, 2008, pp. 1619-1633), and framework regions resulting from screening of FR libraries (see, e.g., Vasa et al., J. Biol. Chem. 272, 1997, pp. 10678-10684 and Rosok et al., J Biol Chem 271 (1996) 22611-22618).

4. Human antibodies.

In some embodiments, the antibody of the present invention is a human antibody. Human antibodies can be generated using a variety of techniques known in the art. Human antibodies are described in general by van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5, 2001, p. 368-374 and Lonberg, Curr. Opin. Immunol. 20, 2008, p. 450-459.

Human antibodies can be generated by administering an immunogen to a transgenic animal modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. These animals typically contain all or part of the human immunoglobulin loci instead of endogenous immunoglobulin loci that are either present outside the chromosomes or integrated arbitrarily into the chromosomes of the animal. In these transgenic mice, endogenous immunoglobulin loci are usually inactivated. For a review of methods for generating human antibodies in transgenic animals, see Lonberg, Nat. Biotech. 23, 2005, p. 1117-1125 (see, for example, also US No. 6075181 and US No. 6150584 for the XENOMOUSE™ Technology; US No. 5770429 for the HUMAB® Technology; US No. 7041870 for the K-M MOUSE® Technology and US Patent Application Publication 2007/0061900 description of VELOCIMOUSE® technology). Human variable regions from intact antibodies derived from these animals can be further modified, for example by combining with another human constant region.

Human antibodies can be generated using hybridoma-based methods. Human myeloma and murine-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (see, for example, Kozbor, J. Immunol. 133, 1984, c. 3001-3005; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, 1987, pp. 51-63 and Boerner et al., J. Immunol. 147, 1991, pp. 86-95). Human antibodies generated using human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. sci. USA 103, 2006, p. 3557-3562. Additional methods include those described, for example, in US No. 7189826 (description of obtaining monoclonal human antibodies of the IgM type from hybridoma cell lines), and in Ni, Xiandai Mianyixue 26, 2006, c. 265-268 (description of human-human hybridomas). Human hybridoma technology (Trioma technology) is also described by Vollmers and Brandlein, Histology and Histopathology 20, 2005, c. 927-937 and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology 27, 2005, p. 185-191.

Human antibodies can also be generated by isolating the variable domain sequences of Fv clones selected from phage display libraries that have been generated using human antibodies. These variable domain sequences can then be combined with the desired human constant domain. Methods for selecting human antibodies from antibody libraries are described below.

5. Antibodies obtained from libraries.

Antibodies of the invention can be isolated by screening combinatorial libraries of 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 said libraries for antibodies having the desired binding characteristics. These methods are summarized in, for example, Hoogenboom et al., Methods in Molecular Biology 178, 2001, p. 1-37 and are also described, for example, in McCafferty et al., Nature 348, 1990, c. 552-554; Clackson et al., Nature 352, 1991, p. 624-628; Marks et al., J. Mol. Biol. 222, 1992, p. 581-597; Marks and Bradbury, Methods in Molecular Biology 248, 2003, p. 161-175; Sidhu et al., J. Mol. Biol. 338, 2004, p. 299-310; Lee et al., J. Mol. Biol. 340, 2004, p. 1073-1093; Fellouse, Proc. Natl. Acad. sci. USA 101, 2004, p. 12467-12472 and Lee et al., J. Immunol. Methods 284, 2004, p.

- 41 040713

119-132.

In some phage display techniques, populations of VH and VL genes are cloned individually by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which are then screened for antigen-binding phage according to the method described by Winter et al., Ann . Rev. Immunol. 12, 1994, p. 433-455. The phage typically displays antibody fragments either as single chain Fv fragments (scFv) or as Fab fragments. Libraries derived from immunized sources include antibodies with high affinity for the immunogen without the need for hybridomas. Alternatively, an untreated (naive) population (e.g. from a human body) can be cloned, producing a single source of antibodies to a wide range of foreign as well as self antigens without any immunization, as described in Griffiths et al., EMBO J, 12, 1993, p. 725-734. Finally, naïve libraries can also be generated synthetically by cloning non-rearranged V-gene segments from stem cells and using random sequence PCR primers to encode CDR3 hypervariable regions and to perform in vitro rearrangement according to the method described by Hoogenboom. and Winter, J. Mol. Biol. 227, 1992, p. 381-388. Human antibody phage libraries are described, for example, in the following patent publications: US 5750373 and US Patent Application Publications 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936 and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody libraries are considered to be human antibodies or human antibody fragments.

6. Multispecific antibodies.

In some embodiments of the invention, the antibody specified in the present description is a multispecific antibody, for example, bispecific antibody. Multispecific antibodies are monoclonal antibodies that have specificities that bind to at least two different sites. In some embodiments, one of the binding specificities allows binding to myostatin and the other to any other antigen. In some embodiments, the bispecific antibodies can bind to two different myostatin epitopes. Bispecific antibodies can also be used to localize cytotoxic agents in cells that express myostatin. Bispecific antibodies can be obtained as full length antibodies or antibody fragments.

Techniques for generating multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305, 1983, pp. 537-540, WO 93/08829 and Traunecker et al., EMBO J. 10,1991, pp. 3655-3659), and knob-in-hole technology (providing a boss-to-trough interaction, see, for example, US No. 5731168). Multispecific antibodies can also be obtained by creating controlled electrostatic influences to obtain antibody molecules with heterodimeric Fc (WO 2009/089004); cross-linking two or more antibodies or fragments (see, for example, US No. 4676980 and Brennan and others, Science 229, 1985, S. 81-83); the use of leucine zippers to produce bispecific antibodies (see, for example, Kostelny et al., J. Immunol. 148, 1992, pp. 1547-1553); the use of dimeric antibody technology (diabodies) to generate bispecific antibody fragments (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA 90, 1993, pp. 6444-6448); and the use of single chain Fv (sFv) dimers (see, for example, Gruber et al., J. Immunol. 152, 1994, pp. 5368-5374); and obtaining trispecific antibodies according to the method described, for example, Tutt and others, J. Immunol. 147, 1991, p. 60-69).

Also within the scope of the present invention are engineered antibodies with three or more functional antigen-binding sites, including octopus antibodies (see, for example, US 2006/0025576A1).

The antibody or fragment thereof may also include a dual-acting Fab or DAF containing an antigen-binding site that binds to myostatin as well as another antigen other than it (see, for example, US 2008/0069820).

7. Antibody variants.

Some embodiments of the invention relate to variants of the amino acid sequences of the antibodies presented in the present description. For example, it may be desirable to increase the binding affinity and/or other biological properties of an antibody. Variants of the amino acid sequence of an antibody can be obtained by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by synthesizing peptides. These modifications include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequences of the antibody. Any combination of deletions, insertions, and substitutions can be used to produce the final construct, as long as the final construct has the desired characteristics, such as the ability to bind to an antigen.

a) Obtained by substitution, insertion and deletion variants.

- 42 040713

Some embodiments of the invention are antibody variants that have one or more amino acid substitutions. Sites of interest for displacement mutagenesis include HVR and FR. Conservative substitutions are presented below in table. 1 under the heading preferred substitutions and are described below with reference to the classes of amino acid side chains. More important substitutions are presented below in Table. 1 under the heading exemplified substitutions and are described below with reference to the amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the products screened for desired activity, eg, retention/increase in antigen binding, reduced immunogenicity, or increased ADCC or CDC.

Table 1

original balance Substitutions cited as an example Preferred Substitutions Ala (A) Val; Leu; Not Val Arg(R) Lys; gin; Asn Lys Asn(N) gin; His; Asp, Lys; Arg gin Asp(g) Glu; Asn Glu Cys (C) Ser; Ala Ser Gin (Q) asn; Glu Asn Glu(d) asp; gin asp Gly (G) Ala Ala His(H) asn; gin; Lys; Arg Arg He(I) Leu; Val; met; Ala; Ph; norleucine Leu Leu(L) norleucine; Ile; Val; met; Ala; Phe ile Lys (K) Arg; gin; Asn Arg Met(M) Leu; Ph; ile Leu Phe(e) 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; Ph; Thr; Ser Phe Val(V) Ile; Leu; met; Ph; Ala; norleucine Leu

Amino acids can be grouped based on common side chain properties as follows: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) main: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions mean the replacement of a representative of one of the specified classes with a representative from another class.

One type of substitution-derived variant involves substitution of one or more hypervariable region residues of the parent antibody (eg, humanized or human antibody). In general, the resulting variant(s) selected for further study should have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody, and /or must (s) have practically retained certain biological properties of the parent antibody. An example of a substitution variant is an affinity matured antibody, which can be generated, for example, using phage display-based affinity maturation technologies, such as those described herein. In general, the method is as follows: one or more HVR residues are mutated and antibody variants are displayed on phage and screened for a particular biological activity (eg, binding affinity).

Changes (eg substitutions) can be made in the HVR, eg to increase the affinity of the antibody. These changes can be made in hot (active) spots in HVR, i.e. residues encoded by codons that mutate at a high rate during somatic maturation (see, for example, Chowdhury, Methods Mol. Biol. 207, 2008, c. 179-196), and/or in residues that come into contact with antigen, followed by testing the VH or VL variant for binding affinity. Affinity maturation by building and reselecting from secondary libraries is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178, 2002, c. 1-37. In some embodiments of the affinity maturation process, diversity is introduced into the variable region genes selected for maturation using any of a wide variety of methods (eg, reduced precision PCR, strand shuffling, or site-directed mutagenesis using oligonucleotides). Then a secondary library is created. The library is then screened to identify any antibody variants with the desired affinity. Another method used to introduce diversity includes HVR-targeting approaches that randomize multiple HVR residues (eg, 4-6 residues at a time). HVR residues involved in antigen binding can be specifically identified, for example, using alanine-scanning mutagenesis or modeling. Often the targets are primarily CDR-H3 and CDRL3.

- 43 040713

In some embodiments, the substitutions, insertions, or deletions may affect one or more HVRs, as long as the changes do not significantly reduce the ability of the antibody to bind to the antigen.

For example, conservative changes can be made to the HVR (eg conservative substitutions as described herein) that do not significantly reduce binding affinity. These changes, for example, may be outside of the HVR residues involved in contacting the antigen. In some embodiments, in the VH and VL variant sequences above, each HVR is either unchanged or contains no more than one, two, or three amino acid substitutions.

A valuable method for identifying antibody residues or regions that can be mutated is the so-called alanine-scanning mutagenesis described in Cunningham and Wells, Science 244, 1989, c. 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 with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to decide whether whether this will affect the interaction of the antibody with the antigen. Additional substitutions can be introduced at amino acid positions that have been shown to be functionally sensitive to the initial substitutions. Alternatively or additionally, the crystal structure of the antigen-antibody complex is examined to identify points of contact between the antibody and antigen. These contacting residues and neighboring residues can be considered as targets or excluded from consideration as candidates for replacement. Variants can be screened to determine if they have the desired properties.

Insertions into amino acid sequences include amino- and/or carboxy-terminal fusions ranging in length from a single residue to polypeptides containing one hundred or more residues, as well as insertions of one or more amino acid residues into the sequence. An example of the result of end insertions is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion of the N- or C-terminus of the antibody with an enzyme (eg, for ADEPT (antibody-directed enzyme prodrug therapy) or a polypeptide that prolongs the serum half-life of the antibody.

b) Variants of glycosylation.

In some embodiments of the invention, the antibody of the invention is modified to increase or decrease the degree of glycosylation of the antibody. The addition or deletion of glycosylation sites in an antibody can be easily accomplished by altering the amino acid sequence in such a way that one or more glycosylation sites are created or removed.

If the antibody contains an Fc region, then you can change the carbohydrate attached to it. Native antibodies that are produced by mammalian cells typically contain a branched biantenna oligosaccharide that is typically N-linked to the Asn297 CH2 domain of the Fc region (see, for example, Wright et al., TIBTECH 15, 1997 , pp. 26-32). The oligosaccharide may include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to the GlcNAc in the stem of the biantenna oligosaccharide structure. In some embodiments, modifications to the oligosaccharide in an antibody of the invention can be made to create antibody variants with certain improved properties.

One of the embodiments of the invention are antibody variants having a carbohydrate structure in which the content of fucose attached (directly or indirectly) to the Fco region is reduced. For example, the fucose content in said Fc region may be 1 to 80%, 1 to 65%, 5 to 65%, or 20 to 40%. The fucose content is determined by calculating the average sugar chain fucose content per Asn297 relative to the sum of all glycostructures attached to Asn297 (e.g. complex, hybrid and high mannose structures), which is measured by MALDI-TOF mass spectrometry according to the method described, for example, in WO 2008/077546. Asn297 denotes an asparagine residue present at approximately position 297 in the Fc region (EU numbering of Fc region residues); however, due to minor sequence variations, the Asn297 antibody can also be located at a position about +3 amino acids upstream or downstream from position 297, i.e. between positions 294 and 300. These fucosylated variants may have improved ADCC function (see, for example, US 2003/0157108 (Presta L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd.). defucosylated or fucose-deficient antibody variants are: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; ; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; Jazakiol et al. Biol 336, 2004, pp. 1239-1249; Yamane-Ohnuki et al., Biotech Bioeng 87, 2004, pp. 614-622). Examples of cell lines that have the ability to produce defucosylated antibodies are Lec13 CHO cells deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249, 1986, c. 533-545; US 2003/0157108 (Presta L.) and WO 2004/056312 (Adams et al., see especially Example 11), and gene knockout cell lines, e.g. ., for example, Yamane-Ohnuki et al., Biotech Bioeng. 87, 2004, pp. 614-622), Kanda et al., Biotechnol. Bioeng. WO 2003/085107).

In addition, variants of antibodies with bisected oligosaccharides are described, for example, in which the bisected oligosaccharide attached to the Fc region of the antibody is bisected with GlcNAc. These antibody variants may have reduced fucosylation and/or increased ADCC function. Examples of these antibody variants are described, for example, in WO 2003/011878 (JeanMairet et al.); US No. 6602684 (Umana et al.) and US 2005/0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fco region are also proposed. These antibody variants may have increased CDC function. These antibody variants are described, for example, in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju S.); and WO 1999/22764 (Raju S.).

c) Fc region variants.

According to specific embodiments of the invention, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby creating a variant Fc region. A variant Fc region may comprise a human Fc region sequence (eg, human IgG1, IgG2, IgG3, or IgG4 Fc regions) comprising an amino acid modification (eg, substitution) at one or more amino acid positions.

Some embodiments of the invention relate to an antibody variant that has some but not all effector functions, making it a promising candidate for applications where the in vivo half-life of the antibody is important, but some effector functions (such as CDC and ADCC) are not are necessary or harmful. In vitro and/or in vivo cytotoxicity assays can be performed to confirm the reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks the ability to bind to Fc-gamma R (and therefore likely lacks ADCC activity), but retains the ability to bind to FcRn. Primary cells mediating ADCC, ie. NK cells express only Fc-gamma RIII while monocytes express Fc-gamma RI, Fc-gamma RII and Fc-gamma RIII. Data on FcR expression on hematopoietic cells are summarized in Table 1. 3 on p. 464 from Ravetch and Kinet, Annu. Rev. Immunol. 9, 1991, p. 457-492. Examples of in vitro assays for ADCC activity of a molecule of interest include, but are not limited to, those described in US No. 5500362 (see, e.g., Hellstrom et al., Proc. Nail. Acad. Sci. USA 83, 1986, 7059-7063 and Hellstrom et al., Proc. Nat'l Acad. Sci. USA 82, 1985, pp. 1499-1502); US No. 5821337 (see Bruggemann et al., J. Exp. Med. 166, 1987, c. 1351-1361). Alternatively, non-radioactive assays can be used (see, for example, non-radioactive flow cytometry-based ACTI™ cytotoxicity assay (CellTechnology, Inc. Mountain View, Calif.); and non-radioactive CytoTox 96® cytotoxicity assay (Promega, Madison). , Wisconsin.) Suitable effector cells for such assays are peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of a molecule of interest can be assessed in vivo, for example, in an animal model , for example, as described in Clynes et al., Proc. Natl. Acad. Sci. USA 95, 1998, pp. 652-656 C1q binding assays can also be performed to confirm that the antibody cannot bind C1q and therefore has no CDC activity (see, for example, WO 2006/029879 and WO 2005/100402 ELISA for assessing C1q and C3c binding). b CDC analysis (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202, 1996, p. 163-171; Cragg et al., Blood 101, 2003, pp. 1045-1052; and Cragg, Blood 103, 2004, p. 2738-2743). Determination of FcRn binding and in vivo clearance/half-life can also be carried out using methods known in the art (see, for example, Petkova et al., Int'l. Immunol. 18(12), 2006, c. 1759- 1769).

Antibodies with reduced effector function include antibodies with the replacement of one or more of the following residues of the Fc region, such as 238, 265, 269, 270, 297, 327 and 329 (see, for example, US No. 6737056). These Fc mutants include Fc mutants with substitutions in two or more of the following amino acid positions: 265, 269, 270, 297 and 327, including the so-called DANA Fc region mutant carrying the replacement of residues 265 and 297 with alanine (US No. 7332581 ).

Some variants of antibodies with increased or decreased ability to bind to FcR are described (see, for example, US No. 6737056; WO 2004/056312 and Shields and others, J. Biol. Chem. 276, 2001, c. 65916604).

In specific embodiments, the antibody variant comprises an Fc-45 040713 region with one or more amino acid substitutions that increase ADCC, such as substitutions at positions 298, 333, and/or 334 of the Fc region (EU residue numbering).

In some embodiments of the invention, changes are made in the Fc region that result in an altered (i.e., either increased or decreased) ability to bind C1q and/or complement dependent cytotoxicity (CDC), as described, for example, in US No. 6194551, WO 1999/51642 and Idusogie et al., J. Immunol. 164, 2000, p. 4178-4184.

Antibodies with extended half-life and increased ability to bind to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the embryo (Guyer et al., J. Immunol. 117, 1976, c. 587-593 and Kim et al. , J. Immunol. 24, 1994, pp. 2429-2434), described in US 2005/0014934 A1 (Hinton et al.). These antibodies contain an Fc region with one or more substitutions that increase the binding of the Fc region to FcRn. These Fc region variants include variants with substitutions of one or more of the following Fc region residues: 378, 380, 382, 413, 424 or 434, for example, substitution of residue 434 in the F region (US No. 7371826). Additional examples regarding other variants of the Fc region are also described in Duncan, Nature 322, 1988, c. 738-740; in US No. 5648260 and 5624821 and in WO 1994/29351.

d) Cysteine engineered antibody variants.

In some embodiments of the invention, it may be desirable to create cysteine-engineered antibodies, such as thioMAbs, in which one or more residues in the antibody are replaced with cysteine residues. In specific embodiments of the invention, the substituted residues are located at accessible sites on the antibody. By replacing these residues with cysteine, reactive thiol groups are placed at accessible sites on the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or drug linker moieties, to generate an immunoconjugate as indicated below in this description. In some embodiments, any one or more of the following residues may be replaced by a cysteine: V205 (Cabot numbering) light chain; A118 (EU number) of the heavy chain; and S400 (EU number) of the heavy chain Fc region. Antibodies constructed with cysteine can be generated according to the method, for example, described in US No. 7521541.

e) Derivatives of antibodies.

In some embodiments, an antibody provided herein can be further modified to contain additional non-protein fragments known in the art and readily available. Fragments suitable for antibody derivatization include, but are not limited to, water soluble polymers. Examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, copolymer ethylene/maleic anhydride, polyamino acids (either homopolymers or random copolymers) and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (eg glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde may have a manufacturing advantage 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 an antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. In general, the amount and/or type of polymers used for derivatization can be determined with regard to, but not limited to, specific properties or functions of the antibody to be improved, whether the antibody derivative is intended to be used in therapy under certain conditions, etc.

Another embodiment of the invention are conjugates of an antibody and a non-protein fragment, which can be selectively heated by exposing it to radiation. In one embodiment, the non-protein fragment is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102, 2005, pp. 11600-11605). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that do not damage normal cells, but that heat the non-protein fragment to a temperature that kills cells closest to the conjugate: antibody-non-protein fragment.

8. Variants of Fc-regions.

One of the objects of the invention is an isolated polypeptide containing a variant of the Fc region with increased Fc-gamma RIIb-binding activity. In some aspects of the invention, the polypeptide is an antibody. In some aspects of the invention, the polypeptide is an Fc-containing fusion protein. In some embodiments, the variant Fc region contains at least one amino acid residue change (e.g., substitution) from the corresponding native Fc region sequence or reference variant sequence (in some cases referred to collectively as the parent Fc region). In some embodiments, the variant Fc region of the invention has increased binding activity for the RIIb simian Fc-gamma relative to the parental Fc region. In specific embodiments, the RIIb simian Fc-gamma is Cynomolgus monkey Fc-gamma RIIb (SEQ ID NO: 223).

In some embodiments, the ratio [KD value of the parent Fc region for simian Fc-gamma RIIb)]/[KD value of the variant Fc region for simian Fc-gamma RIIb] may be 2.0 or more, 3.0 or more, 4, 0 or more, 5.0 or more, 6.0 or more, 7.0 or more, 8.0 or more, 9.0 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, or 50 or more. In other embodiments, the Fc region variant has reduced binding activity to the simian Fc-gamma RIIIa. In some embodiments, the ratio [KD value of the parent Fc region for simian Fc-gamma RIIIa]/[KD value of the variant Fc region for simian Fc-gamma RIIIa] may be 0.50 or less, 0.40 or less, 0 .30 or less, 0.20 or less, 0.10 or less, 0.09 or less, 0.08 or less, 0.07 or less, 0.06 or less, 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, or 0.01 or less. In some embodiments, the simian Fc-gamma RIIb has the sequence of SEQ ID NO: 223 (cynomolgus monkeys). In some embodiments, the simian Fc-gamma RIIIa has the sequence of SEQ ID NO: 224 (cynomolgus monkeys).

In other embodiments, the Fc region variant has increased binding activity to human Fc-gamma RIIb. In some embodiments, the ratio [KD value of parental Fc region for human Fc-gamma RIIb]/[KD value of variant Fc region for human Fc-gamma RIIb] may be 2.0 or more, 3.0 or more, 4 0 or more, 5.0 or more, 6.0 or more, 7.0 or more, 8.0 or more, 9.0 or more, 10 or more, 15 or more, 20 or more, 25 or more , 30 or more, 40 or more, or 50 or more. In other embodiments, the Fc region variant has reduced binding activity to human Fc-gamma RIIIa. In some embodiments, the ratio [KD value of parental Fc region for human Fc-gamma RIIIa]/[KD value of variant Fc region for human Fc-gamma RIIIa] may be 0.50 or less, 0.40 or less, 0 .30 or less, 0.20 or less, 0.10 or less, 0.09 or less, 0.08 or less, 0.07 or less, 0.06 or less, 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, or 0.01 or less. In some embodiments, the human Fc-gamma RIIb has the sequence SEQ ID NO: 212, 213, or 214. In some embodiments, the human Fc-gamma RIIIa has the sequence SEQ ID NO: 215, 216, 217, or 218.

In other embodiments, the Fc region variant has reduced binding activity to human Fc-gamma RIIa (H-type). In some embodiments, the ratio [KD value of parental Fc region for human Fc-gamma RIIa (H-type)]/[KD value of variant Fc region for human Fc-gamma RIIa (H-type)] may be 5.0 or less , 4.0 or less, 3.0 or less, 2.0 or less, 1.0 or less, 0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, 0 .5 or less, 0.4 or less, 0.3 or less, 0.2 or less, or 0.1 or less. In other embodiments, the Fc region variant has reduced binding activity to human Fc-gamma RIIa (R-type). In some embodiments, the ratio [KD value of the parental Fc region for human Fc-gamma RIIa (R-type)]/[KD value of the variant Fc region for human Fc-gamma RIIa (R-type)] may be 5.0 or less , 4.0 or less, 3.0 or less, 2.0 or less, 1.0 or less, 0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, 0 .5 or less, 0.4 or less, 0.3 or less, 0.2 or less, or 0.1 or less. In some embodiments, the human Fc-gamma RIIa (H-type) has the sequence SEQ ID NO: 211. In some embodiments, the human Fc-gamma RIIa (R-type) has the sequence SEQ ID NO: 210.

In some embodiments, the ratio [KD value of the parent Fc region for simian Fc-gamma RIIa]/[KD value of the variant Fc region for simian Fc-gamma RIIa] may be 2.0 or more, 3.0 or more, 4.0 5.0 or more, 6.0 or more, 7.0 or more, 8.0 or more, 9.0 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, or 50 or more. In some embodiments, the simian Fc-gamma RIIa is selected from simian Fc-gamma RIIa1 (Fc-gamma RIIa1 cynomolgus (SEQ ID NO: 220)), simian Fc-gamma RIIa2 (e.g., Fc-gamma RIIa2 cynomolgus (SEQ ID NO: 221)) and simian Fc-gamma RIIa3 (eg Fc-gamma RIIa3 cynomolgus (SEQ ID NO: 222).

In another embodiment, the KD value of the Fc region variant for the simian Fc-gamma RIIb may be 1.0x10'6M or less, ^9.0x10'7M or less, ^8.0x10'7M or less, 7.0x10' 7M or less, 6.0x10'7M or less, 5.0x10'7M or less, 4.0x10'7M or less, 3.0x10'7M or less, 2.0x10'7M or less, or 1.0x10' ⁷ M or less. In another embodiment, the KD value of the Fc region variant for the simian Fc-gamma RIIIa may be 5.0x10'7 M or more, 6.0x10'7 M or more, 7.0x10'7 M or more, 8.0x10' 7M or more, 9.0x10'7M or more, 1.0x10'6M

- 47 040713 or more, 2.0x10 ^-6 M or more, 3.0x10 ^-6 M or more, 4.0x10 ^-6 M or more, 5.0x10 ^-6 M or more, 6.0x10 ^-6 M or more , 7.0x10 ^-6 M or more, 8.0x10 ^-6 M or more, 9.0x10 ^-6 M or more, or 1.0x10 ^-5 M or more. In another embodiment, the KD value of the Fc region variant for human Fc-gamma RIIb may be 2.0 x 10 ^-6 M or less, 1.0 x ^{10 -6} M or less, 9.0 x 10 ^-7 M or less, 8.0 x 10 ^{- 7} M or less, 7.0x10 ^-7 M or less, 6.0x10 ^-7 M or less, 5.0x10 ^-7 M or less, 4.0x10 ^-7 M or less, 3.0x10 ^-7 M or less, 2.0x10 ^-7 M or less or 1.0x10 ^-7 M or less. In another embodiment, the KD value of the Fc region variant for human Fc-gamma RIIIa may be 1.0 x 10 ^-6 M or more, 2.0 x ^{10 -6} M or more, 3.0 x 10 ^-6 M or more, 4.0 x 10 ^{- 6} M or more, 5.0x10 ^-6 M or more, 6.0x10 ^-6 M or more, 7.0x10 ^-6 M or more, 8.0x10 ^-6 M or more, 9.0x10 ^-6 M or more, 1.0 x 10 ^-5 M or more, 2.0 x 10 ^-5 M or more, 3.0 x ^{10 -5} M or more, 4.0 x 10 ^-5 M or more, or 5.0 x 10 ⁵ M or more. In another embodiment, the KD value of the Fc region variant for human Fc-gamma RIIa (H-type) may be 1.0 x 10 ^-7 M or more, 2.0 x 10 ^-7 M or more, 3.0 x 10 ^-7 M or more , 4.0x10 ^-7 M or more, 5.0x10 ^-7 M or more, 6.0x10 ^-7 M or more, 7.0x10 ^-7 M or more, 8.0x10 ^-7 M or more, 9.0x10 ^{- 7} M or more, 1.0 x 10 ^-6 M or more, 2.0 x ^{10 -6} M or more, 3.0 x ^{10 -6} M or more, 4.0 x 10 ^-6 M or more, or 5.0 x 10 ^-6 M or more. In another embodiment, the KD value of the Fc region variant for human Fc-gamma RIIa (R-type) may be 2.0 x ^{10 -7} M or more, 3.0 x ^{10 -7} M or more, 4.0 x 10 ^-7 M or more , 5.0x10 ^-7 M or more, 6.0x10 ^-7 M or more, 7.0x10 ^-7 M or more, 8.0x10 ^-7 M or more, 9.0x10 ^-7 M or more, 1.0x10 ^{- 6} M or more, 2.0 x 10 ^-6 M or more, 3.0 x ^{10 -6} M or more, 4.0 x ^{10 -6} M or more, or 5.0 x 10 ^-6 M or more.

In another embodiment, the KD value of the Fc region variant for the simian Fc-gamma RIIa may be 1.0 x 10 ^-6 M or less, 9.0 x ^{10 -7} M or less, 8.0 x 10 ^-7 M or less, 7.0 x 10 ^{- 7} M or less, 6.0x10 ^-7 M or less, 5.0x10 ^-7 M or less, 4.0x10 ^-7 M or less, 3.0x10 ^-7 M or less, 2.0x10 ^-7 M or less, or 1.0x10 ^-7 M or less. In some embodiments, the simian Fc-gamma RIIa can be selected from one of the following receptors: simian Fc-gamma RIIa1, simian Fc-gamma RIIa2, and simian Fc-gamma RIIa3.

When developing a pharmaceutical product for the treatment of diseases in humans, evaluating its efficacy and safety in monkeys is important because of their biological similarity to humans. With this in mind, the pharmaceutical product to be developed should preferably be cross-reactive to both humans and monkeys in terms of target binding activity.

Fc-gamma receptors (in the context of the present description referred to as Fc-gamma receptors, Fcgamma R or FcgR) are receptors that can bind to the Fc region of monoclonal antibodies of the isotype IgG1, IgG2, IgG3 and IgG4, and above all is any a member of the family of proteins encoded by the Fc-gamma receptor genes. In humans, this family includes, but is not limited to, Fc-gamma RI (CD64), including the isoforms Fc-gamma RIa, Fc-gamma Rib, and Fc-gamma Ric; Fc-gamma RII (CD32), including isoforms Fc-gamma RIIa (including allotypes H131 (type H) and R131 (type R)), Fc-gamma RIIb (including Fc-gamma RIIb-1 and Fc-gamma RIIb-2), and Fc-gamma RIIc; and Fcgamma RIII (CD16), including the isoforms Fc-gamma RIIIa (including allotypes V158 and F158), and Fc-gamma RIIIb (including allotypes Fc-gamma RIIIb-NA1 and Fc-gamma RIIIb-NA2), and any human Fc -gamma R, Fc-gamma R isoforms or allotypes that have not yet been discovered. Fc-gamma RIIb1 and Fc-gamma RIIb2 are described as splicing variants of human Fc-gamma RIIb. In addition, a splicing variant has been described, designated Fc-gamma RIIb3 (J Exp Med, 170, 1989, c. 1369-1385). In addition to these splicing variants, the human Fc-gamma RIIb includes all splicing variants registered with the NCBI, which are NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1 and NP_003992.3. In addition, human RIIb Fc-gamma includes each of the previously described gene polymorphisms as well as RIIb Fc-gamma (Arthritis Rheum. 48, 2003, c. 3242-3252; Kono et al., Hum. Mol. Genet. 14, 2005 , cc.2881-2892 and Kyogoju et al., Arthritis Rheum 46, 2002, pp. 1242-1254), and each gene polymorphism to be described in the future.

Fc-gamma RIIa has two allotypes, one in which the amino acid at position 131 of Fcgamma RIIa is histidine (type H) and the other in which the amino acid at position 131 is changed to arginine (type R) (Warrmerdam, J. Exp. Med. 172, 1990, pp. 19-25).

Fc-gamma R includes (but is not limited to) Fc-gamma R derived from humans, mice, rats, rabbits and monkeys and may be of any organism. Mouse Fc-gamma Rs include, but are not limited to, Fc-gamma RI (CD64), Fc-gamma RII (CD32), Fc-gamma RIII (CD16), and Fc-gamma RIII-2 (CD16-2) and any murine Fc-gamma R or Fc-gamma R isoforms. Unless specifically stated otherwise, simian Fc-gamma R or a variation thereof refers to Fc-gamma RIIa1 (SEQ ID NO: 220), Fc-gamma RIIa2 (SEQ ID NO: 221), Fc-gamma RIIa3 (SEQ ID NO: 222), Fc-gamma RIIb (SEQ ID NO: 223) or Fc-gamma RIIIaS (SEQ ID NO: 224) of cynomolgus monkeys.

The polynucleotide sequence of the human Fc-gamma RI is shown in SEQ ID NO: 199 (NM_000566.3); the polynucleotide sequence of the human Fc-gamma RIIa is shown in SEQ

- 48 040713

ID NO: 200 (BC020823.1) or SEQ ID NO: 201 (NM_001136219.1); the polynucleotide sequence of human Fc-gamma RIIb is shown in SEQ ID NO: 202 (BC146678.1) or SEQ ID NO: 203 (NM_004001.3); the polynucleotide sequence of the human Fc-gamma RIIIa is presented in

SEQ ID NO: 204 (BC033678.1) or SEQ ID NO: 205 (NM_001127593.1) and the human RHIb Fc-gamma polynucleotide sequence is shown in SEQ ID NO: 206 (BC128562.1).

The amino acid sequence of the human Fc-gamma RI is shown in SEQ ID NO: 207 (NP_000557.1); the amino acid sequence of human Fc-gamma RIIa is shown in SEQ ID NO: 208 (AAH20823.1), SEQ ID NO: 209, SEQ ID NO: 210, or SEQ ID NO: 211; the amino acid sequence of human Fc-gamma RIIb is shown in SEQ ID NO: 212 (AAI46679.1), SEQ ID NO: 213 or SEQ ID NO: 214; the amino acid sequence of human RIIIa Fc-gamma is shown 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 RIIIb Fc-gamma is shown in SEQ ID NO : 219 (AAI28563.1).

The amino acid sequence of simian (cynomolgus) Fc-gamma RIIa is shown in SEQ ID NO: 220 (Fc-gamma RUa1), SEQ ID NO: 221 (Fc-gamma RIIa2) or SEQ ID NO: 222 (Fc-gamma RIIa3); the amino acid sequence of simian (cynomolgus) Fc-gamma RIIb is shown in SEQ ID NO: 223 and the amino acid sequence of simian (cynomolgus) Fc-gamma RIIIa is shown in SEQ ID NO: 224.

One subject of the invention is polypeptides that contain variants of Fc regions with increased Fc-gamma RIIb-binding activity compared to the corresponding Fc-gamma RIIb-binding reference polypeptide. In other aspects of the invention, the polypeptide of the invention contains at least one amino acid change 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 some embodiments, the RIIb Fc-gamma has the cynomolgus Fc-gamma RIIb sequence (SEQ ID NO: 223). In some embodiments, the RIIb Fc-gamma has the sequence of a human RIIb Fc-gamma (eg, SEQ ID NO: 212, 213, or 214).

One of the objects of the invention are polypeptides containing variants of Fc regions with increased Fc-gamma RIIb-binding activity, which contain at least two amino acid changes, including: (a) one amino acid change at position 236 and (b) at least one amino acid change at at least one position selected from the group consisting of 231, 232, 233, 234, 235, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327 , 328, 330, 331, 332, 334, and 396, according to EU numbering. In some embodiments, the Fcgamma RIIb has the sequence Fc-gamma RIIb of cynomolgus monkeys (SEQ ID NO: 223). In some embodiments, the RIIb Fc-gamma has the sequence of a human RIIb Fc-gamma (eg, SEQ ID NO: 212, 213, or 214).

One of the objects of the invention are polypeptides containing a variant of the Fc region with increased Fc-gamma RIIb-binding activity, which contains an amino acid change at position 236 according to the EU numbering.

One of the objects of the invention are polypeptides containing a variant of the Fc region with increased Fc-gamma RIIb-binding activity, which contains at least two amino acid changes, including (a) one amino acid change at position 236 and (b) at least one amino acid a change in at least one position selected from the group consisting of 231, 232, 235, 239, 268, 295, 298, 326, 330 and 396, according to the EU numbering. In another embodiment, the Fc region variant contains an amino acid change at at least one position selected from the group consisting of 231, 232, 235, 239, 268, 295, 298, 326, 330, and 396, according to EU numbering. In a further embodiment of the invention, the variant Fc region contains an amino acid change at at least one position selected from the group consisting of 268, 295, 326 and 330 according to EU numbering. In some embodiments, the Fcgamma RIIb has the cynomolgus monkey sequence Fc-gamma RIIb of the cynomolgus monkey (SEQ ID NO: 223). In some embodiments, the RIIb Fc-gamma has the sequence of a human RIIb Fc-gamma (eg, SEQ ID NO: 212, 213, or 214).

Another object of the invention are polypeptides containing variants of Fc regions with increased Fc-gamma RIIb-binding activity, which contain amino acid changes in one of the following positions (1)-(37): (1) positions 231, 236, 239, 268 and 330; (2) regulations 231, 236, 239, 268, 295 and 330; (3) provisions 231, 236, 268 and 330; (4) provisions 231, 236, 268, 295 and 330; (5) provisions 232, 236, 239, 268, 295 and 330; (6) provisions 232, 236, 268, 295 and 330; (7) provisions 232, 236, 268 and 330; (8) provisions 235, 236, 268, 295, 326 and 330; (9) provisions 235, 236, 268, 295 and 330; (10) provisions 235, 236, 268 and 330; (11) provisions 235, 236, 268, 330 and 396; (12) provisions 235, 236, 268 and 396; (13) provisions 236, 239, 268, 295, 298 and 330; (14) regulations 236, 239, 268, 295, 326 and 330; (15) provisions 236, 239, 268, 295 and 330; (16) provisions 236, 239, 268, 298 and 330; (17) provisions 236, 239, 268, 326 and 330; (18) provisions 236, 239, 268 and 330; (19) provisions 236, 239, 268, 330 and 396; (20) provisions 236, 239, 268 and 396; (21) provisions 236 and 268; (22) provisions 236, 268 and 295; (23) provisions 236, 268, 295, 298 and 330;

- 49 040713 (24) regulations 236, 268, 295, 326 and 330; (25) provisions 236, 268, 295, 326, 330 and 396; (26) provisions 236, 268, 295 and 330; (27) provisions 236, 268, 295, 330 and 396; (28) provisions 236, 268, 298 and 330; (29) provisions 236, 268, 298 and 396; (30) provisions 236, 268, 326 and 330; (31) provisions 236, 268, 326, 330 and 396; (32) provisions 236, 268 and 330; (33) provisions 236, 268, 330 and 396; (34) provisions 236, 268 and 396; (35) provisions 236 and 295; (36) provisions 236, 330 and 396; and (37) provisions 236 and 396, according to the EU numbering. In some embodiments, the RIIb Fc-gamma has the cynomolgus Fc-gamma RIIb sequence (SEQ ID NO: 223). In some embodiments, the RIIb Fc-gamma has the sequence of a human RIIb Fc-gamma (eg, SEQ ID NO: 212, 213, or 214).

In a further embodiment of the invention, the Fc region variant with enhanced Fc-gamma RIIb 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; (6) 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; (m) Ala, His, Leu at position 267; (m) Asp, Glu at position 268; (o) Asp, Glu, Gly at position 271; (p) Leu at position 295; (p) Leu at position 298; (c) Glu, Phe, Ile, Leu at position 325; (r) Thr at position 326; (y) Ile, Asn at position 327; (f) Thr at position 328; (x) Lys, Arg at position 330; (c) Glu at position 331; (h) Asp at position 332; (w) 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 some embodiments, the RIIb Fc-gamma has the cynomolgus Fc-gamma RIIb sequence (SEQ ID NO: 223). In some embodiments, the RIIb Fc-gamma has the sequence of a human RIIb Fc-gamma (eg, SEQ ID NO: 212, 213, or 214).

In a further embodiment of the invention, the Fc region variant with increased Fc-gamma RIIb binding activity comprises at least one amino acid change (eg, 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; (e) 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 of the invention, a variant Fc region with increased Fc-gamma RIIb binding activity contains amino acid changes (e.g. substitutions): Asn at position 236, Glu at position 268, Lys at position 330 and Met at position 396 according to EU numbering . In a further embodiment of the invention, a variant Fc region with increased Fc-gamma RIIb binding activity contains amino acid changes (eg substitutions): Asn at position 236, Asp at position 268 and Lys at position 330 according to EU numbering. In a further embodiment of the invention, a variant Fc region with increased Fc-gamma RIIb-binding activity contains amino acid changes (e.g., substitutions): Asn at position 236, Asp at position 268, Leu at position 295, and Lys at position 330 according to EU numbering . In a further embodiment of the invention, a variant Fc region with increased Fc-gamma RIIb binding activity contains amino acid changes (eg substitutions): Thr at position 236, Asp at position 268 and Lys at position 330 according to EU numbering. In a further embodiment of the invention, a variant Fc region with increased Fc-gamma RIIb binding activity contains amino acid changes (e.g., substitutions): Asn at position 236, Asp at position 268, Leu at position 295, Thr at position 326, and Lys at position 330 according to EU numbers. In a further embodiment of the invention, a variant of the Fc region with increased Fc-gamma RIIb-binding activity contains amino acid changes (e.g. substitutions): Trp at position 235, Asn at position 236, Asp at position 268, Leu at position 295, Thr at position 326, and Lys at position 330 according to the EU numbering.

One of the objects of the invention are polypeptides containing variants of Fc regions with increased Fc-gamma RIIb-binding activity, which contain an amino acid change at position 238 according to the EU numbering.

One of the objects of the invention are polypeptides containing variants of Fc regions with increased Fc-gamma RIIb-binding activity, which contain at least one amino acid change in at least one position selected from the group consisting of 234, 238, 250, 264 , 267, 307, and 330, according to the EU numbering. In other embodiments, the polypeptide contains at least one amino acid change at at least one position selected from the group consisting of 234, 250, 264, 267, 307, and 330, according to EU numbering. In some embodiments, the RIIb Fc-gamma has the cynomolgus Fc-gamma RIIb sequence (SEQ ID NO: 223). In some embodiments, Fcgamma RIIb has the sequence of human Fcgamma RIIb (eg, SEQ ID NO: 212, 213, or 214).

- 50 040713

Another object of the invention are polypeptides containing variants of Fc regions with increased Fc-gamma RIIb-binding activity, which contain amino acid changes in one of the following positions (1)-(9): (1) positions 234, 238, 250, 307 and 330; (2) provisions 234, 238, 250, 264, 307 and 330; (3) provisions 234, 238, 250, 264, 267, 307 and 330; (4) provisions 234, 238, 250, 267, 307 and 330; (5) provisions 238, 250, 264, 307 and 330; (6) provisions 238, 250, 264, 267, 307 and 330; (7) provisions 238, 250, 267, 307 and 330; (8) provisions 238, 250 and 307; and (9) EU provisions 238, 250, 307 and 330. In some embodiments, the RIIb Fc-gamma has the cynomolgus Fc-gamma RIIb sequence (SEQ ID NO: 223). In some embodiments, the RIIb Fc-gamma has the sequence of a human RIIb Fc-gamma (eg, SEQ ID NO: 212, 213, or 214).

In a further embodiment of the invention, the Fc region variant with increased Fc-gamma RIIb binding activity comprises at least one amino acid change (eg, substitution) selected from the group consisting of (a) Tyr at position 234; (b) Asp at position 238; (c) Val at position 250, (d) Ile at position 264; (e) Ala at position 267; (f) Pro at position 307 and (g) Lys at position 330; according to EU numbering. In another embodiment of the invention, a variant of the Fc region with increased Fc-gamma RIIb-binding activity contains amino acid changes (eg substitutions): Asp at position 238 according to EU numbering. In another embodiment of the invention, a variant Fc region with increased Fc-gamma RIIb-binding activity contains amino acid changes (eg substitutions): Asp at position 238, Val at position 250, and Pro at position 307; according to EU numbering. In another embodiment of the invention, a variant Fc region with increased Fc-gamma RIIb-binding activity contains amino acid changes (eg, substitutions): Asp at position 238, Val at position 250, Pro at position 307, and Lys at position 330; according to EU numbering. In another embodiment of the invention, a variant Fc region with increased Fc-gamma RIIb binding activity contains amino acid changes (e.g., substitutions): Asp at position 238, Val at position 250, Ile at position 264, Pro at position 307, and Lys at position 330; according to EU numbering. In another embodiment, a variant Fc region with increased Fc-gamma RIIb binding activity contains amino acid changes (e.g., substitutions): Asp at position 238, Val at position 250, Ala at position 267, Pro at position 307, and Lys at position 307. position 330; according to EU numbering. In another embodiment, a variant Fc region with increased Fc-gamma RIIb-binding activity contains amino acid changes (eg, substitutions): Tyr at position 234, Asp at position 238, Val at position 250, Pro at position 307, and Lys at position 330; according to EU numbering. In another embodiment of the invention, a variant Fc region with increased Fc-gamma RIIb-binding activity contains amino acid changes (e.g. substitutions): Tyr at position 234, Asp at position 238, Val at position 250, Ala at position 267, Pro at position 307 and Lys at position 330; according to EU numbering. In another embodiment of the invention, a variant of the Fc region with increased Fc-gamma RIIb-binding activity contains amino acid changes (e.g. substitutions): Asp at position 238, Val at position 250, Ile at position 264, Ala at position 267, Pro at position 307 and Lys at position 330; according to EU numbering. In another embodiment, a variant Fc region with increased Fcgamma RIIb binding activity contains amino acid changes (e.g., substitutions): Tyr at position 234, Asp at position 238, Val at position 250, Ile at position 264, Pro at position 307, and Lys at position 330; according to EU numbering. In another embodiment of the invention, a variant of the Fc region with increased Fc-gamma RIIb-binding activity contains amino acid changes (e.g., substitutions): Tyr at position 234, Asp at position 238, Val at position 250, Ile at position 264, Ala at position 267, Pro at position 307 and Lys at position 330; according to EU numbering. In some embodiments, the RIIb Fc-gamma has the cynomolgus Fc-gamma RIIb sequence (SEQ ID NO: 223). In some embodiments, Fcgamma RIIb has the sequence of human Fcgamma RIIb (eg, SEQ ID NO: 212, 213, or 214).

Another object of the invention are selected polypeptides that contain variants of Fc regions with increased isoelectric point (pI). In some embodiments, the Fc region variant described herein contains at least two amino acid changes in the parent Fc region. In some embodiments, each of the amino acid changes increases the isoelectric point (pI) of the variant Fc region relative to the parent Fc region. These polypeptides are based on the knowledge that elimination of an antigen from plasma can be accelerated by an antibody whose pI is increased by modifying at least two amino acid residues, for example when the antibody is administered in vivo.

According to the present invention pI can be either theoretical or experimentally determined pI. The value of pI can be determined, for example, using isoelectric focusing, known to experts in this field. The theoretical pI value can be calculated, for example, using gene and amino acid sequence analysis software (Genetyx et al.).

In one of the embodiments of the invention, the pI value can be increased, for example, by at least 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 or more, at least 0.6, 0.7, 0.8, 0.9 or more, at least 1.0, 1.1, 1.2, 1.3, 1.4, 1, 5 or more or at least 1.6, 1.7, 1.8, 1.9,

2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0 or more compared to the version before modification.

In some embodiments, the pI-increasing amino acid may be displayed on the surface of a variant Fc region. According to the present invention, the amino acid that can be exposed on the surface, as a rule, is an amino acid residue localized on the surface of the polypeptide, forming a variant Fc region. An amino acid residue localized on the surface of a polypeptide means an amino acid residue whose side chain may be in contact with solvent molecules (most commonly water molecules). However, the side chain need not be wholly in contact with the solvent molecules, and even if only part of the side chain is in contact with the solvent molecules, the amino acid is considered to be an amino acid residue localized on the surface. Amino acid residues localized on the surface of a polypeptide also include amino acid residues localized near the surface, and which as a result can affect the electrical charge of another amino acid residue, even if the side chain of which is only partially in contact with solvent molecules. Those skilled in the art can create a homologous model of a polypeptide, for example, using commercially available software. Alternatively, methods known to those skilled in the art can be used, such as, for example, X-ray crystallography. The amino acid residues that can be exposed on the surface are determined, for example, using the coordinates of a three-dimensional model using a computer program such as the InsightII program (Accelrys). Sites that can be exposed on a surface can be determined using algorithms known in the art (for example, see Lee and Richards, J. Mol. Biol. 55, 1971, pp. 379-400; Connolly, J. Appl. Cryst 16, 1983, pp. 548-558). Sites that can be exposed on a surface can be determined with protein modeling software and 3D structure information. Suitable software for this purpose includes, for example, SYBYL Biopolymer Module software (Tripos Associates). When the algorithm requires a user-defined size parameter, the sample size used in the calculation can be specified as a radius of approximately 1.4 A or less. In addition, methods for determining areas that can be exposed on a surface are described in Pacios (Comput. Chem. 18(4), 1994, c. 377-386; J. Mol. Model. 1, 1995, c. 46-53) . Based on the above information, suitable amino acid residues located on the surface of the polypeptide that form the variant Fc region can be selected.

In some embodiments, the polypeptide contains both a variant Fc region and an antigen-binding domain. In other embodiments, the antigen is a soluble antigen. In one embodiment, the antigen is present in the biological fluids (eg, plasma, interstitial fluid, lymphatic fluid, ascitic fluid, and pleural fluid) of individuals. The antigen may also be a membrane antigen.

In other embodiments of the invention, the antigen-binding activity of the antigen-binding domain varies depending on the level of ion concentration. In one embodiment, the ion concentration is not particularly limited, and means a hydrogen ion concentration (pH) or a metal ion concentration. In the context of the present description, metal ions are ions of group I elements excluding hydrogen, such as alkali metals and copper group elements, group II elements, such as alkaline earth metals and zinc group elements, group III elements excluding boron, group IV elements excluding carbon and silicon, group VIII elements such as iron group and platinum group elements, elements belonging to subgroup A of groups V, VI and VII, and metal elements such as antimony, bismuth and polonium. According to the present invention, metal ions include, for example, calcium ions as described in WO 2012/073992 and WO 2013/125667. In one of the embodiments of the invention, the level of ion concentration may be influencing the biological behavior of the antigen-binding domain level between low ion concentration and high ion concentration. In addition, the concept of antigen-binding activity of antigen-binding domains varies depending on the level of ion concentration means that the antigen-binding activity of antigen-binding domains changes depending on whether the concentration of ions is low or high (in this case, the antigen-binding domain is referred to as ion concentration-dependent antigen-binding domain). The antigen-binding activity of antigen-binding domains at a high level of ion concentration may be higher (stronger) or lower (weaker) than at a low level of ion concentration. In one embodiment of the invention, ion concentration dependent antigen binding domains (such as pH dependent antigen binding domains or calcium ion concentration dependent antigen binding domains) can be obtained using known methods, such as those described in WO 2009/125825, WO 2012/073992 and WO 2013/046722.

According to the present invention, the antigen-binding activity of an antigen-binding domain at a high level of calcium ion concentration may be higher than at a low level of calcium ion concentration. The high calcium ion concentration is not limited to specific values, but may be a concentration selected from the range of 100 µM to 10 mM, 200 µM to 5 mM, 400 µM to 3 mM, 200 µM to 2 mM, 400 µM to 1 mm or from 500 μm to 2.5 mm, which is preferred due to the proximity to the concentration of calcium ions in plasma (blood) in vivo. In addition, the low calcium ion concentration is not limited to specific values, but may be a concentration selected from the range of 0.1 to 30, 0.2 to 20, 0.5 to 10, 1 to 5, or 2 to 4 μM, which is preferred due to its proximity to the concentration of calcium ions in early endosomes in vivo.

In one embodiment, the ratio between antigen-binding activities at a low calcium ion level and a high calcium ion level is not limited to specific values, but the ratio of the dissociation constant (KD) at a low calcium ion level and KD at a high calcium ion level i.e. e. KD (low calcium ion level)/KD (high calcium ion level) is 2 or more, 10 or more, or 40 or more. The upper limit of the ratio may be 400, 1000 or 10000 if said antigen-binding domain can be obtained using techniques known to those skilled in the art. Alternatively, for example, the dissociation reaction rate constant (kd) can be used instead of KD. In this case, the ratio of kd at a low level of calcium ion concentration and kd at a high level of calcium ion concentration, i.e. kd (low calcium ion level)/kd (high calcium ion level) 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 if said antigen-binding domain can be obtained using techniques known to those skilled in the art.

According to the present invention, the antigen-binding activity of an antigen-binding domain at a low concentration of hydrogen ions (at neutral pH) can be higher than at a high concentration of hydrogen ions (at acidic pH). The acidic pH may be, for example, a pH selected from a pH range of 4.0 to 6.5, 4.5 to 6.5, 5.0 to 6.5, or 5.5 to 6.5, which is preferred due to proximity to in vivo pH in early endosomes. The acidic pH can also be, for example, pH 5.8 or pH 6.0. In specific embodiments, the acidic pH is pH 5.8. In addition, the neutral pH may be, for example, a pH selected from a pH range of 6.7 to 10.0, 6.7 to 9.5, 7.0 to 9.0, or 7.0 to 8 0, which is preferred due to its proximity to the in vivo pH in plasma (blood). Neutral pH can also be, for example, pH 7.4 or pH 7.0. In a specific embodiment of the invention, the neutral pH is pH 7.4.

In one embodiment of the invention, the ratio between antigen-binding activities under acidic pH conditions and under neutral pH conditions is not limited to specific values, but the ratio of dissociation constant (KD) under acidic pH conditions and KD under neutral pH conditions, i.e. KD (acidic pH conditions)/KD (neutral pH conditions) is 2 or more, 10 or more, or 40 or more. The upper limit of the ratio may be 400, 1000 or 10000 if said antigen-binding domain can be obtained using techniques known to those skilled in the art. Alternatively, for example, the dissociation reaction rate constant (kd) can be used instead of KD. In this case, the ratio of kd under acidic pH conditions and kd under neutral pH conditions, i.e. kd (acidic pH conditions)/kd (neutral pH conditions) is 2 or more, 5 or more, 10 or more, or 30 or more. The upper limit of the ratio may be 50, 100 or 200 if said antigen-binding domain can be obtained using techniques known to those skilled in the art.

In one embodiment, for example, at least one amino acid residue is replaced with an amino acid residue with a side chain pKa of 4.0-8.0 and/or at least one amino acid residue with a side chain pKa of 4.0-8.0 is inserted into an antigen binding domain as described in WO 2009/125825. An amino acid may be substituted and/or inserted at any site if the antigen-binding activity of the antigen-binding domain becomes weaker under acidic pH conditions compared to neutral pH conditions relative to the variant prior to 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. Those skilled in the art can appropriately determine the amount of amino acids substituted or inserted; and this number can be 1 or more. Amino acids with a side chain pKa of 4.0-8.0 can be used to alter the antigen-binding activity of an antigen-binding domain depending on the level of hydrogen ion concentration. These amino acids include, for example, naturally occurring amino acids such as His (H) and Glu (E), and non-naturally occurring amino acids such as histidine analogs (US 2009/0035836), m-NOrTyr (pKa 7, 45), 3.5-Br ₂ -Tyr (pKa 7.21), and 3.5-I ₂ -Tyr (pKa 7.38) (Heyl et al.,

- 53 040713

Bioorg. Med. Chem. 11(17), 2003, p. 3761-3768). It is also possible to use amino acids with a side chain pKa of 6.0-7.0, which include, for example, His (H).

In another embodiment, the preferred antigen binding domains for the elevated pI variant Fc region are known and can be obtained using the methods described in Japanese Patent Applications JP2015-021371 and JP2015-185254.

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

In other embodiments, the elevated pI variant Fc region contains at least two amino acid changes at at least two positions selected from the group consisting of 311, 341, 343, 384, 399, 400, 401, 402, and 413 , according to EU-numbering.

Another object of the invention are polypeptides containing variants of Fc regions with increased pI, which contain amino acid changes in one of the following positions (1)-(10): (1) positions 311 and 341; (2) provisions 311 and 343; (3) provisions 311, 343 and 413; (4) provisions 311, 384 and 413; (5) provisions 311 and 399; (6) provisions 311 and 401; (7) provisions 311 and 413; (8) provisions 400 and 413; (9) provisions 401 and 413; and (10) provisions 402 and 413; according to EU numbering.

The method for reducing the pI of a protein involves, for example, reducing the amount of amino acids with a negatively charged side chain (for example, aspartic acid and glutamic acid) and/or increasing the amount of amino acids with a positively charged side chain (for example, arginine, lysine and histidine) under neutral pH conditions. Amino acids with a negatively charged side chain have a negative charge corresponding to -1 under pH conditions, which is well above the pKa value of the side chain, according to theory well known to those skilled in the art. For example, the theoretical pKa value for the aspartic acid side chain is 3.9, and the side chain has a negative charge corresponding to -1 under neutral pH conditions (eg, in a pH 7.0 solution). Conversely, amino acids with a positively charged side chain have a positive charge corresponding to +1 under pH conditions that are sufficiently lower than the pKa value of their side chain. For example, the theoretical pKa value for the arginine side chain is 12.5, and the side chain has a positive charge corresponding to +1 under neutral pH conditions (eg, pH 7.0 solution). Moreover, as is known, amino acids, the side chains of which do not have a charge under neutral pH conditions (for example, in a solution with pH 7.0), include 15 types of naturally occurring amino acids, i.e. alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan and tyrosine. It goes without saying that the amino acids that can be used to increase pI may be non-naturally occurring amino acids.

Based on the above, using the method of increasing the pI of a protein under neutral pH conditions (for example, in a solution with pH 7.0), it is possible to change the charge of the protein of interest by +1, for example, by replacing amino acids with uncharged side chains of aspartic acid or glutamic acid (whose side chain has a negative charge of -1) in the amino acid sequence of a protein. In addition, a +1 charge change can be effected in a protein, for example, by substituting arginine or lysine (whose side chain has a +1 positive charge) for amino acids with uncharged side chains. In addition, a +2 charge change of the protein can be carried out simultaneously by substituting arginine or lysine (whose side chain has a +1 positive charge) 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 uncharged side chains and/or preferably amino acids with positively charged side chains can be added or inserted into the amino acid sequence of the protein, or amino acids with uncharged side chains and/or preferably amino acids with negatively charged side chains present in the amino acid sequence of a protein can be removed by deletion. Obviously, for example, the N-terminal and C-terminal amino acid residues of a protein have the charge inherent in their main chain (NH3+ amino groups at the N-terminus and COO ^- carbonyl group at the C-terminus) in addition to the charges inherent in their side chain. Thus, the pI of a protein can be increased by certain addition, deletion, substitution or insertion in the functional groups of the main chain.

Substituting an amino acid to increase pI includes, for example, substituting an amino acid with an uncharged side chain for an amino acid with a negatively charged side chain, substituting an amino acid with a positively charged side chain for an amino acid with an uncharged side chain, and substituting an amino acid with a positively charged side chain for an amino acid with a negatively charged side chain in an amino acid. the sequence of the parent Fc region, which is carried out individually or in appropriate combination.

Insertion or addition of an amino acid to increase pI includes, for example, insertion or addition of an amino acid with an uncharged side chain and/or insertion or addition of an amino acid with a positively charged side chain into the amino acid sequence of the parent Fc-54 040713 region, which is carried out individually or in an appropriate combination.

Deletion of an amino acid to increase pI includes, for example, deletion of an amino acid with an uncharged side chain and/or deletion of an amino acid with a negatively charged side chain in the amino acid sequence of the parent Fc region, which is carried out individually or in an appropriate combination.

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

Another object of the invention are isolated polypeptides containing variants of Fc regions with increased Fc-gamma RIIb-binding activity and increased pI. In some embodiments, the Fc region variant described herein contains at least two amino acid changes in the parent Fc region.

One of the objects of the invention are polypeptides containing variants of Fc regions with increased Fc-gamma RIIb-binding activity and increased pI, which contain at least three amino acid changes, which are: (a) at least one amino acid change in 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 changes 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.

One of the objects of the invention are polypeptides containing variants of Fc regions with increased Fc-gamma RIIb-binding activity and increased pI, which contain at least three amino acid changes, which are: (a) at least one amino acid change in 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 changes of at least in two positions selected from the group consisting of 311, 341, 343, 384, 399, 400, 401, 402 and 413, according to EU numbering.

Another object of the invention are polypeptides containing variants of Fc regions with increased Fc-gamma RIIb-binding activity and increased pI, which contain amino acid changes in one of the following positions (1)-(9): (1) positions 235, 236, 268 , 295, 311, 326, 330 and 343; (2) regulations 236, 268, 295, 311, 326, 330 and 343; (3) provisions 236, 268, 295, 311, 330 and 413; (4) regulations 236, 268, 311, 330, 396 and 399; (5) provisions 236, 268, 311, 330 and 343; (6) provisions 236, 268, 311, 330, 343 and 413; (7) provisions 236, 268, 311, 330, 384 and 413; (8) provisions 236, 268, 311, 330 and 413; and (9) regulations 236, 268, 330, 396, 400 and 413; according to EU numbering. In some embodiments, the RIIb Fc-gamma has the cynomolgus Fc-gamma RIIb sequence (SEQ ID NO: 223). In some embodiments, the RIIb Fc-gamma has the sequence of a human RIIb Fc-gamma (eg, SEQ ID NO: 212, 213, or 214).

One of the objects of the invention are polypeptides containing variants of Fc regions with increased Fc-gamma RIIb-binding activity and increased pI, which contain at least three amino acid changes, which are: (a) at least one amino acid change in 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 changes 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, 4θ1, 402, 413, 420, 422, and 431, according to EU numbering. In other embodiments, the polypeptides contain at least two amino acid changes 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 some embodiments, the RIIb Fc-gamma has the cynomolgus Fc-gamma RIIb sequence (SEQ ID NO: 223). In some embodiments, the RIIb Fc-gamma has the sequence of a human RIIb Fc-gamma (eg, SEQ ID NO: 212, 213, or 214).

Another object of the invention are polypeptides containing variants of Fc regions with increased Fc-gamma RIIb-binding activity and increased pI, which contain amino acid changes in one of the following positions (1)-(16): (1) positions 234, 238, 250 , 264, 307, 311, 330 and 343; (2) provisions 234, 238, 250, 264, 307, 311, 330 and 413; (3) provisions 234, 238, 250, 264, 267, 307, 311,

330 and 343; (4) provisions 234, 238, 250, 264, 267, 307, 311, 330 and 413; (5) provisions 234, 238, 250,267,

307, 311, 330 and 343; (6) regulations 234, 238, 250, 267, 307, 311, 330 and 413; (7) provisions 234, 238,250,

307, 311, 330 and 343; (8) provisions 234, 238, 250, 307, 311, 330 and 413; (9) provisions 238, 250, 264,267,

- 55 040713

307, 311, 330 and 343; (10) provisions 238, 250, 264, 267, 307, 311, 330 and 413; (11) provisions 238, 250, 264,

307, 311, 330 and 343; (12) regulations 238, 250, 264, 307, 311, 330 and 413; (13) provisions 238, 250, 267, 307,

311, 330 and 343; (14) provisions 238, 250, 267, 307, 311, 330 and 413; (15) provisions 238, 250, 307, 311, 330 and

343; and (16) EU provisions 238, 250, 307, 311, 330 and 413.

In a further embodiment of the invention, the Fc region variant contains amino acid changes selected from any of the single changes, combinations of single changes, or combinations of changes listed in Table 1. 14-30.

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

Another embodiment of the invention is a polypeptide containing one of the amino acid sequences of SEQ ID NO: 229-381.

In the context of the present description, the term parent Fc-region refers to the Fc-region before the introduction of the amino acid(s) change(s) specified(s) in the present description. Preferred examples of a parental Fc region are Fc regions from native antibodies. Antibodies include, for example, IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4) and IgM or the like. The antibodies may be of human or simian origin (eg cynomolgus, rhesus monkey, marmoset, chimpanzee or baboon). Native antibodies may also include naturally occurring mutations. A variety of allotypic IgG sequences associated with genetic polymorphisms are described in Sequences proteins of immunological interest, NIH Publication No. 913242, and any of them can be used in the present invention. In particular, in the case of human IgG1, the amino acid sequence at positions 356-358 (EU numbering) can be either DEL or EEM. Preferred examples of the parental Fc region include Fc regions from the heavy chain constant region of human IgG1 (SEQ ID NO: 195), human IgG2 (SEQ ID NO: 196), human IgG3 (SEQ ID NO: 197), and human IgG4 (SEQ ID NO: 198). Another preferred example of a parental Fc region is the Fc region from the SGI heavy chain constant region (SEQ ID NO: 9). In addition, the parent Fc region may be an Fc region obtained by adding an amino acid(s) change(s) other than the amino acid(s) change(s) specified herein for the Fc region of native antibody.

In addition, amino acid changes that are made for other purpose(s) can be combined in the Fc region variant described herein. For example, amino acid substitutions can be added that increase FcRn binding activity (Hinton et al., J. Immunol. 176(1), 2006, c. 346-356; Dall'Acqua et al., J. Biol. Chem. 281( 33), 2006, pp. 23514-23524; Petkova et al., Intl. Immunol. 18(12), 2006, pp. 1759-1769; Zalevsky et al., Nat. Biotechnol. 28(2), 2010, p. 157-159; WO 2006/019447; WO 2006/053301; and WO 2009/086320), and amino acid substitutions that increase antibody heterogeneity or stability (WO 2009/041613). Alternatively, polypeptides having the ability to enhance antigen clearance as described in WO 2011/122011, WO 2012/132067, WO 2013/046704 or WO 2013/180201, polypeptides having the ability to specifically bind to a target tissue as described in WO 2013 /180200, polypeptides having the ability to re-bind to multiple antigen molecules as described in WO 2009/125825, WO 2012/073992 or WO 2013/047752 can be combined with the Fc region variant described herein. Alternatively, in order to confer the ability to bind to other antigens, the amino acid changes described in EP 1752471 and EP 1772465 can be combined into the CH3 variant of the Fc region specified in the present description. Alternatively, amino acid changes that reduce the pI of the constant region (WO 2012/016227) in the Fc region variant described herein can be combined to increase plasma retention. Alternatively, amino acid changes that increase the pI of the constant region (WO 2014/145159), in the Fc region variant described herein, can be combined to increase cellular uptake. Alternatively, amino acid changes that increase the pI of the constant region (Japanese Patent Applications JP2015-021371 and JP2015-185254) in the Fc region variant described herein can be combined to enhance elimination of the target molecule from plasma. In one of the embodiments of the invention, the specified change may include, for example, the replacement of at least one position selected from the group consisting of 311, 343, 384, 399, 400 and 413 according to the EU numbering. In another embodiment of the invention, said substitution may be an amino acid substitution with Lys or Arg at each position.

Amino acid changes resulting in increased ability to bind to human FcRn at acidic pH can also be combined in the Fc region variant described herein. In particular, such changes may include, for example, a change to Leu Met at position 428 and a change

- 56 040713 on Ser Asn at position 434, according to EU numbering (Zalevsky et al., Nat. Biotechnol. 28, 2010, p. 157159); substitution for Ala Asn at position 434 (Deng et al., Metab. Dispos. 38(4), 2010, c. 600-605); a Tyr Met substitution at position 252, a Thr Ser substitution at position 254, and a Glu Thr substitution at position 256 (Dall'Acqua et al., J. Biol. Chem. 281, 2006, c. 23514-23524); a change to Gln Thr at position 250 and a change to Leu Met at position 428 (Hinton et al., J. Immunol. 176(1), 2006, c. 346-356); change to His Asn at position 434 (Zheng et al., Clin. Pharmacol. Ther. 89(2), 2011, c. 283-290), and the changes 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 2004/03WO 2092 Such changes may include, for example, at least one change selected from the group consisting of a change to Leu Met at position 428, a change to Ala Asn at position 434, and a change to Thr Tyr at position 436. These changes also include a change to Arg Gln at position 438 and/or a change to Glu Ser at position 440 (Japanese patent applications JP2015-021371 and JP2015185254).

Two or more polypeptides containing an Fc region variant as described herein may be included in a single molecule in which the two Fc region variant polypeptides are in an association that closely resembles that of an antibody. The type of antibody is not limited, and IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4) and IgM or the like can be used.

Two associated polypeptides containing variants of the Fc regions may be polypeptides that contain variants of the Fc regions into which the same amino acid change(s) has been introduced (hereinafter referred to as homologous variants in the context of the present description). Fc regions), or polypeptides that contain variants of Fc regions into which different amino acid(s) change(s) have been introduced, or alternatively, polypeptides that contain variants of Fc regions, characterized in that the amino acid ( s) the change(s) is(are) introduced into only one of the Fc regions ((hereinafter referred to as heterologous polypeptides of Fc region variants). One of the preferred amino acid changes is a change in the loop structure from position 233 to 239 ( EU numbering) in the CH2 domain of the Fc region which is involved in binding to Fc-gamma RIIb and Fc-gamma RIIa Preferably a change that increases Fc-gamma RIIb-fr binding activity and/or selectivity is introduced into the loop structure of the CH2 domain of one of the Fc regions, and another change is introduced into the loop structure of the CH2 domain of the other Fc region, which destabilizes c. Examples of amino acid changes that can destabilize the loop structure of the CH2 domain may be the substitution of at least one amino acid selected from amino acids at positions 235, 236, 237, 238 and 239 with a different amino acid. In particular, destabilization can be carried out, for example, by changing the amino acid at position 235 to Asp, Gln, Glu or Thr, changing the amino acid at position 236 to Asn, changing the amino acid at position 237 to Phe or Trp, changing the amino acid at position 238 to Glu, Gly or Asn and amino acid changes at position 239 to Asp or Glu, according to EU numbering.

For the association of heterologous polypeptides containing variant Fc regions, the technique of suppressing unwanted association of homologous polypeptides containing variant Fc regions by introducing electrostatic repulsion into the interface of the CH2 or CH3 domain of the Fc region described in WO 2006/106905 can be used.

Examples of amino acid residues contacting at the interface of the CH2 or CH3 domain of the Fc region include residue at position 356 (EU numbering), residue at position 439 (EU numbering), residue at position 357 (EU numbering), residue at position 370 ( EU numbering), the remainder at position 399 (EU numbering), and the remainder at position 409 (EU numbering) in the CH3 domain.

More specifically, for example, it is possible to obtain an Fc region in which 1-3 pairs of amino acid residues selected from (1) to (3) below have the same charge: (1) amino acid residues at positions 356 and 439 (EU -numbering) in the CH3 domain; (2) amino acid residues at positions 357 and 370 (EU numbering) in the CH3 domain; and (3) amino acid residues at positions 399 and 409 (EU numbering) in the CH3 domain.

In addition, heterologous polypeptides can be prepared containing Fc region variants in which 1-3 pairs of amino acid residues selected from (1) to (3) above have the same charge in the CH3 domain of the first Fc region, and pairs of amino acid residues selected in the above 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.

In the above 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).

Other known techniques can also be used to associate heterologous polypeptides containing Fc region variants. In particular, this technique is carried out by replacing the amino acid side chain in one of the Fc regions with a larger side chain (protrusion; which

- 57 040713 referred to as a bulge) and by replacing the side chain of the amino acid present in the Fco region with a smaller side chain (depression; referred to as a cavity), placing the protrusion in the cavity. This can facilitate efficient association between Fc region containing polypeptides that have different amino acid sequences from each other (WO 1996/027011;

Ridgway et al., Prot. Eng. 9, 1996, p. 617-621; Merchant et al., Nat.Biotech. 16, 1998, p. 677-681).

In addition, other known techniques can also be used to associate heterologous polypeptides containing variant Fc regions. The association of polypeptides containing an Fc region can be efficiently induced using a chain-exchange generated CH3 domain of heterodimers (Davis et al., Prot. Eng. Des. & Sel., 23, 2010, pp. 195-202). This technique can also be used to efficiently induce association between Fc region-containing polypeptides with different amino acid sequences.

In addition, methods for producing heterodimerized antibodies that use the association of CH1 and CL antibodies and the association of VH and VL described in WO 2011/028952 can also be used.

Similar to the method described in WO 2008/119353 and WO 2011/131746, it is also possible to apply the technique of obtaining heterodimerized antibodies by first creating two types of homodimerized antibodies, incubating the antibodies under reducing conditions to dissociate them and allowing them to re-associate.

Similar to the method described by Strop, J. Mol. Biol. 420, 2012, p. 204-219, a technique for making heterodimerized antibodies by introducing charged residues such as Lys, Arg, Glu, and Asp can also be used to introduce electrostatic repulsion into the CH3 domains.

In addition, similar to the method described in WO 2012/058768, you can also apply the method of obtaining heterodimerized antibodies by introducing changes in the CH2 and CH3 domains.

When two polypeptides containing an Fc region variant that have different amino acid sequences are simultaneously expressed to produce polypeptides containing heterologous Fc region variants, polypeptides containing homologous Fc region variants are generally formed as impurities. In such cases, polypeptides containing heterologous Fc region variants can be efficiently obtained by separating them and purifying them from polypeptides containing homologous Fc region variants using known techniques. A method for efficiently separating and purifying heterodimerized antibodies from homodimerized antibodies based on the use of ion exchange chromatography by introducing amino acid changes into the variable regions of two types of antibody heavy chains to create a difference in isoelectric points between homodimerized antibodies and heterodimerized antibodies is described (WO 2007/114325). Another method is described for purifying heterodimerized antibodies using protein A chromatography by constructing a heterodimerized antibody containing two types of heavy chains from mouse IgG2a that bind to protein A and rat IgG2b that does not bind to protein A (WO 1998/050431 and WO 1995/ 033844).

In addition, a heterodimerized antibody can be efficiently purified by protein A chromatography by replacing the amino acid residues at positions 435 and 436 (EU numbering), which are located at the protein A binding site in the heavy chain of the antibody, with amino acids such as Tyr or His , resulting in different binding affinities for protein A.

In the present invention, an amino acid change means any of such changes as substitution, deletion, addition, insertion and modification, or a combination thereof. In the present invention, an amino acid change may be referred to as an amino acid mutation.

When substituting amino acid residues, substitution with another amino acid residue can be carried out in order to change the characteristics indicated in subparagraphs (a) to (c) below: (a) the structure of the polypeptide backbone of the region of the folded structure or helical structure; (b) electrical charge or hydrophobicity at the target site; or (c) side chain size.

Amino acid residues are classified into the following groups based on common 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) main: His, Lys and Arg; (e) residues that affect chain orientation: Gly and Pro; and (e) aromatic: Trp, Tyr and Phe.

Amino acid changes are obtained using a variety of methods known to those skilled in the art. These methods include, but are not limited to, the site-directed mutagenesis method (Hashimoto-Gotoh et al., Gene 152, 1995, pp. 271-275; Zoller, Meth. Enzymol. 100, 1983, pp. 468-500; Kramer et al., Nucleic Acids Res. 12, 1984, pp. 9441-9456; Kramer and Fritz, Methods Enzymol. 154, 1987, pp. 350-367 and Kunkel, Proc. Natl. Acad. Sci. USA 82, 1985, pp. 488-492), a method based on PCR mutagenesis and cassette mutagenesis.

The number of amino acid changes introduced from the Fc region is not limited. In some embodiments, it may be 1, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 8 or less, 10 or less, 12 or less, 14 or less, 16 or less, 18 or less or 20 or less.

Amino acid modification includes post-translational modification. Specific

- 58 040713 post-translational modification may be the addition or deletion of the sugar chain. For example, the amino acid residue at position 297 (EU numbering) in the IgG1 constant region can be modified with a sugar chain. The structure of the sugar chain is not limited to a particular structure. For example, sialic acid can be added to the sugar chain of the Fc region (MAbs 2(5), September/October 2010, pp. 519-527). Typically, antibodies that are expressed in eukaryotic cells contain a glycosylation site in the constant region. For example, it is known that several types of sugar chains are normally added to antibodies expressed in cells, such as naturally occurring antibody-producing mammalian cells or eukaryotic cells transformed with an expression vector that contains the DNA encoding the antibody.

In the context of the present description, eukaryotic cells include yeast and animal cells. For example, CHO cells and HEK293 cells are representative animal cells that are used for transformation with an expression vector containing DNA that encodes an antibody. On the other hand, constant regions without glycosylation also fall within the scope of the present invention. Antibodies whose constant regions are not glycosylated can be generated by expression of an antibody-encoding gene in prokaryotic cells such as Escherichia coli.

In addition, the polypeptide containing the variant Fc region of the present invention can be chemically modified with various molecules such as polyethylene glycol (PEG) and cytotoxic substances. Methods for said chemical modification of polypeptides have been developed in the art.

One object of the invention is an isolated polypeptide containing a variant Fc region with increased Fc-gamma RIIb-binding activity. In some aspects of the invention, the polypeptide is an antibody. In some aspects of the invention, the polypeptide is an Fc-containing fusion protein. One object of the invention is an isolated polypeptide containing a variant Fc region with increased Fc-gamma RIIb-binding activity. In some aspects of the invention, the polypeptide is an antibody. In some aspects of the invention, the antibody is a chimeric antibody or a humanized antibody. The origin of the antibody is not limited to a particular origin, but examples include human antibody, mouse antibody, rat antibody, and rabbit antibody. In some aspects of the invention, the polypeptide is an Fc-containing fusion protein.

Antibody variable regions that contain the Fc region variant as described herein and protein-binding motifs of Fc-containing fusion proteins that contain the Fc region variant can recognize any antigen. Examples of antigens that can be bound by such antibodies and fusion proteins include, but are not limited to, ligands (cytokines, chemokines, etc.), receptors, cancer antigens, MHC antigens, differentiation antigens, immunoglobulins, and immune complexes, in part including immunoglobulins.

Examples of cytokines that can be bound by an antibody or fusion protein containing an Fc region variant of the invention and/or recombinantly fused to a polypeptide that contains said Fc region variant include, but are not limited to, colony-stimulating interleukins 118. factors (G-CSF, M-CSF, GM-CSF, etc.), interferons (IFN-alpha, IFNbeta, IFN-gamma, etc.), growth factors (EGF, FGF, IGF, NGF, PDGF , TGF, HGF, etc.), tumor necrosis factors (TNF-alpha and TNF-beta), lymphotoxin, erythropoietin, leptin, SCF, TPO, MCAF and BMP.

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

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

Examples of specific receptors belonging to the above receptor families are

- 59 040713 include human or mouse erythropoietin (EPO) receptors (Jones et al., Blood 76(1), 1990, c. 31-35; D'Andrea et al., Cell 57(2), 1989, c. 277 -285), human or mouse granulocyte colony stimulating factor (G-CSF) receptors (Fukunaga et al., Proc. Natl. Acad. Sci. USA 87(22), 1990, c. 8702-8706), mG-CSFR (Fukunaga et al., Cell 61(2), 1990, pp. 341-350), human or murine thrombopoietin receptors (TPO) (Vigon et al., Proc. Natl. Acad. Sci. USA. 89(12), 1992, pp. 5640-5644; Skoda et al., EMBO J. 12(7), 1993, pp. 2645-2653), human or murine insulin receptors (Ullrich et al., Nature 313(6005), 1985, p. 756 -761), human or mouse receptors for Flt-3 ligand (Small et al., Proc. Natl. Acad. Sci. USA. 91(2), 1994, pp. 459-463), human or mouse platelet growth factor receptors (PDGF) (Gronwald et al., Proc. Natl. Acad. Sci. USA. 85(10), 1988, c. 3435-3439), human or mouse interferon receptors (IFN)-alpha and beta (Uze et al. ., Cell 60(2), 1990, p. 225-234 (1990); Novick et al., Cell 77(3), 1994, p. 391-400), 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 inhibitory leukemia factor (LIF), and human or mouse ciliary neurotrophic factor (CNTF) receptors.

Cancer antigens are antigens that are expressed on cells that become cancerous and are also referred to as tumor-specific antigens. Abnormal sugar chains that appear on the cell surface or on protein molecules when cells become cancerous are also referred to as cancer antigens and are also called sugar chain cancer antigens. Examples of cancer antigens that can be bound by an antibody or fusion protein containing an Fc region variant of the invention include, but are not limited to, GPC3, which is a receptor belonging to the above family of GPI-anchored receptors and is also expressed in some cancers, including liver cancer (Midorikawa et al., Int. J. Cancer 103(4), 2003, pp. 455-465), as well as EpCAM, which is expressed in some cancers, including lung cancer (Linnenbach et al. , Proc Natl Acad Sci USA 86(1), 1989, c.2731), CA19-9, CA15-3 and sialyl-SSEA-1 (SLX).

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

Examples of differentiation antigens that can be bound by an antibody or fusion protein containing an Fc region variant of the invention and/or recombinantly fused to a polypeptide that contains said Fc region variant include, but are not limited to, CD1, CD2 , CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15s, CD16, CD18, CD19, CD20, CD21, CD23, CD25, CD28, CD29, CD30, CD32, CD33, CD34 , CD35, CD38, CD40, CD41a, CD41b, CD42a, CD42b, CD43, CD44, CD45, CD45RO, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD51, CD54, CD55, CD56, CD57, CD58, CD61 , CD62E, CD62L, CD62P, CD64, CD69, CD71, CD73, CD95, CD102, CD106, CD122, CD126 and CDwl30.

Immunoglobulins include IgA, IgM, IgD, IgG and IgE. The immune complexes include a component of at least any of the immunoglobulins.

Other examples of antigens that can be bound by an antibody or fusion protein containing an Fc region variant of the invention and/or recombinantly fused to a polypeptide that contains said Fc region variant include, but are not limited to, 17-IA , 41BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK , ALK-1, ALK-7, alpha-1 antitrypsin, alpha-V/beta antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic peptide, av/b3-integrin, Ax1, L2M, B7-1, B7-2, B7-H, B-lymphocyte stimulating factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R , Bag-1, BAK, Bax, BCA1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, B LK, BMP, BMP-2 BMP-2a, osteogenin BMP-3, 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 neurotrophic factor, BPDE, BPDE-DNA, BTC, complement system factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C/DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR11, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30 , CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80

- 60 040713 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin, Clostridium perfringens toxin, CKb8-l, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5 , CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, tumor cytokeratin-associated antigen, DAN, DCC, DcR3, DC-SIGN, complement regulating factor (dissociation accelerator factor), des(1-3)-IGF-I (IGF-1 brain), Dhh, digoxin, DNAM-1, Dcase, 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 IIa, factor VII, factor VIIIc, factor IX, fibroblast-activating protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3 , FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF1, 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-alpha1, GFR-alpha2, GFRalpha3, GITR, glucagon, Glut4, glycoprotein IIb/IIIa (GPIIb/IIIa), GM-CSF, gp130, gp72, GRO, growth hormone releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV envelope glycoprotein gB, envelope glycoprotein HCMV gH, 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) envelope glycoprotein gB, HSV glycoprotein gD, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3-loops, HLA, HLA-DR, HM1.24, HMFG PEM , HRG, Hrk, human heart myo zine, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (IFN)-alpha , IFN-beta, IFN-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin like growth factor 1, integrin alpha2, integrin alpha3, integrin alpha4, integrin alpha4/beta1, integrin alpha4/beta7, integrin alpha5 (alpha V ), integrin alpha5/beta1, integrin alpha5/beta3, integrin alpha6, integrin beta1, integrin beta2, interferon gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF- 1bp 1, LBP, LDGF, LECT2, Lefty factor, Lewis-Y antigen, Lewis-associated antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT -a, LT-b, LTB4, LTBP-1, lung surface antigen, luteinizing hormone, lymphotoxin receptor beta, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCK2, MCP, M-CSF, MDC, Mer, metalloprotease, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP -13, MMP-14, MMP15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, Mucin (Mucl), MUC18, Inhibitor Mueller, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA 90, NCAM, neprilysin, neurotrophin-3, -4 or -6, neuroturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin , PCNA, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A chain , relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF/KL, SDF-1, serine, serum albumin, sFRP-3, Shh , SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3 , T cell receptor (eg, T cell receptor alpha/beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, PLAP-like testicular alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan-specific, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF- beta5, thrombin, thymus Ck-1, thyroid stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNFalpha, TNF-alpha,beta, TN F-beta2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 ( HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p5560), TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA),

- 61 040713

TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL Rl TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2) , TNFSF11 (TRANCE/RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A/VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-a conectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 ( LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 ( CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE , transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated CA125 antigen, tumor-associated antigen expressing Lewis-Y antigen-associated carbohydrates, TWEAK, TXB2, Ung, uP AR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, A-beta, CD81, CD97, CD98, DDR1, DKK1, EREG, Hsp90, IL-17/IL-17R, IL-20/IL-20R, oxidized LDL, PCSK9, prekallikrein, RON, TMEM16F, SOD1, chromogranin A, chromogranin B, tau, VAP1, high molecular weight kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, factor B, factor D, factor H, properdin, sclerostin, fibrinogen, fibrin, prothrombin, thrombin, tissue factor, factor V, factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor thor X, factor Xa, factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3 , syndecan-1, syndecan-2, syndecan-3, syndecan-4, LPA and S1P; and receptors for hormones and growth factors.

As stated in the present description, the presence of changes in one or more amino acid residues in amino acid sequences that form variable regions is acceptable if their antigen-binding activity is maintained. Changes affecting the amino acid sequence of the variable region are not limited to the site at which the change is made and the number of amino acids changed. For example, the amino acids present in the CDR and/or FR can be modified accordingly. When amino acids in the variable region are changed, the binding activity is preferably maintained without particular limitation; and, for example, compared to the activity before the change, the binding activity can be 50% or more, 80% or more, and 100% or more. In addition, binding activity can be increased due to amino acid changes. For example, the binding activity may be 2-fold, 5-fold, 10-fold, or the like. compared to activity before the change. The amino acid sequence change may be a substitution, addition, deletion or modification of at least one amino acid residue.

For example, modification of the N-terminal glutamine of the variable region, resulting in the formation of pyroglutamic acid by pyroglutamylation, is well known to those skilled in the art. Thus, when the N-terminus of the heavy chain is glutamine, the antibodies described herein may contain variable regions in which glutamine is modified to form pyroglutamic acid.

The variable regions of the antibodies described herein may be of any sequence and may be the variable regions of antibodies of any origin, e.g. mouse antibodies, rat antibodies, rabbit antibodies, goat antibodies, camel antibodies, humanized antibodies obtained by humanizing said non-human antibodies, and human antibodies. In addition, these antibodies may have various amino acid substitutions introduced into their variable regions to improve their antigen binding, pharmacokinetic characteristics, stability, and immunogenicity. Variable regions may have the ability to re-bind to antigens due to their pH dependent antigen binding (WO 2009/125825).

In the constant regions of the light chains of antibodies, the kappa chain and the lambda chain are present, either of which is acceptable. In addition, they may have amino acid changes such as substitutions, deletions, additions and/or insertions.

In addition, polypeptides that contain variants of the Fc regions described herein can form Fc-containing fusion proteins by binding to other proteins, such as physiologically active peptides. Said fusion proteins may be multimers of at least two polypeptides containing variant Fc regions. Examples of other proteins are (but are not limited to) receptors, adhesion molecules, ligands, and enzymes.

Examples of Fc-containing fusion proteins include proteins fused via an Fc region to a receptor that binds to a target molecule, including TNFR-Fc fusion protein, IL1R-62 fusion protein 040713

Fc, VEGFR-Fc fusion protein and CTLA4-Fc fusion protein (Economides et al. Nat. Med. 9(1), 2003, pp. 47-52; Dumont et al., BioDrugs. 20(3), 2006, pp.151-160). In addition, the proteins to be fused can be other molecules that have the ability to bind to the target, such as scFv (WO 2005/θ37989), single domain antibodies (WO 2004/058821; WO 2003/002609), antibody-like molecules (Davinder, Curr Op Biotech 17 2006 653-658 Current Opinion in Biotechnology 18 2007 110 Nygren et al Curr Op Struct Biol 7 1997 463-469 and Hoss Protein Science 15, 2006, pp. 14-27 (2006), such as DARPin (WO 2002/020565), Affibody (WO 1995/001937), Avimer (WO 2004/044011; WO 2005/040229) and Adnectin (WO 2θθ2/032925).Furthermore, antibodies and Fc-containing fusion proteins can be multispecific and can bind to several types of target molecules or epitopes.

B. Methods of recombination and composition.

Antibodies can be generated using recombination and composition techniques, such as those described in US No. 4,816,567. One embodiment of the invention is an isolated nucleic acid encoding an anti-myostatin antibody described herein. Another embodiment of the invention is an isolated nucleic acid that encodes a polypeptide containing a variant Fc region or parental Fc region as defined herein. Said nucleic acid may encode an amino acid sequence containing the VL and/or an amino acid sequence containing the VH of an antibody (eg, antibody light and/or heavy chains). The following embodiment of the invention is(are) one or more vectors (eg, expression vectors), which(e) contains(at) the specified nucleic acid. Another embodiment of the invention is a host cell that contains the specified nucleic acid. In one of these embodiments of the invention, the host cell contains (for example, as a result of transformation with these vectors): (1) a vector containing a nucleic acid that encodes an amino acid sequence containing the VL of the antibody, and an amino acid sequence containing the VH of the antibody, or (2) the first a vector containing a nucleic acid that encodes an amino acid sequence containing the VL of an antibody, and a second vector containing a nucleic acid that encodes an amino acid sequence containing the VH of an antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (eg, Y0, NS0, Sp20 cell). One of the embodiments of the invention is a method for producing an antibody to myostatin, which consists in cultivating a host cell containing a nucleic acid that encodes the antibody described above under conditions suitable for expressing the antibody, and optionally isolating the antibody from the host cell ( or host cell culture medium). Another embodiment of the invention is a method for producing a polypeptide that contains a variant Fc region or a parent Fc region, which consists in cultivating a host cell that contains nucleic acid(s) encoding(s) a polypeptide, such as an antibody, Fc region or Fc region variant as defined above under conditions suitable for expression of the polypeptide, and optionally recovering the polypeptide from the host cell (or host cell culture medium).

For recombinant production of an anti-myostatin antibody, antibody-encoding nucleic acids, such as those described above, are 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, a 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. The specified nucleic acid can be easily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that have the ability to specifically bind to the genes encoding the heavy and light chains of antibodies).

Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc-related effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, US Pat. Nos. 5,648,237; 248, 2003, pp. 245-254 (description of the expression of antibody fragments in E. coli.) After expression, the antibody can be isolated from the bacterial cell paste as a soluble fraction and can be further purified.

In addition to prokaryotic organisms, eukaryotic microorganisms such as filamentous fungi or yeasts, including strains of fungi and yeasts whose glycosylation pathways have been humanized, can be used as hosts suitable for cloning or expressing plasmids that encode antibodies, which makes it possible to obtain an antibody with partially or completely human glycosylation scheme (see Gerngross, Nat. Biotech. 22, 2004, pp. 1409-1414 and Li et al., Nat. Biotech. 24, 2006, pp. 210-215).

Host cells that can be used to express the glycosylated antibody are also obtained from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells are insect cells, and plant cells can also be used. Numerous strains of baculoviruses and corresponding suitable insect cells as hosts have been identified, primarily for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts (see, for example, US Pat.

Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to growth in suspension can be used. Other examples of suitable mammalian host cell lines are the CV1 monkey kidney cell line transformed with OB40 (COS-7); a human embryonic kidney cell line (HEK293 cells or 293 cell line described, for example, in Graham et al., J. Gen. Virol., 36, 1977, pp. 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells described, for example, in Mather, Biol. Reprod., 23, 1980, c. 243-251); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma (HELA) cells; dog kidney cells (MDCK); bovine rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells as described, for example, in Mather et al., Annals N.Y. Acad. Sci. 383, 1982, p. 44-68); MRC 5 cells and FS4 cells. Other valuable mammalian host cell lines are Chinese Hamster Ovary (CHO) cells, including DHFR CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA, 77, 1980, c. 4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of specific mammalian host cell lines that can be used to produce antibodies, see, for example, Yazaki and Wu, in: Methods in Molecular Biology, ed. V.K.S. Lo, Humana Press, Totowa, NJ, vol. 248, 2003, p. 255-268.

Antibodies with pH dependent characteristics can be obtained using screening and/or mutagenesis methods, such as those described in WO 2009/125825. Screening methods may include any process that identifies an antibody with pH-dependent binding characteristics in a population of antibodies specific for a particular antigen. In some embodiments, screening methods may involve measuring one or more binding parameters (eg, KD or kd) of individual antibodies in an initial antibody population, both at acidic and neutral pH. Antibody binding parameters can be measured, for example, using surface plasmon resonance or any other analytical method that allows you to quantitatively or qualitatively evaluate the binding characteristics of an antibody to a particular antigen. In some embodiments, screening methods may involve identifying an antibody whose binding to an antigen is characterized by an acid/neutral pH KD ratio of 2 or more. In other embodiments, screening methods may include identifying an antibody whose binding to an antigen has a KD ratio at pH 5.8/pH 7.4 of 2 or more. Alternatively, screening methods may involve identifying an antibody whose binding to an antigen is characterized by an acid/neutral pH kd ratio of 2 or more. In other embodiments, screening methods may include identifying an antibody whose binding to an antigen has a kd ratio at pH 5.8/pH 7.4 of 2 or more.

In another embodiment of the invention, mutagenesis methods may involve the deletion, substitution or addition of an amino acid in the heavy and/or light chain of an antibody to enhance the pH-dependent binding of the antibody to the antigen. In some embodiments, one or more variable domains of an antibody, such as one or more HVRs (eg, CDRs), can be mutated. For example, mutagenesis can result in the replacement of an amino acid in one or more HVRs (eg, CDRs) of an antibody with a different amino acid. In some embodiments of the invention, mutagenesis can result in the replacement of an amino acid in at least one HVR (eg, CDR) of the antibody with histidine. In some embodiments, increased pH dependent binding means that the mutant version of the antibody has a higher acid/neutral pH KD ratio, or a higher acid/neutral pH KD ratio, than the original parent (i.e., less dependent from pH) the version of the antibody before mutagenesis. In some embodiments of the invention, the mutant version of the antibody is characterized by a KD ratio at acidic/neutral pH of 2 or more. In some embodiments, the mutant version of the antibody has a KD ratio at pH 5.8/pH 7.4 of 2 or more. Alternatively, the mutant version of the antibody has an acid/neutral pH kd ratio of 2 or more. In some embodiments of the invention, the mutant version of the antibody is characterized by a kd ratio at pH 5.8/pH 7.4 of 2 or more.

Polyclonal antibodies are preferably produced in animals after several subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antibody and adjuvant. It is possible to conjugate the relevant antigen to a protein immunogenic for the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional or derivatizing agent, such as maleimidobenzoyl sulfosuccinimide ester (conjugation via residues cysteine), Nhydroxysuccinimide (conjugation via lysine residues), glutaraldehyde, succinic anhydride, SOCl2 or R1N=C=NR, where R and R ¹ are different alkyl groups.

Animals (typically non-human mammals) are immunized with the antigen, immunogenic conjugates or derivatives by combining, for example, 100 or 5 μg of protein or conjugate (for rabbits and mice, respectively) with 3 volumes of complete Freund's adjuvant, and the solution is administered intradermally into several places. After 1 month, the animals are subjected to booster vaccination (revaccination) using from 1/5 to 1/10 of the original amount of the peptide or conjugate in complete Freund's adjuvant by subcutaneous injection at several sites. After 7-14 days, blood samples of the animals are obtained and the serum antibody titer is assessed. Animals are revaccinated until the titer reaches a plateau. Preferably, the animals are revaccinated using a conjugate containing the same antigen but conjugated to a different protein and/or through a different cross-linking agent. Conjugates can also be obtained in recombinant cell culture as protein fusions. In addition, aggregating agents such as alum can be used to enhance the immune response.

Monoclonal antibodies are derived from a population of substantially homogeneous antibodies, ie. the individual antibodies in a population are identical except for possible naturally occurring mutations and/or post-translational modifications (eg, isomerization, amidation), which may be present in minor amounts. The adjective monoclonal indicates that the antibody is not present in a mixture of different antibodies.

For example, monoclonal antibodies can be made using the hybridoma method first described by Kohler et al., Nature 256(5517), 1975, c. 495-497.

In the hybrid method, a mouse or other suitable host animal such as a hamster is immunized according to the method described above to produce lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro.

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

Immortalized cell lines are generally transformed mammalian cells, primarily rodent, bovine and human myeloma cells. Typically, rat or mouse myeloma cell lines are used. The hybridoma cells thus obtained are seeded and grown in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), then the culture medium for hybridomas typically includes hypoxanthine, aminopterine, and thymidine (HAT medium), i.e. substances that interfere with the growth of HGPRT-deficient cells.

Preferred immortalized myeloma cells are cells that can be efficiently fused, that maintain a stable high level of antibody production by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, murine myeloma lines such as those derived from MOPC-21 and MPC-11 murine tumors, which are available from the Salk Institute Cell Distribution Center, San Diego, PA, are preferred. California, USA, and SP-2 cells (and their derivatives, eg X63-Ag8653), which can be obtained from the American Type Culture Collection, Manassas, pc. Virginia, USA. Also described are human myeloma and mouse-human heteromyeloma cell lines that can be used to produce human monoclonal antibodies (Kozbor et al., J. Immunol. 133(6), 1984, c. 3001-3005; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, 1987, pp. 51-63).

The culture medium in which the hybridoma cells are grown is evaluated for the production of monoclonal antibodies against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). These techniques and assays are known in the art. For example, binding affinity can be determined using the Scatchard analysis described in Munson, Anal. Bio

- 65 040713 chem. 107(1), 1980, p. 220-239.

Once hybridoma cells are found to produce antibodies of the desired specificity, affinity and/or activity, clones can be subcloned using serial dilution procedures and grown using standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as tumors in mammals.

Monoclonal antibodies secreted by subclones can be separated from the culture medium, ascitic fluid or serum using conventional immunoglobulin purification procedures, such as chromatography for protein A-sepharose, hydroxyapatite, gel electrophoresis, dialysis or affinity chromatography.

Antibodies can be obtained by immunizing a suitable host animal with an antigen. In one embodiment, the antigen is a full-length myostatin-containing polypeptide. In one of the embodiments of the invention, the antigen is a polypeptide containing latent myostatin. In one embodiment, the antigen is a polypeptide containing a myostatin propeptide. In one embodiment, the antigen is a polypeptide containing a region corresponding to amino acids at positions 21-100 of the myostatin propeptide (SEQ ID NO: 78). In one embodiment, the antigen is a polypeptide containing amino acids at positions 21-80, 41-100, 21-60, 41-80, 61-100, 21-40, 41-60, 61-80 or 81-100 myostatin propeptide (SEQ ID NO: 78). The present invention also relates to antibodies obtained by immunizing an animal with an antigen. Antibodies may have singly or in combination any of the features described above in the Examples of Myostatin Antibodies section.

The Fc region can be obtained by re-eluting the protein A adsorbed fraction after partial digestion of monoclonal antibodies as IgG1, IgG2, IgG3, IgG4 or the like. with a protease such as pepsin. A protease is not limited to a particular protease as long as it can cleave a full-length antibody to form Fab and F(ab')2 in a limited manner under appropriate enzymatic reaction conditions such as pH, and examples include pepsin and papain.

In addition, the present invention provides a method for producing a polypeptide containing an Fc region variant with increased Fc gamma RIIb binding activity compared to a polypeptide containing a parental Fc region, which includes the introduction of at least one amino acid change in the parent Fc region . In some aspects of the invention, the resulting polypeptide is an antibody. In some embodiments, the antibody is a chimeric antibody or a humanized antibody. In some aspects of the invention, the resulting polypeptide is an Fc-containing fusion protein.

In some embodiments of the invention, the method of obtaining is that the following steps are carried out: (a) a polypeptide containing the parent Fc region is obtained; (b) introducing at least one amino acid change into the parent Fc region in the polypeptide; (c) measuring the binding activity to simian Fc-gamma RIIb of the polypeptide that contains the parental Fc region and the polypeptide that contains the Fc region obtained in step (b); and (d) selecting a polypeptide containing a variant Fc region with increased binding activity to simian Fc-gamma RIIb compared to a polypeptide containing the parental Fc region.

In some embodiments of the invention, the method of obtaining includes the following stages, in which (a) receive a polypeptide containing the parental Fc region; (b) introducing at least one amino acid change into the parent Fc region in the polypeptide; (c) simian Fc-gamma RIIIa binding activity of the polypeptide that contains the parental Fc region and the polypeptide that contains the Fc region obtained in step (b) is measured; and (d) selecting a polypeptide containing a variant Fc region with reduced simian Fc-gamma RIIIa binding activity compared to a polypeptide containing the parental Fc region.

In some embodiments of the invention, the method of obtaining is that the following steps are carried out: (a) a polypeptide containing the parent Fc region is obtained; (b) introducing at least one amino acid change into the parent Fc region in the polypeptide; (c) measure the activity of binding to human Fc-gamma RIIb polypeptide, which contains the parental Fc region, and the polypeptide, which contains the Fc region obtained in stage (b); and (d) selecting a polypeptide containing a variant Fc region with increased binding activity to the human Fc-gamma RIIb compared to a polypeptide containing the parental Fc region.

In some embodiments of the invention, the method of obtaining includes the following stages, in which (a) receive a polypeptide containing the parental Fc region; (b) introducing at least one amino acid change into the parent Fc region in the polypeptide; (c) measuring the activity of binding to human Fc-gamma RIIIa polypeptide, which contains the parental Fc region, and the polypeptide, which contains the Fc region obtained in stage (b); and (d) selecting a polypeptide containing a variant Fc region with reduced binding activity to human Fcgamma RIIIa compared to a polypeptide containing the parental Fc region.

- 66 040713

In some embodiments of the invention, the method of obtaining is that the following steps are carried out: (a) a polypeptide containing the parent Fc region is obtained; (b) introducing at least one amino acid change into the parent Fc region in the polypeptide; (c) measuring the binding activity to human Fc-gamma RIIa (H-type) of the polypeptide that contains the parental Fc region and the polypeptide that contains the Fc region obtained in step (b); and (d) selecting a polypeptide containing a variant Fc region with reduced binding activity to the human Fc gamma RIIa (H-type) compared to a polypeptide containing the parental Fc region.

In some embodiments of the invention, the method of obtaining is that the following steps are carried out: (a) a polypeptide containing the parent Fc region is obtained; (b) introducing at least one amino acid change into the parent Fc region in the polypeptide; (c) measuring the activity of binding to human Fc-gamma RIIa (R-type) of the polypeptide that contains the parental Fc region and the polypeptide that contains the Fc region obtained in step (b); and (d) selecting a polypeptide containing a variant Fc region with reduced binding activity to the human Fc gamma RIIa (R-type) compared to a polypeptide containing the parental Fc region.

In one aspect of the invention, at least one amino acid is altered in accordance with the method described above for producing a polypeptide containing an Fc region variant with increased Fcgamma RIIb binding activity at at least one position selected from the group consisting of 231, 232, 233, 234, 235, 236, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334 and 396, according to EU numbering . In some embodiments, the Fcgamma RIIb has the sequence Fc-gamma RIIb of cynomolgus monkeys (SEQ ID NO: 223). In some embodiments, the RIIb Fc-gamma has the sequence of a human RIIb Fc-gamma (eg, SEQ ID NO: 212, 213, or 214).

In another aspect of the invention, one amino acid is changed in accordance with the method described above for producing a polypeptide containing an Fc region variant with increased Fc-gamma RIIb binding activity, at position 236.

In another aspect of the invention, at least two amino acids are changed in accordance with the method described above for producing a polypeptide containing an Fc region variant with increased Fcgamma RIIb binding activity, where the changes are: (a) one amino acid change at position 236 and (b) at least one amino acid change at at least one position selected from the group consisting of 231, 232, 233, 234, 235, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325 , 326, 327, 328, 330, 331, 332, 334 and 396, according to the EU numbering. In some embodiments, the RIIb Fc-gamma has the cynomolgus Fc-gamma RIIb sequence (SEQ ID NO: 223). In some embodiments, the RIIb Fc-gamma has the sequence of a human RIIb Fc-gamma (eg, SEQ ID NO: 212, 213, or 214).

In another aspect of the invention, at least two amino acids are changed in accordance with the method described above for producing a polypeptide containing an Fc region variant with increased Fcgamma RIIb binding activity, where the changes are: (a) one amino acid change at position 236, and (b ) at least one amino acid change 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 some embodiments, the RIIb Fc-gamma has the cynomolgus Fc-gamma RIIb sequence (SEQ ID NO: 223). In some embodiments, the RIIb Fc-gamma has the sequence of a human RIIb Fc-gamma (eg, SEQ ID NO: 212, 213, or 214).

In another aspect of the invention, at least two amino acids are changed in accordance with the method described above for producing a polypeptide containing an Fc region variant with increased Fcgamma RIIb binding activity, where the changes are: (a) one amino acid change at position 236 and (b) at least one amino acid change at at least one position selected from the group consisting of 268, 295, 326, and 330, according to EU numbering. In some embodiments, the RIIb Fc-gamma has the cynomolgus Fc-gamma RIIb sequence (SEQ ID NO: 223). In some embodiments, Fcgamma RIIb has the sequence of human Fcgamma RIIb (eg, SEQ ID NO: 212, 213, or 214).

In another aspect of the invention, the amino acids are changed in accordance with the method described above for obtaining a polypeptide containing an Fc region variant with increased Fc-gamma RIIb binding activity, at one of the following positions (1)-(37): (1) at positions 231, 236, 239, 268 and 330; (2) in regulations 231, 236, 239, 268, 295 and 330; (3) in regulations 231, 236, 268 and 330; (4) in regulations 231, 236, 268, 295 and 330; (5) in regulations 232, 236, 239, 268, 295 and 330; (6) in regulations 232, 236, 268, 295 and 330; (7) in regulations 232, 236, 268 and 330; (8) in regulations 235, 236, 268, 295, 326 and 330; (9) in regulations 235, 236, 268, 295 and 330; (10) in regulations 235, 236, 268 and 330; (11) in regulations 235, 236, 268, 330 and 396; (12) in regulations 235, 236, 268 and 396; (13) in regulations 236, 239, 268, 295, 298 and

- 67 040713

330; (14) in regulations 236, 239, 268, 295, 326 and 330; (15) in regulations 236, 239, 268, 295 and 330; (16) in regulations 236, 239, 268, 298 and 330; (17) in regulations 236, 239, 268, 326 and 330; (18) in regulations 236, 239, 268 and 330; (19) in regulations 236, 239, 268, 330 and 396; (20) in regulations 236, 239, 268 and 396; (21) in regulations 236 and 268; (22) in regulations 236, 268 and 295; (23) in regulations 236, 268, 295, 298 and 330; (24) in regulations 236, 268, 295, 326 and 330; (25) in regulations 236, 268, 295, 326, 330 and 396; (26) in regulations 236, 268, 295 and 330; (27) in regulations 236, 268, 295, 330 and 396; (28) in regulations 236, 268, 298 and 330; (29) in regulations 236, 268, 298 and 396; (30) in regulations 236, 268, 326 and 330; (31) in regulations 236, 268, 326, 330 and 396; (32) in regulations 236, 268 and 330; (33) in regulations 236, 268, 330 and 396; (34) in regulations 236, 268 and 396; (35) in regulations 236 and 295; (36) in regulations 236, 330 and 396; and (37) in regulations 236 and 396 according to the EU numbering. In some embodiments, the RIIb Fc-gamma has the cynomolgus Fc-gamma RIIb sequence (SEQ ID NO: 223). In some embodiments, the RIIb Fc-gamma has the sequence of a human RIIb Fc-gamma (eg, SEQ ID NO: 212, 213, or 214).

In a further aspect of the invention, an amino acid change in accordance with the method described above for producing a polypeptide containing an Fc region variant with increased Fc-gamma RIIb binding activity is selected at each of the positions 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; (6) 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; (m) Ala, His, Leu at position 267; (m) Asp, Glu at position 268; (o) Asp, Glu, Gly at position 271; (p) Leu at position 295; (p) Leu at position 298; (c) Glu, Phe, Ile, Leu at position 325; (r) Thr at position 326; (y) Ile, Asn at position 327; (f) Thr at position 328; (x) Lys, Arg at position 330; (c) Glu at position 331; (h) Asp at position 332; (w) Asp, Ile, Met, Val, Tyr at position 334 and (n) 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 some embodiments, the RIIb Fc-gamma has the cynomolgus Fc-gamma RIIb sequence (SEQ ID NO: 223). In some embodiments, the RIIb Fc-gamma has the sequence of a human RIIb Fc-gamma (eg, SEQ ID NO: 212, 213, or 214).

In a further aspect of the invention, an amino acid change in accordance with the method described above for producing a polypeptide comprising an Fc region variant with increased Fc-gamma RIIb binding activity is selected at each of the positions 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; (e) 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 some embodiments, the RIIb Fc-gamma has the cynomolgus Fc-gamma RIIb sequence (SEQ ID NO: 223). In some embodiments, the RIIb Fc-gamma has the sequence of a human RIIb Fc-gamma (eg, SEQ ID NO: 212, 213, or 214).

In a further aspect of the invention, the amino acid changes according to the method described above for producing a polypeptide containing an Fc region variant with increased Fc-gamma RIIb 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 another aspect of the invention, the amino acid changes in accordance with the method described above for producing a polypeptide containing an Fc region variant with increased Fc-gamma RIIb binding activity are: Asn at position 236, Asp at position 268 and Lys at position 330 according to EU- numbering. In a further aspect of the invention, the amino acid changes according to the method described above for producing a polypeptide containing an Fc region variant with increased Fc-gamma RIIb binding activity are: Asn at position 236, Asp at position 268, Leu at position 295, and Lys at position 330 according to the EU numbering. In a further aspect of the invention, the amino acid changes according to the method described above for producing a polypeptide containing an Fc region variant with increased Fc-gamma RIIb binding activity are: Thr at position 236, Asp at position 268 and Lys at position 330 according to EU- numbering. In a further aspect of the invention, the amino acid changes according to the method described above for producing a polypeptide containing an Fc region variant with increased Fc-gamma RIIb 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 of the invention, the amino acid changes according to the method described above for producing a polypeptide containing an Fc region variant with enhanced Fc-gamma RIIb 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.

- 68 040713

In one aspect of the invention, at least one amino acid is altered in accordance with the method described above for producing a polypeptide containing an Fc region variant with increased Fcgamma RIIb binding activity at at least one position selected from the group consisting of 234, 238, 250, 264, 267, 307 and 330, according to the EU numbering. In some embodiments, the RIIb Fc-gamma has the cynomolgus Fc-gamma RIIb sequence (SEQ ID NO: 223). In some embodiments, the RIIb Fc-gamma has the sequence of a human RIIb Fc-gamma (eg, SEQ ID NO: 212, 213, or 214).

In another aspect of the invention, one amino acid is changed in accordance with the method described above for obtaining a polypeptide containing an Fc region variant with increased Fc-gamma RIIb binding activity, at position 238.

In another aspect of the invention, at least two amino acids are changed in accordance with the method described above for producing a polypeptide containing an Fc region variant with increased Fcgamma RIIb binding activity, where the changes are: (I) one amino acid change at position 238 and (II) at least one amino acid change at at least one position selected from the group consisting of 234, 250, 264, 267, 307 and 330, according to EU numbering. In some embodiments, the RIIb Fc-gamma has the cynomolgus Fc-gamma RIIb sequence (SEQ ID NO: 223). In some embodiments, the RIIb Fc-gamma has the sequence of a human RIIb Fc-gamma (eg, SEQ ID NO: 212, 213, or 214).

In another aspect of the invention, the amino acids are altered in accordance with the method described above for producing a polypeptide containing an Fc region variant with increased Fc-gamma RIIb binding activity, at one of the following positions (1)-(9): (1) at positions 234, 238, 250, 307 and 330; (2) in regulations 234, 238, 250, 264, 307 and 330; (3) in regulations 234, 238, 250, 264, 267, 307 and 330; (4) in regulations 234, 238, 250, 267, 307 and 330; (5) in regulations 238, 250, 264, 307 and 330; (6) in regulations 238, 250, 264, 267, 307 and 330; (7) in regulations 238, 250, 267, 307 and 330; (8) in regulations 238, 250 and 307; and (9) in provisions 238, 250, 307 and 330 according to the EU numbering.

In a further aspect of the invention, an amino acid change according to the method described above for producing a polypeptide comprising an Fc region variant with increased Fc-gamma RIIb binding activity is selected at each of the positions 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; (e) Pro at position 307; and (g) Lys at position 330; according to EU numbering. In a further aspect of the invention, the amino acid changes according to the method described above for producing a polypeptide containing an Fc region variant with increased Fc-gamma RIIb binding activity are: Asp at position 238 according to EU numbering. In a further aspect of the invention, the amino acid changes according to the method described above for producing a polypeptide containing an Fc region variant with increased Fc-gamma RIIb binding activity are: Asp at position 238, Val at position 250 and Pro at position 307 according to EU numbering. In a further aspect of the invention, the amino acid changes according to the method described above for producing a polypeptide containing an Fc region variant with increased Fc-gamma RIIb binding activity are: Asp at position 238, Val at position 250, Pro at position 307, and Lys at position 330 according to EU numbering. according to EU numbering. In a further aspect of the invention, the amino acid changes according to the method described above for producing a polypeptide containing an Fc region variant with increased Fc-gamma RIIb 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 of the invention, the amino acid changes according to the method described above for producing a polypeptide containing an Fc region variant with increased Fc-gamma RIIb 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 of the invention, the amino acid changes according to the method described above for producing a polypeptide containing an Fc region variant with enhanced Fcgamma RIIb 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 of the invention, the amino acid changes according to the method described above for producing a polypeptide containing an Fc region variant with enhanced Fc-gamma RIIb binding activity are: Tyr at position 234, Asp at position 238, Val at position 250, Ala at position 267, Pro at position 307 and Lys at position 330 according to the EU numbering. In a further aspect of the invention, the amino acid changes according to the method described above for producing a polypeptide containing an Fc region variant with increased Fc-gamma RIIb 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 the EU numbering. In a further aspect of the invention, the amino acid changes in accordance with the method described above for producing a polypeptide containing an Fc region variant with increased Fc-gamma RIIb binding activity are

- 69 040713 is: 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 the EU numbering. In a further aspect of the invention, the amino acid changes according to the method described above for producing a polypeptide containing an Fc region variant with increased Fc-gamma RIIb 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 some embodiments, the RIIb Fc-gamma has the cynomolgus Fc-gamma RIIb sequence (SEQ ID NO: 223). In some embodiments, the RIIb Fc-gamma has the sequence of a human RIIb Fc-gamma (eg, SEQ ID NO: 212, 213, or 214).

In addition, the present invention provides a method for producing a polypeptide containing an Fc region variant with an increased pI compared to a polypeptide containing a parental Fc region, which consists in introducing at least two amino acid changes into the parent Fc region.

In some embodiments of the invention, a method for obtaining a polypeptide containing a variant Fc region presented in the present description, is that the following steps are carried out: (a) a polypeptide containing a parental Fc region is obtained; (b) introducing at least two amino acid changes into the parent Fc region in the polypeptide; (c) measure the pI of the polypeptide that contains the parent Fc region and the polypeptide that contains the Fc region obtained in step (b); and (d) selecting a polypeptide containing an Fc region variant with an increased pI compared to a polypeptide containing the parental Fc region.

In one aspect of the invention, at least two amino acids are modified in accordance with the method described above to obtain a polypeptide containing a variant Fc region with increased pI, 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 another aspect of the invention, the resulting polypeptide comprises an elevated pI Fc region variant containing changes 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 another aspect of the invention, at least two amino acids are modified in accordance with the method described above to obtain a polypeptide containing a variant Fc region with increased pI, in one of the following positions (1)-(10): (1) at positions 311 and 341 ; (2) in regulations 311 and 343; (3) in regulations 311, 343 and 413; (4) in regulations 311, 384 and 413; (5) in regulations 311 and 399; (6) in regulations 311 and 401; (7) in regulations 311 and 413; (8) in regulations 400 and 413; (9) in regulations 401 and 413 and (10) in regulations 402 and 413 according to the EU numbering.

In another aspect of the invention, amino acid changes in accordance with the method described above to obtain a polypeptide containing a variant Fc region with increased pI, are: Arg at position 400 and Lys at position 413 according to EU numbering. In another aspect of the invention, amino acid changes in accordance with the method described above to obtain a polypeptide containing a variant Fc region with increased pI, are: Arg at position 311 and Lys at position 413 according to EU numbering. In another aspect of the invention, amino acid changes in accordance with the method described above to obtain a polypeptide containing a variant Fc region with increased pI, are: Arg at position 311 and Arg at position 399 according to EU numbering. In another aspect of the invention, amino acid changes in accordance with the method described above to obtain a polypeptide containing a variant Fc region with increased pI, are: Arg at position 311 and Arg at position 343 according to the EU numbering. In another aspect of the invention, amino acid changes in accordance with the method described above to obtain a polypeptide containing a variant Fc region with increased pI, are: Arg at position 311 and Arg at position 413 according to EU numbering. In another aspect of the invention, amino acid changes in accordance with the method described above to obtain a polypeptide containing 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 another aspect of the invention, amino acid changes in accordance with the method described above to obtain a polypeptide containing 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.

In a further aspect of the invention, amino acid changes in accordance with the method described above to obtain a polypeptide containing a variant Fc region with increased pI is selected from Lys or Arg at each position.

In the following aspect of the invention, the amino acid changes according to the preparation methods described above are selected from any of: a single change, a combination of single changes, or a combination of changes described in Table 1. 14-30.

Optionally, the following additional steps may be included in the methods described above for producing a polypeptide comprising a variant Fc region, wherein the polypeptide further comprises an antigen-binding domain: (e) assessing the plasma pharmacokinetics of the antigen following administration of the polypeptide comprising the parental Fc region, and a polypeptide containing a variant Fco region in animals such as mice, rats, rabbits, dogs, monkeys and humans; and (e) selecting a polypeptide containing a variant Fc region with an increased ability to eliminate antigen from plasma, compared to a polypeptide containing the parental Fc region.

Polypeptides containing a variant Fc region obtained using any of the above methods or other methods known in this field are included in the present invention.

B. Analyzes.

The anti-myostatin antibodies provided herein can be identified, screened for, or characterized for their physical/chemical properties and/or biological activities using a variety of assays known in the art.

The Fc region variants provided herein can be identified, screened for, or characterized for their physical/chemical properties and/or biological activities using a variety of assays known in the art.

1. Binding Assays and Other Assays.

In one aspect of the invention, an antibody of the invention is tested for antigen-binding activity using known methods such as ELISA, Western blotting, BIACORE®, etc. In one aspect of the invention, a polypeptide containing a variant Fco region of the invention is tested for binding activity to its Fc receptor using known methods such as BIACORE®, etc.

According to another aspect of the invention, competition assays can be used to identify an antibody that competes for binding to myostatin with any of the anti-myostatin antibodies described herein. In some embodiments of the invention, when the specified competitive antibody is present in excess, it blocks (for example, reduces) the binding of the 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 cases, binding is inhibited by at least 80%, 85%, 90%, 95% or more. In some embodiments, said competing antibody binds to the same epitope (e.g., a linear or conformational epitope) that the anti-myostatin antibody described herein binds (e.g., the anti-myostatin antibody described in Tables 2a, 11a, or 13) . According to other objects of the invention, the reference antibody has a pair of VH and VL, described in table. 2a, 11a, or 13. For a detailed description of exemplary methods for mapping an epitope to which an antibody binds, see Morris, Epitope Mapping Protocols, in Methods in Molecular Biology, vol. 66, Humana Press, Totowa, NJ, 1996.

As an example of a competition assay, an immobilized latent myostatin or myostatin propeptide is incubated in a solution containing a first labeled antibody that binds to myostatin and a second unlabeled antibody to be tested for its ability to compete with the first antibody for binding to myostatin. The second antibody may be present in the hybridoma supernatant. As a control, immobilized myostatin is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions suitable for the binding of the first antibody to myostatin, the excess of unbound antibody is removed and the amount of label associated with the immobilized myostatin is measured. If the amount of label associated with immobilized myostatin is significantly reduced in the test sample compared to the control sample, then this indicates that the second antibody competes with the first antibody for binding to myostatin (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988).

Assays for determining binding activity of a polypeptide containing a variant Fc region to one or more Fc-gamma R members are provided herein or are known in the art. Such binding assays include, but are not limited to, the BIACORE® assay, which utilizes the Surface Plasmon Resonance (SPR) phenomenon, Amplified Luminescent Proximity Homogeneous Assay (ALPHA), ELISA, and cell sorting by fluorescence intensity (FACS) (Lazar et al., Proc. Natl. Acad. Sci. USA 103(11), 2006, c. 4005-4010).

In one embodiment, the BIACORE® assay can be used to decide whether the binding activity of a polypeptide containing a variant Fc region is increased, or is maintained at the same level, or is reduced against a particular member of the Fc-gamma R family. For example, for this purpose, it is determined whether the value of the dissociation constant (KD) decreases or increases, which is determined using sensorgram analysis when various Fc-gamma R are reacted as an analyte with polypeptides containing an Fc region variant immobilized or captured on a sensor chip, using known methods and reagents, such as protein A, protein L, protein A/G, protein G, antibodies to the lambda chain, antibodies to the kappa chain, antigenic peptides, antigenic proteins. Changes in binding activity can also be determined by comparing changes in the number of resonance units (RU) on the sensorgram before and

- 71 040713 after one or more Fc-gamma R types are reacted as analytes with captured polypeptides containing the variant Fc region. Alternatively, the Fc-gamma R can be immobilized or captured on sensor chips and polypeptides containing the variant Fc region used as the analyte.

When performing BIACORE® assays, one of the substances (ligand) intended to study the interaction is immobilized on a thin layer of gold on a sensor chip and illuminated by light from the rear surface of the sensor chip in such a way that there is a complete reflection at the interface between the thin layer of gold and glass, a region with reduced intensity (SPR signal) is formed in part of the reflected light. Prepare another substance (analyte) intended to study the interaction, for injection on the surface of the sensor chip; and when the ligand binds to the analyte, the mass of the immobilized ligand molecule increases and the refractive index of the solvent on the surface of the sensor chip changes. As a result of said refractive index change, the position of the SPR signal shifts (and vice versa, the position of the signal returns to its original position if dissociation of said binding occurs). With the BIACORE® system, the level of the above-described shift, or more specifically the change in mass versus time, is determined by plotting the change in mass on the surface of the sensor chip along the vertical axis, and thus obtaining quantitative data (sensogram). The amount of analyte bound to the ligand immobilized on the surface of the sensor chip is determined from the sensorgram. Kinetic parameters such as association rate constant (ka) and dissociation rate constant (kd) are determined from the sensory curves, and affinity (KD) is determined as the ratio of these constants. As a method for inhibition assay, the BIACORE® method is preferably used. Examples of such a method for inhibition assays are described in Lazar et al., Proc. Natl. Acad. sci. USA 103(11), 2006, p. 4005-4010.

ALPHA-based screening is based on ALPHA technology, which is based on the principle described below, using two types of beads, donors and acceptors. Luminescent signals are detectable only when the molecules associated with the donor beads physically interact with the molecules associated with the acceptor beads and when both beads are in close proximity to each other. Excited by a laser beam, the photosensitizer in donor granules converts environmental oxygen into excited singlet oxygen. When singlet oxygen diffuses from the donor granules and reaches the acceptor granules located in close proximity, a chemiluminescent reaction is induced in the granules, which ultimately leads to the emission of light. If the molecules bound to the donor beads do not interact with the molecules bound to the acceptor beads, then the chemiluminescent reaction does not occur because the singlet oxygen that is produced by the donor beads does not reach the acceptor beads.

For example, a biotinylated polypeptide complex is coupled to donor beads, and a glutathione-S-transferase (GST) labeled Fcgamma receptor is coupled to acceptor beads. In the absence of a competing polypeptide complex containing a variant Fc region, the polypeptide complex containing the parent Fc region interacts with the Fc gamma receptor and generates signals at a wavelength of 520 to 620 nm. A polypeptide complex containing an unlabeled Fc region variant competes with a polypeptide complex containing a parent Fc region for binding to the Fc gamma receptor. Relative binding affinity can be assessed by quantifying the decrease in fluorescence due to competition. Methods for biotinylating polypeptide complexes such as antibodies with sulfo-NHS-biotin or the like are well known. Method for Expressing Fc-gamma Receptor and GST in a Cell Carrying a Fusion Gene Obtained by Fusing a Polynucleotide That Encodes an Fc-gamma Receptor in Frame with a Polynucleotide That Encodes GST in an Expression Vector and Performing Purification with a glutathione-Containing Column , are adapted accordingly, obtaining a method of labeling the Fc-gamma receptor with GST. The induced signals can be analyzed, for example, by fitting to a single site competition model based on non-linear regression analysis using software such as GRAPHPAD PRISM (GraphPad; San Diego).

The term variant Fc region with reduced Fc-gamma R binding activity refers to an Fc region that binds to an Fc-gamma R with significantly lower binding activity than the parental Fc region when assays are performed using substantially the same amounts of the corresponding parental Fc region. Fc region and variant Fc region. In addition, the term Fc region variant with increased Fc gamma R binding activity refers to an Fc region that binds to an Fc gamma R with substantially stronger binding activity than the parent Fc region when assays are performed using substantially equal amounts of the corresponding a parent Fc region and a variant Fc region. The term variant Fc region with retained Fc-gamma R binding activity refers to an Fc region that binds to an Fc-gamma R with activity equivalent to or substantially the same as that of the parent Fc region, when implemented assays using substantially equal amounts of the corresponding parental Fc region and the polypeptide containing the variant Fc region.

Whether the binding activity of the Fc region to various Fc-gamma Rs increases or decreases can be determined by the increase or decrease in the level of binding of various Fc-gamma Rs to the Fc region when determined using the measurement method described above. In the context of the present description, the level of binding of various Fc-gamma Rs to the Fc region can be estimated as a value obtained by dividing the difference in RU levels in the sensorgrams before and after the interaction of various Fc-gamma Rs, which are used as an analyte, with the Fc region, by the difference in RU levels on sensorgrams, which changed before and after capturing Fc regions on sensor chips.

According to the present invention, increased Fc-gamma RIIb binding activity preferably means, for example, that the ratio [KD value of the parent Fc region for Fc-gamma RIIb]/[KD value of the variant Fc region for Fc-gamma RIIb] when measured as values The KD measured using the above method is preferably 2.0 or more, 3.0 or more, 4.0 or more, 5.0 or more, 6.0 or more, 7.0 or more, 8.0 or more, 9.0 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or even 50 or more, 55 or more, 60 or more more than, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, 95 or more, or 100 or more.

The KD value (mol/L) of the Fc region variant described herein for Fc-gamma RIIb is preferably lower than the parental Fc region for Fc-gamma RIIb, and may be, for example, 2.0 x 10'6 M or less , 1.0x10'6M or less, ^9.0x10'7M or less, ^8.0x10'7M or less ^, 7.0x10'7M or less, ^6.0x10'7M or less, 5.0x10' ^7M or less, ^4.0x10'7M or less, 3.0x10'7M or less, ^{2.0x10'7M or} less, ^1.0x10'7M or less, ^9.0x10'8M or less, ^8.0x10'8M or less, ^7.0x10'8M or less, ^6.0x10'8M or less, ^5.0x10'8M or less.

In addition, a variant Fc region with increased binding selectivity to Fc-gamma RIIb compared to Fc-gamma RIIa means an Fc region in which: (a) Fc-gamma RIIb-binding activity is increased and Fc-gamma RIIa-binding activity remains at this level or decreases, (b) Fc-gamma RIIb-binding activity increases and Fc-gamma RIIa-binding activity also increases, but the degree of increase in Fc-gamma RIIa-binding activity is lower than the degree of increase in Fc-gamma RIIb- binding activity; or (c) Fc-gamma RIIb binding activity is reduced and Fc-gamma RIIa binding activity is also reduced, but the reduction rate of Fc-gamma RIIb binding activity is lower than the reduction rate of Fc-gamma RIIa binding activity. Whether the Fc region variant has increased binding selectivity for the Fc gamma RIIb relative to the Fc gamma RIIa can be determined, for example, by comparing the ratio of the KD value for the Fc gamma RIIa to the KD value for the Fc gamma RIIb variant Fc region (ratio [KD value of Fc-region variant for Fc-gamma RIIa]/[KD value of Fc-region variant for Fc-gamma RIIb]), with the ratio of the KD value for Fc-gamma RIIa and the KD value for Fc-gamma RIIb of the parent Fc -region (the ratio of [KD value of the parent Fc region for Fc-gamma RIIa]/[KD value of the parent Fc region for Fc-gamma RIIb]), which is determined according to the above examples. In particular, when the KD ratio of the Fc region variant is higher than that of the parent Fc region, then the Fc region variant can be considered to have increased binding selectivity for Fc gamma RIIb relative to Fc gamma RIIa relative to the parent Fc region. . With respect to human receptors, Fc-gamma RIIb binding activity is likely to correlate with Fc-gamma RIIa (R-type) binding activity rather than Fc-gamma RIIa (H-type), since the amino acid sequence of Fc-gamma RIIb is more identical to Fc- gamma RIIa (R-type) than with Fc-gamma RIIa (H-type). Thus, the identified amino acid change(s) that can(may) increase binding selectivity to human Fc-gamma RIIb compared to human Fc-gamma RIIa (R-type) is(are) ) important(s) for increasing the selectivity of binding to Fc-gamma RIIb compared to Fc-gamma RIIa in humans.

When a variant Fc region of the present invention has increased binding activity to Fc-gamma RIIb and reduced binding activity to Fc-gamma RIII (such as Fc-gamma RIIIa or Fc-gamma RIIIb) compared to the parental Fc region, then the Fc region variant can be considered to be Fc-gamma RIIb-specific.

2. Activity assays.

One of the objects of the invention are assays designed to identify antibodies to myostatin, which have biological activity. The biological activity may include, for example, inhibition of myostatin activation, blockade of the release of mature myostatin from latent myostatin, inhibition of proteolytic cleavage of latent myostatin, blockade of the availability of latent myostatin to protease, and the like. Also provided are antibodies having said biological activity in vivo and/or in vitro. In some embodiments, an antibody of the invention is tested for a specified biological activity.

In some embodiments, whether a test antibody has the ability to inhibit latent myostatin cleavage is determined by detecting the latent myostatin cleavage product (myostatin) using a method known in the art such as electrophoresis, chromatography, immunoblot analysis, enzyme-linked immunosorbent assay (ELISA) or mass spectrometry, after contacting a protease that can cleave latent myostatin with latent myostatin with or without the test antibody (see, for example, Thies et al., Growth Factors 18(4), 2001, pp. 251-259 ( 2001)). When a reduced amount of latent myostatin cleavage product (eg, human myostatin propeptide) is detected in the presence of (or after contact with) the test antibody, the test antibody is identified as an antibody that can inhibit the breakdown of latent myostatin. In some embodiments, whether a test antibody has the ability to block access to latent myostatin is determined using methods to evaluate protein interactions between protease and latent myostatin, for example, using ELISA or BIACORE®. When a decrease in the interaction between the protease and latent myostatin is detected in the presence of (or after contact with) the test antibody, the test antibody is identified as an antibody that can block the access of the protease to latent myostatin.

In some embodiments, whether a test antibody has the ability to block the release of mature myostatin from latent myostatin is determined by determining the activity of mature myostatin, e.g., myostatin receptor binding activity, or the ability to mediate signal transduction in a cell expressing the myostatin receptor (e.g., ActRIIb ). Cells that can be used in such an assay may be cells that express an endogenous receptor for myostatin, such as L6 myocytes, or may be genetically modified, transiently or stably, such that they express a transgene encoding a receptor for myostatin. , a receptor for activin, such as the type II activin receptor (Thies et al., supra). The binding of myostatin to the myostatin receptor can be assessed using a receptor binding assay. Myostatin-mediated signal transduction can be determined at any level in the signal transduction pathway, for example, by measuring Smad polypeptide phosphorylation, measuring the expression of a myostatin-regulated gene, including a reporter gene, or measuring the proliferation of myostatin-dependent cells. By reducing the activity of mature myostatin in the presence of the test antibody (or after contact with it), the test antibody is identified as an antibody that can block the release of mature myostatin from latent myostatin.

Inhibition of myostatin activation can also be detected and/or measured using the methods set forth as an example in the described embodiments of the invention. Using these assays, or other suitable assay types, test antibodies can be screened for their ability to inhibit myostatin activation. In some embodiments, the inhibition of myostatin activation comprises a reduction in myostatin activation of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% or more in this assay compared to a negative control under the same conditions. In some embodiments, it includes inhibiting myostatin activation by at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more.

Another object of the invention are assays designed to identify antibodies to myostatin, which form an immune complex (ie, an antigen-antibody complex) with myostatin. Also provided are antibodies having said biological activity in vivo and/or in vitro.

In some embodiments, an antibody of the invention is tested for a specified biological activity.

In some embodiments of the invention, the formation of the immune complex is assessed using a method such as gel filtration chromatography, ultracentrifugation, light scattering, electron microscopy or mass spectrometry (Mol. Immunol. 39, 2002, c. 77-84, Mol. Immunol. 47, 2009, pp. 357-364). The possibility of implementing these methods is based on the fact that the immune complex is a larger molecule than an individual antibody or individual antigen. If a large complex containing two or more antibodies and two or more antigens (for example, myostatin molecules) is detected in the presence of a test antibody and an antigen, then the test antibody is identified as an antibody that can form an immune complex containing two or more antibodies. and two or more myostatin molecules. In another embodiment, immune complex formation is assessed by a method such as ELISA, FACS, or SPR (Surface Plasmon Resonance Analysis; e.g., BIACORE™) (Shields et al., J. Biol. Chem. 276(9) , 2001, pp. 65916604; Singh et al., J. Immunol. Methods 50, 1982, pp. 109-114; Suzuki et al., J. Immunol. 184(4), 2010, pp. 19681976; Luo et al. ., mAbs 1(5), 2009, pp. 491-504). The applicability of these methods is based on

- 74 040713 that an immune complex containing two or more antibodies and two or more antigens can bind more strongly to an Fc receptor or component of the complement system than an antibody or antigen when used alone. If increased binding to the Fc receptor or a component of the complement system is found in the presence of both the test antibody and the antigen compared to the presence of the antibody alone, then the test antibody is identified as an antibody that can form an immune complex containing two or more antibodies and two or more number of myostatin molecules. In another embodiment of the invention, immune complex formation is assessed by administering a test antibody to an animal (eg a mouse) and measuring the clearance of the antigen from plasma. As described above, an antibody that forms an immune complex containing two or more antibodies with two or more antigens is expected to accelerate the elimination of antigens from plasma. Thus, if accelerated clearance of myostatin from plasma is found in mice injected with a test antibody compared to mice injected with a reference antibody, then the test antibody is identified as an antibody that can form an immune complex containing two or more antibodies and two or more myostatin molecules, more effective than the reference antibody.

D. Immunoconjugates.

Some embodiments of the invention are immunoconjugates comprising the myostatin antibody described herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active bacterial, fungal, , vegetable or animal origin or their fragments) or radioactive isotopes.

In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including (but not limited to) maytansinoid (see US No. 5208020, 5416064 and EP 0425235 IN 1); auristatin, for example, fragments DE and DF of monomethylauristatin (MMAE and MMAF) (see US No. 5635483, 5780588 and 7498298); dolastatin; calicheamicin or its derivatives (see US No. 5712374, 5714586, 5739116, 5767285, 5770701, 5770710, 5773001 and 5877296; Hinman and others, Cancer Res. 53, 1993, c. 3336-3342; and Lode and others. Res. 58, 1998, pp. 2925-2928); an anthracycline such as daunomycin or doxorubicin (see Kratz et al., Curr. Med. Chem. 13, 2006, c. 477-523; Jeffrey et al., Bioorg. Med. Chem. Lett. 16, 2006, c. 358-362; Torgov et al., Bioconjug. Chem. 16, 2005, pp. 717-721; Nagy et al., Proc. Natl. Acad. Sci. USA 97, 2000, pp. 829-834; Dubowchik et al. ., Bioorg. & Med. Chem. Letters 12, 2002, pp. 1529-1532; King et al., J. Med. Chem. 45, 2002, pp. 4336-4343; and US No. 6630579); methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel and orthotaxel; trichothecene and CC1065.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including (but not limited to) diphtheria toxin A chain, non-binding diphtheria toxin active moieties, exotoxin A chain ( from Pseudomonas aeruginosa), ricin A-chain, abrin A-chain, modecin A-chain, alpha-sarcin, Aleurites fordii proteins, diantine proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-5), inhibitor from Momordica charantia, curcin , crotin, an inhibitor from Sapaonaria officinalis, gelonin, mitogellin, restrictocin, fenomycin, enomycin, and trichothecenes.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A wide variety of radioactive isotopes can be used to prepare radioconjugates.

Examples include ²¹¹ At, ¹³¹ I, ¹²⁵ I, ⁹⁰ Y, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹² Bi, ³² P, ²¹² Pb and radioactive isotopes of Lu. When the radioconjugate is used for detection, it may contain a radioactive atom for scintigraphy, such as TC ^99m or I ¹²³ , or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron. Antibody-cytotoxic agent conjugates can be prepared using a wide variety of bifunctional protein-binding agents such as N-succinimidyl-3(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) , iminothiolane (IT), difunctional imidoester derivatives (such as dimethyl adipimidateχHCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bisazido derivatives (such as bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (such such as bis(para-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene-2,6-diisocyanate), and dual activity fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, the ricin immunotoxin can be prepared according to the method described in Vitetta et al., Science 238, 1987, p. 1098-1104. Labeled with C ¹⁴ 1-isothiocyanatebenzyl

- 75 040713

3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an example of a chelating agent used to conjugate a radionucleotide to an antibody (see WO 94/11026). The linker may be a cleavable linker that facilitates the release of the cytotoxic drug. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Res. 52, 1992, c. 127-131; US No. 5,208,020).

In the context of the present description, immunoconjugates or ADCs mean (but not limited to) these conjugates obtained using cross-linking reagents, which include (but not limited to) BMPS, EMCS, GMBS, HBVS, LCSMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfoKMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB, and SVSB (succinimidyl(4vinylsulfone)benzoate), which commercially available (for example, from Pierce Biotechnology, Inc., Rockford, Illinois, USA).

E. Methods and compositions for diagnosis and detection.

In some embodiments, any of the anti-myostatin antibodies provided herein can be used to detect the presence of myostatin in a biological sample. The concept of detection (detection) in the context of the present description provides for quantitative or qualitative detection. In one embodiment, the biological sample comprises a cell or tissue, e.g., serum, whole blood, plasma, biopsy sample, tissue sample, cell suspension, saliva, sputum, oral fluid, cerebrospinal fluid, amniotic fluid, ascitic fluid, milk, colostrum, mammary secretion, lymph, urine, sweat, lacrimal fluid, gastric juice, synovial fluid, peritoneal fluid, lens fluid or mucus.

One embodiment of the invention is an anti-myostatin antibody provided herein for use in a diagnostic or detection method. Another object of the invention relates to a method for detecting the presence of latent myostatin or myostatin propeptide in a biological sample. In another embodiment, the method comprises contacting a biological sample with an anti-myostatin antibody provided herein under conditions that allow the anti-myostatin antibody to bind to myostatin and detecting the formation of a complex between the anti-myostatin antibody and myostatin. Said method may be an in vitro or in vivo method. In one embodiment, the anti-myostatin antibody is used to select individuals who can be treated with the anti-myostatin antibody, for example where myostatin is a biomarker for patient selection.

Some embodiments of the invention are labeled anti-myostatin antibodies. Labels include, but are not limited to, labels or moieties that can be detected directly (such as fluorescent, chromophoric, electron dense, chemiluminescent, and radioactive labels) as well as moieties such as enzymes or ligands that are indirectly detectable, such as through an enzymatic reaction or molecular interaction. Examples of labels include, but are not limited to, ³² P, ¹⁴ C, ¹²⁵ I, ³ H, and ¹³¹ I radioisotopes, fluorophores such as rare earth metal chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases , e.g., firefly luciferase and bacterial luciferase (US No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose -6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase linked to an enzyme that uses hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals and so on.

E. Pharmaceutical compositions.

Pharmaceutical compositions of the anti-myostatin antibody provided herein are prepared by mixing said antibody, having the required degree of purity, with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th ed. Ed. Osol A., 1980) in the form of lyophilized compositions or aqueous solutions.

Pharmaceutical compositions of a polypeptide containing an Fc region variant as described herein are prepared by mixing said polypeptide, having the desired purity, with one or more optional pharmaceutically acceptable carriers in the form of lyophilized compositions or aqueous solutions.

Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and include (but are not limited to): buffers such as phosphate, citrate, and other organic acid buffers; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or

- 76 040713 benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and meta-cresol); low molecular weight polypeptides (containing less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as poly(vinylpyrrolidone); 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 counterions such as sodium; metal-containing complexes (eg Zn-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG). As used herein, examples of pharmaceutically acceptable carriers also include interstitial drug dispersing agents such as soluble neutrally active hyaluronidase glycoproteins (sHASEGP), e.g. human soluble hyaluronidase PH-20 glycoproteins such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Some examples of sHASEGP and methods of using them, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. According to one aspect of the invention, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

Examples of lyophilized antibody compositions are described in US No. 6267958. Aqueous antibody compositions include those described in US No. 6171586 and WO 2006/044908, the latter compositions include histidine acetate buffer.

The composition according to the invention may also contain more than one active substance, if necessary for the specific indication to be treated, preferably substances with complementary activities that do not adversely affect each other. Such active substances must be present in combination in amounts effective to achieve the intended purpose.

The active substances can also be included in microcapsules, for example, obtained by coacervation or interfacial polymerization methods, for example, in hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, in liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsion. Such methods are described in Remington's Pharmaceutical Sciences, 16th ed., ed. Osol A., 1980.

Sustained release formulations can be prepared. Suitable examples of sustained release preparations are semi-permeable matrices of solid hydrophobic polymers containing an antibody or conjugate, such matrices are shaped articles such as films or microcapsules.

Compositions intended for use in vivo, as a rule, must be sterile. Sterility can easily be achieved, for example, by filtration through sterile filter membranes.

G. Therapeutic Methods and Compositions.

Any of the anti-myostatin antibodies provided herein can be used in therapeutic methods. Likewise, any of the polypeptides containing the Fc region variant provided herein can be used in therapeutic methods.

One object of the invention is an antibody to myostatin, presented in the present description, for use as a medicine. Another aspect of the invention is an anti-myostatin antibody provided herein for use in the treatment of a disease. In some embodiments, an anti-myostatin antibody is provided for use in a method of treating a subject, comprising administering to the subject an effective amount of the myostatin antibody. In one of these embodiments of the invention, the method also consists in administering to the individual in an effective amount of at least one additional therapeutic agent. The individual in the context of any of the above embodiments of the invention is preferably a human.

An anti-myostatin antibody of the present invention may have pH dependent binding characteristics. In other embodiments, the invention is an anti-myostatin antibody for use in increasing the clearance of myostatin from plasma. In some embodiments, the invention is an anti-myostatin antibody for use in a method of increasing the plasma clearance of myostatin in a subject, comprising administering to the subject an effective amount of the anti-myostatin antibody to increase the clearance of myostatin from plasma. In one embodiment, an anti-myostatin antibody with pH-dependent binding characteristics increases the clearance of myostatin from plasma compared to a canonical anti-myostatin antibody that does not have pH-dependent binding characteristics. In a further embodiment of the invention, an anti-myostatin antibody with pH-dependent binding characteristics, having the ability to bind at a pH between pH 5.8 and pH 7.4, increases the clearance of myostatin from plasma compared to a canonical anti-myostatin antibody that does not

- 77 040713 has pH dependent binding characteristics. The individual in the context of any of the above embodiments of the invention is preferably a human.

The next object of the invention is the use of antibodies to myostatin for the preparation or production of drugs. In one of the embodiments of the invention, the drug is intended for the treatment of a disease. In another embodiment, the drug is used in a method for treating a disease, comprising administering to an individual having the disease an effective amount of the drug. In one of these embodiments of the invention, the method also consists in administering to the individual in an effective amount of at least one additional therapeutic agent. An individual in the context of any of the above embodiments of the invention may be a human.

An anti-myostatin antibody of the present invention may have pH dependent binding characteristics. In a further embodiment of the invention, the drug is intended to increase the clearance of myostatin from plasma. In another embodiment, the drug is for use in a method for increasing plasma myostatin clearance in a subject, comprising administering to the subject an effective amount of the drug to increase plasma myostatin clearance. In one embodiment, an anti-myostatin antibody with pH-dependent binding characteristics increases the clearance of myostatin from plasma compared to a canonical anti-myostatin antibody that does not have pH-dependent binding characteristics. In another embodiment, the anti-myostatin antibody has different pH dependent binding characteristics between pH 5.8 and pH 7.4. In one embodiment, the subject is a human.

Another object of the invention is a method for treating a disease. In one of the embodiments of the invention, the method consists in administering to an individual having the specified disease in an effective amount of the antibody to myostatin, presented in the present description. In one of the embodiments of the invention, the method also includes the introduction to the individual in an effective amount of at least one additional therapeutic agent. An individual in the context of any of the above embodiments of the invention may be a human.

An anti-myostatin antibody of the present invention may have pH dependent binding characteristics. Another embodiment of the invention is a method for increasing the plasma clearance of myostatin in an individual. In one embodiment, the method comprises administering to the subject an effective amount of an anti-myostatin antibody provided herein to increase clearance of myostatin from plasma. In one embodiment, an anti-myostatin antibody with pH-dependent binding characteristics increases the clearance of myostatin from plasma compared to a canonical anti-myostatin antibody that does not have pH-dependent binding characteristics. In another embodiment, the anti-myostatin antibody has different pH dependent binding characteristics between pH 5.8 and pH 7.4. The individual in the context of any of the above embodiments of the invention is preferably a human.

Another object of the invention are pharmaceutical compositions containing any of the antibodies to myostatin presented in the present description, which are intended, for example, for use in any of the above therapeutic methods. In one embodiment, the pharmaceutical composition comprises any of the anti-myostatin antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises any of the anti-myostatin antibodies provided herein and at least one additional therapeutic agent.

The next object of the invention is a pharmaceutical composition intended for the treatment of a disease. An anti-myostatin antibody of the present invention may have pH dependent binding characteristics. In a further embodiment of the invention, the pharmaceutical composition is for increasing the clearance of myostatin from plasma. In one of the embodiments of the invention, the pharmaceutical composition is administered to an individual who has a disease. The individual in the context of any of the above embodiments of the invention is preferably a human.

A further aspect of the invention are methods of preparing a medicament or pharmaceutical composition comprising mixing any of the anti-myostatin antibodies provided herein with a pharmaceutically acceptable carrier, for example, for use in any of the above therapeutic methods. In one of the embodiments of the invention, methods for preparing a drug or pharmaceutical composition further include adding at least one additional therapeutic agent to the drug or pharmaceutical composition.

Any of the polypeptides containing the Fc region variant provided herein

- 78 040713 can be used in therapeutic methods. A further object of the invention are pharmaceutical compositions containing a polypeptide that contains any of the polypeptides containing variants of the Fc regions presented in the present description, for example, for use in therapeutic methods. In one of the embodiments of the invention, the pharmaceutical composition includes a polypeptide that contains any of the polypeptides containing variants of the Fc regions presented in the present description, and a pharmaceutically acceptable carrier. In another embodiment of the invention, the pharmaceutical composition contains a polypeptide that contains any of the variants of the Fc regions presented in the present description, and at least one additional therapeutic agent.

One of the objects of the invention is a polypeptide containing a variant Fc region intended for use as a drug. Another aspect of the invention is a polypeptide containing a variant Fc region for the treatment of a disorder. In some embodiments, the invention is a polypeptide containing a variant Fc region for use in a method of treatment. In some embodiments, the invention is a polypeptide comprising a variant Fc region for use in a method of treating an individual who has a disorder, comprising administering to the individual in an effective amount a polypeptide comprising a variant Fc region as described herein. In one of the embodiments of the invention, the method further includes the introduction to the individual in an effective amount of at least one additional therapeutic agent. In one embodiment, the subject is a human.

Another aspect of the invention is the use of a polypeptide containing a variant Fco region for the preparation or production of a medicament. In one embodiment, the drug is for the treatment of a disorder. In some aspects of the invention, the polypeptide is an antibody. In some aspects of the invention, the polypeptide is an Fc-containing fusion protein. In another embodiment of the invention, the drug is for use in a method for treating a disorder, comprising administering to an individual who has the disorder being treated in an effective amount of the drug. In one of the embodiments of the invention, the method further includes the introduction to the individual in an effective amount of at least one additional therapeutic agent. In one embodiment, the subject is a human.

The next object of the invention is a method for treating disorders. In one embodiment of the invention, the method comprises administering to an individual who has said disorder an effective amount of a polypeptide containing a variant Fc region. In one of the embodiments of the invention, the method further includes the introduction to the individual in an effective amount of at least one additional therapeutic agent. In one embodiment, the subject is a human.

The following object of the invention are pharmaceutical compositions that contain a polypeptide containing a variant Fc region, presented in the present description, intended for use in a therapeutic method, such as any of the therapeutic methods presented in the present description. In one of the embodiments of the invention, the pharmaceutical composition contains a polypeptide that contains a variant of the Fc region presented in the present description, and a pharmaceutically acceptable carrier. In another embodiment of the invention, the pharmaceutical composition contains a polypeptide that contains a variant Fc region, presented in the present description, and at least one additional therapeutic agent.

According to a further aspect of the invention, the pharmaceutical composition is for the treatment of a disorder. In one embodiment of the invention, the pharmaceutical composition is administered to an individual who has a disorder. In one embodiment, the subject is a human.

Antibodies that have been modified to increase binding activity to Fc-gamma R, including Fc-gamma RIIb, by modifying at least one amino acid residue, may enhance antigen clearance from plasma, as described or suggested, for example, in WO 2013/047752, WO 2013/125667, WO 2014/030728 or WO 2014/163101.

Without wishing to be bound by a particular theory, an antibody having increased Fc-gamma R-binding activity under neutral pH conditions rapidly enters cells along with the antigen complexed with the antibody, and as a result, elimination of the antigen from plasma can be enhanced when the antibody is administered in vivo.

Antibodies that have been modified to have an increased pI, by modifying at least one amino acid residue that can be displayed on the surface of the antibody, may be more rapidly incorporated into cells, or may enhance antigen clearance from plasma, as described or suggested, for example in WO 2007/114319, WO 2009/041643, WO 2014/145159 or WO 2012/016227.

- 79 040713

Without going into a particular theory, it can be assumed that the pH of biological fluids (eg plasma) is in the range of neutral pH values. In biological fluids, the net positive charge of an antibody with increased pI increases due to the increase in pI, and as a result, the antibody is attracted with greater force through physicochemical Coulomb interaction to the endothelial cell surface, which has a net negative charge, compared to an antibody that does not have an increased pI. . This means that through such non-specific binding, cell penetration of the antibody in combination with its antigen is increased, resulting in increased elimination of the antigen from the plasma. It is assumed that this phenomenon is widespread in vivo regardless of cell type, tissue type, organ type, etc.

In the context of the present description, increased elimination of an antigen from plasma means, for example, that the rate of elimination of an antigen from plasma is increased compared to the rate when a polypeptide is administered that contains the corresponding but not similarly modified Fco region. The increase in antigen elimination from plasma can be assessed, for example, by measuring the plasma concentration of the antigen. Various methods are known in the art for measuring plasma antigen concentration.

In other embodiments, the invention is a polypeptide containing a variant Fco region for use in enhancing antigen clearance from plasma. In some embodiments, the invention is a polypeptide that contains an Fc region variant for use in a method of enhancing elimination of an antigen from plasma in an individual, comprising administering to the individual an effective amount of a polypeptide that contains an Fc region variant to enhance elimination of the antigen. from plasma. In these embodiments, it is preferred that the polypeptide further comprises an antigen-binding domain. The individual in the context of any of the above embodiments of the invention is preferably a human.

Another object of the invention is the use of a polypeptide that contains a variant Fc region for the manufacture or production of a drug. In some aspects of the invention, the polypeptide is an antibody. In some aspects of the invention, the polypeptide is an Fc-containing fusion protein. In another embodiment of the invention, the drug is intended to enhance the elimination of antigen from plasma. In a further embodiment of the invention, the drug is for use in a method for enhancing plasma antigen clearance in an individual, comprising administering to the subject an effective amount of the drug to enhance plasma antigen clearance. In these embodiments, it is preferred that the polypeptide further comprises an antigen-binding domain. An individual in the context of any of the above embodiments of the invention may be a human.

Another embodiment of the invention is a method for enhancing the elimination of an antigen from plasma in an individual. In one embodiment of the invention, the method comprises administering to an individual an effective amount of a polypeptide that contains a variant Fc region to enhance clearance of the antigen from plasma. In these embodiments, it is preferred that the polypeptide further comprises an antigen-binding domain. In one embodiment, the subject is a human.

The following object of the invention are pharmaceutical compositions that contain any of the polypeptides containing the variant Fc region presented in the present description. The next embodiment of the invention is a pharmaceutical composition designed to enhance the elimination of antigen from plasma. In this embodiment, it is preferred that the polypeptide further comprises an antigen binding domain.

In one of the embodiments of the invention, compared with the variant before the amino acid modification, an antibody that has an Fc region variant of the present invention increases the elimination of antigen from plasma, for example, at least 1.1, 1.25, 1.5 , 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4 .75, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times or more at administration of the antibody in vivo.

A further aspect of the invention are methods of preparing a medicament or pharmaceutical composition comprising mixing any polypeptides containing the Fc region variants described herein with a pharmaceutically acceptable carrier, for example, for use in any of the above therapeutic methods. In one of the embodiments of the invention, methods for preparing a drug or pharmaceutical composition further include adding at least one additional therapeutic agent to the drug or pharmaceutical composition.

The antibodies of the invention can be used for treatment either alone or in combination with other agents. For example, an antibody of the invention may be co-administered with at least one additional therapeutic agent.

Similarly, polypeptides containing an Fc region variant of the invention

- 80 040713 for treatment can be used either alone or in combination with other agents. For example, a polypeptide containing a variant Fc region of the invention may be co-administered with at least one additional therapeutic agent.

Such combination therapies as mentioned above include co-administration (when two or more therapeutic agents are included in the same or different compositions) and separate administration, in each case administration of an antibody or polypeptide containing an Fc region variant of the invention, may be performed before, concurrently and/or after administration of the additional therapeutic agent or agents. In one embodiment, administration of the anti-myostatin antibody and administration of the additional therapeutic agent may be within about a month, or within about 1, 2, or 3 weeks, or within about 1, 2, 3, 4, 5, or 6 days of each other. friend.

In another embodiment of the invention, the administration of the polypeptide containing the Fco region variant and the administration of the additional therapeutic agent can be within about a month, or within about 1, 2, or 3 weeks, or within about 1, 2, 3, 4, 5, or 6 days. relative to each other.

An antibody or polypeptide containing an Fc region variant of the invention (and optionally any additional therapeutic agent) can be administered by any suitable route, including parenteral, intrapulmonary and intranasal, and, if necessary, used for topical treatment, injection into damage. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal or subcutaneous administration. Dosing may be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending, inter alia, on whether the treatment is short-term or chronic. Various dosing regimens may be used, including (but not limited to) single administration or multiple administrations at different time points, bolus administration, and pulsatile infusion.

Antibodies or polypeptides containing the Fc region variant of the invention may be formulated, dosed, and administered in accordance with good clinical practice. Factors considered 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 administration of the agent, the method of administration, the schedule of administration, and other factors known to medical practitioners. The antibody may, but is not required, be included in the composition in combination with one or more other agents currently used to prevent and treat the disorder in question. The effective amount of these other agents depends on the amount of antibody present in the composition, the type of disorder or treatment, and other factors mentioned above. They are generally used in the same doses and using the same routes of administration as specified in the present description, or in an amount ranging from 1 to 99% of the dose indicated in the present description, or in any dose and with any pathways that are empirically/clinically judged to be suitable.

For the prevention or treatment of a disease, the acceptable dose of an antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) should depend on the type of disease being treated, the type of antibody, the type of polypeptide containing the variant Fc region , severity and course of disease, whether the antibody or polypeptide containing the Fc region variant is used for preventive or therapeutic purposes, prior therapy, the patient's medical history and response to the antibody or polypeptide containing the Fc region variant, as well as the prescription of the attending physician. An antibody or polypeptide containing a variant Fc region can be administered to a patient at a single time or as a series of treatments. Depending on the type and severity of the disease, from about 1 μg/kg to 15 mg/kg (e.g., 0.5-10 mg/kg) of the antibody may represent the initial possible dose to be administered to the patient, e.g., via one or more separate treatments. or by continuous infusion. One example of a typical daily dose may be from about 1 μg/kg to 100 mg/kg or more, depending on the above factors. When repeated administrations are used over a period of several days or a longer period of time, depending on the condition, treatment should generally be continued until suppression of the symptoms of the disease is achieved. One example of doses of an antibody or polypeptide containing a variant Fc region is from about 0.05 to about 10 mg/kg. Thus, one or more of the following doses may be administered to a patient: about 0.5, 2.0, 4.0, or 10 mg/kg (or any combination thereof). These doses can be administered in fractions, such as every week or every three weeks (eg, so that the patient receives from about two to about twenty, for example, about six doses of an antibody or polypeptide containing a variant Fc region). An initial increased loading dose may be used followed by one or more lower doses. Using conventional methods and assays, the effect of said therapy can be easily monitored.

Obviously, any of the above compositions or therapeutic methods can be used using an immunoconjugate of the invention, instead of or in addition to an anti-myostatin antibody.

Obviously, any of the above compositions or therapeutic methods can be used using the immunoconjugate of the invention, instead of or in addition to the polypeptide containing the variant Fc region presented in the present description.

3. Products.

Another object of the invention is a product that contains products used for the treatment, prevention and/or diagnosis of the above disorders. The product is a container and a label or package insert placed on or inside the container. Acceptable containers are, for example, jars, vials, syringes, intravenous solution bags, etc. Containers can be made from various materials such as glass or plastic. The container contains a composition that alone or in combination with another composition is effective in treating, preventing, and/or diagnosing a condition, and may have a sterile access port (for example, the container may be an intravenous solution bag or vial provided with a stopper that can be pierced using a hypodermic needle). At least one active ingredient in the composition is an antibody of the invention. The label or package insert indicates that the composition is used to treat the selected condition. In addition, the product may include (a) a first container containing the composition, where the composition contains the antibody proposed in the present invention; and (b) a second container containing the composition, wherein the composition contains an additional cytotoxic or other therapeutic agent. According to this embodiment of the invention, the product may contain a package insert leaflet that contains information that the compositions can be used to treat a particular condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container with a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BVI), phosphate buffered saline, Ringer's solution and dextrose solution. In addition, it may include other products required from a commercial and consumer point of view, in particular other buffers, diluents, filters, needles and syringes.

As should be obvious, any of the above products may include the immunoconjugate of the invention, instead of or in addition to the antibody to myostatin.

Examples

Below are examples of methods and compositions proposed in the invention. As should be obvious, various other embodiments of the invention can be practiced in view of the general description of the invention presented above.

Example 1

Expression and purification of latent and mature forms of human myostatin, cynomolgus monkeys and mice.

Human latent myostatin (herein also referred to as the latent form of human myostatin) (SEQ ID NO: 1) was transiently expressed using FreeStyle293-F cells (FS293-F cells) (Thermo Fisher, Carlsbad, CA, USA) . Conditioned media containing the expressed latent form of human myostatin were acidified to pH 6.8 and diluted 1/2 volume with milliQ water before being loaded onto a Q-sepharose FF ion exchange column (GE healthcare, Uppsala, Sweden). The pH of the permeate fraction was adjusted to pH 5.0 and loaded onto a HP SP Sepharose cation exchange column (GE healthcare, Uppsala, Sweden) and then eluted using a NaCl gradient. Fractions containing latent human myostatin were collected and then subjected to gel filtration on a Superdex 200 column (GE healthcare, Uppsala, Sweden) equilibrated with 1x PBS. Then the fractions containing the latent form of human myostatin were pooled and stored at -80°C.

Human mature myostatin (herein also referred to as mature human myostatin) (SEQ ID NO: 2) was purified from purified latent human myostatin. Latent human myostatin was acidified by adding 0.1% trifluoroacetic acid (TFA) and applied to a Vydac 214TP C4 reverse phase column (Grace, Deerfield, IL, USA) and eluted using a TFA/CH ₃ CH gradient. containing human mature myostatin were combined, dried and stored at -80°C. For reconstitution, human mature myostatin was dissolved in 4 mM HCl.

Expression and purification of latent and mature myostatin from cynomolgus monkeys (cynomolgus or cyno) (SEQ ID NOS: 3 and 4, respectively) and mice (SEQ ID NOS: 5 and 6, respectively) was carried out in all cases in exactly the same manner as in the case of human copy. With respect to sequence homology, mature human, cyno, and mouse myostatin were 100% identical, so any mature myostatin, regardless of species, was used as mature myostatin in all experiments performed.

- 82 040713

Example 2

Identification of antibodies to latent myostatin.

Antibodies to latent myostatin were obtained, selected and analyzed as follows.

New Zealand White Rabbits (NZW) aged 12-16 weeks were immunized intradermally with mouse latent myostatin and/or human latent myostatin (50-100 μg/dose/rabbit). Treatment with the indicated dose was repeated 3-4 times within 1 month. Spleens and blood samples from the immunized rabbit were collected 1 week after the last immunization. Antigen-specific B cells were stained with labeled antigen, sorted using an FCM cell sorter (FACS aria III, BD) and plated in 96-well plates at a density of 1 cell/well along with 25,000 EL4 cells/well (cells from the European Cellular Collection). cultures) and together with conditioned medium for rabbit T-cells, diluted 20 times, and cultured for 7-12 days. EL4 cells were pretreated with mitomycin C (Sigma) for 2 hours and washed 3 times. Rabbit T cell conditioned medium was prepared by culturing rabbit thymocytes in RPMI-1640 medium containing phytohemagglutinin-M (Roche), phorbol-12-myristate-13-acetate (Sigma) and 2% FBS. After cultivation, B cell culture supernatants were collected for further analysis and the debris was cryopreserved.

An ELISA assay was used to evaluate the specificity of antibodies in the B cell culture supernatant. MAXISorp 384-well plates (Nunc) were sensitized with streptavidin (GeneScript) at 50 nM in PBS for 1 hour at room temperature. The plates were then blocked with Blocking One reagent (Nacalai Tesque) diluted 5 times. Human or mouse latent myostatin was labeled with NHS-PEG4-biotin (PIERCE) and added to 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 loaded onto ELISA plates, incubated for 1 h and washed with TBS-T. Binding was detected with goat anti-rabbit IgG conjugated to horseradish peroxidase (BETHYL) with ABTS (KPL).

A total of 17818 B cell lines were screened for specific binding to mouse and/or human latent myostatin, 299 lines were selected and designated as MST0255-287, 630-632, 677-759, 910, 932-1048, 1050-1055 , 1057-1066, 1068, 1070-1073, 1075-1110, 11131119. RNA was purified from appropriate cell debris using ZR-96 Quick-RNA kits (ZYMO RESEARCH).

DNA encoding the heavy and light chain variable regions of each selected cell line was amplified by reverse transcription PCR and cloned into expression vectors with the Glm heavy chain constant region sequence (SEQ ID NO: 50 (amino acid sequence shown in SEQ ID NO: 7) ) and with the light chain constant region sequence k0MTC or k0MC (SEQ ID NO: 53 or 194 (amino acid sequence (same in both cases) shown in SEQ ID NO: 8)) respectively. Recombinant antibodies were transiently expressed using FreeStyle FS293-F and 293 fectin cells (Life technologies) according to the manufacturer's instructions. Culture supernatant or recombinant antibodies were used for screening. Recombinant antibodies were purified with protein A (GE Healthcare) and eluted in D-PBS or His buffer (20 mM histidine, 150 mM NaCl, pH 6.0). Additionally, if necessary, gel filtration was performed to remove the high molecular weight and/or low molecular weight component.

Example 3

Characterization of antibodies to latent myostatin (HEK-Blue-analysis (BMP 1-activation)).

Reporter gene analysis was used to assess the biological activity of active myostatin in vitro. HEK-Blue™ TGF-beta cells (Invivogen), which express Smad3/4-binding element (SBE)-indicated SEAP (Secreted Alkaline Phosphatase) reporter genes, allow for the detection of bioactive myostatin by monitoring receptor activation for Type 1 and Type 1 Activin. 2.

Active myostatin stimulates the production of SEAP, which is secreted into the cell supernatant. The amount of SEAP secreted is then determined using QUANTIBlue™ (Invivogen).

HEK-Blue™ TGF-beta cells were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum, 50 µg/ml streptomycin, 50 U/ml penicillin, 100 µg/ml Normocin™, 30 µg/ml blasticidin, 200 µg/ml HygroGold™ and 100 µg/ml Zeocin™. When performing a functional assay, cell medium was changed to assay medium (DMEM supplemented with 0.1% bovine serum albumin, streptomycin, penicillin, and Normocin™) and plated in a 96-well plate. Human, cynomolgus or mouse latent myostatin were incubated with recombinant human BMP1 (R&D Systems) and anti-latent myostatin antibody at 37° C. overnight. Sample mixtures were transferred into cells. After 24 hours of incubation, cell supernatants were mixed with QUAN-83 040713

TIBlue™ and measured absorbance at 620 nm in a colorimetric plate reader. As shown in FIG. 1, mAb MST1032-Glm (see Table 2a for amino acid and nucleotide sequence) interfered with protease-mediated activation of human, cynomolgus, and mouse latent myostatin, as evidenced by reduced concentrations of secreted SEAP. It was demonstrated that MST1032-Glm had almost comparable inhibitory activity against human latent myostatin, cynomolgus and mice.

Table 2a

Antibodies to latent myostatin and their DNA and amino acid sequences (their SEQ ID NO: are indicated)

Antibody designation Reduction , Variable region Constant region Heavy Light Heavy Light cnc protein DNA protein DNA protein DNA protein MS_MST1032Ha-Glm/MST1032La-k0MC MST1032-Glm 039 012 041 014 050 007 194 008 MS_MST1032Ha-F760/MST1032La-k0MC MST1032-F760 039 012 041 014 051 011 194 008 MS_M103205H000-SGl/M103202L000-SKl MS1032LO00-SG1 or AbOOl 040 013 042 015 052 009 054 010 MS_M 103205 H000-F760/M103 202L000-5K1 MS1032LO00-F76O 040 013 042 015 051 011 054 010 M S_M 103205 H 714-5 G1/ M103202L861-SK1 MS1O32LOO1-5G1 043 032 046 035 052 009 054 010 M S_M 103205 H 781-S G1/ M103202L719-SK1 MS1O32LOO2-SG1 044 033 047 036 052 009 054 010 MS_M1O32O5H781-SG1/M1O32O2L813-5K1 MS1O32LOO3-SG1 044 033 048 037 052 009 054 010 M S_M 103205 H 707-5 G1/ M103202L802-SK1 MS1032L004-SG1 045 034 049 038 052 009 054 010 MS_M103205H714-F760/M103202L861-SK1 MS1032LO01-F760 043 032 046 035 051 011 054 010 MS_M103205H781-F760/M103202L719-5K1 M51032LO02-F760 044 033 047 036 051 011 054 010 MS_M103205H781-F760/M103202L813-5K1 MS1032LO03-F760 044 033 048 037 051 He 054 010 M5_M103205H707-F760/M103202L802-SK1 M51032LO04-F760 045 034 049 038 051 He 054 010 Table 2b Amino acid sequences of HRV antibodies to latent myostatin (their SEQ ID NO: are indicated) Hypervariable region (HRV) Antibody designation Reduction H1 H2 NZ L1 L2 L3 MS_MST1032Ha-Glm/MST1032La-k0MC MST1032-Glm 055 058 061 065 070 073 MS_MST1032Ha-F760/MST1032La-k0MC MST1032-F760 055 058 061 065 070 073 MSM103205H000-SG1/M103202L000-SK1 MS1032LO00-SG1 or AbOOl 055 058 061 065 070 073 MS_M 103205 H000-F760/M103202L000-SK1 MS1032LO00-F760 055 058 061 065 070 073 MS_M103205H714-SGl/M103202L861-SKl MS1032LO01-SG1 057 058 053 067 071 074 MS_M103205H781-SGl/M103202L719-SKl MS1032LO02-SG1 057 060 063 066 072 073 MS_M103205H781 SG1/M103202L813 SKI MS1032LO03 SGI 057 060 063 068 070 074 MS_M103205H707-SGl/M103202L802-SKl MS1032LO04-SG1 056 058 063 069 071 073 MS_M103205H714-F760/M103202L861-SKl MS1032LO01-F760 057 058 063 067 071 074 MS_M103205H781-F760/M103202L719-SKl MS1032LO02-F760 057 060 063 066 072 073 MS_M103205H781-F760/M103202L813-SKl MS1032LO03-F760 057 060 063 068 070 074 MS_M103205H707-F760/M103202L802-SKl MS1032LO04-F760 056 058 063 069 071 073

Example 4

Characterization of anti-latent myostatin antibody (HEK Blue assay (spontaneous activation)).

Human, cynomolgus or mouse latent myostatin were incubated with anti-latent myostatin antibody at 37° C. overnight and the sample mixture was loaded into HEK-Blue TGF-beta cells. After a 24 hour incubation, the cell supernatant was mixed with QUANTIBlue™ and the absorbance was measured at 620 nm in a colorimetric plate reader. As shown in FIG. 2, after incubation at 37° C., active myostatin was released from the latent form. The presence of the mAb MST1032-Glm inhibited myostatin activation and, as a result, a lower level of SEAP was detected in the cell supernatant. MST1032-Glm inhibited spontaneous activation of latent myostatin and showed almost comparable inhibitory activity against human, cynomolgus and mice latent myostatin.

Example 5

Characterization of antibodies to latent myostatin (ELISA).

Unsensitized ELISA plates (NUNC-IMMUNO plate, MAXISORP surface, Nalge Nunc International) were coated with 20 μl of 50 nM streptavidin (GenScript) for 1 hour at room temperature. The plates were then washed three times with PBS and blocked overnight with 50 μl of 20% Blocking One (Nacalai Tesque). The following 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 chimeric protein (R&D Systems) at 4 nM/well/20 µl for 2 hours. After washing 20 μl of antibody sample was added to the wells and the plates were incubated for 1 h. The plates were washed and 20 μl of anti-human IgG conjugated with horseradish peroxidase (HRP) (Abcam) diluted in HEPES buffered saline was added and the plates were incubated for another 1 hour. The plates were then washed again and then 50 μl of ABTS (KPL) were 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 experiment on

- 84 040713 binding are shown in FIG. 3. MST1032-Glm binds to latent myostatin (ie, a non-covalent complex of mature myostatin and propeptides) and propeptides, but does not bind to mature myostatin. These results indicate that MST1032-Glm specifically binds to myostatin propeptide (eg, the propeptide portion), but does not bind to active myostatin (eg, the mature region).

Example 6

Characterization of Anti-Latent Myostatin Antibody (Western Blotting) Mouse latent myostatin was incubated with recombinant human BMP1 (R&D Systems) with or without MST1032-Glm antibody at 37° C. overnight. The samples were then mixed with 4x SDS-PAGE reconstitution sample buffer (Wako) and held at 95° C. for 5 minutes and then loaded onto the SDS gel electrophoresis system. Proteins were transferred to the membrane using a Trans-Blot Turbo transfer system (Bio-rad). Myostatin propeptide was detected using a sheep anti-mouse GDF8 propeptide antibody (R&D Systems), which was then detected using an anti-sheep IgG-HRP antibody (Santa Cruz). The membrane was incubated with ECL substrate and visualized using an ImageQuant LAS 4000 (GE Healthcare). As shown in FIG. 4, propeptide cleavage by BMP1 was inhibited by MST1032-Glm.

Example 7

Characterization of an antibody to latent myostatin (BIACORE®).

The kinetic parameters of latent myostatin antibodies against human latent myostatin, cynomolgus (supo) monkeys and mice were evaluated at 37° C. at pH 7.4 using a BIACORE® T200 device (GE Healthcare). ProA/G (Protein A/G) (Pierce) was immobilized on all flow cells of the CM4 chip using an amine coupling kit (GE Healthcare). Anti-latent myostatin antibody and analytes were prepared in ACES, pH 7.4 (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl ₂ , 0.05% Tween 20, 0.005% NaN ₃ ). The antibody was captured on the surface of the sensor chip using ProA/G. Antibody immobilization levels typically corresponded to 150-220 resonance units (RU). Human, supo or mouse latent myostatin was then injected at concentrations ranging from 3.125 to 50 nM, which were obtained by two-fold serial dilutions and then allowed to dissociate. 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® T200 Evaluation software version 2.0 (GE Healthcare). The sensorgrams are shown in Fig. 5. Data on the rate of association (ka), rate of dissociation (kd) and binding affinity (KD) are presented in table. 3. The kinetic parameters of MST1032-Glm, which characterize the binding to latent myostatin in humans, supo, and mice, were comparable.

Table 3

The values of ka, kd and KD of the antibody to latent myostatin MST1032-Glm

ka (M s ) kd (with ) KD (M) Human latent myostatin 1.13E+06 8.65Е-05 7.66E-11 Mouse latent myostatin 1.41E+06 8.16Е-05 5.79E-11 Monkey (supo) latent myostatin 1.09E+06 7.79Е-05 7.17E-11

Example 8

Humanization of antibodies to latent myostatin.

Variable region amino acid residues were numbered according to Cabot (Kabat et al., Sequence of proteins of immunological interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

Heavy and light chain variable regions were created for humanized MST1032 antibodies. Some of the humanized MST1032 antibodies contained a reverse mutation in the framework region. The polynucleotides of the designed heavy and light chain variable regions were synthesized by GenScript Inc. and cloned into expression vectors containing the SG1 heavy chain constant region sequence (SEQ ID NO: 52 (amino acid sequence shown in SEQ ID NO: 9)) and SK1 light chain constant region sequence (SEQ ID NO: 54 (amino acid sequence shown in SEQ ID NO: 10)) respectively. Humanized antibodies were transiently expressed in FS293-F cells and HEK Blue and BIACORE® assays were performed as described above. As shown in FIG. 6 and in table. 4 and when compared with FIG. 1 and 2, the humanized antibody (MS1032LO00-SG1) was found to have inhibitory activity and affinity comparable to the chimeric antibody (MST1032-Glm).

- 85 040713

Table 4

Kinetic parameters of a humanized antibody to latent myostatin

ka (M'L' ¹ ) kd (c' ¹ ) KD (Μ) MST1032-Glm 1.13E+06 8.65E-05 7.66E-11 MS1032LO00-SG1 1.27E+06 9.52E-05 7.50E-11

Example 9

Creation of a pH-dependent antibody to latent myostatin.

To create pH-dependent antibodies to latent myostatin, histidine-scanning mutagenesis of all CDRs of mAb MS1032LO00-SG1 was performed. Each amino acid in the CDR was individually replaced with histidine using an In-Fusion HD cloning kit (Clontech Inc. or Takara Bio company) according to the manufacturer's instructions. After sequencing confirmed the correct mutation in each variant, the variants were transiently expressed and purified as described above. All histidine substitution variants were evaluated using the BIACORE® assay modified from the assay described above. In general, the method was as follows: in the BIACORE® assay, an additional dissociation phase at pH 5.8 was carried out immediately after the dissociation phase at pH 7.4. This was done to evaluate the pH-dependent dissociation of antibody (Ab) and antigen (Ag) from complexes prepared at pH 7.4 compared to the corresponding dissociation at pH 5.8. The dissociation rate in pH 5.8 buffer was determined by data processing and fitting using Scrubber 2.0 curve fitting software (BioLogic Software).

As shown in FIG. 7, the parent antibody (Ab001) showed no decrease in binding response at pH 5.8 compared to the dissociation phase at pH 7.4. Several single histidine substitutions resulted in a moderate to strong reduction in binding response at pH 5.8 compared to the dissociation phase at pH 7.4. The dissociation rate at pH 5.8 for each variant with a single replacement for histidine is presented in Table. 5. As shown in Table. 5, for Ab002, the fastest rate of dissociation from the Ab/Ag complex at pH 5.8 was found, exceeding the rate for the parent antibody (Ab001) by more than 200 times. Then created antibodies with a combination of these mutations in the CDR. The CDR sequences of antibodies containing these various mutations are shown in Table 1. 6.

Table 5

Dissociation rate at pH 5.8 of antibodies to latent myostatin with single substitutions for histidine

Variable region designation Reduction kd (1/s) heavy chain light chain M103205 H000 M103202L000 AL001 2.92E-05 М103205Н001 M103202L000 AL002 6.09Е-03 М103205Н009 M1O3202LOOO ALOOD 8.8DE-04 М103205Н026 M103202L000 AL004 1.86E-04 М103205Н028 M103202L000 AL005 7.63Е-05 М103205Н000 M103202L008 ALOOB 8.58Е-05 М103205Н028 M103202L008 AL007 4.15E-04

Table 6

CDR sequences and their SEQ ID NOs of antibodies to latent myostatin with substitutions for histidine

heavy chain

Antibody designation Variab. region Hypervariab. region (HRV) 34 35 50 51 52 53 54 55 56 Cabot numbering H1 "2 NZ 3i 32 33 57 53 59 60 61 62 53 64 65 95 96 97 S3 No. 100 too* ioc® loot ιοί 102 М10М05НМ0 013 055........._ oea 061 5 ¥0 ) 5 eleven $ γ A 0 5 T ¥ Y A S W A K 6G V ^p . A ¥_ _$__T_.....£ _G___0 J. M1032O5NO01 016 056 055 061 Η Y D ί s eleven S VA G S T Y Y A S W A K G G V P A Y S T G G 0 1 M1O32O5NOO9 017 055__ 059_ 061 s ¥D ί 5 I 1 s H A 0s T ¥ Ύ A S WAX G G v P A ¥ _5 _T G G D L M1O32O5NO26 _ _ °!? ^_055_ 05 V 062 ¥0 I S ! 1 s ¥ A G5 T ¥ ¥ A S W A K G G A S _T G_ G_ P_L M1O32OSHO28 019 055___ 053_ __ 063 J___ 1 5 1 t 5 ¥ A "s T ¥ Y.....A $ W A K G G V A_ ... ...7 __ ^L - M10320StW33 020 086"" 059 061 Η ¥0 ί s 1 t s H A S5 τ ¥ ¥ A S WAX S G V P 'a' ¥ S T G G 0 L — *---...-4-4-----. -^,.ΙΪ. -1·..- ,.-.,,,--,-... ..... .. - ._.| - . . 1 - I - I - I 4 . I-I. .. ----*. .... .-.. .... ,. _u ____, . ..-- ,,.____ . .... ... .. . ..,. ..,,,,....1. , ._ ,__., . _ . - -., M10320SH034 021 055_ 059 062 h Y 0 ! s ίΪ 5 H A G'4 T Y Y A S WAX G G V P A H $ T G G _0 L М10320514035 022 056 058 062 Η ¥D 1 s ! $ S VA G5 T Y ¥ A 5 WAX G G V P A H S 7 G G D L M1O32O5NO36 023 ~056^7 059 062 ....... Η „G A i 5 I 1 s H A G S T Y Y A S WAX G G V 9 A H........S T G _G C- L_ M1O32OSH7O7 _024 056_ 053 063 Η Y 0 ί 5 I 1 5 VA _G S T Y ¥ A S W _A X S G_ V ₽ A ¥ S_H G G_D L M10320bHJ8 O2b □b 059 063 5 V D ί S eleven S H A G S T ¥ Ύ A S WAX G G V P A ¥ S H G G U L Μ1Ο32Ο5Η7Ο9 026 055 058 064 S Y 0 ί s eleven 5 Y A G S T ί Y A S WAX G G >¥ P A H S H G G 0 L М1О32О5Н71О _O27 __O56 059_ 063_ _ _Y 0 j s 1 j J H^A G i T _Y Y_ A 2 W_A G7- ^G V p A _¥_ S H G__G _C _ L M1D32O5H711 028 , , -, ^O55 . 059 064 5_ Y 0 I s 1J 5 H A G.S T Y Y A S W A K G G^ V P A U S H G G 0 L М1О32О5Н712 329 056 0=8 064 Η ¥D G 9 eleven 5 Y A G S T Ύ Y A 5 WAX G G V p A H 9 H G G 0 L II - , -, .... . i. LI.-. .J-IJ-. 111 - .. I U -. - .111.1. 1..——J-1. .1 .11. - . ".,1 - -1..-1-..41.. 1 I". . 1 II II I 1 -IJ .. — 111--1-4=- I......1:.,.,,4-.. _--,.-11-.-.1 1,,,14, . .... .. ... .., ..... ,.·...·· -.1-1 Ш11-1--:-..=1- 1. --,---- "- - ---- ,, ", --- - - ........ -11..- 4. - I-I - ......- -- I 1 1, ... 1—1 ,. , -eleven- " -.,·,.,··.,........ -1. .,.. ,,-. . 4 1. L1—1JI-1...,. ...1. .4*^ M1O3205H713 030 056 059 064 Η Y 0 3 i! 3 H A G3 T ¥ ¥ A 3 W A K G G V P A H 5 H G G P L light chain Designation Variab. region Gpervariab . region (HRV) Cabot numbering antibodies U 12 . L3 24 25 26 27 27a 27b 25 29 thirty 31 32 33 34 50 51 52 S3 54 55 56 89 90 91 92 .93 94 95 95* 96 97 М1032021000 ^_015 065 070 073 Q JQ ¥___D N W I s W A 5 T I A S ......A G Y _G__G___L____Y A m 103202100a 032 066 070 073 α 5 5 Q 5V ¥H N N W L S W A 5 T 1A$ A G G Y G G G I YA

Example 10

Increasing the affinity of pH-dependent antibodies to latent myostatin.

To identify mutations that increase affinity at pH 7.4 and/or in vitro inhibitory activity, more than 500 heavy and light chain variants, respectively, were generated using at least one variant generated in Example 9 as a template. In these embodiments, each amino acid in the CDR was replaced with 18 other amino acids, with the exception of the original amino acid and cysteine. The binding capacity of the variants to human latent myostatin was evaluated at 37° C. at pH 7.4 using a BIACORE® 4000 device (GE Healthcare). The culture supernatant containing variants in FS293-F cells was prepared in ACES buffer, pH 7.4, containing 10 mg/ml BSA and 0.1 mg/ml carboxymethyl dextran (CMD). Each antibody was captured on the flow cells until an immobilization level of approximately 200 RU was reached. Then 25 nM human latent myostatin was injected into the flow cells. In the BIACORE® assay, an additional dissociation phase was performed at pH 5.8 immediately after the dissociation phase at pH 7.4. In this additional dissociation phase, the pH-dependent dissociation of the antibody (Ab) and antigen (Ag) from the complexes obtained at pH 7.4 was evaluated. The flow cell surface was regenerated with 25 mM NaOH. Kinetic parameters were determined using BIACORE™ 4000 Evaluation software version 2.0 (GE Healthcare). Analysis of additional dissociation at pH 5.8 was carried out using Scrubber2 (software from BioLogic).

Variants with increased affinity at pH 7.4 and/or in vitro inhibitory activity were selected and combined with the pH enhancing mutations identified in Example 9. After combining these mutations, 4 variants (MS1032LO01, 02, 03 and expressed with SG1 or F760 (SEQ ID NOs: 11 and 51) for in vitro and/or in vivo kinetic binding assays with BIACORE®. Both SG1 and F760 are human heavy chain constant regions. SG1 binding affinities to human Fc-gamma R are comparable to the constant region affinity of naturally occurring IgG1, but F760 affinity is eliminated due to Fc modification (referred to herein also as Fc silencing). Amino acid and nucleotide sequences of four variants of antibodies to myostatin are presented in table. 2a.

Example 11.

Characterization of pH dependent MS1032 variants (HEK Blue analysis (BMP1 and spontaneous activation)).

All experimental conditions were as described in Examples 3 and 4. As shown in FIG. 8, all variants exhibit inhibitory activity comparable to MS1032LO00-SG1 against human latent myostatin.

Example 12.

Characterization of pH dependent variants of MS1032 (BIACORE®).

All experimental conditions were as described in Example 7, except that measurements were also made in ACES buffer, pH 5.8, in addition to ACES conditions, pH 7.4. The activity of some antibodies was evaluated only against human latent myostatin. The antibody immobilization level should correspond to 185 RU and 18.5 RU for avidity and affinity analysis, respectively. Kinetic parameters were determined by fitting the data to a 1:1 binding model using BIACORE® T200 Evaluation software version 2.0 (GE Healthcare). Sensorgrams for all anti-human latent myostatin antibodies obtained under the conditions used to evaluate avidity are shown in FIG. 9, a ka, kd, and KD calculated for various conditions are presented in Table. 7-10. Under the conditions used to evaluate avidity, all antibodies except MS1032LO00-SG1 showed a faster rate of dissociation at acidic pH than at neutral pH. Under the conditions used to evaluate the affinity, all antibodies except MS1032LO00-SG1 showed little interaction with latent myostatin at acidic pH and therefore kinetic parameters could not be determined.

Table 7

ka, kd and KD values obtained under the conditions used for avidity analysis at neutral pH

people latent myostatin mice. latent myostatin supo latent myostatin ka (M 's ¹ ) kd (with ¹ ) KD (M) ka (M 's ¹ ) kd (with ¹ ) KD (M) ka (M 's ¹ ) kd (with ¹ ) KD (M) MS1032LO00-SG1 1.27E+06 9.52E-O5 7.50E-11 1.52E+06 7.18E-O5 4.71E-11 1.21E+O5 8.08Е-05 6.66E-11 MS1032LO01-SG1 1.27E+06 2.16E-04 1.70E-1O 1.45E+06 2.17E-04 1.49E-10 1.19E+06 1.97E-04 1.65E-10 MS1032LO02-SG1 8.36E+05 2.49E-O4 2.98E-1O N/T N/T N/T N/T N/T N/T MS1032LO03-SG1 9.81E+O5 1.92E-O4 1.96E-1O N/T N/T N/T N/T N/T N/T MS1032LO04-SG1 1.33E+06 2.17E-04 1.63E 10 N/T N/T N/T N/T N/T N/T

N/Τ; not tested

- 87 040713

Table 8

ka, kd and KD values obtained under the conditions used for the analysis of avidity at acidic pH

people latent myostatin mice. latent myostatin supo latent myostatin ka (M 's ¹ ) kd (with ') KD (M) ka (M 's ¹ ) kd (with ') KD (M) ka (M 's ¹ ) kd (with ') KD (M) MS1032LO00-SG1 1.60E+06 1.28E-04 7.97E-11 2.32E+O6 2.O2E-O5 8.7ΏΕ-12 1.79E+O6 1.58E-O5 8.83Ё-12 MS1032LO01-SG1 1.22E+06 2.42E-O2 L99E-08 1.02E+06 6.25E-03 6.15E-09 7.64E+05 6.80E-03 8.90E-09 MS1O32LO02SG1 1.70E+06 4.14E 02 2.43E-08 NT N/T NT N/T N/T N/T MS1O32LO03-SG1 5.43E+0S S.24E-O3 9.66E09 NT N/T NT N/T N/T N/T MS1032LO04-SG1 7.81E+05 7.13E-O3 9.13Е-09 NT N/T NT N/T NT NT

N/Τ: not tested

Table 9 ka, kd and KD values obtained under the conditions used for affinity analysis at neutral pH human. mouse latent myostatin, latent myostatin supo-latent myostatin

ka (M 's ¹ ) kd (c' ¹ ) KD (M) ka (M's') kd (c' ¹ ) KD (Μ) ka (M 'c ¹ ) kd (c ¹ ) KD(M) MS1032LO0O-SG1 1.07E+06 2.45E-04 2. Zoe-yu 1.31E+06 1.60E-04 1.22E-10 1.02E+06 2.13E-04 2.O9E-1O MS1Q32LO01-SG1 1.64E+06 1.29E-03 7.88E-1O 1.BOE+06 1.07Е-03 7.13E-10 1.15E+06 1.10E-03 9.54E-10 MS1032LO02-SG1 1.09E+06 1.79E-03 1.64Е-09 N/T NT N/T N/T N/T N/T MS1032LO03-SG1 1.08E+06 1.17E-03 1.08E-09 N/T N/T N/T N/T N/T N/T MS1032LO04-SG1 1.S4E+O6 1.25E-03 8.12E-1O N/T NT N/T N/T N/T N/T

N/Τ: not tested

Table 10

The values of ka, kd and KD obtained under the conditions used for the analysis of affinity at acidic pH j people. mouse latent myostatin. ^{latent myostatin supolatent myostatin _ _} )

MS 1032LOGO SGI 1Ό9Ε+06 2.35E04 2.ISE-10 1.62E+06 7.08E-05 4.38E11 1.41E+06 1.96E-04 1.39E-10 MS1O32LO01-SG1 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. MS 1032LO 02-SGI n.d. n.d. n.d. N/T N/T N/T NT N/T N/T MS1032LO03-SG1 n.d. n.d. n.d. N/T N/T N/T NT N/T NT MS1032LO04SG1 n.d. n.d. n.d. N/T N/T N/T NT N/T NT

n.d.: not determined, N/Τ: not tested

Example 13

Comparison of plasma concentrations of total myostatin in mice using antibodies with the ability to bind to Fc-gamma R and those with an eliminated ability to bind to Fc-gamma R.

An in vivo experiment using C.B-17 SCID mice.

The accumulation of endogenous myostatin was assessed in vivo after administration of an antibody to latent myostatin to C.B-17 SCID mice (In Vivos, Singapore). Antibody to latent myostatin (3 mg/ml) was administered as a single dose of 10 ml/kg into the caudal vein. Blood samples were collected at 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 immediately centrifuged at 14,000 rpm at 4° C. for 10 minutes to separate the plasma. The separated plasma was stored at -80° C. or below until analysis. MS1032LO00-SG1 and MS1032LO00-F760 were used as antibodies to latent myostatin.

Measurement of plasma concentration of total myostatin by electrochemiluminescence (ECL).

The concentration of total myostatin in the plasma of mice was measured using ECL. Anti-mature myostatin antibody immobilized plates were prepared by applying anti-mature myostatin antibody RK35 (described in WO 2009/058346) to a 96-well MULTI-ARRAY plate (Meso Scale Discovery) and incubating overnight at 4°C. Mature myostatin samples were prepared to obtain a calibration curve and plasma samples diluted 40 times or more. Samples were mixed in an acidic solution (0.2 M glycine-HCl, pH 2.5) to separate by dissociation of mature myostatin from its binding protein (such as a propeptide). The samples were then loaded onto a plate immobilized with anti-mature myostatin antibody and allowed to bind for 1 hour at room temperature, followed by washing. The SULFO-labeled anti-mature myostatin antibody RK22 (described in WO 2009/058346) was then added and the plate was incubated for 1 hour at room temperature followed by washing. Read Buffer T (x4) (Meso Scale Discovery) was immediately added to the plate, and signal detection was performed using a SECTOR Imager 2400 (Meso Scale Discovery). Mature myostatin concentration was calculated from the response to the standard curve using SOFTmax PRO analytical software (Molecular Devices). The time dependence of the plasma concentration of total myostatin after intravenous administration of antibodies to latent myostatin, determined using this method, is shown in Fig. 10.

- 88 040713

Effect of Fc-gamma R binding on myostatin accumulation in vivo Following administration of the MS1032LO00-F760 antibody, the plasma concentration of total myostatin accumulated by day 28 was 248 times the plasma concentration of total myostatin after 5 minutes. In contrast, after administration of MS1032LO00-SG1, the plasma concentration of total myostatin accumulated by day 28 was 37 times the plasma concentration of total myostatin after 5 minutes. Approximately 7-fold difference in plasma total myostatin concentration on day 28 was found between MS1032LO00-F760 (silent Fc) and MS1032LO00-SG1 as a result of binding to Fc-gamma R. In the case of human soluble IL-6R (hsIL-6R), no significant differences in plasma concentration of hsIL-6R when using antibodies to hsIL-6R F760 (silent Fc) and SG1, as described in WO 2013/125667. Since hsIL-6R is a monomeric antigen, the antibody-hsIL-6R complex contains only one Fc. Thus, the results obtained in WO 2013/125667 suggest that the single Fc antibody-antigen complex does not bind to the Fc-gamma R in vivo to such an extent that the immune complex is taken up by the cells. On the other hand, a multimeric antigen (such as myostatin) can form a large immune complex with an antibody, and the antibody-antigen complex contains more than two Fc. Thus, there may be a significant difference in plasma antigen concentration between F760 and SG1 due to the strong avidity of binding to Fc-gamma R. This result suggests that MST1032 may form a large immune complex with myostatin that contains more than two antibodies.

Example 14

Comparison of plasma concentrations of total myostatin when using a pH-independent antibody to latent myostatin and a pH-dependent antibody to latent myostatin in mice.

An in vivo experiment using C.B-17 SCID mice.

Endogenous myostatin accumulation was assessed in vivo after administration of anti-myostatin latent antibody to C.B-17 SCID mice (In Vivos, Singapore) according to the method described in Example 13. Anti-myostatin latent antibodies MS1032LO01-SG1 and MS1032LO01-F760 were used.

Measurement of plasma concentration of total myostatin using ECL.

The plasma concentration of total myostatin in mice was measured by ECL according to the method described in Example 13. The time dependence of the plasma concentration of total myostatin after intravenous administration of anti-latent myostatin antibody, determined using this method, is shown in FIG. eleven.

Effect of pH dependent myostatin binding on myostatin accumulation in vivo.

The pH-dependent antibodies to latent myostatin (MS1032LO01-SG1 and MS1032LO01-F760) were tested in vivo and plasma concentrations of total myostatin were compared. Total myostatin concentration data after administration of MS1032LO00-SG1 and MS1032LO00-F760 are shown in Example 13 and also in FIG. 11. MS1032LO00 is a pH independent antibody and MS1032LO01 is a pH dependent antibody. As shown in FIG. 11, total myostatin concentrations after administration of MS1032LO01-F760 decreased compared to MS1032LO00-F760 due to pH dependent binding. In addition, total myostatin concentrations after administration of MS1032LO01SG1 decreased dramatically compared to MS1032LO00-SG1 due to pH-dependent binding and increased cell uptake resulting from Fc-gamma R binding. MS1032LO01-SG1 can be expected to have a very high ability to increase clearance of myostatin from plasma as a Sweeping® antibody (i.e., an antibody that has the ability to actively eliminate soluble antigens from the bloodstream.

Example 15

Expression and purification of human and mouse latent GDF11.

Human GDF11 with a Flag tag at the N-terminus (also referred to herein as Flag-hGDF11, human latent GDF11, hGDF11 or human GDF11, SEQ ID NO: 85) was transiently expressed using FreeStyle293-F cells (Thermo Fisher). Conditioned media expressing Flag-hGDF11 were used to pack a column with anti-Flag M2 affinity resin (Sigma) and eluted with Flag peptide (Sigma). Fractions containing Flag-hGDF11 were collected and then subjected to gel filtration on a Superdex 200 column (GE healthcare) equilibrated with 1×PBS. Fractions containing Flag-hGDF11 were then pooled and stored at -80°C.

Example 16

Characterization of antibodies to latent myostatin (ELISA).

Non-sensitized ELISA plates (NUNC-IMMUNO plate, MAXISORP surface, Nalge Nunc International) were coated with 20 μl of 50 nM streptavidin (GenScript) for 2 hours at room temperature. The plates were then washed three times with PBS and blocked overnight with 50 μl of 20% Blocking One (Nacalai Tesque). The next day, each well of the plate was incubated with biotinylated human latent myostatin at 2 nM/well/20 µl or biotinylated human latent GDF11 at 20 nM/well/20 µl for 2 hours. After washing, 20 µl of antibody sample was added to the wells and tablets withstand

- 89 040713 for 1 hour. The plates were washed and 20 μl of anti-human IgG conjugated with horseradish peroxidase (HRP) (Abcam) diluted in HEPES buffered saline was added, and the plates were incubated for another 1 hour. Then the plates again washed and then added to each well with 50 μl of ABTS (KPL) and the plates were incubated for 1 hour. Signal detection was performed at 405 nm in a colorimetric plate reader. The results of the binding experiment are shown in FIG. 12. MST1032-Glm was found to bind to latent myostatin (ie, a non-covalent complex of mature myostatin and propeptides) but not to hGDF11.

These results demonstrate that MST1032-Glm specifically binds to myostatin but does not bind to hGDF11.

Example 17.

Characterization of anti-latent myostatin antibody (HEK Blue assay (BMP1 and spontaneous activation)).

Reporter gene analysis was used to assess the biological activity of active GDF11 in vitro. HEK-Blue™ TGF-beta cells (Invivogen), which express Smad3/4 binding element (SBE)-indicated SEAP reporter genes, allow for the detection of bioactive GDF11 by monitoring activin type 1 and type 2 activin receptor activation. Active GDF11 stimulates production and secretion of SEAP into the cell supernatant. The amount of SEAP secreted is then determined using QUANTIBlue™ (Invivogen).

HEK-Blue™ TGF-beta cells were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum, 50 µg/ml streptomycin, 50 U/ml penicillin, 100 µg/ml Normocin™, 30 µg/ml blasticidin, 200 µg/ml HygroGold™ and 100 µg/ml Zeocin™. When performing a functional assay, cell medium was changed to assay medium (DMEM supplemented with 0.1% bovine serum albumin, streptomycin, penicillin, and Normocin™) and plated in a 96-well plate. Human GDF11 was incubated with or without recombinant human BMP1 (Calbiochem) and latent myostatin antibody (MST1032-Glm) at 37°C overnight. Sample mixtures were transferred into cells. After a 24 hour incubation, cell supernatants were mixed with QUANTIBlue™ and absorbance at 620 nm was measured in a colorimetric plate reader. As shown in FIG. 13, MST1032-Glm did not interfere with protease-mediated activation or spontaneous activation of human GDF11 and therefore did not inhibit SEAP secretion.

Example 18.

Additional optimization of MS1032 variants to increase the elimination of antigen from the bloodstream (sweeping effect).

Since the pH dependent antibody, MS1032LO01-SG1, has been shown to have very high potency, when studied in mice, further optimizations were made to increase pH dependence, increase cell uptake, increase stability, etc. by introducing mutations in the CDR of the antibody. or by changing the frame sections. Over a thousand variants were evaluated using the BIACORE® and/or HEK Blue assays described above and generated 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. Amino acid and nucleotide sequences of these created antibodies are presented in table. eleven.

Table 11a

MS1032 variants and their DNA and amino acid sequences (SEQ ID NO: indicated)

Antibody designation Reduction Variable region __ Constant domain _____ heavy light heavy light DNA protein DNA protein DNA protein DNA protein MS_M103205H795-SG1/M103202L889-SK1 MS1032LO06-SG1 100 86 By 96 052 009 054 010 MS_M 103205 H1004-SG1/M103202 L889-S KI MS1032LO07-SG1 101 87 110 96 052 009 054 010 MS_M 103205 H1046-SG1/M103202 L889-S KI MS1032LO10-SG1 102 88 110 96 052 009 054 010 MS_M 103205H1047 SG 1/M 103202L889-SKI MS1032LO11-SG1 103 89 110 96 052 009 054 010 MS_M 103205 H1057-SG 1/M 103202 L889-S KI MS1032LO12-SG1 104 90 110 96 052 009 054 010 MS_M 103205 H1186-SG 1/M 103202 L889-S KI MS1032LO18-SG1 105 91 110 96 052 009 054 010 MS_M103240H795-5Gl/M103202L1045-SKl MS1032LO19-SG1 106 92 111 97 052 009 054 010 MS_M103245H795-SGl/M103202L1045-SKl MS1032LO21-SG1 107 93 111 97 052 009 054 010 MS_M 103 245H1233-SG1/M1O3202L1O45-S KI MS1032LO23-SG1 108 94 111 97 052 009 054 010 MS_M103205H795-SGl/M103202L1060-SKl MS1032LO24-SG1 100 86 112 98 052 009 054 010 MS_M 103 240H1246-SG1/M1032O2L1045-S KI MS1032LO25-SG1 109 95 111 97 052 009 054 010 MS M 103240H795-SG1/ M103209L1045-S KI MS1032LO26-SG1 106 92 113 99 052 009 054 010

- 90 040713

Table 11b

Amino acid sequences of HRV variants of MS1032 (their SEQ ID NO: are indicated)

Antibody designation Reduction Hl Guinness H2 zariapoor NZ region (HRV) L1 L2 L3 MS_M103205H795-SGl/M103202L889-SKl MS1032LO06-SG1 114 58 63 122 71 74 M5_M103205H1004-SG1/M103202L889-5K1 MS1032LO07-SG1 114 116 121 122 71 74 MS_M103205H1046-SGl/M103202L889-SKl MS1032LO10-SG1 57 117 63 122 71 74 MS_M103205H1047-SGl/M103202L889-SKl MS1032LO11-SG1 57 118 63 122 71 74 MS M103205H1057-SG1/M1D3202L889-SK1 MS1032LO12-SG1 57 119 63 122 71 74 MS_M1O32O5H1186-SG1/M1O32O2L889-SK1 MS1032LO18-SG1 114 118 63 122 71 74 M S_M 103 240 H 795-SG1/ M103202 L1045-S K1 MS1032LO19-SG1 114 58 63 123 71 74 M S_M 103 245 H 795-SG1/ M103202 L1045-S K1 MS1032LO21-SG1 114 58 63 123 71 74 7IS_M103245H1233-SGl/M103202L1045-SKl MS1032LO23-SG1 115 58 63 123 71 74 M S_M 103 205 H 795-SG 1/ M103202 L1060-S K1 MS1032LO24-SG1 114 58 63 124 125 74 4S_M 103240H1246-SG1/M103202L1045-5K1 MS1032LO25-SG1 115 120 63 123 71 74 M S_M 103 240 H 795-SG 1/ M103209 L1045-S K1 MS1032LO26-SG1 114 58 63 123 71 74

The binding affinity of MS1032 variants to latent myostatin in humans, cynomolgus (supo) monkeys, and mice was evaluated at 37° C. at pH 7.4 and pH 5.8 using a BIACORE® T200 device (GE Healthcare) to evaluate the effect of pH on antigen binding. . ProA/G (Pierce) was immobilized on all flow cells of the CM4 chip using an amine coupling kit (GE Healthcare). All antibodies and analytes were prepared in ACES buffer, pH 7.4 or pH 5.8, containing 20 mM ACES, 150 mM NaCl, 1.2 mM CaCl ₂ , 0.05% Tween 20, 0.005% NaN3. Each antibody was captured on the surface of the sensor chip using ProA/G. Antibody immobilization levels typically corresponded to 130-240 resonance units (RU). Human, supo or mouse latent myostatin was injected at concentrations ranging from 3.125 to 50 nM, which were obtained by two-fold serial dilutions and then allowed to dissociate. The sensor surface was regenerated after each cycle with 10 mM glycine-HCl, pH 1.5. Binding affinity was determined by processing and fitting the data to a 1:1 binding model using BIACORE® T200 Evaluation software version 2.0 (GE Healthcare).

Binding affinity (KD) data for MS1032 variants with latent human, supo, and mouse myostatin at pH 7.4 and pH 5.8 are shown in Table 1. 12. For all variants, a ratio of KD values ((KD at pH 5.8)/(KD at pH 7.4)) of more than 5 was obtained, which indicates a pH-dependent binding to latent myostatin.

Table 12

Kinetic parameters of MS1032 variants

Designation At Variable region designation _ heavy chain light chain Human latent myostatin Supo latent myostatin Mouse latent myostatin KD pH 7.4 (M) KD pH 5.8 (M) 1 KD ratio at pH 5.8/pH 7.4 1 KD pH 7.4 , (M) KD pH 5.8 (M) i 1 ratio KD three pH 5.8/pH 7.4 1 KD pH 7.4 , (M) KD pH 5.8 KD ratio (M) at pH 5.8/pH 7.4 MS1032LO06-SG1 М103205Н795 M1O32O2LS89 2.85E-1O 3.40E-08 119 3.43Ε-10 2.59Ε-08 76 2.99E-1O 2.26E-08 76 M51032LO07-5G1 М103205Н1004 M103202L889 2.75E-10 4.62E-O8 168 3.39Ε-1Ο 3.47 E-08 102 3.19E-1O 3.08E-08 97 MS1032LO10-SG1 M103205H1046 M103202L889 3.51E-1O 4.38E-O8 125 4.19Ε-10 1.61 E-08 38 3.14E-1O 1.09E-08 35 MS1032LO11-SG1 M103205H1047 M103202L889 4.19E-10 1.43E-07 341 5.02Ε-10 4.35Ε-08 87 3.62E-1O 2.92E-08 81 MS1032LO12-SG1 М103205Н1057 M1O32O2L889 4.07Е-10 8.49E-O8 209 4.92Ε-10 3.58Ε-08 73 4.13E-1O 2.48E-08 60 MS1032LO18-SG1 М103205Н1186 M103202L889 3.29E-1O n.d. n.d. 3.54Ε-10 8.09 E-08 229 2.72E-1O n.d. n.d. MS1032LO19-SG1 М103240Н795 M103202L1045 2.73E-10 4.58E-08 168 3.26Ε-10 3.56 E-08 109 2.71E-1O 2.87E-08 106 MS1032LO21-SG1 М103245Н795 M103202L1045 2.57E-1O 5.23E-08 204 3.Ο9Ε-10 3.10ΕΌ8 100 2.61E-1O 2.94E-08 113 MS1032LO23-SG1 M103245H1233 M103202L1045 2.60E-10 1.61E-O8 62 2.99Ε-10 7.71Ε-09 26 2.66E-1O 6.40E-09 24 MS1032LO24-SG1 М103205Н795 M103202L1060 1.64E-10 1.52E-09 9 1.89Ε-10 9.29Ε-10 5 1.76E-10 1.02E-09 6 MS1032LO25-SG1 M103240H1246 M103202L1045 2.76E-10 2.55E-08 92 3.40Ε-10 1.41 E-08 41 2.59E-10 9.88E-09 38

n.d.: not determined

Example 19.

Evaluation of the neutralizing activity of the variants obtained as a result of additional optimization using the HEK Blue analysis.

The neutralizing activity of MS1032LO06-SG1, MS1032LO07-SG1, MS1032LO10-SG1, MS1032LO11-SG1, MS1032LO12-SG1, MS1032LO18-SG1, MS1032LO19-SG1, MS1032LO21-SG1, MS1032LO23-SG1, and MS1032LO25-SG1, MS1032LO23-SG1, and MS1032LO25-SG1, MS1032LO23-SG1, and MS1032LO25-SG1, MS1032LO23-SG1, and MS1032LO25 using the method described in Example 3. As shown in FIG. 14, all variants showed activity comparable to that of MS1032LO01-SG1.

Example 20.

Comparison of total myostatin plasma concentration in mice using a pH-independent antibody to latent myostatin and various pH-dependent antibodies to latent myostatin.

- 91 040713

An in vivo experiment using C.B-17 SCID mice.

The accumulation of endogenous myostatin was assessed in vivo after administration of an antibody to latent myostatin to C.B-17 SCID mice (In Vivos, Singapore). Antibody to latent myostatin (3 mg/ml) was administered as a single dose of 10 ml/kg into the caudal vein. Blood samples were collected at 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 immediately centrifuged at 14,000 rpm at 4° C. for 10 minutes to separate the plasma. The separated plasma was stored at -80° C. or below until analysis. As antibodies to latent myostatin, MS1032LO00-SG1, MS1032LO01-SG1, MS1032LO06-SG1, MS1032LO11-SG1,

MS1032LO18-SG1, MS1032LO19-SG1, MS1032LO21-SG1 and MS1032LO25-SG1.

Measurement of plasma concentration of total myostatin by electrochemiluminescence (ECL).

The concentration of total myostatin in the plasma of mice was measured using ECL. Anti-mature myostatin antibody immobilized plates were prepared by applying biotinylated anti-mature myostatin antibody RK35 (described in WO 2009/058346) to a 96-well streptavidin-coated MULTI-ARRAY plate (Meso Scale Discovery) and incubating in blocking buffer for 2 hours at room temperature.

Mature myostatin samples were prepared to obtain a calibration curve and plasma samples diluted 40 times or more. Samples were mixed in an acid solution (0.2 M glycine-HCl, pH 2.5) to separate by dissociation of mature myostatin from its binding protein (such as a propeptide). The samples were then loaded onto a plate immobilized with anti-mature myostatin antibody and allowed to bind for 1 hour at room temperature, followed by washing. The SULFO-labeled anti-mature myostatin antibody RK22 (described in WO 2009/058346) was then added and the plate was incubated for 1 hour at room temperature followed by washing. 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). Mature myostatin concentration was calculated from the response to the standard curve using SOFTmax PRO analytical software (Molecular Devices). The time dependence of the plasma concentration of total myostatin after intravenous administration of antibodies to latent myostatin, determined using this method, is shown in Fig. 15.

Effect of pH-dependent binding to myostatin on myostatin accumulation in mice in vivo.

The effect of pH-dependence on myostatin accumulation in mice was compared using a pH-independent anti-latent myostatin antibody (MS1032LO00-SG1) and various pH-dependent antibodies to latent myostatin (MS1032LO01-SG1, MS1032LO06-SG1, MS1032LO11-SG1, MS1032LO18SG1, MS1032LO19-SG1, MS1032LO21-SG1 and MS1032LO25-SG1). The introduction of pH dependence significantly accelerated the clearance of myostatin from the plasma of SCID mice. As shown in FIG. 15, pH-dependent antibodies to latent myostatin (MS1032LO01-SG1, MS1032LO06-SG1, MS1032LO11-SG1, MS1032LO18-SG1, MS1032LO19-SG1, MS1032LO21-SG1, and MS1032LO25-SG1) could reduce myostatin accumulation compared to a pH-independent antibody to latent myostatin (MS1032LO00-SG1) on day 28.

Example 21.

Comparison of plasma concentrations of total myostatin in cynomolgus monkeys using a pH-independent antibody to latent myostatin and pH-dependent antibodies to latent myostatin having an engineered Fc region.

An in vivo experiment using cynomolgus monkeys.

The accumulation of endogenous myostatin was assessed in vivo after administration of antibodies to latent myostatin to 2-4-year-old Masaca fascicularis (cynomolgus cynomolgus monkey) from Cambodia (Shin Nippon Biomedical Laboratories Ltd., Japan). A dose of 30 mg/kg was injected into the cephalic vein of the forearm using a disposable syringe, an extension tube, an internal needle and an infusion pump. The dosing rate was 30 minutes per animal. Blood samples were collected before the start of dosing and either 5 min, 7 h and 1, 2, 3, 7, 14, 21, 28, 35, 42, 49 and 56 days after the end of dosing, or 5 min 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 taken from the femoral vein using a syringe containing sodium heparin. The blood was immediately cooled on ice and plasma was obtained by centrifugation at 4°C, 1700xg for 10 min. Plasma samples were stored in a deep freezer (acceptable range: -70° C. or below) until analysis. В качестве антител к латентному миостатину применяли MS1032LO00-SG1, MS1032LO06-SG1012, MS1032LO06-SG1016, MS1032LO06-SG1029, MS1032LO06SG1031, MS1032LO06-SG1033 и MS1032LO06-SG1034 (в контексте настоящего описания SG1012, SG1016, SG1029, SG1031, SG1033 и SG1034 обозначают константные heavy chain regions constructed from SG1 according to the method described below).

- 92 040713

Anti-latent myostatin antibodies MS1032LO19SG1079, MS1032LO19-SG1071, MS1032LO19-SG1080, MS1032LO19-SG1074, MS1032LO19-SG0321 (MS1032LO19-SG1080, MS1032LO19-SG1074, in the context of this specification, SG1079, SG1071, SG1080, SG1074, SG1081 and SG1077 denote heavy chain constant regions constructed from SG1 according to the method described below). Blood samples were collected before 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 according to the procedure described above. Plasma samples were stored in a deep freezer (acceptable range: -70° C. or below) until analysis.

Measurement of plasma concentration of total myostatin by electrochemiluminescence (ECL).

Plasma total myostatin concentration in monkeys was measured by ECL according to the method described in Example 20. The time course of plasma total myostatin concentration after intravenous administration of anti-latent myostatin antibody, determined using this method, is shown in FIG. 16.

Measurement of monkey plasma ADA by electrochemiluminescence (ECL).

The biotinylated drug was used to sensitize a 96-well MULTI-ARRAY streptavidin plate (Meso Scale Discovery) and incubated in LowCross® buffer (Candor) for 2 hours at room temperature. Monkey plasma samples were diluted 20-fold in LowCross® buffer prior to plate loading. Samples were incubated overnight at 4°C. The next day, the plate was washed three times with wash buffer before adding a SULFO-labeled anti-monkey IgG secondary antibody (Thermo Fisher Scientific). After incubation for 1 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 detection was performed with a SECTOR Imager 2400 (Meso Scale Discovery).

Effect of pH-dependence and Fc engineering on myostatin accumulation in monkeys in vivo.

Administration of pH-independent antibody (MS1032LO00-SG1) to cynomolgus monkeys resulted in at least a 60-fold increase in myostatin concentrations from baseline on day 28. On day 28, pH-dependent latent myostatin antibodies MS1032LO06-SG1012 and MS1032LO06-SG1033 provided and 8-fold increase from baseline, respectively. The strong swipping effect is mainly associated with an increase in affinity for Fc-gamma RIIb of cynomolgus monkeys. On day 28, pH-dependent latent myostatin antibodies MS1032LO06-SG1029, MS1032LO06-SG1031 and MS1032LO06-SG1034 were able to eliminate the antigen to below baseline levels. The reason for the strong swipping effect in the case of MS1032LO06-SG1029, MS1032LO06-SG1031 and MS1032LO06-SG1034 is an increase in non-specific uptake by the cell due to an increase in the positively charged antibody cluster and an increase in Fc-gamma R mediated cellular uptake due to increased binding to Fc-gamma R .

The introduction of pH-dependent antibodies to latent myostatin MS1032LO19-SG1079, MS1032LO19SG1071, MS1032LO19-SG1080, MS1032LO19-SG1074, MS1032LO19-SG1081 and MS1032LO19-SG1077 reduced the concentration of myostatin to a level below the detection limit (<0.25 ng/ml) 1 in cynomolgus monkeys. On day 14, myostatin concentrations exceeded the detection limit with MS1032LO19-SG1079 and MS1032LO19-SG1071, although myostatin levels remained below the detection limit with MS1032LO19-SG1080, MS1032LO19-SG1074, MS1032LO19-SG1081, and MS1032LO19-SG1077. The weaker suppression found for MS1032LO19-SG1079 and MS1032LO19-SG1071 may be the result of various mutations leading to a change in pI.

The results suggest that a strong elimination of myostatin from plasma can be achieved by mutations that increase binding to Fc-gamma RIIb or combinations of mutations that increase the positive charge of the antibody and increase binding to Fc-gamma RIIb. It can be expected that a strong elimination of myostatin can be achieved in humans by combining mutations that increase the positive charge of the antibody and increase binding to Fc-gamma R.

Example 22.

Epitope mapping of antibodies to latent myostatin.

Additional antibodies to human latent myostatin were designed to map their respective epitopes to which they bind. Two NZW rabbits were immunized and antibodies were obtained according to the method described in Example 2. The 1760 antibody-producing B cell lines identified were further screened for their ability to block BMP1 mediated activation of human latent myostatin. In general, the method was as follows: B-cell supernatant containing secreted antibodies was incubated with human latent myostatin in the presence of recombinant human BMP1 (R&D

- 93 040713

Systems) at 37°C overnight. Then 50 μl of the reaction mixture was transferred into a 96-well MULTI-ARRAY plate from Meso Scale Discovery sensitized with an antibody to mature myostatin RK35 (described in WO 2009/058346). After incubation for 1 hour at room temperature with shaking, the plates were incubated with biotinylated anti-mature myostatin antibody RK22 (described in WO 2009/058346) and then with SULFO-labeled streptavidin. Then, Read Buffer T (x4) (Meso Scale Discovery) was added to the plate, and signal detection was performed using a SECTOR Imager 2400 (Meso Scale Discovery). For subsequent analysis, 94 lines were selected, characterized by different levels of neutralizing activity (MST1495-MST1588). The variable regions of these selected lines were cloned according to the method described in Example 2, except that an expression vector with the heavy chain constant region sequence F1332m (SEQ ID NO: 193) was used.

Inhibitory activity against 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 identifying amino acid sequence numbers of these antibodies are shown in Table 13). As shown in FIG. 17, all antibodies were able to inhibit BMP1 mediated activation of human latent myostatin in a dose dependent manner.

Table 13 Amino acid sequences of antibodies to latent myostatin

SEQIDNO:

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

Four antibodies that showed promise in Western blot analysis were selected for epitope mapping. Fragments of the N-terminal propeptide coding region of human latent myostatin were cloned into the pGEX4.1 vector so as to obtain GST labeled propeptide fragments each of 100 amino acids with 80 overlapping amino acids (FIG. 18A). Protein expression was induced in BL21-transformed competent cells using the Overnight Express Autoinduction System (Merck Millipore) and the protein was extracted with BugBuster Protein Extraction Reagent (Novagen). Expression of the desired 37 kDa protein fragments was confirmed by Western blot analysis as described in Example 6 (anti-GST antibody (Abcam)) (FIG. 18B). When examining antibodies to human latent myostatin indicated in FIG. 18B, while all 4 antibodies could inhibit latent myostatin activation, they recognized different epitopes on human latent myostatin. The MST1032 antibody could detect the first five fragments, no bands were detected after deletion of amino acids 81-100 (SEQ ID NO: 78). Both antibodies MST1538 and MST1572 bound only to the first three fragments, no bands were detected in the absence of amino acids 41-60 of SEQ ID NO: 78. The MST1573 antibody strongly bound only to the first two fragments, which suggests that its epitope lies in amino acids 21-40 SEQ ID NO: 78 (FIG. 18D).

Example 23.

Creation of new Fc variants with increased activity against Fc-gamma RIIb.

This example illustrates the construction of Fc to increase myostatin clearance.

WO 2013/125667 demonstrates that the clearance of a soluble antigen can be increased by administering antigen-binding molecules containing an Fc domain with increased affinity for Fc-gamma RIIb. In addition, Fc variants that have been found to have increased ability to bind to human Fc-gamma RIIb are illustrated in WO 2012/115241 and WO 2014/030728. In addition, it has been demonstrated that these Fc variants can have a selectively increased ability to bind to human Fc-gamma RIIb and a reduced ability to bind to other active Fc-gamma R. This selective increase in binding to Fc-gamma RIIb can have a beneficial effect not only on clearance of soluble antigen, but also to reduce the risk of unwanted effector functions and immune response.

When developing an antibody as a drug, efficacy, pharmacokinetics, and safety should be studied in non-human animals for which the drug has pharmacological activity. If it is only active in humans, alternative approaches should be used, such as using a surrogate antibody (Int. J. Tox. 28, 2009, c. 230-94 040713

253) . However, it is difficult to predict the effects of the interaction between the Fc region and the Fc-gamma R in humans using a surrogate antibody, because the Fc-gamma R expression and/or function patterns in non-human animals are not always the same as in humans. Preferably, the Fc regions of antibodies used as drugs have cross-reactivity with regions of animals other than humans, especially cynomolgus monkeys, which have expression patterns and functions of Fc-gamma R close to those of humans, so that results obtained in animals other than a person can be extrapolated to a person.

Therefore, when creating the present invention created Fc-variants with cross-reactivity between Fc-gamma R human and supo.

Measurement of the affinity of existing Fc variants with increased activity against Fcgamma RIIb for human and simian Fcgamma R.

The gene for the heavy chain variant with increased activity against human Fc-gamma RIIb (in the present description, the term with increased activity against Fc-gamma RIIb means with increased Fc-gamma RIIb-binding activity), described in WO 2012/115241, was created by replacing Pro at position 238 according to EU numbering on Asp in the heavy chain MS1032LO06-SG1, which was designated M103205H795-SG1 (VH, SEQ ID NO: 86; CH, SEQ ID NO: 9). The resulting heavy chain was designated M103205H795-MY009 (VH, SEQ ID NO: 86; CH, SEQ ID NO: 252). M103202L889-SK1 (VL, SEQ ID NO: 96; CL, SEQ ID NO: 10) was used as the light chain of the antibody. The recombinant antibody was expressed according to the method described in Example 30.

The extracellular domains of Fc-gamma R were obtained as follows. Synthesis of human Fc-gamma R extracellular domain genes was performed using methods known to those skilled in the art based on information registered with the NCBI. In particular, the basis for the creation of Fc-gamma RIIa was the NCBI sequence with access code no. Fc-gamma RIIa and Fc-gamma RIIIa allotypes were derived from data on Fc-gamma RIIa (J. Exp. Med. 172, 1990, pp. 19-25) and Fc-gamma RIIIa polymorphisms (J. Clin. Invest. 100(5 ), 1997, pp. 1059-1070) respectively. Synthesis of the cynomolgus Fc-gamma R extracellular domain gene was accomplished by cloning the cDNA of each cynomolgus Fc-gamma R using methods known to those skilled in the art. The amino acid sequences of the engineered extracellular domains of Fc-gamma R are shown in the sequence listing: (SEQ ID NO: 210 for human Fc-gamma RIIaR, SEQ ID NO: 211 for human Fc-gamma RIIaH, SEQ ID NO: 214 for human Fc-gamma 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 RIIa1, 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 RIIIaS). Then a His-tag was added to their C-terminus and each resulting gene was inserted into an expression vector designed for expression in mammalian cells. The expression vector was introduced into FreeStyle293 human embryonic kidney cells (Invitrogen) to express the desired protein. After cultivation, the resulting culture supernatant was filtered and purified mainly by the following four purification steps. Cation exchange chromatography using SP Sepharose FF was performed as the first step, anti-His tag affinity chromatography (HisTrap HP) was performed as the second step, gel filtration on columns (Superdex 200) was used as the third step, and sterilization by filtration was used as the fourth step. stages. The absorbance at 280 nm of the purified proteins was measured with a spectrophotometer and the concentration of the purified protein was determined using the extinction coefficient calculated by the PACE method (Protein Science 4, 1995, c. 2411-2423).

Kinetic analysis of interactions between these antibodies and Fc-gamma R was performed using BIACORE® T200 or BIACORE® 4000 (GE Healthcare). HBS-EP+ (GE Healthcare) was used as running buffer and the measurement temperature was set to 25°C. The chip was prepared by immobilizing protein A, protein A/G or mouse kappa light chain to human IgG (BD Biosciences) on an S series CM4 or CM5 sensor chip (GE Healthcare) using the amine coupling method. An antibody of interest was captured on the surface of the interaction chip with each Fc-gamma R that was diluted in running buffer and binding to the antibody was measured. After the measurement, the antibody immobilized on the chip was washed off, allowing it to react with 10 mM glycine-HCl, pH 1.5, and 25 mM NaOH, resulting in chip regeneration and reuse. The obtained sensograms, which are the results of the measurement, were analyzed using a 1:1 Langmuir binding model using the BIACORE® Evaluation software to calculate the binding rate constant ka (l/mol/s) and the dissociation reaction rate constant kd (1/s), and on Based on these values, the dissociation constant KD (mol/l) was calculated. Since the binding of MS1032LO06-MY009 to human Fc-gamma RIIaH, human Fc-gamma RIIIaV and cynoFc-gamma RIIIaS was weak, kinetic parameters such as KD could not be calculated using the above analytical method. The KD values describing these interactions were calculated using the 1:1 binding model described below in the BIACORE® T100 Handbook BR1006-48 Edition AE software.

The behavior of interacting molecules based on the BIACORE® 1:1 binding model can be described by Equation 1: Req = C x Rmax/(KD+C)+RI where, Req denotes the functional dependence of stationary binding levels on analyte concentration, C denotes concentration, RI is the total refractive index contribution for the sample, and Rmax is the analyte binding capacity of the surface. If this equation is transformed, then KD can be expressed in the form of equation 2: KD = C x Rmax / (Req - RI) - C. KD can be calculated by replacing the parameters Rmax, RI and C in equation 1 or equation 2 with the appropriate values. measurement conditions, you can use the values RI=0, C=2 µmol/l. In addition, the Rmax value obtained by a global fit of the sensogram measured by analyzing the interaction of each Fc-gamma R with IgG1 using a 1:1 Langmuir binding model was divided by the amount of SG1 entrapped, the result was multiplied by the amount of MY009 entrapped, and the resulting value was used as Rmax. This calculation is based on the hypothesis that the limit on the amount of each Fc-gamma R that can be associated with SG1 remains unchanged for all variants obtained by introducing mutations in SG1, and that the value of Rmax at the time of measurement is proportional to the amount of antibody bound with the chip at the time of the measurement. Req was defined as the number of bindings of each Fc-gamma R to each variant on the sensor chip detected at the time of the measurement.

In table. 14 Cross-reactivity of a variant with increased activity against human Fc-gamma RIIb with cynoFc-gamma R (see below) presents the results of a kinetic analysis of the binding of SG1 and MY009 with human and cynoFc-gamma R. KD values in bold cells were calculated using Equation 2 because their affinity was too low to accurately determine it using kinetic analysis. The fold of KD values was calculated by dividing the KD value for SG1 by the KD value for the variant for each Fc-gamma R.

As shown in Table. 14, variant MY009 with enhanced activity against human Fc-gamma RIIb did not show increased binding to cynoFc-gamma RIIb, but increased binding to human Fc-gamma RIIb. Its affinity for cynoFc-gamma RIIb even turned out to be reduced by 0.4 times compared to SG1, which indicates that the MY009 variant with increased activity against human Fc-gamma RIIb does not cross-react to cynoFc-gamma R.

Development of a new Fc variant with increased binding to both human and cynoFc-gamma RIIb.

Ideally, the new Fc variant should have increased selective binding to both human and cynoFc-gamma RIIb and reduced binding to other active Fc-gamma R. However, since the putative residues responsible for Fc-binding in cynoFc-gamma RIIb are fully consistent with the residues in any cynoFc-gamma RIIa allotype (FIG. 19), it is theoretically impossible to selectively increase binding to cynoFc-gamma RIIb relative to cynoFc-gamma RIIa binding. Thus, the novel Fc variant should have selectively increased binding to human Fc-gamma RIIb, cynoFc-gamma RIIb and cynoFc-gamma RIIa.

To generate a new Fc variant characterized by selectively increased binding to both human and cyno-Fc-gamma RIIb, the sweep mutagenesis (brute force amino acid substitution) assay described in WO 2012/11524 was used. In a sweep mutagenesis assay, sweep mutations were introduced at all positions in the Fc-gamma R binding region of IgG1 isotype antibodies, and a complete binding analysis was performed on each Fc-gamma R according to the procedures below.

The variable region of an anti-glypican 3 antibody containing the CDR GpH 7, which is an antibody to glypican 3 with enhanced plasma kinetics, as described in WO 2009/041062, was used as the variable region of the heavy chain of the antibody (GpH 7: SEQ ID NO: 225 ). Similarly, as the light chain of the antibody, GpL16-k0 (SEQ ID NO: 226), the plasma-enhanced anti-glypican 3 antibody described in WO 2009/041062 was used. In addition, the sequence of the B3 antibody (SEQ ID NO: 228) in which the K439E mutation was introduced into Gld (SEQ ID NO: 227) obtained by deleting the C-terminal Gly and Lys of IgG1 was used as the H chain constant region. The specified heavy chain, created by the fusion of GpH 7 and B3, was designated as GpH 7-B3 (VH, SEQ ID NO: 225; CH, SEQ ID NO: 228).

In GpH, 7-B3 amino acid residues that are expected to be involved in binding to Fc-gamma R and their surrounding amino acid residues (positions 234-239, 265-271, 295, 296, 298, 300 and 324-337 according to EU numbering) were replaced by 18 amino acid residues, respectively, excluding the original amino acid residue and Cys. These Fc variants are referred to as OT variants. B3 variants were expressed and the binding of protein A-purified antibodies to each of the Fc-gamma R (Fc-gamma RIIa type H, Fc-gamma RIIa type R, Fc-gamma RIIb, and Fc-gamma RIIIaF) was comprehensively assessed following

- 96 040713 in the same way. Interaction analysis between each modified antibody and Fc-gamma receptor prepared according to the method described above was performed using BIACORE® T100 (GE Healthcare), BIACORE® T200 (GE Healthcare), BIACORE® A100 or BIACORE® 4000. As running buffer HBS-EP+ (GE Healthcare) was used and the measurement temperature was set to 25°C. The chip was prepared by immobilizing Protein A (Thermo Scientific), Protein A/G (Thermo Scientific), or Protein L (ACTIGEN or BioVision) on an S-series CM4 or CM5 sensor chip (GE Healthcare) using the amine coupling method. . After capturing the antibodies of interest on these sensor chips, the Fc-gamma receptor diluted in running buffer was allowed to react and the amount of antibody-bound receptors measured. However, since the number of bound Fc-gamma receptors depended on the number of captured antibodies, the number of bound Fc-gamma receptors was divided by the amount of each captured antibody to obtain corrected values, and these values were compared. In addition, the antibodies immobilized on the chips were washed with 10 mM glycine-HCl, pH 1.5, resulting in chip regeneration and reuse. The level of Fc-gamma R binding to each B3 variant was divided by the level of Fc-gamma R binding of the parent B3 antibody (an antibody having the sequence of naturally occurring human IgG1 at positions 234-239, 265-271, 295, 296, 298, 300, and 324-337 according to the EU numbering). The value obtained by multiplying this level by 100 was used as an indication of the relative Fc-gamma R-binding activity of each variant.

In table. 15 Binding profile of selected substitutions to human Fc-gamma R (see below) shows the binding profile of prospective substitutions selected based on the following criteria: more than 40% higher binding to human Fc-gamma RIIb, more than 100% lower binding to human Fc-gamma RIIaR and Fc-gamma RIIaH, more than 10% reduction in binding to human Fc-gamma RIIIaF compared to the parent antibody B3.

The next step was to assess the cross-reactivity of these substitutions against cynoFcgamma R. 16 of these mutations were introduced into M103205H795-SG1. Variants were expressed using M103202L889-SK1 as light chain and analyzed for their affinity for cynoFcgamma RIIa1, IIa2, IIa3, IIb, IIIaS.

In table. 16 Binding profile of selected substitutions to cynoFc-gamma R (see below) shows the binding profile of these 16 variants to cynoFc-gamma R. The level of Fc-gamma R binding was obtained by dividing the amount of Fc-gamma R bound by the amount of each immobilized antibody. This value was standardized taking the value obtained for SG1 as 100.

Of the 16 Fc variants selected, only MY001 retained 85% binding to cynoFc-gamma RIIb relative to SG1 binding. In addition, MY001 showed reduced binding to cynoFc-gamma RIIIaS, while its binding to cynoFc-gamma RIIa1, IIa2 and IIa3 was preserved. As a result, the G236N mutation was found to be a unique substitution that provides a similar binding profile to both human and cynoFc-gamma R.

Although the G236N substitution is cross-reactive with both human and cynoFcgamma R, when introduced, the affinity for Fc-gamma RIIb is lower compared to SG1 (75% for human Fc-gamma RIIb and 85% for cynoFc-gamma RIIb, respectively). Additional substitutions were evaluated to increase affinity for Fc-gamma RIIb. Specifically, based on the result of the sweep mutagenesis assay (Table 17), substitutions were selected that exhibit increased binding to human Fc-gamma RIIb, reduced binding to human Fc-gamma RIIIa, and increased selectivity for human Fc-gamma RIIb for compared to human Fc-gamma RIIa.

- 97 040713

Table 17

Binding profile of further selected substitutions to human Fc-gamma R

Binding to human FcgRs (for parental OT=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 S239I 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 R271E 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 AZZOK 121 127 96 86 R331E 96 67 100 46 I332D 129 139 198 235 K334I 169 137 174 187 K334V 172 132 184 176 K334Y 157 131 160 188 K334M 151 127 148 183 K334D 97 72 108 155

Among these substitutions, L234W and L234Y were selected to improve the selectivity for human Fcgamma RIIb over human Fcgamma RIIa. In addition, G236A, G236S, A330R, AZZOK and P331E were selected to reduce binding to human Fc-gamma RIIIa. All other substitutions were selected to increase binding to human Fc-gamma RIIb. The cross-reactivity of the selected substitutions to cynoFc-gamma R was evaluated. In addition, the three substitutions P396M, V264I and E233D were also evaluated. Substitutions were introduced into M103205H795-SG1 and variants were expressed using M103202L889-SK1 as the light chain.

In table. 18 Kinetic analysis of binding of variants to human and cynoFc-gamma R (see below) shows the results of the kinetic analysis of selected substitutions, including G236N, in relation to both human and cynoFc-gamma R. In particular, the substitutions were introduced in M103205H795-SG1. Variants were expressed using M103202L889-SK1 as light chain and analyzed for their affinity for cynoFc-gamma RIIa1, IIa2, IIa3, IIb, IIIaS.

The KD values in bold cells were calculated using Equation 2 because their affinity was too low to be accurately determined by kinetic analysis. The fold of KD values was calculated by dividing the KD value for SG1 by the KD value for the variant for each Fc-gamma R.

For G236N, a binding affinity comparable to that of SG1 was found for cynoFc-gamma RIIa1, cynoFc-gamma RIIa2, cynoFc-gamma RIIa3, and cynoFc-gamma RIIb. Although the affinity for human Fc-gamma RIIb decreased to 0.6, the affinity for human Fc-gamma RIIaH and the affinity for human Fc-gamma RIIaR decreased to 0.2 and 0.1, respectively, indicating that this substitution has high selectivity for human Fc-gamma RIIb compared to human Fc-gamma RIIa. In addition, the affinities for cynoFc-gamma RIIIaS and human Fc-gamma RIIIaV were 0.03 and 0.04, respectively, which is preferable for eliminating ADCC activity. Based on this result, G236N has almost perfect cross-reactivity with human and cynoFc-gamma R, although affinity for both human and cynoFc-gamma RIIb may be increased when administered.

Among the additional substitutions studied, S298L, G236A, P331E, E233D, K334Y, K334M, L235W, S239V, K334I, L234W, K328T, Q295L, K334V, K326T, P396M, I332D, H268E, P271G, S267A and H268D resulted in increased binding to human and H268D , and with cynoFc-gamma RIIb. In particular, H268D demonstrated the highest potency (7-fold for human Fc-gamma RIIb and 5.3-fold for cynoFc-gamma RIIb). Regarding the effect of reducing binding to Fc-gamma RIIIa, less than 0.5-fold affinity was found for G236S, A330K and G236D compared to SG1.

To increase the affinity of MY001 for both human and cynoFc-gamma RIIb, combinations of substitutions listed in Table 1 were evaluated. 18. In particular, substitutions were introduced in M103205H795-SG1. Vari

- 98 040713 ants were expressed using M103202L889-SK1 as light chain and analyzed for their affinity. In table. 19 Binding kinetic analysis of variants that contain G236N and additional substitutions (see below) presents the results of a kinetic analysis of binding to human and cynoFc-gamma R. KD values in bold cells were calculated using Equation 2 because their affinity was too low to accurately determine it using kinetic analysis. The fold of KD values was calculated by dividing the KD value for SG1 by the KD value for the variant for each Fc-gamma R.

All variants suppressed affinity for human Fc-gamma RIIIaV by less than 0.26-fold and for cynoFc-gamma RIIIaS by less than 0.42-fold compared to SG1. In addition, all variants except MY047, MY051 and MY141 successfully demonstrated increased binding to both human and cynoFc-gamma RIIb compared to MY001, their binding to human Fc-gamma RIIa was maintained at less than 2-fold by compared to SG1. Among them, MY201, MY210, MY206, MY144, MY103, MY212, MY105, MY205, MY109, MY107, MY209, MY101, MY518, MY198 and MY197 were characterized by increased binding to both human and cynoFc-gamma RIIb, exceeding more than 4 fold binding of SG1. In addition, MY205, MY209, MY198 and MY197 showed more than 7-fold increased binding to both human and cynoFc-gamma RIIb.

WO 2014030728 illustrates that substitutions at position 396 in the CH3 domain increased affinity for human Fc-gamma RIIb. A complete enumeration of amino acid substitutions was introduced at position 396 M1O32O5H795-MYO52, which contains the substitutions G236N/H268E/P396M. The resulting variants were expressed using M103202L889-SK1 as light chain and their affinity for human and cynoFc-gamma R was evaluated (Table 20 Binding Kinetic Analysis of P396 Variants Derived from MV052 (see below). The fold KD values were calculated by dividing the value KD for SG1 by the KD value for the variant for each Fc-gamma R.

Among the substitutions studied, P396I, P396K and P396L maintained more than 5-fold binding to human Fc-gamma RIIb compared to SG1, and only the P396L substitution resulted in increased affinity for human Fc-gamma RIIb compared to MY052. Regarding binding to cynoFc-gamma RIIb, the parental MY052 showed the highest affinity, 3.9 times higher than that of SG1.

Since G236N has ideal cross-reactivity with human and cynoFc-gamma R, other substitutions at position 236 that were not explored in the previous examples were studied. In particular, Asn in M103205H795-MY201 was replaced with Met, His, Val, Gln, Leu, Thr, and Ile. The resulting variants were expressed using M103202L889-SK1 as light chain and their affinity for human and cynoFc-gamma R was evaluated (Table 21 Binding Kinetic Analysis of G236 Variants Derived from MV201 (see below)). The KD values in bold cells were calculated using Equation 2 because their affinity was too low to be accurately determined by kinetic analysis. The fold of KD values was calculated by dividing the KD value for SG1 by the KD value for the variant for each Fc-gamma R.

Although all variants had reduced affinity for both human and cynoFc-gamma RIIb compared to the parent molecule MY201, MY265, which was generated by replacing Asn with Thr at position 236 of MY201, retained a 2.1-fold increased binding to human Fcgamma RIIb and 3.1-fold binding to cynoFc-gamma RIIb, respectively, compared to SG1. The MY265 variant also retained reduced binding to both human and cynoFc-gamma RIIIa. Although the affinity for human Fc-gamma RIIaH and Fc-gamma RHaR increased over 2.5-fold compared to SGI, G236T was the second preferred substitution at position 236.

To improve the selectivity and affinity of MY265 for the human Fc-gamma RIIb, a complete enumeration of amino acid substitutions at positions 231 and 232 was carried out. In particular, the amino acid at positions 231 and 232 of M1O32O5H795-MY265 was replaced with one of 18 amino acid residues, excluding the original amino acid and Cys. The resulting variants were expressed using M103202L889-SK1 as light chain and their affinity for human and cynoFc-gamma R was evaluated (Table 22 Binding Kinetic Analysis of Variants Derived from MV265 (see below). The fold KD values were calculated by dividing the KD value for SG1 by the KD value for the variant for each Fc-gamma R.

Regarding affinity for Fc-gamma RIIb, the addition of A231T, A231M, A231V, A231G, A231F, A231I, A231L, A231, A231W, P232V, P232Y, P232F, P232M, P232I, P232W, P232L increased binding to both human and cynoFc- RIIb compared to the parent molecule MY265. Moreover, the addition of A231T and A231G reduced binding to human Fc-gamma RIIaR compared to MY265.

Combinations of substitutions were studied that were not explored in the previous examples. Tested and potentially promising substitutions were introduced into M103205H795-SG1 in combination. The resulting variants were expressed using M103202L889-SK1 as the light chain and their affinity for human and cynoFc-gamma R was assessed. The results are shown in Table 1. 23A. Kinetic analysis of the binding of other combinations (see below) and in table. 23B. Binding kinetics of other combinations (see below) with KD values in bold cells calculated using Equation 2 because their affinity was too low to be accurately determined by kinetic analysis. The multiplicity of KD values was calculated by dividing the value

KD for SG1 by the KD value for the variant for each Fc-gamma R. For comparison, the values for MY209 are again shown, selected as promising options according to the results presented in table. 19.

Among the variants tested, only MY213 showed a higher binding affinity for both human and cynoFc-gamma RIIb compared to MY209. However, the affinity of MY213 for human Fc-gamma RIIaH and human Fc-gamma RIIaR was higher than for MY209.

Example 24.

Evaluation of myostatin clearance using Fc variants with increased activity against Fc-gamma RIIb in mice transgenic for all human Fc-gamma R.

The effect on myostatin clearance of an Fc variant with enhanced activity against Fc-gamma RIIb, constructed according to the method described in Example 23, was evaluated in mice in which all murine Fc-gamma Rs had been stifled by deletion and human Fc-gamma Rs encoded in as transgenes, were inserted into the mouse genome (Proc. Nail. Acad. Sci., 109, 2012, p. 6181). In these mice, all murine Fc-gamma R is replaced by human receptors, so the effect of increased affinity for human Fc-gamma RIIb on soluble antigen clearance can be assessed in these mice. In addition, pI-increasing substitutions were evaluated in combination with Fc variants with increased activity against Fc-gamma RIIb, which were designed according to the method described in Example 23.

Obtaining and profiling test variants.

Information about 8 tested antibodies and their binding profiles are summarized in table. 24 Binding profile of tested variants in mice transgenic for all human Fc-gamma R (see below). The heavy chain, MS103205H795-PK2, was generated by introducing pI-elevating substitutions (S400R/D413K) in MS103205H795-MY101. MS103205H795-MY351, MS103205H795-MY344, MS103205H795-MY335 were obtained by introducing other pI-increasing substitutions (Q311R/D413K) into MS103205H795-MY201, MS103205H795-MY205, MS103205H795, respectively. All MS1032LO06 variants were expressed with M103202L889-SK1 as light chain according to the method described in Example 23 and their affinity for human and cynoFc-gamma R was assessed using the method described in Example 23.

Based on the SPR analysis, the results of which are summarized in Table. 24, pI-increasing substitutions were confirmed not to affect binding to Fc-gamma R of Fc variants with enhanced Fc-gamma RIIb activity. MY101 and PK2, which were obtained by introducing S400R/D413K into MY101, were found to have 9 and 8 fold increased binding to human Fcgamma RIIb, respectively. The affinity of MY101 and PK2 for human Fc-gamma RIIa remained comparable to that of SG1. For other human and cynoFc-gamma R, an almost similar binding profile was found. Similarly, MY351 has a similar binding profile to the parent MY201 molecule, MY344 to the parent MY205, and MY335 to the parent MY265 (Table 21), respectively. These results indicate that any of the pairs of substitutions that increase pI, S400R/D413K or Q311R/D413K, do not affect the affinity for human and cynoFcgamma R.

PK study conducted in transgenic all human Fc-gamma R mice.

In vivo testing using transgenic all human Fc-gamma R mice.

Elimination of myostatin and anti-latent myostatin antibody was assessed in vivo after co-administration of anti-latent myostatin antibody and human latent myostatin to all human Fc-gamma R transgenic mice (FIGS. 20 and 21). Antibody to latent myostatin (0.3 mg/ml) and latent myostatin (0.05 mg/ml) were administered in a single dose of 10 ml/kg into the caudal vein. Anti-CD4 antibody (1 mg/ml) was administered three times (every 10 days) at a dose of 10 ml/kg into the caudal vein to suppress anti-drug antibodies. Blood samples were collected at 5 min, 15 min, 1 h, 4 h, 7 h, 1 day, 2 days, 7 days, 14 days, 21 days and 28 days after administration. The collected blood was immediately centrifuged at 15,000 rpm at 4° C. for 5 minutes to separate the plasma. The separated plasma was stored at -20° C. or below until analysis. MS1032LO06-SG1, MS1032LO06-MY101, MS1032LO06-PK2, MS1032LO06-MY201, MS1032LO06MY351, MS1032LO06-MY205, MS1032LO06-MY344 and MS1032LO06-MY335 were used as antibodies to latent myostatin.

Measurement of plasma concentration of total myostatin by electrochemiluminescence (ECL).

The concentration of total myostatin in the plasma of mice was measured using ECL. Anti-mature myostatin antibody immobilized plates were prepared by applying anti-mature myostatin antibody RK35 (described in WO 2009/058346) to a 96-well MULTI-ARRAY plate (Meso Scale Discovery) and incubating in blocking buffer for 2 hours at room temperature. Mature myostatin samples were prepared to obtain a calibration curve and plasma samples diluted 40 times or more. Samples were mixed in an acid solution (0.2 M glycine-HCl, pH 2.5) to separate by dissociation of mature myostatin from its binding protein (such as a propeptide). Then samples

- 100 040713 was added to a plate immobilized with an antibody to mature myostatin and allowed to bind for 1 hour at room temperature, after which washing was carried out. Biotin labeled anti-mature myostatin antibody RK22 (described in WO 2009/058346) was then added and the plate was incubated for 1 hour at room temperature followed by washing. Then, SULFO labeled streptavidin (Meso Scale Discovery) was added and the plate was incubated for 1 hour at room temperature, followed by washing. Read Buffer T (x4) (Meso Scale Discovery) was immediately added to the plate, and signal detection was performed using a SECTOR Imager 2400 (Meso Scale Discovery). Mature myostatin concentration was calculated from the response to the standard curve using SOFTmax PRO analytical software (Molecular Devices). The time dependence of total myostatin plasma concentration after intravenous administration of antibodies to latent myostatin and latent myostatin, determined using this method, is shown in Fig. 20.

Measurement of anti-latent myostatin antibody concentration in plasma using high-performance liquid chromatography-tandem spectrometry with electrospray ionization (LC/ESIMC/MS).

Anti-latent myostatin antibody concentration in mouse plasma was measured by LC/ESIMC/MS. Mouse plasma concentrations of calibration standards were 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, and 200 µg/mL. 3 µl of calibration standards and plasma samples were added to 50 µl of AbCapture Mag (ProteNova) and allowed to incubate for 2 hours at room temperature. The magnetic beads were then isolated from the samples and washed twice with 0.2 ml (10 mmol/L) PBS with 0.05% Tween 20. The magnetic beads were then washed with PBS (10 mmol/L) to ensure removal of Tween 20. After washing, the magnetic beads were suspended in 25 µl of a mixture containing urea (7.5 mol/l), dithiothreitol (8 mmol/l) and lysozyme (chicken egg protein) (1 µg/ml), 50 mmol/l ammonium bicarbonate, and the suspended samples were incubated for 45 min at 56°C. Then 2 μl of iodoacetamide (500 mmol/l) was added and the samples were incubated for 30 min at 37° C. in the dark. Lysyl endopeptidase digestion was then carried out by adding 150 μl of a solution containing 0.67 μg/ml of lysyl endopeptidase suitable for biochemical analyzes (Wako) in ammonium bicarbonate (50 mmol/l) and the samples were incubated for 3 hours at 37°C. Trypsin digestion was then carried out by adding 10 μl (10 μg/ml) of sequencing grade modified trypsin (Promega) in ammonium bicarbonate (50 mmol/l). Digestion was allowed to take place in the samples by stirring overnight at 37° C. and 5 μl of 10% trifluoroacetic acid was added to terminate the reaction. 50 μl of digested samples were analyzed by LC/ESI-MS/MS. LC/ESI-MS/MC was performed using a Xevo TQ-S triple quadrupole device (Waters) equipped with 2D-UPLC class I (Waters). Anti-latent myostatin specific peptide YAFGQGTK and lysozyme specific peptide GTDVQAWIR used as an internal standard were monitored using Selected Reaction Monitoring (SRM) mode. The SRM transition was [M+2H]2+ (m/z 436.2) to y8 ion (m/z 637.3) for latent myostatin antibody and [M+2H]2+ (m/z 523, 3) to the y8 ion (m/z 545.3) for lysozyme. An internal standard curve was created using a weighted (1/x or 1/x2) linear regression using the dependence of peak area on concentrations. The plasma concentration in mice was calculated using a calibration curve using Masslynx analytical software, version 4.1 (Waters). The time dependence of plasma antibody concentration after intravenous administration of anti-latent myostatin antibody and latent myostatin measured by this method is shown in FIG. 21.

Effect of pI and Fc-gamma R binding on myostatin concentration in vivo.

After the introduction of MS1032LO06-SG1, the concentration of total myostatin in plasma after 7 hours decreased by 5 times compared with the concentration of total myostatin in plasma after 5 minutes. In contrast, after administration of MS1032LO06-MY101, MS1032LO06-MY201, and MS1032LO06-MY205, plasma total myostatin concentration after 7 hours decreased by 28-200 times compared with plasma total myostatin concentration after 5 minutes. In addition, after the administration of MS1032LO06-PK2, MS1032LO06-MY351, MS1032LO06-MY344, and MS1032LO06-MY335, the concentration of total myostatin in plasma after 7 hours decreased by 361-419 times compared with the concentration of total myostatin in plasma after 5 minutes. On the other hand, the difference in the concentration of each antibody at each time point tested compared to MS1032LO06-SG1 was found to be approximately 2-fold, and the pI variants did not increase antibody elimination from plasma. Substitutions resulting in both increased binding to human Fc-gamma RIIb and increased pI increased myostatin but not antibody elimination.

Variants with high pI had a greater positive charge in plasma. Therefore, the indicated positive charge interacted with the negatively charged cell surface, the antigen-antibody immune complex of the variants with high pI contacted the cell surface more strongly, which led to increased absorption of the antigen-antibody variants antigen-antibody complex by the cell.

- 101 040713 with high pI.

Example 25.

Evaluation of myostatin clearance using Fc variants with increased activity against Fc-gamma RIIb in monkeys.

The effect of increased binding capacity of Fc variants generated according to the method described in Example 23 to cynoFc-gamma RIIb on myostatin elimination was evaluated in cynomolgus monkeys. In addition, the combined effect with substitutions leading to an increase in pI was also evaluated.

Obtaining and profile of evaluated options.

The 14 antibodies tested and their binding profiles are summarized in Table 1. 25 (see below). The presence of an Fc variant with increased ability to bind to FcRn at acidic pH has been reported to increase the half-life of the antibody in vivo (J. Biol. Chem. 281, 2006, c. 23514-23524; Nat. Biotechnol. 28, 2010, cc. 159, Clin Pharm & Thera 89(2), 2011, pp. 283-290). To increase the half-life of the antibody without binding to rheumatoid factor, the invention combined these substitutions with Fc variants with increased activity against Fc-gamma RIIb and increased pI.

Тяжелые цепи MS103205H795-SG1012, MS103205H795-SG1029, MS103205 H795-SG1031, MS103205H795-SG1033, MS103205H795-SG1034 получали путем интродукции замены N434A в MS103205H795-MY101, MS103205 H795-MY344, MS103205H795-MY351, MS103205H795-MY201, MS103205H795-MY335, соответственно . MS103205H795-SG1016 was generated by introducing pI-increasing substitutions (Q311R/D399K) into MS103205H795-SG1012. MS103240H795-SG1071 and MS103240H795-SG1079 were obtained by introducing the M428L/N434A/Y436T/Q438R/S440E substitutions and the N434A/Q438R/S440E substitutions into MS103240 H795-MY344, respectively (MS103240H795: ID NO: 92 SEQ).

MS103240H795-SG1074 and MS1O324OH795-SG1O77 were generated by introducing pI-increasing substitutions Q311R/P343R and substitutions M428L/N434A/Y436T/Q438R/S440E in MS103240H795-MY209 and MS103240H795-MY518, respectively. MS103240H795-SG1080 and MS103240H795-SG1081 were generated by introducing pI-increasing substitutions Q311R/P343R and substitutions N434A/Q438R/S440E in MS103240H795-MY209 and MS103240H795-MY518, respectively. MS103240H795-SG1071 was generated by introducing pI-increasing substitutions Q311R/D413K and substitutions M428L/N434A/Y436T/Q438R/S440E in MS103240H795-MY205. MS103240H795-SG1079 was generated by introducing pI-increasing substitutions Q311R/D413K and substitutions N434A/Q438R/S440E in MS103240H795-MY205. These MS1032LO06 variants and MS1032LO19 variants were expressed with M103202L889-SK1 and M103202L1045-SK1 respectively (M103202L1045: VL, SEQ ID NO: 97) as light chain according to the method described in Example 30, and their affinity for human and cynoFcgamma R was evaluated using the method described in Example 23. In this example, the fold KD value for each Fc variant was calculated by dividing the KD value for the parent SG1 molecule by the KD value for the variant for each Fc-gamma R.

For example, the multiples of KD values for MS1032LO06-SG1012 and MS1032LO19-SG1071 were calculated by dividing the KD values for MS1032LO06-SG1 and MS1032LO19-SG1 by the KD values for MS1032LO06-SG1012 and MS1032LO19-SG1071, respectively.

In table. 25 summarizes the results of the SPR analysis. Of these variants, SG1012, SG1016, SG1074, and SG1080 had the strongest affinity for human Fc-gamma RIIb, with 10-fold higher affinity for human Fc-gamma RIIb compared to SG1. 29.2. PK study in monkeys In vivo testing using cynomolgus monkeys Endogenous myostatin accumulation was assessed in vivo after administration of anti-latent myostatin antibody to 2-4 year old Masaca fascicularis (cynomolgus monkey) from Cambodia (Shin Nippon Biomedical Laboratories Ltd. , Japan). A dose of 30 mg/kg was injected into the cephalic vein of the forearm using a disposable syringe, an extension tube, an internal needle and an infusion pump. The dosing rate was 30 minutes per animal. Blood samples were collected before the start of dosing and either 5 min, 7 h and 1, 2, 3, 7, 14, 21, 28, 35, 42, 49 and 56 days after the end of dosing, or 5 min 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 taken from the femoral vein using a syringe containing sodium heparin. The blood was immediately cooled on ice and plasma was obtained by centrifugation at 4°C, 1700xg for 10 min. Plasma samples were stored in a deep freezer (acceptable range: -70° C. or below) until analysis. В качестве антител к латентному миостатину применяли MS1032LO00-SG1, MS1032LO06-SG1012, MS1032LO06-SG1016, MS1032LO06-SG1029, MS1032LO06SG1031, MS1032LO06-SG1033 и MS1032LO06-SG1034 (в контексте настоящего описания SG1012, SG1016, SG1029, SG1031, SG1033 и SG1034 обозначают константные heavy chain regions constructed from SG1 according to the method described below).

Anti-latent myostatin antibodies MS1032LO19SG1079, MS1032LO19-SG1071, MS1032LO19-SG1080, MS1032LO19-SG1074, MS1032LO19-SG0321 (MS1032LO19-SG1080, MS1032LO19-SG1074, context of the present description SG1079, SG1071, SG1080, SG1074, SG1081 and

- 102 040713

SG1077 denotes heavy chain constant regions constructed from SG1 according to the method described below). Blood samples were 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 according to the procedure described above. Plasma samples were stored in a deep freezer (acceptable range: -70° C. or below) until analysis.

Plasma measurement of total myostatin by electrochemiluminescence (ECL)

Plasma total myostatin concentration in monkeys was measured by ECL according to the method described in Example 20. The time course of plasma total myostatin concentration after intravenous administration of anti-latent myostatin antibody, determined using this method, is shown in FIG. 22.

Measurement of ADA in monkey plasma using electrochemiluminescence (ECL).

The biotinylated drug was used to sensitize a 96-well MULTI-ARRAY streptavidin plate (Meso Scale Discovery) and incubated in LowCross® buffer (Candor) for 2 hours at room temperature. Monkey plasma samples were diluted 20-fold in LowCross® buffer prior to loading into the plate. Samples were incubated overnight at 4°C. The next day, the plate was washed three times with wash buffer before adding a SULFO-labeled anti-monkey IgG secondary antibody (Thermo Fisher Scientific). After incubation for 1 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 detection was performed with a SECTOR Imager 2400 (Meso Scale Discovery).

Effect of pH-dependence and Fc engineering on myostatin accumulation in monkeys in vivo.

Administration of pH-independent antibody (MS1032LO00-SG1) to cynomolgus monkeys resulted in at least a 60-fold increase in myostatin concentration on day 28 from baseline. On day 28, pH-dependent latent myostatin antibodies MS1032LO06-SG1012 and MS1032LO06-SG1033 provided 3- and 8-fold increases from baseline, respectively. The strong sweeping effect is mainly associated with an increase in affinity for Fc-gamma RIIb of cynomolgus monkeys. pH-dependent antibodies to latent myostatin MS1032LO06-SG1029, MS1032LO06-SG1031 and MS1032LO06-SG1034 were able to eliminate the antigen by day 28 to a level below baseline. The reason for the strong sweeping effect induced by MS1032LO06-SG1029, MS1032LO06SG1031 and MS1032LO06-SG1034K) is an increase in non-specific uptake by the cell due to an increase in the positively charged antibody cluster and an increase in Fc-gamma R mediated cellular uptake due to increased binding to Fc-gamma R .

The introduction of pH-dependent antibodies to latent myostatin MS1032LO19-SG1079, MS1032LO19SG1071, MS1032LO19-SG1080, MS1032LO19-SG1074, MS1032LO19-SG1081 and MS1032LO19-SG1077 reduced the concentration of myostatin to a level below the detection limit (<0.25 ng/ml) 1 in cynomolgus monkeys. On day 14, myostatin concentrations exceeded the detection limit with MS1032LO19-SG1079 and MS1032LO19-SG1071, although myostatin levels remained below the detection limit with MS1032LO19-SG1080, MS1032LO19-SG1074, MS1032LO19-SG1081, and MS1032LO19-SG1077. The weaker suppression found for MS1032LO19-SG1079 and MS1032LO19-SG1071 may be the result of various mutations leading to a change in pI.

The results suggest that it is possible to achieve a strong elimination of myostatin from plasma using mutations that increase binding to Fc-gamma RIIb or combinations of mutations that increase the positive charge of the antibody and increase binding to Fc-gamma RIIb. It can be expected that a strong elimination of myostatin can be achieved in humans by combining mutations that increase the positive charge of the antibody and increase binding to Fc-gamma R.

Example 26.

Screening for pI-increasing substitutions to increase myostatin clearance.

In this example, the role of pI-increasing substitutions in the Fc region of an antibody in increasing myostatin clearance was evaluated. The method of introducing amino acid substitutions in the constant region of an antibody to increase pI is not limited to the above method, but for example, it can be carried out according to the method described in WO 2014/145159. As with the variable region, the amino acid substitutions that were introduced into the constant region were preferably substitutions that reduced negatively charged amino acids (such as aspartic acid and glutamic acid) while increasing positively charged amino acids (such as arginine and lysine). In addition, amino acid substitutions can be introduced at any position in the antibody constant region and can be a single amino acid substitution or combinations of several amino acid substitutions. The sites of introduction of amino acid substitutions are preferably, but not limited to, positions at which the side chains

- 103040713 amino acids can be displayed on the surface of the antibody molecule. Most preferred examples include the method of introducing a combination of several amino acid substitutions at positions that can be exposed on the surface of an antibody molecule. Alternatively, the multiple amino acid substitutions introduced in the course of the present invention are preferably positioned so that they are in structural proximity to one another. In addition (but not limited to), several of the amino acid substitutions introduced during the creation of the present invention, preferably were substitutions for positively charged amino acids, so that it preferably leads to a state in which many positive charges are present in structurally similar positions.

Obtaining and profile of studied variants.

Information about the studied antibodies are summarized in table. 26'' Summary of Fc variants with increased pI (see below). The MS103205H795-SG141 heavy chain was generated by introducing pI-increasing Q311R/D399R substitutions into MS103205H795-SG1. Other variants of the heavy chains were also obtained by introducing the appropriate substitutions, presented in table. 26, in MS103205H795SG1. All variants of MS1032LO06 were expressed with M103202L889-SK1 as light chain according to the method described in Example 30.

Evaluation of binding activity to mouse Fc-gamma RII Fc variants with increased pI using BIACORE®.

Whether or not variant-containing Fc-region antibodies were obtained, binding assays between soluble mouse Fc-gamma RII and antigen-antibody complexes were performed using BIACORE® T200 (GE Healthcare). Soluble mouse Fc-gamma RII was obtained in the form of a His labeled molecule using a method known to those skilled in the art. An appropriate amount of anti-His antibody was fixed on a CM5 sensor chip (GE Healthcare) by the amine coupling method using a His capture kit (GE Healthcare) to immobilize mouse Fc-gamma RII. The antibody-antigen complex and running buffer (as reference solution) were then injected and allowed to react with mouse Fc-gamma RII immobilized on the sensor chip. 20 mM N-(2-acetamido)-2-aminoethanesulfonic acid, 150 mM NaCl, 1.2 mM CaCl ₂ and 0.05% (w/v) Tween 20, pH 7.4 were added as running buffer, and an appropriate buffer was also used to dilute soluble mouse Fc-gamma RII. To regenerate the sensor chip, 10 mM glycine-HCl, pH 1.5 was used. All measurements were carried out at 25°C. The analyzes were performed based on the binding (RU) calculated from the sensograms obtained by measurements, and relative values are presented, for which the binding level of SG1 was taken as 1.00. Parameters were calculated using Evaluation BIACORE® T100 software (GE Healthcare).

The results of the SPR analysis are summarized in Table. 26. Several Fc variants were found to have increased affinity for mouse Fc-gamma RII fixed on the BIACORE® sensor chip.

Without being limited by any particular theory, this result can be explained as follows. As is known, the BIACORE® sensor chip is negatively charged, and it can be assumed that such a charged state resembles the state of the cell membrane surface. More specifically, the affinity of the antigen-antibody complex for a mouse Fc-gamma RII fixed on a negatively charged BIACORE® sensor chip would likely resemble the binding of an antigen-antibody complex to a mouse Fc-gamma RII present on a similarly negatively charged cell membrane surface.

As used herein, antibodies obtained by introducing pI-enhancing modifications into the Fc region are antibodies in which the charge of the Fc region is more positive than before the introduction of the modifications. Thus, it can be assumed that the Coulomb interaction between the Fc region (positive charge) and the surface of the sensor chip (negative charge) will be enhanced when using amino acid modifications that increase pI. In addition, similar phenomena are expected to occur on the same negatively charged surface of the cell membrane; therefore, it can also be expected that they reflect the phenomena of increasing the rate of uptake by the cell in vivo.

Of the Fc variants with elevated pI with two amino acid substitutions relative to SG1, the antigen-antibody complex formed by SG141, P1499m, P1501m and P1540m exhibited the highest level of binding to human Fc-gamma RIIb. The amino acid substitutions Q311R/D399R, Q311R/P343R, Q311R/D413R and D401R/D413K were confirmed to have strong charge-driven effects on binding to human Fc-gamma RIIb on the sensor chip.

Cellular uptake of Fc variants with increased pI.

To assess the rate of intracellular uptake of human Fc-gamma RIIb expressing cell line, the following assay was performed. MDCK (Maidin-Derby dog kidney cell line) cells that constitutively express human Fc-gamma RIIb were obtained

- 104 040713 known methods. These cells were used to evaluate the intracellular uptake of antigen-antibody complexes.

In particular, pHrodoRed (Life Technlogies) was used to label human latent myostatin (antigen) according to a known protocol, and antigen-antibody complexes were obtained in culture solution with an antibody concentration of 10 mg/ml and an antigen concentration of 2.5 mg/ml. The culture solution containing the antigen-antibody complexes was added to culture plates with the above MDCK cells that constitutively express the human Fc-gamma RIIb and incubated for 1 hour, and then the fluorescence intensity of the antigen taken up by the cells was quantified using the InCell analyzer 6000 (GE healthcare). The amount of absorbed antigen was expressed as relative values, while the value for SG1 was taken as 1.00.

The quantitative results of cellular uptake are summarized in Table 1. 26.

When using some variants of Fc found a strong fluorescence in the cells inherent in the antigen.

Without being limited by any particular theory, this result can be explained as follows.

The antigen and antibodies added to the cell culture solution form antigen-antibody complexes in the culture solution. Antigen-antibody complexes bind to human Fcgamma RIIb expressed on the cell membrane via the Fc region of the antibody and are taken up by cells in a receptor-dependent manner. The antibodies used in this experiment bind to the antigen in a pH dependent manner; thus, the antibody can dissociate from the antigen in endosomes (acidic pH conditions) within cells. Since the dissociated antigen was labeled with pHrodoRed as described earlier, its fluorescence occurs in endosomes. Thus, the strong intensity of fluorescence inside the cell probably indicates that the absorption of antigen-antibody complexes into cells occurred at a faster rate or in greater quantities.

Among the Fc variants with increased pI with two amino acid substitutions relative to SG1, antigen-antibody complexes formed from SG141, P1375m, P1378m, P1499m, P1524m, P1525m, P1532m, P1533m, P1540m, P1541m and P1543m showed higher uptake of antigen by cells. Amino acid substitutions Q311R/D399R, Q311/D413K, S400R/D413K, Q311R/P343R, Q311R/G341R, Q311R/G341K, Q311R/D401R, Q311R/D401K, D401R/D413K, D411K/D413K, charge-mediated effect on cellular uptake of the antigen-antibody complex.

PK study conducted in transgenic human FcRn mice.

In vivo testing using transgenic human FcRn mice.

Elimination of myostatin and anti-latent myostatin antibody was assessed in vivo after co-administration of anti-latent myostatin antibody and latent myostatin to human FcRn transgenic mice. 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 PK-2 experiment (described in FIG. 23). Antibody to latent myostatin (0.3 mg/ml), human latent myostatin (0.05 mg/ml) and human normal immunoglobulin (CSL Behring AG) (100 mg/ml) were administered in a single dose of 10 ml/kg into the caudal vein in the PK-4 experiment (described in Fig. 24). Blood samples were collected at 5 min, 15 min, 1 h, 4 h, 7 h, 1 day, 2 days, 7 days, 14 days, 21 days and 28 days after administration in the PK-2 experiment. Blood samples were collected at 5 min, 1 h, 4 h, 7 h, 1 day, 7 days, 14 days, 21 days and 30 days after administration in the PK-4 experiment. The collected blood was immediately centrifuged at 15,000 rpm at 4° C. for 5 minutes to separate the plasma. The separated plasma was stored at -20° C. or below until analysis. Antibodies to latent myostatin MS1032LO06-SG1, MS1032LO06-P1375m, MS1032LO06P1378m, MS1032LO06-P1383m were used in the PK-2 experiment, and MS1032LO06-P1375m, MS1032LO06-P1499m were used in the PK-4 experiment.

Plasma measurement of total myostatin by electrochemiluminescence (ECL)

The concentration of total myostatin in the plasma of mice was measured using ECL according to the method described in example 24 (Ravetch PK). Data on the time dependence of the concentration of total myostatin in plasma after intravenous administration of antibodies to latent myostatin and latent myostatin, obtained using this method, are presented in Fig. 23A and 24A.

Measurement of the concentration of antibodies to latent myostatin in plasma using enzyme-linked immunosorbent assay (ELISA).

Anti-latent myostatin antibody concentration in mouse plasma was measured by ELISA in the PK-2 experiment. Anti-human IgG (gamma chain specific) F(ab')2 fragment (Sigma) was applied to a Nunc-Immuno MaxiSorp plate (Nalge Nunc International) and allowed to stand overnight at 4°C to prepare plates with immobilized anti-human IgG. Plasma concentrations of 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, and 0.039 µg/mL and mouse plasma samples diluted 100 or more

- 105 040713 times. Samples were then plated onto immobilized anti-human IgG plates and allowed to stand for 1 hour at room temperature. Goat anti-human IgG (gamma chain specific)-biotin conjugate (Southern Biotech) was then added and allowed to react for 1 hour at room temperature. Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was then added and allowed to react for 1 hour at room temperature, and a chromogenic reaction was performed using TMB One Component HRP Microwell (BioFX Laboratories) as a substrate. After termination of the reaction with 1N. sulfuric acid (Showa Chemical) absorbance was measured at 450 nm using a microplate reader. The plasma concentration in mice was calculated from the absorbance of the calibration curve using SOFTmax PRO analytical software (Molecular Devices). Data on the time dependence of the concentration of antibodies in plasma after intravenous administration of antibodies to latent myostatin and latent myostatin, obtained by this method, are presented in Fig. 23B.

In the PK-4 experiment, the plasma concentration of anti-latent myostatin antibody in mice was measured using the Gyrolab Bioaffy CD method (Gyros). The biotinylated anti-human IgGFc antibody was passed through a column of streptavidin beads in a CD microstructure. Samples were prepared for the calibration curve at plasma concentrations of 0.5, 1, 2, 4, 8, 16 and 32 μg/ml by injecting human normal immunoglobulin (CSL Behring AG) at a concentration of 5 mg/ml and at a concentration of 200 μg/ml mouse latent myostatin, and mouse plasma samples were diluted 25-fold or more by injecting into plasma at a concentration of 200 μg/ml mouse latent myostatin. Samples were then added to CD and passed through a bead column. Then, an Alexa-labeled goat anti-human IgG polyclonal antibody (BETHYL) was added to the CD and passed through a bead column. Mouse plasma concentration was calculated from the calibration curve using the Gyrolab Evaluator software. Data on the time dependence of the concentration of antibodies in plasma after intravenous administration of antibodies to latent myostatin and latent myostatin, obtained by this method, are presented in Fig. 24B.

Effect of pI on myostatin concentration in vivo.

After the introduction of MS1032LO06-SG1, the plasma concentration of total myostatin after 7 hours decreased by 12 times compared with the plasma concentration of total myostatin after 5 minutes in the PK-2 experiment. In contrast, after administration of MS1032LO06-P1375m, MS1032LO06-P1378m, and MS1032LO06P1383m, the plasma concentration of total myostatin after 7 h decreased by 26-67 times compared with the plasma concentration of total myostatin after 5 min in the PK-2 experiment. The concentration of antibodies MS1032LO06-P1378m and MS1032LO06-P1383m decreased by more than 3-fold compared to MS1032LO06-SG1, although the difference in the concentration of MS1032LO06-P1375m at each tested time point compared to MS1032LO06-SG1 was approximately 2-fold in the PK-2 experiment . In the presence of pI variants, including amino acid substitutions Q311R/D413K, an increased elimination of myostatin from plasma was found without an increase in the elimination of antibody from plasma.

In addition, the total myostatin concentration and the antibody concentration were determined after administration of MS1032LO06-P1499m by co-administration of human normal immunoglobulin to mimic human plasma. After the introduction of MS1032LO06-P1375m and MS1032LO06-P1499m, the concentration of total myostatin in plasma after 7 hours decreased by 3.4 and 5.3 times compared with the concentration of total myostatin in plasma after 5 minutes, and the concentration of total myostatin in plasma and the concentration of antibodies in each the tested time point when using MS1032LO06-P1499m was 1.5 times higher than those established for MS1032LO06-P1375m in the PK-4 experiment. In the presence of pI variants, including amino acid substitutions Q311R/P343R, an increased elimination of myostatin from plasma was found without an increase in the elimination of antibody from plasma.

Variants with high pI had a greater positive charge in plasma. Therefore, this positive charge interacted with the negatively charged cell surface, the antigen-antibody immune complex of the high pI variants contacted the cell surface more strongly, resulting in increased uptake of the antigen-antibody complex of the high pI variants by the cell.

Example 27.

Substitutions resulting in an increase in pI, in combination with an Fc variant with increased activity against Fc-gamma RIIb.

The effect on myostatin clearance of an Fc variant with increased activity against Fc-gamma RIIb in combination with other pI-increasing substitutions was evaluated in transgenic human Fc-gamma RIIb mice. Human Fc-gamma RIIb transgenic mice were generated by microinjection of a BAC (bacterial artificial chromosome) vector containing exons of the human FCGR2B gene into the pronucleus of fertilized eggs of C57BL/6N mice using standard techniques (see, for example, J. Immunol., 195 (7), October 1, 1915, pp. 3198-3205). Mouse Fc-gamma RIIb deficient mice were generated using zinc finger nucleases (ZFNs) that were designed to target exon 1. Humanized Fc-gamma RIIb mice were generated by crossing human Fc-gamma RIIb transgenic mice with ( KO)

- 106 040713 mouse Fc-gamma RIIb. Using these mice, the combined effects of increased affinity for human Fc-gamma RIIb and increased pI on soluble antigen clearance can be studied.

Obtaining and profiling test variants.

Information about the 7 tested antibodies and their binding profiles are summarized in table. 27 (see below). heavy chain, MS103205H795-MY352, MS103205H795-PK55, MS103205H795-PK56,

MS103205H795-PK57 were obtained by introducing pI-increasing substitutions Q311R/D413R, Q311R/P343R, Q311R/P343R/D413R, Q311R/N384R/D413R, respectively, in MS103205H795-MY201. All MS1032LO06 variants were expressed using M103202L889-SK1 as light chain according to the method described in Example 30 and their affinity for human and cynoFcgamma R was determined using the method described in Example 23.

Based on the results of the SPR analysis, which are summarized in Table. 27, it was confirmed that pI-increasing substitutions did not affect binding to Fc-gamma R of Fc variants with enhanced Fc-gamma RIIb activity. MY201 and MY351, which were obtained by introducing Q311R/D413K into MY201, were found to have 6-fold and 5-fold increased binding to human Fc-gamma RIIb, respectively. They had almost the same binding profile with other human and cynoFc-gamma R. Similarly, MY352, PK55, PK56 and PK57 showed a similar binding profile to that of the parent MY201 molecule (Table 27). These results indicate that any pair of substitutions resulting in an increase in pI did not affect the affinity for human and cynoFc-gamma R.

PK study conducted in transgenic human FcRn mice.

In vivo testing using transgenic human FcRn mice.

Myostatin and anti-latent myostatin antibody elimination was assessed in vivo after co-administration of anti-latent myostatin antibody and human latent myostatin to human Fc-gamma RIIb transgenic mice in which mouse Fc-gamma RII was replaced with human Fc-gamma RIIb. Antibody to latent myostatin (0.3 mg/ml) and mouse latent myostatin (0.05 mg/ml) were administered in a single dose of 10 ml/kg into the caudal vein. Blood samples were collected at 5 min, 1 h, 4 h, 7 h, 1 day, 7 days, 14 days, 21 days and 28 days after administration. The collected blood was immediately centrifuged at 15,000 rpm at 4° C. for 5 minutes to separate the plasma. The separated plasma was stored at -20° C. or below until analysis. The following latent myostatin antibodies MS1032LO06-SG1, MS1032LO06-MY201, MS1032LO06-MY351, MS1032LO06-MY352, MS1032LO06PK55, MS1032LO06-PK56 and MS1032LO06-PK57 were used.

Measurement of plasma concentration of total myostatin by electrochemiluminescence (ECL).

The plasma concentration of total myostatin in mice was measured by ECL according to the method described in Example 24. The time-dependence data of the plasma concentration of total myostatin after intravenous administration of anti-latent myostatin antibody and latent myostatin obtained using this method are shown in FIG. 25.

Measurement of the concentration of antibodies to latent myostatin in plasma using enzyme-linked immunosorbent assay (ELISA).

The concentration of antibodies to latent myostatin in the plasma of mice was measured using ELISA. An anti-human IgG (gamma chain specific) F(ab')2 fragment (Sigma) was applied to a Nunc-Immuno MaxiSorp plate (Nalge Nunc International) and allowed to stand overnight at 4°C to obtain plates with immobilized anti-human IgG. Plasma concentrations of 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, and 0.039 μg/mL and mouse plasma samples diluted 100-fold or more were prepared. Samples were then plated onto immobilized anti-human IgG plates and allowed to stand for 1 hour at room temperature. Goat anti-human IgG (gamma chain specific)-biotin conjugate (Southern Biotech) was then added and allowed to react for 1 hour at room temperature. Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was then added and allowed to react for 1 hour at room temperature and a chromogenic reaction was performed using TMB One Component HRP Microwell (BioFX Laboratories) as the substrate. After termination of the reaction with 1N. sulfuric acid (Showa Chemical) absorbance was measured at 450 nm using a microplate reader. The plasma concentration in mice was calculated from the absorbance of the calibration curve using SOFTmax PRO analytical software (Molecular Devices). Data on the time dependence of the concentration of antibodies in plasma after intravenous administration of antibodies to latent myostatin and latent myostatin, obtained by this method, are presented in Fig. 26.

Effect of pI and Fc-gamma R-binding on myostatin concentration in vivo.

After the introduction of MS1032LO06-MY201, the plasma concentration of total myostatin after 7 hours decreased by 8 times compared with the plasma concentration of total myostatin after 5 minutes. In contrast, after administration of MS1032LO06-PK57, the plasma concentration of total myostatin after 7 hours decreased by 376 times compared with the plasma concentration of total myostatin after 5 minutes. For others

- 107 040713 variants with high pI found a similar increase in the elimination of myostatin from plasma. The concentration of high pI antibody variants after 28 days decreased by 2-3 times compared with the concentration of the MS1032LO06-SG1 antibody, and analysis for pI variants revealed a slight increase in the elimination of antibodies from plasma.

Variants with high pI had a greater positive charge in plasma. Therefore, this positive charge interacted with the negatively charged cell surface, the antigen-antibody immune complex of the high pI variants contacted the cell surface more strongly, resulting in increased uptake of the antigen-antibody complex of the high pI variants by the cell.

Example 28.

Creation of Fc variants with enhanced activity against human Fc-gamma RIIb.

Fc variants with increased activity against human Fc-gamma RIIb having the variable region of an anti-myostatin antibody were constructed and their affinity for human Fc-gamma R was evaluated. In particular, Fc variants with increased activity against human Fc-gamma RIIb were constructed by introducing substitutions in MS103240H795-SG1 (VH, SEQ ID NO: 92; CH, SEQ ID NO: 9) in combination with substitutions T250V/T307P. In addition, substitutions (Q311R/D413K or Q311R/P343R) were also introduced. Variants were expressed using M103202L1045-SK1 as the light chain, and their affinity for human Fc-gamma R was analyzed according to the method described in example 30. In table. 28 The binding profile of Fc variants with increased activity against human Fc-gamma RIIb (see below) presents the results of kinetic analyzes against human and cynoFc-gamma R. The KD values, which are indicated in the cells of the table in bold, were calculated using the equation 2 because their affinity was too low to be accurately determined by kinetic analysis.

All studied variants showed increased affinity for human Fc-gamma RIIb and reduced ability to bind to human Fc-gamma RIIaR, Fc-gamma RIIaH, Fc-gamma RIIIaV compared to SG1. The affinity for human Fc-gamma of RIIb ranged from 4.1-fold to 30.5-fold compared to SG1. The affinity for human Fc-gamma RIIaR was between 0.02-fold and 0.22-fold. Affinities for human Fc-gamma RIIaH and human Fc-gamma RIIIaV were less than 0.04-fold and 0.012-fold, respectively. It was found that the substitutions (T250V/T307P) did not affect the binding profile to human Fc-gamma R compared to the affinity of MY009 (P238D) and MY214 (P238D/T250V/T307P). On the other hand, both pairs of pI-increasing substitutions (Q311R/D413K and Q311R/P343R) reduced binding affinity by about 0.5-fold compared to variants that did not contain pI-increasing substitutions. For example, although TT14 was found to have a 30.5-fold higher affinity for human Fc-gamma RIIb, only 16.1-fold and 145-fold increases were found when used in combination with Q311R/D413K (TT33) and Q311R/P343R (TT32 ) respectively.

In addition, the substitutions described in Example 25, which extended the half-life of the antibody and reduced binding to rheumatoid factor, were introduced into Fc variants with increased activity against human Fc-gamma RIIb and analyzed for their affinity for human Fc-gamma R (Table 29 Amino acid sequences of heavy chain constant regions of Fc variants with increased activity against human Fc-gamma RIIb (shown as SEQ ID NO:) kappa chain to capture antibodies of interest Table 30 Binding Profile of Fc Variants with Enhanced Activity on Human Fcgamma RIIb (see below) presents the results and where KD values in cells in bold were calculated using Equation 2 , because their affinity was too low to accurately determine it using kinetic anal isa.

In this experiment, SG1 and TT33 were again evaluated, while the capture method differed from that, the results of which are presented in table. 28. As a result, it was found that the values of the multiplicity of KD for TT33 corresponded to those obtained using the method, the results of which are given in Table. 28 (16.1-fold level in Table 28 and 15.7-fold level in Table 30 characterizing binding to human Fc-gamma RIIb). For TT92 and TT93, which were constructed by introducing N434A/Q438R/S440E and M428L/N434A/Y436T/Q438R/S440E into TT33, a binding profile comparable to TT33 was found. Similarly, for TT90 and TT91, which were obtained from TT32, TT72 and TT73, which were obtained from TT31, TT70 and TT71, which were obtained from TT30, TT68 and TT69, which were obtained from TT21, and TT66 and TT67, which were obtained from TT20, found a binding profile comparable to the parental antibodies shown in table. 28. These results indicate that substitutions designed to increase antibody half-life and reduce rheumatoid factor binding do not affect the binding profile to human Fc-gamma R Fc variants with increased activity against human Fc-gamma RIIb.

Example 29.

Cell imaging analysis of Fc variants with increased activity against human Fc-gamma RIIb.

To study the intracellular uptake of an antigen-antibody complex formed with the antibodies of Example 28, the cell imaging assay described in Example 26 was performed. was 1.25 mg/ml and the antigen concentration was 0.34 mg/ml.

The results of the analysis are shown in Fig. 27. With increasing binding affinity to human Fc-gamma RIIb, uptake of antigen-antibody complex by cells increased. In the case of TT14, which has a 30-fold increased binding affinity for human Fc-gamma RIIb compared to SG1, the fluorescence intensity of the antigen inside the cells was 7.5 times higher than the fluorescence intensity of SG1.

Although pI-increasing substitutions (Q311R/D413K and Q311R/P343R) reduced binding affinity for human Fc-gamma RIIb by about 0.5-fold compared to variants that did not contain pI-increasing substitutions, cellular uptake of the antigen-antibody complex with Fc variants containing pI-increasing substitutions increased compared to Fc variants without pI-increasing substitutions. For TT33, a 36-fold increase in cellular uptake of the antigen-antibody complex was found compared to SG1, while for the parent molecule TT14, a 7.5-fold increase in cellular uptake was found. In addition, uptake of the antigen-antibody complex by cells was found for TT33, comparable to the uptake by cells of the SG1071, SG1074, SG1077, SG1079, SG1080, and SG1081 variants, for which a strong sweeping effect was found when studying cynomolgus monkeys. These results suggest that combining pI-increasing substitutions with Fc variants with increased activity against Fc-gamma RIIb is an effective tool for eliminating antigens.

Example 30.

Construction of expression vectors for antibodies, expression and purification of antibodies.

Synthesis of full-length genes encoding the nucleotide sequences of the H chain and L chain of antibody variable regions was carried out using methods of preparation known to those skilled in the art, using PCR used for assembly, and the like. The introduction of amino acid substitutions was carried out according to methods known to those skilled in the art using PCR or the like. The resulting plasmid fragments were inserted into an animal cell expression vector, and an H chain expression vector and an L chain expression vector were obtained. The nucleotide sequence of the resulting expression vector was determined using methods known to those skilled in the art. The resulting plasmids were briefly introduced into the HEK293H cell line derived from human embryonic kidney cancer cells (Invitrogen) or FreeStyle293 cells (Invitrogen) for antibody expression. The resulting culture supernatant was collected and passed through a 0.22 µm MILLEX®-GV filter (Millipore) or through a 0.45 µm MILLEX®-GV filter (Millipore) to obtain a culture supernatant. Antibodies were purified from the obtained culture supernatant using methods known to those skilled in the art using r(recombinant) Protein A-Sepharose Fast Flow (GE Healthcare) or Protein G-Sepharose 4 Fast Flow (GE Healthcare). To determine the concentration of purified antibodies, their absorbance was measured at 280 nm using a spectrophotometer. From these values, the antibody concentration was determined using an extinction coefficient calculated using methods such as PACE (Protein Science 4, 1995, c. 2411-2423).

Although the invention has been described above for a better understanding with some details for the purpose of illustration and examples, the description and examples should not be construed as limiting the scope of the invention. The description of all patent and scientific literature cited in the present description is expressly incorporated herein by reference in its entirety.

Table 14

KD(M) for cynoFc ^RS KD (M) for human FcgRs Symbol heavy chain; Replacement FcgRIIal FcgRIIa 2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 1 M103205H795-SG1 4.2E-06 5.2E-06 1.5E-05 1.7E-06 4.2E-07 9.3E-07 1.4E-06 4.7E-06 1.2E-06 2 M103205H795-MY009 1 ^3238D 2.5E-05 2.6E-05 3.1E-05 4.3E-06 1.0E-04 7.5E-05 2.0E-05 1.4E-06 5.8E-04 KD fold for cynoFcgRs (SGI = 1) The multiplicity of KD in relation to human. FcgRs (SGI = 1) Symbol heavy chain FcgRIIal FcgRIIa 2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 1 M103205H795-SG1 1.0 1.0 1.0 1.0 1.000 1.00 1.0 1.0 1.000 2 M103205H795-MY009 0.2 0.2 0.5 0.4 0.004 0.01 0.1 3.3 0.002

- 109 040713

Table 15

Binding to human FcgRs (for parental OT = 100) Mutation FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV G236N 54 81 75 8 G237Y 77 4 85 0 G237D 74 0 95 0 R238E 21 2 97 1 P238Y 93 9 114 3 R238M 86 9 97 2 P238Q 19 4 43 -1 S239P 49 4 61 -1 V266F 91 8 88 2 S267H 27 3 42 1 S267L 63 3 112 2 N325L 93 13 116 0 N325E 49 13 40 4 N325I 76 8 97 1 N325F 65 6 72 -1 A327I 37 12 46 8

Table 16

Binding to cynoFcgRs (for SGI = 100) Symbol CH Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS SGI 100 100 100 100 100 MY001 G236N 83 86 62 85 15 SG165 G237Y 18 17 thirty thirty 7 SG166 G237D 10 10 17 8 5 SG167 P238Y 18 14 38 22 14 SG168 P238E 21 16 thirty 28 13 SG169 P238M 17 16 32 22 22 SG170 P238Q 16 15 21 18 12 SG171 S239P 17 20 18 20 8 SG172 V266F 14 17 27 16 31 SG173 S267L 13 14 21 12 24 SG174 S267H 13 16 20 eleven 15 SG175 N325F 13 13 17 14 8 SG176 N325I 14 15 19 16 20 SG177 N325L 15 15 25 18 21 SG178 N325E 22 21 29 26 36 SG179 A327I 19 17 28 19 48

- 110 040713

Table 18

KD (M) for cynoFcgRs KD(M) against nea.FcgRs Symbol heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 1 M103205H795-SG1 - 3.2E-06 5.1E-06 1.7E-05 1.8E-06 3.6E-07 6.9E-07 1.1E-06 4.6E-06 8.1E-07 2 M103205H795-MY016 E233D 2.9E-06 3.4E-06 3.2E-06 1.7E-06 2.3E-07 8.5E-07 9.4E-07 2.9E-06 3.7E-07 3 M103205H795-MY006 L234W 2.0E-06 1.9E-06 3.6E-06 9.2E-07 3.2E-07 1.3E-06 1.8E-06 2.3E-06 7.3E-07 4 M103205H795-MY013 L234Y 4.5E-06 4.7E-06 5.2E-05 2.0E-06 3.3E-07 2.6E-06 5.1E-06 3.7E-06 4.1E-07 5 M103205H795-MY070 L235W 2.3E-06 2.8E-06 4.7E-06 1.2E-06 4.9E-07 5.0E-07 7.4E-07 2.6E-06 7.0E-07 6 M103205H795-MY001 G236N 2.9E-06 3.6E-06 1.5E-05 1.8E-06 1.2E-05 3.2E-06 8.2E-06 7.7E-06 1.8E-05 7 M103205H795-MY061 G236D 6.5E-06 6.8E-06 1.8E-05 4.2E-06 3.4E-06 1.4E-06 1.3E-06 2.5E-06 3.0E-06 8 M103205H795-MY071 G236E 5.0E-06 3.7E-06 9.2E-06 2.5E-06 2.8E-07 4.0E-07 4.5E-07 3.1E-06 5.1E-07 9 M103205H795-MY074 G236A 2.0E-06 2.3E-06 6.2E-06 1.1E-06 2.7E-07 9.2E-08 2.1E-07 3.6E-06 5.6E-07 10 M103205H795-MY124 G236S 2.6E-06 3.1E-06 1.2E-05 2.2E-06 1.3E-06 1.4E-07 5.2E-07 9.2E-06 5.2E-06 eleven M103205H795-MY008 S239V 4.2E-06 3.7E-06 2.4E-05 1.7E-06 3.7E-07 1.6E-06 1.8E-06 2.6E-06 6.3E-07 12 M103205H795-MY037 S239N 3.8E-06 4.5E-06 9.6E-06 2.4E-06 2.0E-07 1.1E-06 9.1E-07 2.6E-06 3.7E-07 13 M103205H795-MY038 S239I 3.9E-06 4.5E-06 I.IE-05 1.9E-06 3.1E-07 1.2E-06 1.5E-06 2.6E-06 5.5E-07 14 M103205H795-MY010 V264I 3.5E-06 5.0E-06 1.6E-05 2.0E-06 2.2E-07 1.2E-06 1.8E-06 5.1E-06 2.2E-07 15 M103205H795-MY011 S267A 2.3E-06 2.8E-06 9.9E-06 1.0E-06 1.5E-07 4.9E-07 1.7E-07 6.6E-07 3.4E-07 16 M103205H795-MY017 H268E 2.7E-06 2.9E-06 5.5E-06 6.8E-07 9.8E-08 4.5E-07 2.8E-07 9.4E-07 1.4E-07 17 M103205H795-MY063 H268D 1.4E-06 1.5E-06 2.0E-06 3.4E-07 6.7E-08 2.7E-07 2.1E-07 6.6E-07 1.6E-07 18 M103205H795-MY012 P271G 2.1E-06 2.3E-06 7.9E-06 1.1E-06 2.0E-07 4.4E-07 2.3E-07 9.0E-07 4.8E-07 19 M103205H795-MY040 P271D 5.5E-06 LIE-05 4.4E-06 3.5E-06 3.7E-07 1.8E-06 1.2E-06 3.0E-06 6.1E-07 20 M103205H795-MY041 R271E 4.0E-06 4.1E-06 7.4E-06 3.5E-06 2.6E-07 1.8E-06 1.2E-06 3.1E-06 4.8E-07 KD (M) for cynoFcgRs KD(M) against nea.FcgRs Symbol heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 21 M103205H795-MY073 Q295L 1.2E-06 1.3E-06 5.1E-06 6.7E-07 1.7E-07 2.4E-07 5.0E-07 2.0E-06 3.4E-07 22 M103205H795-MY042 S298L 5.1E-07 3.1E-06 8.0E-06 1.6E-06 4.4E-07 1.0E-06 9.8E-07 4.0E-06 5.3E-07 23 M103205H795-MY069 K326T 1.6E-06 1.6E-06 6.5E-06 7.3E-07 8.6E-08 4.4E-07 3.9E-07 1.6E-06 2.4E-07 24 M103205H795-MY043 A327N 5.8E-06 6.1E-05 1.8E-05 4.4E-06 8.4E-07 2.9E-06 1.5E-06 3.0E-06 1.2E-06 25 M103205H795-MY068 L328T 2.1E-06 2.7E-06 5.7E-06 1.0E-06 5.8E-07 3.7E-07 5.2E-07 2.1E-06 1.4E-06 26 M103205H795-MY014 A330R 2.5E-06 2.5E-06 1.2E-05 1.5E-06 4.0E-07 5.2E-07 9.6E-07 4.8E-06 6.9E-07 27 M103205H795-MY196 AZZOK 2.5E-06 3.0E-06 2.3E-05 2.4E-06 6.0E-07 4.8E-07 1.3E-06 6.2E-06 2.2E-06 28 M103205H795-MY044 R331E 3.2E-06 3.5E-06 5.2E-05 1.6E-06 4.7E-07 1.2E-06 9.2E-07 3.3E-06 9.0E-07 29 M103205H795-MY072 I332D 1.0E-06 1.2E-06 2.6E-06 4.8E-07 5.3E-08 2.5E-07 4.8E-07 1.5E-06 1.2E-07 thirty M103205H795-MY045 K334D 5.7E-06 4.0E-06 6.8E-06 2.2E-06 1.4E-07 1.6E-06 1.6E-06 2.9E-06 2.4E-07 31 M103205H795-MY064 K334I 2.4E-06 2.6E-06 6.9E-06 1.1E-06 1.2E-07 5.0E-07 6.8E-07 2.4E-06 2.2E-07 32 M103205H795-MY065 K334V 2.3E-06 2.8E-06 4.2E-06 1.3E-06 1.1E-07 5.2E-07 5.1E-07 1.9E-06 2.3E-07 33 M103205H795-MY066 K334Y 2.1E-06 2.6E-06 6.7E-06 1.4E-06 1.6E-07 6.4E-07 9.7E-07 2.8E-06 2.9E-07 34 M103205H795-MY067 K334M 2.1E-06 2.3E-06 6.7E-06 1.1E-06 1.0E-07 5.3E-07 7.6E-07 2.8E-06 2.0E-07 35 M103205H795-MY015 R396M 1.3E-06 1.5E-06 4.7E-06 6.0E-07 1.0E-07 3.3E-07 3.2E-07 1.6E-06 2.4E-07

- 111 040713

KD fold for cynoFcgRs (SGI = 1) KD fold in relation to nen.FcgRs (SGI = 1) Symbol heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 1 M103205H795-SG1 - 1.0 1.0 1.0 1.0 1.00 1.0 1.0 1.0 1.00 2 M103205H795-MY016 E233D 1.1 1.5 5.3 1.1 1.57 0.8 1.2 1.6 2.19 3 M103205H795-MY006 L234W 1.6 2.7 4.7 2.0 1.13 0.5 0.6 2.0 1.11 4 M103205H795-MY013 L234Y 0.7 1.1 0.3 0.9 1.09 0.3 0.2 1.2 1.98 5 M103205H795-MY070 L235W 1.4 1.8 3.6 1.5 0.73 1.4 1.5 1.8 1.16 6 M103205H795-MY001 G236N 1.1 1.4 1.1 1.0 0.03 0.2 0.1 0.6 0.04 7 M103205H795-MY061 G236D 0.5 0.8 0.9 0.4 0.11 0.5 0.8 1.8 0.27 8 M103205H795-MY071 G236E 0.6 1.4 1.8 0.7 1.29 1.7 2.4 1.5 1.59 9 M103205H795-MY074 G236A 1.6 2.2 2.7 1.6 1.33 7.5 5.2 1.3 1.45 10 M103205H795-MY124 G236S 1.2 1.6 1.4 0.8 0.28 4.9 2.1 0.5 0.16 eleven M103205H795-MY008 S239V 0.8 1.4 0.7 1.1 0.97 0.4 0.6 1.8 1.29 12 M103205H795-MY037 S239N 0.8 1.1 1.8 0.8 1.80 0.6 1.2 1.8 2.19 13 M103205H795-MY038 S239I 0.8 1.1 1.5 0.9 1.16 0.6 0.7 1.8 1.47 14 M103205H795-MY010 V264I 0.9 1.0 1.1 0.9 1.64 0.6 0.6 0.9 3.68 15 M103205H795-MY011 S267A 1.4 1.8 1.7 1.8 2.40 1.4 6.5 7.0 2.38 16 M103205H795-MY017 H268E 1.2 1.8 3.1 2.6 3.67 1.5 3.9 4.9 5.79 17 M103205H795-MY063 H268D 2.3 3.4 8.5 5.3 5.37 2.6 5.2 7.0 5.06 18 M103205H795-MY012 P271G 1.5 2.2 2.2 1.6 1.80 1.6 4.8 5.1 1.69 19 M103205H795-MY040 P271D 0.6 0.5 3.9 0.5 0.97 0.4 0.9 1.5 1.33 20 M103205H795-MY041 R271E 0.8 1.2 2.3 0.5 1.38 0.4 0.9 1.5 1.69 KD fold for cynoFcgRs (SGI = 1) KD fold in relation to nen.FcgRs (SGI = 1) Symbol heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 21 M103205H795-MY073 Q295L 2.7 3.9 3.3 2.7 2.12 2.9 2.2 2.3 2.38 22 M103205H795-MY042 S298L 6.3 1.6 2.1 1.1 0.82 0.7 1.1 1.2 1.53 23 M103205H795-MY069 K326T 2.0 3.2 2.6 2.5 4.19 1.6 2.8 2.9 3.38 24 M103205H795-MY043 A327N 0.6 0.1 0.9 0.4 0.43 0.2 0.7 1.5 0.68 25 M103205H795-MY068 L328T 1.5 1.9 3.0 1.8 0.62 1.9 2.1 2.2 0.58 26 M103205H795-MY014 A330R 1.3 2.0 1.4 1.2 0.90 1.3 1.1 1.0 1.17 27 M103205H795-MY196 AZZOK 1.3 1.7 0.7 0.8 0.60 1.4 0.8 0.7 0.37 28 M103205H795-MY044 R331E 1.0 1.5 0.3 1.1 0.77 0.6 1.2 1.4 0.90 29 M103205H795-MY072 I332D 3.2 4.3 6.5 3.8 6.79 2.8 2.3 3.1 6.75 thirty M103205H795-MY045 K334D 0.6 1.3 2.5 0.8 2.57 0.4 0.7 1.6 3.38 31 M103205H795-MY064 K334I 1.3 2.0 2.5 1.6 3.00 1.4 1.6 1.9 3.68 32 M103205H795-MY065 K334V 1.4 1.8 4.0 1.4 3.27 1.3 2.2 2.4 3.52 33 M103205H795-MY066 K334Y 1.5 2.0 2.5 1.3 2.25 1.1 1.1 1.6 2.79 34 M103205H795-MY067 K334M 1.5 2.2 2.5 1.6 3.60 1.3 1.4 1.6 4.05 35 M103205H795-MY015 R396M 2.5 3.4 3.6 3.0 3.60 2.1 3.4 2.9 3.38

- 112 040713

Table 19

KD (M) for cynoFcgRs KD (M) for nen.FcgRs Ebozn. heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 1 M103205H795-SG1 3.2E-06 5.1E-06 1.7E-05 1.8E-06 3.6E-07 6.9E-07 LIE-06 4.6E-06 8.1E-07 2 M103205H795-MY001 G236N 2.9E-06 3.6E-06 1.5E-05 1.8E-06 1.2E-05 3.2E-06 8.2E-06 7.7E-06 1.8E-05 3 M103205H795-MY047 G236N/P271G 4.8E-06 3.4E-06 2.4E-05 2.6E-06 7.6E-06 2.8E-06 3.8E-06 3.7E-06 7.7E-06 4 M103205H795-MY048 G236N/P396M 1.4E-06 1.7E-06 7.0Е-06 9.1E-07 5.4E-06 LIE-06 3.1E-06 3.3E-06 1.2E-05 5 M103205H795-MY049 G236N/H268E 2.7E-06 2.3E-06 6.6E-06 8.5E-07 4.6E-06 1.8E-06 7.8E-06 1.5E-06 7.8E-06 6 M103205H795-MY050 G236N/S267A/H268E 4.3E-06 4.2E-06 1.9E-05 1.4E-06 1.7E-06 2.1E-06 1.5E-06 7.5E-07 3.3E-06 7 M103205H795-MY051 G236N/H268E/P271G 4.6E-06 4.8E-06 1.8E-05 1.9E-06 2.1E-06 2.2E-06 2.8E-06 2.0E-06 7.4E-06 8 M103205H795-MY052 G236N/H268E/P396M 1.4E-06 1.5E-06 3.9E-06 4.6E-07 1.5E-06 8. ZE-07 1.4E-06 7.2E-07 4.6E-06 9 M103205H795-MY101 G236N/H268E/A330K/P396M LIE-06 1.2E-06 3.6E-06 3.5E-07 3.2E-06 4.9E-07 8.7E-07 4.9E-07 LIE-05 10 M103205H795-MY103 G236N/H268E/A330R/P396M 1.4E-06 1.ZE-06 3.6E-06 3.8E-07 2.6E-06 4.1E-07 1.2E-06 7.2E-07 4.5E-06 eleven M103205H795-MY105 G236N/S239V/H268E/P396M 1.2E-06 9.0E-07 1.ZE-06 3.9E-07 1.ZE-06 1.5E-06 1.4E-06 5.7E-07 5.9E-06 12 M103205H795-MY107 G236N/S239V/H268E/A330R/P396M 9.0E-07 7.3E-07 1.2E-06 3.0E-07 2.2E-06 9.5E-07 1.4E-06 5.0E-07 5.8E-06 13 M103205H795-MY109 G236N/S239V/H268E/A330K/P396M 8.7E-07 7.1E-07 1.2E-06 2.8E-07 2.0E-06 9.4Е-07 1.3E-06 5.5E-07 4.2E-06 14 M103205H795-MY111 L234W/G23 6N/H268E/A3 3 OK/RZ 96M 1.7E-06 1.8E-06 4.2E-06 5.5E-07 2.3E-06 4.7E-07 2.0E-06 LIE-06 3.1E-06 15 M103205H795-MY113 L234W/G23 6N/H268E/A3 3 0R/P3 96M 1.6E-06 1.7E-06 4.0E-06 5.0E-07 2.0E-06 1. ZE-06 1.8E-06 9.8E-07 3.7E-06 16 M103205H795-MY115 G236N/S267A/H268E/P396M 2.3E-06 2.7E-06 7.6E-06 7.8E-07 8.5E-07 9.6E-07 6.4E-07 3.2E-07 3.6E-06 17 M103205H795-MY117 L234W/G236N/S267A/H268E/P396M 2.6E-06 3.2E-06 9.9Е-06 1.0E-06 1.0E-06 2.2E-06 1.0E-06 6.0E-07 7.9E-06 18 M103205H795-MY141 G236N/S267A/P396M 2.2E-06 2.8E-06 2.0E-05 1.8E-06 4.7E-06 1.2E-06 1.6E-06 LIE-06 1.2E-05 19 M103205H795-MY144 G236N/H268D/P396M 8.9E-07 8.9E-07 3.8E-06 4.4E-07 2.3E-06 6.5E-07 1.3E-06 7.4E-07 7.7E-06 20 M103205H795-MY145 G236N/H268E/P331E/P396M 1.9E-06 2.4E-06 1.0E-05 9.8E-07 3.6E-06 1.8E-06 2.3E-06 1.2E-06 9.1E-06 21 M103205H795-MY146 G236N/H268E/S298L/P396M 1.7E-07 1.8E-06 4.8E-06 8.5E-07 3.1E-06 2.2E-06 2.1E-06 8.9E-07 8.8E-06 22 M103205H795-MY147 L235W/G236N/H268E/P396M 2.2E-06 2.2E-06 6.6E-06 9.6E-07 5.5E-06 1.2E-06 LIE-06 8.3E-07 LIE-05 23 M103205H795-MY197 G236N/H268D/A330K/P396M 5.8E-07 5.6E-07 1.4E-06 1.7E-07 2.1E-06 3.6E-07 7.1E-07 3.9E-07 1.0E-05 24 M103205H795-MY198 G236N/S239V/H268D/A330K/P396M 4.4E-07 3.ZE-07 1.4E-06 2.4E-07 2.7E-06 5.8E-07 8.0E-07 4.2E-07 6.9E-06 KD(M) for cynoFcgRs KD (M) for nen.FcgRs Symbol heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 25 M103205H795-MY199 G236N/H268E/A330K 1.9E-06 2.0E-06 1.2E-05 1.1E-06 4.1E-06 LIE-06 2.8E-06 1.9E-06 1.3E-05 26 M103205H795-MY200 G236N/S239V/H268E/A330K 1.7E-06 1.5E-06 5.0E-06 1.1E-06 4.4E-06 1.7E-06 2.7E-06 1.4E-06 1.5E-05 27 M103205H795-MY201 G236N/H268D/A330K 1.4E-06 1.4E-06 3.9E-06 4.3E-07 5.7E-06 9.1E-07 1.9E-06 LIE-06 2.4E-05 28 M103205H795-MY202 G236N/S239V/H268D/A330K 8.6E-07 6.4E-07 2.2E-06 4.6E-07 4.0E-06 1.2E-06 1.2E-06 7.1E-07 9.4E-06 29 M103205H795-MY204 G236N/S239V/H268E/Q295L/A330K 1.ZE-06 1.4E-06 5.2E-06 8.1E-07 3.5E-06 1.ZE-06 2.8E-06 1.3E-06 LIE-05 thirty M103205H795-MY205 G236N/H268D/Q295L/A330K 9.0E-07 1.1E-06 3.0E-06 2.5E-07 6.0E-06 4.7E-07 1.3E-06 5.6E-07 1.3E-05 31 M103205H795-MY206 G236N/S239V/H268D/Q295L/A330K 8.1E-07 7.9E-07 3.0E-06 4.5E-07 3.ZE-06 1.0E-06 1.4E-06 9.0E-07 8.2E-06 32 M103205H795-MY207 G236N/H268D 1.5E-06 1.4E-06 6.9Е-06 8.2E-07 4.1E-06 1.ZE-06 2.8E-06 1.6E-06 8.0E-06 33 M103205H795-MY208 G236N/H268D/Q295L 1.2E-06 1.5E-06 6.7E-06 6.4E-07 2.8E-06 8.3E-07 2.1E-06 LIE-06 1.0E-05 34 M103205H795-MY209 G236N/H268D/Q295L/K326T/A330K 6.3E-07 6.9E-07 1.9E-06 1.9E-07 2.2E-06 5.7E-07 8.8E-07 5.0E-07 1.4E-05 35 M103205H795-MY210 G23 6N/H268D/K326T/A3 3 0K 9.5E-07 8.4E-07 2.3E-06 2.8E-07 2.4E-06 8.8E-07 1.4E-06 9.8E-07 3.1E-05 36 M103205H795-MY211 G236N/S239V/H268D/K326T/A330K 7.5E-07 5.8E-07 2.7E-06 4.7E-07 3.0E-06 LIE-06 1.3E-06 8.2E-07 4.8E-06 37 M103205H795-MY212 G236N/S239V/H268D/Q295L/K326T/A3 ZOK 5.6E-07 5.ZE-07 2.9E-06 3.7E-07 2.6E-06 8.0E-07 1.3E-06 6.3E-07 6.5E-06 38 M103205H795-MY288 G236N/Q295L 1.5E-06 2.0E-06 1.1E-05 1.2E-06 1.ZE-05 1.4E-06 8.5E-06 5.1E-06 6.8E-05 39 M103205H795-MY289 G236N/A33 OK/RZ 96M 1.ZE-06 1.5E-06 8.6E-06 7.4E-07 5.2E-06 8.3E-07 3.5E-06 2.9E-06 7.7E-06 40 M103205H795-MY350 L23 5W/G23 6N/H268E/A3 3 OK/RZ 96M 2.1E-06 2.2E-06 4.7E-06 6.1E-07 6.6E-06 9.6E-07 9.0E-07 6.7E-07 1.2E-05 41 M103205H795-MY434 L23 5W/G23 6N/H268D/A3 3 0K 2.9E-06 2.4E-06 6.4E-06 7.4E-07 1.1E-05 1.6E-06 1.9E-06 1.4E-06 1.6E-05 42 M103205H795-MY440 L235W/G236N/H268D/Q295L/A330K 1.6E-06 1.8E-06 4.8E-06 4.6E-07 9.ZE-06 9.1E-07 LIE-06 8.0E-07 2.7E-05 43 M103205H795-MY518 L235W/G236N/H268D/Q295L/K326T/A ZZOK 8.9E-07 9.0E-07 2.4E-06 2.6E-07 4.2E-06 8.5E-07 6.7E-07 4.8E-07 2.3E-05

- 113 040713

KD fold for cynoFcgRs (SGI = 1) KD fold in relation to nen.FcgRs (SGI = 1) Symbol heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 1 M103205H795-SG1 1.0 1.0 1.0 1.0 1.00 1.0 1.0 1.0 1.00 2 M103205H795-MY001 G236N 1.1 1.4 1.1 1.0 0.03 0.2 0.1 0.6 0.04 3 M103205H795-MY047 G236N/P271G 0.7 1.5 0.7 0.7 0.05 0.2 0.3 1.2 0.11 4 M103205H795-MY048 G236N/P396M 2.3 3.0 2.4 2.0 0.07 0.6 0.4 1.4 0.07 5 M103205H795-MY049 G236N/H268E 1.2 2.2 2.6 2.1 0.08 0.4 0.1 3.1 0.10 6 M103205H795-MY050 G236N/S267A/H268E 0.7 1.2 0.9 1.3 0.21 0.3 0.7 6.1 0.25 7 M103205H795-MY051 G236N/H268E/P271G 0.7 1.1 0.9 0.9 0.17 0.3 0.4 2.3 0.11 8 M103205H795-MY052 G236N/H268E/P396M 2.3 3.4 4.4 3.9 0.24 0.8 0.8 6.4 0.18 9 M103205H795-MY101 G236N/H268E/A330K/P396M 2.9 4.3 4.7 5.1 0.11 1.4 1.3 9.4 0.07 10 M103205H795-MY103 G236N/H268E/A330R/P396M 2.3 3.9 4.7 4.7 0.14 1.7 0.9 6.4 0.18 eleven M103205H795-MY105 G236N/S239V/H268E/P396M 2.7 5.7 13.1 4.6 0.28 0.5 0.8 8.1 0.14 12 M103205H795-MY107 G23 6N/S23 9V/H268E/A3 3 0R/ R396M 3.6 7.0 14.2 6.0 0.16 0.7 0.8 9.2 0.14 13 M103205H795-MY109 G23 6N/S23 9V/H268E/A3 3 0K/ R396M 3.7 7.2 14.2 6.4 0.18 0.7 0.8 8.4 0.19 14 M103205H795-MY111 L234W/G236N/H268E/A330K /R396M 1.9 2.8 4.0 3.3 0.16 1.5 0.6 4.2 0.26 15 M103205H795-MY113 L234W/G236N/H268E/A330R/ R396M 2.0 3.0 4.3 3.6 0.18 0.5 0.6 4.7 0.22 16 M103205H795-MY115 G236N/S267A/H268E/P396M 1.4 1.9 2.2 2.3 0.42 0.7 1.7 14.4 0.22 17 M103205H795-MY117 L234W/G236N/S267A/H268E/ R396M 1.2 1.6 1.7 1.8 0.36 0.3 1.1 7.7 0.10 18 M103205H795-MY141 G236N/S267A/P396M 1.5 1.8 0.9 1.0 0.08 0.6 0.7 4.2 0.07 19 M103205H795-MY144 G236N/H268D/P396M 3.6 5.7 4.5 4.1 0.16 1.1 0.8 6.2 0.11 20 M103205H795-MY145 G236N/H268E/P331E/P396M 1.7 2.1 1.7 1.8 0.10 0.4 0.5 3.8 0.09 KD fold for cynoFcgRs (SGI = 1) KD fold in relation to nen.FcgRs (SGI = 1) Symbol heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 21 M103205H795-MY146 G236N/H268E/S298L/P396M 18.8 2.8 3.5 2.1 0.12 0.3 0.5 5.2 0.09 22 M103205H795-MY147 L235W/G236N/H268E/P396M 1.5 2.3 2.6 1.9 0.07 0.6 1.0 5.5 0.07 23 M103205H795-MY197 G23 6N/H268D/A3 3 OK/RZ 96M 5.5 9.1 12.1 10.6 0.17 1.9 1.5 11.8 0.08 24 M103205H795-MY198 G23 6N/S23 9V/H268D/A3 3 0K/ R396M 7.3 15.5 12.1 7.5 0.13 1.2 1.4 11.0 0.12 25 M103205H795-MY199 G236N/H268E/A330K 1.7 2.6 1.4 1.6 0.09 0.6 0.4 2.4 0.06 26 M103205H795-MY200 G236N/S239V/H268E/A330K 1.9 3.4 3.4 1.6 0.08 0.4 0.4 3.3 0.05 27 M103205H795-MY201 G236N/H268D/A330K 2.3 3.6 4.4 4.2 0.06 0.8 0.6 4.2 0.03 28 M103205H795-MY202 G236N/S239V/H268D/A330K 3.7 8.0 7.7 3.9 0.09 0.6 0.9 6.5 0.09 29 M103205H795-MY204 G236N/S239V/H268E/Q295L/ AZZOK 2.5 3.6 3.3 2.2 0.10 0.5 0.4 3.5 0.07 thirty M103205H795-MY205 G236N/H268D/Q295L/A330K 3.6 4.6 5.7 7.2 0.06 1.5 0.8 8.2 0.06 31 M103205H795-MY206 G236N/S239V/H268D/Q295L/ AZZOK 4.0 6.5 5.7 4.0 0.11 0.7 0.8 5.1 0.10 32 M103205H795-MY207 G236N/H268D 2.1 3.6 2.5 2.2 0.09 0.5 0.4 2.9 0.10 33 M103205H795-MY208 G236N/H268D/Q295L 2.7 3.4 2.5 2.8 0.13 0.8 0.5 4.2 0.08 34 M103205H795-MY209 G236N/H268D/Q295L/K326T/ AZZOK 5.1 7.4 8.9 9.5 0.16 1.2 1.3 9.2 0.06 35 M103205H795-MY210 G236N/H268D/K326T/A330K 3.4 6.1 7.4 6.4 0.15 0.8 0.8 4.7 0.03 36 M103205H795-MY211 G236N/S239V/H268D/K326T/ AZZOK 4.3 8.8 6.3 3.8 0.12 0.6 0.8 5.6 0.17 37 M103205H795-MY212 G236N/S239V/H268D/Q295L/ K326T/A330K 5.7 9.6 5.9 4.9 0.14 0.9 0.8 7.3 0.12 38 M103205H795-MY288 G236N/Q295L 2.1 2.6 1.5 1.5 0.03 0.5 0.1 0.9 0.01 39 M103205H795-MY289 G236N/A330K/P396M 2.5 3.4 2.0 2.4 0.07 0.8 0.3 1.6 0.11 40 M103205H795-MY350 L23 5W/G23 6N/H268E/A33 0K/R396M 1.5 2.3 3.6 3.0 0.05 0.7 1.2 6.9 0.07 41 M103205H795-MY434 L235W/G236N/H268D/A330K 1.1 2.1 2.7 2.4 0.03 0.4 0.6 3.3 0.05 KD fold for cynoFcgRs (SGI = 1) KD fold in relation to nea.FcgRs (SGI = 1) Symbol heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 42 M103205H795-MY440 L235W/G236N/H268D/Q295L /AZZOK 2.0 2.8 3.5 3.9 0.04 0.8 1.0 5.8 0.03 43 M103205H795-MY518 L235W/G236N/H268D/Q295L /K326T/A330K 3.6 5.7 7.1 6.9 0.09 0.8 1.6 9.6 0.04

- 114 040713

Table 20

KD (M) for cynoFcgRs KD(M) vs. chel-FcgRs Symbol heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 1 M103205H795-SG1 3.2E-06 5.1E-06 1.7E-05 1.8E-06 3.6E-07 6.9E-07 1.1E-06 4.6E-06 8.1E-07 2 M103205H795-MY052 G236N/H268E/P396M 1.4E-06 1.5E-06 3.9E-06 4.6E-07 E5E-06 8.3E-07 E4E-06 7.2E-07 1.5E-06 M103205H795-MY168 G236N/H268E/P396A 2.2E-06 2.5E-06 1.0E-05 E2E-06 4.5E-06 E6E-06 2.8E-06 1.7E-06 8.1E-06 4 M103205H795-MY169 G236N/H268E/P396D 2.3E-06 2.3E-06 9.9E-06 1.0E-06 3.5E-06 E2E-06 2.5E-06 1.3E-06 6.2E-06 5 M103205H795-MY170 G236N/H268E/P396E 2.3E-06 2.4E-06 1.2E-05 1.2E-06 2.9E-06 E7E-06 2.5E-06 1.6E-06 6.3E-06 6 M103205H795-MY171 G236N/H268E/P396F 2.3E-06 2.3E-06 9.2E-06 9.8E-07 3.3E-06 1.2E-06 2.1E-06 1.2E-06 5.9E-06 7 M103205H795-MY172 G236N/H268E/P396G 2.7E-06 2.8E-06 1.1E-05 1.2E-06 3.3E-06 E7E-06 3.1E-06 1.8E-06 6.5E-06 8 M103205H795-MY173 G236N/H268E/P396H 1.7E-06 1.8E-06 8.0E-06 7.8E-07 3.1E-06 1.0E-06 E9E-06 1.0E-06 7.0E-06 9 M103205H795-MY174 G236N/H268E/P396I 1.5E-06 1.6E-06 6.4E-06 6.5E-07 2.5E-06 8.1E-07 E4E-06 8.5E-07 6.8E-06 10 M103205H795-MY175 G236N/H268E/P396K 1.4E-06 1.5E-06 7.5E-06 7.1E-07 2.6E-06 8.6E-07 1.5E-06 8.3E-07 7.6E-06 eleven M103205H795-MY176 G236N/H268E/P396L 1. ZE-06 1.4E-06 6.0E-06 6.1E-07 2.2E-06 7.0E-07 1.3E-06 7.0E-07 6.2E-06 12 M103205H795-MY177 G236N/H268E/P396N 1.9E-06 1.8E-06 8.1E-06 8.3E-07 2.9E-06 1.0E-06 2.0E-06 1.1E-06 6.2E-06 13 M103205H795-MY178 G236N/H268E/P396Q 2.0E-06 2.1E-06 8.7E-06 9.5E-07 3.0E-06 1.2E-06 2.3E-06 1.3E-06 4.5E-06 14 M103205H795-MY179 G236N/H268E/P396R 1.9E-06 2.0E-06 7.9E-06 9.8E-07 2.7E-06 1.1E-06 2.1E-06 1.2E-06 5.9E-06 15 M103205H795-MY180 G236N/H268E/P396S 2.6E-06 2.7E-06 E2E-05 1.3E-06 3.1E-06 E7E-06 3.0E-06 1.9E-06 5.9E-06 16 M103205H795-MY181 G236N/H268E/P396T 2.3E-06 2.3E-06 9.2E-06 1.0E-06 4.1E-06 E3E-06 2.5E-06 1.4E-06 7.3E-06 17 M103205H795-MY182 G236N/H268E/P396V 2.1E-06 2.2E-06 7.8E-06 9.6E-07 3.6E-06 E3E-06 2.1E-06 1.2E-06 7.0E-06 18 M103205H795-MY183 G236N/H268E/P396W 2.1E-06 2.0E-06 8.4E-06 9.7E-07 3.5E-06 1.2E-06 2.1E-06 1.1E-06 7.2E-06 19 M103205H795-MY184 G236N/H268E/P396Y 1.9E-06 2.0E-06 8.4E-06 9.0E-07 3.3E-06 1.1E-06 2.0E-06 1.1E-06 6.8E-06 KD fold for cynoFcgRs (SGI = 1) KD fold in relation to nen.FcgRs (SGI = 1) Symbol heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 1 M103205H795-SG1 - 1.0 1.0 1.0 1.0 1.00 1.0 1.0 1.0 1.00 2 M103205H795-MY052 G236N/H268E/P396M 2.3 3.4 4.4 3.9 0.24 0.8 0.8 6.4 0.54 3 M103205H795-MY168 G236N/H268E/P396A 1.5 2.0 1.7 1.5 0.08 0.4 0.4 2.7 0.10 4 M103205H795-MY169 G236N/H268E/P396D 1.4 2.2 1.7 1.8 0.10 0.6 0.4 3.5 0.13 5 M103205H795-MY170 G236N/H268E/P396E 1.4 2.1 1.4 1.5 0.12 0.4 0.4 2.9 0.13 6 M103205H795-MY171 G236N/H268E/P396F 1.4 2.2 1.8 1.8 0.11 0.6 0.5 3.8 0.14 7 M103205H795-MY172 G236N/H268E/P396G 1.2 1.8 1.5 1.5 0.11 0.4 0.4 2.6 0.12 8 M103205H795-MY173 G236N/H268E/P396H 1.9 2.8 2.1 2.3 0.12 0.7 0.6 4.6 0.12 9 M103205H795-MY174 G236N/H268E/P396I 2.1 3.2 2.7 2.8 0.14 0.9 0.8 5.4 0.12 10 M103205H795-MY175 G236N/H268E/P396K 2.3 3.4 2.3 2.5 0.14 0.8 0.7 5.5 0.11 eleven M103205H795-MY176 G236N/H268E/P396L 2.5 3.6 2.8 3.0 0.16 1.0 0.8 6.6 0.13 12 M103205H795-MY177 G236N/H268E/P396N 1.7 2.8 2.1 2.2 0.12 0.7 0.6 4.2 0.13 13 M103205H795-MY178 G236N/H268E/P396Q 1.6 2.4 2.0 1.9 0.12 0.6 0.5 3.5 0.18 14 M103205H795-MY179 G236N/H268E/P396R 1.7 2.6 2.2 1.8 0.13 0.6 0.5 3.8 0.14 15 M103205H795-MY180 G236N/H268E/P396S 1.2 1.9 1.4 1.4 0.12 0.4 0.4 2.4 0.14 16 M103205H795-MY181 G236N/H268E/P396T 1.4 2.2 1.8 1.8 0.09 0.5 0.4 3.3 0.11 17 M103205H795-MY182 G236N/H268E/P396V 1.5 2.3 2.2 1.9 0.10 0.5 0.5 3.8 0.12 18 M103205H795-MY183 G236N/H268E/P396W 1.5 2.6 2.0 1.9 0.10 0.6 0.5 4.2 0.11 19 M103205H795-MY184 G236N/H268E/P396Y 1.7 2.6 2.0 2.0 0.11 0.6 0.6 4.2 0.12

Table 21

KD (M) for cynoFcgRs KD(M) vs. chel-FcgRs Symbol heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 1 M103205H795-SG1 3.2E-06 5.1E-06 1.7E-05 1.8E-06 3.6E-07 6.9E-07 1.1E-06 4.6E-06 8.1E-07 2 M103205H795-MY201 G236N/H268D/A330K 1.4E-06 1.4E-06 3.9E-06 4.3E-07 5.7E-06 9.1E-07 1.9E-06 1.1E-06 2.4E-05 3 M103205H795-MY260 G236M/H268D/A330K 4.5E-06 3.7E-06 1.4E-05 1.6E-06 6.5E-07 9.1E-07 1.8E-06 6.1E-06 3.0E-06 4 M103205H795-MY261 G23 6H/H268D/A3 3 0K 3.7E-06 4.0E-06 5.5E-06 1.5E-06 2.4E-06 2.4E-07 1.1E-06 5.3E-06 1.1E-05 5 M103205H795-MY262 G236V/H268D/A330K 1.9E-06 1.7E-06 8.0E-06 5.6E-07 2.8E-06 1.4E-07 7.1E-07 6.0E-06 5.0E-06 6 M103205H795-MY263 G236Q/H268D/A330K 3.2E-06 3.0E-06 7.3E-06 1.0E-06 7.8E-07 3.2E-07 7.1E-07 3.5E-06 2.3E-06 7 M103205H795-MY264 G236L/H268D/A330K 5.6E-06 5.5E-06 1.6E-05 2.6E-06 3.7E-06 1.5E-06 2.3E-06 1.2E-05 6.4E-05 8 M103205H795-MY265 G236T/H268D/A330K 2.2E-06 1.9E-06 4.8E-06 5.9E-07 2.0E-06 2.1E-07 4.4E-07 2.2E-06 5.4E-06 9 M103205H795-MY267 G236VH268D/A330K 2.4E-06 2.0E-06 7.3E-06 8.4E-07 2.7E-06 2.0E-07 7.1E-07 5.7E-06 1.5E-O5 KD fold for cynoFcgRs (SGI = 1) Multiplicity of KD in relation to human gRs (SGI = 1) Symbol heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 1 M103205H795-SG1 1.0 1.0 1.0 1.0 1.00 1.0 1.0 1.0 1.00 2 M103205H795-MY201 G236N/H268D/A330K 2.3 3.6 4.4 4.2 0.06 0.8 0.6 4.2 0.03 3 M103205H795-MY260 G236M/H268D/A330K 0.7 1.4 1.2 1.1 0.55 0.8 0.6 0.8 0.27 4 M103205H795-MY261 G236H/H268D/A330K 0.9 1.3 3.1 1.2 0.15 2.9 1.0 0.9 0.07 5 M103205H795-MY262 G236V/H268D/A330K 1.7 3.0 2.1 3.2 0.13 4.9 1.5 0.8 0.16 6 M103205H795-MY263 G236Q/H268D/A330K 1.0 1.7 2.3 1.8 0.46 2.2 1.5 1.3 0.35 7 M103205H795-MY264 G236L/H268D/A330K 0.6 0.9 1.1 0.7 0.10 0.5 0.5 0.4 0.01 8 M103205H795-MY265 G236T/H268D/A330K 1.5 2.7 3.5 3.1 0.18 3.3 2.5 2.1 0.15 9 M103205H795-MY267 G236I/H268D/A330K 1.3 2.6 2.3 2.1 0.13 3.5 1.5 0.8 0.05

- 115 040713

Table 22

KD (M) for cynoFcgRs KD(M) against nea.FcgRs Ebozn. heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV M103205H795-SG1 3.2E-06 5.1E-06 1.7E-05 1.8E-06 3.6E-07 6.9E-07 1.1E-06 4.6E-06 8.1E-07 2 M103205H795-MY265 G236T/H268D/A330K 2.2E-06 1.9E-06 4.8E-06 5.9E-07 2.0E-06 2.1E-07 4.4E-07 2.2E-06 5.4E-06 M103205H795-MY459 A231D/G236T/H268D/A330K 5.0E-06 4.7E-06 7.5E-06 LIE-06 3.2E-06 3.9E-07 5.6E-07 2.5E-06 8.7E-06 4 M103205H795-MY460 A231E/G236T/H268D/A330K 3.1E-06 3.1E-06 5.5E-06 7.9E-07 2.7E-06 2.6E-07 5.5E-07 2.4E-06 6.7E-06 M103205H795-MY461 A231F/G236T/H268D/A330K 1.8E-06 1.8E-06 3.5E-06 4.7E-07 2.1E-06 1.8E-07 3.4E-07 1.7E-06 5.4E-06 6 M103205H795-MY462 A231G/G236T/H268D/A330K 1.9E-06 1.6E-06 4.2E-06 4.7E-07 1.8E-06 2.4E-07 4.9E-07 1.9E-06 5.4E-06 7 M103205H795-MY463 A231H/G236T/H268D/A330K 2.1E-06 1.8E-06 4.4E-06 5.8E-07 1.6E-06 2.2E-07 5.1E-07 2.3E-06 4.8E-06 8 M103205H795-MY464 A231I/G236T/H268D/A330K 1.9E-06 1.5E-06 3.9E-06 5.0E-07 1.7E-06 1.7E-07 2.6E-07 1.6E-06 4.4E-06 9 M103205H795-MY465 A231K/G236T/H268D/A330K 2.0E-06 2.8E-06 5.2E-06 6.3E-07 1.9E-06 2.5E-07 6.0E-07 2.8E-06 5.5E-06 10 M103205H795-MY466 A231L/G236T/H268D/A330K 1.9E-06 1.8E-06 4.4E-06 5.1E-07 1.8E-06 1.7E-07 2.6E-07 1.4E-06 4.3E-06 eleven M103205H795-MY467 A231M/G236T/H268D/A330K 1.9E-06 1.9E-06 4.7E-06 5.1E-07 1.7E-06 1.8E-07 3.6E-07 2.0E-06 4.8E-06 12 M103205H795-MY468 A231N/G236T/H268D/A330K 2.8E-06 2.5E-06 5.2E-06 6.5E-07 2.3E-06 2.8E-07 4.7E-07 2.6E-06 6.8E-06 13 M103205H795-MY469 A231P/G236T/H268D/A330K 2.2E-06 2.1E-06 4.9E-06 6.1E-07 2.2E-06 2.0E-07 4.3E-07 2.4E-06 5.9E-06 14 M103205H795-MY470 A231Q/G236T/H268D/A330K 2.2E-06 1.8E-06 4.9E-06 5.5E-07 1.7E-06 2.1E-07 4.7E-07 2.3E-06 5.1E-06 15 M103205H795-MY471 A231R/G236T/H268D/A330K 2.6E-06 2.1E-06 4.5E-06 7.0E-07 1.8E-06 2.3E-07 4.7E-07 2.8E-06 5.3E-06 16 M103205H795-MY472 A231S/G236T/H268D/A330K 2.4E-06 2.1E-06 5.1E-06 6.6E-07 2.3E-06 2.5E-07 4.9E-07 2.5E-06 6.2E-06 17 M103205H795-MY473 A231T/G236T/H268D/A330K 1.6E-06 2.6E-06 5.5E-06 5.8E-07 1.7E-06 2.2E-07 4.9E-07 2.2E-06 5.2E-06 18 M103205H795-MY474 A231V/G236T/H268D/A330K 2.0E-06 1.8E-06 4.8E-06 5.0E-07 1.6E-06 1.7E-07 3.4E-07 2.0E-06 4.6E-06 19 M103205H795-MY475 A231W/G236T/H268D/A330K 1.0E-06 1.0E-06 2.3E-06 2.7E-07 1.7E-06 9.0E-08 2.1E-07 LIE-06 5.0E-06 20 M103205H795-MY476 A231Y/G236T/H268D/A330K 1.4E-06 1.3E-06 2.6E-06 3.9E-07 2.0E-06 1.4E-07 2.5E-07 1.2E-06 5.5E-06 21 M103205H795-MY441 P232A/G236T/H268D/A330K 3.9E-06 3.3E-06 8.0E-06 8.8E-07 2.6E-06 3.6E-07 6.0E-07 2.8E-06 8.1E-06 22 M103205H795-MY442 P232D/G236T/H268D/A330K 4.4E-06 4.2E-06 6.8E-06 1.1E-06 2.1E-06 4.6E-07 4.2E-07 1.8E-06 4.7E-06 23 M103205H795-MY443 P232E/G236T/H268D/A330K 4.8E-06 5.0E-06 8.8E-06 1.2E-06 2.6E-06 5.3E-07 6.4E-07 2.8E-06 5.9E-06 24 M103205H795-MY444 P232F/G236T/H268D/A330K 1.2E-06 1.1E-06 1.9E-06 3.0E-07 1.6E-06 1.1E-07 1.4E-07 7.4E-07 5.2E-06 25 M103205H795-MY445 P232G/G236T/H268D/A330K 5.6E-06 5.5E-06 1.4E-05 1.4E-06 3.5E-06 5.6E-07 1.1E-06 4.2E-06 9.5E-06 26 M103205H795-MY446 P232H/G236T/H268D/A330K 2.5E-06 2.2E-06 4.7E-06 6.3E-07 2.9E-06 2.6E-07 4.8E-07 2.4E-06 7.1E-06 27 M103205H795-MY447 P232I/G236T/H268D/A3 3 0K 9.9E-07 8.8E-07 1.5E-06 2.3E-07 1.1E-06 9.7E-08 9.8E-08 4.4E-07 4.0E-06 28 M103205H795-MY448 P232K/G236T/H268D/A330K 9.6E-06 7.5E-06 2.1E-05 2.3E-06 6.5E-06 9.3E-07 2.1E-06 8.4E-06 1.3E-05 29 M103205H795-MY449 P232L/G236T/H268D/A330K 8.7E-07 9.4E-07 1.5E-06 2.3E-07 1.0E-06 9.8E-08 7.4E-08 2.9E-07 3.7E-06 thirty M103205H795-MY450 P232M/G236T/H268D/A330K 1.3E-06 1.1E-06 2.2E-06 2.8E-07 1.3E-06 1.3E-07 1.2E-07 4.7E-07 4.0E-06 KD(M) for cynoFcgRs KD(M) against nea.FcgRs Symbol heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 31 M103205H795-MY451 P232N/G236T/H268D/A3 3 0K 3.6E-06 4.2E-06 6.7E-06 1.0E-06 2.7E-06 4.1E-07 6.4E-07 3.0E-06 7.1E-06 32 M103205H795-MY452 P232Q/G236T/H268D/A330K 3.4E-06 3.1E-06 6.5E-06 9.2E-07 2.8E-06 3.6E-07 6.2E-07 2.9E-06 6.1E-06 33 M103205H795-MY453 P232R/G236T/H268D/A330K 6.2E-06 9.9E-06 2.1E-05 2.1E-06 6.5E-06 7.5E-07 1.6E-06 6.5E-06 1.4E-05 34 M103205H795-MY454 P232S/G236T/H268D/A330K 3.8E-06 3.1E-06 7.6E-06 1.0E-06 2.8E-06 4.1E-07 7.6E-07 3.1E-06 5.9E-06 35 M103205H795-MY455 P232T/G236T/H268D/A330K 3.1E-06 2.9E-06 5.4E-06 9.2E-07 2.8E-06 3.1E-07 4.6E-07 2.2E-06 6.6E-06 36 M103205H795-MY456 P232V/G236T/H268D/A330K 1.9E-06 1.7E-06 3.4E-06 5.2E-07 2.0E-06 1.7E-07 2.9E-07 1.1E-06 5.3E-06 37 M103205H795-MY457 P232W/G236T/H268D/A330K 8.1E-07 8.4E-07 1.2E-06 2.0E-07 LIE-06 1.2E-07 7.3E-08 3.3E-07 3.6E-06 38 M103205H795-MY458 P232Y/G236T/H268D/A3 3 0K 1.8E-06 1.6E-06 3.0E-06 4.3E-07 1.9E-06 1.6E-07 1.7E-07 9.9E-07 5.3E-06 KD fold for cynoFcgRs (SGI = 1) KD fold in relation to nen.FcgRs (SGI = 1) Symbol heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIt FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 1 M103205H795-SG1 - 1.0 1.0 1.0 1.( 1.0 1.0 1.0 1.0 1.0 2 M103205H795-MY265 G236T/H268D/A330K 1.5 2.7 3.5 34 0.2 3.3 2.5 2.1 0.2 3 M103205H795-MY459 A231D/G236T/H268D/A330K 0.6 1.1 2.3 1.( 0.1 1.8 2.0 1.8 0.1 4 M103205H795-MY460 A231E/G236T/H268D/A330K 1.0 1.6 3.1 22. 0.1 2.7 2.0 1.9 0.1 5 M103205H795-MY461 A231F/G236T/H268D/A330K 1.8 2.8 4.9 33 0.2 3.8 3.2 2.7 0.2 6 M103205H795-MY462 A231G/G236T/H268D/A330K 1.7 3.2 4.0 33 0.2 2.9 2.2 2.4 0.2 7 M103205H795-MY463 A231H/G236T/H268D/A330K 1.5 2.8 3.9 3.] 0.2 3.1 2.2 2.0 0.2 8 M103205H795-MY464 A231I/G236T/H268D/A330K 1.7 3.4 4.4 3.( 0.2 4.1 4.2 2.9 0.2 9 M103205H795-MY465 A231K/G236T/H268D/A330K 1.6 1.8 3.3 2.( 0.2 2.8 1.8 1.6 0.1 10 M103205H795-MY466 A231L/G236T/H268D/A330K 1.7 2.8 3.9 32 0.2 4.1 4.2 3.3 0.2 eleven M103205H795-MY467 A231M/G236T/H268D/A330K 1.7 2.7 3.6 35 0.2 3.8 3.1 2.3 0.2 12 M103205H795-MY468 A231N/G236T/H268D/A330K 1.1 2.0 3.3 23 0.2 2.5 2.3 1.8 0.1 13 M103205H795-MY469 A231P/G236T/H268D/A330K 1.5 2.4 3.5 3.( 0.2 3.5 2.6 1.9 0.1 14 M103205H795-MY470 A231Q/G236T/H268D/A330K 1.5 2.8 3.5 32. 0.2 3.3 2.3 2.0 0.2 15 M103205H795-MY471 A231R/G236T/H268D/A330K 1.2 2.4 3.8 2.( 0.2 3.0 2.3 1.6 0.2 16 M103205H795-MY472 A231S/G236T/H268D/A330K 1.3 2.4 3.3 2.' 0.2 2.8 2.2 1.8 0.1 17 M103205H795-MY473 A231T/G236T/H268D/A330K 2.0 2.0 3.1 34 0.2 3.1 2.2 2.1 0.2 18 M103205H795-MY474 A231V/G236T/H268D/A330K 1.6 2.8 3.5 3.( 0.2 4.1 3.2 2.3 0.2 19 M103205H795-MY475 A231W/G236T/H268D/A330K 3.2 5.1 7.4 62 0.2 7.7 5.2 4.2 0.2 20 M103205H795-MY476 A231Y/G236T/H268D/A330K 2.3 3.9 6.5 4.( 0.2 4.9 4.4 3.8 0.1 21 M103205H795-MY441 P232A/G236T/H268D/A330K 0.8 1.5 2.1 2.( 0.1 1.9 1.8 1.6 0.1 22 M103205H795-MY442 P232D/G23 6T/H268D/A3 3 0K 0.7 1.2 2.5 1.( 0.2 1.5 2.6 2.6 0.2 23 M103205H795-MY443 P232E/G236T/H268D/A330K 0.7 1.0 1.9 1.5 0.1 1.3 1.7 1.6 0.1 24 M103205H795-MY444 P232F/G236T/H268D/A330K 2.7 4.6 8.9 6.( 0.2 6.3 7.9 6.2 0.2 25 M103205H795-MY445 P232G/G236T/H268D/A330K 0.6 0.9 1.2 17 0.1 1.2 1.0 1.1 0.1 26 M103205H795-MY446 P232H/G236T/H268D/A330K 1.3 2.3 3.6 2.( 0.1 2.7 2.3 1.9 0.1 27 M103205H795-MY447 P232I/G236T/H268D/A330K 3.2 5.8 11.3 73 0.3 7.1 11.2 10.5 0.2 28 M103205H795-MY448 P232K/G236T/H268D/A330K 0.3 0.7 0.8 03 0.1 0.7 0.5 0.5 0.1

- 116 040713

KD fold for cynoFcgRs (SGI = 1) KD fold in relation to nen.FcgRs (SGI = 1) Symbol heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 29 M103205H795-MY449 P232L/G236T/H268D/A330K 3.7 5.4 11.3 7.8 0.4 7.0 14.9 15.9 0.2 thirty M103205H795-MY450 P232M/G236T/H268D/A330K 2.5 4.6 7.7 6.4 0.3 5.3 9.2 9.8 0.2 31 M103205H795-MY451 P232N/G236T/H268D/A330K 0.9 1.2 2.5 1.8 0.1 1.7 1.7 1.5 0.1 32 M103205H795-MY452 P232Q/G236T/H268D/A330K 0.9 1.6 2.6 2.0 0.1 1.9 1.8 1.6 0.1 33 M103205H795-MY453 P232R/G236T/H268D/A330K 0.5 0.5 0.8 0.9 0.1 0.9 0.7 0.7 0.1 34 M103205H795-MY454 P232S/G236T/H268D/A330K 0.8 1.6 2.2 1.8 0.1 1.7 1.4 1.5 0.1 35 M103205H795-MY455 P232T/G236T/H268D/A330K 1.0 1.8 3.1 2.0 0.1 2.2 2.4 2.1 0.1 36 M103205H795-MY456 P232V/G236T/H268D/A330K 1.7 3.0 5.0 3.5 0.2 4.1 3.8 4.2 0.2 37 M103205H795-MY457 P232W/G236T/H268D/A330K 4.0 6.1 14.2 9.0 0.3 5.8 15.1 13.9 0.2 38 M103205H795-MY458 P232Y/G236T/H268D/A330K 1.8 3.2 5.7 4.2 0.2 4.3 6.5 4.6 0.2

Table 23A

KD (M) for cynoFcgRs KD(M) for nen.FcgRs Symbol heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV M103205H795-SG1 3.2E-06 5.1E-06 1.7E-05 1.8E-06 3.6E-07 6.9E-07 1.1E-06 4.6E-06 8.1E-07 2 M103205H795-MY209 G236N/H268D/Q295L/K326T/A330K 6.3E-07 6.9E-07 1.9E-06 1.9E-07 2.2E-06 5.7E-07 8.8E-07 5.0E-07 1.4E-05 3 M103205H795-MY142 G236E/H268E/P396M 4.1E-06 4.9E-06 2.5E-05 1.8E-06 1.4E-07 1.9E-07 1.7E-07 9.5E-07 5.8E-07 4 M103205H795-MY143 G236S/H268E/P396M 1.1E-06 1.ZE-06 2.9E-06 4.6E-07 2.8E-07 3.7E-08 8.3E-08 6.8E-07 1.6E-06 5 M103205H795-MY269 G236M/H268D/A330K/P396K 2.3E-06 2.6E-06 6.4E-06 6.5E-07 2.2E-07 2.5E-07 7.2E-07 2.1E-06 LIE-06 6 M103205H795-MY270 G236H/H268D/A330K/P396K 1.8E-06 2.1E-06 2.6E-06 5. ZE-07 LIE-06 7.3E-08 3.7E-07 2.6E-06 3.7E-06 7 M103205H795-MY271 G236V/H268D/A330K/P396K 7.6E-07 7.0E-07 3.1E-06 1.9E-07 8.7E-07 4.4E-08 2.3E-07 2.1E-06 2.3E-06 8 M103205H795-MY272 G236Q/H268D/A330K/P396K 1.7E-06 1.9E-06 6.2E-06 4.3E-07 2.9E-07 9.2E-08 2.5E-07 1.2E-06 8.7E-07 9 M103205H795-MY273 G236L/H268D/A330K/P396K 3.7E-06 3.5E-06 1.1E-05 1.2E-06 1.7E-06 5.1E-07 1.0E-06 5.0E-06 9.7E-06 10 M103205H795-MY274 G236T/H268D/A330K/P396K 8.8E-07 8.0E-07 2.0E-06 2.3E-07 6.2E-07 8.1E-08 1.7E-07 9.5E-07 2.5E-06 eleven M103205H795-MY276 G236I/H268D/A330K/P396K 1.1E-06 1.1E-06 4.5E-06 3.1E-07 1.2E-06 5.ZE-08 2.5E-07 2.4E-06 5.7E-06 12 M103205H795-MY278 G236M/H268D/Q295L/A330K 3.0E-06 3.6E-06 1.0E-05 1.1E-06 7.4E-07 5.ZE-07 1.5E-06 4.4E-06 3.4E-06 13 M103205H795-MY279 G236H/H268D/Q295L/A330K 2.8E-06 Z.ZE-06 4.7E-06 9.1E-07 2.4E-06 1.4E-07 7.2E-07 4.6E-06 7.0E-06 14 M103205H795-MY280 G236V/H268D/Q295L/A330K 1.0E-06 1.2E-06 4.6E-06 3. ZE-07 1.8E-06 8.1E-08 4.5E-07 2.9E-06 4.4E-06 15 M103205H795-MY281 G236Q/H268D/Q295L/A330K 1.9E-06 2.5E-06 9.1E-06 6.2E-07 7.4E-07 1.ZE-07 3.9E-07 1.8E-06 2.1E-06 16 M103205H795-MY285 G236I/H268D/Q295L/A330K 1.6E-06 1.7E-06 6.8E-06 5.8E-07 2.9E-06 1.1E-07 5.6E-07 3.9E-06 1.6E-05 17 M103205H795-MY213 G236N/H268D/A330K/P396K 5.ZE-07 5.2E-07 1.ZE-06 1.6E-07 1.9E-06 3.5E-07 6.9E-07 3.6E-07 LIE-05 18 M103205H795-MY242 P238I/H268D/A330K/P396K 4.5E-06 4.2E-06 5.9E-06 1.1E-06 5.ZE-07 8.2E-07 6.3E-07 1.9E-06 3.2E-06 19 M103205H795-MY219 G237Y/H268D/A330K 3.5E-06 4.0E-06 5.5E-06 1. ZE-06 6.2E-06 2.8E-06 1.6E-07 3.4E-07 9.1E-05 20 M103205H795-MY227 G237Y/H268D/A330K/P396K 1.9E-06 2.5E-06 2.5E-06 5.1E-07 3.9E-06 1.ZE-06 6.2E-08 1.3E-07 1.8E-05 KD(M) for cynoFcgRs KD (M) for nen.FcgRs Symbol heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 21 M103205H795-MY228 G237Y/H268D/Q295L/A330K 3.0E-06 3.1E-06 3.6E-06 1.1E-06 9.7E-06 2.2E-06 1.6E-07 3.0E-07 1.4E-04 22 M103205H795-MY229 G237Y/H268D/Q295L/A330K/P396K 1.6E-06 2.2E-06 1.9E-06 4.7E-07 4.2E-06 9.8E-07 8.1E-08 1.5E-07 3.7E-05 23 M103205H795-MY292 G237Y/H268D/Q295L/A330K/P396M 1.5E-06 2.6E-06 2.2E-06 4.5E-07 4.0E-06 9.5E-07 7.6E-08 1.4E-07 2.8E-05 24 M103205H795-MY293 G237Y/H268E/Q295L/A330K/P396K 2.8E-06 Z.ZE-06 3.9E-06 8.6E-07 4.2E-06 1.ZE-06 7.3E-08 1.5E-07 3.9E-05 25 M103205H795-MY308 G236T/H268E/A330K Z.ZE-06 4.6E-06 1.1E-05 1.5E-06 2.3E-06 4.5E-07 8.6E-07 4.1E-06 8.8E-06 26 M103205H795-MY309 G236T/H268E/A330K/P396M 2.5E-06 2.5E-06 7.0Е-06 7.1E-07 1.2E-06 1.4E-07 2.6E-07 1.6E-06 4.3E-06 27 M103205H795-MY310 G236T/H268E/K326T/A330K 3.0E-06 2.9E-06 7.2E-06 9.5E-07 1.8E-06 4.0E-07 5.5E-07 2.9E-06 6.9E-06 28 M103205H795-MY311 G236T/H268E/Q295L/A330K 2.3E-06 3.4E-06 8.6E-06 7.8E-07 2.4E-06 2.0E-07 4.5E-07 2.1E-06 7.1E-06 29 M103205H795-MY312 G236T/H268E/K326T/A330K/P396M 1.4E-06 1.5E-06 3.7E-06 4.0E-07 6.5E-07 1.1E-07 1.7E-07 1.1E-06 2.8E-06 thirty M103205H795-MY316 G236L/H268E/Q295L/A330K/P396K 5.1E-06 8.6E-06 2.2E-05 2.0E-06 2.4E-06 4.4E-07 1.1E-06 4.0E-06 1.7E-05 31 M103205H795-MY318 G236L/H268E/K326T/A330K/P396M 7.9Е-06 7.7E-06 3.2E-05 2.2E-06 1.5E-06 1.1E-06 1.3E-06 4.8E-06 8.7E-06 32 M103205H795-MY321 G236H/H268D/A330K/P396M 2.3E-06 2.2E-06 2.9E-06 6.4E-07 1.1E-06 7.1E-08 3.8E-07 2.6E-06 3.4E-06 33 M103205H795-MY325 G236D/H268D/A330K 4.0E-06 4.4E-06 5.8E-06 9.8E-07 4.0E-06 5.1E-07 3.0E-07 2.6E-07 8.1E-06 34 M103205H795-MY327 G236D/H268E/Q295L/A330K 3.4E-06 4.8E-06 5.ZE-06 1.0E-06 4.5E-06 3.5E-07 2.0E-07 1.7E-07 9.9E-06 35 M103205H795-MY329 G236D/H268D/Q295L/A330K 1.9E-06 3.1E-06 3.7E-06 5.9E-07 2. ZE-06 2.7E-07 2.1E-07 1.6E-07 6.4E-06

- 117 040713

KD fold for cynoFcgRs (SGI = 1) KD fold in relation to nen.FcgRs (SGI = 1) Symbol heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 1 M103205H795-SG1 1.0 1.0 1.0 1.0 1.00 1.0 1.0 1.0 1.00 2 M103205H795-MY209 G236N/H268D/Q295L/K326T/A330K 5.1 7.4 8.9 9.5 0.16 1.2 1.3 9.2 0.06 3 M103205H795-MY142 G236E/H268E/P396M 0.8 1.0 0.7 1.0 2.57 3.6 6.5 4.8 1.40 4 M103205H795-MY143 G236S/H268E/P396M 2.9 3.9 5.9 3.9 1.29 18.6 13.3 6.8 0.51 5 M103205H795-MY269 G236M/H268D/A330K/P396K 1.4 2.0 2.7 2.8 1.64 2.8 1.5 2.2 0.74 6 M103205H795-MY270 G23 6H/H268D/A3 3 OK/RZ 96K 1.8 2.4 6.5 3.4 0.33 9.5 3.0 1.8 0.22 7 M103205H795-MY271 G236V/H268D/A330K/P396K 4.2 7.3 5.5 9.5 0.41 15.7 4.8 2.2 0.35 8 M103205H795-MY272 G236Q/H268D/A330K/P396K 1.9 2.7 2.7 4.2 1.24 7.5 4.4 3.8 0.93 9 M103205H795-MY273 G236L/H268D/A330K/P396K 0.9 1.5 1.5 1.5 0.21 1.4 1.1 0.9 0.08 10 M103205H795-MY274 G236T/H268D/A330K/P396K 3.6 6.4 8.5 7.8 0.58 8.5 6.5 4.8 0.32 eleven M103205H795-MY276 G236I/H268D/A330K/P396K 2.9 4.6 3.8 5.8 0.30 13.0 4.4 1.9 0.14 12 M103205H795-MY278 G236M/H268D/Q295L/A330K 1.1 1.4 1.7 1.6 0.49 1.3 0.7 1.0 0.24 13 M103205H795-MY279 G236H/H268D/Q295L/A330K 1.1 1.5 3.6 2.0 0.15 4.9 1.5 1.0 0.12 14 M103205H795-MY280 G236V/H268D/Q295L/A330K 3.2 4.3 3.7 0.20 8.5 2.4 1.6 0.18 15 M103205H795-MY281 G236Q/H268D/Q295L/A330K 1.7 2.0 1.9 2.9 0.49 5.3 2.8 2.6 0.39 16 M103205H795-MY285 G236I/H268D/Q295L/A330K 2.0 3.0 2.5 3.1 0.12 6.3 2.0 1.2 0.05 17 M103205H795-MY213 G23 6N/H268D/A3 3 OK/RZ 96K 6.0 9.8 13.1 11.3 0.19 2.0 1.6 12.8 0.07 18 M103205H795-MY242 P238I/H268D/A330K/P396K 0.7 1.2 2.9 1.6 0.68 0.8 1.7 2.4 0.25 19 M103205H795-MY219 G237Y/H268D/A330K 0.9 1.3 3.1 1.4 0.06 0.2 6.9 13.5 0.01 KD fold for cynoFcgRs (SGI = 1) KD fold in relation to nen.FcgRs (SGI = 1) Symbol heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 20 M103205H795-MY227 G23 7Y/H268D/A3 3 OK/RZ 96K 1.7 2.0 6.8 3.5 0.09 0.5 17.7 35.4 0.04 21 M103205H795-MY228 G237Y/H268D/Q295L/A330K 1.1 1.6 4.7 1.6 0.04 0.3 6.9 15.3 0.01 22 M103205H795-MY229 G237Y/H268D/Q295L/A330K/P396K 2.0 2.3 8.9 3.8 0.09 0.7 13.6 30.7 0.02 23 M103205H795-MY292 G237Y/H268D/Q295L/A330K/P396M 2.1 2.0 7.7 4.0 0.09 0.7 14.5 32.9 0.03 24 M103205H795-MY293 G237Y/H268E/Q295L/A330K/P396K 1.1 1.5 4.4 2.1 0.09 0.5 15.1 30.7 0.02 25 M103205H795-MY308 G236T/H268E/A330K 1.0 1.1 1.5 1.2 0.16 1.5 1.3 1.1 0.09 26 M103205H795-MY309 G236T/H268E/A330K/P396M 1.3 2.0 2.4 2.5 0.30 4.9 4.2 2.9 0.19 27 M103205H795-MY310 G236T/H268E/K326T/A330K 1.1 1.8 2.4 1.9 0.20 1.7 2.0 1.6 0.12 28 M103205H795-MY311 G236T/H268E/Q295L/A330K 1.4 1.5 2.0 2.3 0.15 3.5 2.4 2.2 0.11 29 M103205H795-MY312 G236T/H268E/K326T/A330K/P396M 2.3 3.4 4.6 4.5 0.55 6.3 6.5 4.2 0.29 thirty M103205H795-MY316 G236L/H268E/Q295L/A330K/P396K 0.6 0.6 0.8 0.9 0.15 1.6 1.0 1.2 0.05 31 M103205H795-MY318 G236L/H268E/K326T/A330K/P396M 0.4 0.7 0.5 0.8 0.24 0.6 0.8 1.0 0.09 32 M103205H795-MY321 G23 6H/H268D/A3 3 OK/RZ 96M 1.4 2.3 5.9 2.8 0.33 9.7 2.9 1.8 0.24 33 M103205H795-MY325 G236D/H268D/A330K 0.8 1.2 2.9 1.8 0.09 1.4 3.7 17.7 0.10 34 M103205H795-MY327 G236D/H268E/Q295L/A330K 0.9 1.1 3.2 1.8 0.08 2.0 5.5 27.1 0.08 35 M103205H795-MY329 G236D/H268D/Q295L/A330K 1.7 1.6 4.6 3.1 0.16 2.6 5.2 28.8 0.13

- 118 040713

Table 23B

KD (M) for cynoFcgRs KD(M) against nea.FcgRs Ebozn. heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV М103205Н795- SG1 3.2E-06 5.1E-06 1.7E-05 1.8E-06 3.6E-07 6.9E-07 1.1E-06 4.6E-06 8.1E-07 ² М103205Н795- MY209 G236N/H268D/Q295L/K326T/A330K 6.3E-07 6.9E-07 1.9E-06 1.9E-07 2.2E-06 5.7E-07 8.8E-07 5.0E-07 1.4E-05 М103205Н795- MY340 G236L/H268D/Q295L/A330K/P396K 4.3E-06 4.9E-06 2.3E-05 1.1E-06 2.3E-06 3.1E-07 8.3E-07 3.3E-06 1.7E-05 4 М103205Н795- MY341 G236L/H268D/Q295L/K326T/A330K /R396C 2.6E-06 2.9E-06 8.8E-06 6.0E-07 9.4E-07 2.6E-07 4.2E-07 1.8E-06 7.0E-06 М103205Н795- MY342 G236L/H268D/Q295L/A330K/P396M 5.1E-06 8.5E-06 5.1E-05 1.3E-06 2.6E-06 3.7E-07 1.0E-06 3.9E-06 2.5E-05 6 М103205Н795- MY283 G236T/H268D/Q295L/A330K 1.4E-06 1.6E-06 4.4E-06 4.2E-07 2.2E-06 1.4E-07 3.5E-07 1.8E-06 5.7E-06 7 М103205Н795- MY339 G236L/H268D/K326T/A330K/P396K 3.4E-06 3.0E-06 9.8E-06 7.5E-07 7.6E-07 4.1E-07 6.3E-07 2.8E-06 5.6E-06 8 М103205Н795- MY380 G236N/H268E/A330K/P396K 9.3Е-07 1.0E-06 3.3E-06 2.9E-07 2.8E-06 4.5E-07 8.5E-07 4.8E-07 1.0E-05 9 М103205Н795- MY386 G236L/H268D/Q295L/K326T/A330K 5.2E-06 5.6E-06 1.6E-05 1.6E-06 2.5E-06 8.1E-07 1.7E-06 4.6E-06 2.4E-05 10 М103205Н795- MY388 G236T/S239I/H268D/A330K 2.2E-06 1.7E-06 1.3E-06 7.6E-07 1.6E-06 3.3E-07 2.3E-07 1.5E-06 4.3E-06 eleven М103205Н795- MY389 G236T/S239L/H268D/A330K 2.0E-06 1.6E-06 1.4E-06 7.0E-07 1.5E-06 2.7E-07 1.9E-07 1.1E-06 3.5E-06 12 М103205Н795- MY400 G236T/H268D/A330K/P331E 1.8E-06 2.2E-06 3.8E-06 6.3E-07 2.4E-06 4.2E-07 7.4E-07 2.5E-06 7.8E-06 13 М103205Н795- MY392 G236T/H268D/S298L/A330K 3.0E-07 2.3E-06 3.9E-06 7.0E-07 4.0E-06 4.3E-07 4.1E-07 1.7E-06 9.3E-06 14 М103205Н795- MY387 G236T/S239V/H268D/A330K 2.3E-06 1.9E-06 1.4E-06 8.3E-07 2.5E-06 2.4E-07 2.0E-07 1.6E-06 4.7E-06 15 М103205Н795- G236T/S239V/H268D/S298L/A330K 2.6E-07 1.6E-06 1.5E-06 7.6E-07 4.4E-06 3.5E-07 1.6E-07 6.9E-07 8.8E-06 KD (M) for cynoFcgRs KD(M) against nea.FcgRs Ebozn. heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV MY514 16 М103205Н795- MY515 G236T/H268D/Q295L/S298L/A330K 1.4E-07 1.6E-06 2.9E-06 3.9E-07 3.8E-06 2.2E-07 2.6E-07 1.2E-06 9.0E-06 17 М103205Н795- MY516 G236T/S239V/H268D/Q295L/S298L/ A330K 1.5E-07 1.3E-06 7.9E-07 4.5E-07 3.0E-06 2.2E-07 8.5E-08 5.1E-07 6.7E-06 18 М103205Н795- MY520 P232D/G236T/S239V/H268D/Q295L/ AZZOK 2.5E-06 2.3E-06 2.1E-06 9.6E-07 2.0E-06 4.9E-07 3.4E-07 1.4E-06 3.5E-06 19 М103205Н795- MY521 P232D/G236T/H268D/Q295L/A330K 2.5E-06 3.3E-06 4.4E-06 8.0E-07 2.2E-06 2.9E-07 3.0E-07 1.5E-06 5.1E-06 20 М103205Н795- MY522 A231G/G236T/S239V/H268D/A330K 1.7E-06 1.2E-06 9.3E-07 5.3E-07 1.9E-06 2.4E-07 1.7E-07 1.1E-06 5.7E-06 21 М103205Н795- MY523 A231G/G236T/S239V/H268D/Q295L/ AZZOK 1.4E-06 1.3E-06 1.1E-06 4.8E-07 2.1E-06 1.9E-07 2.1E-07 1.1E-06 5.5E-06 22 М103205Н795- MY524 A231G/G236T/H268D/Q295L/A330K 1.0E-06 1.1E-06 2.7E-06 3.1E-07 2.0E-06 1.1E-07 2.8E-07 1.0E-06 4.5E-06 23 М103205Н795- MY525 A231T/G236T/S239V/H268D/A330K 1.6E-06 1.1E-06 1.1E-06 5.1E-07 1.4E-06 2.1E-07 1.6E-07 1.2E-06 4.3E-06 24 М103205Н795- MY526 A231T/G236T/S239V/H268D/Q295L/ AZZOK 1.ZE-06 1.2E-06 1.3E-06 4.6E-07 1.6E-06 1.7E-07 2.1E-07 1.2E-06 4.3E-06 25 М103205Н795- MY527 A231T/G236T/H268D/Q295L/A330K 1.1E-06 1.3E-06 3.4E-06 3.4E-07 1.5E-06 1.1E-07 2.9E-07 1.4E-06 4.5E-06 26 М103205Н795- MY528 L234W/G236T/H268D/A330K 1.4E-06 1.5E-06 3.2E-06 4.1E-07 2.0E-06 3.5E-07 5.6E-07 2.2E-06 4.2E-06 27 М103205Н795- MY529 L234W/G236T/H268D/Q295L/A330K 1.0E-06 1.4E-06 3.2E-06 3.0E-07 2.2E-06 2.3E-07 5.3E-07 1.4E-06 4.0E-06 28 М103205Н795- MY530 G236T/H268D/K326T/A330K 1.3E-06 1.1E-06 2.5E-06 3.2E-07 8.6E-07 1.7E-07 2.7E-07 1.6E-06 3.6E-06 29 М103205Н795- MY531 L234W/G23 6T/H268D/K326T/A3 3 0K 1.1E-06 9.2E-07 2.2E-06 2.7E-07 1.0E-06 2.9E-07 4.1E-07 1.5E-06 2.9E-06 thirty М103205Н795- MY532 L234W/G23 6T/S23 9V/H268D/A3 3 0K 7.7E-07 6.0E-07 7.4E-07 2.3E-07 8.6E-07 2.1E-07 1.9E-07 8.3E-07 1.7E-06 KD(M) for cynoFcgRs KD(M) against nea.FcgRs Symbol heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 31 М103205Н795- MY533 L234W/G236T/S239V/H268D/Q295L /AZZOK 8.0E-07 7.3E-07 1.2E-06 2.5E-07 LIE-06 2.1E-07 2.0E-07 9.5E-07 1.8E-06 32 М103205Н795- MY534 G236T/S239V/H268D/Q295L/A330K 1.3E-06 1.3E-06 1.2E-06 4.2E-07 1.7E-06 1.7E-07 1.9E-07 1.2E-06 4.3E-06 33 М103205Н795- MY535 A231G/L234W/G236T/H268D/A330 TO 1.5E-06 1.4E-06 3.0E-06 4.1E-07 2.1E-06 3.9E-07 6.4E-07 1.9E-06 5.2E-06 34 М103205Н795- MY536 A231G/L234W/G236T/S239V/H268D /AZZOK 1.2E-06 1.1E-06 9.8E-07 4.0E-07 1.6E-06 2.9E-07 2.2E-07 9.9E-07 2.7E-06 35 М103205Н795- MY537 A231G/L234W/G236T/S239V/H268D /Q295L/A330K 1.2E-06 1.1E-06 1.4E-06 3.2E-07 1.5E-06 2.8E-07 3.7E-07 1.0E-06 3.3E-06 36 М103205Н795- MY538 A231G/L234W/G236T/H268D/Q295L /AZZOK 9.6E-07 1.1E-06 3.0E-06 2.8E-07 2.4E-06 2.5E-07 6.2E-07 1.3E-06 5.7E-06

- 119 040713

KD fold for cynoFcgRs (SGI = 1) KD fold in relation to human FcgRs (SGI = 1) Symbol heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 1 М103205Н795- SG1 1.0 1.0 1.0 1.0 1.00 1.0 1.0 1.0 1.00 2 М103205Н795- MY209 G236N/H268D/Q295L/K326T/A330K 5.1 7.4 8.9 9.5 0.16 1.2 1.3 9.2 0.06 3 М103205Н795- MY340 G236L/H268D/Q295L/A330K/P396K 0.7 1.0 0.7 1.6 0.16 2.2 1.3 1.4 0.05 4 М103205Н795- MY341 G236L/H268D/Q295L/K326T/A330K/ R396K 1.2 1.8 1.9 3.0 0.38 2.7 2.6 2.6 0.12 5 М103205Н795- MY342 G236L/H268D/Q295L/A330K/P396M 0.6 0.6 0.3 1.4 0.14 1.9 1.1 1.2 0.03 6 М103205Н795- MY283 G236T/H268D/Q295L/A330K 2.3 3.2 3.9 4.3 0.16 4.9 3.1 2.6 0.14 7 М103205Н795- MY339 G236L/H268D/K326T/A330K/P396K 0.9 1.7 1.7 2.4 0.47 1.7 1.7 1.6 0.14 8 М103205Н795- MY380 G236N/H268E/A330K/P396K 3.4 5.1 5.2 6.2 0.13 1.5 1.3 9.6 0.08 9 М103205Н795- MY386 G236L/H268D/Q295L/K326T/A330K 0.6 0.9 1.1 1.1 0.14 0.9 0.6 1.0 0.03 10 М103205Н795- MY388 G236T/S239I/H268D/A330K 1.5 3.0 13.1 2.4 0.23 2.1 4.8 3.1 0.19 eleven М103205Н795- MY389 G236T/S239L/H268D/A330K 1.6 3.2 12.1 2.6 0.24 2.6 5.8 4.2 0.23 12 М103205Н795- MY400 G236T/H268D/A330K/P331E 1.8 2.3 4.5 2.9 0.15 1.6 1.5 1.8 0.10 13 М103205Н795- MY392 G236T/H268D/S298L/A330K 10.7 2.2 4.4 2.6 0.09 1.6 2.7 2.7 0.09 14 М103205Н795- MY387 G236T/S239V/H268D/A330K 1.4 2.7 12.1 2.2 0.14 2.9 5.5 2.9 0.17

KD fold for cynoFcgRs (SGI = 1) KD fold in relation to human FcgRs (SGI = 1) Symbol heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 15 M103205H795- MY514 G236T/S239V/H268D/S298L/A330K 12.3 3.2 11.3 2.4 0.08 2.0 6.9 6.7 0.09 16 M103205H795- MY515 G236T/H268D/Q295L/S298L/A330K 22.9 3.2 5.9 4.6 0.09 3.1 4.2 3.8 0.09 17 M103205H795- MY516 G236T/S239V/H268D/Q295L/S298L/ AZZOK 21.3 3.9 21.5 4.0 0.12 3.1 12.9 9.0 0.12 18 M103205H795- MY520 P232D/G236T/S239V/H268D/Q295L/ AZZOK 1.3 2.2 8.1 1.9 0.18 1.4 3.2 3.3 0.23 19 M103205H795- MY521 P232D/G236T/H268D/Q295L/A330K 1.3 1.5 3.9 2.3 0.16 2.4 3.7 3.1 0.16 20 M103205H795- MY522 A231G/G236T/S239V/H268D/A330K 1.9 4.3 18.3 3.4 0.19 2.9 6.5 4.2 0.14 21 M103205H795- MY523 A231G/G236T/S239V/H268D/Q295L/ AZZOK 2.3 3.9 15.5 3.8 0.17 3.6 5.2 4.2 0.15 22 M103205H795- MY524 A231G/G236T/H268D/Q295L/A330K 3.2 4.6 6.3 5.8 0.18 6.3 3.9 4.6 0.18 23 M103205H795- MY525 A231T/G236T/S239V/H268D/A330K 2.0 4.6 15.5 3.5 0.26 3.3 6.9 3.8 0.19 24 M103205H795- MY526 A231T/G236T/S239V/H268D/Q295L/ AZZOK 2.5 4.3 13.1 3.9 0.23 4.1 5.2 3.8 0.19 25 M103205H795- MY527 A231T/G236T/H268D/Q295L/A330K 2.9 3.9 5.0 5.3 0.24 6.3 3.8 3.3 0.18 26 M103205H795- MY528 L234W/G236T/H268D/A330K 2.3 3.4 5.3 4.4 0.18 2.0 2.0 2.1 0.19 27 M103205H795- MY529 L234W/G236T/H268D/Q295L/A330K 3.2 3.6 5.3 6.0 0.16 3.0 2.1 3.3 0.20 28 M103205H795- MY530 G236T/H268D/K326T/A330K 2.5 4.6 6.8 5.6 0.42 4.1 4.1 2.9 0.23 29 M103205H795- MY531 L234W/G23 6T/H268D/K326T/A3 3 0K 2.9 7.7 6.7 0.36 2.4 2.7 3.1 0.28

KD fold for cynoFcgRs (SGI = 1) KD fold in relation to human FcgRs (SGI = 1) Symbol heavy chain Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV thirty M103205H795- MY532 L234W/G23 6T/S23 9V/H268D/A3 3 OK 4.2 8.5 23.0 7.8 0.42 3.3 5.8 5.5 0.48 31 M103205H795- MY533 L234W/G236T/S239V/H268D/Q295L/ AZZOK 4.0 7.0 14.2 7.2 0.33 3.3 5.5 4.8 0.45 32 M103205H795- MY534 G236T/S239V/H268D/Q295L/A330K 2.5 3.9 14.2 4.3 0.21 4.1 5.8 3.8 0.19 33 M103205H795- MY535 A231G/L234W/G236T/H268D/A330K 2.1 3.6 5.7 4.4 0.17 1.8 1.7 2.4 0.16 34 M103205H795- MY536 A231G/L234W/G236T/S239V/H268D/ AZZOK 2.7 4.6 17.3 4.5 0.23 2.4 5.0 4.6 0.30 35 M103205H795- MY537 A231G/L234W/G236T/S239V/H268D/ Q295L/A330K 2.7 4.6 12.1 5.6 0.24 2.5 3.0 4.6 0.25 36 M103205H795- MY538 A231G/L234W/G236T/H268D/Q295L/ AZZOK 3.3 4.6 5.7 6.4 0.15 2.8 1.8 3.5 0.14

- 120 040713

Table 24

KD (M) for cynoFcgRs KD(M) for nen.FcgRs Symbol heavy chain CH SEQ ID NO Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 1 М103205Н7 95-SG1 SG1 9 3.2E-06 5.1E-06 1.7E-05 1.8E-06 3.6E-07 6.9E-07 1.1E-06 4.6E-06 8.1E-07 2 М103205Н7 95-MY101 MY101 229 G236N/H268E/A330K/P396 M 1.1E-06 1.2E-06 3.6E-06 3.5E-07 3.2E-06 4.9E-07 8.7E-07 4.9E-07 LIE-05 3 М103205Н7 95-RK2 RK2 230 G236N/H268E/A330K/P396 M/S400R/D413K 1.ZE-06 1.ZE-06 3.8E-06 3.8E-07 3.9E-06 5.7E-07 1.1E-06 5.9E-07 1.3E-05 4 М103205Н7 95-MY201 MY201 231 G236N/H268D/A330K 1.4E-06 1.4E-06 3.9E-06 4.3E-07 5.7E-06 9.1E-07 1.9E-06 1.1E-06 2.4E-05 5 М103205Н7 95-MUZ 51 MY351 232 G236N/H268D/Q311R/A330 K/D413K 9.2E-07 7.6E-07 1.9E-06 3.5E-07 5.2E-06 1.0E-06 1.6E-06 1.2E-06 1.4E-05 6 М103205Н7 95-MY205 MY205 233 G236N/H268D/Q295L/A330 TO 9.0E-07 1.1E-06 3.0E-06 2.5E-07 6.0E-06 4.7E-07 1.3E-06 5.6E-07 1.3E-05 7 М103205Н7 95-MY344 MY344 234 G236N/H268D/Q295L/Q311 R/A330K/D413K 8.9E-07 1.1E-06 2.2E-06 3.1E-07 4.9E-06 6.7E-07 1.2E-06 7.2E-07 1.9E-05 8 М103205Н7 95-MUZ 35 MY335 235 G236T/H268D/Q311R/A330 K/D413K 2.0E-06 1.7E-06 2.7E-06 6.5E-07 1.5E-06 2.9E-07 2.9E-07 2.3E-06 5.0E-06 KD fold for cynoFcgRs (SGI = 1) KD fold in relation to nen.FcgRs (SGI = 1) Symbol heavy chain CH SEQID NO Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV М103205Н79 5-SG1 SG1 9 1.0 1.0 1.0 1.0 1.00 1.0 1.0 1.0 1.00 ² М103205Н79 5-MY101 MY101 229 G236N/H268E/A330K/P396 M 2.9 4.3 4.7 5.1 0.11 1.4 1.3 9.4 0.07 М103205Н79 5-RK2 RK2 230 G236N/H268E/A330K/P396 M/S400R/D413K 2.5 3.9 4.5 4.7 0.09 1.2 1.0 7.8 0.06 4 М103205Н79 5-MY201 MY201 231 G236N/H268D/A330K 2.3 3.6 4.4 4.2 0.06 0.8 0.6 4.2 0.03 М103205Н79 5-MY351 MY351 232 G236N/H268D/Q311R/A330 K/D413K 3.5 6.7 8.9 5.1 0.07 0.7 0.7 3.8 0.06 6 М103205Н79 5-MY205 MY205 233 G236N/H268D/Q295L/A330 TO 3.6 4.6 5.7 7.2 0.06 1.5 0.8 8.2 0.06 7 М103205Н79 5-MY344 MY344 234 G236N/H268D/Q295L/Q311 R/A330K/D413K 3.6 4.6 7.7 5.8 0.07 1.0 0.9 6.4 0.04 8 М103205Н79 5-MY335 MY335 235 G236T/H268D/Q311R/A330 K/D413K 1.6 3.0 6.3 2.8 0.24 2.4 3.8 2.0 0.16

Table 25

KD(M) for cynoFcgRs KD(M) for nen.FcgRs Symbol heavy chain CH SEQID NO mutation FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 1 M103205H 795-SG1 SGI 9 4.8E-06 4.7E-06 8.6E-06 1.9E-06 2.8E-07 8.9E-07 1.0E-06 4.7E-06 6.2E-07 2 M103205H 795SG1012 SG1012 236 G236N/H268E/A330K/P 396M/N434A 1.1E-06 1.0E-06 2.3E-06 2.7E-07 2.1E-06 5.0E-07 7.5E-07 4.9E-07 5.8E-06 3 M103205H 795SG1016 SG1016 237 G236N/H268E/Q311R/A 330K/P396M/D399R/N4 34A 1.3E-06 1.1E-06 2.2E-06 3.3E-07 2.4E-06 6.0E-07 8.2E-07 5.5E-07 1.0E-05 4 M103205H 795SG1029 SG1029 238 G236N/H268D/Q295L/ Q311R/A330K/D413K/ N434A 1.4E-06 1.5E-06 3.1E-06 4.0E-07 5.3E-06 7.9E-07 1.3E-06 9.0E-07 2.0E-05 5 M103205H 795SG1031 SG1031 239 G236N/H268D/Q311R/ A330K/D413K/N434A 1.8E-06 1.5E-06 2.8E-06 5.0E-07 4.4E-06 1.3E-06 8.0E-07 1.2E-06 1.5E-05 6 M103205H 795SG1033 SG1033 240 G236N/H268D/A330K/ N434A 1.8E-06 1.5E-06 3.0E-06 5.0E-07 4.4E-06 1.2E-06 1.0E-06 1.2E-06 1.2E-05 7 M103205H 795SG1034 SG1034 241 G236T/H268D/Q311R/A 330K/D413K/N434A 3.1E-06 2.7E-06 4.2E-06 8.8E-07 2.2E-06 3.1E-07 2.5E-07 2.6E-06 7.1E-06 8 M103240H 795-SG1 SGI 9 3.8E-06 4.1E-06 6.6E-06 1.7E-06 2.6E-07 9.7E-07 9.6E-07 3.8E-06 5.8E-07 9 M103240H 795SG1071 SG1071 242 G236N/H268D/Q295L/ Q311R/A330K/D413K/ M428L/N434A/Y436I7 Q438R/S440E 1.2E-06 1.3E-06 2.1E-06 3.3E-07 3.8E-06 6.9E-07 1.2E-06 6.8E-07 1.4E-05 10 M103240H 795SG1074 SG1074 243 G236N/H268D/Q295L/ Q311R/K326T/A330K/P 343R/M428L/N434A/Y4 7.0E-07 6.8E-07 1.1E-06 1.6E-07 1.4E-06 4.5E-07 5.4E-07 3.8E-07 4.5E-06

- 121 040713

Symbol severe SEQ ID CH NO chains eleven М103240Н 795- SG1077 SG1077 244 12 М103240Н 795- SG1079 SG1079 245 13 М103240Н 795- SG1080 SG1080 246 14 М103240Н 795- SG1081 SG1081 247 mutation 36T/Q438R/S440E L235W/G236N/H268D/ Q295L/Q311R/K326T/A 330K/P343R/M428L/N4 34A/Y436T/Q438R/S44 0E G236N/H268D/Q295L/ Q311R/A330K/D413K/ N434A/Q438R/S440E G236N/H268D/Q295L/ Q311R/K326T/A330K/P 343R/N434A/Q438R/S4 40E L235W/G236N/H268D/ Q295L/Q311R/K326T/A 330K/P343R/N434A/Q4 38R/S440E KD(M) for cynoFcgRs FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS 1.4E-06 1.3E-06 2.0E-06 3.2E-07 2.9E-06 I.IE-06 1.2E-06 2.1E-06 3.0E-07 3.3E-06 6.5E-07 6.8E-07 LIE-06 1.4E-07 1.4E-06 1.4E-06 1.3E-06 2.1E-06 2.9E-07 2.9E-06 KD(M) for nen.FcgRs FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 9.3E-07 6.9E-07 5.1E-07 3.4E-06 6.7E-07 1.2E-06 6.9E-07 3.2E-06 4.5E-07 5.6E-07 3.7E-07 4.6E-06 9.9E-07 6.8E-07 5.0E-07 3.4E-06 KD fold for cynoFcgRs (SGI = 1) KD fold in relation to nen.FcgRs (SGI = 1) Symbol heavy chain CH SEQID NO mutation FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 1 М103205Н 795-SG1 SG1 9 1.0 1.0 1.0 1.0 1.00 1.0 1.0 1.0 1.00 2 М103205Н 795SG1012 SG1012 236 G236N/H268E/A330K/P 396M/N434A 4.4 4.7 3.8 7.2 0.13 1.8 1.4 9.6 0.11 3 М103205Н 795SG1016 SG1016 237 G236N/H268E/Q311R/A 33 OK/RZ 96M/D399R/N4 34A 3.8 4.1 3.9 6.0 0.12 1.5 1.2 8.7 0.06 4 М103205Н 795SG1029 SG1029 238 G236N/H268D/Q295L/ Q311R/A330K/D413K/ N434A 3.5 3.1 2.8 4.9 0.05 1.1 0.8 5.3 0.03 5 М103205Н 795SG1031 SG1031 239 G236N/H268D/Q311R/ A330K/D413K/N434A 2.6 3.1 3.1 3.9 0.06 0.7 1.3 3.8 0.04 6 М103205Н 795SG1033 SG1033 240 G236N/H268D/A330K/ N434A 2.6 3.0 2.9 3.9 0.06 0.7 1.0 3.9 0.05 7 М103205Н 795SG1034 SG1034 241 G236T/H268D/Q311R/A 330K/D413K/N434A 1.5 1.7 2.0 2.2 0.13 2.9 4.1 1.8 0.09 8 М103240Н 795-SG1 SG1 9 1.0 1.0 1.0 1.0 1.00 1.0 1.0 1.0 1.00 9 М103240Н 795SG1071 SG1071 242 G236N/H268D/Q295L/ Q311R/A330K/D413K/ M428L/N434A/Y436T/ Q438R/S440E 3.0 3.2 3.1 5.3 0.07 1.4 0.8 5.6 0.04 10 М103240Н 795- SG1074 243 G236N/H268D/Q295L/ Q311R/K326T/A330K/P 5.4 6.1 6.2 11.1 0.19 2.2 1.8 10.2 0.13 KD fold for cynoFcgRs (SGI = 1) KD fold in relation to nen.FcgRs (SGI = 1) Symbol heavy chain CH SEQID NO mutation FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV SG1074 343R/M428L/N434A/Y4 36T/Q438R/S440E eleven М103240Н 795SG1077 SG1077 244 L235W/G236N/H268D/Q295L/Q311R/K326T/A 330K/P343R/M428L/N4 34A/Y436T/Q438R/S44 0E 2.7 3.1 3.3 5.4 0.09 1.0 1.4 7.5 0.17 12 М103240Н 795SG1079 SG1079 245 G236N/H268D/Q295L/ Q311R/A330K/D413K/ N434A/Q438R/S440E 3.4 3.4 3.2 5.7 0.08 1.4 0.8 5.6 0.18 13 М103240Н 795SG1080 SG1080 246 G236N/H268D/Q295L/ Q311R/K326T/A330K/P 343R/N434A/Q438R/S4 40E 5.8 6.1 6.1 12.6 0.19 2.1 1.7 10.5 0.13 14 М103240Н 795SG1081 SG1081 247 L235W/G236N/H268D/ Q295L/Q311R/K326T/A 330K/P343R/N434A/Q4 38R/S440E 2.7 3.2 3.1 5.9 0.09 1.0 1.4 7.7 0.17

- 122 040713

Table 26

Symbol heavy chain 1 change Cage lodging IC bonding (Biacore® method) 1 M103205H795-SG1 1.00 1.00 2 M103205H795-SG141 5311R/D399R 4.42 1.64 3 М103205Н795-Р1375t ( 5311R/D413K 3.43 1.21 4 М103205Н795-Р1378т‘ 5400R/D413K 3.86 1.23 5 М103205Н795-Р1383t ( 5311R/S400R/D413K 5.68 1.41 6 М103205Н795-Р1484t( 5311R/D312R 0.00 1.33 7 М103205Н795-Р1485t ( 5311R/N315R 1.88 1.29 8 М103205Н795-Р1486t( 5311R/N315K 1.52 1.20 9 М103205Н795-Р1487t( 5311R/N384R 1.23 1.46 10 М103205Н795-Р1488t( 5311R/N384K 1.45 1.43 eleven М103205Н795-Р1489t ( 5311R/E318R 1.68 1.24 12 М103205Н795-Р1490t ( 5311R/E318K 1.33 1.28 13 М103205Н795-Р1491t ( 5311R/E333R 1.37 1.05 14 М103205Н795-Р1492t( 5311R/E333K 1.13 0.88 15 М103205Н795-Р1493t ( 5311R/T335R 0.99 1.10 16 М103205Н795-Р1494t ( 5311R/T335K 1.12 1.14 17 М103205Н795-Р1495t ( 5311R/S337R 1.76 1.30 18 М103205Н795-Р1496t( 5311R/S337K 1.69 1.28 19 М103205Н795-Р1497t( 3311R/Q342R 1.38 1.49 20 М103205Н795-Р1498t( 5311R/Q342K 1.56 1.38 21 М103205Н795-Р1499t ( 5311R/P343R 3.61 1.98 22 М103205Н795-Р1500t( 5311R/P343K 1.82 1.32 23 М103205Н795-Р1501t( 5311R/D413R 2.40 1.82 24 М103205Н795-Р1522t( 5311R/H285R 2.13 1.24 25 М103205Н795-Р1523t( 5311R/H285K 1.91 1.19 26 М103205Н795-Р1524t( 5311R/G341R 3.66 1.40 27 М103205Н795-Р1525t( 5311R/G341K 3.55 1.31 28 М103205Н795-Р1526t( 5311R/G385R 1.20 1.04 29 М103205Н795-Р1527t( 5311R/G385K 0.92 0.98 thirty М103205Н795-Р1528t Q311R/E388R 3.02 1.12 31 М103205Н795-Р1529t Q311R/E388K 2.86 1.08 32 М103205Н795-Р1530t Q311R/N390R 3.12 1.09 33 М103205Н795-Р1531t Q311R/N390K 2.35 1.01 34 М103205Н795-Р1532t Q311R/D401R 4.16 1.23 35 М103205Н795-Р1533t Q311R/D401K 3.52 1.18 36 М103205Н795-Р1534t Q311R/G402R 3.12 1.12 37 М103205Н795-Р1535t Q311R/G402K 2.49 1.11 38 М103205Н795-Р1536t Q311R/G420R 2.05 1.16 39 М103205Н795-Р1537t Q311R/V422R 2.77 1.32 40 М103205Н795-Р1538t Q311R/V422K 1.84 1.11 41 М103205Н795-Р1539t Q311R/A431R 3.20 0.90 42 М103205Н795-Р1540t D401R/D413K 7.64 1.73 43 М103205Н795-Р1541t D401K/D413K 7.17 1.57 44 М103205Н795-Р1542t G402R/D413K 3.32 1.21 45 М103205Н795-Р1543t G402K/D413K 3.70 1.19 46 М103205Н795-Р1544t Q311R/D401R/D413K 7.66 2.04 47 М103205Н795-Р1545t Q311R/D401K/D413K 7.73 1.80 48 М103205Н795-Р1546t Q311R/G402R/D413K 5.10 1.41 49 М103205Н795-Р1547t Q311R/G402K/D413K 4.85 1.33 50 М103205Н795-Р1548t Q311R/G385R/D401R 2.30 1.37 51 М103205Н795-Р1549t Q311R/G385R/G402R 2.03 1.22 52 М103205Н795-Р1550t Q311R/E388R/D401R 6.43 1.63 53 М103205Н795-Р1551t Q311R/E388R/G402R 2.91 1.33 54 М103205Н795-Р1552t G385R/D401R/D413K 4.44 2.19 55 М103205Н795-Р1553t G385R/G402R/D413K 2.86 1.47 56 М103205Н795-Р1555t E388R/G402R/D413K 3.71 2.01

- 123 040713

Table 27

KD(M) for cynoFcgRs KD (M) vs. human FcgRs Symbol heavy chain CH SEQ ID NO Replacement FcgRIIal FcgRIIal .FcgRIIa/ FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 1 М103205Н79 5-SG1 SG1 9 4.8E-06 4.7E-0( 8.6E-0( 1.9E-06 2.8E-07 8.9E-07 1.0E-06 4.7E-06 6.2E-07 2 М103205Н79 5-MY201 MY201 231 3236N/H268D/A330K 1.5E-06 1.2E-0( 1.8E-01 3.3E-0' 3.7E-06 9.8E-07 1.2E-06 8.5E-07 3.7E-05 3 М103205Н79 5-MY351 MUZ 51 232 3236N/H268D/Q311R/ A330K/D413K 1.6E-06 1.3E-0( 2.2E-01 3.8E-0' 4.1E-06 1.2E-06 1.6E-06 9.7E-07 7.4E-06 4 М103205Н79 5-MY352 MUZ 52 248 3236N/H268D/Q311R/ A330K/D413R 1.7E-06 1.3E-0( 2.2E-01 4.2E-0' 4.0E-06 1.2E-06 1.6E-06 1.0E-06 1.1E-05 5 М103205Н79 5-RK55 RK55 249 3236N/H268D/Q311R/ A330K/P343R 1.4E-06 1.2E-0( 2.0E-01 3.6E-0' 3.3E-06 8.3E-07 1.5E-06 7.6E-07 1.3E-05 6 М103205Н79 5-RK56 RK56 250 3236N/H268D/Q311R/ A330K/P343R/D413R 1.8E-06 1.5E-0( 2.5E-01 5.0E-0' 4.4E-06 1.2E-06 1.9E-06 1.0E-06 1.0E-05 7 М103205Н79 5-RK57 RK57 251 3236N/H268D/Q311R/ A330K/N384R/D413R 1.9E-06 1.7E-0( 3.1E-01 5.2E-0' 4.3E-06 1.3E-06 2.2E-06 1.2E-06 2.3E-05 KD fold for cynoFcgRs (SGI = 1) KD fold in relation to human FcgRs (SGI = 1) Symbol heavy chain । :n SEQID NO Replacement FcgRIIal FcgRIIa2 FcgRIIa3 FcgRIIb FcgRIIIaS FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 1 М103205Н 795-SG1 5G1 9 1.0 1.0 1.0 1.0 1.00 1.0 1.0 1.0 1.00 2 М103205Н 795-MY201 JS20 231 G236N/H268D/A330K 3.2 4.0 4.9 5.8 0.08 0.9 0.8 5.6 0.02 3 М103205Н 795-MY351 4Y35 232 G236N/H268D/Q311R/ A330K/D413K 3.0 3.6 4.0 5.0 0.07 0.7 0.6 4.9 0.08 4 М103205Н 795-MY352; 4Y35 248 G236N/H268D/Q311R/ A330K/D413R 2.7 3.5 3.9 4.7 0.07 0.7 0.6 4.6 0.06 5 М103205Н 795-RK55 ³ K55 249 G236N/H268D/Q311R/ A330K/P343R 3.3 4.1 4.3 5.4 0.08 1.1 0.7 6.3 0.05 6 М103205Н 795-RK56 ³ K56 250 G236N/H268D/Q311R/ A330K/P343R/D413R 2.6 3.1 3.5 3.9 0.06 0.8 0.5 4.7 0.06 7 М103205Н 795-RK57 >K57 251 G236N/H268D/Q311R/ A330K/N384R/D413R 2.5 2.8 2.7 3.8 0.06 0.7 0.5 4.1 0.03

Table 28

KD(M) against nea.FcgRs KD fold against nea.FcgRs (SGI = 1) Symbol heavy chain CH SEQID NO Replacement FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 1 MS103240H795- SG1 SGI 9 - 7.5E-07 1.1E-06 5.8E-06 8.1E-07 1.000 1.00 1.0 1.000 2 MS103240H795- MY009 MY009 252 P238D 7.3E-O5 2.3E-05 1.3E-06 2.2E-04 0.010 0.05 4.5 0.004 3 MS103240H795- MY214 MY214 253 P238D/T250V/T307P 3.2E-05 2.1E-05 1.0E-06 1.5E-04 0.023 0.05 5.8 0.005 4 MS103240H795- TT08 GT08 254 P238D/T250V/T307P/ AZZOK 5.5E-05 1.6E-05 6.8E-07 9.6E-05 0.014 0.07 8.5 0.008 MS103240H795- TT09 GT09 255 P238D/T250V/V264ET 307P/A330K 4.4E-O5 1.4E-05 2.9E-07 1.7E-04 0.017 0.08 20.0 0.005 6 MS103240H795- TT10 GT10 256 P238D/T250V/S267A/ T307P/A330K 5.6E-05 8.7E-06 6.3E-07 7.9E-04 0.013 0.13 9.2 0.001 7 MS103240H795- TT11 GT11 257 L234Y/P238D/T250V/ T307P/A330K 2.4E-05 1.0E-05 5.2E-07 1.7E-04 0.031 0.11 11.2 0.005 8 MS103240H795- TT12 GT12 258 L234Y/P238D/T250V/ S267A/T307P/A330K 2.0E-05 5.0E-06 4.9E-07 1.3E-04 0.038 0.22 11.8 0.006 9 MS103240H795- TT13 GT13 259 P238D/T250V/V264ES 267A/T307P/A330K 4.5E-O5 9.8E-06 3.2E-07 1.4E-04 0.017 0.11 18.1 0.006 10 MS103240H795- TT14 GT14 260 L234Y/P238D/T250V/ V264I/T307P/A330K 1.8E-05 7.6E-06 1.9E-07 1.5E-04 0.042 0.14 30.5 0.005 eleven MS103240H795- TT15 GT15 261 L234Y/P238D/T250V/ V264I/S267A/T307P/A 2.9E-05 6.1E-06 2.5E-07 1.3E-04 0.026 0.18 23.2 0.006 12 MS103240H795- TT20 GT20 262 P238D/T250V/T307P/ Q311R/A330K/P343R 9.1E-05 2.8E-05 1.4E-06 8.4E-05 0.008 0.04 4.1 0.010 13 MS103240H795- TT21 GT21 263 P238D/T250V/T307P/ Q311R/A330K/D413K 5.6E-05 2.0E-05 1.3E-06 8.5E-05 0.013 0.06 4.5 0.010

- 124 040713

14 MS103240H795- TT22 GT22 264 P238D/T250V/V264I/T3 07P/Q311R/A330K/P343 R 6.0Е-05 2.1E-05 5.9E-07 7.2E-05 0.013 0.05 9.8 0.011 15 MS103240H795- TT23 GT23 265 P238D/T250V/V264I/T3 07P/Q311R/A330K/D413K 1.0E-04 4.8E-05 5.5E-07 4.5E-04 0.007 0.02 10.5 0.002 16 MS103240H795- TT24 GT24 266 P23 8D/T250V/S267A/TK 07P/Q311R/A330K/P343 R 4.9E-05 1. ZE-05 1.2E-06 2.6E-04 0.015 0.08 4.8 0.003 17 MS103240H795- TT25 GT25 267 P23 8D/T250V/S267A/TK 07P/Q311R/A330K/D413K 2.2E-05 1. ZE-05 1.0E-06 1.4E-04 0.034 0.08 5.8 0.006 18 MS103240H795- TT26 GT26 268 L234Y/P238D/T250V/T3 07P/Q311R/A330K/P343 R 6.9Е-05 2.1E-05 1.1E-06 2.0E-04 0.011 0.05 5.3 0.004 19 MS103240H795- TT27 GT27 269 L234Y/P238D/T250V/T3 07P/Q311R/A330K/D413K 5.2E-05 1.4E-05 8.2E-07 1.3E-04 0.014 0.08 7.1 0.006 20 MS103240H795- TT28 GT28 270 L234Y/P238D/T250V/S2 67A/T307P/Q311R/A330K/P343R 4.8E-05 7.7E-06 8.5E-07 8.2E-05 0.016 0.14 6.8 0.010 21 MS103240H795- TT29 GT29 271 L234Y/P238D/T250V/S2 67A/T307P/Q311R/A330K/D413K 3.7E-05 7.5E-06 8.1E-07 8.9Е-05 0.020 0.15 7.2 0.009 22 MS103240H795- TTZO GTZO 272 P238D/T250V/V264I/S26 7A/T307P/Q311R/A330K/P343R 1.5E-04 1.4E-05 5.7E-07 6.6E-05 0.005 0.08 10.2 0.012 23 MS103240H795- TT31 GT31 273 P238D/T250V/V264I/S26 7A/T307P/Q311R/A330K/D413K 1.1E-04 1.9E-05 5.6E-07 2.9E-04 0.007 0.06 10.4 0.003 24 MS103240H795- TT32 GT32 274 L234Y/P238D/T250V/V2 64I/T307P/Q311R/A330K/P343R 5.0Е-05 1.2E-05 4.0E-07 1.1E-04 0.015 0.09 14.5 0.007 25 MS103240H795ttzz gzz 275 L234Y/P238D/T250V/ V264I/T307P/Q311R/ A330K/D413K 4.2E-05 1.2E-05 3.6E-07 4.4E-04 0.018 0.09 16.1 0.002 26 MS103240H795- TT34 GT34 276 L234Y/P238D/T250V/ V264I/S267A/T307P/Q 2.2E-05 8.9Е-06 4.9E-07 9.6E-05 0.034 0.12 11.8 0.008 311R/A330K/P343R 27 MS103240H795- TT35 TT35 277 L234Y/P238D/T250V/V264I/S267A/T307P/Q311R/A330K/D413K 2.5E-05 9.0Е-06 4.3E-07 1.8E-04 0.030 0.12 13.5 0.004

Table 29

CH - +N434A +Q438R +S440E +N434A +Y436T +Q438R +S440E +M428L +N434A +Y436T +Q438R +S440E +M428L +N434A +Q438R +S440E MY009 252 278 304 330 356 MY214 253 279 305 331 357 TT08 254 280 306 332 358 TT09 255 281 307 333 359 TT10 256 282 308 334 360 TT11 257 283 309 335 361 TT12 258 284 310 336 362 TT13 259 285 311 337 363 TT14 260 286 312 338 364 TT15 261 287 313 339 365 TT20 262 288 314 340 366 TT21 263 289 315 341 367 GT22 264 290 316 342 368 TT23 265 291 317 343 369 TT24 266 292 318 344 370 TT25 267 293 319 345 371 TT26 268 294 320 346 372 TT27 269 295 321 347 373 TT28 270 296 322 348 374 TT29 271 297 323 349 375 GTZO 272 298 324 350 376 TT31 273 299 325 351 377 TT32 274 300 326 352 378 GTZZ 275 301 327 353 379 TT34 276 302 328 354 380 TT35 277 303 329 355 381

- 125 -

Claims (9)

Table 30 KD (M) for nen.FcgRs KD fold in relation to nen.FcgRs (SGI = 1) Symbol heavy chain CH SEQID NO Replacement FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV FcgRIIaH FcgRIIaR FcgRIIb FcgRIIIaV 1 MS103240H79 5-SG1 SG1 9 9.7E-07 1.2E-06 4.7E-06 7.2E-07 1.000 1.00 1.0 1.000 2 MS103240H79 5-TTZZ TTZZ 275 L234Y/P238D/T250V/V264I/T307P/ Q311R/A330K/D413K 3.5E-05 2.3E-05 3.0E-07 4.0E-04 0.028 0.05 15.7 0.002 3 MS103240H79 5-TT66 TT66 288 P238D/T250V/T307P/Q311R/A330K/ P343R/N434A/Q438R/S440E 1.3E-04 3.2E-05 1.0E-06 5.9E-04 0.007 0.04 4.7 0.001 4 MS103240H79 5-TT67 TT67 340 P238D/T250V/T307P/Q311R/A330K/ P343R/M428L/N434A/Y436T/Q438R S440E 1.2E-04 2.8E-05 1.2E-06 6.1E-04 0.008 0.04 3.9 0.001 5 MS103240H79 5-TT68 TT68 289 P238D/T250V/T307P/Q311R/A330K/ D413K/N434A/Q438R/S440E 2.1E-04 6.1E-05 9.6E-07 4.8E-04 0.005 0.02 4.9 0.001 6 MS103240H79 5-TT69 TT69 341 P238D/T250V/T307P/Q311R/A330K/ D4I3K/M428L/N434A/Y436T/Q438 R/S440E 1.3E-04 3.4E-05 1.1E-06 5.3E-04 0.008 0.04 4.3 0.001 7 MS103240H79 5-TT70 TT70 290 P238D/T250V/V264I/T307P/Q311R/ A330K/P343R/N434A/Q438R/S440E 7.2E-05 4.0Е-05 3.7E-07 3.9E-04 0.013 0.03 12.7 0.002 8 MS103240H79 5-TT71 TT71 342 P238D/T250V/V264I/T307P/Q311R/ A330K/P343R/M428L/N434A/Y436 T/Q438R/S440E 9.6E-05 2.7E-05 4.2E-07 4.0E-04 0.010 0.04 11.2 0.002 9 MS103240H79 5-TT72 TT72 291 P238D/T250V/V264I/T307P/Q311R/ AZ 30K/D413K/N434A/Q43 8R/S440E 7.9Е-05 2.4E-05 4.0E-07 5.0E-04 0.012 0.05 11.8 0.001 10 MS103240H79 5-TT73 TT73 343 P238D/T250V/V264I/T307P/Q311R/AZ 30K/D413K/M428L/N434A/Y436T/Q438R/S440E 7.4E-05 1.9E-05 4.2E-07 5.6E-04 0.013 0.06 11.2 0.001 eleven MS103240H79 5-TT90 TT90 300 L234Y/P238D/T250V/V264I/T307P/ Q311R/A330K/P343R/N434A/Q438R S440E 2.9E-05 2.5E-05 3.0E-07 3.6E-04 0.033 0.05 15.7 0.002 12 MS103240H79 5-TT91 TT91 352 L234Y/P238D/T250V/V264I/T307P/Q311R/A330K/P343R/M428L/N434A/Y436T/Q438R/S440E 4.6E-05 3.0E-05 3.2E-07 4.5E-04 0.021 0.04 14.7 0.002 13 MS103240H79 5-TT92 TT92 301 L234Y/P238D/T250V/V264I/T307P/Q311R/A330K/D413K/N434A/Q438 4.0Е-05 2.0E-05 2.8E-07 4.9E-04 0.024 0.06 16.8 0.001 R/S440E 14 MS103240H79 5-TT93 TT93 353 L234Y/P238D/T250V/V264I/T307P/ Q311R/A330K/D413K/M428L/N434 A/Y436T/Q438R/S440E 5.2E-05 2.4E-05 3.1E-07 4.7E-04 0.019 0.05 15.2 0.002 CLAIM 1. An isolated antibody that binds to latent myostatin, wherein the antibody inhibits myostatin activation and binds to an epitope that is represented by amino acids 81-100 of SEQ ID NO: 78. 2. The antibody according to claim 1, where the antibody: (a) blocks the release of mature myostatin from latent myostatin; (b) blocks the proteolytic release of mature myostatin or (c) blocks the spontaneous release of mature myostatin. 3. An antibody according to one of claims 1 or 2 which binds latent myostatin with a higher affinity at neutral pH than at acidic pH. 4. The antibody of claim 3, wherein the antibody binds latent myostatin with higher affinity at pH 7.4 than at pH 5.8. 5. An antibody according to one of claims 1 to 4, which is (a) a monoclonal antibody, (b) a human, humanized or chimeric antibody; (c) a full-length IgG-type antibody; or (d) an antibody fragment that binds to myostatin. 6. An isolated nucleic acid encoding an antibody according to one of claims 1-5. 7. A host cell containing a nucleic acid according to claim 6. 8. A method for producing antibodies to myostatin, including: (a) culturing the host cell according to claim 7 so as to obtain an antibody; or (b) immunizing the animal with a polypeptide, wherein the polypeptide comprises a region corresponding to positions 81-100 of the myostatin polypeptide (SEQ ID NO: 78). 9. Pharmaceutical composition for increasing the clearance of myostatin from plasma, containing an antibody according to one of claims 1 to 5 and a pharmaceutically acceptable carrier. - 126 -