CN108473562B - Anti-myostatin antibodies, polypeptides comprising variant FC regions, and methods of use - Google Patents

Abstract

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

Description

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

Technical Field

The present invention relates to anti-myostatin antibodies and methods of using the same. The invention also relates to polypeptides comprising variant Fc regions and methods of use thereof.

Background

Myostatin, also known as growth differentiation factor-8 (GDF8), is a secreted protein and is a member of the transforming growth factor-beta (TGF- β) protein superfamily. Members of this superfamily have growth-regulating and morphogenetic properties (see, e.g., NPL 1, NPL 2, and PTL 1). Myostatin is expressed primarily in developing and adult skeletal muscle and functions as a negative regulator of muscle growth. Systemic overexpression of myostatin in adult mice results in muscle wasting (see, e.g., NPL 3), while, conversely, myostatin knockout mice are characterized by hypertrophy and hyperplasia of skeletal muscle, which results in two to three times higher muscle mass compared to their wild-type counterparts (see, e.g., NPL 4).

Similar to other members of the TGF- β family, myostatin is synthesized as a large precursor protein containing an N-terminal propeptide domain and a C-terminal domain that is considered to be an active molecule (see, e.g., NPL 5; PTL 2). Two myostatin precursor molecules are covalently linked via a single disulfide bond in the C-terminal growth factor domain. Through a multi-step proteolytic process, active mature myostatin (a disulfide-bonded homodimer consisting of C-terminal growth factor domains) is released from myostatin precursors. In the first step of the myostatin activation pathway, the peptide bond between the N-terminal propeptide domain and the C-terminal growth factor domain, Arg266-Asp267, in both chains of the homodimeric precursor is cleaved by furin-type proprotein convertase. The resulting three peptides (two pro-peptides and one mature myostatin (i.e., disulfide-bonded homodimer consisting of growth factor domains)) remain linked to form a non-covalent inactive complex known as "latent myostatin". Mature myostatin can then be released from latent myostatin via degradation of the propeptide. Members of the bone morphogenetic protein 1(BMP1) family of metalloproteinases cleave a single peptide bond within the propeptide, Arg98-Asp99, which is accompanied by the release of mature active muscle growth inhibitory factor as a homodimer (see, e.g., NPL 6). Furthermore, latent myostatin can be activated in vitro by dissociating the complex with acid or heat treatment (see, e.g., NPL 7).

Myostatin acts through a family of transmembrane serine/threonine kinase heterotetrameric receptors, the activation of which enhances receptor phosphotransfer, resulting in stimulation of serine/threonine kinase activity. It has been demonstrated that the myostatin pathway involves active myostatin dimers binding with high affinity to activin receptor type IIB (ActRIIB), which then recruits and activates phosphotransfer of low affinity receptor activin-like kinase 4(ALK4) or activin-like kinase 5(ALK 5). It has also been demonstrated that the proteins Smad 2 and Smad 3 are subsequently activated and form complexes with Smad 4, which are then transferred to the nucleus for activation of target gene transcription. ActRIIB has been shown to mediate the effects of myostatin in vivo, as the expression of the dominant negative form of ActRIIB in mice is similar to myostatin gene knock-out (see, e.g., NPL 8).

Several diseases or disorders 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, weakness, Congestive Obstructive Pulmonary Disease (COPD), sarcopenia (sarcopenia), and cachexia resulting from cancer or other diseases, as well as kidney, heart failure or heart disease, and liver disease. Patients will benefit from an increase in muscle mass and/or muscle strength; however, the currently available treatments for these conditions are limited. Thus, due to its role as a negative regulator of skeletal muscle growth, myostatin is a suitable target for therapeutic or prophylactic intervention for such diseases or disorders, or for monitoring the progression of such diseases or disorders. In particular, agents that inhibit myostatin activity can be therapeutically beneficial.

Inhibition of myostatin expression results in muscle hypertrophy and hyperplasia (NPL 4). Following injury, myostatin negatively regulates muscle regeneration and the lack of myostatin in myostatin-free mice results in accelerated muscle regeneration (see, e.g., NPL 9). Anti-myostatin (GDF8) antibodies described in, for example, PTL 3, PTL 4, PTL 5, PTL 6, and PTL 7, and PTL 8, PTL 9, and PTL 10 have been shown to bind myostatin and inhibit myostatin activity, including that associated with negative regulation of skeletal muscle mass, in vitro and in vivo. Myostatin neutralizing antibodies increase body weight, skeletal muscle mass, and muscle size and strength in skeletal muscle in wild-type mice (see, e.g., NPL 10) as well as mdx mice (see, e.g., NPL 11; NPL 12) that are models of muscular dystrophy. However, these prior art antibodies are specific for mature myostatin and not for latent myostatin, and the described strategies for inhibiting myostatin activity have utilized antibodies that can bind to and neutralize mature myostatin.

Antibodies are of interest as drugs because of their high stability in blood and fewer side effects (see, e.g., NPL 13 and NPL 14). Almost all therapeutic antibodies currently marketed are of the human IgG1 subclass. One of the known functions of IgG class antibodies is antibody-dependent cell-mediated cytotoxicity (hereinafter, ADCC activity) (see, for example, NPL 15). For an antibody to exhibit ADCC activity, the antibody Fc region must bind to Fc γ receptors (hereinafter referred to as fcyr) that are antibody binding receptors present on the surface of effector cells such as killer cells, natural killer cells, and activated macrophages.

In humans, Fc γ RIa (CD64A), Fc γ RIIa (CD32A), Fc γ RIIb (CD32B), Fc γ RIIIa (CD16A) and Fc γ RIIIb (CD16B) isoforms have been reported as Fc γ R protein families, and the corresponding allotypes have also been reported (see, e.g., NPL 16). Fc γ RIa, Fc γ RIIa and Fc γ RIIIa are referred to as activating Fc γ R because they have an immunologically active function, while Fc γ RIIb is referred to as inhibiting Fc γ R because it has an immunosuppressive function (see, e.g., NPL 17).

In the binding between the Fc region and Fc γ R, several amino acid residues in the hinge and CH2 domains of the antibody, as well as the sugar chain attached to Asn at position 297(EU numbering) that binds to the CH2 domain, have proven important (see, e.g., NPL 18, NPL 19 and NPL 20). A number of variants with Fc γ R binding properties have been studied, mainly antibodies with mutations introduced at these sites; and Fc region variants having higher binding activity to activating Fc γ R have been obtained (see, for example, PTL 11, PTL 12, PTL 13, and PTL 14).

When the activating Fc γ R cross-links with immune complexes, it phosphorylates an immunoreceptor tyrosine-based activation motif (ITAM) contained in the intracellular domain or FcR consensus γ -chain (interaction partner), activates the signal transducer SYK, and triggers an inflammatory immune response by initiating an activation signaling cascade (see, e.g., NPL 21).

Fc γ RIIb is the only Fc γ R expressed on B cells (see, e.g., NPL 22). Interaction of the antibody Fc region with Fc γ RIIb reportedly suppresses the primary immune response of B cells (see, e.g., NPL 23). Furthermore, it has been reported that B cell activation and antibody production by B cells are inhibited when Fc γ RIIb and B Cell Receptor (BCR) on B cells are crosslinked via immune complexes in blood (see, for example, NPL 24). In this immunosuppressive signaling mediated by BCR and Fc γ RIIb, an immunoreceptor tyrosine-based inhibitory motif (ITIM) contained in the intracellular domain of Fc γ RIIb is necessary (see, e.g., NPL 25 and NPL 26). When ITIMs are phosphorylated following signal transduction, SH 2-containing inositol polyphosphate 5-phosphatase (SHIP) is recruited, transduction of other activating Fc γ R signaling cascades is inhibited, and immune responses are inhibited (see, e.g., NPL 27). Furthermore, it was reported that aggregation of Fc γ RIIb alone transiently inhibited calcium influx in a BCR-independent manner without causing apoptosis of IgM-producing B cells due to BCR cross-linking and B cell proliferation (see, e.g., NPL 28).

Fc γ RIIb is also expressed on dendritic cells, macrophages, activated neutrophils, mast cells and basophils. Fc γ RIIb inhibits the functions of the activating Fc γ R in these cells, such as phagocytosis and release of inflammatory cytokines, and inhibits the inflammatory immune response (see, e.g., NPL 17).

The importance of Fc γ RIIb immunosuppressive function has now been elucidated by studies using Fc γ RIIb knockout mice. It has been reported that humoral immunity is not appropriately regulated in Fc γ RIIb knockout mice (see, e.g., NPL 29), sensitivity to collagen-induced arthritis (CIA) is increased (see, e.g., NPL 30), lupus-like symptoms are exhibited, and Goodpasture's syndrome-like symptoms are exhibited (see, e.g., NPL 31).

In addition, under-regulation of Fc γ RIIb has been reported to be associated with autoimmune diseases in humans. For example, a relationship between genetic polymorphisms of transmembrane and promoter regions of Fc γ RIIb and the frequency of occurrence of Systemic Lupus Erythematosus (SLE) has been reported (see, for example, NPL 32, NPL 33, NPL 34, NPL 35, and NPL 36), and a decrease in Fc γ RIIb expression on the surface of B cells in SLE patients (see, for example, NPL 37 and NPL 38).

From the mouse model and thus clinical findings, Fc γ RIIb is thought to play a role in controlling autoimmune and inflammatory diseases by specifically involving B cells, and is a promising target molecule for controlling autoimmune and inflammatory diseases.

IgG1, which is mainly used as a commercially available therapeutic antibody, is known to bind not only Fc γ RIIb, but also strongly to activate Fc γ R (see, e.g., NPL 39). It is possible to develop therapeutic antibodies with greater immunosuppressive properties than that of IgG1 by utilizing Fc regions with enhanced or increased Fc γ RIIb binding or Fc γ RIIb binding selectivity compared to activating Fc γ rs. For example, it has been suggested that B cell activation can be inhibited using antibodies having variable regions that bind BCR and Fc with enhanced Fc γ RIIb binding (see, e.g., NPL 40). It has been reported that cross-linking Fc γ RIIb on B cells with IgE that binds to B cell receptors inhibits differentiation of B cells into plasma cells, which consequently leads to inhibition of IgE production; and in mice transplanted with human PBMCs, human IgG and IgM concentrations were maintained while human IgE concentrations were reduced (see, e.g., NPL 41). In addition to IgE, it has been reported that when antibodies cross-link Fc γ RIIB and CD79B, which is a constituent molecule of the B-cell receptor complex, B-cell proliferation is inhibited in vitro and arthritis symptoms are alleviated in a collagen arthritis model (see, for example, NPL 42).

In addition to B cells, it was reported that cross-linking of fcyriib and fcyriib on mast cells with molecules in which the Fc portion of IgG with enhanced fcyriib binding is fused to the Fc portion of IgE that binds the IgE receptor fcyri results in phosphorylation of fcyriib, thereby inhibiting fcyriib-dependent calcium influx. This suggests that by enhancing Fc γ RIIb binding, it is possible to inhibit degranulation stimulated via Fc γ RIIb (see, e.g., NPL 43).

Thus, antibodies to Fc with improved Fc γ RIIb-binding activity are shown to be promising as therapeutic agents for inflammatory diseases such as autoimmune diseases.

Furthermore, it has been reported that activation of macrophages and dendritic cells via Toll-like receptor 4 due to LPS stimulation is inhibited in the presence of antibody-antigen immune complexes, and this effect is also considered to be that of immune complexes via Fc γ RIIb (see, for example, NPL 44 and NPL 45). Thus, the use of antibodies with enhanced Fc γ RIIb binding is expected to enhance TLR-mediated activation signaling-inhibition; thus, it was shown that such antibodies are promising as therapeutic agents for inflammatory diseases such as autoimmune diseases.

Furthermore, mutants with enhanced Fc γ RIIb binding have been proposed as promising cancer therapeutics, as well as therapeutics for inflammatory diseases such as autoimmune diseases. Now, it has been found that Fc γ RIIb plays an important role in the agonist activity of agonist antibodies against the anti-TNF receptor superfamily. Specifically, it has been proposed that agonist activity against antibodies against CD40, DR4, DR5, CD30, and CD137 included in the TNF receptor family requires interaction with Fc γ RIIb (see, e.g., NPL 46, NPL 47, NPL 48, NPL 49, NPL 50, NPL 51, and NPL 52). NPL 46 showed that the anti-tumor effect of the anti-CD 40 antibody was enhanced using an antibody with enhanced Fc γ RIIb binding. Thus, antibodies with enhanced Fc γ RIIb are expected to have the effect of enhancing agonist activity of agonist antibodies including antibodies directed against the anti-TNF receptor superfamily.

Furthermore, it has been demonstrated that cell proliferation is inhibited when antibodies recognizing Kit, a Receptor Tyrosine Kinase (RTK), are used to crosslink Fc γ RIIb and Kit on Kit expressing cells. Similar effects have been reported even in cases where the Kit is constitutively activated and has mutations that lead to tumor formation (see, e.g., NPL 53). Thus, it is expected that the use of antibodies with enhanced Fc γ RIIb binding may enhance inhibition on cells expressing RTKs with constitutively active mutations.

Antibodies with Fc having increased Fc γ RIIb-binding activity have been reported (see, e.g., NPL 40). In this document, the Fc γ RIIb-binding activity is increased by adding modifications to the antibody Fc region such as S267E/L328F, G236D/S267E, and S239D/S267E. Among these, the antibody introduced with the S267E/L328F mutation bound most strongly to Fc γ RIIb and maintained the same level of binding to Fc γ RIa and Fc γ RIIa H type (where the residue at position 131 of Fc γ RIIa is His as with naturally occurring IgG 1). However, another report shows that this alteration enhances binding to Fc γ RIIa type R (where the residue at position 131 of Fc γ RIIa is Arg) by several hundred-fold to the same level of Fc γ RIIb binding, which means that Fc γ RIIb-binding selectivity is not improved compared to type R Fc γ RIIa (see, e.g., PTL 15).

The effect of enhancing Fc γ RIIa binding without enhancing Fc γ RIIb binding is only believed to have an effect on cells expressing Fc γ RIIa without expressing Fc γ RIIb, such as platelets (see, e.g., NPL 17). For example, it is known that a patient group to which bevacizumab (an antibody against VEGF) is administered has an increased risk of thromboembolism (see, e.g., NPL 54). In addition, thromboembolism was observed in a similar manner in clinical development trials of antibodies against CD40 ligand, and the clinical studies were discontinued (see, e.g., NPL 55). In the case of these two antibodies, later studies using animal models and the like have shown that the administered antibodies aggregate platelets via Fc γ RIIa bound on the platelets and form blood clots (see, for example, NPL 56 and NPL 57). In systemic lupus erythematosus, which is an autoimmune disease, platelets are activated via an Fc γ RIIa-dependent mechanism, and it is reported that platelet activation is associated with the severity of symptoms (see, for example, NPL 58). Administration of antibodies with enhanced Fc γ RIIa binding to such patients already at high risk of developing thromboembolism would increase the risk of developing thromboembolism and thus be extremely dangerous.

Furthermore, antibodies with enhanced Fc γ RIIa binding have been reported to enhance macrophage-mediated antibody-dependent cellular phagocytosis (ADCP) (see, e.g., NPL 59). When the antigen to which the antibody is bound is phagocytosed by macrophages, the antibody itself is considered to be phagocytosed at the same time. When an antibody is administered as a drug, it is presumed that a peptide fragment derived from the administered antibody may also be presented as an antigen, thereby increasing the risk of producing an antibody against a therapeutic antibody (anti-therapeutic antibody). More specifically, enhancing Fc γ RIIa binding will increase the risk of producing antibodies against the therapeutic antibody, and this will significantly reduce its value as a medicament. Furthermore, it has been proposed that Fc γ RIIb on dendritic cells aids in peripheral tolerance by inhibiting dendritic cell activation by immune complexes formed between antigen and antibody, or by inhibiting antigen presentation to T cells via activation of Fc γ receptors (see, e.g., NPL 60). Since Fc γ RIIa is also expressed on dendritic cells, when an antibody having Fc with enhanced selective binding to Fc γ RIIb is used as a drug, the antigen is not easily presented by dendritic cells and the like due to the enhanced selective binding to Fc γ RIIb, and the risk of anti-drug antibody production can be relatively reduced. Such antibodies are also useful in this regard.

More specifically, when Fc γ RIIa binding is enhanced, resulting in an increased risk of thrombus formation via platelet aggregation and an increased risk of anti-therapeutic antibody production due to increased immunogenicity, the value as a drug will be significantly reduced.

From this point of view, the aforementioned Fc variants with enhanced Fc γ RIIb binding show significantly enhanced R-type Fc γ RIIa binding compared to naturally occurring IgG 1. Therefore, its value as a drug for patients with R-type Fc γ RIIa is significantly reduced. Fc γ RIIa of type H and R is observed at approximately the same frequency in caucasian and african americans (see, e.g., NPL 61 and NPL 62). Thus, when the Fc variant is used to treat autoimmune disease, the number of patients who can safely use it while enjoying its pharmacological effects will be limited.

Furthermore, dendritic cells have been reported to mature in dendritic cells lacking Fc γ RIIb or in dendritic cells in which the interaction between Fc γ RIIb and the Fc portion of the antibody is inhibited by anti-Fc γ RIIb antibodies (see, e.g., NPL 63 and NPL 64). This report suggests that Fc γ RIIb actively inhibits maturation of dendritic cells at steady state (no inflammation etc. and no activation). In addition to Fc γ RIIb, Fc γ RIIa is also expressed on the surface of dendritic cells; thus, even if binding to inhibitory Fc γ RIIb is enhanced and if binding to an activating Fc γ R such as Fc γ RIIa is also enhanced, maturation of dendritic cells can still be promoted accordingly. More specifically, it is considered that increasing not only the Fc γ RIIb-binding activity but also the ratio of the Fc γ RIIb-binding activity to the Fc γ RIIa-binding activity is important in providing an antibody having an immunosuppressive effect.

Thus, when considering the production of medicaments that exploit Fc γ RIIb binding-mediated immunosuppression, there is a need for Fc variants that not only have enhanced Fc γ RIIb-binding activity, but are also capable of binding to both isoforms of Fc γ RIIa H-type and R-type (either maintained at similar levels to naturally occurring IgG1 or reduced to lower levels compared to naturally occurring IgG 1).

Meanwhile, an example has been reported so far in which amino acid changes are introduced into the Fc region to increase Fc γ RIIb-binding selectivity (see, for example, NPL 65). However, all variants as reported in this document allegedly having improved Fc γ RIIb selectivity show reduced Fc γ RIIb binding compared to naturally occurring IgG 1. It is therefore believed that these variants are in fact difficult to elicit a more potent Fc γ RIIb-mediated immunosuppressive response than IgG 1.

Furthermore, since Fc γ RIIb plays an important role in the above-mentioned agonist antibodies, it is expected that enhancing its binding activity will enhance the agonist activity. However, when Fc γ RIIa binding is similarly enhanced, undesirable activities such as ADCC activity and ADCP activity will be exhibited, and this may cause side effects. Also, from this viewpoint, it is preferable to be able to selectively enhance the Fc γ RIIb-binding activity.

From these results, in the preparation of therapeutic antibodies using Fc γ RIIb for the treatment of autoimmune diseases and cancer, it is important that the activity of binding to both Fc γ RIIa allotypes is maintained or reduced and Fc γ RIIb binding is enhanced compared to naturally occurring IgG. However, the extracellular region of Fc γ RIIb has 93% sequence identity with the extracellular region of Fc γ RIIa, one of the activating Fc γ rs, and it is very similar in structure. Allotypes, H-type and R-type, of Fc γ RIIa exist in which the amino acid at position 131 is His (H-type) or Arg (R-type), and each of which reacts differently with antibodies (see, e.g., NPL 66). Thus, a difficult problem may be to prepare Fc region variants with enhanced selective Fc γ RIIb binding compared to various allotypes of Fc γ RIIa, which involves distinguishing sequences with high homology between Fc γ RIIa and Fc γ RIIb. Despite the difficulties, several Fc region variants having selective binding activity to Fc γ RIIb compared to Fc γ RIIa have been identified so far by performing comprehensive amino acid modification analysis in the Fc region (see, for example, PTL 16, PTL 17, PTL 18, PTL 19, and PTL 20).

There have been reports of Fc region variants with binding selectivity for Fc γ RIIb associated with human Fc γ R, while there have been no reports of Fc region variants with binding selectivity for Fc γ RIIb associated with monkey Fc γ R. In the absence of such Fc variants, the effect of selective binding of Fc variants to Fc γ RIIb has not been thoroughly tested in monkeys.

In addition to the above, it has been reported that it is possible to regulate the half-life of an antibody in blood by changing the charge of amino acid residues that may be exposed on the surface of the antibody so as to increase or decrease the isoelectric point (pI) of the antibody (see, for example, PTL 21 and PTL 22). It shows that it is possible to prolong the plasma half-life of an antibody by reducing the pI of the antibody, and vice versa.

Furthermore, it has been reported that antigen binding into cells can be promoted by specifically changing the charge of a specified amino acid residue in its CH3 domain to thereby increase the pI of an antibody (see, for example, PTL 23). Furthermore, it has been reported that changing the charge of amino acid residues in the constant region of an antibody (mainly the CH1 domain) to lower the pI can prolong the half-life of the antibody in plasma (see, for example, PTL 24).

Reference list

Patent document

PTL 1: U.S. Pat. No. 5,827,733

PTL 2WO 1994/021681

PTL 3: U.S. Pat. No. 6,096,506

PTL 4: U.S. Pat. No. 7,261,893

PTL 5: U.S. Pat. No. 7,320,789

PTL 6: U.S. Pat. No. 7,807,159

PTL 7: U.S. Pat. No. 7,888,486

PTL 8：WO 2005/094446

PTL 9：WO 2007/047112

PTL 10：WO 2010/070094

PTL 11：WO 2000/042072

PTL 12：WO 2006/019447

PTL 13：WO 2004/099249

PTL 14：WO 2004/029207

PTL 15: U.S. application publication No. US2009/0136485

PTL 16：WO 2012/115241

PTL 17：WO 2013/047752

PTL 18：WO 2013/125667

PTL 19：WO 2014/030728

PTL 20：WO 2014/163101

PTL 21：WO 2007/114319

PTL 22：WO 2009/041643

PTL 23：WO 2014/145159

PTL 24：WO 2012/016227

Non-patent document

NPL 1: kingsley et al, Genes Dev.8(2):133-146(1994)

NPL 2: hoodless et al, curr. Top. Microbiol. Immunol.228:235-272(1998)

NPL 3: zimmers et al, Science 296(5572):1486-1488(2002)

NPL 4: McPherron et al, Nature 387(6628):83-90(1997)

NPL 5: McPherron and Lee, Proc.Natl.Acad.Sci.USA

94(23):12457-12461(1997)

NPL 6: szlama et al, FEBS J280 (16):3822-3839(2013)

NPL 7：Lee,PloS One 3(2):e1628(2008)

NPL 8：Lee,Proc.Natl.Acad.Sci.USA 98(16):9306-9311(2001)

NPL 9: McCroskery et al, J Cell Sci.118(15):3531-3541(2005)

NPL 10: whittemore et al, biochem. Biophys. Res. Commun.

300(4):965-971(2003)

NPL 11: bogdannovich et al, Nature 420(6914):418-

NPL 12：Wagner.,Ann.Neurol.52(6):832-836(2002)

NPL 13: reichert et al, nat. Biotechnol.23:1073-1078(2005)

NPL 14: pavlou et al, Eur.J.Pharm.Biopharm.59:389-396(2005)

NPL 15: clark et al, chem.Immunol.65:88-110(1997)

NPL 16: jefferis et al, immunol. Lett.82:57-65(2002)

NPL 17: smith et al, nat. Rev. Immunol.10:328-343(2010)

NPL 18: radaev et al, J.biol.chem.276:16478-16483(2001)

NPL 19: greenwood et al, Eur.J.Immunol.23:1098-1104(1993)

NPL 20: morgan et al Immunology 86:319-

NPL 21: nimmerjahn et al, nat. Rev. Immunol.8:34-47(2008)

NPL 22: amigorena et al, Eur.J.Immunol.19:1379-1385(1989)

NPL 23：Sinclair,J.Exp.Med.129:1183-1201(1969)

NPL 24：Heyman,Immunol.Lett.88:157-161(2003)

NPL 25: amigorena et al, Science 256:1808-1812(1992)

NPL 26: muta et al, Nature 368:70-73(1994)

NPL 27：Ravetch,Science 290:84-89(2000)

NPL 28: fournier et al, J.Immunol.181:5350-5359(2008)

NPL 29:J.Immunol.163:618-622(1999)

NPL 30: Yuasa et al, J.Exp.Med.189:187-194(1999)

NPL 31: Nakamura et al, J.Exp.Med.191:899-906(2000)

NPL 32:Blank,Hum.Genet.117:220-227(2005)

NPL 33: Olferiev et al, J.biol.chem.282:1738-1746(2007)

NPL 34: Chen et al, Arthritis Rheum.54:3908-3917(2006)

NPL 35: Floto et al, nat. Med.11:1056-

NPL 36: Li et al, J.Immunol.176:5321-5328(2006)

NPL 37: Mackay et al, J.Exp.Med.203:2157-

NPL 38: Yang et al, J.Immunol.178:3272-3280(2007)

NPL 39: Bruhns et al, Blood 113:3716-

NPL 40: Chu et al, mol.Immunol.45:3926-

NPL 41: Chu et al, J.allergy Clin.Immunol.129:1102-1115(2012)

NPL 42: Veri et al, Arthritis Rheum.62:1933-

NPL 43: Cemerski et al, Immunol.Lett.143:34-43(2012)

NPL 44: Wenink et al, J.Immunol.183:4509-4520(2009)

NPL 45: Zhang et al, J.Immunol.182:554-

NPL 46:Ravetch,Science 333:1030-1034(2011)

NPL 47: Wilson et al, Cancer Cell 19:101-113(2011)

NPL 48: Kohrt et al, J.Clin.Invest.122:1066-1075(2012)

NPL 49: Xu et al, J.Immunol.171:562-568(2003)

NPL 50: Zhang et al, Blood 108:705-

NPL 51: Chuntharapai et al, J.Immunol.166:4891-4898(2001)

NPL 52: ravatch et al, Proc. Natl. Acad. Sci. USA 109:10966-

NPL 53 Malbec et al, Immunol.Lett.143:28-33(2012)

NPL 54 Scappacicii et al, J.Natl.cancer.Inst.99:1232-1239(2007)

NPL 55:Arthritis Rheum.48:719-727(2003)

NPL 56: Meyer et al, J.Thromb.Haemost.7:171-

NPL 57: Robles-Carrillo et al, J.Immunol.185:1577-1583(2010)

NPL 58: Duffau et al, Sci.Transl.Med.2:47ra63(2010)

NPL 59:Richards et al,Mol.Cancer Ther.7:2517-2527(2008)

NPL 60: Desai et al, J.Immunol.178:6217-

NPL 61: Salmonon et al, J.Clin.Invest.97:1348-

NPL 62: Manger et al, Arthritis Rheum.41: 1181-K1189 (1998)

NPL 63: Boruchov et al, J.Clin.invest.115:2914-2923(2005)

NPL 64: Dhodapkar et al, Proc. Natl. Acad. Sci. USA 102:2910-2915(2005)

NPL 65: Armour et al, mol.Immunol.40:585-

NPL 66: Warmerdam et al, J.exp.Med.172:19-25(1990)

Summary of The Invention

Technical problem

It is an object of the present invention to provide anti-myostatin antibodies, polypeptides comprising variant Fc regions, and methods of using the same.

Means for solving the problems

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

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

In some embodiments, an isolated anti-myostatin antibody of the invention binds latent myostatin with a higher affinity at neutral pH than at acidic pH. In some embodiments, the anti-myostatin antibody binds latent myostatin with a higher affinity at ph7.4 than at ph 5.8. In some embodiments, an isolated anti-myostatin antibody of the invention binds to a polypeptide fragment consisting of amino acids 21-100 of the myostatin pro peptide (SEQ ID NO:78) with higher affinity at pH7.4 than at pH 5.8. In some embodiments, the antibody binds to the same myostatin epitope at neutral pH with higher affinity than at acidic pH as an antibody described in table 13. In a further embodiment, the anti-myostatin antibody binds the same epitope at ph7.4 with greater affinity than an antibody described in table 13 binds at ph 5.8. In a further embodiment, the antibody binds the same epitope at ph7.4 with higher affinity than at ph5.8 as an antibody comprising a VH and VL pair as set forth in table 13. In some embodiments, the antibody binds the same myostatin epitope at neutral pH with higher affinity than at acidic pH as the antibody described in table 2 a. In some embodiments, the antibody binds the same myostatin epitope at ph7.4 with greater affinity than the antibody described in table 2a binds to at ph 5.8. In a further embodiment, the antibody binds the same epitope at ph7.4 with higher affinity than at ph5.8 as an antibody comprising a VH and VL pair as described in table 2 a. In further embodiments, the anti-myostatin antibody binds to the same epitope as an antibody described in table 11a at neutral pH with higher affinity than at acidic pH. In a further embodiment, the antibody binds the same myostatin epitope as an antibody described in table 11a with greater affinity at ph7.4 than at ph 5.8. In a further embodiment, the antibody binds to the same epitope as an antibody comprising a VH and VL pair as described in table 11a with higher affinity at ph7.4 than at ph 5.8. In further embodiments, the anti-myostatin antibody binds to the same epitope as an antibody described in table 2a, 11a, or 13 at neutral pH with higher affinity than at acidic pH. In a further embodiment, the antibody binds the same myostatin epitope as an antibody described in tables 2a, 11a, or 13 with greater affinity at ph7.4 than at ph 5.8. In further embodiments, the antibody binds to the same epitope as an antibody comprising a VH and VL pair described in table 2a, 11a, or 13 with higher affinity at ph7.4 than at ph 5.8.

In some embodiments, an isolated anti-myostatin antibody of the invention competes for binding to latent myostatin with an antibody provided herein. In some embodiments, an isolated anti-myostatin antibody of the invention competes for binding to latent myostatin with an antibody described in table 13. In some embodiments, an isolated anti-myostatin antibody of the invention competes for binding to latent myostatin with an antibody comprising a VH and VL pair described in table 13. In some embodiments, the antibody competes for binding to latent myostatin with an antibody described in table 2 a. In some embodiments, an isolated anti-myostatin antibody of the invention competes for binding to latent myostatin with an antibody comprising a VH and VL pair described in table 2 a. In some embodiments, the antibody competes for binding to latent myostatin with an antibody described in table 11 a. In some embodiments, an isolated anti-myostatin antibody of the invention competes for binding to latent myostatin with an antibody comprising a VH and VL pair described in table 11 a. In additional embodiments, the anti-myostatin antibody competes for binding to latent myostatin with an antibody described in table 2a, 11a, or 13. In additional embodiments, the anti-myostatin antibody competes for binding to latent myostatin with an antibody comprising a VH and VL pair described in table 2a, 11a, or 13. In additional embodiments, the anti-myostatin antibody binds latent myostatin at neutral pH with a higher affinity than at acidic pH. In another embodiment, the anti-myostatin antibody binds latent myostatin with a higher affinity at ph7.4 than at ph 5.8. In another embodiment, the anti-myostatin antibody binds to a polypeptide fragment consisting of amino acids 21-100 of the myostatin pro peptide (SEQ ID NO:78) with higher affinity at pH7.4 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 antibodies of the invention are monoclonal antibodies. In some embodiments, the isolated anti-myostatin antibodies of the invention are human, humanized, or chimeric antibodies. In some embodiments, an isolated anti-myostatin antibody of the invention is an antibody fragment that binds myostatin. In some embodiments, an isolated anti-myostatin antibody of the invention is an antibody fragment that binds latent myostatin. In some embodiments, an isolated anti-myostatin antibody of the invention is an antibody fragment that binds to a polypeptide fragment consisting of amino acids 21-100 of a myostatin pro peptide (SEQ ID NO: 78). In some embodiments, an isolated anti-myostatin antibody of the invention is a full length IgG antibody.

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

(a) (i) HVR-H3, said HVR-H3 comprising the amino acid sequence GVPAX₁SX₂GGDX₃Wherein X is₁Is Y or H, X₂Is T or H, X₃Is L or K (SEQ ID NO:128), (ii) HVR-L3, said HVR-L3 comprising the amino acid sequence AGGYGGGX ₁YA, wherein X₁Is L or R (SEQ ID NO:131), and (iii) HVR-H2, said HVR-H2 comprising amino acid sequence IISX₁AGX₂X₃YX₄X₅X₆WAKX₇Wherein X is₁Is Y or H, X₂Is S or K, X₃Is T, M or K, X₄Is Y or K, X₅Is A, M or E, X₆Is S or E, X₇Is G or K (SEQ ID NO: 127);

(b) (i) HVR-H1, said HVR-H1 comprising amino acid sequence X₁X₂DIS, wherein X₁Is S or H, X₂Is Y, T, D or E (SEQ ID NO:126), (ii) HVR-H2, said HVR-H2 comprising the amino acid sequence IISX₁AGX₂X₃YX₄X₅X₆WAKX₇Wherein X is₁Is Y or H, X₂Is S or K, X₃Is T, M or K, X₄Is Y or K, X₅Is A, M or E, X₆Is S or E, X₇Is G or K (SEQ ID NO:127), and (iii) HVR-H3, said HVR-H3 comprising the amino acid sequence GVPAX₁SX₂GGDX₃Wherein X is₁Is Y or H, X₂Is T or H, X₃Is L or K (SEQ ID NO: 128);

(c) (i) HVR-H1, said HVR-H1 comprising amino acid sequence X₁X₂DIS, wherein X₁Is S or H, X₂Is Y, T, D or E (SEQ ID NO:126), (ii) HVR-H2, said HVR-H2 comprising the amino acid sequence IISX₁AGX₂X₃YX₄X₅X₆WAKX₇Wherein X is₁Is Y or H, X₂Is S or K, X₃Is T, M or K, X₄Is Y or K, X₅Is A, M or E, X₆Is S or E, X₇Is G or K (SEQ ID NO:127), (iii) HVR-H3, said HVR-H3 comprising the amino acid sequence GVPAX₁SX₂GGDX₃Wherein X is₁Is Y or H, X₂Is T or H, X₃Is L or K (SEQ ID NO:128), (iv) HVR-L1, said HVR-L1 comprising amino acid sequence X ₁X₂SQX₃VX₄X₅X₆NWLS, wherein X₁Is Q or T, X₂Is S or T, X₃Is S or E, X₄Is Y or F, X₅Is D or H, X₆Is N, D, A or E (SEQ ID NO: 129); (v) HVR-L2, the HVR-L2 comprising the amino acid sequence WAX₁TLAX₂Wherein X is₁Is S or E, X₂Is S, Y, F or W (SEQ ID NO: 130); and (vi) HVR-L3, said HVR-L3 comprising the amino acid sequence AGGYGGGX₁YA, wherein X₁Is L or R (SEQ ID NO: 131);

(d) (i) HVR-L1, said HVR-L1 comprising amino acid sequence X₁X₂SQX₃VX₄X₅X₆NWLS, wherein X₁Is Q or T, X₂Is S or T, X₃Is S or E, X₄Is Y or F, X₅Is D or H, X₆Is N, D, A or E (SEQ ID NO: 129); (ii) HVR-L2, the HVR-L2 comprising the amino acid sequence WAX₁TLAX₂Wherein X is₁Is S or E, X₂Is S, Y, F or W (SEQ ID NO: 130); and (iii) HVR-L3, said HVR-L3 comprising the amino acid sequence AGGYGGGX₁YA, wherein X₁Is L or R (SEQ ID NO: 131). In some embodiments, the antibody of (b) further comprises: the heavy chain variable domain framework FR1, wherein the FR1 comprises the amino acid sequence of any one of SEQ ID NO: 132-134; FR2, wherein the FR2 comprises the amino acid sequence of any one of SEQ ID NOs 135-136; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 137; and FR4, the FR4 comprising the amino acid sequence of SEQ ID NO: 138. In some embodiments, the antibody of (d) further comprises: light chain variable domain framework FR1, a process for its preparation FR1 contains the amino acid sequence of SEQ ID NO: 139; FR2, wherein the FR2 comprises the amino acid sequence of any one of SEQ ID NO: 140-141; FR3, wherein the FR3 comprises the amino acid sequence of any one of SEQ ID NO: 142-143; and FR4, the FR4 comprising the amino acid sequence of SEQ ID NO: 144.

In some embodiments, an isolated anti-myostatin antibody of the invention comprises (a) HVR-H3, said HVR-H3 comprising the amino acid sequence GVPAX₁SX₂GGDX₃Wherein X is₁Is Y or H, X₂Is T or H, X₃Is L or K (SEQ ID NO:128), (b) HVR-L3, said HVR-L3 comprising the amino acid sequence AGGYGGGX₁YA, wherein X₁Is L or R (SEQ ID NO:131), and (c) HVR-H2, said HVR-H2 comprising the amino acid sequence IISX₁AGX₂X₃YX₄X₅X₆WAKX₇Wherein X is₁Is Y or H, X₂Is S or K, X₃Is T, M or K, X₄Is Y or K, X₅Is A, M or E, X₆Is S or E, X₇Is G or K (SEQ ID NO: 127).

In some embodiments, an isolated anti-myostatin antibody of the invention comprises (a) HVR-H1, said HVR-H1 comprising amino acid sequence X₁X₂DIS, wherein X₁Is S or H, X₂Is Y, T, D or E (SEQ ID NO:126), (b) HVR-H2, said HVR-H2 comprising the amino acid sequence IISX₁AGX₂X₃YX₄X₅X₆WAKX₇Wherein X is₁Is Y or H, X₂Is S or K, X₃Is T, M or K, X₄Is Y or K, X ₅Is A, M or E, X₆Is S or E, X₇Is G or K (SEQ ID NO:127), and (c) HVR-H3, said HVR-H3 comprising the amino acid sequence GVPAX₁SX₂GGDX₃Wherein X is₁Is Y or H, X₂Is T or H, X₃Is L or K (SEQ ID NO: 128). In further embodiments, the antibody comprises the heavy chain variable domain framework FR1, the FR1 comprises the amino acid sequence of any one of SEQ ID NO: 132-134; FR2, wherein the FR2 comprises the amino acid sequence of any one of SEQ ID NOs 135-136; FR3, thereforFR3 contains an amino acid sequence of SEQ ID NO: 137; and FR4, the FR4 comprising the amino acid sequence of SEQ ID NO: 138. In additional embodiments, the antibody further comprises (a) HVR-L1, said HVR-L1 comprising amino acid sequence X₁X₂SQX₃VX₄X₅X₆NWLS, wherein X₁Is Q or T, X₂Is S or T, X₃Is S or E, X₄Is Y or F, X₅Is D or H, X₆Is N, D, A or E (SEQ ID NO: 129); (b) HVR-L2, the HVR-L2 comprising the amino acid sequence WAX₁TLAX₂Wherein X is₁Is S or E, X₂Is S, Y, F or W (SEQ ID NO: 130); and (c) HVR-L3, the HVR-L3 comprising the amino acid sequence AGGYGGGX₁YA, wherein X₁Is L or R (SEQ ID NO: 131).

In some embodiments, an isolated anti-myostatin antibody of the invention comprises (a) HVR-L1, said HVR-L1 comprising amino acid sequence X ₁X₂SQX₃VX₄X₅X₆NWLS, wherein X₁Is Q or T, X₂Is S or T, X₃Is S or E, X₄Is Y or F, X₅Is D or H, X₆Is N, D, A or E (SEQ ID NO: 129); (b) HVR-L2, the HVR-L2 comprising the amino acid sequence WAX₁TLAX₂Wherein X is₁Is S or E, X₂Is S, Y, F or W (SEQ ID NO: 130); and (c) HVR-L3, the HVR-L3 comprising the amino acid sequence AGGYGGGX₁YA, wherein X₁Is L or R (SEQ ID NO: 131). In another embodiment, the antibody further comprises: light chain variable domain framework FR1, the FR1 comprising the amino acid sequence of SEQ ID NO: 139; FR2, wherein the FR2 comprises the amino acid sequence of any one of SEQ ID NO: 140-141; FR3, wherein the FR3 comprises the amino acid sequence of any one of SEQ ID NO: 142-143; and FR4, the FR4 comprising the amino acid sequence of SEQ ID NO: 144.

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

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

The invention also provides isolated nucleic acids encoding the anti-myostatin antibodies of the invention. The invention also provides host cells comprising a nucleic acid of the invention. The invention also provides a method of producing the antibody, the method comprising culturing the host cell of the invention to produce the antibody.

In some aspects, the invention provides methods of making an anti-myostatin antibody, comprising: (a) culturing the host cell of the invention to thereby produce the antibody; or (b) immunizing the animal against a polypeptide, wherein the polypeptide comprises a region corresponding to amino acids at positions 21 to 100 of the myostatin pro peptide (SEQ ID NO: 78).

The invention also provides methods of making anti-myostatin antibodies. In some embodiments, the method comprises immunizing an animal against a polypeptide, wherein the polypeptide comprises a region corresponding to amino acids at positions 21-100 of the myostatin pro peptide (SEQ ID NO: 78).

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

The anti-myostatin antibodies of the invention can be used as a medicament. In some embodiments, the antibody is used in the preparation of a medicament for: (a) treating a muscle wasting disease; (b) increasing the mass of muscle tissue; (c) increasing the strength of the muscle tissue; or (d) reducing body fat accumulation. In some embodiments, the anti-myostatin antibodies of the invention can be used to treat muscle wasting diseases. The anti-myostatin antibodies of the invention can be used to increase the mass of muscle tissue. The anti-myostatin antibodies of the invention can be used to increase muscle tissue strength. The anti-myostatin antibodies of the invention can be used to reduce body fat accumulation.

In some embodiments, the anti-myostatin antibodies provided herein have utility in: (a) treating a muscle wasting disease; (b) increasing the mass of muscle tissue; (c) increasing the strength of the muscle tissue; or (d) reducing body fat accumulation.

The anti-myostatin antibodies of the invention can be used for the preparation of a medicament. In some embodiments, the antibody is used in the preparation of a medicament for: (a) treating a muscle wasting disease; (b) increasing muscle tissue mass; (c) increasing the strength of the muscle tissue; or (d) reducing body fat accumulation. In some embodiments, the medicament is for treating a muscle wasting disease. In some embodiments, the medicament is for increasing the mass of muscle tissue. In some embodiments, the medicament is for increasing the strength of muscle tissue. In some embodiments, the medicament is for reducing body fat accumulation.

The invention also provides methods of treating an individual having a muscle wasting disorder. In some embodiments, the method comprises administering to the individual an effective amount of an anti-myostatin antibody of the invention. The invention also provides a method of increasing the mass of muscle tissue in an individual. In some embodiments, the method comprises administering to the individual an effective amount of an anti-myostatin antibody of the invention to increase muscle tissue mass. The invention also provides a method of increasing the strength of muscle tissue in an individual. In some embodiments, the method comprises administering to the individual an effective amount of an anti-myostatin antibody of the invention to increase muscle tissue strength. The invention also provides methods of reducing body fat accumulation in an individual. In some embodiments, the method comprises administering to the individual an effective amount of an anti-myostatin antibody of the invention to reduce body fat accumulation.

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

In one embodiment, the invention provides Fc γ RIIB-binding polypeptides comprising a variant Fc region and methods of use thereof. In some embodiments, a variant Fc region of the invention having enhanced fcyriib-binding activity comprises at least one amino acid alteration in a parent Fc region. In further embodiments, the KD value of the [ parent Fc region for monkey Fc γ RIIb]/[ KD value for monkey Fc γ RIIb for variant Fc region]The ratio of (A) to (B) is 2.0 or more. In further embodiments, the KD value of the [ parent Fc region for monkey Fc γ RIIIa]/[ KD value for monkey Fc γ RIIIa for variant Fc region]The ratio of (A) to (B) is 0.5 or less. In further embodiments, the KD value of the [ parent Fc region for human Fc γ RIIb]/[ KD value for human Fc γ RIIb for variant Fc region]The ratio of (A) to (B) is 2.0 or more. In further embodiments, the KD value of the [ parent Fc region for human Fc γ RIIIa]/[ KD value for human Fc γ RIIIa for variant Fc region]The ratio of (A) to (B) is 0.5 or less. In further embodiments, the KD value of the [ parent Fc region for human Fc γ RIIa (type H)]/[ KD values for human Fc γ RIIa (type H) for the variant Fc region]The ratio of (A) to (B) is 5.0 or less. In further embodiments, the KD value of the [ parent Fc region for human Fc γ RIIa (R type) ]/[ KD value for human Fc γ RIIa (R-type) for variant Fc region]The ratio of (A) to (B) is 5.0 or less. In another embodiment, a variant FcThe KD value of the region for monkey Fc gamma RIIb is 1.0x10^-6M or less. In another embodiment, the variant Fc region has a KD value for monkey Fc γ RIIIa of 5.0x10^-7M or greater. In another embodiment, the variant Fc region has a KD value for human fcyriib of 2.0x10^-6M or less. In another embodiment, the variant Fc region has a KD value for human fcyriiia of 1.0x10^-6M or greater. In another embodiment, the variant Fc region has a KD value for human fcyriia (type H) of 1.0x10^-7M or greater. In another embodiment, the variant Fc region has a KD value for human fcyriia (type R) of 2.0x10^-7M or greater.

In some embodiments, the variant Fc region of the invention having enhanced fcyriib-binding activity comprises 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 additional embodiments, the variant Fc region having enhanced fcyriib-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 least one position selected from the group consisting of: (i) positions 231, 232, 233, 234, 235, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334, and 396; (ii) positions 231, 232, 235, 239, 268, 295, 298, 326, 330 and 396; or (iii) positions 268, 295, 326 and 330 (according to EU numbering).

In additional embodiments, the variant Fc region having enhanced fcyriib-binding activity comprises at least two amino acid alterations comprising: (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, 233, 234, 235, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334 and 396 (numbering according to EU).

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

In some embodiments, the variant Fc region of the invention having enhanced Fc γ 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; (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) a Phe at position 266; (l) Ala, His, Leu at position 267; (m) Asp, Glu at position 268; (n) Asp, Glu, Gly at position 271; (o) Leu at position 295; (p) Leu at position 298; (q) Glu, Phe, Ile, Leu at position 325; (r) Thr at position 326; (s) Ile, Asn at position 327; (t) Thr at position 328; (u) Lys, Arg at position 330; (v) glu at position 331; (w) Asp at position 332; (x) Asp, Ile, Met, Val, Tyr at position 334; and (y) Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 396 (numbering according to EU).

In further embodiments, the variant Fc region having enhanced Fc γ 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; (f) asp, Glu at position 268; (g) leu at position 295; (h) leu at position 298; (i) thr at position 326; (j) lys, Arg at position 330, and (k) Lys, Met at position 396 (according to EU numbering).

In another embodiment, the invention provides polypeptides comprising a variant Fc region having an increased isoelectric point (pI) and methods of use thereof. In some embodiments, a polypeptide comprising a variant Fc region having an increased pI comprises at least two amino acid changes in a parent Fc region. In further embodiments, each of the amino acid changes increases the isoelectric point (pI) of the variant Fc region as compared to the parent Fc region. In additional embodiments, the amino acid may be exposed on the surface of the variant Fc region. In further embodiments, the polypeptide comprises a variant Fc region and an antigen binding domain. In further embodiments, the antigen binding activity of the antigen binding domain varies depending on the ionic concentration conditions. In further embodiments, the variant Fc-region of the invention with increased pI comprises 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). In further embodiments, the variant Fc-region with increased pI comprises Arg or Lys at each position selected.

In some embodiments, a variant Fc region of the invention comprises an amino acid alteration described in tables 14-30.

In some embodiments, the polypeptide comprises a variant Fc region of the invention. In further embodiments, the parent Fc region is derived from human IgG 1. In further embodiments, the polypeptide is an antibody. In further embodiments, the polypeptide is an Fc fusion protein.

The present invention provides a polypeptide comprising the amino acid sequence of any one of SEQ ID NO 229-381.

The invention also provides isolated nucleic acids encoding polypeptides comprising the variant Fc regions of the invention. The invention also provides host cells comprising a nucleic acid of the invention. The invention also provides a method of producing a polypeptide comprising a variant Fc region, the method comprising culturing a host of the invention such that the polypeptide is produced.

The invention also provides a pharmaceutical formulation comprising a polypeptide comprising a variant Fc region of the invention and a pharmaceutically acceptable carrier.

Brief Description of Drawings

[ FIG. 1]

Figure 1 is a graph showing that anti-latent myostatin antibodies inhibit proteolytic activation of latent myostatin, as described in example 3. The activity of active myostatin released from latent myostatin by BMP1 protease was measured using the HEK Blue assay in the presence of anti-latent myostatin antibodies.

[ FIG. 2]

Figure 2 is a graph showing that anti-latent myostatin antibodies inhibit spontaneous activation of latent myostatin, as described in example 4. The activity of active myostatin released by incubation with latent myostatin at 37 ℃ was measured using the HEK Blue assay in the presence of anti-latent myostatin antibodies.

[ FIG. 3]

Figure 3 is a graphical representation of the binding of anti-latent myostatin antibodies to propeptide domains, as described in example 5.

[ FIG. 4]

FIG. 4 is a graphical representation of a Western blot analysis directed to myostatin pro peptide, as described in example 6. Proteolytic cleavage of myostatin pro peptide by BMP1 was measured in the presence and absence of anti-latent myostatin antibodies.

[ FIG. 5]

FIGS. 5A-5C are graphs showing BIACORE (registered trademark) sensorgrams of anti-latent myostatin antibody MST1032-G1m for human latent myostatin (A), cynomolgus monkey latent myostatin (B), and mouse latent myostatin (C), as described in example 7.

[ FIG. 6A ]

Figure 6A is a graph illustrating the in vivo efficacy of anti-latent myostatin and anti-mature myostatin antibodies on muscle mass and fat mass, as described in example 8. Anti-latent myostatin antibody (MST1032-G1 m; depicted in the figure as MST1032) or anti-mature myostatin antibody (41C1E4) was administered to SCID mice, and total lean body mass (or lean body mass: LBM) was measured.

[ FIG. 6B ]

Figure 6B is a graph illustrating the in vivo efficacy of anti-latent myostatin and anti-mature myostatin antibodies on muscle mass and fat mass, as described in example 8. Anti-latent myostatin antibody (MST1032-G1 m; depicted in the figure as MST1032) or anti-mature myostatin antibody (41C1E4) was administered to SCID mice, and changes in systemic body fat mass from day 0 to day 14 were measured.

[ FIG. 6C ]

Figure 6C is a graph illustrating the in vivo efficacy of anti-latent myostatin and anti-mature myostatin antibodies on muscle mass and fat mass, as described in example 8. Anti-latent myostatin antibody (MST1032-G1 m; depicted in the figure as MST1032) or anti-mature myostatin antibody (41C1E4) was administered to SCID mice, and gastrocnemius and quadriceps masses were measured.

[ FIG. 7A ]

FIG. 7A is a graph depicting a comparison of in vivo potency between several anti-myostatin antibodies, as described in example 9. Anti-latent myostatin antibody (MST1032-G1 m; depicted in the figure as MST1032) or anti-mature myostatin antibody (41C1E4, REGN, OGD, or MYO-029) was administered to SCID mice, and total lean body mass was measured.

[ FIG. 7B ]

Figure 7B is a graph showing a comparison of in vivo potency between several anti-myostatin antibodies, as described in example 9. Anti-latent myostatin antibody (MST1032-G1 m; depicted in the figure as MST1032) or anti-mature myostatin antibody (41C1E4, REGN, OGD, or MYO-029) was administered to SCID mice and grip strength was measured.

[ FIG. 7C ]

Figure 7C is a graph showing a comparison of in vivo potency between several anti-myostatin antibodies, as described in example 9. Anti-latent myostatin antibody (MST1032-G1 m; depicted in the figure as MST1032) or anti-mature myostatin antibody (41C1E4, REGN, OGD, or MYO-029) was administered to SCID mice, and changes in systemic body fat mass from day 0 to day 14 were measured.

[ FIG. 8]

Figure 8 is a graph depicting the inhibition of proteolysis and spontaneous activation of latent myostatin by a humanized anti-latent myostatin antibody, as described in example 10. The activity of active myostatin released by latent myostatin was measured using the HEK Blue assay in the presence of anti-latent myostatin antibodies, either by BMP1 protease (proteolysis) or by incubation at 37 ℃ in the absence of BMP1 (spontaneous).

[ FIG. 9]

FIG. 9 is a graphical representation of the BIACORE (registered trademark) sensorgram for histidine-substituted variants of the anti-latent myostatin antibody, as described in example 11. The antibody/antigen complex was allowed to dissociate at pH7.4, followed by further dissociation (indicated by arrows) at pH5.8 to assess pH-dependent interactions. The antibodies tested in this experiment were: ab001 (black solid curve), Ab002 (black short dashed curve), Ab003 (black dot curve), Ab004 (gray short dashed curve), Ab005 (gray solid curve), Ab006 (gray long dashed curve), and Ab007 (black long dashed curve).

[ FIG. 10]

Figure 10 is a graph showing that pH-dependent anti-latent myostatin antibody inhibits proteolysis and spontaneous activation of latent myostatin, as described in example 13. The activity of active myostatin released by latent myostatin was measured using the HEK Blue assay in the presence of anti-latent myostatin antibodies, either by BMP1 protease (proteolysis) or by incubation at 37 ℃ in the absence of BMP1 (spontaneous). Antibodies MS1032LO01-SG1, MS1032LO02-SG1, MS1032LO03-SG1, and MS1032LO04-SG1 are depicted in the figures as MSLO-01, MSLO-02, MSLO-03, and MSLO-04, respectively. MS1032LO01-SG1, MS1032LO02-SG1, MS1032LO03-SG1, and MS1032LO04-SG1 achieved comparable inhibition of proteolysis and spontaneous activation of latent myostatin to MS1032LO00-SG 1.

[ FIG. 11]

FIGS. 11A-11F are graphs showing BIACORE (registered trademark) profiles of pH-dependent anti-latent myostatin antibodies, as described in example 14. Kinetic parameters of MST1032-SG1(A), MS1032LO00-SG1(B), MS1032LO01-SG1(C), MS1032LO02-SG1(D), MS1032LO03-SG1(E), and MS1032LO04-SG1(F) were measured at neutral and acidic pH.

[ FIG. 12]

Figure 12 is a graphical representation of the time course of plasma myostatin concentration following intravenous administration of anti-myostatin antibodies in mice, as described in example 15. The effect of Fc γ R-mediated cellular uptake of the antibody/antigen complex on muscle growth inhibitor clearance in vivo was assessed by comparing anti-muscle growth inhibitor antibodies with Fc γ R binding (MS1032LO00-SG1) and with disrupted Fc γ R binding (MS1032LO 00-F760).

[ FIG. 13]

Figure 13 is a graphical representation of the time course of plasma myostatin concentration following intravenous administration of anti-myostatin antibodies in mice, as described in example 16. The effect of pH-dependent binding of anti-myostatin antibodies on myostatin clearance in vivo was assessed by comparing pH-dependent anti-myostatin antibodies (MS1032LO01-SG1 or MS1032LO01-F760) and pH-independent anti-myostatin antibodies (MS1032LO00-SG1 or MS1032LO 00-F760).

[ FIG. 14]

Figures 14A-14E are graphs illustrating the in vivo efficacy of pH-dependent and pH-independent anti-latent myostatin antibodies, as described in example 17. A pH-dependent anti-latent myostatin antibody (MS1032LO01-SG 1; depicted in the figure as MSLO1) or a non-pH-dependent anti-latent myostatin antibody (MS1032LO00-SG 1; depicted in the figure as MSLO0) was administered to SCID mice, and the total lean body mass (a), total body fat mass (B), quadriceps mass (C), gastrocnemius mass (D), and grip strength (E) were measured.

[ FIG. 15]

Figure 15 is a graph depicting the binding activity of anti-latent myostatin antibody MST1032 with latent myostatin and GDF11, as described in example 19.

[ FIG. 16]

Figure 16 illustrates the inhibitory activity of anti-latent myostatin antibody MST1032 against proteolysis and spontaneous activation of GDF11, as described in example 20. The activity of the released active GDF11 was measured using the HEK Blue assay in the presence of anti-latent myostatin antibodies, either by BMP1 protease (proteolysis) or by incubation at 37 ℃ in the absence of BMP1 (spontaneous).

[ FIG. 17]

Figure 14 is a graph showing that anti-latent myostatin antibodies inhibit proteolytic activation of latent myostatin, as described in example 22. The activity of active myostatin released from latent myostatin by BMP1 protease was measured using the HEK Blue assay in the presence of anti-latent myostatin antibodies.

[ FIG. 18]

Figure 18 illustrates the time course of plasma myostatin concentration following intravenous administration of anti-latent myostatin antibody in mice, as described in example 23. The effect of pH-dependence on in vivo muscle growth inhibitor clearance was assessed by comparing a pH-independent anti-latent muscle growth inhibitor antibody (MS1032LO00-SG1) with a different pH-dependent anti-latent muscle growth inhibitor antibody (MS1032LO01-SG1, MS1032LO06-SG1, MS1032LO11-SG1, MS1032LO18-SG1, MS1032LO19-SG1, MS1032LO21-SG1 and MS1032LO25-SG 1).

[ FIG. 19]

Figures 19A and 19B illustrate the time course of plasma myostatin concentration following intravenous administration of anti-latent myostatin antibody in cynomolgus monkeys, as described in example 24. (A) The effects of pH-dependence and Fc engineering on the clearance of myostatin in vivo were assessed by comparing pH-independent anti-latent myostatin antibodies (MS1032LO00-SG1) and pH-dependent anti-latent myostatin antibodies with Fc engineering (MS1032LO06-SG1012, MS1032LO06-SG1016, MS1032LO06-SG1029, MS1032LO06-SG1031, MS1032LO06-SG1033, MS1032LO06-SG 1034). (B) The effect of Fc engineering on myostatin clearance in vivo was assessed by comparing anti-latent myostatin antibodies (MS1032LO19-SG1079, MS1032LO19-SG1071, MS1032LO19-SG1080, MS1032LO19-SG1074, MS1032LO19-SG1081, and MS1032LO19-SG 1077).

[ FIG. 20A ]

Figure 20A, together with figures 20B-20I, illustrates the in vivo efficacy of anti-latent myostatin antibody (MS1032 variant) on Lean Body Mass (LBM), grip strength, and body fat mass, as described in example 25. MS1032LO06-SG1, MS1032LO11-SG1 and MS1032LO18-SG1 were administered to Scid mice, and lean body mass (A) was measured.

[ FIG. 20B ]

Figure 20B, together with figures 20A, 20C-20I, illustrates in vivo efficacy of anti-latent myostatin antibody (MS1032 variant) on Lean Body Mass (LBM), grip strength, and body fat mass, as described in example 25. MS1032LO06-SG1, MS1032LO19-SG1 and MS1032LO25-SG1 were administered to Scid mice, and lean body mass was measured (B).

[ FIG. 20C ]

Figure 20C, together with figures 20A-B, 20D-I, illustrates in vivo efficacy of anti-latent myostatin antibody (MS1032 variant) on Lean Body Mass (LBM), grip strength, and body fat mass, as described in example 25. MS1032LO01-SG, MS1032LO06-SG1 and MS1032LO11-SG1 were administered to Scid mice and lean body mass (C) was measured.

[ FIG. 20D ]

Figure 20D, together with figures 20A-C, 20E-I, illustrates in vivo efficacy of anti-latent myostatin antibody (MS1032 variant) on Lean Body Mass (LBM), grip strength, and body fat mass, as described in example 25. MS1032LO06-SG1, MS1032LO11-SG1 and MS1032LO18-SG1 were administered to Scid mice, and the grip strength (D) was measured.

[ FIG. 20E ]

Figure 20E, along with figures 20A-D, 20F-I, illustrates in vivo efficacy of anti-latent myostatin antibody (MS1032 variant) on Lean Body Mass (LBM), grip strength, and body fat mass, as described in example 25. MS1032LO06-SG1, MS1032LO19-SG1 and MS1032LO25-SG1 were administered to Scid mice, and the grip strength (E) was measured.

[ FIG. 20F ]

Figure 20F, along with figures 20A-E, 20G-I, illustrates in vivo efficacy of anti-latent myostatin antibody (MS1032 variant) on Lean Body Mass (LBM), grip strength, and body fat mass, as described in example 25. MS1032LO01-SG, MS1032LO06-SG1 and MS1032LO11-SG1 were administered to Scid mice, and the grip strength (F) was measured.

[ FIG. 20G ]

Figure 20G, together with figures 20A-F, 20H-I, graphically illustrate in vivo efficacy of anti-latent myostatin antibody (MS1032 variant) on Lean Body Mass (LBM), grip strength, and body fat mass as described in example 25. MS1032LO06-SG1, MS1032LO11-SG1 and MS1032LO18-SG1 were administered to Scid mice, and body fat mass (G) was measured.

[ FIG. 20H ]

Figure 20H, together with figures 20A-G, 20I, illustrates in vivo efficacy of anti-latent myostatin antibodies (MS1032 variant) on Lean Body Mass (LBM), grip strength, and body fat mass, as described in example 25. MS1032LO06-SG1, MS1032LO19-SG1 and MS1032LO25-SG1 were administered to Scid mice, and body fat mass (H) was measured.

[ FIG. 20I ]

Figure 20I, together with figures 20A-H, illustrates in vivo efficacy of anti-latent myostatin antibodies (MS1032 variant) on Lean Body Mass (LBM), grip strength, and body fat mass, as described in example 25. MS1032LO01-SG, MS1032LO06-SG1 and MS1032LO11-SG1 were administered to Scid mice and body fat mass (I) was measured.

[ FIG. 21]

Figure 21 is a graph showing the inhibitory activity of anti-latent myostatin antibodies on latent myostatin activation, as described in example 26. The amount of mature myostatin released by latent myostatin by BMP1 protease was measured in the presence of anti-latent myostatin antibodies (MST1032, MST1504, MST1538, MST1551, MST1558, MST1572, and MST 1573).

[ FIG. 22A ]

Figure 22A illustrates a schematic of latent myostatin fragments of 100 amino acids each designed for epitope mapping of anti-latent myostatin antibodies, as described in example 26.

[ FIG. 22B ]

FIG. 22B illustrates a Western blot analysis by anti-GST antibody against GST-tagged human latent myostatin fragment (GST-hMSTN), as described in example 26. Each lane indicates: 1, GST-hMSTN 1-100 aa; 2, GST-hMSTN 21-120 aa; 3, GST-hMSTN 41-140 aa; 4, GST-hMSTN 61-160 aa; 5, GST-hMSTN 81-180 aa; 6, GST-hMSTN 101-200 aa; 7, GST-hMSTN 121-220 aa; 8, GST-hMSTN 141-241 aa; 9, GST control.

[ FIG. 22C ]

Figure 22C illustrates western blot analysis for GST-tagged human latent myostatin fragment (GST-hMSTN) by anti-latent myostatin antibodies (MST1032, MST1538, MST1572, and MST1573) as described in example 26. Each lane indicates: 1, GST-hMSTN 1-100 aa; 2, GST-hMSTN 21-120 aa; 3, GST-hMSTN 41-140 aa; 4, GST-hMSTN 61-160 aa; 5, GST-hMSTN 81-180 aa; 6, GST-hMSTN 101-200 aa; 7, GST-hMSTN 121-220 aa; 8, GST-hMSTN 141-241 aa; 9, GST control; human latent myostatin (100 ng).

[ FIG. 22D ]

Figure 22D illustrates a summary result of western blot analysis and putative epitope positions of anti-latent myostatin antibodies (MST1032, MST1538, MST1572, and MST1573) as described in example 26.

[ FIG. 23]

Figure 23 illustrates an alignment of amino acid sequences of cynomolgus monkey (cyno) Fc γ RIIa1, Fc γ RIIa2, Fc γ RIIa3, Fc γ RIIb, human Fc γ RIIaH, Fc γ RIIaR, and Fc γ RIIb. Boxed regions indicate putative residues that interact with the Fc domain.

[ FIG. 24]

Figure 24 illustrates the time course of total myostatin concentration in plasma following intravenous administration of anti-myostatin antibodies with Fc variants having enhanced Fc γ RIIb in fully human Fc γ R transgenic mice, as described in example 28. Fc variants with enhanced Fc γ RIIb were evaluated for their effect on antigen elimination by human Fc γ RIIb.

[ FIG. 25]

Figure 25 illustrates the time course of antibody concentration in plasma after intravenous administration of anti-myostatin antibodies with Fc variants having enhanced Fc γ RIIb in fully human Fc γ R transgenic mice, as described in example 28. The effect of Fc γ RIIb-enhanced Fc variants on antibody pharmacokinetics was evaluated.

[ FIG. 26]

Figures 26A and 26B illustrate the time course of plasma myostatin concentration following intravenous administration of anti-latent myostatin antibody in cynomolgus monkeys, as described in example 29. (A) The effects of pH-dependence and Fc engineering on the clearance of myostatin in vivo were assessed by comparing pH-independent anti-latent myostatin antibodies (MS1032LO00-SG1) and pH-dependent anti-latent myostatin antibodies with Fc engineering (MS1032LO06-SG1012, MS1032LO06-SG1016, MS1032LO06-SG1029, MS1032LO06-SG1031, MS1032LO06-SG1033, MS1032LO06-SG 1034). (B) The effect of Fc engineering on myostatin clearance in vivo was assessed by comparing anti-latent myostatin antibodies (MS1032LO19-SG1079, MS1032LO19-SG1071, MS1032LO19-SG1080, MS1032LO19-SG1074, MS1032LO19-SG1081, and MS1032LO19-SG 1077).

[ FIG. 27A ]

Figure 27A illustrates the time course of total myostatin concentration in plasma after intravenous administration of an anti-myostatin antibody with an Fc variant having an increased pI in human FcRn transgenic mice, as described in example 30. The effect of the pI-enhanced Fc variants on antigen elimination was evaluated.

[ FIG. 27B ]

Figure 27B illustrates the time course of antibody concentration in plasma after intravenous administration of anti-myostatin antibodies with Fc variants with increased pI in human FcRn transgenic mice, as described in example 30. The effect of the Fc variants with increased pI on antibody pharmacokinetics was evaluated.

[ FIG. 28A ]

Figure 28A illustrates the time course of total myostatin concentration in plasma after intravenous administration of an anti-myostatin antibody with an Fc variant having an increased pI in human FcRn transgenic mice, as described in example 30. The effect of the pI-enhanced Fc variants on antigen elimination was evaluated. In this assay, an excess of human normal immunoglobulin was co-administered with an anti-myostatin antibody to mimic the situation of human plasma.

[ FIG. 28B ]

Figure 28B illustrates the time course of antibody concentration in plasma after intravenous administration of anti-myostatin antibodies with Fc variants with increased pI in human FcRn transgenic mice, as described in example 30. The effect of the Fc variants with increased pI on antibody pharmacokinetics was evaluated. In this assay, an excess of human normal immunoglobulin was co-administered with an anti-myostatin antibody to mimic the situation of human plasma.

[ FIG. 29]

Figure 29 illustrates the time course of total myostatin concentration in plasma following intravenous administration of anti-myostatin antibodies with Fc variants with Fc γ RIIb enhancement in human Fc γ RIIb transgenic mice, as described in example 31. The effect of Fc γ RIIb-enhanced Fc variants on antigen elimination by human Fc γ RIIb was evaluated.

[ FIG. 30]

Figure 30 illustrates the time course of antibody concentration in plasma following intravenous administration of anti-myostatin antibodies with Fc γ RIIb enhanced Fc variants in human Fc γ RIIb transgenic mice, as described in example 31. The effect of Fc γ RIIb-enhanced Fc variants on antibody pharmacokinetics was evaluated.

[ FIG. 31]

Figure 31 is a graphical representation of the results of cellular imaging analysis of anti-myostatin antibodies with Fc variants having enhanced Fc γ RIIb, as described in example 33. Each antibody was complexed with fluorescently labeled myostatin and intracellular uptake of the antigen-antibody complex into cells expressing human Fc γ RIIb was measured.

Detailed description of the preferred embodiments

The techniques and methods described or referenced herein are generally well understood and routinely used by those skilled in the art using conventional methodologies, such as, for example, the widely used method described in Sambrook et al, Molecular Cloning, A Laboratory Manual 3 rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; current Protocols in Molecular Biology (F.M. Ausubel et al, eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, organism Manual, and Animal Cell Culture (R.I. Freeshine, ed. (1987)); oligonucleotide Synthesis (m.j. gate, ed., 1984); methods in Molecular Biology, human Press; cell Biology A Laboratory Notebook (J.E.Cellis, ed.,1998) Academic Press; animal Cell Culture (r.i. freshney), ed., 1987); introduction to Cell and Tissue Culture (J.P.Mather and P.E.Roberts,1998) Plenum Press; cell and Tissue Culture Laboratory Procedures (A.Doyle, J.B.Griffiths, and D.G.Newell, eds.,1993-8) J.Wiley and Sons; handbook of Experimental Immunology (d.m.weir and c.c.blackwell, eds.); gene Transfer Vectors for Mammalian Cells (j.m.miller and m.p.calos, eds., 1987); PCR The Polymerase Chain Reaction, (Mullis et al, eds., 1994); current Protocols in Immunology (J.E.Coligan et al, eds., 1991); short Protocols in Molecular Biology (Wiley & Sons, 1999); immunobiology (c.a. janeway and p.travers, 1997); antibodies (p.finch, 1997); antibodies A Practical Approach (D.Catty., ed., IRL Press, 1988-; monoclonal Antibodies A Practical Approach (P.shepherd and C.dean, eds., Oxford University Press, 2000); use Antibodies, available Manual (E.Harlow and D.Lane, Cold Spring Harbor Laboratory Press, 1999); the Antibodies (m.zanetti and j.d.capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T.Devita et al, eds., J.B.Lippincott Company, 1993).

I. Definition of

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

For purposes of interpreting this application, the following definitions will apply and where appropriate, terms used in the singular will also include the plural and vice versa. It is to be understood that the technology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. In the event that any of the definitions set forth below conflict with any document incorporated herein by reference, the definitions set forth below control.

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

"affinity" refers to the sum of the forces of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

An "affinity matured" antibody is one that has one or more alterations in one or more hypervariable regions (HVRs) as compared to a parent antibody not having such alterations that result in an increase in the affinity of the antibody for an antigen.

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

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

An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that binds to an 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(ii) a A diabody; a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

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

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

"Classification" of an antibody refers to the type of constant domain or constant region that the heavy chain has. There are mainly five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these may be further divided into subclasses (allotypes), e.g., IgG₁，IgG₂，IgG₃，IgG₄，IgA₁And IgA, and₂. The heavy chain constant domains corresponding to different types of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At)²¹¹，I¹³¹，I¹²⁵，Y⁹⁰，Re¹⁸⁶，Re¹⁸⁸，Sm¹⁵³，Bi²¹²，P³²，Pb²¹²And radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate), doxorubicin (adriamycin), vinca alkaloid (vinca alkaloid) (vincristine), vinblastine (vinblastine), etoposide (etoposide)), doxorubicin (doxorubicin), melphalan (melphalan), mitomycin c (mitomycin c), chlorambucil (chlorembucil), daunorubicin (daunorubicin), or other chimeric agents); a growth inhibitor; enzymes and fragments thereof such as nucleic acid hydrolases; (ii) an antibiotic; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various antitumor agents or anticancer agents disclosed below.

"Effector function" refers to those biological activities attributable to the Fc region of an antibody that vary with antibody allotype. Examples of antibody effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

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

The term "epitope" includes any determinant capable of being bound by an antibody. An epitope is the region of an antigen that is bound by an antibody targeted to the antigen and includes specific amino acids in direct contact with the antibody. Epitopic determinants may include chemically active surface clusters of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and may have specific three-dimensional structural characteristics, and/or specific charge characteristics. Generally, an antibody specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and/or macromolecules.

"Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR is one that binds an IgG antibody (gamma receptor) and includes receptors of the Fc γ RI, Fc γ RII, and Fc γ RIII subclasses, including allelic variants and alternatively spliced forms of the receptors. Fc γ RII receptors include Fc γ RIIA ("activating receptor") and Fc γ RIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor Fc γ RIIA comprises in its cytoplasmic domain an immunoreceptor tyrosine-based activation motif (ITAM). The inhibitory receptor Fc γ RIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (see, e.g., Daeron, Annu. Rev. Immunol.15:203-234 (1997)) FcRs are reviewed, e.g., in Ravetch and Kinet, Annu. Rev. Immunol.9:457-92 (1991); capel et al, immunolmethods 4:25-34 (1994); and de Haas et al, J.Lab.Clin.Med.126:330-41 (1995). The term "FcR" herein encompasses other fcrs, including those determined in the future.

The term "Fc receptor" or "FcR" also includes the neonatal receptor, FcRn, which is responsible for transfer of maternal IgG to the fetus (Guyer et al, J.Immunol.117:587(1976) and Kim et al, J.Immunol.24:249(1994)) and regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and ward, immunol. today 18(12):592-598 (1997); Ghetie et al, Nature Biotechnology 15(7):637-640 (1997); Hinton et al, j. biol. chem.279(8):6213-6216 (2004); WO 2004/92219(Hinton et al); in vivo binding to human FcRn and serum half-life of human FcRn high affinity binding polypeptides can be determined, e.g., in transgenic mice or 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 binding to FcR see, e.g., shield et al, j. biol. chem.9(2):6591-6604 (6604)).

Herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a 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 carboxy-terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (residue 446-447) of the Fc region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also referred to as the EU index, as defined in Kabat et al, Sequences of Proteins of Immunological Interest,5th Ed.

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

"framework" or "FR" refers to variable domain residues that are different from the hypervariable region (HVR) residues. The FRs of a variable domain typically consist of four FR domains: FR1, FR2, FR3 and FR 4. Thus, in VH (or VL) the HVR and FR sequences typically occur in the following order: FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.

The terms "full length antibody", "intact antibody" and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain containing an Fc region as defined herein.

The "functional Fc region" has the "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors; BCR), and the like. Such effector functions typically require an Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using, for example, the various assays disclosed in the definitions section herein.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to a cell into which an exogenous nucleic acid is introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include transformed primary cells and progeny derived therefrom (regardless of the number of passages). The nucleic acid content of the progeny may not be identical to the parent cell and may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell.

A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from an antibody of non-human origin using a human antibody repertoire or other human antibody coding sequences. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues.

A "human consensus framework" is a framework that represents the most common amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. Typically, the subset of Sequences is a subset as in Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, NIH publication 91-3242, Bethesda MD (1991), volumes 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I as in Kabat et al, supra. In one embodiment, for the VH, the subgroup is subgroup III as in Kabat et al above.

A "humanized" antibody is a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one (and typically, two) variable domain, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to HVRs of a non-human antibody, and all or substantially all of the FRs correspond to FRs of a human antibody. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. "humanized forms" of antibodies, e.g., non-human antibodies, refer to antibodies that have been humanized.

The term "hypervariable region" or "HVR" as used herein refers to regions of an antibody variable domain which are hypervariable in sequence ("complementarity determining regions" or "CDRs") and/or form structurally defined loops ("hypervariable loops") and/or contain residues which contact antigen ("antigen contacts"). Typically, an antibody comprises six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include: (a) the hypervariable loops which occur at amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53-55(H2), and 96-101(H3) (Chothia and Lesk, J.mol.biol.196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34(L1), 50-56(L2), 89-97(L3), 31-35b (H1), 50-65(H2), and 95-102(H3) (Kabat et al, Sequences of Proteins of Immunological Interest,5th Ed. public Health Service, NIH, Bethesda, MD (1991)); (c) antigen contacts occurring at amino acid residues 27c-36(L1), 46-55(L2), 89-96(L3), 30-35b (H1), 47-58(H2), and 93-101(H3) (MacCallum et al, J.mol.biol.262:732-745 (1996)); and (d) combinations of (a), (b), and/or (c) comprising HVR amino acid residues 46-56(L2), 47-56(L2), 48-56(L2), 49-56(L2), 26-35(H1), 26-35b (H1), 49-65(H2), 93-102(H3), and 94-102 (H3).

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

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

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

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

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

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

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

By "naked antibody" is meant an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. Naked antibodies may be present in pharmaceutical formulations.

"native antibody" refers to a naturally occurring immunoglobulin molecule having a variety of structures. For example, a native IgG antibody is a heterologous tetraglycan protein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains bonded by disulfide bonds. From N-terminus to C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH 3). Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), also referred to as a variable light chain domain or light chain variable domain, followed by a light chain Constant (CL) domain. The light chain of an antibody can be assigned to one of two types, called κ (κ) and λ (λ), based on the amino acid sequence of its constant domain.

A "native sequence Fc region" comprises an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1Fc regions (non A and A allotypes); a native sequence human IgG2Fc region; native sequence human IgG3Fc region; and the native sequence human IgG4Fc region and naturally occurring variants thereof.

The term "package insert" is used to refer to instructions for use, typically contained in commercial packaging for a therapeutic product, that contain an indication, use, dosage, administration, combination therapy, contraindications and/or warnings of use for such therapeutic product.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in the reference polypeptide sequence after the sequences are aligned and gaps (gaps) introduced, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be achieved in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, megalign (dnastar) software or GENETYX (registered trademark) (GENETYX co., Ltd.). One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared. The author of the ALIGN-2 sequence comparison computer program was Genentech, inc, and the source code has been submitted with the user file to the us copyright office, Washington d.c.,20559, which is registered with us copyright registration number TXU 510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or the program may be compiled from source code. The ALIGN-2 program should be compiled for use with a UNIX operating system, including the digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and need not be changed.

In the case where ALIGN-2 is used for amino acid sequence comparison, the% amino acid sequence identity of a given amino acid sequence a with or relative to a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence a with or comprising a particular% amino acid sequence identity with or relative to a given amino acid sequence B) is calculated as follows: 100 times a fraction X/Y; wherein X is the number of amino acid residues scored as an identical match by sequence alignment program ALIGN-2 in the program alignment of A and B, and Y is the total number of amino acid residues in B. It is understood that when the length of amino acid sequence a is not equal to the length of amino acid sequence B, the% amino acid sequence identity of a to B will not be equal to the% amino acid sequence identity of B to a. Unless otherwise specifically indicated, all% amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

The term "pharmaceutical formulation" refers to a formulation having a form that allows the biological activity of the active ingredient contained therein to be effective, and which is free of other components having unacceptable toxicity to the subject to which the formulation is to be administered.

By "pharmaceutically acceptable carrier" is meant an ingredient of a pharmaceutical formulation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

The term "myostatin" as used herein can refer to any natural myostatin from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). Unless otherwise indicated, the term "myostatin" refers to a human myostatin protein having the amino acid sequence shown in SEQ ID NO 1 and containing the terminal propeptide domain of human myostatin shown in SEQ ID NO 75 or 78. The term encompasses "full-length" unprocessed myostatin as well as any form of myostatin that results from processing in a cell. The term also encompasses naturally occurring variants of myostatin, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human myostatin (prepromagnesia) is shown in SEQ ID NO 1. The amino acid sequence of an exemplary C-terminal growth factor domain of human myostatin is shown in SEQ ID NO 2. The amino acid sequence of an exemplary N-terminal propeptide domain of human myostatin is shown in SEQ ID NO 75 or 78. Active mature myostatin is a disulfide-bonded homodimer consisting of two C-terminal growth factor domains. Inactive latent myostatin is a non-covalently bound complex of two pro peptides and mature myostatin. As disclosed herein, the antibodies of the invention bind inactive latent myostatin, but do not bind mature active myostatin homodimer. In some embodiments, the antibodies of the invention bind to an epitope within a fragment consisting of amino acids 21-100 of the myostatin pro peptide (SEQ ID NO:78), but do not bind to mature active myostatin homodimer. The amino acid sequences of exemplary cynomolgus monkey and murine myostatin (pro myostatin) are shown in SEQ ID NOS: 3 and 5, respectively. The amino acid sequences of exemplary C-terminal growth factor domains of cynomolgus monkey and murine myostatin are shown in SEQ ID NOS 4 and 6, respectively. The amino acid sequences of exemplary N-terminal propeptide domains of cynomolgus monkey and murine myostatin are shown in SEQ ID 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- β superfamily. Similar to myostatin, GDF11 is first synthesized as a precursor polypeptide and then cleaved into an N-terminal prodomain and a C-terminal mature GDF 11. The amino acid sequence of human GDF11 (precursor) is shown in SEQ ID NO: 81. The amino acid sequence of C-terminal mature human GDF11 is shown in SEQ ID NO: 82. The amino acid sequence of the N-terminal prodomain of human GDF11 is shown in SEQ ID NO 83 or 84. The amino acid sequences of SEQ ID NOS 1, 3, 5, 78, 79, 80, 81 and 84 include signal sequences. Amino acids 1 to 24 thereof correspond to the signal sequence and are removed during processing in the cell.

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

The term "variable region" or "variable domain" refers to a domain of an antibody heavy or light chain that is involved in antigen binding of the antibody. The heavy and light chain variable domains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved Framework Regions (FR) and three hypervariable regions (HVRs). (see, e.g., Kindt et al, Kuby Immunology,6^th ed.,W.H.Freeman&Co, page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. Furthermore, antibodies that bind a particular antigen can be isolated using screening libraries of complementary VL or VH domains, respectively, from antibodies that bind the antigen. See, e.g., Portolano et al, J.Immunol.150: 880-; clarkson et al, Nature 352: 624-.

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

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors which are self-replicating nucleic acid structures as well as vectors which are incorporated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operatively linked. Such vectors are referred to herein as "expression vectors".

Compositions and methods

In one aspect, the invention is based, in part, on anti-myostatin antibodies and uses thereof. In certain embodiments, antibodies that bind myostatin are provided. The antibodies of the invention are useful, for example, in the diagnosis or treatment of muscle wasting diseases.

In another aspect, the invention is based, in part, on polypeptides comprising a variant Fc region and uses thereof. In one embodiment, a polypeptide comprising a variant Fc region having enhanced Fc γ RIIb-binding activity is provided. In another embodiment, a polypeptide comprising a variant Fc region having an increased pI is provided. In particular embodiments, the polypeptide of the invention is an antibody. The polypeptides of the invention comprising a variant Fc region are useful, for example, in the diagnosis or treatment of disease.

A. Exemplary anti-myostatin antibodies and polypeptides comprising variant Fc regions

In one aspect, the invention provides an isolated antibody that binds myostatin. In certain embodiments, an anti-myostatin antibody of the invention binds latent myostatin. In additional embodiments, an anti-myostatin antibody of the invention binds to a myostatin pro peptide (human: SEQ ID NO:75 or 78; cynomolgus monkey: SEQ ID NO:76 or 79; mouse: SEQ ID NO:77 or 80). In a further embodiment, the antibody binds to an epitope within a fragment consisting of amino acids 21-100 of the myostatin pro peptide (SEQ ID NO: 78). As described above, the propeptide is included as one of the components in the latent myostatin. In certain embodiments, an anti-myostatin antibody of the invention inhibits myostatin activation. In certain embodiments, the anti-myostatin antibody blocks release of mature myostatin from latent myostatin. Mature myostatin is reportedly released via proteolytic and non-proteolytic processes from latent myostatin. The anti-myostatin antibodies of the invention can block proteolytic and/or non-proteolytic release of mature myostatin from latent myostatin. In certain embodiments, the anti-myostatin antibody blocks proteolytic cleavage of latent myostatin. In certain embodiments, the anti-myostatin antibody blocks access of proteases to latent myostatin (particularly, to the proteolytic cleavage site of latent myostatin (Arg98-Asp 99)). In another embodiment, the protease may be a BMP1/TLD family metalloprotease such as BMP1, TED, tolloid-like protein-1 (TLL-1) or tolloid-like protein-2 (TLL-2). In another embodiment, the anti-myostatin antibody blocks non-proteolytic release of mature myostatin from latent myostatin. Non-proteolytic release as used herein 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, release of mature myostatin by incubating latent myostatin, e.g., at 37 ℃, in the absence of a protease that cleaves latent myostatin. In certain embodiments, the anti-myostatin antibodies of the invention do not bind mature myostatin. In some embodiments, the anti-myostatin antibody binds to the same epitope as an antibody described in table 2 a. In some embodiments, the anti-myostatin antibody competes for binding to latent myostatin with an antibody described in table 2 a. In additional embodiments, the anti-myostatin antibody competes for binding to latent myostatin with an antibody comprising a VH and VL pair described in table 2 a. In some embodiments, the anti-myostatin antibody competes with an antibody described in Table 2a for binding to a fragment consisting of amino acids 21-100 of the myostatin pro peptide (SEQ ID NO: 78). In further embodiments, the anti-myostatin antibody binds to the same epitope as an antibody described in table 11a or 13. In some embodiments, the anti-myostatin antibody competes for binding to latent myostatin with an antibody described in table 11a or 13. In some embodiments, the anti-myostatin antibody competes with an antibody described in Table 11a or 13 for binding to a fragment consisting of amino acids 21-100 of the myostatin pro peptide (SEQ ID NO: 78).

In some embodiments, an anti-myostatin antibody of the invention binds to latent myostatin and inhibits activation of myostatin. In further embodiments, the antibody: (a) blocking release of mature myostatin from latent myostatin; (b) blocking proteolytic release of mature muscle growth inhibitory factor; (c) blocking the spontaneous release of mature muscle growth inhibitory factor; or (d) does not bind mature myostatin; or binds to an epitope within a fragment consisting of amino acids 21-100 of the myostatin pro peptide (SEQ ID NO: 78). In further embodiments, the antibody competes for binding to latent myostatin with an antibody comprising a VH and VL pair described in table 2a, 11a, or 13, or binds to the same epitope as an antibody comprising a VH and VL pair described in table 2a, 11a, or 13. In further embodiments, the antibody binds latent myostatin with a higher affinity at neutral pH (e.g., pH7.4) than at acidic pH (e.g., pH 5.8). In additional embodiments, the antibody is (a) a monoclonal antibody, (b) a human, humanized, or chimeric antibody; (c) a full length IgG antibody or (d) an antibody fragment that binds to latent myostatin or myostatin pro peptide.

In another embodiment, the anti-myostatin antibodies of the invention do not bind GDF 11. In certain embodiments, the anti-myostatin antibodies of the invention do not inhibit activation of GDF 11. In certain embodiments, the anti-myostatin antibody does not block release of mature GDF11 from latent GDF 11. The anti-myostatin antibodies of the invention do not block the hydrolytic or non-proteolytic release of mature GDF11 from latent GDF 11. In certain embodiments, the anti-myostatin antibody does not block proteolytic cleavage of latent GDF 11. In certain embodiments, the anti-myostatin antibody does not block access of the protease to latent GDF11 (particularly, to the proteolytic cleavage site of latent GDF 11). In another embodiment, the protease may be a BMP1/TLD family metalloprotease such as BMP1, TED, tolloid-like protein-1 (TLL-1) or tolloid-like protein-2 (TLL-2). Non-proteolytic release as used herein means the spontaneous release of mature GDF11 from latent GDF11, which is not accompanied by proteolytic cleavage of latent GDF 11. Non-proteolytic release includes, for example, release of mature GDF11 by incubating latent GDF11, e.g., at 37 ℃, in the absence of a protease that cleaves latent GDF 11. Most of the currently known anti-myostatin antibodies are not specific for myostatin. These antibodies also have high affinity for other members of the TGF- β superfamily (e.g., GDF11) and neutralize their biological activity. GDF11 plays an important role during embryogenesis and is responsible for homeotic transformation (homeotic transformation) of the axial skeleton. Homozygous GDF11 knockout mice are perinatal lethal, and mice with one copy of the wild-type GDF11 gene survive but are skeletal defective. Because GDF11 plays an important role in embryogenesis, antagonists that inhibit GDF11 pose a theoretical safety risk that may be manifested as toxicity in the treated patient or reproductive toxicity in, for example, women with the potential to become pregnant. Thus, there is a need for specific inhibition of myostatin activity in the treatment of myostatin related disorders, which require increased muscle mass, size, strength, etc., particularly in women with a potential for pregnancy.

In another aspect, the invention provides anti-myostatin antibodies that exhibit pH-dependent binding properties. As used herein, the expression "pH-dependent binding" means that the antibody "shows a reduced binding to myostatin at acidic pH compared to its binding at neutral pH" (for the present disclosure, both expressions may be used interchangeably). For example, an antibody that "has pH-dependent binding properties" includes an antibody that binds myostatin at neutral pH with a higher affinity than at acidic pH. In certain embodiments, the antibodies of the invention bind myostatin at neutral pH with at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times greater affinity than at acidic pH. In some embodiments, the antibody binds myostatin (e.g., latent myostatin or pro-peptide myostatin) with a higher affinity at ph7.4 than at ph 5.8. In further embodiments, the antibody binds myostatin at ph7.4 with an affinity that is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times higher than ph 5.8.

When the antigen is a soluble protein, binding of the antibody to the antigen may result in an extended half-life of the antigen in plasma (i.e., reduced clearance of the antigen from the plasma) because the half-life of the antibody in plasma may be longer than the antigen itself and may serve as a carrier for the antigen. This is due to the recycling of antigen-antibody complexes by FcRn via the endosomal pathway in cells (Roopenian, nat. Rev. Immunol.7(9):715-725 (2007)). However, antibodies with pH-dependent binding properties, which bind their antigen in a neutral extracellular environment and release the antigen into an acidic endosomal compartment upon entry into the cell, are expected to have superior properties with respect to antigen neutralization and clearance compared to their pH-independent binding counterparts (Igawa et al, Nature Biotechnol.28(11): 1203; 1207 (2010); Devanabaoyina et al, mAbs 5(6): 851; 859 (2013); WO 2009/125825).

For this disclosureIn addition, the "affinity" of an antibody for myostatin is expressed as the KD of the antibody. The KD of an antibody refers to the equilibrium dissociation constant of an antibody-antigen interaction. The greater the KD value for an antibody binding to its antigen, the weaker its binding affinity for that particular antigen. Thus, as used herein, the expression "higher affinity at neutral pH than at acidic pH" (or equivalently "pH-dependent binding") means that the KD for the antibody to bind myostatin at acidic pH is higher than the KD for the antibody to bind myostatin at neutral pH. For example, in the context of the present invention, an antibody is considered to have a higher affinity for binding myostatin at neutral pH than at acidic pH if the KD for the antibody to bind myostatin is at least 2 times higher than the KD for the antibody to bind myostatin at neutral pH. Thus, the invention includes antibodies that bind myostatin at an acidic pH with a KD that is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times higher than the KD for the antibody to bind myostatin at a neutral pH. In another embodiment, the antibody may have a KD value of 10 at neutral pH ^-7M，10^-8M，10^-9M，10^-10M，10^-11M，10^-12M is less than or equal to M. In another embodiment, the antibody may have a KD value of 10 at acidic pH^-9M，10^-8M，10^-7M，10^-6M is more than M.

In further embodiments, an antibody is considered to bind myostatin (e.g., latent myostatin or pro peptide myostatin) with a higher affinity at neutral pH than at acidic pH if the antibody binds myostatin at pH5.8 with a KD at least 2-fold higher than the antibody binds myostatin at pH 7.4. In some embodiments, an antibody is provided that binds myostatin with a KD at ph5.8 that is at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times higher than the KD for the antibody to bind myostatin at ph 7.4. In thatIn another embodiment, the antibody may have a KD value of 10 at pH7.4^-7M，10^-8M，10^-9M，10^-10M，10^-11M，10^-12M is less than or equal to M. In another embodiment, the antibody may have a KD value of 10 at pH5.8^-9M，10^-8M，10^-7M，10^-6M or greater.

The binding properties of an antibody to a particular antigen can also be expressed as kd of the antibody. Kd of an antibody refers to the dissociation rate constant of an antibody with respect to a particular antigen and is in the reciprocal of a unit of second (i.e., sec) ^-1) And (4) showing. An increase in kd indicates that the antibody binds weakly to its antigen. The invention therefore includes antibodies that bind myostatin at acidic pH with higher kd values than at neutral pH. The invention includes antibodies that bind myostatin kd at acidic pH at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times higher than the antibody binds myostatin kd at neutral pH. In another embodiment, the antibody may have a kd value of 10 at neutral pH ^-2 1/s，10^-3 1/s，10^-4 1/s，10^-5 1/s，10^-61/s or less. In another embodiment, the antibody may have a kd value of 10 at acidic pH ^-3 1/s，10^-2 1/s，10^-11/s or more. The invention also includes antibodies that bind myostatin (e.g., latent myostatin or pro-peptide myostatin) at ph5.8 with a higher kd value than at ph 7.4. The invention includes antibodies that bind myostatin kd at ph5.8 that is at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times higher than the antibody binds myostatin kd at ph 7.4. In another embodiment, the antibody may have a kd value of 10 at pH7.4 ^-21/s，10^-3 1/s，10^-4 1/s，10^-5 1/s，10^-61/s or less. In another embodiment, the antibody may have a kd value of 10 at pH5.8^-3 1/s，10^-2 1/s，10^-11/s or more.

In certain instances, "binding of myostatin at acidic pH is reduced compared to its binding at neutral pH" is expressed as the ratio of the KD of the antibody binding myostatin at acidic pH to the KD of the antibody binding myostatin at neutral pH (or vice versa). For example, for the present invention, an antibody can be considered to exhibit "reduced binding to myostatin at acidic pH as compared to its binding at neutral pH" if the antibody exhibits an acidic/neutral KD ratio of 2 or greater. In certain embodiments, the anti-myostatin antibodies of the invention have a pH5.8/pH7.4KD ratio of 2 or greater. In certain exemplary embodiments, the antibodies of the invention can have an acidic/neutral KD ratio of 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or greater. In another embodiment, the antibody may have a KD value of 10 at neutral pH^-7M，10^-8M，10^-9M，10^-10M，10^-11M，10^-12M is less than or equal to M. In another embodiment, the antibody may have a KD value of 10 at acidic pH ^-9M，10^-8M，10^-7M，10^-6M is more than M. In other cases, an antibody may be considered to exhibit "reduced binding to a myostatin (e.g., latent myostatin) at acidic pH compared to its binding at neutral pH" if the antibody exhibits a pH5.8/pH7.4kd ratio of 2 or greater. In certain exemplary embodiments, the antibody may have a ph5.8/ph7.4kd ratio of 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or greater. In another embodiment, the antibody may have a KD value of 10 at pH7.4^-7M，10^-8M，10^-9M，10^-10M，10^-11M，10^-12M is less than or equal to M. In another embodiment, the antibody may have a KD value of 10 at pH5.8^-9M，10^-8M，10^-7M，10^-6M is more than M.

In certain instances, "binding of myostatin at acidic pH is reduced compared to its binding at neutral pH" is expressed as the ratio of kd for an antibody binding myostatin at acidic pH to kd for an antibody binding myostatin at neutral pH (or vice versa). For example, for the present invention, an antibody can be considered to exhibit "reduced binding to myostatin at acidic pH as compared to its binding at neutral pH" if the antibody exhibits an acidic/neutral kd ratio of 2 or greater. In certain exemplary embodiments, the antibodies of the invention have a pH5.8/pH7.4kd ratio of 2 or greater. In certain exemplary embodiments, the acidic/neutral kd ratio of an antibody of the invention can be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or greater. In another embodiment, the antibody may have a kd value of 10 at neutral pH ^-2 1/s，10^-3 1/s，10^-4 1/s，10^-5 1/s，10^-61/s or less. In another embodiment, the antibody may have a kd value of 10 at acidic pH ^-3 1/s，10^-2 1/s，10^-11/s or more. In certain exemplary embodiments, the antibodies of the invention may have a ph5.8/ph7.4kd ratio of 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 antibody may have a kd value of 10 at pH7.4^-2 1/s，10^-3 1/s，10^-4 1/s，10^-5 1/s，10^-61/s or less. In another embodiment, the antibody may have a kd value of 10 at pH5.8^-3 1/s，10^-2 1/s，10^-11/s or more.

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

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

As expressed herein, KD values as well as KD values can be determined using surface plasmon resonance based biosensors to characterize antibody-antigen interactions. (see, e.g., example 7 herein). The KD and KD values can be determined at 25 ℃ or 37 ℃.

In certain embodiments, an anti-myostatin antibody of the invention binds myostatin from more than one species. In additional embodiments, the anti-myostatin antibody binds myostatin from a human and a non-human animal. In additional embodiments, the anti-myostatin antibody binds myostatin from a human, mouse, and monkey (e.g., cynomolgus monkey, rhesus monkey, marmoset monkey, chimpanzee, or baboon).

In certain embodiments, an anti-myostatin antibody of the invention binds latent myostatin from more than one species. In additional embodiments, the anti-myostatin antibody binds latent myostatin from a human and a non-human animal. In additional embodiments, the anti-myostatin antibody binds latent myostatin from a human, mouse, and monkey.

In certain embodiments, an anti-myostatin antibody of the invention binds a pro peptide myostatin from more than one species. In additional embodiments, the anti-myostatin antibody binds to pro-peptide myostatin from a human and a non-human animal. In additional embodiments, the anti-myostatin antibody binds to a pro-peptide myostatin from a human, mouse, and monkey.

In another aspect, the invention provides anti-myostatin antibodies that form immune complexes (i.e., antigen-antibody complexes) with myostatin. In certain embodiments, two or more anti-myostatin antibodies bind to two or more myostatin molecules to form an immune complex. This is possible because myostatin exists as a homodimer containing two myostatin molecules, while antibodies have two antigen binding sites. The anti-myostatin antibodies can bind to the same epitope on the myostatin molecule or can bind to different epitopes on the myostatin molecule, much like bispecific antibodies. Generally, when two or more antibodies form immune complexes with two or more antigens, the resulting immune complexes can strongly bind to Fc receptors present on the cell surface due to the affinity action through the Fc region of the antibodies in the complexes, and then can be taken into cells with high efficiency. Thus, the anti-myostatin antibodies described above, which are capable of forming an immune complex comprising two or more anti-myostatin antibodies and two or more myostatin molecules, can result in rapid clearance of myostatin from plasma in vivo via strong binding to Fc receptors due to the effects of affinity.

Furthermore, antibodies with pH-dependent binding properties are believed to have superior properties with respect to their pH-independent binding counterparts in antigen neutralization and clearance (Igawa et al, Nature Biotech.28(11):1203-1207 (2010); Devianobina et al mAbs 5(6):851-859 (2013); WO 2009/125825). It is therefore expected that antibodies having the above-mentioned two properties, i.e., antibodies having pH-dependent binding properties and forming immune complexes with two or more antigens with two or more antibodies, have even better properties for highly accelerated elimination of antigens from plasma (WO 2013/081143).

In another aspect, the invention provides an anti-myostatin antibody comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1, wherein the HVR-H1 comprises the amino acid sequence of any one of SEQ ID NO:55-57, 114-115, 126; (b) HVR-H2, wherein the HVR-H2 comprises the amino acid sequence of any one of SEQ ID NO 58-60, 116-120, 127; (c) HVR-H3, the HVR-H3 comprises an amino acid sequence of any one of SEQ ID NOs 61-64, 121, 128; (d) HVR-L1, wherein the HVR-L1 comprises the amino acid sequence of any one of SEQ ID NO 65-69, 122-124, 129; (e) HVR-L2, the HVR-L2 comprises an amino acid sequence of any one of SEQ ID NOs 70-72, 125, 130; and (f) HVR-L3, said HVR-L3 comprises the amino acid sequence of any one of SEQ ID NOS: 73-74, 131.

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

In one aspect, the invention provides an anti-myostatin antibody comprising at least one, two, three, four, five or six hypervariable regions (HVRs) selected from: (a) HVR-H1, wherein the HVR-H1 comprises the amino acid sequence of any one of SEQ ID NO: 114-115; (b) HVR-H2, wherein the HVR-H2 comprises the amino acid sequence of any one of SEQ ID NO: 116-120; (c) HVR-H3, the HVR-H3 comprises the amino acid sequence of SEQ ID NO: 121; (d) HVR-L1, wherein the HVR-L1 comprises the amino acid sequence of any one of SEQ ID NO. 122-124; (e) HVR-L2, the HVR-L2 comprises the amino acid sequence of SEQ ID NO 125; and (f) HVR-L3, said HVR-L3 comprises the amino acid sequence of any one of SEQ ID NOS: 73-74.

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

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

In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1, wherein the HVR-H1 comprises the amino acid sequence of any one of SEQ ID NO:55-57, 114-115, 126; (b) HVR-H2, wherein the HVR-H2 comprises the amino acid sequence of any one of SEQ ID NO 58-60, 116-120, 127; (c) HVR-H3, the HVR-H3 comprises an amino acid sequence of any one of SEQ ID NOs 61-64, 121, 128. In one embodiment, the antibody comprises HVR-H3, wherein HVR-H3 comprises the amino acid sequence of any one of SEQ ID NOS 61-64, 121, 128. In another embodiment, the antibody comprises HVR-H3, wherein HVR-H3 comprises the amino acid sequence of any one of SEQ ID Nos. 61-64, 121, 128; and HVR-L3, the HVR-L3 comprises the amino acid sequence of any one of SEQ ID NOs 73-74, 131. In another embodiment, the antibody comprises HVR-H3, wherein HVR-H3 comprises the amino acid sequence of any one of SEQ ID Nos. 61-64, 121, 128; HVR-L3, the HVR-L3 comprises an amino acid sequence of any one of SEQ ID NOs 73-74, 131; and HVR-H2, wherein the HVR-H2 comprises the amino acid sequence of any one of SEQ ID NO 58-60, 116-120, 127. In another embodiment, the antibody comprises (a) an HVR-H1, wherein the HVR-H1 comprises the amino acid sequence of any one of SEQ ID NOS: 55-57, 114-115, 126; (b) HVR-H2, wherein the HVR-H2 comprises the amino acid sequence of any one of SEQ ID NO 58-60, 116-120, 127; and (c) HVR-H3, wherein HVR-H3 comprises the amino acid sequence of any one of SEQ ID NOS: 61-64, 121, 128.

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

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

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

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

In another aspect, the invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1, said HVR-L1 comprises the amino acid sequence of any one of SEQ ID NO 65-69, 122-124, 129; (b) HVR-L2, the HVR-L2 comprises an amino acid sequence of any one of SEQ ID NOs 70-72, 125, 130; and (c) HVR-L3, said HVR-L3 comprises the amino acid sequence of any one of SEQ ID NOS: 73-74, 131. In one embodiment, the antibody comprises (a) HVR-L1, wherein HVR-L1 comprises the amino acid sequence of any one of SEQ ID NOS 65-69, 122-124, 129; (b) HVR-L2, the HVR-L2 comprises an amino acid sequence of any one of SEQ ID NOs 70-72, 125, 130; and (c) HVR-L3, said HVR-L3 comprises the amino acid sequence of any one of SEQ ID NOS: 73-74, 131.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In one aspect, the invention provides an anti-myostatin antibody comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1, wherein the HVR-H1 comprises the amino acid sequence of any one of SEQ ID NO 157-162; (b) HVR-H2, wherein the HVR-H2 comprises the amino acid sequence of any one of SEQ ID NO 163-168; (c) HVR-H3, wherein the HVR-H3 comprises the amino acid sequence of any one of SEQ ID NO 169-174; (d) HVR-L1, wherein the HVR-L1 comprises the amino acid sequence of any one of SEQ ID NO 175-180; (e) HVR-L2, wherein the HVR-L2 comprises the amino acid sequence of any one of SEQ ID NO 181-186; and (f) HVR-L3, wherein the HVR-L3 comprises the amino acid sequence of any one of SEQ ID NO: 187-192.

In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1, wherein the HVR-H1 comprises the amino acid sequence of any one of SEQ ID NO: 157-162; (b) HVR-H2, wherein the HVR-H2 comprises the amino acid sequence of any one of SEQ ID NO 163-168; and (c) HVR-H3, wherein the HVR-H3 comprises the amino acid sequence of any one of SEQ ID NO: 169-174. In one embodiment, the antibody comprises HVR-H3, wherein HVR-H3 comprises the amino acid sequence of any one of SEQ ID NO 169-174. In another embodiment, the antibody comprises HVR-H3, the HVR-H3 comprises the amino acid sequence of any one of SEQ ID NO:169-174, and HVR-L3, the HVR-L3 comprises the amino acid sequence of any one of SEQ ID NO: 187-192. In another embodiment, the antibody comprises HVR-H3, the HVR-H3 comprises the amino acid sequence of any one of SEQ ID NO 169-174, HVR-L3, the HVR-L3 comprises the amino acid sequence of any one of SEQ ID NO 187-192; and HVR-H2, wherein the HVR-H2 comprises the amino acid sequence of any one of SEQ ID NO 163-168. In another embodiment, the antibody comprises (a) an HVR-H1, wherein the HVR-H1 comprises the amino acid sequence of any one of SEQ ID NOs 157-162; (b) HVR-H2, wherein the HVR-H2 comprises the amino acid sequence of any one of SEQ ID NO 163-168; and (c) HVR-H3, wherein the HVR-H3 comprises the amino acid sequence of any one of SEQ ID NO: 169-174.

In another aspect, the invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1, wherein HVR-L1 comprises the amino acid sequence of any one of SEQ ID NO 175-180; (b) HVR-L2, wherein the HVR-L2 comprises the amino acid sequence of any one of SEQ ID NO 181-186; and (c) HVR-L3, wherein the HVR-L3 comprises the amino acid sequence of any one of SEQ ID NO: 187-192. In one embodiment, the antibody comprises (a) HVR-L1, wherein HVR-L1 comprises the amino acid sequence of any one of SEQ ID NOs 175-180; (b) HVR-L2, wherein the HVR-L2 comprises the amino acid sequence of any one of SEQ ID NO 181-186; and (c) HVR-L3, wherein the HVR-L3 comprises the amino acid sequence of any one of SEQ ID NO: 187-192.

In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1, said HVR-H1 comprising the amino acid sequence of any one of SEQ ID NO:157-162, (ii) HVR-H2, said HVR-H2 comprising the amino acid sequence of any one of SEQ ID NO:163-168, and (iii) HVR-H3, said HVR-H3 comprising the amino acid sequence of any one of SEQ ID NO: 169-174; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1, said HVR-L1 comprising the amino acid sequence of any one of SEQ ID NO:175-180, (ii) HVR-L2, said HVR-L2 comprising the amino acid sequence of any one of SEQ ID NO:181-186, and (iii) HVR-L3, said HVR-L3 comprising the amino acid sequence of any one of SEQ ID NO: 187-192.

In another aspect, the invention provides an antibody comprising (a) HVR-H1, said HVR-H1 comprises the amino acid sequence of any one of SEQ ID NO: 157-162; (b) HVR-H2, wherein the HVR-H2 comprises the amino acid sequence of any one of SEQ ID NO 163-168; (c) HVR-H3, wherein the HVR-H3 comprises the amino acid sequence of any one of SEQ ID NO 169-174; (d) HVR-L1, wherein HVR-L1 comprises the amino acid sequence of any one of SEQ ID NO 175-180; (e) HVR-L2, wherein the HVR-L2 comprises the amino acid sequence of any one of SEQ ID NO 181-186; and (f) HVR-L3, wherein the HVR-L3 comprises the amino acid sequence of any one of SEQ ID NO: 187-192.

In another aspect, an anti-myostatin antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence of any of SEQ ID NOs 13, 16-30, 32-34, and 86-95. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises a substitution (e.g., a conservative substitution), insertion, or deletion relative to a reference sequence, but an anti-myostatin antibody comprising the sequence retains the ability to bind myostatin. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in any of SEQ ID NOs 13, 16-30, 32-34 and 86-95. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-myostatin antibody comprises a VH sequence of any one of SEQ ID NOs 13, 16-30, 32-34, and 86-95, including post-translational modifications of the sequence. In particular embodiments, the VH comprises one, two or three HVRs selected from: (a) HVR-H1, said HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOS: 55-57, 114-. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamate acylation.

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

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

In another aspect, an anti-myostatin antibody comprises a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 86. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises a substitution (e.g., a conservative substitution), insertion, or deletion relative to a reference sequence, but an anti-myostatin antibody comprising the sequence retains the ability to bind myostatin. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO 86. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-myostatin antibody comprises the VH sequence of SEQ ID NO 86, which includes a post-translational modification of the sequence. In particular embodiments, the VH comprises one, two or three HVRs selected from: (a) HVR-H1, said HVR-H1 comprising the amino acid sequence of SEQ ID NO:114, (b) HVR-H2, said HVR-H2 comprising the amino acid sequence of SEQ ID NO:58, and (c) HVR-H3, said HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamate acylation. In another aspect, an anti-myostatin antibody comprises a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 92. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises a substitution (e.g., a conservative substitution), insertion, or deletion relative to a reference sequence, but an anti-myostatin antibody comprising the sequence retains the ability to bind myostatin. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO 92. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-myostatin antibody comprises the VH sequence of SEQ ID NO 92, which includes a post-translational modification of said sequence. In particular embodiments, the VH comprises one, two or three HVRs selected from: (a) HVR-H1, said HVR-H1 comprising the amino acid sequence of SEQ ID NO:114, (b) HVR-H2, said HVR-H2 comprising the amino acid sequence of SEQ ID NO:58, and (c) HVR-H3, said HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamate acylation.

In another aspect, an anti-myostatin antibody is provided, wherein the antibody comprises a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence of any one of SEQ ID NOs 15, 31, 35-38, and 96-99. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises a substitution (e.g., a conservative substitution), insertion, or deletion relative to a reference sequence, but an anti-myostatin antibody comprising said sequence retains the ability to bind myostatin. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in any of SEQ ID NOs 15, 31, 35-38 and 96-99. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVRs (i.e., in the FRs). Optionally, the anti-myostatin antibody comprises a VL sequence of any one of SEQ ID NOs 15, 31, 35-38, and 96-99, including post-translational modifications of the sequence. In particular embodiments, the VL comprises one, two, or three HVRs selected from: (a) HVR-L1, said HVR-L1 comprises the amino acid sequence of any one of SEQ ID NO 65-69, 122-124, 129; (b) HVR-L2, the HVR-L2 comprises an amino acid sequence of any one of SEQ ID NOs 70-72, 125, 130; and (c) HVR-L3, said HVR-L3 comprises the amino acid sequence of any one of SEQ ID NOS: 73-74, 131. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamate acylation.

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

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

In another aspect, an anti-myostatin antibody is provided, wherein the antibody comprises a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 96. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises a substitution (e.g., a conservative substitution), insertion, or deletion relative to a reference sequence, but an anti-myostatin antibody comprising the sequence retains the ability to bind myostatin. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO 96. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR. Optionally, the anti-myostatin antibody comprises the VL sequence of SEQ ID NO 96, including post-translational modifications of the sequence. In particular embodiments, the VL comprises one, two or three HVRs selected from: (a) HVR-L1, the HVR-L1 comprises the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2, the HVR-L2 comprises the amino acid sequence of SEQ ID NO: 71; and (c) HVR-L3, said HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamate acylation. In another aspect, an anti-myostatin antibody is provided, wherein the antibody comprises a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 97. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises a substitution (e.g., a conservative substitution), insertion, or deletion relative to a reference sequence, but an anti-myostatin antibody comprising the sequence retains the ability to bind myostatin. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO 97. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVRs. Optionally, the anti-myostatin antibody comprises the VL sequence of SEQ ID NO:97, including post-translational modifications of the sequence. In particular embodiments, the VL comprises one, two, or three HVRs selected from: (a) HVR-L1, the HVR-L1 comprises the amino acid sequence of SEQ ID NO: 123; (b) HVR-L2, the HVR-L2 comprises the amino acid sequence of SEQ ID NO: 71; and (c) HVR-L3, said HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamate acylation.

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

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

In another aspect, the anti-myostatin antibody comprises a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NO 12, 145-150. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises a substitution (e.g., a conservative substitution), insertion, or deletion relative to a reference sequence, but an anti-myostatin antibody comprising the sequence retains the ability to bind myostatin. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in any of SEQ ID NO 12, 145-150. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-myostatin antibody comprises a VH sequence of any one of SEQ ID NO 12, 145-150, including post-translational modifications of the sequence. In particular embodiments, the VH comprises one, two or three HVRs selected from: (a) HVR-H1, said HVR-H1 comprising the amino acid sequence of any one of SEQ ID NO:55, 157-162, (b) HVR-H2, said HVR-H2 comprising the amino acid sequence of any one of SEQ ID NO:58, 163-168, and (c) HVR-H3, said HVR-H3 comprising the amino acid sequence of any one of SEQ ID NO:61, 169-174. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamate acylation.

In another aspect, anti-myostatin antibodies are provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NO:14, 151-156. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises a substitution (e.g., a conservative substitution), insertion, or deletion relative to a reference sequence, but an anti-myostatin antibody comprising the sequence retains the ability to bind myostatin. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in any of SEQ ID NO 14, 151 and 156. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-myostatin antibody comprises the VL sequence of any one of SEQ ID NO 14, 151 and 156, including post-translational modifications of said sequence. In particular embodiments, the VL comprises one, two or three HVRs selected from: (a) HVR-L1, wherein the HVR-L1 comprises the amino acid sequence of any one of SEQ ID NO. 65, 175 and 180; (b) HVR-L2, wherein the HVR-L2 comprises the amino acid sequence of any one of SEQ ID NO 70, 181-186; and (c) HVR-L3, wherein the HVR-L3 comprises the amino acid sequence of any one of SEQ ID NO:73, 187-192. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamate acylation.

In another aspect, there is provided an anti-myostatin antibody, wherein said antibody comprises a VH as in any of the embodiments above and a VL as in any of the embodiments above. In one embodiment, the antibody comprises the VH and VL sequences of any one of SEQ ID NO 12, 145-150 and any one of SEQ ID NO 14, 151-156, respectively, including post-translational modifications of the sequences. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamate acylation.

In certain embodiments, an anti-myostatin antibody of the invention comprises a VH as described in any of the embodiments above and a heavy chain constant region comprising the amino acid sequence of any of SEQ ID NO 7, 9, 11, 193, 195-198, 227, 228, 229-381. In certain embodiments, an anti-myostatin antibody of the invention comprises a VL as in any of the embodiments described above and a light chain constant region comprising an amino acid sequence of any of SEQ ID NOs 8 and 10.

In another aspect, the invention provides antibodies that bind to the same epitope as the anti-myostatin antibodies provided herein. In another aspect, the invention provides an antibody that binds to the same epitope as an antibody described in table 2 a. In another aspect, the invention provides an antibody that binds to the same epitope as an antibody described in table 11a or 13. In certain embodiments, antibodies are provided that bind to an epitope within a fragment of myostatin pro peptide consisting of amino acids 21-100 of SEQ ID NO: 78. Alternatively, the antibody binds to a myostatin pro peptide fragment consisting of amino acids 21-80, 41-100, 21-60, 41-80, 61-100, 21-40, 41-60, 61-80, or 81-100 of SEQ ID NO: 78.

In another aspect of the invention, the anti-myostatin antibody according to any of the above embodiments is a monoclonal antibody, including chimeric, humanized, or human antibodies. In one embodiment, the anti-myostatin antibody is an antibody fragment, e.g., Fv, Fab, Fab ', scFv, diabody, or F (ab')₂And (3) fragment. In another embodiment, the antibody is a full length IgG antibody, e.g., a complete IgG1 or IgG4 antibody or other antibody class or isotype as defined herein.

In another aspect, an anti-myostatin antibody according to any of the above embodiments can bind to any of the features described in sections 1-7 below (alone or in combination).

1. Affinity of antibody

In certain embodiments, an antibody provided herein has a dissociation constant (Kd) of 1 μ M or less, 100nM or less, 10nM or less, 1nM or less, 0.1nM or less, 0.01nM or less, or 0.001nM or less (e.g., 10nM or less)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M)。

In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using a Fab form of the antibody of interest and its antigen. For example, solution binding affinity of Fab to antigen is measured by: (iii) with minimum concentration in the presence of a titration series of unlabelled antigen ¹²⁵I) The labeled antigen equilibrates the Fab, and the bound antigen is then captured with an anti-Fab antibody coated plate (see, e.g., Chen et al, J.Mol.biol.293:865-881 (1999)). To determine the measurement conditions, MICROTITER (registered trademark) multi-well plate (Thermo Scientific)) Coated overnight with 5. mu.g/ml of capture anti-Fab antibody (Cappel Labs) in 50mM sodium carbonate (pH9.6) and subsequently blocked with 2% (w/v) fetal bovine serum albumin in PBS for two to five hours at room temperature (about 23 ℃). In a non-absorbent plate (Nunc #269620), 100pM or 26pM [ alpha ] amino acid is prepared¹²⁵I]Mixing of antigen with serial dilutions of Fab of interest (e.g.in accordance with the evaluation of anti-VEGF antibodies, Fab-12, in Presta et al, Cancer Res.57:4593-4599 (1997)). Then incubating the target Fab overnight; however, incubation may be continued for a longer period of time (e.g., about 65 hours) to ensure that equilibrium is achieved. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (e.g., for one hour). The solution was then removed and the plate was washed eight times with 0.1% polysorbate 20(TWEEN-20 (registered trademark)) in PBS. When the plates had dried, 150. mu.l/well of scintillator (MICROSCINT-20) was added^TM(ii) a Packard) and plates were mounted on TOPCOUNT ^TMCount on a gamma counter (Packard) for ten minutes. The concentration of each Fab that resulted in less than or equal to 20% of maximal binding was selected for competitive binding assays.

According to another embodiment, Kd is measured using BIACORE (registered trademark) surface plasmon resonance assay. For example, the measurement using BIACORE (registered trademark) -2000 or BIACORE (registered trademark) -3000(BIACORE (registered trademark), inc., Piscataway, NJ) was performed at 25 ℃ using an immobilized antigen CM5 chip at-10 Response Units (RU). In one embodiment, the carboxymethylated dextran biosensor chip (CM5, BIACORE (registered trademark), Inc.) is activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen was diluted to 5 μ g/ml (. about.0.2. mu.M) with 10mM sodium acetate pH4.8 before injection at a flow rate of 5 μ l/min to achieve approximately 10 Response Units (RU) of conjugated protein. After injection of the antigen, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions (0.78nM to 500nM) of Fab were injected at 25 ℃ with 0.05% polysorbate 20 (TWEEN-20) at a flow rate of about 25. mu.l/min ^TM) Surfactant in pbs (pbst). (BIACORE) using a simple one-to-one Langmuir (Langmuir) binding modelRegistered trademark) Evaluation Software version 3.2) the association rate (k) was calculated by simultaneously fitting association and dissociation sensorgrams_on) And dissociation Rate (k)_off). Equilibrium dissociation constant (Kd) as k_off/k_onThe ratio of the ratios is calculated. See, e.g., Chen et al, J.mol.biol.293:865-881 (1999). If the binding rate measured by the above surface plasmon resonance assay exceeds 10⁶M^-1s^-1The binding rate can then be determined by: using measurements such as at a spectrometer such as a spectrophotometer (Aviv Instruments) equipped with a stop-flow or 8000-series SLM-AMINCO with a stir chamber^TMFluorescence quenching technique (excitation 295 nM; emission 340nM, 16nM band pass) with an increase or decrease in fluorescence emission intensity of 20nM anti-antigen antibody (Fab form) in PBS, ph7.2 at 25 ℃, in the presence of increasing concentrations of antigen, measured in a spectrophotometer (ThermoSpectronic).

2. Antibody fragments

In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab ', Fab ' -SH, F (ab ')₂Fv and scFv fragments, as well as other fragments described below. For a review of specific antibody fragments, see Hudson et al nat. Med.9:129-134 (2003). For an overview of scFv fragments see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, vol.113, edited by Rosenburg and Moore, (Springer-Verlag, New York), pp.269-315 (1994); see also, WO 1993/16185; and U.S. patent nos. 5,571,894 and 5,587,458. For Fab and F (ab') containing salvage receptor binding epitope residues and having increased half-life in vivo ₂See U.S. Pat. No. 5,869,046 for a discussion of fragments.

Diabodies are antibody fragments with two antigen binding sites, which may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; hudson et al, nat. Med.9: 129-; and Hollinger et al, Proc. Natl. Acad. Sci. USA 90: 6444-. Tri-and tetrabodies are also described in Hudson et al, nat. Med.9:129-134 (2003).

A single domain antibody is an antibody fragment comprising all or part of a heavy chain variable domain or all or part of a light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516B 1).

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

3. Chimeric and humanized antibodies

In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Pat. nos. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In further examples, a chimeric antibody is a "class switch" antibody, wherein the class or subclass has been altered by the class or subclass of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

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

Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro, Front.biosci.13:1619-1633(2008), and further described, for example, in Riechmann et al, Nature 332:323-329 (1988); queen et al, Proc.nat' l Acad.Sci.USA 86:10029-10033 (1989); U.S. Pat. nos. 5,821,337,7,527,791,6,982,321, and 7,087,409; kashmiri et al, Methods 36:25-34(2005) (describes Specificity Determining Region (SDR) grafting); padlan, mol.Immunol.28:489-498(1991) (describing "surface reconstruction"); dall' Acqua et al, Methods 36:43-60(2005) (describing "FR shuffling"); and Osbourn et al, Methods 36:61-68(2005) and Klimka et al, Br.J. cancer,83: 252-.

Human framework regions that may be used for humanization include, but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims et al J. Immunol.151:2296 (1993)); framework regions derived from consensus sequences of human antibodies having particular subsets of light or heavy chain variable regions (see, e.g., Carter et al, Proc. Natl. Acad. Sci. USA 89:4285 (1992); and Presta et al J. Immunol.151:2623 (1993); human mature (somatomerism) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, front.biosci.13:1619-1633 (2008)); and framework regions derived from FR library screening (see, e.g., Baca et al, J.biol.chem.272:10678-10684(1997) and Rosok et al, J.biol.chem.271:22611-22618 (1996)).

4. Human antibodies

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

Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce a fully human antibody or a fully antibody with human variable regions in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus, which replaces an endogenous immunoglobulin locus, or which is present outside the chromosome or randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin loci have typically been inactivated. For an overview of the method for obtaining human antibodies from transgenic animals, see Lonberg, nat. Biotech.23:1117-1125 (B) 2005). See also, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584, which describe XeNOMOUSE^TMA technique; U.S. patent No. 5,770,429, which describes HUMAB (registered trademark) technology; U.S. patent No. 7,041,870, which describes K-M MOUSE (registered trademark) technology, and U.S. patent application publication No. US 2007/0061900, which describes VELOCIMOUSE (registered trademark) technology). The human variable regions from intact antibodies produced by such animals may be further modified, for example, by combination with different human constant regions.

Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines for use in the preparation of human monoclonal antibodies have been described. (see, e.g., Kozbor J.Immunol.,133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp.51-63(Marcel Dekker, Inc., New York, 1987); and Boerner et al, J.Immunol.147:86 (1991)). Human antibodies prepared via human B-cell hybridoma technology are also described in Li et al, Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006). Additional methods include those described in, for example, U.S. Pat. No. 7,189,826 (describing the preparation of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue 26(4):265-268(2006) (describing human-human hybridomas). The human hybridoma technique (Trioma technique) is also described in Vollmers and Brandlein, Histology and Histopathlogy 20(3): 927-.

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

5. Antibodies derived from libraries

Antibodies of the invention can be isolated by screening combinatorial libraries of antibodies having a desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening the libraries for antibodies with desired binding properties. Such Methods are reviewed, for example, in Hoogenboom et al, Methods in Molecular Biology 178:1-37 (2000); o' Brien et al, ed., Human Press, Totowa, NJ,2001) and is further described, for example, in McCafferty et al, Nature 348: 552-; clackson et al, Nature 352: 624-; marks et al, J.mol.biol.222:581-597 (1992); marks and Bradbury, Methods in Molecular Biology 248:161-175(Lo, ed., Human Press, Totowa, NJ, 2003); sidhu et al, J.mol.biol.338(2):299-310 (2004); lee et al, J.mol.biol.340(5):1073-1093 (2004); fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-; and Lee et al, J.Immunol.methods 284(1-2):119-132 (2004).

In some phage display methods, VH and VL gene libraries are separately cloned by Polymerase Chain Reaction (PCR) and randomly recombined in phage libraries, which can then be screened against antigen-binding phage, as described by Winter et al, Ann. Rev. Immunol.12:433-455 (1994). Phage typically display antibody fragments as single chain fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, a natural (nave) library (e.g., by humans) can be cloned to provide a single source of antibody to multiple non-self antigens as well as to self antigens without the need for any immunization, as described by Griffiths et al, EMBO J,12: 725-. Finally, natural libraries can also be prepared synthetically by: unrearranged V-gene segments were cloned from stem cells and PCR primers containing random sequences were used to encode the hypervariable CDR3 regions and to effect rearrangement in vitro as described by Hoogenboom and Winter, J.Mol.biol.227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373 and U.S. publication nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936 and 2009/0002360.

Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

6. Multispecific antibodies

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

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see, Milstein and Cuello, Nature 305:537(1983)), WO 1993/08829, and Traunecker et al, EMBO J.10:3655(1991)), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be prepared by engineering electrostatic targeting for the preparation of antibody Fc-heterodimeric molecules (WO 2009/089004a 1); crosslinking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al, Science,229:81 (1985)); the use of leucine zippers to prepare bispecific antibodies (see, e.g., Kostelny et al, J.Immunol.148(5):1547-1553 (1992)); the "diabody" technique is used for the preparation of bispecific antibody fragments (see, e.g., Hollinger et al, Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); and the use of single chain fv (sFv) dimers (see, e.g., Gruber et al, J.Immunol.152:5368 (1994)); and making trispecific antibodies as described, for example, in Tutt et al, J.Immunol.147:60 (1991).

Also included herein are engineered antibodies with more than three functional antigen binding sites, including "octopus antibodies" (see, e.g., US 2006/0025576a 1).

Antibodies or fragments herein also include "dual action fabs" or "DAFs" comprising an antigen binding site that binds myostatin as well as another, different antigen (see, e.g., US 2008/0069820).

7. Antibody variants

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

a. Substitution, insertion and deletion variants

In certain embodiments, antibody variants are provided having one or more amino acid substitutions. Sites of interest for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in table 1 under the heading of "preferred substitutions". Further changes are provided under the heading of "exemplary substitutions" in table 1 and as further described below with respect to amino acid side chain classifications. Amino acid substitutions may be introduced into the antibody of interest and the product screened for the desired activity (e.g., maintained/increased antigen binding, reduced immunogenicity or increased ADCC or CDC).

[ Table 1]

Initial residue	Exemplary permutations	Preference is given to substitution
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Asp，Lys；Arg	Gln
Asp(D)	Glu；Asn	Glu
			Cys(C)	Ser；Ala	Ser
Gln(Q)	Asn；Glu	Asn
			Glu(E)	Asp；Gln	Asp
Gly(G)	Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe；Norleucine	Leu
			Leu(L)	Norleucine；Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Trp；Leu；Val：Ile；Ala；Tyr	Tyr
			Pro(p)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Val；Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala；Norleucine	Leu

Amino acids can be grouped into groups based on common side chain properties: (1) hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilicity: cys, Ser, Thr, Asn, Gln; (3) acidity: asp, Glu; (4) alkalinity: his, Lys, Arg; (5) residues that influence chain orientation: gly, Pro; and (6) aromaticity: trp, Tyr, Phe. Non-conservative substitutions entail exchanging a member of one of these groups for a member of the other group.

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

Alterations (e.g., substitutions) may be made in HVRs, for example, to improve antibody affinity. Such changes can be made in HVR "hot spots", i.e., residues encoded by codons that are mutated at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods mol. biol.207:179-196(2008)), and/or residues that contact the antigen, and the resulting variant VH or VL is tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, for example, in Hoogenboom et al, Methods in Molecular Biology 178:1-37(O' Brien et al, ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). Secondary libraries were then generated. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity includes HVR-directed methods, in which several HVR residues (e.g., 4-6 residues at the same time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are generally targeted.

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

A method that can be used to identify antibody residues or regions that can be targeted for mutagenesis is referred to as "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science,244: 1081-1085. In this method, a residue or set 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 determine whether to affect the interaction of an antibody with an antigen. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or additionally, the crystal structure of the antigen-antibody complex may be analyzed to determine the contact points between the antibody and the antigen. Such contact residues and adjacent residues may be targeted or excluded as candidates for replacement. Variants can be screened to determine if they have the desired properties.

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

b. Glycosylation variants

In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree to which the antibody is glycosylated. The addition of glycosylation sites to an antibody or deletion of glycosylation sites can be readily accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

When the antibody comprises an Fc region, the carbohydrate to which it is attached may be altered. Natural antibodies produced by mammalian cells typically comprise a branched, bifurcated (biantennary) oligosaccharide, which is usually attached to Asn297 of the CH2 domain of the Fc region by an N-linkage. See, for example, Wright et al, TIBTECH 15:26-32 (1997). Oligosaccharides may include a variety of carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to GlcNAc in the "stem" of a bifurcated oligosaccharide structure. In some embodiments, modifications of oligosaccharides in the antibodies of the invention can be made to produce antibody variants with specific improved properties.

In one embodiment, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the sum of all sugar structures (e.g. complex, hybrid and high mannose structures) associated with Asn297, as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to an aspartic acid residue located near position 297 of the Fc region (Eu numbering of Fc region residues); however, due to small sequence variations in the antibody, Asn297 may also be located about +/-3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have an improved ADCC function. See, e.g., U.S. publication No. US 2003/0157108(Presta, L.); US 2004/0093621(Kyowa Hakko Kogyo co., Ltd). Disclosed example variants involving "defucosylated" or "fucose-deficient" antibodies include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; okazaki et al, J.mol.biol.336:1239-1249 (2004); Yamane-Ohnuki et al, Biotech.Bioeng.87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include protein fucosylation deficient Lec13CHO cells (Ripka et al, Arch. biochem. Biophys.249:533-545 (1986); U.S. patent application No. US 2003/0157108A 1, Presta, L; and WO 2004/056312, Adams et al, especially example 11), and knock-out cell lines, such as the alpha-1, 6-fucosyltransferase gene, FUT8, knock-out CHO cells (see, e.g., Yamane-Ohnuki et al, Biotech. Bioeng.87:614 (2004); Kanda et al, Biotechnol. Bioeng.94(4):680-688 (2006); and WO 2003/085107).

Antibody variants having bisected oligosaccharides, for example, wherein a bisected oligosaccharide connected to the Fc region of the antibody is bisected by GlcNAc, are also provided. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878(Jean-Mairet et al); U.S. Pat. No. 6,602,684(Umana et al); and US 2005/0123546(Umana et al). Also provided are antibody variants having at least one galactose residue in an oligosaccharide attached to an Fc region. Such antibody variants may have increased CDC function. Such antibody variants are described, for example, in WO 1997/30087(Patel et al); WO 1998/58964(Raju, S.); and WO 1999/22764(Raju, S.).

Fc region variants

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

In certain embodiments, the invention contemplates antibody variants that have some, but not all, effector functions, which make them ideal candidates for applications where the in vivo half-life of the antibody is important and where certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays may be performed to confirm the reduction/elimination of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays may be performed to ensure that the antibody lacks fcyr binding (and therefore may lack ADCC activity), but retains FcRn binding ability. The primary cell mediating ADCC, NK cell, expresses only Fc γ RIII, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of ravatch and Kinet, Annu.Rev.Immunol.9:457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom et al Proc. nat 'l Acad. Sci. USA 83:7059-7063(1986)) and Hellstrom, I et al, Proc. nat' l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann et al, J.Exp.Med.166: 1351-. Alternatively, a non-radioactive assay may be used (see, e.g., ACT1 for flow cytometry ^TMNon-radioactive cytotoxicity assay (CellTechnology, inc. mountain View, CA); and CytoTox 96 (registered trademark) non-radioactive cytotoxicity assay (Promega, Madison, WI)). Effector cells that can be used in such assays include Peripheral Blood Mononuclear Cells (PBMCs) and Natural Killer (NK) cells. Alternatively, or in addition, the ADCC activity of the molecule of interest can be assessed in vivo, for example in an animal model as described in Clynes et al Proc. nat' l Acad. Sci. USA 95: 652-. A C1q binding assay may also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, for example, WO 2006/029879 and WO 2005/100402 for C1q and C3C binding ELISA. To assess complement activation, CDC assays can be performed (see, e.g., Gazzano-Santoro et al, J.Immunol. methods 202:163 (1996); Cragg et al, Blood 101:1045-1052 (2003); and Cragg, Blood 103:2738-2743(2004)). FcRn binding and in vivo clearance/half-life assays can also be performed using methods known in the art (see, e.g., Petkova et al, Int' l. immunol.18(12): 1759-.

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

Certain antibody variants with increased or decreased binding to FcR are described. (see, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, and Shields et al, J.biol.chem.9(2):6591-6604 (2001))

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

In some embodiments, alterations are made in the Fc region that result in altered (i.e., increased or decreased) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. Nos. 6,194,551, WO 1999/51642, and Idusogene et al, J.Immunol.164: 4178-.

Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn) responsible for the transfer of maternal IgG to the fetus (J.Immunol.117:587 (1976)) and Kim et al, J.Immunol.24:249(1994)) are described in US2005/0014934A1(Hinton et al). The antibody comprises an Fc region having one or more substitutions therein that increase binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, for example, a substitution of residue 434 in the Fc region (U.S. patent No. 7,371,826). See also, Duncan, Nature 322:738-40 (1988); U.S. Pat. nos. 5,648,260 and 5,624,821; and WO 1994/29351 which relates to other examples of variants of the Fc region.

d. Cysteine engineered antibody variants

In certain embodiments, it may be desirable to make cysteine engineered antibodies, e.g., "thiomabs," in which one or more residues of the antibody are substituted with a cysteine residue. In particular embodiments, the substituted residue occurs at an access site of the antibody. By replacing the residue with cysteine, a reactive thiol group is thereby placed at the access site of the antibody and can be used to conjugate the antibody to other moieties, such as a drug moiety or linker-drug moiety, thereby producing an immunoconjugate, as further described herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: v205 of the light chain (Kabat numbering); a118 of the heavy chain (EU numbering); and S400 of the heavy chain Fc region (EU numbering). Cysteine engineered antibodies can be produced as described, for example, in U.S. patent No. 7,521,541.

e. Antibody derivatives

In certain embodiments, the antibodies provided herein can be further modified to contain additional non-protein moieties known in the art and readily available. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-tris-cyclopentane

Alkanes, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polyoxypropylene/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in preparation due to its stability in water. The polymer may have an arbitrary compositionQuantum, and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, it can be the same or different molecules. In general, since the amount and/or type of derivatized polymer can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative is useful for therapy under defined conditions, and the like.

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

8. Variant Fc regions

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

In certain embodiments, the ratio of [ KD value of parent Fc region to monkey Fc γ RIIb ]/[ KD value of variant Fc region to monkey Fc γ RIIb ] can be 2.0 or more, 3.0 or more, 4.0 or more, 5.0 or more, 6.0 or more, 7.0 or more, 8.0 or more, 9.0 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, or 50 or more. In further embodiments, the variant Fc region has reduced binding activity to monkey Fc γ RIIIa. In certain embodiments, the ratio of [ KD value of parent Fc region to monkey Fc γ RIIIa ]/[ KD value of variant Fc region to monkey Fc γ 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 certain embodiments, the sequence of monkey Fc γ RIIb is SEQ ID NO:223 (cynomolgus monkey). In certain embodiments, the sequence of monkey Fc γ RIIIa is SEQ ID NO:224 (cynomolgus monkey).

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

In further embodiments, the variant Fc region has reduced binding activity to human Fc γ RIIa (type H) as compared to binding activity to human Fc γ RIIb. In certain embodiments, the [ KD value of the parent Fc region for human Fc γ RIIa (type H) ]/[ KD value of the variant Fc region for human Fc γ RIIa (type H) ] can be 5.0 or less, 4.0 or less, 3.0 or less, 2.0 or less, 1.0 or less, 0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, or 0.1 or less. In further embodiments, the variant Fc region has reduced binding activity to human Fc γ RIIa (R-type) as compared to binding activity to human Fc γ RIIb. In certain embodiments, the [ KD value of the parent Fc region to human Fc γ RIIa (type R) ]/[ KD value of the variant Fc region to human Fc γ RIIa (type R) ] can be 5.0 or less, 4.0 or less, 3.0 or less, 2.0 or less, 1.0 or less, 0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, or 0.1 or less. In certain embodiments, the sequence of human Fc γ RIIa (form H) is SEQ ID NO 211. In certain embodiments, the sequence of human Fc γ RIIa (R-type) is SEQ ID NO 210.

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

In another embodiment, the variant Fc region can have a KD value for monkey Fc γ RIIb of 1.0x10^-6M < 9.0x10^-7M below, 8.0x10^-7M < 7.0x10^-7M below, 6.0x10^-7M below, 5.0x10^-7M below, 4.0x10^-7M below, 3.0x10^-7M below, 2.0x10^-7Under M, or 1.0x10^-7M is less than or equal to M. In another embodiment, the variant Fc region can have a KD value for monkey Fc γ RIIIa of 5.0x10^-7M above, 6.0x10^-7M above, 7.0x10^-7M above, 8.0x10^-7M above, 9.0x10^-7M above, 1.0x10^-6M above, 2.0x10^-6M above, 3.0x10^-6M above, 4.0x10^-6M above, 5.0x10^-6M above, 6.0x10^-6M above, 7.0x10 ^-6M above, 8.0x10^-6M above, 9.0x10^-6M or more, or 1.0x10^-5M is more than M. In another embodiment, the variant Fc region can have a KD value of 2.0x10 for human fcyriib^-6M below, 1.0x10^-6M below, 9.0x10^-7M below, 8.0x10^-7M below, 7.0x10^-7M below, 6.0x10^-7M below, 5.0x10^-7M below, 4.0x10^-7M below, 3.0x10^-7M below, 2.0x10^-7Under M, or 1.0x10^-7M is less than or equal to M. In another embodiment, the variant Fc region can have a KD value for human fcyriiia of 1.0x10^-6M above, 2.0x10^-6M above, 3.0x10^-6M above, 4.0x10^-6M above, 5.0x10^-6M above, 6.0x10^-6M above, 7.0x10^-6M above, 8.0x10^-6M above, 9.0x10^-6M above, 1.0x10^-5M above, 2.0x10^-5M above, 3.0x10^-5M above, 4.0x10^-5M or more, or 5.0x10^-5M is more than M. In another embodiment, the variant Fc region can have a KD value of 1.0x10 for human fcyriia (type H)^-7M above, 2.0x10^-7M above, 3.0x10^-7M above, 4.0x10^-7M above, 5.0x10^-7M above, 6.0x10^-7M above, 7.0x10^-7M above, 8.0x10^-7M above, 9.0x10^{- 7}M above, 1.0x10^-6M above, 2.0x10^-6M above, 3.0x10^-6M above, 4.0x10^-6M or more, or 5.0x10^-6M is more than M. In another embodiment, the variant Fc region can have a KD value of 2.0x10 for human fcyriia (type R) ^-7M above, 3.0x10^-7M above, 4.0x10^-7M above, 5.0x10^-7M above, 6.0x10^-7M above, 7.0x10^-7M above, 8.0x10^-7M above, 9.0x10^-7M above, 1.0x10^-6M above, 2.0x10^-6M above, 3.0x10^-6M above, 4.0x10^-6M or more, or 5.0x10^-6M is more than M.

In another embodiment, the variant Fc region can have a KD value for monkey Fc γ RIIa of 1.0x10^-6M below, 9.0x10^-7M below, 8.0x10^-7M below, 7.0x10^-7M below, 6.0x10^-7M below, 5.0x10^-7M below, 4.0x10^-7M below, 3.0x10^-7M below, 2.0x10^-7Under M, or 1.0x10^-7M is less than or equal to M. In certain embodiments, monkey Fc γRIIa may be selected from any of monkey Fc γ RIIa1, monkey Fc γ RIIa2 and monkey Fc γ RIIa 3.

When we develop a pharmaceutical product for the treatment of human diseases, it is important to assess its efficacy and safety in monkeys, since the biology of monkeys is close to that of humans. From this point of view, the drug product to be developed preferably has cross-reactivity to both human and monkey in terms of target binding activity.

"Fc γ receptor" (referred to herein as Fc γ receptor, Fc γ R or FcgR) refers to a receptor that can bind to the Fc region of IgG1, IgG2, IgG3 and IgG4 monoclonal antibodies, and indeed refers to any member of the family of proteins encoded by the Fc γ receptor gene. In humans, this family includes Fc γ RI (CD64), including isoforms Fc γ RIa, Fc γ RIb, and Fc γ RIc; fc γ RII (CD32), including isoforms Fc γ RIIa (including allotype H131(H type) and R131(R type)), Fc γ RIIb (including Fc γ RIIb-1 and Fc γ RIIb-2), and Fc γ RIIc; and Fc γ RIII (CD16), including isoforms Fc γ RIIIa (including allotype V158 and F158), and Fc γ RIIIb (including allotype Fc γ RIIIb-NA1 and Fc γ RIIIb-NA2), and any human Fc γ R, not yet discovered Fc γ R isoform or allotype, but not limited thereto. Fc γ RIIb1 and Fc γ RIIb2 are reported to be splice variants of human Fc γ RIIb. In addition, a splice variant called Fc γ RIIb3 has been reported (J Exp Med,1989,170: 1369-1385). In addition to these splice variants, human Fc γ RIIb includes all splice variants registered in NCBI, which are NP _001002273.1, NP _001002274.1, NP _001002275.1, NP _001177757.1, and NP _ 003992.3. In addition, human Fc γ RIIb includes each genetic polymorphism previously reported, as well as Fc γ RIIb (Arthritis Rheum.48:3242-3252 (2003); Kono et al, hum. mol. Genet.14:2881-2892 (2005); and Kyogoju et al, Arthritis Rheum.46:1242-1254(2002)), and each genetic polymorphism to be reported in the future.

In Fc γ RIIa, there are two allotypes, in one allotype the amino acid at position 131 of Fc γ RIIa is histidine (type H), and in the other allotype the amino acid at position 131 is replaced with arginine (type R) (Warrmerdam, j.exp.med.172:19-25 (1990)).

The Fc γ R includes Fc γ rs derived from human, mouse, rat, rabbit and monkey but is not limited thereto, and may be derived from any organism. Mouse Fc γ rs include, but are not limited to, Fc γ RI (CD64), Fc γ RII (CD32), Fc γ RIII (CD16), and Fc γ RIII-2(CD16-2), and any mouse Fc γ R, or Fc γ R isoform. Unless otherwise indicated, the term "monkey Fc γ R" or variants thereof refers to cynomolgus monkey Fc γ RIIa1(SEQ ID NO:220), Fc γ RIIa2(SEQ ID NO:221), Fc γ RIIa3(SEQ ID NO:222), Fc γ RIIb (SEQ ID NO:223), or Fc γ RIIIaS (SEQ ID NO: 224).

The polynucleotide sequence of human Fc γ RI is shown in SEQ ID NO:199 (NM-000566.3); the polynucleotide sequence of human Fc γ RIIa is shown in SEQ ID NO 200(BC020823.1) or SEQ ID NO 201 (NM-001136219.1); the polynucleotide sequence of human Fc γ RIIb is shown in SEQ ID NO 202(BC146678.1) or SEQ ID NO 203 (NM-004001.3); the polynucleotide sequence of human Fc γ RIIIa is shown in SEQ ID NO 204(BC033678.1) or SEQ ID NO 205 (NM-001127593.1); the polynucleotide sequence of human Fc γ RIIIb is shown in SEQ ID NO 206(BC 128562.1).

The amino acid sequence of human Fc γ RI is shown in SEQ ID NO 207 (NP-000557.1); the amino acid sequence of human Fc γ 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 γ 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 Fc γ RIIIa is shown in SEQ ID NO 215(AAH33678.1), SEQ ID NO 216, SEQ ID NO 217 or SEQ ID NO 218; the amino acid sequence of human Fc γ RIIIb is shown in SEQ ID NO:219(AAI 28563.1).

The amino acid sequence of cynomolgus monkey Fc γ RIIa is shown in SEQ ID NO 220(Fc γ RIIa1), SEQ ID NO 221(Fc γ RIIa2) or SEQ ID NO 222(Fc γ RIIa 3); the amino acid sequence of cynomolgus monkey Fc γ RIIb is shown in SEQ ID NO: 223; the amino acid sequence of cynomolgus monkey Fc γ RIIIa is shown in SEQ ID NO 224.

In one aspect, the invention provides polypeptides comprising a variant Fc region having enhanced Fc γ RIIb-binding activity as compared to a corresponding reference Fc γ RIIb-binding polypeptide. In a further aspect, the polypeptide of the invention comprises 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). In certain embodiments, the Fc γ RIIb has the sequence of cynomolgus monkey Fc γ RIIb (SEQ ID NO: 223). In certain embodiments, Fc γ RIIb has the sequence of human Fc γ RIIb (e.g., SEQ ID NOS:212, 213, or 214).

In one aspect, the present invention provides a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity, said variant Fc region comprising at least two amino acid alterations comprising: (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, 233, 234, 235, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334 and 396 (according to EU numbering). In certain embodiments, the Fc γ RIIb has the sequence of cynomolgus monkey Fc γ RIIb (SEQ ID NO: 223). In certain embodiments, the Fc γ RIIb has the sequence of human Fc γ RIIb (e.g., SEQ ID NO:212, 213, or 214).

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

In one aspect, the present invention provides a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity, said variant Fc region comprising at least two amino acid alterations comprising: (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 another embodiment, the variant Fc region comprises an 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 another embodiment, the variant Fc region comprises an amino acid change at least one position selected from the group consisting of: 268. 295, 326 and 330 (according to EU numbering). In certain embodiments, the Fc γ RIIb has the sequence of cynomolgus monkey Fc γ RIIb (SEQ ID NO: 223). In certain embodiments, Fc γ RIIb has the sequence of human Fc γ RIIb (e.g., SEQ ID NOS: 212, 213, or 214).

In another aspect, the present invention provides a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity, the variant Fc region comprising an amino acid change in any one of (1) to (37) below: (1) positions 231, 236, 239, 268, and 330; (2) positions 231, 236, 239, 268, 295, and 330; (3) positions 231, 236, 268, and 330; (4) positions 231, 236, 268, 295, and 330; (5) positions 232, 236, 239, 268, 295, and 330; (6) positions 232, 236, 268, 295, and 330; (7) positions 232, 236, 268, and 330; (8) positions 235, 236, 268, 295, 326 and 330; (9) positions 235, 236, 268, 295, and 330; (10) positions 235, 236, 268, and 330; (11) positions 235, 236, 268, 330 and 396; (12) positions 235, 236, 268, and 396; (13) positions 236, 239, 268, 295, 298 and 330; (14) positions 236, 239, 268, 295, 326 and 330; (15) positions 236, 239, 268, 295, and 330; (16) positions 236, 239, 268, 298 and 330; (17) positions 236, 239, 268, 326 and 330; (18) positions 236, 239, 268, and 330; (19) positions 236, 239, 268, 330 and 396; (20) positions 236, 239, 268 and 396; (21) positions 236 and 268; (22) positions 236, 268, and 295; (23) positions 236, 268, 295, 298 and 330; (24) positions 236, 268, 295, 326 and 330; (25) positions 236, 268, 295, 326, 330 and 396; (26) positions 236, 268, 295, and 330; (27) positions 236, 268, 295, 330 and 396; (28) positions 236, 268, 298 and 330; (29) positions 236, 268, 298 and 396; (30) positions 236, 268, 326 and 330; (31) positions 236, 268, 326, 330 and 396; (32) positions 236, 268, and 330; (33) positions 236, 268, 330 and 396; (34) positions 236, 268 and 396; (35) positions 236 and 295; (36) positions 236, 330 and 396; and (37) positions 236 and 396 (according to EU numbering). In certain embodiments, the Fc γ RIIb has the sequence of cynomolgus monkey Fc γ RIIb (SEQ ID NO: 223). In certain embodiments, the Fc γ RIIb has the sequence of human Fc γ RIIb (e.g., SEQ ID NO:212, 213, or 214).

In another embodiment, the variant Fc region having enhanced fcyriib-binding activity comprises at least one amino acid selected from the group consisting of: (a) asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 231; (b) ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 232; (c) asp at position 233; (d) trp, Tyr at position 234; (e) trp at position 235; (f) ala, Asp, Glu, His, Ile, Leu, Met, Asn, Gln, Ser, Thr, Val at position 236; (g) asp, Tyr at position 237; (h) glu, Ile, Met, Gln, Tyr at position 238; (i) ile, Leu, Asn, Pro, Val at position 239; (j) ile at position 264; (k) a Phe at position 266; (l) Ala, His, Leu at position 267; (m) Asp, Glu at position 268; (n) Asp, Glu, Gly at position 271; (o) Leu at position 295; (p) Leu at position 298; (q) Glu, Phe, Ile, Leu at position 325; (r) Thr at position 326; (s) Ile, Asn at position 327; (t) Thr at position 328; (u) Lys, Arg at position 330; (v) glu at position 331; (w) Asp at position 332; (x) Asp, Ile, Met, Val, Tyr at position 334; and (y) Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 396 (numbering according to EU). In certain embodiments, the Fc γ RIIb has the sequence of cynomolgus monkey Fc γ RIIb (SEQ ID NO: 223). In certain embodiments, the Fc γ RIIb has the sequence of human Fc γ RIIb (e.g., SEQ ID NO:212, 213, or 214).

In another embodiment, the variant Fc region having enhanced Fc γ RIIb-binding activity comprises at least one amino acid alteration (e.g., substitution) selected from the group consisting of: (a) gly, Thr at position 231; (b) asp at position 232; (c) trp at position 235; (d) asn, Thr at position 236; (e) val at position 239; (f) asp, Glu at position 268; (g) leu at position 295; (h) leu at position 298; (i) thr at position 326; (j) lys, Arg at position 330; and (k) Lys, Met at position 396 (numbering according to EU). In another embodiment, the variant Fc region having enhanced Fc γ RIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): asn at position 236, Glu at position 268, Lys at position 330, and Met at position 396 (according to EU numbering). In another embodiment, the variant Fc region having enhanced Fc γ RIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): asn at position 236, Asp at position 268, and Lys at position 330 (according to EU numbering). In another embodiment, the variant Fc region having enhanced Fc γ RIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): asn at position 236, Asp at position 268, Leu at position 295, and Lys at position 330 (according to EU numbering). In another embodiment, the variant Fc region having enhanced Fc γ RIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): thr at position 236, Asp at position 268, and Lys at position 330 (according to EU numbering). In another embodiment, the variant Fc region having enhanced Fc γ RIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): asn at position 236, Asp at position 268, Leu at position 295, Thr at position 326, and Lys at position 330 (according to EU numbering). In another embodiment, the variant Fc region having enhanced Fc γ RIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): trp at position 235, Asn at position 236, Asp at position 268, Leu at position 295, Thr at position 326, and Lys at position 330 (according to EU numbering).

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

In one aspect, the present invention provides a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity, said variant Fc region comprising at least one amino acid alteration at least one position selected from the group consisting of: 234, 238, 250, 264, 267, 307, and 330 (according to EU numbering). In further embodiments, the polypeptide comprises at least one amino acid change at least one position selected from the group consisting of: 234, 250, 264, 267, 307, and 330 (according to EU numbering). In certain embodiments, the Fc γ RIIb has the sequence of cynomolgus monkey Fc γ RIIb (SEQ ID NO: 223). In certain embodiments, the Fc γ RIIb has the sequence of human Fc γ RIIb (e.g., SEQ ID NO:212, 213, or 214).

In another aspect, the present invention provides a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity, the variant Fc region comprising an amino acid change in any one of the following (1) to (9): (1) positions 234, 238, 250, 307, and 330; (2) positions 234, 238, 250, 264, 307, and 330; (3) positions 234, 238, 250, 264, 267, 307, and 330; (4) positions 234, 238, 250, 267, 307, and 330; (5) positions 238, 250, 264, 307, and 330; (6) positions 238, 250, 264, 267, 307, and 330; (7) positions 238, 250, 267, 307, and 330; (8) positions 238, 250, and 307; and (9) positions 238, 250, 307, and 330 (according to EU numbering). In certain embodiments, the Fc γ RIIb has the sequence of cynomolgus monkey Fc γ RIIb (SEQ ID NO: 223). In certain embodiments, Fc γ RIIb has the sequence of human Fc γ RIIb (e.g., SEQ ID NOS: 212, 213, or 214).

In another embodiment, the variant Fc region having enhanced Fc γ RIIb-binding activity comprises at least one amino acid alteration (e.g., substitution) selected from the group consisting of: (a) tyr at position 234; (b) asp at position 238; (c) val at position 250, (d) Ile at position 264; (e) ala at position 267; (f) pro at position 307; and (g) Lys at position 330 (according to EU numbering). In another embodiment, the variant Fc region having enhanced Fc γ RIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): asp at position 238 (numbering according to EU). In another embodiment, the variant Fc region having enhanced Fc γ RIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): asp at position 238, Val at position 250, and Pro at position 307 (numbering according to EU). In another embodiment, the variant Fc region having enhanced Fc γ RIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): asp at position 238, Val at position 250, Pro at position 307, and Lys at position 330 (numbering according to EU). In another embodiment, the variant Fc region having enhanced Fc γ RIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): asp at position 238, Val at position 250, Ile at position 264, Pro at position 307 and Lys at position 330 (numbering according to EU). In another embodiment, the variant Fc region having enhanced Fc γ RIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): asp at position 238, Val at position 250, Ala at position 267, Pro at position 307, and Lys at position 330 (numbering according to EU). In another embodiment, the variant Fc region having enhanced Fc γ RIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): tyr at position 234, Asp at position 238, Val at position 250, Pro at position 307, and Lys at position 330 (numbering according to EU). In another embodiment, the variant Fc region having enhanced Fc γ RIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): tyr at position 234, Asp at position 238, Val at position 250, Ala at position 267, Pro at position 307, and Lys at position 330 (numbering according to EU). In another embodiment, the variant Fc region having enhanced Fc γ RIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): asp at position 238, Val at position 250, Ile at position 264, Ala at position 267, Pro at position 307, and Lys at position 330 (numbering according to EU). In another embodiment, the variant Fc region having enhanced Fc γ RIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): tyr at position 234, Asp at position 238, Val at position 250, Ile at position 264, Pro at position 307, and Lys at position 330 (numbering according to EU). In another embodiment, the variant Fc region having enhanced Fc γ RIIb-binding activity comprises the following amino acid alterations (e.g., substitutions): tyr at position 234, Asp at position 238, Val at position 250, Ile at position 264, Ala at position 267, Pro at position 307, and Lys at position 330 (numbering according to EU). In certain embodiments, the Fc γ RIIb has the sequence of cynomolgus monkey Fc γ RIIb (SEQ ID NO: 223). In certain embodiments, the Fc γ RIIb has the sequence of human Fc γ RIIb (e.g., SEQ ID NO:212, 213, or 214).

In another aspect, the invention provides an isolated polypeptide comprising a variant Fc region having an increased isoelectric point (pI). In certain embodiments, the variant Fc region described herein comprises at least two amino acid alterations in the parent Fc region. In certain embodiments, each amino acid change results in an increase in the isoelectric point (pI) of the variant Fc region as compared to the isoelectric point (pI) of the parent Fc region. It is based on the finding that elimination of antigen from plasma can be promoted with an antibody having an increased pI by modifying at least two amino acid residues, for example, when the antibody is administered in vivo.

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

In one embodiment, the pI value may be increased, e.g., 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, as compared to before modification.

In certain embodiments, the amino acid that increases the pI may be exposed at the surface of the variant Fc region. In the present invention, amino acids that can be exposed to the surface generally refer to amino acid residues located at the surface of the polypeptide constituting the variant Fc region. Amino acid residues located on the surface of a polypeptide are amino acid residues having side chains that can be brought into contact with solvent molecules, which are usually mostly water molecules. However, the side chain does not necessarily need to be in complete contact with the solvent molecule, and an amino acid is defined as a "surface-located amino acid residue" even when only a portion of the side chain contacts the solvent molecule. Amino acid residues located on the surface of the polypeptide also include amino acid residues located close to the surface and may therefore be affected by the charge from another side chain even from amino acid residues that are only partially in contact with solvent molecules. Homology models for polypeptides can be prepared by those skilled in the art, for example, using commercially available software. Alternatively, it is possible to use methods known to the person skilled in the art, such as X-ray crystallography. For example, the coordinates from a three-dimensional model using a computer program such as the insight ii program (Accelrys) are used to determine amino acid residues that may be exposed to the surface. The surface exposable sites can be determined using algorithms known in the art (e.g., Lee and Richards (J.mol. biol.55:379-400(1971)), Connolly (J.appl. Crystal.16: 548-558 (1983). the "size" of the probe used for calculation can be set to a radius of about 1.4 angstroms (A) when the algorithm requires user input of size parameters.

In certain embodiments, the polypeptide comprises both a variant Fc region and an antigen binding domain. In further embodiments, the antigen is a soluble antigen. In one embodiment, the antigen is present in a biological fluid (e.g., plasma, interstitial fluid, lymph, ascites fluid, and pleural fluid) of the subject. The antigen may also be a membrane antigen.

In further embodiments, the antigen binding activity of the antigen binding domain varies depending on the ionic concentration conditions. In one embodiment, the ion concentration is not particularly limited and refers to hydrogen ion concentration (pH) or metal ion concentration. Herein, the metal ion means an ion of a group I element other than hydrogen, such as an alkali metal and a copper group element, a group II element, such as an alkaline earth metal and a zinc group element, a group III element other than boron, a group IV element other than carbon and silicon, a group VIII element, such as an iron group and a platinum group element, an element belonging to subgroup a of groups V, VI and VII, and a metal element, such as antimony, bismuth and polonium. In the present invention, the metal ion includes, for example, calcium ion as described in WO 2012/073992 and WO 2013/125667. In one embodiment, an "ionic concentration condition" may be a condition that focuses on the difference in biological performance of the antigen binding domain between a low ionic concentration and a high ionic concentration. Further, "the antigen binding activity of the antigen binding domain changes depending on the ion concentration condition" means that the antigen binding activity of the antigen binding domain changes between a low ion concentration and a high ion concentration (such an antigen binding domain is referred to herein as "ion concentration-dependent antigen binding domain"). The antigen binding domain may have a higher (stronger) or lower (weaker) antigen binding activity under high ionic concentration conditions than under low ionic concentration conditions. In one embodiment, the ionic concentration-dependent antigen-binding domain (such as a pH-dependent antigen-binding domain or a calcium ion concentration-dependent antigen-binding domain) may be obtained by known methods as described in, for example, WO 2009/125825, WO 2012/073992, and WO 2013/046722.

In the present invention, the antigen-binding activity of the antigen-binding domain under the condition of high calcium ion concentration may be higher than that under the condition of low calcium ion concentration. The high calcium ion concentration is not particularly limited but may be a concentration selected between 100. mu.M to 10mM, between 200. mu.M to 5mM, between 400. mu.M to 3mM, between 200. mu.M to 2mM, between 400. mu.M to 1mM, or between 500. mu.M to 2.5mM, which is preferably close to the plasma (blood) concentration of calcium ions in vivo. Meanwhile, the low calcium ion concentration is not particularly limited but may be a concentration selected between 0.1. mu.M to 30. mu.M, 0.2. mu.M to 20. mu.M, 0.5. mu.M to 10. mu.M, 1. mu.M to 5. mu.M, or 2. mu.M to 4. mu.M, which is preferably close to the calcium ion concentration in the endosome in the early stage in vivo.

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

In the present invention, the antigen binding activity of the antigen binding domain at a low hydrogen ion concentration (neutral pH) can be higher than that at a high hydrogen ion concentration (acidic pH). The acidic pH may be, for example, a pH selected from pH4.0 to pH6.5, from pH4.5 to pH6.5, from pH5.0 to pH6.5, or from pH5.5 to pH6.5, preferably close to the pH in the endosome in vivo. The acidic pH can also be, for example, pH5.8 or pH 6.0. In a particular embodiment, the acidic pH is pH 5.8. Meanwhile, the neutral pH may be, for example, a pH selected from pH6.7 to pH10.0, from pH6.7 to pH9.5, from pH7.0 to pH9.0, or from pH7.0 to pH8.0, which is preferably close to the pH in plasma (blood) in vivo. The neutral pH may also be, for example, pH7.4 or pH 7.0. In a particular embodiment, the neutral pH is pH 7.4.

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

In one embodiment, for example, at least one amino acid residue is substituted with an amino acid residue having a side chain pKa of 4.0 to 8.0, and/or at least one amino acid having a side chain pKa of 4.0 to 8.0 is inserted into the antigen binding domain, as described in WO 2009/125825. The amino acid may be substituted and/or inserted at any site as long as the antigen binding activity of the antigen binding domain is weaker under acidic pH conditions than under neutral pH conditions as compared to before the substitution or insertion. When the antigen binding domain has a variable region or CDR, the site may be within the variable region or CDR. The number of amino acids to be substituted or inserted may be appropriately determined by those skilled in the art; and the number may be more than one. Depending on the hydrogen ion concentration conditions, amino acids with side chain pKa of 4.0-8.0 may be used to alter the antigen binding activity of the antigen binding domain. Such amino acids include, for example, natural amino acids such as His (H) and Glu (E), and unnatural amino acids such as histidine analogs (US2009/0035836), m-NO2-Tyr (pKa 7.45), 3,5-Br2-Tyr (pKa 7.21), and 3,5-I2-Tyr (pKa 7.38) (Heyl et al, bioorg. Med. chem.11(17):3761-3768 (2003)). Amino acids with side chain pKa of 6.0-7.0 can also be used, including, for example, his (h).

In another embodiment, preferred antigen binding domains for variant Fc regions with increased pI are described in japanese patent applications JP2015-021371 and JP2015-185254 and can be obtained by the methods described therein.

In certain embodiments, the variant Fc region having an increased pI comprises 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).

In further embodiments, the variant Fc region having an increased pI comprises 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 another aspect, the present invention provides a polypeptide comprising a variant Fc-region having an increased pI, the variant Fc-region comprising an amino acid change of any one of the following (1) to (10): (1) positions 311 and 341; (2) positions 311 and 343; (3) positions 311, 343 and 413; (4) positions 311, 384 and 413; (5) positions 311 and 399; (6) positions 311 and 401; (7) positions 311 and 413; (8) positions 400 and 413; (9) positions 401 and 413; and (10) positions 402 and 413 (according to EU numbering).

Methods for increasing the pI of a protein are, for example, reducing the number of amino acids having negatively charged side chains (e.g., aspartic acid and glutamic acid) at neutral pH conditions and/or increasing the number of amino acids having positively charged side chains (e.g., arginine, lysine and histidine) at neutral pH conditions. An amino acid with a negatively charged side chain has a negative charge denoted-1 at a sufficiently higher pH than its side chain pKa, a theory known to those skilled in the art. For example, the theoretical pKa of the side chain of aspartic acid is 3.9, and the side chain has a negative charge expressed as-1 at neutral pH conditions (e.g., in solution at pH 7.0). Conversely, an amino acid with a positively charged side chain has a positive charge denoted as +1 at a sufficiently lower pH than its side chain pKa. For example, the theoretical pKa of the side chain of arginine is 12.5, and the side chain has a positive charge expressed as +1 at neutral pH conditions (e.g., in a solution at pH 7.0). Meanwhile, amino acids known to have no charge in the side chain under neutral pH conditions (e.g., in a solution at pH 7.0) include 15 kinds of natural amino acids, i.e., alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, aspartic acid, proline, glutamine, serine, threonine, valine, tryptophan, and tyrosine. Of course, it is understood that the amino acids used to increase the pI may be unnatural amino acids.

From the above, a method for increasing the pI of a protein under neutral pH conditions (e.g., in a solution at pH 7.0) can impart a charge change of +1 to a protein of interest, for example, by replacing aspartic acid or glutamic acid (whose side chain has a negative charge of-1) with an amino acid having an uncharged side chain in the amino acid sequence of the protein. Further, for example, by replacing an amino acid having no charge in the side chain with arginine or lysine (whose side chain has a positive charge of + 1), a charge change of +1 can be given to the protein. Further, by replacing aspartic acid or glutamic acid (the side chain of which has a minus 1) with arginine or lysine (the side chain of which has a plus 1 charge)Charge) can be given to the protein +2 at once. Alternatively, in order to increase the pI of the protein, an amino acid having a side chain without charge and/or an amino acid preferably having a positively charged side chain may be added or inserted into the amino acid sequence of the protein, or an amino acid having a side chain without charge and/or an amino acid preferably having a negatively charged side chain may be deleted in the amino acid sequence of the protein. It is understood, for example, that the N-terminal and C-terminal amino acid residues of a protein have charges derived from the main chain (NH of the amino group at the N-terminal) in addition to the charges derived from the side chains thereof ₃ ⁺And COO of a carbonyl group at the C-terminus^-). Therefore, the pI of the protein can also be increased by adding, deleting, replacing, or inserting some of the functional groups derived from the main chain.

Amino acid substitutions that increase the pI include, for example, in the amino acid sequence of the parent Fc region, amino acid substitutions having a side chain without a charge for an amino acid having a negatively charged side chain, amino acid substitutions having a positively charged side chain for an amino acid having a side chain without a charge, and amino acid substitutions having a positively charged side chain for an amino acid having a negatively charged side chain, either individually or in suitable combinations.

Amino acid insertions or additions that increase the pI include, for example, insertions or additions of amino acids with uncharged side chains and/or insertions or additions of amino acids with positively charged side chains, either alone or in suitable combinations, in the amino acid sequence of the parent Fc region.

Amino acid deletions to increase the pI include, for example, deletion of amino acids with side chains without charge and/or deletion of amino acids with negatively charged side chains in the amino acid sequence of the parent Fc region, either alone or in suitable combination.

In one embodiment, the natural amino acids used to increase pI can be categorized as follows: (a) the amino acid having a negatively charged side chain may be glu (e) or asp (d); (b) amino acids without charge in the side chains 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 amino acid having a positively charged side chain may be His (H), Lys (K), or Arg (R). In one embodiment, the modified amino acid insertion or substitution is lys (k) or arg (r).

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

In one aspect, the invention provides a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity and increased pI, said variant Fc region comprising at least three amino acid changes comprising: (a) 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), 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).

In one aspect, the invention provides a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity and increased pI, and comprising at least three amino acid changes comprising: (a) at least one amino acid change 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 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, the present invention provides a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity and increased pI, the variant Fc region comprising an amino acid change in any one of the following (1) to (9): (1) positions 235, 236, 268, 295, 311, 326, 330 and 343; (2) positions 236, 268, 295, 311, 326, 330 and 343; (3) positions 236, 268, 295, 311, 330 and 413; (4) positions 236, 268, 311, 330, 396 and 399; (5) positions 236, 268, 311, 330 and 343; (6) positions 236, 268, 311, 330, 343 and 413; (7) positions 236, 268, 311, 330, 384 and 413; (8) positions 236, 268, 311, 330 and 413; and (9) positions 236, 268, 330, 396, 400 and 413 (according to EU numbering). In certain embodiments, the Fc γ RIIb has the sequence of cynomolgus monkey Fc γ RIIb (SEQ ID NO: 223). In certain embodiments, the Fc γ RIIb has the sequence of human Fc γ RIIb (e.g., SEQ ID NO:212, 213, or 214).

In one aspect, the invention provides a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity and increased pI, said variant Fc region comprising at least three amino acid changes comprising: (a) at least one amino acid change 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, 401, 402, 413, 420, 422 and 431 (according to EU numbering). In further embodiments, the polypeptide comprises 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 certain embodiments, the Fc γ RIIb has the sequence of cynomolgus monkey Fc γ RIIb (SEQ ID NO: 223). In certain embodiments, the Fc γ RIIb has the sequence of human Fc γ RIIb (e.g., SEQ ID NO:212, 213, or 214).

In another aspect, the invention provides a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity and increased pI, said variant Fc region comprising an amino acid change in any one of (1) to (16) below: (1) positions 234, 238, 250, 264, 307, 311, 330, and 343; (2) positions 234, 238, 250, 264, 307, 311, 330, and 413; (3) positions 234, 238, 250, 264, 267, 307, 311, 330, and 343; (4) positions 234, 238, 250, 264, 267, 307, 311, 330, and 413; (5) positions 234, 238, 250, 267, 307, 311, 330, and 343; (6) positions 234, 238, 250, 267, 307, 311, 330, and 413; (7) positions 234, 238, 250, 307, 311, 330, and 343; (8) positions 234, 238, 250, 307, 311, 330, and 413; (9) positions 238, 250, 264, 267, 307, 311, 330, and 343; (10) positions 238, 250, 264, 267, 307, 311, 330, and 413; (11) positions 238, 250, 264, 307, 311, 330, and 343; (12) positions 238, 250, 264, 307, 311, 330, and 413; (13) positions 238, 250, 267, 307, 311, 330, and 343; (14) positions 238, 250, 267, 307, 311, 330, and 413; (15) positions 238, 250, 307, 311, 330, and 343; and (16) positions 238, 250, 307, 311, 330 and 413 (according to EU numbering).

In another embodiment, the variant Fc region comprises any single alteration, combination of single alterations, or combination of alterations selected from those recited in tables 14-30.

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

In another embodiment, the present invention provides a polypeptide comprising the amino acid sequence of any one of SEQ ID NO 229-381.

As used herein, "parent Fc region" refers to an Fc region prior to the introduction of the amino acid alterations described herein. Preferred examples of parent Fc regions include Fc regions derived from natural antibodies. Antibodies include, for example, IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), and IgM, among others. The antibody may be derived from a human or monkey (e.g., cynomolgus monkey, rhesus monkey, marmoset monkey, chimpanzee, or baboon). Natural antibodies may also include naturally occurring mutations. Various allotypic Sequences of IgG due to genetic polymorphisms are described in "Sequences of proteins of immunological interest", NIH publication No.91-3242, and any of them can be used in the present invention. In particular, for human IgG1, the amino acid sequence at positions 356 to 358(EU numbering) may be DEL or EEM. Preferred examples of parent Fc regions include Fc regions derived 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 parent Fc region is the Fc region derived from heavy chain constant region SG 1(SEQ ID NO: 9). Furthermore, a parent Fc region may be an Fc region produced by adding amino acid changes to an Fc region derived from a native antibody that differ from the amino acid changes described herein.

In addition, amino acid changes made for other purposes may be incorporated into the variant Fc regions described herein. For example, amino acid substitutions that increase FcRn-binding activity (Hinton et al, J.Immunol.176(1):346-356 (2006); Dall' Acqua et al, J.biol.chem.281(33):23514-23524 (2006); Petkova et al, Intl.Immunol.18(12):1759-1769 (2006); Zalevsky et al, nat.Biotechnol.28(2):157-159 (2010); WO 2006/019447; WO 2006/053301; and WO 2009/086320) and amino acid substitutions for increasing antibody heterogeneity or stability (WO 2009/041613) may be added. Alternatively, polypeptides having the property of promoting antigen clearance described in WO 2011/122011, WO 2012/132067, WO 2013/046704 or WO 2013/180201, polypeptides having the property of specifically binding to a target tissue described in WO 2013/180200, polypeptides having the property of repeatedly binding to multiple antigenic molecules described in WO 2009/125825, WO 2012/073992 or WO 2013/047752, may bind to a variant Fc region described herein. Alternatively, the amino acid changes disclosed in EP1752471 and EP1772465 may be incorporated into CH3 of the variant Fc regions described herein in order to confer binding capacity to other antigens. Alternatively, to increase plasma retention, amino acid changes that decrease the pI of the constant region (WO 2012/016227) can be incorporated into the variant Fc regions described herein. Alternatively, to facilitate uptake into cells, amino acid changes that increase the pI of the constant region (WO 2014/145159) can be incorporated into the variant Fc regions described herein. Alternatively, to facilitate elimination of the target molecule from plasma, amino acid changes that increase the pI of the constant region (japanese patent application nos. JP2015-021371 and JP2015-185254) may be incorporated into the variant Fc regions described herein. In one embodiment, such changes may include, for example, substitution at least one position selected from the group consisting of: 311, 343, 384, 399, 400, and 413 (according to EU numbering). In another embodiment, such a substitution may be a replacement of an amino acid with Lys or Arg at each position.

Amino acid changes that enhance human FcRn-binding activity at acidic pH can also be incorporated into the variant Fc regions described herein. Specifically, such changes may include, for example, substitution of Leu for Met at position 428 and Ser for Asn at position 434 according to EU numbering (Zalevsky et al, nat. Biotechnol.28: 157-; ala substitution of Asn at position 434 (Deng et al, Metab. Dispos.38(4):600-605 (2010)); substitution of Met by Tyr at position 252, Ser by Thr at position 254, and Thr by Glu at position 256 (Dall' Acqua et al, J.biol.chem.281:23514-23524 (2006)); gln at position 250 for Thr and Leu at position 428 for Met (Hinton et al, J.Immunol.176(1):346-356 (2006)); his at position 434 for Asn (Zheng et al, Clin. Pharmacol. Ther.89(2): 283) -290(2011), 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 2004/035752, or WO 2002/060919.

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

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

For binding heterologous polypeptides comprising variant Fc regions, techniques can be applied that inhibit binding of undesired homologous polypeptides comprising variant Fc regions by introducing electrostatic repulsion in the interface of the CH2 or CH3 domains of the Fc region, as described in WO 2006/106905.

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

More specifically, for example, an Fc region in which one to three pairs of amino acid residues selected from (1) to (3) shown below have the same charge can be prepared: (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.

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

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

In addition, other known techniques may be used for binding of heterologous polypeptides comprising variant Fc regions. Specifically, such a technique proceeds by: the amino acid side chains present in one Fc region are replaced with larger side chains (knob; which refers to a "bulge") and the amino acid side chains present in the Fc region are replaced with smaller side chains (hole; which refers to an "empty") to place the knob within the hole. This may facilitate efficient binding of Fc region-containing polypeptides having different amino acid sequences to each other (WO 1996/027011; Ridgway et al, prot. Eng.9:617-621 (1996); Merchant et al, nat. Biotech.16,677-681 (1998)).

In addition, other known techniques may also be used for heterologous binding of polypeptides comprising variant Fc regions. Binding of a polypeptide comprising an Fc region can be efficiently introduced using a strand-exchange engineered domain CH3 heterodimer (Davis et al, prot. Eng. Des. & Sel.,23:195-202 (2010)). This technique can also be used to efficiently introduce binding between polypeptides having Fc-containing regions with different amino acid sequences.

In addition, the heterodimeric antibody production techniques described in WO 2011/028952, which utilize the binding of antibodies CH1 and CL and the binding of VH and VL, can also be used.

With regard to the methods described in WO 2008/119353 and WO 2011/131746, it is also possible to use a technique of preparing heterodimeric antibodies by preparing two types of homodimeric antibodies in advance, incubating the antibodies under reducing conditions to dissociate them, and allowing them to recombine.

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

Furthermore, with respect to the method described in WO 2012/058768, it is also possible to use a technique for the preparation of heterodimeric antibodies by adding modifications to the CH2 and CH3 domains.

Polypeptides comprising homologous variant Fc regions are also typically produced as impurities when two polypeptides comprising variant Fc regions having different amino acid sequences are simultaneously expressed to produce a polypeptide comprising a heterologous variant Fc region. In this case, the polypeptide comprising a heterologous variant Fc-region may be efficiently obtained by isolating it from a polypeptide comprising a homologous variant Fc-region using known techniques. A method for efficiently separating and purifying a heterodimeric antibody from a homodimeric antibody by introducing amino acid changes into the variable regions of heavy chains of two types of antibodies to generate isoelectric point differences between the homodimeric antibody and the heterodimeric antibody using ion exchange chromatography has been reported (WO 2007/114325). Another method for purifying heterodimeric antibodies by constructing heterodimeric antibodies comprising two heavy chains derived from mouse IgG2a that binds protein a and rat IgG2b that does not bind protein a using protein a chromatography has been reported (WO 1998/050431 and WO 1995/033844).

In addition, heterodimeric antibodies can be efficiently purified using protein a chromatography by substituting amino acid residues at positions 435 and 436(EU numbering) in the protein a binding site of the heavy chain of the antibody with amino acids such as Tyr or His to yield different protein a binding affinities.

In the present invention, amino acid changes mean any of substitutions, deletions, additions, insertions and modifications, or a combination thereof. In the present invention, amino acid changes may be expressed as amino acid mutations.

When the amino acid residues are substituted, the substitution into different amino acid residues may be performed in order to change the following aspects such as (a) to (c): (a) a polypeptide backbone structure in a folded sheet structure or a helical structure region; (b) charge or hydrophobicity at the target site; or (c) the size of the side chain.

Amino acid residues are classified into the following groups based on their overall side chain properties: (a) hydrophobic: norleucine, Met, Ala, Val, Leu, and Ile; (b) neutral hydrophilic: cys, Ser, Thr, Asn, and Gln; (c) acidic: asp and Glu; (d) basic: his, Lys, and Arg; (e) residues that influence chain positioning: gly and Pro; and (f) aromatic: trp, Tyr, and Phe.

Amino acid changes are made by a variety of methods known to those skilled in the art. The methods include site-directed mutagenesis (Hashimoto-Gotoh et al, Gene 152:271-275 (1995); Zoller, meth. enzymol.100: 468-94500 (1983); Kramer et al, Nucleic Acids Res.12:9441-9456 (1984)); kramer and Fritz, Methods Enzymol.154:350-367 (1987); and Kunkel, Proc.Natl.Acad.Sci.USA 82:488-492(1985)), PCR mutation method and cassette (cassette) mutation method, but are not limited thereto.

The number of amino acid changes introduced into the Fc region is not limited. In certain 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 modifications include post-translational modifications. Specific post-translational modifications may be the addition or deletion of sugar chains. For example, the amino acid residue at position 297(EU numbering) in the constant region of IgG1 can be sugar chain modified. The sugar chain structure used for modification is not limited. For example, sialic acid can be added to the sugar chain of the Fc region (MAbs 2010 Sep-Oct, 2 (5): 519-. Typically, antibodies expressed in eukaryotic cells contain glycosylation in the constant region. For example, it is known that some types of sugar chains are generally added to antibodies expressed in cells such as naturally occurring mammalian antibody-producing cells or eukaryotic cells transformed with expression vectors containing DNA encoding the antibodies.

Eukaryotic cells shown herein include yeast and animal cells. For example, CHO cells and HEK293 cells are representative animal cells for transformation with an expression vector comprising DNA encoding an antibody. On the other hand, constant regions without glycosylation are also included in the present invention. Antibodies in which the constant region is not glycosylated can be obtained by expressing the antibody-encoding gene in prokaryotic cells such as E.coli.

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

In one aspect, the invention provides an isolated polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In certain embodiments, the antibody is a chimeric antibody or a humanized antibody. The source of the antibody is not particularly limited, but examples include human antibodies, mouse antibodies, rat antibodies and rabbit antibodies. In some aspects, the polypeptide is an Fc fusion protein.

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

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

Examples of chemokines that can be bound by an antibody or fusion protein comprising a variant Fc region of the present invention and/or recombinantly fused to a polypeptide comprising the disclosed variant Fc region include, but are not limited to, CC chemokines such as CCL1 through CCL28, CXC chemokines such as CXCL1 through CXCL17, C chemokines such as XCL1 through XCL2, and CX3C chemokines such as CX3CL 1.

Examples of receptors that can be bound by antibodies or fusion proteins containing the variant Fc regions of the present invention and/or recombinantly fused to polypeptides comprising the disclosed variant Fc regions include, but are not limited to, receptors belonging to a family of receptors 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. Receptors belonging to these receptor families and their characteristics have been described in many documents, such as Cooke, ed.New comprehensive Biochemistry Vol.18B "horrons and the action Part II" pp.1-46(1988) Elsevier Science Publishers BV; patthy (Cell 61(1):13-14 (1990)); ullrich (Cell 61(2): 203-; massague (Cell 69(6): 1067-; miyajima et al (Annu. Rev. Immunol.10:295-331 (1992)); taga et al (FASEB J.6:3387-3396 (1992)); fantl et al (Annu. Rev. biochem.62:453-481 (1993)); smith et al (Cell 76(6):959-962 (1994)); and Flower (Biochim. Biophys. acta 1422(3):207-234 (1999)).

Examples of specific receptors belonging to the above-mentioned receptor family include the human or mouse Erythropoietin (EPO) receptor (Jones et al, Blood 76(1):31-35 (1990); D' Andrea et al, Cell 57(2):277-285(1989)), the human or mouse granulocyte-colony stimulating factor (G-CSF) receptor (Fukunaga et al, Proc. Natl. Acad. Sci. USA 87(22):8702-8706(1990), mG-CSFR; Fukunaga et al, Cell 61(2):341 (1990)), the human or mouse Thrombopoietin (TPO) receptor (Vigon et al, Proc. Natl. Acad. Sci. USA 89(12): 5640) -5644 (1992)), Skoda et al, EMBO J.12(7): 2645) Asn et al (1993)), or the human insulin receptor (Natl. Sci. USA.459.USA.22): 5640) -5644(1992), the Skoda et al, EMBO J.12 (USA) receptor (19891), the human or mouse insulin receptor (USA 5. Sal-459.22) (Proc. Sci., USA) receptor (19891 ), the human insulin receptor (19891), the human or the human erythropoietin receptor (20) (USA 22, 19891) receptor (19891) of human granulocyte, human or mouse platelet-derived growth factor (PDGF) receptor (Gronwald et al, Proc. Natl. Acad. Sci. USA.85(10):3435-3439(1988)), human or mouse Interferon (IFN) -alpha and beta receptors (Uze et al, Cell 60 (2): 225-234 (1990); Novick et al, Cell 77(3):391-400(1994)), human or mouse leptin receptor, human or mouse Growth Hormone (GH) receptor, human or mouse Interleukin (IL) -10 receptor, human or mouse insulin-like growth factor (IGF) -I receptor, human or mouse Leukemia Inhibitory Factor (LIF) receptor, and human or mouse hairy neurotrophic factor (CNTF) receptor.

Cancer antigens are antigens that are expressed when cells become malignant, and are also referred to as tumor-specific antigens. Abnormal sugar chains appearing on the cell surface or on protein molecules when cells become cancer cells are also cancer antigens, and they are also called sugar chain cancer antigens. Examples of cancer antigens that can be bound by antibodies or fusion proteins comprising the variant Fc regions of the present invention include, but are not limited to, GPC3, which is a receptor belonging to the GPI-anchored receptor family mentioned above, and is also expressed in a variety of cancers including liver cancer (Midorikawa et al, int.J. cancer 103(4):455-465(2003)), and EpCAM, which is expressed in a variety of cancers including lung cancer (Linnenbach et al, Proc. Natl.Acad.Sci.USA 86(1):27-31(1989)), CA19-9, CA15-3, and sialylSSEA-1 (SLX).

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

Examples of differentiation antigens that can be bound by antibodies or fusion proteins comprising the variant Fc regions of the present invention and/or recombinantly fused to polypeptides comprising the disclosed variant Fc regions include, but are not limited to, CD11, CD15, CD41, CD42, CD45RO, CD49, CD106, CD122, CD126, and CDw 130.

Immunoglobulins include IgA, IgM, IgD, IgG and IgE. The immune complex includes at least any immunoglobulin component.

Other examples of antigens that can be bound by antibodies or fusion proteins comprising a variant Fc region of the present invention and/or recombinantly fused to a polypeptide comprising the disclosed variant Fc region include, but are not limited to, 17-IA, 4-1BB, 4Dc, 6-keto-PGF 1a, 8-iso-PGF 2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin A, activin AB, activin B, activin C, activin RIA, activin RIAALK-2, activin RIAALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, AM, ALP-7, ALK-7, alpha-1-ALK-V-beta-trypsin antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic peptide, av/B3 integrin, Axl, B2M, B7-1, B7-2, B7-H, B-lymphocyte stimulating factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, B-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BACE, BMP-2a, BMP-3 osteogenin, BMP-4, BMP-2B, BMP-5, BMP-6 VPR-1, BMP-7(OP-1), BMP-8 (ALP-8, ALK-2 a), ALK-2-3, GRIA (PR-PR 3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, B-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complementary 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, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL18, CCL18, CCL18, CCL18, CCL18, CCL-E, cathepsin H-E-H-D, cathepsin E-E, and other elements, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR11, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD4, CD4, CD4, CD4, CD11 4, CD11 4, CD4, CD 6856856856856854, CD 6856856856856856856856856856856854, CD 6856856856856856856856856854, CD 6856856856854, CD4, CD4, CD4, CD 685137, CD4, CD4, CD4, CD4, CD4, CD4, CD4 (4) 4, CD4, CD4, CD4, CD4, CD4, CD4, CD4, CD4, CD4, CD4, CD 4-4, CD4, CD4, C, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, a cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, complement regulatory factor (decay accelerating factor), des (1-3) -IGF-I (cerebroing-1), Dhh, digoxin, DNAM-1, DNAase, Dpp, DPPIV/CD 3, 3, ECAD, FcRAD, EDA-A3, EDA-A3, EDAR, EGF, EGFR (ErbB-1), EMMPR, ENA, Fasina, endothelin receptor, brain eosinophil, peptidase, 3, EPO-A3, EphEC-A3, EPO-activating factor, EPO-C-1, EPO-C-2, EPO-C-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle stimulating hormone, chemokine CX3(fractalkine), FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3(Vgr-2), GDF-5(BMP-14, CDMP-1), GDF-6(BMP-13, CDMP-2), GDF-7(BMP-12, CDMP-3), GDF8 (muscle growth inhibitory factor), GDF-9, GDF-15(MIC-1), GDNF, GFAP, GFRa-1, GFR-2, GITR-4, GITR-TR- α -2, GITR 638, glycoprotein IIb/IIIa (GPIIb/IIIa), GM-CSF, gp130, gp72, GRO, growth hormone releasing hormone, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, Hematopoietic Growth Factor (HGF), hepB gp120, heparanase, Her2, Her2/neu (ErbB-2), Her3(ErbB-3), Her4(ErbB-4), Herpes Simplex Virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, Human Cytomegalovirus (HCMV), Human Growth Hormone (HGH), EM, I-309, ICAM, ICAM 1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, Interferon (IFN) - α, IFN- β, IFN- γ, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, integrin α 2, integrin α 3, integrin α 4/β 1, integrin α 4/β 7, integrin α 5(α V), integrin α 5/β 1, integrin α 5/β 3, integrin α 6, integrin β 1, integrin β 2, interferon γ, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1bp1, LBP, LDGF, LECT2, lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloprotease S, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, IF, MPF, Mpo, MSK, MSP, mucin (Muc1), C18, Mullerian inhibitory substance, Mug, Mulleian, Musk, NAP, NCSK, NCAM, NCAD 90, NCAD-A, renin (neprilysin), neurotrophic factor-3, -4 or-6, neurturin, Nerve Growth Factor (NGF), NGFR, NGF- β, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, P150, P95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDK-1, PECAM, PEME, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIP, PP14, Pro-insulin, prorelaxin (proRerelaxin), protein C, PS, PSA, PSCA, Prostate Specific Membrane Antigen (PSMA), PTsyncytial N, PTRSV, PTN 62, RANR 2, RSV, RPF, RSV A, RSV B chain 7375, RSV, rF, RSV chain relaxing factor (S) and K-1, 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 receptors (e.g., T-cell receptor alpha/beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-Pan Specific, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, Ctk-thrombin, thyroid stimulating hormone, tie, TIMP, TIQ, tissue factor, TMEFF, Tmpo, TMPRSS, TNF, TNF- α, TNF- α β, TNF- β 2, TNFac, TNF-RI, TNF-RII, TNFRSF10 (TRAIL Apo-2, DR), TNFRSF10 (TRAIL DR, KILLER, TRICK-2A, TRICK-B), TNFRSF10 (TRAIL DcR, LIT, TRID), TNFRSF10 (TRAIL DcR, TRUNDD), TNFRSF11 (RANK ODF R, NCE R), TNFRSF11 (OPG IF, TRTR), TNFRSF (TWEAK R FN), TNFRSF13 (TACI), TNFRSF13 (TNFRSF), TNFRSF (HVEM, HveA, GHLIGHT, TNFRSF (NGFR 75), TNFRSF (BCMA), TNFRSF (GITRROSF), TNFRSF (TRAFLR 120, TNFRSF (TNFRSF) and TNFRSF (TNFRSR) including TNFRSF, TNFRSF 80, TNFRSF (TNFRSF II), TNFRSF (TNFRSF-R, TNFRSF (TNFRSF-80, TNFRSF-R), TXGP1), TNFRSF (CD40 p), TNFRSF (Fas Apo-1, APT, CD), TNFRSF6 (DcR M, TR), TNFRSF (CD), TNFRSF (CD), TNFRSF (4-1BB CD137, ILA), TNFRSF (DR), TNFRSF (DcTRAIL R TNFRRH), TNFRST (DcTRAIL TNFRRH), TNFRSF (DR Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFRSF (TRAIL Apo-2 ligand, TL), TNFRSF (TRANCE/RANK ligand, ODF, OPG ligand), TNFRSF (TWEAK Apo-3 ligand, DR ligand), TNFRSF (), TNFRSF13 (BAFF, TALL, THANK, TNFRSF), TNFRSF (ligand), TNFRSF (TL 1/VEGFGI ligand), TNFRSF (GITR ligand, TL ligand, TNFRSF1 (TNF-factor-Con), TNFRSF (TNFRSF, TNFRSF-154, TNFRSF-P ligand, TNFRSF (LTSF-2 ligand, TNFRSF (LTSF, LTSF-G ligand, gp39, HIGM1, IMD3, TRAP), TNFSF6(Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7(CD27 ligand CD70), TNFSF8(CD30 ligand CD153), TNFSF9(4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expressing Lewis-Y-associated carbohydrate, TWEAK, TXB2, TXB Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin-2, VEFGR-1(flt-1), VEGF, VEGFR, VLT-3, VEGFR-4, VIGAP, VNA-4, VN-A-1, VN-A-V-A, VN-A-1, VN-A-and VEGFR, WIF-1, WNT, WNT, WNT 2/13, WNT, WNT3, WNT, WNT5, WNT5, WNT, WNT7, WNT7, WNT8, WNT8, WNT9, WNT9, WNT10, WNT10, WNT, WNT, XCL, XCR, XCR, XEDAR, XIAP, XPD, HMGB, IgA, Abeta, CD, CD, DDR, DKK, EREG, IL-17/IL-17R, IL-20/IL-20R, oxidized LDL, PCSK, prekallikrein, RON, TMEM16, SOD, chromogranin A, chromogranin B, tau, VAP, high molecular weight kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, WNT 1.6, Nav1.7, NavC 1.4, NavC 1.5, NavC 1.3, NavC 4, NavC 1, NavC 3, NavC 4, NavC 3, NavC 4, NavC 3, and C4, prothrombin, thrombin, tissue factor, factor V, factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X, factor Xa, factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, syndecan-1, syndecan-2, syndecan-3, syndecan-4, LPA, and S1P; and receptors for hormones and growth factors.

As described herein, one or more amino acid residues are allowed to be changed in the amino acid sequence constituting the variable region as long as the antigen-binding activity thereof is maintained. When the variable region amino acid sequence is changed, there is no particular limitation on the site of the change and the number of changed amino acids. For example, the amino acids present in the CDRs and/or FRs may be suitably altered. When changing amino acids in the variable region, the binding activity is preferably maintained without particular limitation; and, for example, the binding activity can be 50% or more, 80% or more, and 100% or more, as compared to before the alteration. In addition, amino acid changes may result in increased binding activity. For example, the binding activity may be 2-fold, 5-fold, 10-fold, etc. prior to the alteration. The change in the amino acid sequence may be at least one of amino acid residue substitution, addition, deletion and modification.

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

The antibody variable regions described herein may have any sequence, and may be antibody variable regions of any origin, such as mouse antibodies, rat antibodies, rabbit antibodies, goat antibodies, camel antibodies, humanized antibodies produced by humanizing these non-human antibodies, and human antibodies. In addition, these antibodies may incorporate various amino acid substitutions in their variable regions to improve their antigen binding, pharmacokinetics, stability and immunogenicity. The variable region may be capable of repeatedly binding antigen due to its pH dependence in antigen binding (WO 2009/125825).

Kappa and lambda chains are present in the antibody light chain constant region and either is acceptable. In addition, it may have some amino acid changes such as substitutions, deletions, additions, and/or insertions.

In addition, polypeptides comprising the variant Fc regions described herein can be made into Fc fusion proteins by linking to other proteins, such as peptides having physiological activity. Such fusion proteins can be multimers of at least two polypeptides comprising a variant Fc region. Examples of other proteins include, but are not limited to, receptors, adhesion molecules, ligands, and enzymes.

Examples of Fc fusion proteins include proteins fused to receptors that bind target molecules using an Fc region, including TNFR-Fc fusion proteins, IL1R-Fc fusion proteins, VEGFR-Fc fusion proteins, and CTLA4-Fc fusion proteins (Econoids et al, nat. Med.9(1):47-52 (2003); Dumont et al, BioDrugs.20(3):151-60 (2006)). Furthermore, the proteins to be fused may be other molecules having target binding activity, for example, scFv (WO 2005/037989), single domain antibodies (WO 2004/058821; WO 2003/002609), antibody-like molecules (Davinder, Current. Op. Biotech.17: 653-. Furthermore, antibodies and Fc fusion proteins can be multispecific and can bind to multiple types of target molecules or epitopes.

B. Recombinant methods and compositions

Antibodies can be prepared using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid is provided that encodes an anti-myostatin antibody as described herein. In another embodiment, an isolated nucleic acid is provided that encodes a polypeptide described herein comprising a variant Fc region or a parent Fc region. Such nucleic acids may encode an amino acid sequence comprising an antibody VL and/or an amino acid sequence comprising an antibody VH (e.g., a light chain and/or a heavy chain of an antibody). In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, host cells comprising such nucleic acids are provided. In one embodiment, the host cell comprises (e.g., is transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VL and an amino acid sequence comprising an antibody VH, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VL and a second vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VH. In one embodiment, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or a lymphocyte (e.g., Y0, NS0, and Sp20 cells). In one embodiment, a method of making an anti-myostatin antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody as described above, and optionally recovering the antibody from the host cell (or host cell culture medium). In another embodiment, a method of producing a polypeptide comprising a variant Fc region or a parent Fc region is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the polypeptide under conditions suitable for expression of a polypeptide as described above, such as an antibody, Fc region or variant Fc region, and optionally recovering the polypeptide from the host cell (or host cell culture medium).

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

Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be made in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. nos. 5,648,237, 5,789,199, and 5,840,523. (see also, Charlton, Methods in Molecular Biology, Vol.248(B.K.C.Lo, ed., Humana Press, Totowa, NJ,2003), pp.245-254, which describes the expression of antibody fragments in E.coli). After expression, the antibody can be isolated from the bacterial cell paste into a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized" resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, nat. Biotech.22: 1409-.

Host cells suitable for expression of glycosylated antibodies also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Various baculovirus strains have been identified which can be used with insect cells, particularly for transfecting Spodoptera frugiperda (Spodoptera frugiperda) cells.

Plant cell cultures may also be used as hosts. See, for example, U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 (which describe PLANTIBODIIES for antibody production in transgenic plants^TMA technique).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for suspension culture may be useful. Other examples of useful mammalian host cell lines are the SV40(COS-7) transformed monkey kidney CV1 cell line; human embryonic kidney cell lines (293 or 293 cells as described, for example, in Graham et al, J.Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells, as described, for example, in Mather, biol. reprod.23:243-251 (1980)); monkey kidney cells (CV 1); VERO cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK; buffalo mouse hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); TRI cells as described, for example, in Mather et al, Annals N.Y.Acad.Sci.383:44-68 (1982); MRC 5 cells; and FS4 cells Hamster Ovary (CHO) cells including DHFR^-CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp 2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol.248(B.K.C.Lo, ed., Humana Press, Totowa, NJ), pp.255-268 (2003).

Antibodies with pH-dependent characteristics can be obtained using screening methods and/or mutagenesis methods, e.g. as described in WO 2009/125825. The screening method may include any method of identifying antibodies having pH-dependent binding properties in a population of antibodies specific for a particular antigen. In certain embodiments, the screening method can include measuring more than one binding parameter (e.g., KD or KD) of individual antibodies within the initial antibody population at acidic pH and neutral pH. The binding parameters of the antibody can be measured using, for example, the following methods: surface plasmon resonance, or any other analytical method that allows quantitative or qualitative assessment of the binding properties of an antibody to a particular antigen. In certain embodiments, the screening method can include identifying antibodies that bind to the antigen with an acidic/neutral KD ratio of 2 or greater. In another embodiment, the screening method may comprise identifying antibodies that bind antigen with a pH5.8/pH7.4KD ratio of 2 or greater. Alternatively, the screening method may comprise identifying antibodies that bind antigen with an acidic/neutral kd ratio of 2 or greater. In another embodiment, the screening method may comprise identifying antibodies that bind antigen at a pH5.8/pH7.4kd ratio of 2 or greater.

In another embodiment, the mutagenesis method may comprise introducing amino acid deletions, substitutions or additions within the heavy and/or light chain of the antibody to enhance the pH-dependent binding of the antibody to the antigen. In certain embodiments, mutagenesis can be performed within one or more variable domains of an antibody, e.g., within one or more HVRs (e.g., CDRs). For example, mutagenesis can include replacing an amino acid within one or more HVRs (e.g., CDRs) of an antibody with another amino acid. In certain embodiments, mutagenesis can include substitution of one or more amino acids in at least one HVR (e.g., CDR) of the antibody to histidine. In certain embodiments, "enhanced pH-dependent binding" refers to a mutated form of an antibody that exhibits a greater acidic/neutral KD ratio or a greater acidic/neutral KD ratio than the original "parent" (i.e., less pH-dependent) form of the antibody prior to mutagenesis. In certain embodiments, the acidic/neutral KD ratio of the mutant form of the antibody is 2 or greater. In certain embodiments, the antibody in the mutated form has a pH5.8/pH7.4KD ratio of 2 or greater. Alternatively, the acidic/neutral kd ratio of the mutated form of the antibody is 2 or more. In another embodiment, the antibody in a mutated form has a pH5.8/pH7.4kd ratio of 2 or greater.

Polyclonal antibodies are preferably prepared in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. It may be useful to use bifunctional or derivatizing reagents, for example, maleimidobenzoate succinimido ester (conjugated via a cysteine residue), N-hydroxysuccinimido (conjugated via a lysine residue), glutaraldehyde, succinic anhydride, SOCl₂Or R is¹N ═ C ═ NR, where R and R¹Are different alkyl groups, and the relevant antigen is conjugated to a protein that is immunogenic in the species to be immunized (e.g., keyhole limpet hemocyanin (keyhole limpet hemocyanin), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor).

Animals (typically non-human mammals) are immunized against an antigen, immunogenic conjugate or derivative by combining, for example, 100 μ g or 5 μ g of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, animals were boosted with 1/5 to 1/10 of the original amount of peptide or conjugate in freund's complete adjuvant by subcutaneous injection at multiple sites. After 7 to 14 days, the animals were bled and the serum was assayed for antibody titer. Animals were boosted until titer plateaus. Preferably, the animal is boosted with conjugates of the same antigen conjugated to different proteins and/or conjugated through different cross-linking agents. Conjugates can also be prepared as protein fusions in recombinant cell culture. In addition, aggregating agents such as alum are useful for enhancing immune responses.

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

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

The immunological agent will typically comprise an antigenic protein or a fusion variant thereof. Typically, Peripheral Blood Lymphocytes (PBLs) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian origin are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusion agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103).

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

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

For the production of monoclonal antibodies against the antigen, the medium in which the hybridoma cells are cultured is assayed. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. For example, binding affinity can be determined by Scatchard analysis of Munson, anal. biochem.107(1):220-239 (1980).

After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and/or activity, the clones can be subcloned by limiting dilution methods and cultured by standard methods (Goding, supra). A medium suitable for this purpose includes, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells can be cultured in vivo in mammals as tumors.

The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid or serum by conventional immunoglobulin purification methods such as, for example, protein a-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

Antibodies can be prepared by immunizing a suitable host animal against an antigen. In one embodiment, the antigen is a polypeptide comprising full length myostatin. In one embodiment, the antigen is a polypeptide comprising latent myostatin. In one embodiment, the antigen is a polypeptide comprising a myostatin pro peptide. In one embodiment, the antigen is a polypeptide comprising a region corresponding to amino acids at positions 21 to 100 of the myostatin pro peptide (SEQ ID NO: 78). In one embodiment, the antigen is a polypeptide comprising an amino acid at position 21-80, 41-100, 21-60, 41-80, 61-100, 21-40, 41-60, 61-80, or 81-100 of the myostatin pro peptide (SEQ ID NO: 78). Also included in the invention are antibodies prepared by immunizing an animal against an antigen. The antibodies may bind any feature, alone or in combination, as described above in the "exemplary anti-myostatin antibodies".

The Fc region can be obtained by: the fractions adsorbed on the protein a column were re-eluted after partial digestion of IgG1, IgG2, IgG3, IgG4 monoclonal antibodies, etc. with proteases such as pepsin. The protease is not particularly limited as long as it can digest a full-length antibody by appropriately setting enzyme reaction conditions such as pH to produce Fab and F (ab')2 in a limited manner, and examples include pepsin and papain.

Furthermore, the present invention provides a method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity as compared to a polypeptide comprising a parent Fc region, the method comprising introducing at least one amino acid alteration to the parent Fc region. In some aspects, the polypeptide produced is an antibody. In certain embodiments, the antibody is a chimeric antibody or a humanized antibody. In some aspects, the polypeptide produced is an Fc fusion protein.

In certain embodiments, the method of preparation comprises the steps of: (a) preparing a polypeptide comprising a parent Fc region; (b) introducing at least one amino acid change to a parent Fc region within the polypeptide; (c) measuring monkey Fc γ RIIb-binding activity of the polypeptide comprising the parent Fc region and the polypeptide comprising the Fc region of step (b); and (d) selecting a polypeptide comprising a variant Fc region that has enhanced monkey Fc γ RIIb-binding activity compared to a polypeptide comprising a parent Fc region.

In certain embodiments, the method of preparation comprises the steps of: (a) preparing a polypeptide comprising a parent Fc region; (b) introducing at least one amino acid change to a parent Fc region within the polypeptide; (c) measuring monkey Fc γ RIIIa-binding activity of the polypeptide comprising the parent Fc region and the polypeptide comprising the Fc region of step (b); and (d) selecting a polypeptide comprising a variant Fc region that has reduced monkey Fc γ RIIIa-binding activity as compared to a polypeptide comprising a parent Fc region.

In certain embodiments, the method of preparation comprises the steps of: (a) preparing a polypeptide comprising a parent Fc region; (b) introducing at least one amino acid change to a parent Fc region within the polypeptide; (c) measuring human Fc γ RIIb-binding activity of the polypeptide comprising the parent Fc region and the polypeptide comprising the Fc region in step (b); and (d) selecting a polypeptide comprising a variant Fc region that has enhanced human fcyriib-binding activity as compared to a polypeptide comprising a parent Fc region.

In certain embodiments, the method of preparation comprises the steps of: (a) preparing a polypeptide comprising a parent Fc region; (b) introducing at least one amino acid change to a parent Fc region within the polypeptide; (c) measuring human Fc γ RIIIa-binding activity of the polypeptide comprising the parent Fc region and the polypeptide comprising the Fc region in step (b); and (d) selecting a polypeptide comprising a variant Fc region that has reduced human fcyriiia-binding activity as compared to a polypeptide comprising a parent Fc region.

In certain embodiments, the method of preparation comprises the steps of: (a) preparing a polypeptide comprising a parent Fc region; (b) introducing at least one amino acid change to a parent Fc region within the polypeptide; (c) measuring human Fc γ RIIa (type H) -binding activity of the polypeptide comprising the parent Fc region and the polypeptide comprising the Fc region in step (b); and (d) selecting a polypeptide comprising a variant Fc region that has reduced human Fc γ RIIa (type H) -binding activity as compared to a polypeptide comprising a parent Fc region.

In certain embodiments, the method of preparation comprises the steps of: (a) preparing a polypeptide comprising a parent Fc region; (b) introducing at least one amino acid change to a parent Fc region within the polypeptide; (c) measuring human Fc γ RIIa (R-type) -binding activity of the polypeptide comprising the parent Fc region and the polypeptide comprising the Fc region in step (b); and (d) selecting a polypeptide comprising a variant Fc region having reduced human Fc γ RIIa (R-type) -binding activity compared to a polypeptide comprising a parent Fc region.

In one aspect, in the above-mentioned method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity, at least one amino acid at least one position selected from the group consisting of: 231. 232, 233, 234, 235, 236, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334 and 396 (according to EU numbering). In certain embodiments, the Fc γ RIIb has the sequence of cynomolgus monkey Fc γ RIIb (SEQ ID NO: 223). In certain embodiments, Fc γ RIIb has the sequence of human Fc γ RIIb (e.g., SEQ ID NOS: 212, 213, or 214).

In another aspect, in the above-mentioned method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity, one amino acid at position 236 is altered.

In another aspect, in the above-mentioned method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity, at least two amino acids are altered, the alteration comprising: (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, 233, 234, 235, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334 and 396 (according to EU numbering). In certain embodiments, the Fc γ RIIb has the sequence of cynomolgus monkey Fc γ RIIb (SEQ ID NO: 223). In certain embodiments, the Fc γ RIIb has the sequence of human Fc γ RIIb (e.g., SEQ ID NO:212, 213, or 214).

In another aspect, in the above-mentioned method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity, at least two amino acids are altered, said alterations comprising: (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 certain embodiments, the Fc γ RIIb has the sequence of cynomolgus monkey Fc γ RIIb (SEQ ID NO: 223). In certain embodiments, the Fc γ RIIb has the sequence of human Fc γ RIIb (e.g., SEQ ID NO:212, 213, or 214).

In another aspect, in the above-mentioned method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity, at least two amino acids are altered, said alterations comprising: (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: 268. 295, 326 and 330 (according to EU numbering). In certain embodiments, the Fc γ RIIb has the sequence of cynomolgus monkey Fc γ RIIb (SEQ ID NO: 223). In certain embodiments, the Fc γ RIIb has the sequence of human Fc γ RIIb (e.g., SEQ ID NO:212, 213, or 214).

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

In another aspect, in the above-mentioned method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity, the amino acid change is selected at each position from the group consisting of: (a) asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 231; (b) ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 232; (c) asp at position 233; (d) trp, Tyr at position 234; (e) trp at position 235; (f) ala, Asp, Glu, His, Ile, Leu, Met, Asn, Gln, Ser, Thr, Val at position 236; (g) asp, Tyr at position 237; (h) glu, Ile, Met, Gln, Tyr at position 238; (i) ile, Leu, Asn, Pro, Val at position 239; (j) ile at position 264; (k) a Phe at position 266; (l) Ala, His, Leu at position 267; (m) Asp, Glu at position 268; (n) Asp, Glu, Gly at position 271; (o) Leu at position 295; (p) Leu at position 298; (q) Glu, Phe, Ile, Leu at position 325; (r) Thr at position 326; (s) Ile, Asn at position 327; (t) Thr at position 328; (u) Lys, Arg at position 330; (v) glu at position 331; (w) Asp at position 332; (x) Asp, Ile, Met, Val, Tyr at position 334; and (y) Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 396 (numbering according to EU). In certain embodiments, the Fc γ RIIb has the sequence of cynomolgus monkey Fc γ RIIb (SEQ ID NO: 223). In certain embodiments, Fc γ RIIb has the sequence of human Fc γ RIIb (e.g., SEQ ID NOS: 212, 213, or 214).

In another aspect, in the above-mentioned method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity, the amino acid change is selected at each position from the group consisting of: (a) gly, Thr at position 231; (b) asp at position 232; (c) trp at position 235; (d) asn, Thr at position 236; (e) val at position 239; (f) asp, Glu at position 268; (g) leu at position 295; (h) leu at position 298; (i) thr at position 326; (j) lys, Arg at position 330, and (k) Lys, Met at position 396 (according to EU numbering). In certain embodiments, the Fc γ RIIb has the sequence of cynomolgus monkey Fc γ RIIb (SEQ ID NO: 223). In certain embodiments, the Fc γ RIIb has the sequence of human Fc γ RIIb (e.g., SEQ ID NO:212, 213, or 214).

In another aspect, the above-mentioned amino acid changes in the method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity are: an Asn at position 236, a Glu at position 268, a Lys at position 330, and a Met at position 396 (numbering according to EU). In another aspect, the above-mentioned amino acid changes in the method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity are: asn at position 236, Asp at position 268, and Lys at position 330 (according to EU numbering). In another aspect, the above-mentioned amino acid changes in the method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity are: asn at position 236, Asp at position 268, Leu at position 295, and Lys at position 330 (according to EU numbering). In another aspect, the above-mentioned amino acid changes in the method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity are: thr at position 236, Asp at position 268, and Lys at position 330 (according to EU numbering). In another aspect, the above-mentioned amino acid changes in the method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-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 another aspect, the above-mentioned amino acid changes in the method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-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).

In one aspect, in the above-mentioned method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity, at least one amino acid at least one position selected from the group consisting of: 234, 238, 250, 264, 267, 307, and 330 (according to EU numbering). In certain embodiments, the Fc γ RIIb has the sequence of cynomolgus monkey Fc γ RIIb (SEQ ID NO: 223). In certain embodiments, the Fc γ RIIb has the sequence of human Fc γ RIIb (e.g., SEQ ID NO:212, 213, or 214).

In another aspect, in the above-mentioned method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity, one amino acid at position 238 is altered.

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

In another aspect, in the above-mentioned method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity, the amino acid at any one of the following positions (1) to (9) is changed: (1) at positions 234, 238, 250, 307 and 330; (2) at positions 234, 238, 250, 264, 307 and 330; (3) at positions 234, 238, 250, 264, 267, 307, and 330; (4) at positions 234, 238, 250, 267, 307 and 330; (5) at positions 238, 250, 264, 307 and 330; (6) at positions 238, 250, 264, 267, 307, and 330; (7) at positions 238, 250, 267, 307 and 330; (8) at positions 238, 250 and 307; and (9) at positions 238, 250, 307, and 330 (according to EU numbering).

In another aspect, in the above-mentioned method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity, the amino acid change is selected at each position from the group consisting of: (a) tyr at position 234; (b) asp at position 238; (c) val at position 250; (d) ile at position 264; (e) ala at position 267; (f) pro at position 307; and (g) Lys at position 330 (according to EU numbering). In another aspect, the above-mentioned amino acid changes in the method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity are: asp at position 238 (numbering according to EU). In another aspect, the above-mentioned amino acid changes in the method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity are: asp at position 238, Val at position 250, and Pro at position 307 (numbering according to EU). In another aspect, the above-mentioned amino acid changes in the method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity are: asp at position 238, Val at position 250, Pro at position 307, and Lys at position 330 (numbering according to EU). In another aspect, the amino acid changes mentioned above for use in the method of preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity are: asp at position 238, Val at position 250, Ile at position 264, Pro at position 307 and Lys at position 330 (numbering according to EU). In another aspect, the above-mentioned amino acid changes in the method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity are: asp at position 238, Val at position 250, Ala at position 267, Pro at position 307, and Lys at position 330 (numbering according to EU). In another aspect, the above-mentioned amino acid changes in the method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity are: tyr at position 234, Asp at position 238, Val at position 250, Pro at position 307, and Lys at position 330 (numbering according to EU). In another aspect, the above-mentioned amino acid changes in the method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-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 (numbering according to EU). In another aspect, the above-mentioned amino acid changes in the method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-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 (numbering according to EU). In another aspect, the above-mentioned amino acid changes in the method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-binding activity are: tyr at position 234, Asp at position 238, Val at position 250, Ile at position 264, Pro at position 307, and Lys at position 330 (numbering according to EU). In another aspect, the above-mentioned amino acid changes in the method for preparing a polypeptide comprising a variant Fc region having enhanced fcyriib-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 (numbering according to EU). In certain embodiments, the Fc γ RIIb has the sequence of cynomolgus monkey Fc γ RIIb (SEQ ID NO: 223). In certain embodiments, the Fc γ RIIb has the sequence of human Fc γ RIIb (e.g., SEQ ID NO:212, 213, or 214).

Furthermore, the present invention provides a method for preparing a polypeptide comprising a variant Fc region having an increased pI as compared to a polypeptide comprising a parent Fc region, the method comprising introducing at least two amino acid alterations to the parent Fc region.

In certain embodiments, the preparation method for preparing a polypeptide comprising a variant Fc region described herein comprises the steps of: (a) preparing a polypeptide comprising a parent Fc region; (b) introducing at least two amino acid changes to a parent Fc region within the polypeptide; (c) measuring the pI of the polypeptide comprising the parent Fc region and the polypeptide comprising the Fc region in step (b); and (d) selecting a polypeptide comprising a variant Fc region having an increased pI as compared to a corresponding polypeptide comprising a parent Fc region.

In one aspect, in the above-mentioned method for preparing a polypeptide comprising a variant Fc region with an increased pI, at least two amino acids 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, the prepared polypeptide comprises a variant Fc region having an increased pI comprising 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 another aspect, in the above-mentioned method for preparing a polypeptide comprising a variant Fc region with an increased pI, the amino acid at any one of the following (1) to (10) positions is changed: (1) at positions 311 and 341; (2) at positions 311 and 343; (3) at positions 311, 343 and 413; (4) at positions 311, 384 and 413; (5) at positions 311 and 399; (6) at positions 311 and 401; (7) at positions 311 and 413; (8) at positions 400 and 413; (9) at positions 401 and 413; and (10) at positions 402 and 413 (according to EU numbering).

In another aspect, the amino acid changes mentioned above for use in the method of preparing a polypeptide comprising a variant Fc region with an increased pI are: arg at position 400 and Lys at position 413 (according to EU numbering). In another aspect, the amino acid changes mentioned above for use in the method of preparing a polypeptide comprising a variant Fc region with an increased pI are: arg at position 311 and Lys at position 413 (according to EU numbering). In another aspect, the amino acid changes mentioned above for use in the method of preparing a polypeptide comprising a variant Fc region with an increased pI are: arg at position 311 and Arg at position 399 (according to EU numbering). In another aspect, the amino acid changes mentioned above for use in the method of preparing a polypeptide comprising a variant Fc region with an increased pI are: arg at position 311 and Arg at position 343 (according to EU numbering). In another aspect, the amino acid changes mentioned above for use in the method of preparing a polypeptide comprising a variant Fc region with an increased pI are: arg at position 311 and Arg at position 413 (according to EU numbering). In another aspect, the amino acid changes mentioned above for use in the method of preparing a polypeptide comprising a variant Fc region with an increased pI are: arg at position 311, Arg at position 343, and Arg at position 413 (according to EU numbering). In another aspect, the amino acid changes mentioned above for use in the method of preparing a polypeptide comprising a variant Fc region with an increased pI are: arg at position 311, Arg at position 384, and Arg at position 413 (according to EU numbering).

In another aspect, the amino acid change in the above-mentioned method for preparing a polypeptide comprising a variant Fc-region with increased pI is selected from Lys or Arg at each position.

In another aspect, the amino acid changes in the above-mentioned methods of preparation are selected from any of the individual changes, combinations of individual changes, or combination changes described in tables 14-30.

Optionally, when the polypeptide further comprises an antigen binding domain, the following additional steps may be included in the above-mentioned method for preparing a polypeptide comprising a variant Fc region: (e) evaluating the pharmacokinetics of the antigen in plasma after administration of a polypeptide comprising a parent Fc region and a polypeptide comprising a variant Fc region in animals such as mice, rats, rabbits, dogs, monkeys, and humans; and (f) selecting a polypeptide comprising a variant Fc region that has an enhanced ability to eliminate antigen from plasma compared to a polypeptide comprising a parent Fc region.

Polypeptides comprising a variant Fc region prepared by any of the above-mentioned methods or other methods known in the art are encompassed by the present invention.

C. Measurement of

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

The variant Fc regions provided herein can be identified, screened for, or characterized for their physical/chemical properties and/or biological activity by a variety of assays described herein or known in the art.

1. Binding assays and other assays

In one aspect, the antigen binding activity of the antibody of the present invention is tested by known methods such as ELISA, western blot, BIACORE (registered trademark), and the like. In one aspect, polypeptides comprising a variant Fc region of the invention are tested for Fc receptor binding activity by known methods, such as BIACORE (registered trademark) and the like.

In another aspect, a competition assay can be used to identify antibodies that compete with any of the anti-myostatin antibodies described herein for binding to myostatin. In certain embodiments, such a competing antibody blocks (e.g., reduces) 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 when present in excess. In some cases, binding is inhibited by at least 80%, 85%, 90%, 95% or more. In certain embodiments, the epitope (e.g., linear or conformational epitope) to which such a competing antibody binds is the same as the epitope to which an anti-myostatin antibody described herein (e.g., an anti-myostatin antibody described in table 2a, 11a, or 13) binds. In further aspects, the reference antibody has a VH and VL pair described in table 2a, 11a, or 13. Detailed exemplary Methods for Mapping the Epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols," Methods in Molecular Biology vol.66(Humana Press, Totowa, NJ).

In an exemplary competition assay, immobilized latent myostatin or myostatin pro peptide is incubated in a solution comprising a first labeled antibody that binds myostatin and a second unlabeled antibody that is 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, the immobilized myostatin was incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind myostatin, excess unbound antibody is removed and the amount of label bound to immobilized myostatin is measured. If the amount of label bound to the immobilized myostatin is significantly reduced in the test sample relative to the control sample, this indicates that the second antibody competes with the first antibody for binding to myostatin. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14(Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

Assays for determining the binding activity of a polypeptide comprising a variant Fc region to one or more fcyr family members are described herein or are known in the art. Such binding assays include, but are not limited to, BIACORE (registered trademark) analysis using the Surface Plasmon Resonance (SPR) phenomenon, Amplified luminescence Proximity Homogeneous Assay (Amplified luminescence Proximity Homogeneous Assay) (ALPHA-LPHA) screening, ELISA, and Fluorescence Activated Cell Sorting (FACS) (Lazar et al, Proc. Natl. Acad. Sci. USA (2006)103(11): 4005-.

In one embodiment, BIACORE (registered trademark) assays can be used to assess whether the binding activity of a polypeptide comprising a variant Fc region is enhanced, or maintained or reduced, for a particular fcyr family member, e.g., by observing whether the dissociation constant (KD) value obtained from a sensorgram assay is decreased or increased, wherein a plurality of fcyr's interact as analytes with a polypeptide comprising a variant Fc region immobilized or captured on a sensor chip using known methods and reagents (e.g., protein a, protein L, protein a/G, protein G, anti- λ chain antibodies, anti- κ chain antibodies, antigenic peptides, antigenic proteins). Changes in binding activity can also be determined by comparing changes in Resonance Unit (RU) values on the sensorgram before and after interaction of one or more fcyr as an analyte with a captured polypeptide comprising a variant Fc region. Alternatively, the Fc γ R may be immobilized or captured on a sensor chip and a polypeptide comprising a variant Fc region is used as the analyte.

In BIACORE (registered trademark) analysis, in observation of the interaction, one of the substances (ligand) is immobilized on the gold thin film on the sensor chip, and a part of the reduced reflection intensity is formed by the reflected light (SPR signal) by emitting light from the reverse side of the sensor chip so that total reflection occurs at the interface between the gold thin film and the glass. When another substance (analyte) in the observation of the interaction is caused to flow on the sensor chip surface and the ligand binds to the analyte, the mass of the immobilized ligand molecule increases and the refractive index of the solvent on the sensor chip surface changes. The position of the SPR signal shifts with this change in refractive index (on the other hand, when the binding is dissociated, the signal position returns). The BIACORE (registered trademark) system indicates the amount of displacement mentioned above, or more specifically, indicates a time variable of mass on the surface of the sensor chip as measurement data (sensorgram) by plotting the mass change on the coordinate. The amount of analyte bound to the captured ligand on the sensor chip surface is determined from the sensorgram. Kinetic parameters such as the association rate constant (ka) and dissociation rate constant (KD) are determined from the sensorgram curves, and the dissociation constant (KD) is determined from the ratio of these constants. In the BIACORE (registered trademark) method, a method for measuring inhibition is preferably used. Examples of methods for measuring inhibition are described in Lazar et al, Proc. Natl. Acad. Sci. USA 103(11): 4005-.

Screening was performed by the ALPHA technique, which utilizes two beads, donor and acceptor, based on the following principle. The luminescent signal is only detected when the molecule bound to the donor bead and the molecule bound to the acceptor bead physically interact and the two beads are in close proximity to each other. The laser-excited photosensitizer in the donor bead converts the ambient oxygen to excited singlet (singlet) oxygen. Singlet oxygen is dispersed throughout the donor bead and when it encounters an adjacent acceptor bead, a chemiluminescent reaction is initiated in the bead and light is ultimately emitted. When the molecule bound to the donor bead does not interact with the molecule bound to the acceptor bead, no chemiluminescent reaction occurs because the singlet oxygen generated by the donor bead does not touch the acceptor bead.

For example, a biotinylated polypeptide complex is bound to a donor bead and an Fc γ receptor with a Glutathione S Transferase (GST) tag is linked to an acceptor bead. In the absence of a competing polypeptide complex comprising a variant Fc region, the polypeptide complex comprising the parent Fc region interacts with the Fc γ receptor and generates a 520-620nm signal. A polypeptide complex comprising an unlabeled variant Fc region competes with a polypeptide complex comprising a parent Fc region for interaction with an fey receptor. Relative binding activity can be determined by quantifying the decrease in fluorescence observed as a result of competition. Biotinylation of a polypeptide complex such as an antibody using Sulfo-NHS-biotin or the like is known. A method of expressing Fc γ receptor and GST in a cell carrying a fusion gene prepared by fusing a polynucleotide encoding Fc γ receptor and a polynucleotide encoding GST in frame in an expressible vector, and performing purification using a glutathione column is suitably used as a method for making the Fc γ receptor have a GST tag. The signals obtained are preferably analyzed, for example, by: this was fitted to a single point competition model using non-linear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).

A variant Fc region having reduced fcyr-binding activity refers to an Fc region that binds fcyr with significantly weaker binding activity than the parent Fc region (when measured using substantially the same amount of the corresponding parent and variant Fc regions). Furthermore, a variant Fc-region having enhanced fcyr-binding activity refers to an Fc-region that binds fcyr significantly more strongly than the corresponding parent Fc-region (when measured using substantially the same amount of the corresponding parent and variant Fc-regions). A variant Fc-region having retained Fc γ R-binding activity refers to an Fc-region that binds Fc γ R with the same or no significant difference from the binding activity of the parent Fc-region (when measured using substantially the same amount of the corresponding parent and variant Fc-regions).

Whether the binding activity of the Fc region to the different fcyr is enhanced or attenuated may be determined by an increase or decrease in the amount of binding of the different fcyr to the Fc region, which is determined according to the above-mentioned measurement method. Here, the amount of binding of the different Fc γ rs to the Fc region may be estimated as a value obtained by dividing the difference between the RU values of the sensorgrams changed before and after the interaction of the different Fc γ rs with the Fc region as the analyte by the difference between the RU values of the sensorgrams changed before and after the capturing of the Fc region to the sensor chip.

In the present invention, the enhanced Fc γ RIIb-binding activity preferably means, for example, that the ratio of [ KD value of parent Fc region to Fc γ RIIb ]/[ KD value of variant Fc region to Fc γ RIIb ] in the KD values measured by the above-mentioned measurement method preferably becomes 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 even 50 or more, 55 or more, 60 or more, 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 variant Fc regions described herein preferably have a KD value (mol/L) for fcyriib that is less than the KD value for fcyriib of the parent Fc region, and can be, for example, 2.0x10^-6M below, 1.0x10^-6M below, 9.0x10^-7M below, 8.0x10^-7M < 7.0x10^-7M below, 6.0x10^-7M below, 5.0x10^-7M < 4.0x10^-7M below, 3.0x10^-7M below, 2.0x10^{- 7}M below, 1.0x10^-7M below, 9.0x10^-8M below, 8.0x10^-8M below, 7.0x10^-8M below, 6.0x10^-8M below, 5.0x10^-8M is less than or equal to M.

Further, a variant Fc region that has increased binding selectivity for Fc γ RIIb over Fc γ RIIa refers to an Fc region wherein: (a) fc γ RIIb-binding activity is enhanced and Fc γ RIIa-binding activity is maintained or reduced; (b) fc γ RIIb-binding activity is enhanced and Fc γ RIIa-binding activity is also enhanced, but the degree of enhancement of Fc γ RIIa-binding activity is less than the degree of enhancement of Fc γ RIIb-binding activity; or (c) the Fc γ RIIb-binding activity is reduced and the Fc γ RIIa-binding activity is also reduced, but the degree of reduction of the Fc γ RIIb-binding activity is less than the degree of reduction of the Fc γ RIIa-binding activity. Whether a variant Fc region has improved binding selectivity to Fc γ RIIb but not Fc γ RIIa can be determined, for example, by comparing the ratio of the KD value for Fc γ RIIa to the KD value for Fc γ RIIb for the variant Fc region determined according to the above-mentioned examples ([ KD value for variant Fc region to Fc γ RIIa ]/[ KD value for variant Fc region to Fc γ RIIb ]) to the ratio of the KD value for Fc γ RIIa to the KD value for Fc γ RIIb for the parent Fc region ([ KD value for parent Fc region to Fc γ RIIa ]/[ KD value for parent Fc region to Fc γ RIIb ]). In particular, when the KD ratio of the variant Fc region is greater than the KD ratio of the parent Fc region, it can be determined that the variant Fc region has improved binding selectivity to Fc γ RIIb over Fc γ RIIa as compared to the parent Fc region. In humans in particular, Fc γ RIIb-binding activity may be correlated with binding activity to Fc γ RIIa (type R) rather than with binding activity to Fc γ RIIa (type H), because the amino acid sequence of Fc γ RIIb has a higher identity to Fc γ RIIa (type R) than to Fc γ RIIa (type H). Thus, it was found that amino acid changes that can enhance the binding selectivity for human Fc γ RIIb compared to human Fc γ RIIa (R-type) are important for enhancing the binding selectivity for Fc γ RIIb compared to Fc γ RIIa in humans.

A variant Fc region of the invention may be said to be Fc γ RIIb-specific when it has higher binding activity to Fc γ RIIb and lower binding activity to Fc γ RIII (e.g., Fc γ RIIIa or Fc γ RIIIb) than the parent Fc region.

2. Activity assay

In one aspect, assays are provided for identifying anti-myostatin antibodies having biological activity. Biological activities may include, for example, inhibiting activation of myostatin, blocking release of mature myostatin from latent myostatin, inhibiting proteolytic cleavage of latent myostatin, blocking access of proteases to latent myostatin, and the like. Also provided are antibodies having such biological activity in vivo and/or in vitro. In certain embodiments, the antibodies of the invention are tested for said biological activity.

In certain embodiments, whether a test antibody inhibits cleavage of latent myostatin is determined by detecting the cleavage product of latent myostatin (myostatin) using methods 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 in the presence or absence of the test antibody (see, e.g., Thies et al, Growth Factors 18(4): 251-. When a decrease in the amount of cleavage product of latent myostatin (e.g., human myostatin pro peptide) is detected in the presence of (or after contact with) the test antibody, the test antibody is identified as an antibody that can inhibit cleavage of latent myostatin. In certain embodiments, whether the test antibody blocks access of the protease to the latent myostatin is determined by a method for detecting a protein interaction between the protease and the latent myostatin, such as ELISA or BIACORE (registered trademark). When a decrease in the interaction between the protease and the 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 access of the protease to the latent myostatin.

In certain embodiments, whether a test antibody blocks release of mature myostatin from latent myostatin is determined by detecting mature myostatin activity, e.g., activity that binds to a myostatin receptor, or activity that mediates signaling in a cell expressing a myostatin receptor (e.g., ActRIIb). Cells useful in the assay may be those expressing endogenous myostatin receptors, e.g., L6 muscle cells, or may be those genetically modified, transiently or stably, to express a transgene encoding a myostatin receptor (e.g., an activin receptor such as an activin type II receptor) (Thies et al, supra). Binding of myostatin to a myostatin receptor can be detected using a receptor binding assay. Myostatin-mediated signal transduction can be detected at any level in the signal transduction pathway, for example, by detecting phosphorylation of a Smad polypeptide, detecting expression of a myostatin regulator gene (including a reporter gene), or measuring proliferation of myostatin-dependent cells. When a decrease in mature myostatin activity is detected in the presence of (or after contact with) the test antibody, the test antibody is identified as an antibody that can block release of mature myostatin from latent myostatin.

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

In another aspect, assays are provided for identifying anti-myostatin antibodies that form immune complexes (i.e., antigen-antibody complexes) with myostatin. Also provided are antibodies having such biological activity in vivo and/or in vitro.

In certain embodiments, the antibodies of the invention are tested for said biological activity.

In certain embodiments, immune complex formation is assessed by methods such as size exclusion (gel filtration) chromatography, ultracentrifugation, light scattering, electron microscopy or mass spectrometry (mol. Immunol.39:77-84(2002), mol. Immunol.47:357-364 (2009)). These methods take advantage of the property that immune complexes are larger molecules than either the antibody alone or the antigen alone. When a large complex comprising two or more antibodies and two or more antigens (e.g., myostatin molecules) is detected in the presence of the test antibody and antigen, the test antibody is identified as an antibody that can form an immune complex comprising two or more antibodies and two or more myostatin molecules. In another embodiment, the formation of immune complexes is assessed by methods such as ELISA, FACS or SPR (surface plasmon resonance assay; e.g., using BIACORE (registered trademark)) (Shields et al, J.biol. chem.276(9): 6591-. These methods take advantage of the property that immune complexes containing more than two antibodies and more than two antigens can bind Fc receptors or complement components more strongly than antibodies or antigens alone. A test antibody is identified as an antibody that can form an immune complex comprising two or more antibodies and two or more myostatin molecules when the ratio of binding to Fc receptors or complement components detected in the presence of both the test antibody and antigen increases in the presence of the antibody alone. In another embodiment, immune complex formation is assessed by administering a test antibody to an animal (e.g., a mouse) and measuring clearance of antigen from plasma. As described above, it is expected that the antibody forming the immune complex containing two or more antibodies and two or more antigens accelerates the elimination of the antigen from the plasma. Thus, when the observed elimination of myostatin from plasma in a mouse administered with a test antibody is accelerated compared to a mouse administered with a reference antibody, the test antibody is identified as an antibody that can form an immune complex comprising two or more antibodies and two or more myostatin molecules more efficiently than the reference antibody.

D. Immunoconjugates

In some embodiments, the invention provides immunoconjugates comprising an anti-myostatin antibody herein conjugated to one or more cytotoxic agents such as a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope.

In some embodiments, the present invention provides immunoconjugates comprising a polypeptide comprising a variant Fc region herein conjugated to one or more cytotoxic agents such as a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope.

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 maytansinoids (see U.S. Pat. nos. 5,208,020, 5,416,064 and european patent EP 0425235B 1); auristatins such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. nos. 5,635,483 and 5,780,588, and 7,498,298); dolastatin (dolastatin); calicheamicin (calicheamicin) or a derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296; Hinman et al, Cancer Res.53:3336-3342 (1993); and Lode et al, Cancer Res.58:2925-2928 (1998)); anthracyclines (anthracyclines) such as daunomycin (daunomycin) or doxorubicin (doxorubicin) (see Kratz et al, Current Med. chem.13: 477-; methotrexate (methotrexate); vindesine (vindesine); taxanes (taxanes) such as docetaxel (docetaxel), paclitaxel (paclitaxel), larotaxel (larotaxel), tesetaxel (tesetaxel) and otetaxel (ortataxel); crescent toxin (trichothecene); and CC 1065.

In another embodiment, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria a chain, a non-binding active fragment of diphtheria toxin, exotoxin a chain (from Pseudomonas aeruginosa), ricin a chain, abrin a chain, anemonin a chain, α -sarcina, Aleurites fordii protein, dianthin protein, phytolacca Americana protein (PAPI, PAPII, and PAP-S), momordica charantia (momordia) inhibitor, leprosy toxin protein, croton toxin protein, saponaria officinalis (sapaonaria officinalis) inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and triene toxin (tricothecene).

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for making radioconjugates. Examples include At²¹¹，I¹³¹，I¹²⁵，Y⁹⁰，Re¹⁸⁶，Re¹⁸⁸，Sm¹⁵³，Bi²¹²，P³²，Pb²¹²And radioactive isotopes of Lu. When the radioconjugate is used for detection, it may contain radioactive atoms for scintigraphic studies, for example tc99m or I123, or spin labels for Nuclear Magnetic Resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123 (again), iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese and iron.

Conjugates of the antibody and cytotoxic agent can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane hydrochloride (IT), bifunctional derivatives of imidoesters (such as dimethyl adipate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), diazide compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazo derivatives (such as bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene). For example, ricin immunotoxins may be prepared as described in Vitetta et al, Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionuclides to antibodies. See WO 1994/11026. The linker may be a "cleavable linker" which aids in the release of the cytotoxic drug in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al, Cancer Res.52:127-131 (1992); U.S. Pat. No. 5,208,020).

Immunoconjugates or ADCs herein expressly contemplate, but are not limited to, such conjugates prepared using crosslinker agents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, thio-EMCS, thio-GMBS, thio-KMUS, thio-MBS, thio-SIAB, thio-SMCC and thio-SMPB, and SVSB (succinimidyl- (4-vinylsulfone) benzoate), which are commercially available (e.g., from Pierce Biotechnology, inc., Rockford, il., u.s.a.).

E. Methods and compositions for diagnosis and detection

In certain embodiments, any of the anti-myostatin antibodies provided herein can be used to detect the presence of myostatin in a biological sample. As used herein, the term "detecting" includes quantitative or qualitative detection. In certain embodiments, the biological sample comprises a cell or tissue, such as serum, whole blood, plasma, biopsy sample, tissue sample, cell suspension, saliva, sputum, oral fluid, cerebrospinal fluid, amniotic fluid, ascites fluid, breast milk, colostrum, mammary secretion, lymph fluid, urine, sweat, tears, gastric fluid, synovial fluid, peritoneal fluid, ocular lens fluid, or mucus.

In one embodiment, anti-myostatin antibodies are provided for use in a diagnostic or detection method. In another aspect, a method is provided for detecting the presence of latent myostatin or myostatin pro peptide in a biological sample. In certain embodiments, the method comprises contacting the biological sample with an anti-myostatin antibody as described herein under conditions that allow the anti-myostatin antibody to bind myostatin, and detecting whether a complex is formed between the anti-myostatin antibody and myostatin. Such methods may be in vitro or in vivo. In one embodiment, the anti-myostatin antibody is used to select a subject suitable for treatment with the anti-myostatin antibody, e.g., wherein myostatin is a biomarker used to select patients.

Exemplary disorders that can be diagnosed using the antibodies of the invention include, but are not limited to, muscle dystrophy (MD; including duchenne muscular dystrophy), Amyotrophic Lateral Sclerosis (ALS), muscle atrophy, organ atrophy, carpal tunnel syndrome, weakness, Congestive Obstructive Pulmonary Disease (COPD), sarcopenia, cachexia, muscle wasting syndrome, HIV-induced muscle wasting, type 2 diabetes, impaired glucose tolerance, metabolic syndrome (including syndrome X), insulin resistance (including resistance caused by trauma (e.g., burns or nitrogen imbalance)), adipose tissue disorders (e.g., obesity, dyslipidemia, non-alcoholic steatoliver, etc.), osteoporosis, osteopenia, osteoarthritis, and metabolic bone disease (including low bone mass, premature gonadal failure, androgen suppression, vitamin D deficiency, secondary hyperparathyroidism, nutritional deficiencies, and anorexia nervosa).

In certain embodiments, labeled anti-myostatin antibodies are provided. Labels include, but are not limited to, labels or moieties that are directly detectable (e.g., fluorescent, chromophoric, electron density, chemiluminescent, and radioactive labels), as well as moieties that are indirectly detectable, e.g., as enzymes or ligands, e.g., by enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes³²P，¹⁴C，¹²⁵I，³H, and¹³¹fluorophores such as rare earth chelates or luciferin and derivatives thereof, rhodamine and derivatives thereof, dansyl, umbelliferone, luciferase, for example, firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2, 3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, for example, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with enzymes using hydrogen peroxideWith oxidative dye precursors such as HRP, lactose peroxidase, or microperoxidase, biotin/avidin, spin labels, phage labels, stable free radicals, and the like.

F. Pharmaceutical preparation

Pharmaceutical formulations of anti-myostatin antibodies as described herein are prepared in lyophilized or aqueous solution form by mixing the antibody with the desired degree of purity with one or more optional Pharmaceutical carriers (Remington's Pharmaceutical Sciences 16 th edition, Osol, a.ed. (1980)).

Pharmaceutical formulations of polypeptides comprising a variant Fc region as described herein are prepared in lyophilized formulations or in aqueous solution by admixing the polypeptide with the desired degree of purity and one or more optional pharmaceutically acceptable carriers.

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 acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g. octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl p-hydroxybenzoate esters such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, aspartic acid, 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 complexes (e.g., Zn-protein complexes); and/or a non-ionic surfactant such as polyethylene glycol (PEG). Exemplary pharmaceutical carriers herein also include interstitial drug dispersing agents such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, e.g., rHuPH20(HYLENEX (registered trademark), Baxter International, Inc.). Certain exemplary shasegps and methods of use, including rHuPH20, are described in U.S. publication nos. 2005/0260186 and 2006/0104968. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO 2006/044908, the latter formulations containing a histidine-acetic acid buffer.

When desired, the formulations herein may also contain more than one active ingredient for a particular indication of treatment, preferably those having complementary activities that do not adversely affect each other. Such active ingredients are suitably present in the combination in an amount effective for the intended use.

The active ingredients can be encapsulated, for example, in microcapsules prepared by coacervation techniques or by interfacial polymerization of, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. This technique is disclosed in Remington's Pharmaceutical Sciences 16 th edition, Osol, a.ed. (1980).

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

Formulations useful for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

G. Therapeutic methods and compositions

Any of the anti-myostatin antibodies provided herein can be used in a method of treatment. Similarly, any of the polypeptides comprising a variant Fc region provided herein can be used in a method of treatment.

In one aspect, an anti-myostatin antibody for use as a medicament is provided. In a further aspect, anti-myostatin antibodies are provided for use in treating a muscle wasting disease. In certain embodiments, anti-myostatin antibodies are provided for use in a method of treatment. In certain embodiments, the invention provides anti-myostatin antibodies for use in a method of treating a subject having a muscle wasting disease, comprising administering to the subject an effective amount of an anti-myostatin antibody. In one embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In another embodiment, the invention provides anti-myostatin antibodies for use in increasing the mass of muscle tissue. In certain embodiments, the invention provides an anti-myostatin antibody for use in a method of increasing mass of muscle tissue in an individual, comprising administering to the individual an effective amount of an anti-myostatin antibody to increase the mass of muscle tissue. In another embodiment, the invention provides anti-myostatin antibodies for use in increasing the strength of muscle tissue. In certain embodiments, the present invention provides an anti-myostatin antibody for use in a method of increasing muscle tissue strength in an individual, comprising administering to the individual an effective amount of an anti-myostatin antibody to increase muscle tissue strength. In another embodiment, the invention provides anti-myostatin antibodies for use in reducing body fat accumulation. In certain embodiments, the invention provides an anti-myostatin antibody for use in a method of reducing body fat accumulation in an individual, comprising administering to the individual an effective amount of an anti-myostatin antibody to reduce body fat accumulation. An "individual" according to any of the above embodiments is preferably a human.

The anti-myostatin antibodies of the invention can exhibit pH-dependent binding properties. In additional embodiments, the invention provides anti-myostatin antibodies for use in enhancing myostatin clearance from plasma. In certain embodiments, the invention provides anti-myostatin antibodies for use in a method of enhancing clearance of myostatin from plasma in an individual, comprising administering to the individual an effective amount of an anti-myostatin antibody to enhance clearance of myostatin from plasma. In one embodiment, an anti-myostatin antibody having a pH-dependent binding property enhances clearance of myostatin from plasma compared to a conventional anti-myostatin antibody not having a pH-dependent binding property. In another embodiment, an anti-myostatin antibody having a pH-dependent binding property between binding at pH5.8 and pH7.4 enhances clearance of myostatin from plasma compared to a conventional anti-myostatin antibody having no pH-dependent binding property. An "individual" according to any of the above embodiments is preferably a human.

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

The anti-myostatin antibodies of the invention can exhibit pH-dependent binding properties. In another embodiment, the medicament is for enhancing clearance of myostatin from plasma. In another embodiment, the medicament is for use in a method of enhancing clearance of myostatin from plasma in an individual, the method comprising administering to the individual an effective amount of the medicament to enhance clearance of myostatin from plasma. In one embodiment, an anti-myostatin antibody having a pH-dependent binding property enhances clearance of myostatin from plasma compared to a conventional anti-myostatin antibody not having a pH-dependent binding property. In another embodiment, the anti-myostatin antibody exhibits different pH-dependent binding properties between pH5.8 and pH 7.4. An "individual" according to any of the above embodiments is preferably a human.

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

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

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

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

The anti-myostatin antibodies of the invention can exhibit pH-dependent binding properties. In another embodiment, the present invention provides a method for enhancing clearance of myostatin from plasma in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-myostatin antibody provided herein to enhance myostatin clearance from plasma. In one embodiment, an anti-myostatin antibody having a pH-dependent binding property enhances clearance of myostatin from plasma compared to a conventional anti-myostatin antibody not having a pH-dependent binding property. In another embodiment, the anti-myostatin antibody exhibits different pH-dependent binding properties between pH5.8 and pH 7.4. In one embodiment, the "individual" is a human.

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

In another aspect, the pharmaceutical formulation is for use in treating a muscle wasting disease. In another embodiment, the pharmaceutical formulation is for increasing the mass of muscle tissue. In another embodiment, the pharmaceutical formulation is for increasing the strength of muscle tissue. In another embodiment, the pharmaceutical formulation is for reducing body fat accumulation. The anti-myostatin antibodies of the invention can exhibit pH-dependent binding properties. In another embodiment, the pharmaceutical formulation is for use in enhancing clearance of myostatin from plasma. In one embodiment, the pharmaceutical formulation is administered to an individual having a muscle wasting disease. An "individual" according to any of the above embodiments is preferably a human.

In another aspect, the invention provides a method for preparing a medicament or pharmaceutical formulation, the method comprising admixing any of the anti-myostatin antibodies provided herein with a pharmaceutically acceptable carrier, e.g., for use in any of the above-described methods of treatment. In one embodiment, the method for preparing a medicament or pharmaceutical formulation further comprises adding at least one additional therapeutic agent to the medicament or pharmaceutical formulation.

In certain embodiments, the muscle wasting disease is selected from the group consisting of: muscle dystrophy (MD; including duchenne muscular dystrophy), Amyotrophic Lateral Sclerosis (ALS), muscle atrophy, organ atrophy, carpal tunnel syndrome, frailty, Congestive Obstructive Pulmonary Disease (COPD), sarcopenia, cachexia, muscle wasting syndrome, HIV-induced muscle wasting, type 2 diabetes mellitus, impaired glucose tolerance, metabolic syndrome (including syndrome X), insulin resistance (including resistance caused by trauma (e.g., burns or nitrogen imbalance)), adipose tissue disorders (e.g., obesity, dyslipidemia, non-alcoholic fatty liver disease, etc.), osteoporosis, osteopenia, osteoarthritis, and metabolic bone disease (including low bone mass, premature gonadal failure, androgen suppression, vitamin D deficiency, secondary hyperparathyroidism, nutritional deficiencies, and anorexia nervosa).

Any of the polypeptides comprising a variant Fc region provided herein can be used in a method of treatment. In another aspect, the invention provides a pharmaceutical formulation, e.g., for use in a method of treatment, comprising a polypeptide comprising any one of the polypeptides comprising a variant Fc region provided herein. In one embodiment, the pharmaceutical formulation comprises a polypeptide comprising any one of the polypeptides comprising a variant Fc region provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises a polypeptide comprising any of the variant Fc regions provided herein and at least one additional therapeutic agent.

In one aspect, a polypeptide comprising a variant Fc region for use as a medicament is provided. In a further aspect, a polypeptide comprising a variant Fc region for use in the treatment of a disease is provided. In certain embodiments, polypeptides comprising a variant Fc region for use in a method of treatment are provided. In certain embodiments, the present invention provides a polypeptide comprising a variant Fc region for use in a method of treating an individual with a disease, the method comprising administering to the individual an effective amount of a polypeptide comprising a variant Fc region provided herein. In one embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In one embodiment, an "individual" is a human.

In another aspect, the invention provides the use of a polypeptide comprising a variant Fc region in the manufacture or formulation of a medicament. In one embodiment, the medicament is for treating a disease. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In another embodiment, the medicament is for use in a method of treating a disease, the method comprising administering to an individual having a disease to be treated an effective amount of the medicament. In one embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In one embodiment, the "individual" is a human.

In another aspect, the invention provides methods for treating a disease. In one embodiment, the method comprises administering to an individual having such a disease an effective amount of a polypeptide comprising a variant Fc region. In one embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In one embodiment, the "individual" is a human.

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

In another aspect, the pharmaceutical formulation is for use in the treatment of a disease. In one embodiment, the pharmaceutical formulation is administered to an individual with the disease. In one embodiment, an "individual" is a human.

In another embodiment, the present invention provides a polypeptide comprising a variant Fc region for use in inhibiting the activation of B cells, mast cells, dendritic cells and/or basophils. In certain embodiments, the present invention provides a polypeptide comprising a variant Fc region for use in a method of inhibiting activation of a B cell, mast cell, dendritic cell and/or basophil in an individual, the method comprising administering to the individual an effective amount of a polypeptide comprising a variant Fc region to inhibit activation of the B cell, mast cell, dendritic cell and/or basophil. In one embodiment, an "individual" is a human.

In another aspect, the invention provides the use of a polypeptide comprising a variant Fc region in the manufacture or formulation of a medicament. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In another embodiment, the medicament is for inhibiting the activation of B cells, mast cells, dendritic cells and/or basophils. In another embodiment, the medicament is for use in a method of inhibiting activation of B cells, mast cells, dendritic cells and/or basophils in an individual, the method comprising administering to the individual an effective amount of the medicament to inhibit activation of B cells, mast cells, dendritic cells and/or basophils. In one embodiment, the "individual" is a human.

In another aspect, the invention provides methods for inhibiting the activation of B cells, mast cells, dendritic cells and/or basophils in an individual. In one embodiment, the method comprises administering to the individual an effective amount of a polypeptide comprising a variant Fc region to inhibit activation of B cells, mast cells, dendritic cells, and/or basophils. In one embodiment, an "individual" is a human.

In another aspect, the invention provides a pharmaceutical formulation comprising a polypeptide comprising a variant Fc region. In another embodiment, the pharmaceutical formulation is for use in inhibiting the activation of B cells, mast cells, dendritic cells and/or basophils.

The polypeptides of the invention comprising a variant Fc region are capable of inhibiting the activation of B cells, mast cells, dendritic cells and/or basophils. Without wishing to be bound by theory, it is believed that this inhibited activation is a result of the provided selective binding of the variant Fc region to Fc γ RIIb without activating the activating Fc γ R. As used herein, "B cell activation" includes proliferation, IgE production, IgM production, and IgA production. Without wishing to be bound by theory, it is believed that inhibited B cell activation is a result of the ability of the variant Fc region provided on a polypeptide of the invention comprising the variant Fc region to crosslink Fc γ RIIb with IgE to inhibit IgE production by B cells, to crosslink Fc γ RIIb with IgM to inhibit IgM production by B cells, and to crosslink Fc γ RIIb with IgA to inhibit IgA production. In addition to the above, similar inhibitory effects to those mentioned above are exhibited by directly or indirectly crosslinking Fc γ RIIb to such molecules: the molecules are expressed on B cells and comprise or interact with an ITAM domain (e.g., BCR, CD19, and CD79B) within the cell. As used herein, "activation of mast cells" includes proliferation, activation by IgE, and degranulation. Without wishing to be bound by theory, it is believed that inhibited mast cell activation is the result of the variant Fc region provided on the polypeptides of the invention comprising a variant Fc region crosslinking Fc γ RIIb directly or indirectly to IgE receptor molecules expressed on mast cells and comprising or interacting with ITAM domains (e.g., fceri, DAP12, and CD200R 3). As used herein, "activation of basophils" includes proliferation and degranulation of basophils. Without wishing to be bound by theory, it is believed that inhibited basophil activation is the result of the variant Fc region provided on the polypeptides of the invention comprising a variant Fc region directly or indirectly crosslinking Fc γ RIIb to a molecule on a cell membrane that comprises or interacts with an ITAM domain within the cell. As used herein, "activation of dendritic cells" includes proliferation and degranulation of dendritic cells. Without wishing to be bound by theory, it is believed that inhibited dendritic cell activation is the result of the variant Fc region provided on the polypeptides of the invention comprising a variant Fc region directly or indirectly crosslinking Fc γ RIIb to a molecule on a cell membrane that comprises or interacts with an ITAM domain within the cell.

In another embodiment, the present invention provides a polypeptide comprising a variant Fc region as provided herein for use in the treatment of an immunoinflammatory disorder. In certain embodiments, the present invention provides a polypeptide comprising a variant Fc region for use in a method of treating an immunoinflammatory disorder in an individual, the method comprising administering to the individual an effective amount of a polypeptide comprising a variant Fc region to treat the immunoinflammatory disorder. In one embodiment, an "individual" is a human.

In another aspect, the invention provides the use of a polypeptide comprising a variant Fc region as provided herein in the manufacture or formulation of a medicament. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In another embodiment, the medicament is for treating an immunoinflammatory disorder. In another embodiment, the medicament is for use in a method of treating an immunoinflammatory disorder in an individual, the method comprising administering to the individual an effective amount of the medicament to treat the immunoinflammatory disorder. An "individual" according to any of the above embodiments may be a human.

In another aspect, the invention provides a method for treating an immunoinflammatory disorder in an individual. In one embodiment, the method comprises administering to the individual an effective amount of a polypeptide comprising a variant Fc region to treat an immunoinflammatory disorder. In one embodiment, the "individual" is a human.

In another aspect, the invention provides pharmaceutical formulations comprising a polypeptide comprising a variant Fc region provided herein. In another embodiment, the pharmaceutical formulation is for use in treating an immunoinflammatory disorder.

As described above, since the polypeptide comprising a variant Fc region of the present invention can inhibit the activation of B cells, mast cells, dendritic cells and/or basophils, administration of the polypeptide comprising a variant Fc region of the present invention can thus treat or prevent an immunoinflammatory disease.

In certain embodiments, the immunoinflammatory disorder treated is selected from the group consisting of: rheumatoid arthritis, autoimmune hepatitis, autoimmune thyroiditis, autoimmune blistering diseases, autoimmune adrenocorticism, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, megaloblastic anemia, autoimmune atrophic gastritis, autoimmune neutropenia, autoimmune orchitis, autoimmune encephalomyelitis, autoimmune receptor diseases, autoimmune infertility, chronic active hepatitis, glomerulonephritis, interstitial pulmonary fibrosis, multiple sclerosis, Paget's disease, osteoporosis, multiple myeloma, uveitis, acute and chronic spondylitis, gouty arthritis, inflammatory bowel disease, Adult Respiratory Distress Syndrome (ARDS), psoriasis, Crohn's disease, baserow's disease, Juvenile diabetes, Addison's disease, myasthenia gravis, crystalline uveitis, systemic lupus erythematosus, allergic rhinitis, allergic dermatitis, ulcerative colitis, hypersensitivity, muscular degeneration, cachexia, systemic scleroderma, Sjogren's syndrome, Behcet's disease, Reiter's syndrome, type I and type II diabetes, bone resorption disorders, graft-versus-host reaction, ischemic reperfusion injury, atherosclerosis, brain injury, brain type malaria, sepsis, septic shock, toxic shock syndrome, fever, stain-induced myalgia (malgias), aplastic anemia, hemolytic anemia, idiopathic thrombocytopenia, Goodpasture's syndrome, Guillain-Barre syndrome (Guillain-Barre syndrome), Hashimoto's thyroiditis, pemphigus, IgA nephropathy, pollinosis, antiphospholipid syndrome, polymyositis, Wegener's granulomatosis, nodular arteritis, mixed connective tissue disease, fibromyalgia, asthma, atopic dermatitis, chronic atrophic gastritis, primary biliary cirrhosis, primary sclerosing cholangitis, autoimmune pancreatitis, aortosis syndrome, accelerated glomerulonephritis, megaloblastic anemia, idiopathic thrombocytopenic purpura, primary hypothyroidism, idiopathic ehydic disease, insulin-dependent diabetes mellitus, chronic discoid lupus erythematosus, pemphigoid, herpes gestationis, IgA bullous dermatosis, acquired epidermolysis bullosa, alopecia areata, vitiligo vulgaris, Sutton's leukoplakia after centrifugation, primary field disease (Harada's disease), Autoimmune optic neuropathy, idiopathic azoospermia, habitual abortion, hypoglycemia, chronic urticaria, ankylosing spondylitis, psoriatic arthritis, enteropathic arthritis, reactive arthritis, spondyloarthropathies, onset and death (entheopathy), irritable bowel syndrome, chronic fatigue syndrome, dermatomyositis, inclusion body myositis, Schmidt's syndrome, Graves ' disease, pernicious anemia, lupus-like hepatitis, presenile dementia, Alzheimer's disease, demyelinating disease, amyotrophic lateral sclerosis, hypoparathyroidism, derlescent syndrome (Dressler's syndrome), myasthenia-syndrome (easy-Lambert syndrome), dermatitis herpetiformis, alopecia, progressive systemic sclerosis, CREST syndrome (calcinosis, raynaud's phenomenon, esophageal disorder, scleroderma, and CREST syndrome, Telangiectasia), sarcoidosis, rheumatic fever, erythema multiforme, Cushing's syndrome, transfusion reactions, Hansen's disease, Takayasu arteritis, polymyalgia rheumatica, temporal arteritis, giant cell arteritis, eczema, lymphomatoid granuloma, Kawasaki disease, endocarditis, endocardial fibrosis, endophthalmitis, fetal erythroblastosis, eosinophilic fasciitis, ferti syndrome, hensche-schoein purpura, transplant rejection, parotitis, cardiomyopathy, suppurative arthritis, familial mediterranean fever, Muckle-weidi syndrome (Muckle-Wells syndrome), and hyper-IgD syndrome.

In another embodiment, the invention provides a polypeptide comprising an Fc region for use in the treatment or prevention of an autoimmune disease that may be caused by or associated with the production of antibodies to autoantigens (autoantibodies). In certain embodiments, the present invention provides a polypeptide comprising a variant Fc region for use in a method of treating or preventing an immunoinflammatory disorder in an individual that may be caused by or associated with the production of antibodies to autoantigens (autoantibodies), the method comprising administering to the individual an effective amount of a polypeptide comprising a variant Fc region to treat or prevent an autoimmune disorder. An "individual" according to any of the above embodiments is preferably a human.

In another aspect, the invention provides the use of a polypeptide comprising a variant Fc region in the manufacture or formulation of a medicament. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In another embodiment, the medicament is for the treatment or prevention of an autoimmune disease that may be caused by or associated with the production of antibodies to autoantigens (autoantibodies). In another embodiment, the medicament is for use in a method of treating or preventing an immunoinflammatory disorder in an individual that may be caused by or associated with the production of antibodies to self-antigens (autoantibodies), the method comprising administering to the individual an effective amount of the medicament to treat or prevent an autoimmune disorder. An "individual" according to any of the above embodiments may be a human.

In another aspect, the invention provides methods for treating or preventing an autoimmune disease in an individual that may be caused by or associated with the production of antibodies to autoantigens (autoantibodies). In one embodiment, the method comprises administering to the individual an effective amount of a polypeptide comprising a variant Fc region to treat or prevent an autoimmune disease. In one embodiment, the "individual" is a human.

In another aspect, the invention provides a pharmaceutical formulation comprising a polypeptide comprising a variant Fc region provided herein. In another embodiment, the pharmaceutical formulation is for use in treating or preventing an autoimmune disease that may be caused by or associated with the production of antibodies to autoantigens (autoantibodies).

The polypeptides of the invention comprising variant Fc regions are capable of treating or preventing autoimmune diseases that may be caused by or associated with the production of antibodies to self-antigens (autoantibodies) by inhibiting the production of those antibodies. It has been reported that the use of a fusion molecule of an antibody Fc portion with AchR (autoantigen of myasthenia gravis) inhibits proliferation and induces apoptosis of B cells expressing AchR recognizing BCR (j.neurohimnol 227:35-43 (2010)). The use of fusion proteins formed between the variant Fc region of the invention and an antigen recognized by an autoantibody enables cross-linking of Fc γ RIIb to BCR on B cells directed against the autoantigen, which results in inhibition of B cell proliferation and/or induction of B cell apoptosis.

In certain embodiments, the autoimmune disease that can be treated or prevented is selected from the group consisting of: Guillain-Barre syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, autoimmune pancreatitis, aortolitis syndrome, goodpasture's syndrome, accelerated glomerulonephritis, megaloblastic anemia, autoimmune hemolytic anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Barcelado's disease, hashimoto's thyroiditis, primary hypothyroidism, idiopathic Addison's disease, insulin-dependent diabetes mellitus, chronic discoid lupus erythematosus, scleroderma, pemphigus, pemphigoid, gestational herpes, IgA bullous skin disease, acquired epidermolysis bullosa, alopecia areata, leukoplakia vulgaris, postcentrifugation leukoplakia Sutton, Protoyota, autoimmune neuropathy, autoimmune optic neuropathy, herpes, IgA macroangiopathy, IgA macroangiosis, Alzheimer's disease, multiple sclerosis, hepatitis B, and hepatitis B, and hepatitis B, and hepatitis B, Idiopathic azoospermia, habitual abortion, type II diabetes, hypoglycemia, and chronic urticaria.

In another embodiment, the invention provides a polypeptide comprising an Fc region for use in treating a disease associated with a deficiency in a biologically essential protein. In certain embodiments, the present invention provides a polypeptide comprising a variant Fc region for use in a method of treating a disease associated with a deficiency in a biologically essential protein in an individual, the method comprising administering to the individual an effective amount of a polypeptide comprising a variant Fc region to treat the disease. An "individual" according to any of the above embodiments is preferably a human.

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

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

In another aspect, the invention provides a pharmaceutical formulation comprising a polypeptide comprising a variant Fc region provided herein. In another embodiment, the pharmaceutical formulation is used to treat a disease associated with a deficiency in a biologically essential protein.

For diseases lacking biologically essential proteins, therapeutic methods of administering and supplementing proteins as agents are used. However, since the patient lacks the protein from the beginning, the externally supplied protein is recognized as a foreign substance, and an antibody against the protein is produced. Therefore, the protein becomes easy to remove, and the effect as a drug is reduced. The use of a fusion protein comprising this protein and a variant Fc region of the invention enables cross-linking between fcyriib and BCRs on B cells that recognize the protein, which allows the inhibition of antibody production against the protein.

In certain embodiments, the protein to be supplemented is selected from the group consisting of: factor VIII, factor IX, TPO, EPO, alpha-iduronidase, iduronate sulfatase, heparinoid type A N-sulfatase, alpha-N-acetylglucosaminidase type B, acetyl CoA type C, alpha-glucosaminidase acetyltransferase, N-acetylglucosamine 6-sulfatase type D, galactose 6-sulfatase, N-acetylgalactosamine 4-sulfatase, beta-glucuronidase, alpha-galactosidase, acid alpha-galactosidase and glucocerebrosidase. These proteins can be supplemented for diseases such as hemophilia, idiopathic thrombocytopenic purpura, renal anemia, and lysosomal disease (mucopolysaccharidosis), Fabry's disease, Pompe disease, Gaucher's disease, etc., but are not limited thereto.

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

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

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

In another aspect, the invention provides a pharmaceutical formulation comprising a polypeptide comprising a variant Fc region provided herein. In another embodiment, the pharmaceutical formulation is for use in treating a viral infection.

An anti-viral antibody comprising a variant Fc region of the invention can inhibit the antibody-dependent potentiation observed in conventional anti-viral antibodies. The antibody-dependent potentiation is a phenomenon in which: wherein the virus bound to the antibody is phagocytosed via the activating Fc γ R such that infection of the cell by the virus is enhanced. Binding of anti-dengue virus antibodies to Fc γ RIIb was reported to play an important role in inhibiting antibody-dependent potentiation (proc. natl. acad. sci. usa 108:12479-12484, (2011)). Cross-linking of immune complexes against dengue virus and dengue virus with Fc γ RIIb inhibits Fc γ R-mediated phagocytosis, which results in inhibition of antibody-dependent potentiation. Examples of such viruses include, but are not limited to, dengue virus (DENV1, DENV2, DENV3, and DENV4) and HIV.

In another embodiment, the present invention provides a polypeptide comprising a variant Fc region for use in the prevention or treatment of arteriosclerosis. In certain embodiments, the present invention provides a polypeptide comprising a variant Fc region for use in a method of preventing or treating arteriosclerosis in an individual, the method comprising administering to the individual an effective amount of a polypeptide comprising a variant Fc region to prevent or treat arteriosclerosis. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. An "individual" according to any of the above embodiments is preferably a human.

In another aspect, the invention provides the use of a polypeptide comprising a variant Fc region in the manufacture or formulation of a medicament. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In another embodiment, the medicament is for the prevention or treatment of arteriosclerosis. In another embodiment, the medicament is for use in a method of preventing or treating arteriosclerosis in an individual, the method comprising administering to the individual an effective amount of the medicament to prevent or treat arteriosclerosis. An "individual" according to any of the above embodiments may be a human.

In another aspect, the invention provides a method for preventing or treating atherosclerosis in an individual. In one embodiment, the method comprises administering to the individual an effective amount of a polypeptide comprising a variant Fc region to prevent or treat arteriosclerosis. In one embodiment, the "individual" is a human.

In another aspect, the invention provides a pharmaceutical formulation comprising a polypeptide comprising a variant Fc region provided herein. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In another embodiment, the pharmaceutical formulation is for use in the prevention or treatment of arteriosclerosis.

Antibodies against oxidized LDL (i.e. the cause of arteriosclerosis) comprising the variant Fc region of the invention can prevent Fc γ RIIa-dependent adhesion of inflammatory cells. It was reported that while anti-oxidized LDL antibodies inhibit the interaction of oxidized LDL with CD36, anti-oxidized LDL antibodies bind to endothelial cells and monocytes recognize their Fc portion in an Fc γ RIIa-dependent or Fc γ RI-dependent manner (immunol. lett.108:52-61, (2007)). Use of antibodies comprising the variant Fc regions of the invention can inhibit Fc γ RIIa-dependent binding and inhibit monocyte adhesion through Fc γ RIIb-mediated inhibitory signals.

In another embodiment, the present invention provides a polypeptide comprising a variant Fc region for use in the prevention or treatment of cancer. In certain embodiments, the present invention provides a polypeptide comprising a variant Fc region for use in a method of preventing or treating cancer in an individual, the method comprising administering to the individual an effective amount of a polypeptide comprising a variant Fc region to prevent or treat cancer. An "individual" according to any of the above embodiments is preferably a human.

In another aspect, the invention provides the use of a polypeptide comprising a variant Fc region as provided herein in the manufacture or formulation of a medicament. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In another embodiment, the medicament is for preventing or treating cancer. In another embodiment, the medicament is for use in a method of preventing or treating cancer in an individual, the method comprising administering to the individual an effective amount of the medicament to prevent or treat cancer. An "individual" according to any of the above embodiments may be a human.

In another aspect, the invention provides a method for preventing or treating cancer in an individual. In one embodiment, the method comprises administering to the individual an effective amount of a polypeptide comprising a variant Fc region to prevent or treat cancer. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In one embodiment, an "individual" is a human.

In another aspect, the invention provides a pharmaceutical formulation comprising a polypeptide comprising a variant Fc region provided herein. . In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In another embodiment, the pharmaceutical formulation is for use in the prevention or treatment of cancer.

As described above, it is known that enhancing Fc γ RIIb binding increases the agonist activity of agonist antibodies and enhances the anti-tumor effect of the antibodies. Thus, agonist antibodies comprising the variant Fc regions of the invention are useful for treating or preventing cancer. In particular, the variant Fc regions of the invention enhance agonist activity of antibodies directed to: for example, receptors of the TNF receptor family, such as CD120a, CD120b, lymphotoxin beta receptor, CD134, CD40, FAS, TNFRSF6B, CD27, CD30, CD137, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, RANK, osteoprotegerin, TNFRSF12A, TNFRSF13B, TNFRSF13C, TNFRSF14, nerve growth factor receptor, TNFRSF17, TNFRSF18, TNFRSF19, TNFRSF21, TNFRSF25 and ectoderm dysplasia 2 receptors, and can be used to treat or prevent cancer. Furthermore, agonist activity is also enhanced for agonistic antibodies directed against other molecules that exhibit agonist activity through interaction with Fc γ RIIb. In addition, by incorporating a variant Fc region of the invention into an antibody directed against a Receptor Tyrosine Kinase (RTK) such as Kit that inhibits cell proliferation after cross-linking with fcyriib, the inhibitory effect of the antibody on cell proliferation can be enhanced.

In some embodiments, the present invention provides methods for preventing or treating cancer, including but not limited to members selected from the group consisting of: small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), carcinoma of the large intestine, rectum, colon, breast, liver, stomach, pancreas, kidney, prostate, ovary, thyroid, bile duct, peritoneum, mesothelioma, squamous cell, cervix, endometrium, bladder, esophagus, head and neck, nasopharynx, salivary gland, thymus, skin, basal cell, malignant melanoma, anus, penis, testis, Wilms' tumor, acute myelocytic leukemia (including acute myelogenous leukemia, acute primitive myelogenous leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, and acute monocytic leukemia), chronic myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and small cell leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma (Burkitt's lymphoma), chronic lymphocytic leukemia, mycosis fungoides, mantle cell lymphoma, follicular lymphoma, diffuse large cell lymphoma, marginal zone lymphoma, hairy cell leukemia plasmacytoma (pilocytic leukemia plasmacytoma), peripheral T cell lymphoma, and adult T cell leukemia/lymphoma), Langerhans cell histiocytosis (Langerhans cell histiocytosis), multiple myeloma, myelodysplastic syndrome, brain tumors (including gliomas, astrocytomas, glioblastomas, meningiomas, and ependymomas), neuroblastomas, retinoblastomas, osteosarcomas, Kaposi's sarcoma (Kaposi's sarcoma), Ewing's sarcoma, angiosarcoma, and hemangioblastoma.

Antibodies that have been modified by modification of at least one amino acid residue to have enhanced binding activity to Fc γ R (including Fc γ RIIb) may promote elimination of antigen 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 enhanced Fc γ R-binding activity under neutral pH conditions is rapidly taken into cells together with an antigen forming a complex therewith, and thus, when the antibody is administered in vivo, elimination of the antigen from plasma can be promoted.

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

Without wishing to be bound by a particular theory, it is believed that the pH of the biological fluid (e.g., plasma) is in the neutral pH range. In biological fluids, the net positive charge of antibodies with increased pI increases due to the increased pI, and thus, the antibodies are attracted more strongly to the endothelial cell surface with net negative charge by physiochemical coulombic interactions than antibodies without increased pI. This means that, via this non-specific binding, the uptake of the antibody complexed with its antigen into the cells is enhanced, which leads to an enhanced elimination of the antigen from the plasma. These phenomena are expected to occur universally in vivo regardless of cell type, tissue type, organ type, and the like.

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

In additional embodiments, the present invention provides polypeptides comprising a variant Fc region for promoting elimination of an antigen from plasma. In certain embodiments, the present invention provides a polypeptide comprising a variant Fc region for use in a method of promoting elimination of an antigen from plasma in an individual, the method comprising administering to the individual an effective amount of a polypeptide comprising a variant Fc region to promote elimination of the antigen from plasma. From these embodiments, it is preferred that the polypeptide further comprises an antigen binding domain. An "individual" according to any of the above embodiments is preferably a human.

In another aspect, the invention provides the use of a polypeptide comprising a variant Fc region in the manufacture or formulation of a medicament. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In another embodiment, the medicament is for promoting elimination of an antigen from plasma. In another embodiment, the medicament is for use in a method of promoting elimination of an antigen from plasma in an individual, the method comprising administering to the individual an effective amount of the medicament to promote elimination of the antigen from plasma. In these embodiments, it is preferred that the polypeptide further comprises an antigen binding domain. An "individual" according to any of the above embodiments may be a human.

In another embodiment, the present invention provides a method for promoting elimination of an antigen from plasma in an individual. In one embodiment, the method comprises administering to the individual an effective amount of a polypeptide comprising a variant Fc region to promote elimination of the antigen from plasma. In these embodiments, it is preferred that the polypeptide further comprises an antigen binding domain. In one embodiment, the "individual" is a human.

In another aspect, the invention provides a pharmaceutical formulation comprising any of the polypeptides comprising a variant Fc region provided herein. In another embodiment, the pharmaceutical formulation is used to promote elimination of antigen from plasma. In such embodiments, it is preferred that the polypeptide further comprises an antigen binding domain.

In one embodiment, an antibody having a variant Fc region of the invention enhances elimination of an antigen from plasma, e.g., by at least 1.1 fold, 1.25 fold, 1.5 fold, 1.75 fold, 2.0 fold, 2.25 fold, 2.5 fold, 2.75 fold, 3.0 fold, 3.25 fold, 3.5 fold, 3.75 fold, 4.0 fold, 4.25 fold, 4.5 fold, 4.75 fold, 5.0 fold, 5.5 fold, 6.0 fold, 6.5 fold, 7.0 fold, 7.5 fold, 8.0 fold, 8.5 fold, 9.0 fold, 9.5 fold, or more than 10.0 fold when the antibody is administered in vivo as compared to prior to amino acid modification.

In another aspect, the invention provides a method for preparing a medicament or pharmaceutical formulation, the method comprising admixing any of the polypeptides comprising a variant Fc region provided herein with a pharmaceutically acceptable carrier, e.g., for any of the methods of treatment described above. In one embodiment, the method for preparing a medicament or pharmaceutical formulation further comprises adding at least one additional therapeutic agent to the medicament or pharmaceutical formulation.

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

Similarly, polypeptides comprising a variant Fc region of the invention can be used in therapy alone or in combination with other agents. For example, a polypeptide comprising a variant Fc region of the invention can be co-administered with at least one additional therapeutic agent.

Such combination therapies described above include both combined administration (where two or more therapeutic agents are contained in the same or separate formulations) and separate administration, in which case administration of the antibody or polypeptide comprising a variant Fc region of the invention can occur prior to, concurrently with, and/or after administration of the additional therapeutic agent or agents. In one embodiment, the administration of the anti-myostatin antibody and the administration of the additional therapeutic agent occur within about one month of each other, or within about one week, two weeks, or three weeks, or within about one day, two days, three days, four days, five days, or six days.

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

The antibody or polypeptide comprising a variant Fc region of the invention (and optionally any additional therapeutic agent) may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if required for local treatment, intralesional administration. Parenteral injection includes intramuscular administration, intravenous administration, intraarterial administration, intraperitoneal administration, or subcutaneous administration. Administration may be by any suitable route, e.g., by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is transient or chronic. Various dosing regimens are contemplated herein, including but not limited to a single administration or multiple administrations at multiple time points, bolus administration, and pulsed infusion.

The antibodies or polypeptides of the invention comprising a variant Fc region may be formulated, administered and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular disease being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause, the site of agent delivery, the method of administration, the timing of administration, and other factors known to medical practitioners. The antibodies need not be, but are optionally, formulated with one or more agents currently used to prevent or treat the target disease. The effective amount of such other agents will depend on the amount of antibody present in the formulation, the type of disease or treatment, and other factors discussed above. These are generally used at the same dosages and routes of administration as described herein, or at about 1 to 99% of the dosages described herein, or at any dosage and any route empirically/clinically determined to be appropriate.

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

It is to be understood that any of the above formulations or methods of treatment may be carried out using the immunoconjugates of the invention (either instead of or in addition to the anti-myostatin antibodies).

It is also understood that any of the above-described formulations or methods of treatment may be performed using the immunoconjugates of the invention (in place of or in addition to the polypeptides comprising variant Fc regions provided herein).

H. Article of manufacture

In another aspect of the invention, articles of manufacture are provided which comprise materials useful in the treatment, prevention and/or diagnosis of the above-mentioned diseases. The article comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be made of a variety of materials, such as glass or plastic. The container holds a composition, either alone or in combination with another composition effective for treating, preventing and/or diagnosing a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody of the invention. The indicia or package insert indicates that the composition is for use in treating the selected condition. Further, the article of manufacture may comprise (a) a first container having a composition contained therein, wherein the composition comprises an antibody of the invention; and (b) a second container having a composition contained therein, wherein the composition further comprises a cytotoxic or other therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the composition may be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution and dextrose solution. It may also contain other materials as desired by the commercial or user, including other buffers, diluents, fillers, needles and syringes.

It is to be understood that any of the above-described preparations may include an immunoconjugate of the invention in place of, or in addition to, an anti-myostatin antibody.

Examples

The following are examples of the methods and compositions of the present invention. It is to be understood that various other embodiments may be practiced given the general description provided above.

Example 1

Expression and purification of latent and mature forms of human, cynomolgus and mouse myostatin

Human latent myostatin (also referred to herein as latent form of human myostatin) was transiently expressed using FreeStyle293-F cells (FS293-F cells) (Thermo Fisher, Carlsbad, Calif., USA) (SEQ ID NO: 1). Conditioned media containing the latent form of human myostatin expressed was acidified to ph6.8 and diluted with 1/2 volumes of milliQ water before being applied to a Q-sepharose FF anion exchange column (GE healthcare, Uppsala, Sweden). The flow-through fractions were adjusted to ph5.0 and applied to a SP-sepharose HP cation exchange column (GE healthcare, Uppsala, Sweden) and then eluted with a NaCl gradient. The fractions containing the latent form of human myostatin were collected and subsequently applied to a Superdex 200 gel filtration column equilibrated with 1x PBS (GE healthcare, Uppsala, Sweden). The fractions containing the latent form of human myostatin were then pooled and stored at-80 ℃.

Mature human myostatin (also referred to herein as the mature form of human myostatin) (SEQ ID NO:2) was purified from the purified latent form of human myostatin. The latent form of human myostatin was acidified by addition of 0.1% trifluoroacetic acid (TFA) andapplied to a Vydac 214TP C4 reverse phase column (Grace, Deerfield, IL, USA) with TFA/CH₃And (3) performing gradient elution by CN. The fractions containing human mature myostatin were pooled, dried and stored at-80 ℃. Human mature myostatin was reconstituted by dissolving in 4mM HCl.

The expression and purification of latent and mature forms of myostatin from cynomolgus monkeys (cynomolgus or cyno) (SEQ ID NOS: 3 and 4, respectively) and mice (SEQ ID NOS: 5 and 6, respectively) was performed in exactly the same manner as the human counterpart. The sequence homology in the mature form between human, cynomolgus monkey and mouse is 100% identical, and therefore any mature myostatin (whatever the species) can be used as a mature myostatin in all necessary experiments.

Example 2

Identification of anti-latent myostatin antibodies

Anti-latent myostatin antibodies were prepared, selected, and assayed as follows.

NZW rabbits 12 to 16 weeks old were immunized intradermally with mouse latent myostatin and/or human latent myostatin (50-100 μ g/dose/rabbit). This dose was repeated 3-4 times over a month period. One week after the final immunization, spleens and blood from immunized rabbits were collected. Antigen-specific B-cells were stained with labeled antigen, sorted with FCM cell sorter (FACS aria III, BD), and plated at a density of one cell/well in 96-well plates with 25,000 cells/well of EL4 cells (european collection of cell cultures) and rabbit T-cell conditioned medium diluted 20-fold, and cultured for 7-12 days. EL4 cells were treated with mitomycin c (sigma) for 2 hours and washed 3 times in advance. Rabbit T-cell conditioned media was prepared by culturing rabbit thymocytes in RPMI-1640 with phytohemagglutinin-M (Roche), phorbol 12-myristate 13-acetate (Sigma) and 2% FBS. After incubation, the B-cell culture supernatant was collected for further analysis and the cell pellet (pellet) was cryopreserved.

An ELISA assay was used to test the specificity of the antibodies in the B-cell culture supernatant. Streptavidin (GeneScript) at 50nM in PBS was coated on 384-well maxisorp (nunc) for 1 hour at room temperature. The plate was then blocked with 5-fold dilution of Blocking One (Nacalai Tesque). Human or mouse latent myostatin was labeled with NHS-PEG 4-biotin (PIERCE) and added to the blocked ELISA plates, incubated for 1 hour, and washed with Tris buffered saline (TBS-T) containing 0.05% Tween-20. The B-cell culture supernatant was added to the ELISA plates, incubated for 1 hour, and washed with TBS-T. Binding was detected by goat anti-rabbit IgG-horseradish peroxidase (BETHYL) followed by addition of abts (kpl).

A total of 17,818B-cell lines were screened for binding specificity to mouse and/or human latent myostatin, and 299 cell lines were selected and designated MST0255-287, 630-632, 677-759, 910, 932-1048, 1050-1055, 1057-1066, 1068, 1070-1073, 1075-1110, 1113-1119. RNA was purified from the corresponding cell pellet using the ZR-96 Quick-RNA kit (ZYMO RESEARCH).

The DNA encoding the variable regions of the heavy and light chains of each selected cell line was amplified by reverse transcription PCR and cloned into expression vectors having the heavy chain constant region G1m sequence (SEQ ID NO:50 (amino acid sequence shown in SEQ ID NO: 7)) and the light chain constant region k0MTC or k0MC sequence (SEQ ID NO:53 or 194 (amino acid sequences (both identical) shown in SEQ ID NO: 8)), respectively. Recombinant antibodies were transiently expressed using FreeStyle FS293-F cells and 293fectin (life technologies) according to the manufacturer's instructions. Culture supernatants or recombinant antibodies were used for screening. The recombinant antibody was purified with protein A (GE healthcare) and eluted in D-PBS or His buffer (20mM histidine, 150mM NaCl, pH 6.0). Size exclusion chromatography is further performed to remove high molecular weight and/or low molecular weight components, if desired.

Example 3

Characterization of anti-latent myostatin antibodies (HEK Blue assay (BMP1 activated))

Reporter gene assays are used to assess the biological activity of in vitro active muscle growth inhibitory factor. Expression of Smad 3/4-binding element (S)BE) inducible HEK-Blue of SEAP reporter gene^TMTGF-beta cells (Invivogen) allow for the detection of biologically active myostatin by monitoring activation of activin type 1 and type 2 receptors. Active myostatin stimulates production of SEAP secreted into the cell supernatant. Then using QUANTIBlue^TM(Invivogen) the amount of SEAP secreted was assessed.

HEK-Blue^TMTGF-beta cells were maintained in a supplemented state with 10% fetal bovine serum, 50. mu.g/mL streptomycin, 50U/mL penicillin, 100. mu.g/mL Normocin ^TM30. mu.g/mL Blasticidin (Blasticidin), 200. mu.g/mL Hygrogold^TMAnd 100. mu.g/mL Zeocin^TMIn DMEM medium (Gibco). During the functional assay, cells were changed to assay medium (with 0.1% bovine serum albumin, streptomycin, penicillin and Normocin)^TMDMEM) and seeded into 96-well plates. Human, cynomolgus monkey or mouse latent muscle growth inhibitory factor and recombinant human BMP1 (R)&D Systems) and anti-latent antibody were incubated overnight at 37 ℃. The sample mixture is transferred to the cells. After 24 mice incubation, cell supernatants were incubated with QUANTIBlue ^TMMix and measure the optical density at 620nm in a colorimetric plate reader. As shown in figure 1, mAb MST1032-G1m (amino acid and nucleotide sequence see table 2a) prevented protease mediated activation of human, cynomolgus monkey and mouse latent myostatin as reflected by reduced secreted SEAP concentrations. MST1032-G1m showed nearly equivalent inhibitory activity against human, cynomolgus and mouse latent myostatin.

[ Table 2a ]

[ Table 2b ]

Example 4

Anti-latent myostatinCharacterization of the antibodies (HEK Blue assay (spontaneous activation))

Human, cynomolgus monkey or mouse latent myostatin and anti-latent myostatin antibody were incubated overnight at 37 ℃ and the sample mixture was transferred to HEK-Blue^TMTGF-beta cells. After 24 hours incubation, cell supernatants were incubated with QUANTIBlue^TMThe optical density at 620nm was mixed and measured in a colorimetric plate reader. As shown in FIG. 2, the release of active myostatin from its latent form was detected after incubation at 37 ℃. In the presence of mAb MST1032-G1m, myostatin activation was inhibited and therefore lower SEAP levels were detected in the cell supernatant. MST1032-G1m inhibited spontaneous activation of latent myostatin and showed nearly comparable inhibitory activity against human, cynomolgus monkey and mouse latent myostatin.

Example 5

Characterization of anti-latent myostatin antibodies (ELISA)

Uncoated ELISA plates (NUNC-IMMUNO plate MAXISORP surface, Nalge Nunc International) were coated with 20. mu.l of 50nM streptavidin (GenScript) for 1 hour at room temperature. The plates were then washed three times with PBST and blocked overnight with 50. mu.l of 20% Blocking One (Nacalai Tesque). On the next day, each well of each plate was incubated with biotinylated mature myostatin, human or mouse biotinylated latent myostatin, or biotinylated recombinant mouse myostatin pro peptide-Fc chimeric protein (R & D Systems) at 4 nM/well/20 μ l for 2 hours. After washing, 20. mu.l of the antibody sample was added to the well and the plate was allowed to stand for 1 hour. The plate was washed and 20 μ l of anti-human IgG-horseradish peroxidase (HRP) (Abcam) diluted in HEPES buffered saline was added, and the plate was allowed to stand for an additional one hour. The plates were then washed again, then 50 μ Ι ABTS (KPL) was added to each well and the plates were incubated for 1 hour. The signal was detected at 405nm in a colorimetric plate reader. The results of the binding experiments are shown in figure 3. MST1032-G1m binds to latent myostatin (i.e., a non-covalent complex of mature myostatin and a propeptide) and the propeptide, but does not bind to mature myostatin. These results show that MST1032-G1m specifically binds to the myostatin pro peptide (e.g., the pre (prp) peptide portion), but does not bind to the active myostatin (e.g., the maturation region).

Example 6

Characterization of anti-latent myostatin antibodies (Western blot)

Mouse latent muscle growth inhibitory factor and recombinant human BMP1 (R)&D Systems) was incubated overnight at 37 ℃ in the presence or absence of MST1032-G1 m. The samples were then mixed with 4x reducing SDS-PAGE sample buffer (Wako) and heated at 95 ℃ for 5 minutes before loading for SDS gel electrophoresis. Then Turbo by Trans-Blot (registered trademark)^TMThe Transfer System (Bio-rad) transfers proteins to membranes. Using sheep anti-mouse GDF8 propeptide antibody (R)&D Systems) detection of myostatin pro peptide, which pro peptide antibody is then detected by anti-sheep IgG-hrp (santa cruz). Membranes were incubated with ECL substrate and imaged using ImageQuant LAS 4000(GE healthcare). As shown in FIG. 4, propeptide cleavage by BMP1 was inhibited by MST1032-G1 m.

Example 7

Characterization of anti-latent myostatin antibody (BIACORE (registered trademark))

Anti-latent myostatin antibodies were evaluated against kinetic parameters of human, cynomolgus monkey (cyno), and mouse latent myostatin at 37 ℃ at ph7.4 using BIACORE (registered trademark) T200 instrument (GE healthcare). ProA/G (Pierce) was immobilized on all flow chambers of a CM4 chip using an amine coupling kit (GE healthcare). At ACES pH7.4(20mM ACES, 150mM NaCl, 1.2mM CaCl) ₂，0.05％Tween 20，0.005％NaN₃) Preparing anti-latent myostatin antibodies and analytes. The antibody was captured to the sensor surface by ProA/G. Antibody capture levels are typically 150 to 220 Resonance Units (RU). Then 3.125 to 50nM human, food prepared by two-fold serial dilution was injectedLatent myostatin in cynomolgus monkeys or mice, then isolated. The sensor surface was regenerated with 25mM NaOH. Kinetic parameters were determined by processing and fitting the data to a 1:1 binding model using BIACORE (registered trademark) T200Evaluation software, version 2.0 (GE healthcare). The induction diagram is shown in fig. 5. The on-rate (ka), off-rate (KD), and binding affinity (KD) are listed in table 3. The kinetic parameters of MST1032-G1m for latent myostatin in humans, cynomolgus monkeys, and mice were comparable.

[ Table 3]

Ka, KD and KD of anti-latent myostatin antibody MST1032-G1m

Example 8

In vivo efficacy of anti-latent myostatin antibodies on muscle mass and fat mass

The in vivo efficacy of mAb MST1032-G1m was evaluated in mice. Anti-mature myostatin antibody 41C1E4 (as described in U.S. patent No. 7,632,499) was used as a positive control in this study. To avoid potential immunomodulation due to mouse anti-human antibody responses, in vivo studies were performed in Severely Combined Immunodeficient (SCID) mice that were immunodeficient. Five-week-old SCID (c.b-17 SCID) mice (Charles River Laboratories Japan, Inc. (Kanagawa, Japan)) were treated with different doses of monoclonal antibody or vehicle (PBS) (administered intravenously once a week for two weeks). On days 0, 7 and 14, mice were evaluated for total body lean body mass and fat mass by Nuclear Magnetic Resonance (NMR) (small nuclear magnetic LF-50, Bruker Bio Spin, (Kanagawa, JAPAN)). The animals were euthanized on day 14, and gastrocnemius and quadriceps muscles were isolated and weighed.

Statistical significance was determined by ANOVA, schuentt test (Student's t-test), and Dunnett test (Dunnett's test) using JMP 9 software (SAS, Inc.). p values less than 0.05 were considered significant.

The results of this experiment are shown in FIGS. 6A-C. Both antibodies (MST1032-G1m and 41C1E4) increased lean body mass in a dose-dependent manner, and MST1032-G1m (when administered at 40 mg/kg) significantly reduced fat mass compared to the vehicle (PBS) group at day 14.

Two weeks after treatment, antibody administered at 10mg/kg and 40mg/kg resulted in a significant increase in wet weight of the quadriceps and gastrocnemius muscles relative to the vehicle group.

Example 9

Comparison of in vivo potency of various myostatin-related antibodies

All experimental settings used for comparison were the same as in example 8. Various myostatin-related antibodies (41C1E4, REGN, OGD, and MYO-029) were tested in five-week-old SCID mice for two weeks. REGN, OGD and MYO-029 are anti-mature muscle growth inhibitor antibodies described as H4H1657N2 in WO 2011/150008, OGD1.0.0 in WO 2013/186719 and MYO-029 in WO 2004/037861, respectively. These antibodies were administered intravenously once a week. Lean body mass and fat mass were examined by whole body NMR scanning on days 0 and 14. Grip strength was measured on day 14 using a grip tester (e.g., GPM-100B, MELQUEST ltd., (Toyama, JAPAN)). As shown by the results presented in FIGS. 7A-C, these antibodies all increased lean body mass, except MYO-029 (P > 0.05). The grip strength of the group administered MST1032-G1m was significantly increased (P <0.05) at 10mg/kg relative to the vehicle (PBS) group. 41C1E4 and REGN also significantly increased grip strength compared to vehicle (PBS). MST1032-G1m at 10mg/kg tended to reduce the total body fat mass. The fat mass reduction efficacy of MST1032-G1m was greater compared to other anti-mature myostatin antibodies.

Example 10

Humanization of anti-latent myostatin antibodies

Amino acid residues in the variable region of an antibody are numbered according to Kabat (Kabat et al, Sequence of proteins of immunological interest, 5)^th Ed.,Public Health Service,National Institutes of Health,Bethesda,MD(1991))。

The variable regions of the heavy and light chains of the humanized MST1032 antibody were designed. Some humanized MST1032 antibodies contain back mutations in the framework regions. The designed polynucleotides for the heavy and light chain variable regions were synthesized by GenScript Inc. and cloned into expression vectors containing the heavy chain constant region SG1 sequence (SEQ ID NO:52 (amino acid sequence shown in SEQ ID NO: 9)) and the light chain constant region SK1 sequence (SEQ ID NO:54 (amino acid sequence shown in SEQ ID NO: 10)), respectively. Humanized antibody was transiently expressed in FS293-F cells, and HEK Blue assay and BIACORE (registered trademark) analysis were performed as described above. As shown in fig. 8 and table 4, and compared to fig. 1 and 2, the humanized antibody (MS1032LO00-SG1) showed comparable inhibitory activity and affinity to the chimeric antibody (MST1032-G1 m).

[ Table 4]

Kinetic parameters of humanized anti-latent myostatin antibodies

Example 11

production of pH-dependent anti-latent myostatin antibodies

To prepare pH-dependent anti-latent myostatin antibodies, all CDRs of mAb MS1032LO00-SG1 were subjected to histidine-scanning mutagenesis. Each amino acid In the CDR was mutated individually to histidine using the In-Fusion HD Cloning Kit (Clontech Inc. or Takara Bio company) according to the manufacturer's instructions. After each variant was confirmed to be correctly mutated by sequencing, the variant was transiently expressed and purified by the above method. All variants of histidine substitutions were evaluated by a BIACORE (registered trademark) assay modified compared to the above. Briefly, an additional dissociation phase at ph5.8 was integrated into the BIACORE (registered trade mark) assay, which immediately follows the dissociation phase at ph 7.4. This was to assess pH-dependent dissociation between the antibody (Ab) and antigen (Ag) of the pair from the complex formed at pH7.4 at the corresponding dissociation phase of pH 5.8. The dissociation rate in ph5.8 buffer was determined by processing and fitting the data using Scrubber 2.0(BioLogic Software) curve fitting Software.

As shown in figure 9, the parent antibody (Ab001) showed no reduction in binding response at ph5.8 compared to the dissociation phase at ph 7.4. Several single-histidine substitutions resulted in a moderate to strong reduction in the binding response at ph5.8 compared to the dissociation phase at ph 7.4. The off-rates at pH5.8 for the individual mono-histidine substitution variants are shown in Table 5. As shown in table 5, Ab002 showed the fastest Ab/Ag complex dissociation rate at ph5.8, 200 times faster than the parent antibody (Ab 001). Antibodies with combinations of these mutations in the CDRs were then prepared. The CDR sequences of the antibodies containing these different mutations are shown in table 6.

[ Table 5]

Single amino acid substitution anti-latent myostatin antibodies with a pH of 5.8 dissociation rate

[ Table 6]

Example 12

Affinity enhancement of pH-dependent anti-latent myostatin antibodies

To identify mutations that increase affinity and/or inhibitory activity at ph7.4 in vitro, more than 500 variants of the heavy and light chains, respectively, were prepared, using at least one of the variants prepared in example 11 as template. Each amino acid in the CDRs of these variants was replaced with 18 other amino acids, excluding the original amino acids and cysteines. The binding capacity of the variants to human latent myostatin was assessed at 37 ℃ at ph7.4 using BIACORE (registered trademark) 4000 equipment (GE healthcare). Culture supernatants containing the variants expressed in FS293-F cells were prepared using ACES pH7.4 buffer containing 10mg/ml BSA and 0.1mg/ml carboxymethyl dextran (CMD). Each antibody was captured onto the flow cell until the capture level reached approximately 200 RU. 25nM human latent myostatin was then injected into the flow chamber. An additional dissociation phase at pH5.8 was integrated into the BIACORE (registered trade Mark) assay, immediately following the dissociation phase at pH 7.4. This additional dissociation phase assesses pH-dependent dissociation between the antibody (Ab) and the antigen (Ag) from the complex formed at pH 7.4. The flow cell surfaces were regenerated with 25mM NaOH. Kinetic parameters were determined using BIACORE (registered trademark) 4000Evaluation software, version 2.0 (GE healthcare). Additional analysis of dissociation at pH5.8 was performed using Scrubber2(BioLogic Software).

Variants with increased affinity at pH7.4 and/or in vitro inhibitory activity were selected and combined with the pH-dependent increasing mutations identified in example 11. After combination of these mutations, four variants (MS1032LO01, 02, 03 and 04) were selected and transiently expressed with SG1 or F760(SEQ ID NOS: 11 and 51) for BIACORE (registered trademark) binding kinetics analysis, in vitro and/or in vivo assays. SG1 and F760 are both human heavy chain constant regions. SG1 has a binding affinity for human fcyr that is comparable to that of native IgG1 constant region, while Fc modification results in the elimination of the binding affinity of F760 (also referred to herein as silent Fc). The amino acid and nucleotide sequences of the four anti-myostatin variants are shown in table 2 a.

Example 13

Characterization of the pH-dependent MS1032 variant (HEKBlue assay (BMP1 and spontaneous activation))

All experimental settings were the same as in examples 3 and 4. As shown in figure 10, all variants showed comparable inhibitory activity against human latent myostatin as MS1032LO00-SG 1.

Example 14

Characterization of pH-dependent MS1032 variant (BIACORE (registered trademark))

All experimental settings were the same as in example 7, except that the measurements were also carried out in ACES pH5.8 buffer, except for the ACES pH7.4 condition. Some antibodies were measured against human latent myostatin only. For avidity and affinity assays, antibody capture levels were targeted at 185RU and 18.5RU, respectively. Kinetic parameters were determined using BIACORE (registered trademark) T200 Evaluation software, version 2.0 (GE healthcare) using a 1:1 binding fit. The sensorgram and avidity conditions for all antibodies against human latent myostatin are shown in fig. 11, and ka, KD, and KD calculated from the different conditions are listed in tables 7-10. Under avidity conditions, all antibodies (except MS1032LO00-SG 1) showed a faster off-rate at acidic pH than at neutral pH. Under affinity conditions, all antibodies (except MS1032LO00-SG 1) showed weak interaction with latent myostatin at acidic pH, and therefore, no kinetic parameters were determined.

[ Table 7]

Ka, KD and KD determined under conditions of affinity at neutral pH

N/T: not tested

[ Table 8]

Ka, KD and KD determined under conditions of affinity at acidic pH

N/T: not tested

[ Table 9]

Ka, KD and KD determined under affinity conditions at neutral pH

N/T: not tested

[ Table 10]

Ka, KD and KD determined under affinity conditions at acidic pH

n.d.: not determined, N/T: not tested

Example 15

Fcyr junctions with binding and elimination of plasma total myostatin concentration to Fc γ R between antibodies in mice Comparison of results

In vivo testing using C.B-17 SCID mice

Accumulation of endogenous myostatin was assessed In vivo after administration of anti-latent myostatin antibodies In c.b-17 SCID mice (In vivo, Singapore). Anti-latent myostatin antibody (3mg/ml) was administered in a single dose of 10ml/kg into the tail vein. Blood was 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 14,000rpm at 4 ℃ for 10 minutes to separate plasma. The separated plasma was stored below-80 ℃ until measured. The anti-latent myostatin antibodies used were MS1032LO00-SG1 and MS1032LO 00-F760.

Measurement of total myostatin concentration in plasma by Electrochemiluminescence (ECL)

The concentration of total myostatin in mouse plasma was measured by ECL. Anti-mature myostatin immobilized plates were prepared by dispensing anti-mature myostatin antibody RK35 (as described in WO 2009/058346) onto MULTI-ARRAY 96 well plates (Meso Scale Discovery) and incubating overnight at 4 ℃. Mature myostatin calibration curve samples and mouse plasma samples diluted 40-fold or more were prepared. The sample was mixed in an acidic solution (0.2M glycine-HCl, pH2.5) to dissociate mature myostatin from its binding protein (e.g., the pro peptide). Subsequently, the samples were added to the plates immobilized with anti-mature myostatin and allowed to bind at room temperature for 1 hour, followed by washing. Next, the SULFO TAG-labeled anti-mature myostatin antibody RK22 (as described in WO 2009/058346) was added and the plates were incubated at room temperature for 1 hour, followed by washing. Read buffer T (x4) (Meso Scale Discovery) was immediately added to the plate and the signal detected by SECTOR Imager 2400(Meso Scale Discovery). Mature myostatin concentrations were calculated based on the response of the calibration curve using the analytical software SOFTmax PRO (Molecular Devices). The time course of total myostatin concentration in plasma after intravenous administration of anti-latent myostatin antibody measured by this method is shown in fig. 12.

Effect of in vivo Fc Gamma R binding on the accumulation of myostatin

Following administration of antibody MS1032LO00-F760, the plasma total myostatin concentration at 28 days accumulated 248-fold compared to the plasma total myostatin concentration at 5 minutes. In contrast, the plasma total myostatin concentration at day 28 accumulated 37-fold after administration of MS1032LO00-SG1 compared to the plasma total myostatin concentration at 5 minutes. Due to Fc γ R binding, an approximately 7-fold difference in plasma total myostatin concentration was observed between MS1032LO00-F760 (silent Fc) and MS1032LO00-SG1 at day 28. In human soluble IL-6R (hsIL-6R), no significant difference in plasma hsIL-6R concentration was observed between anti-hsIL-6R antibody-F760 (silent Fc) and-SG 1 (as described in WO 2013/125667). Because hsIL-6R is a monomeric antigen, the antibody-hsIL-6R complex contains only 1 Fc. Thus, the results in WO 2013/125667 indicate that an antibody-antigen complex with 1 Fc does not significantly bind Fc γ R in vivo to accelerate cellular uptake of immune complexes. On the other hand, using multimeric antigens (such as myostatin), antibodies can form large immune complexes and the antibody-antigen complexes contain more than 2 Fc. Thus, a significant difference in plasma antigen concentration was observed between F760 and SG1 due to strong avidity binding for Fc γ R. The results indicate that MST1032 can form a large immune complex with myostatin with more than 2 antibodies.

Example 16

PH-independent anti-latent myostatin antibodies and PH-dependent anti-latent muscle in mice Comparison of plasma Total myostatin concentrations between somatostatin antibodies

In vivo testing Using C.B-17 SCID mice

In vivo accumulation of endogenous mouse myostatin was assessed after administration of anti-latent myostatin antibodies In c.b-17 SCID mice (In vivo, Singapore), as described In example 15. The anti-latent myostatin antibodies used were MS1032LO01-SG1 and MS1032LO 01-F760.

Measurement of Total myostatin concentration in plasma by ECL

Total myostatin concentration in mouse plasma was measured by ECL as described in example 15. The time course of plasma total myostatin concentration after intravenous administration of anti-latent myostatin antibody measured by this method is shown in fig. 13.

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

In vivo tests of pH-dependent anti-latent myostatin antibodies (MS1032LO01-SG1 and MS1032LO01-F760) and comparisons of total myostatin plasma concentrations were performed. The results of total myostatin concentration measurements after administration of MS1032LO00-SG1 and MS1032LO00-F760 described in example 15 are also shown in fig. 13. MS1032LO00 is a pH independent antibody and MS1032LO01 is a pH dependent antibody. As shown in figure 13, total myostatin concentration after administration of MS1032LO01-F760 was reduced compared to MS1032LO00-F760 due to pH-dependent binding. Furthermore, the total myostatin concentration following administration of MS1032LO01-SG1 was significantly reduced compared to MS1032LO00-SG1 due to pH-dependent binding and increased cellular uptake by Fc γ R binding. MS1032LO01-SG1 was expected to have excellent properties to enhance clearance of myostatin from plasma as a "scavenger antibody".

Example 17

pH-dependent anti-latencyIn vivo efficacy of sex myostatin antibodies

All experimental settings were the same as in example 8. As shown in figure 14, MS1032LO00-SG1 (non-scavenging antibody) and MS1032LO01-SG1 (scavenging antibody) increased skeletal muscle mass in a dose-dependent manner after 2 weeks when administered intravenously to SCID mice at doses of 0.2, 1 and 5 mg/kg. MS1032LO01-SG1 significantly increased quadriceps wet weight and grip strength at 1 and 5mg/kg, and significantly increased lean body mass and gastrocnemius wet weight at 5mg/kg relative to vehicle group (PBS). MS1032LO01-SG1 also significantly reduced fat mass at 1 and 5mg/kg relative to vehicle group (PBS). MS1032LO00-SG1 did not show an increase in lean body mass at 0.2 mg/kg. On the other hand, MS1032LO01-SG1 increased significantly lean body mass, quadriceps wet weight, gastrocnemius wet weight and grip strength at 0.2 mg/kg. Thus, pH-dependent binding antibodies exhibit greater muscle mass growth and muscle strength enhancement relative to pH-independent binding antibodies.

Example 18

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 medium expressing Flag-hdgf 11 was applied to a column packed with anti-Flag M2 affinity resin (Sigma) and eluted with Flag peptide (Sigma). The Flag-hdgf 11 containing fractions were collected and subsequently applied to a Superdex 200 gel filtration column (GE healthcare) equilibrated with 1x PBS. The Flag-hdgf 11 containing fractions were then pooled and stored at-80 ℃.

Example 19

Characterization of anti-latent myostatin antibodies (ELISA)

Uncoated ELISA plates (NUNC-IMMUNO plate MAXISORP surface, Nalge Nunc International) were coated with 20. mu.l of 50nM streptavidin (GenScript) for 2 hours at room temperature. The plates were then washed three times with PBST and blocked overnight with 50. mu.l of 20% Blocking One (Nacalai Tesque). On the next day, each well of the plate was incubated with 2 nM/well/20 μ l of biotinylated human latent myostatin or 20 nM/well/20 μ l of biotinylated human latent GDF11 for 2 hours. After washing, 20 μ l of antibody sample was added to the wells and the plate was left to stand for 1 hour. The plate was washed and 20 μ l of anti-human IgG-horseradish peroxidase (HRP) (Abcam) diluted in HEPES buffered saline was added, and the plate was allowed to stand for an additional one hour. The wells were washed again, then 50 μ Ι ABTS (KPL) was added to each well and the plates were incubated for 1 hour. The signal was detected at 405nm in a colorimetric plate reader. The results of the binding experiments are shown in fig. 15. MST1032-G1m binds latent myostatin (i.e., a non-covalent complex of mature myostatin and a propeptide), but does not bind hdf 11. These results show that MST1032-G1m specifically binds myostatin, but not hdf 11.

Example 20

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

Reporter assays are used to assess the biological activity of GDF11 in vitro. HEK-Blue expressing Smad 3/4-binding element (SBE) -inducible SEAP reporter^TMTGF-beta cells (Invivogen) allow detection of biologically active GDF11 by monitoring activation of activin type 1 and type 2 receptors. Active GDF11 stimulates SEAP production and secretion into the cell supernatant. Then using QUANTIBlue^TM(Invivogen) the amount of SEAP secreted was assessed.

HEK-Blue^TMTGF-beta cells were maintained in a medium supplemented with 10% fetal bovine serum, 50. mu.g/mL streptomycin, 50U/mL penicillin, 100. mu.g/mL Normocin^TM30. mu.g/mL blasticidin, 200. mu.g/mL Hygrogold^TMAnd 100. mu.g/mL Zeocin^TMIn DMEM medium (Gibco). During the functional assay, cells were changed to assay medium (containing 0.1% bovine serum albumin, streptomycin, penicillin and Normocin)^TMDMEM) and seeded into 96-well plates. Human GDF11 was incubated overnight at 37 ℃ in the presence or absence of recombinant human BMP1(Calbiochem) and anti-latent antibody (MST1032-G1 m). The sample mixture is transferred to the cells. After the incubation for the 24 hours, the cells were, Mixing the cell supernatant with QUANTIBlue^TMThe optical density at 620nm was mixed and measured in a colorimetric plate reader. As shown in fig. 16, MST1032-G1m did not prevent protease-mediated or spontaneous activation of human GDF11 and thus failed to inhibit SEAP secretion.

Example 21

Further optimization of MS1032 variants to enhance sweeping effect

As a pH-dependent antibody, MS1032LO01-SG1, showed excellent efficacy in mice, was further optimized to increase pH-dependence by introducing mutations into antibody CDRs or by changing framework regions, enhanced uptake into cells, increased stability, and the like. More than one thousand variants were evaluated in the BIACORE (registered trademark) and/or HEK Blue assay as described above, and MS1032LO06-SG1, MS1032LO07-SG1, MS1032LO10-SG1, MS1032LO11-SG1, MS1032LO12-SG1, MS1032LO18-SG1, MS1032LO19-SG1, MS1032LO21-SG1, MS1032LO23-SG1, MS1032LO24-SG1, MS1032LO25-SG1, and MS1032LO26-SG1 were prepared. The amino acid and nucleotide sequences of these prepared antibodies are shown in table 11.

[ Table 11a ]

[ Table 11b ]

The binding affinity of the MS1032 variant to human, cynomolgus monkey (cyno) or mouse latent myostatin at pH7.4 and pH5.8 was determined using BIACORE (registered trademark) T200 instrument (GE Healthcare) at 37 ℃ to assess the effect of pH on antigen binding. ProA/G (Pierce) was immobilized to all flow chambers of a CM4 chip using an amine coupling kit (GE Healthcare). In the presence of 20mM ACES, 150mM NaCl, 1.2mM CaCl ₂，0.05％Tween 20，0.005％NaN₃All antibodies and analytes were prepared in ACES pH7.4 or pH5.8 buffer. Capture of each antibody by proA/GIs obtained on the sensor surface. Antibody capture levels are typically 130 to 240 Resonance Units (RU). Human, cynomolgus and mouse latent myostatin prepared by two-fold serial dilution was injected at 3.125 to 50nM, followed by dissociation. The sensor surface was regenerated with 10mM glycine HCl ph1.5 for each cycle. Binding affinity was determined by processing and fitting the data to a 1:1 binding model using BIACORE (registered trademark) T200 Evaluation software, version 2.0 (GE Healthcare).

The binding affinities (KD) of the MS1032 variants to human, cynomolgus monkey (cyno) and mouse latent myostatin at ph7.4 and ph5.8 are shown in table 12. All variants showed KD ratios above 5 ((KD of pH 5.8)/(KD of pH 7.4)) indicating pH-dependent binding to latent myostatin.

[ Table 12]

Example 22

Evaluation of neutralizing Activity of further optimized variants in HEK Blue assay

We evaluated 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 against human latent muscle growth inhibitors as described in example 3. As shown in fig. 17, all variants showed activity comparable to MS1032LO01-SG 1.

Example 23

PH-independent anti-latent myostatin antibodies and different PH-dependent anti-latent myostatin antibodies in mice Comparison of plasma Total myostatin concentration between Volvin myostatin antibodies

In vivo testing using C.B-17 SCID mice

In vivo accumulation of endogenous myostatin was assessed following administration of anti-latent myostatin antibodies In c.b-17 SCID mice (In vivo, Singapore). Anti-latent myostatin antibody (3mg/ml) was administered in a single dose of 10ml/kg into the tail vein. Blood was 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 14,000rpm at 4 ℃ for 10 minutes to separate plasma. The separated plasma was stored below-80 ℃ until measured. The anti-latent myostatin antibodies tested were MS1032LO00-SG1, MS1032LO01-SG1, MS1032LO06-SG1, MS1032LO11-SG1, MS1032LO18-SG1, MS1032LO19-SG1, MS1032LO21-SG1 and MS1032LO25-SG 1.

Measurement of total myostatin concentration in plasma by Electrochemiluminescence (ECL)

The concentration of total myostatin in mouse plasma was measured by ECL. Anti-mature myostatin immobilized plates were prepared by dispensing biotinylated anti-mature myostatin antibody RK35 (as described in WO 2009/058346) onto MULTI-ARRAY 96-well streptavidin plates (Meso Scale Discovery) and incubating in blocking buffer for 2 hours at room temperature. Mature myostatin calibration curve samples and mouse plasma samples diluted 40-fold or more were prepared. The sample was mixed in an acidic solution (0.2M glycine-HCl, pH2.5) to dissociate mature myostatin from its binding protein (e.g., propeptide). Subsequently, the samples were added to the plates immobilized with anti-mature myostatin and allowed to bind at room temperature for 1 hour, followed by washing. Next, a SULFO TAG-labeled anti-mature myostatin antibody RK22 (as described in WO 2009/058346) was added and the plates were incubated at room temperature for 1 hour, followed by washing. Read Buffer T (x4) (Meso Scale Discovery) was immediately added to the plate and the signal detected by SECTOR Imager 2400(Meso Scale Discovery). Mature myostatin concentrations were calculated based on the response of the calibration curves using the analytical software SOFTmax PRO (Molecular Devices). The time course of total myostatin concentration in plasma after intravenous administration of anti-latent myostatin antibody measured by this method is shown in fig. 18.

PH-dependent myostatin binding versus myostatin accumulation in vivo studies in mice Function of (2)

The effect of pH-dependence on myostatin accumulation in mice was compared using a pH-independent anti-latent myostatin antibody (MS1032LO00-SG1) and different pH-dependent anti-latent myostatin antibodies (MS1032LO01-SG1, MS1032LO06-SG1, MS1032LO11-SG1, MS1032LO18-SG1, MS1032LO19-SG1, MS1032LO21-SG1, and MS1032LO25-SG 1). The introduction of pH-dependence significantly accelerated the clearance of myostatin from the plasma of SCID mice. As shown in fig. 18, on day 28, pH-dependent anti-latent myostatin antibodies (MS1032LO01-SG1, MS1032LO06-SG1, MS1032LO11-SG1, MS1032LO18-SG1, MS1032LO19-SG1, MS1032LO21-SG1, and MS1032LO25-SG1) can reduce myostatin accumulation compared to pH-independent anti-latent myostatin antibodies (MS1032LO00-SG 1).

Example 24

PH-independent anti-latent myostatin antibodies and methods of use thereof Comparison of plasma Total myostatin concentration between anti-latent myostatin antibodies

In vivo testing using cynomolgus monkeys

The in vivo accumulation of endogenous myostatin was assessed after administration of anti-latent myostatin antibody in 2-4 years old bundled monkeys (Macaca fascicularis, cynomolgus monkeys) (Shin Nippon biological Laboratories ltd., japan) from cambodia. The 30mg/kg dose level was injected into the cephalic vein of the forearm using a disposable syringe, extension tube, indwelling needle, and infusion pump. The rate of administration was 30 minutes per body. Blood was collected before the start of administration and at 5 minutes, 7 hours and 1, 2, 3, 7, 14, 21, 28, 35, 42, 49 and 56 days after the end of administration or at 5 minutes and 2, 4 and 7 hours and 1, 2, 3, 7, 14, 21, 28, 35, 42, 49 and 56 days after the end of administration. Blood was drawn from the femoral vein using a syringe containing heparin sodium. The blood was immediately cooled on ice and plasma was obtained by centrifugation at 1700x g for 10 minutes at 4 ℃. Plasma samples were stored in a deep-freezing refrigerator (acceptable range: -70 ℃ or lower) until measurement. The anti-latent myostatin antibodies used were MS1032LO00-SG1, MS1032LO06-SG1012, MS1032LO06-SG1016, MS1032LO06-SG1029, MS1032LO06-SG1031, MS1032LO06-SG1033, and MS1032LO06-SG1034 (herein, SG1012, SG1016, SG1029, SG1031, SG1033, and SG1034 are heavy chain constant regions constructed based on SG1, as described below).

Anti-latent myostatin antibodies MS1032LO19-SG1079, MS1032LO19-SG1071, MS1032LO19-SG1080, MS1032LO19-SG1074, MS1032LO19-SG1081, and MS1032LO19-SG1077 (herein, SG1079, SG1071, SG1080, SG1074, SG1081, and SG1077 are heavy chain constant regions constructed based on SG1, as described below) were administered at a dose level of 2mg/kg into the cephalic or saphenous vein of the forearm using a disposable syringe and a retaining needle. Blood was collected before the start of administration and at 5 minutes and 2, 4, and 7 hours and 1, 2, 3, 7, 14 days after the end of administration. Blood was treated as described above. Plasma samples were stored in a deep-freezing refrigerator (acceptable range: -70 ℃ or lower) until measurement.

Measurement of total myostatin concentration in plasma by Electrochemiluminescence (ECL)

The concentration of total myostatin in monkey plasma was measured by ECL as described in example 23. The time course of plasma total myostatin concentration after intravenous administration of anti-latent myostatin antibody measured by this method is shown in fig. 19.

Measurement of ADA in monkey plasma using Electrochemiluminescence (ECL)

Biotinylated drugs were coated on a MULTI-ARRAY 96-well streptavidin plate (Meso Scale Discovery) and incubated in low cross buffer (Candor) for 2 hours at room temperature. Monkey plasma samples were diluted 20-fold in low cross buffer before addition to the plates. The samples were incubated overnight at 4 ℃. The next day, the plates were washed three times with wash buffer before adding the SULFO TAG-labeled anti-monkey IgG secondary antibody (Thermo Fisher Scientific). After one hour incubation at room temperature, the plates were washed three times with wash buffer. Read Buffer T (x4) (Meso Scale Discovery) was immediately added to the plate and signals were detected using SECTOR Imager 2400(Meso Scale Discovery).

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

In cynomolgus monkeys, administration of a pH-independent antibody (MS1032LO00-SG1) resulted in at least a 60-fold increase in myostatin concentration from baseline at day 28. On day 28, the pH-dependent anti-latent myostatin antibodies MS1032LO06-SG1012 and MS1032LO06-SG1033 resulted in 3-fold and 8-fold increases from baseline, respectively. Strong clearance is mainly due to an increase in affinity for cynomolgus monkey Fc γ RIIb. On day 28, the pH-dependent anti-latent myostatin antibodies MS1032LO06-SG1029, MS1032LO06-SG1031, and MS1032LO06-SG1034 can clear antigen below baseline. The strong clearance of MS1032LO06-SG1029, MS1032LO06-SG1031 and MS1032LO06-SG1034 is due to an increase in non-specific uptake in cells due to an increase in positive charge clusters of the antibody and an increase in Fc γ R mediated cellular uptake due to enhanced binding to Fc γ R.

In cynomolgus monkeys, administration of the pH-dependent anti-latent myostatin antibodies MS1032LO19-SG1079, MS1032LO19-SG1071, MS1032LO19-SG1080, MS1032LO19-SG1074, MS1032LO19-SG1081 and MS1032LO19-SG1077 reduced the myostatin concentration below the detection limit (<0.25ng/mL) from day 1. On day 14, the concentration of myostatin increased above the detection limit for MS1032LO19-SG1079 and MS1032LO19-SG1071, while for MS1032LO19-SG1080, MS1032LO19-SG1074, MS1032LO19-SG1081 and MS1032LO19-SG1077, the concentration of myostatin remained below the detection limit. The weaker inhibition of MS1032LO19-SG1079 and MS1032LO19-SG1071 may be due to differences in pI mutations.

The data indicate that strong clearance of myostatin from plasma can be achieved by mutations that increase binding to Fc γ RIIb or mutations that will increase the positive charge of the antibody and increase binding to Fc γ RIIb. It is expected that strong clearance of myostatin in humans can be achieved by binding mutations that increase the positive charge of the antibody and improve binding to Fc γ R.

Example 25

In vivo efficacy of other optimized variants in mice

In vivo efficacy was assessed in Scid mice using MS1032LO06-SG1, MS1032LO11-SG1, MS1032LO18-SG1, MS1032LO19-SG1 and MS1032LO25-SG1 as described in example 8. Three independent studies were performed and MS1032LO01 was used as a control.

The results of these experiments are shown in FIGS. 20A-20I. All antibodies (MS1032LO06-SG1, MS1032LO11-SG1, MS1032LO18-SG1, MS1032LO19-SG1 and MS1032LO25-SG1) showed dose-dependent increases in Lean Body Mass (LBM) and limb grip strength. Antibodies also showed a decrease in body fat mass in a dose-dependent manner.

Example 26

Epitope mapping of anti-latent myostatin antibodies

Additional anti-human latent myostatin antibodies were prepared for mapping of their corresponding binding epitopes. Two NZW rabbits were immunized and harvested as described in example 2. The identified 1760 antibody-producing B-cell lines were further screened for their ability to block BMP 1-mediated activation of human latent myostatin. Briefly, the B-cell supernatants containing the secreted antibodies were incubated with human latent myostatin in the presence of recombinant human BMP1(R & D Systems) overnight at 37 ℃. Then 50. mu.l of the reaction mixture was transferred to a Meso Scale Discovery multiple-ARRAY 96 well plate coated with anti-mature myostatin antibody RK35 (as described in WO 2009/058346). After 1 hour incubation at room temperature with shaking, the plates were incubated with biotinylated anti-mature myostatin antibody RK22 (as described in WO 2009/058346) followed by incubation with SULFO-tagged streptavidin. Read Buffer T (x4) (Meso Scale Discovery) was then added to the plate and ECL signals were detected by SECTOR Imager 2400(Meso Scale Discovery). 94 cell lines showing different levels of neutralizing activity were selected for downstream analysis (MST1495-MST 1588). The variable regions of these selected cell lines were cloned as described in example 2, except that an expression vector having the heavy chain constant region F1332m sequence (SEQ ID NO:193) was used.

A set of 7 anti-latent myostatin antibodies (MST1032, MST1504, MST1538, MST1551, MST1558, MST1572 and MST 1573; sequence identifiers of the amino acid sequences of these antibodies are shown in Table 13) was further evaluated for inhibitory activity on human latent myostatin activation. As shown in figure 21, all antibodies were able to inhibit BMP 1-mediated activation of human latent myostatin in a dose-dependent manner.

[ Table 13]

Amino acid sequence of anti-latent myostatin antibodies

4 antibodies that performed well in western blotting were selected for epitope mapping. A fragment of the N-terminal propeptide coding region of human latent myostatin was cloned into pGEX4.1 vector to prepare 100 amino acid GST-tagged propeptide fragments each having an 80 amino acid overlap (FIG. 22A). Protein expression was induced in transformed BL21 competent cells using the Overnight Express Autoindexing System (Merck Millipore) and protein was extracted using the BugBuster protein extraction reagent (Novagen). Expression of the desired protein fragment of approximately 37kDa (anti-GST antibody (Abcam)) was confirmed by western blot analysis as described in example 6 (fig. 22B). When tested with anti-human latent myostatin antibodies, all 4 antibodies recognized different epitopes on human latent myostatin, as shown in fig. 22C, although they were able to inhibit latent myostatin activation. The MST1032 antibody can detect the first five fragments, with NO detectable band after removal of amino acids 81-100(SEQ ID NO: 78). Both the MST1538 and MST1572 antibodies bound only the first three fragments, and NO band was detected in the absence of amino acids 41-60 of SEQ ID NO: 78. Antibody MST1573 bound strongly to only the first two fragments, indicating that its epitope is within amino acids 21-40 of SEQ ID NO:78 (FIG. 22D).

Example 27

Development of novel Fc γ RIIb-enhanced Fc variants

In this example, Fc engineering for enhancing myostatin clearance is illustrated.

It has been demonstrated in WO 2013/125667 that clearance of soluble antigens can be enhanced by its administration of an antigen binding molecule comprising an Fc domain showing improved affinity for Fc γ RIIb. Furthermore, Fc variants that may show enhanced binding to human Fc γ RIIb have been exemplified in WO 2012/115241 and WO 2014/030728. Also exemplified are Fc variants that can selectively display enhanced binding to human fcyriib and reduced binding to other active fcyr. This selective Fc γ RIIb binding enhancement may be beneficial not only for clearance of soluble antigens but also for reducing the risk of undesired effector functions and immune responses.

To develop an antibody drug, efficacy, pharmacokinetics and safety should be evaluated in a non-human animal to which the drug has pharmacological activity. Alternative approaches such as the use of alternative antibodies (int.j. tox.28:230-253(2009)) have to be considered if they are active only in humans. However, it is not easy to accurately predict the effect of the interaction between the Fc region and the Fc γ R in humans using surrogate antibodies, because the expression pattern and/or function of Fc γ R in non-human animals is not always the same as in humans. Preferably, the Fc region of the antibody drug is cross-reactive with a non-human animal (particularly a cynomolgus monkey having an expression pattern and function of Fc γ R similar to that of a human), so that the results obtained in the non-human animal can be extrapolated to humans.

Thus, Fc variants that show cross-reactivity to human and cynomolgus monkey Fc γ rs were developed in this study.

Affinity measurements for human and monkey Fc γ rs for existing Fc γ RIIb-enhanced Fc variants

The heavy chain genes of the human Fc γ RIIb enhancer variant disclosed in WO 2012/115241 (herein, "Fc γ RIIb enhancer" means "having enhanced Fc γ RIIb-binding activity") were prepared by substituting Asp for Pro at EU numbering position 238 in the heavy chain of MS1032LO06-SG1, 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). As the antibody light chain, M103202L889-SK1(VL, SEQ ID NO: 96; CL, SEQ ID NO:10) was used. Recombinant antibodies were expressed according to the method shown in example 34.

The extracellular domain of Fc γ R was prepared in the following manner. Synthesis of human Fc γ R extracellular domain genes was performed by methods known to those skilled in the art based on the information registered in NCBI. Specifically, Fc γ RIIa is based on NCBI numbering NM _001136219.1 sequence, Fc γ RIIb is based on NM _004001.3, Fc γ RIIIa is based on NM _001127593.1, respectively. Allotypes of Fc γ RIIa and Fc γ RIIIa were prepared according to reports on polymorphisms of Fc γ RIIa (J.Exp.Med.172:19-25(1990)) and Fc γ RIIIa (J.Clin.invest.100(5): 1059-. The synthesis of cynomolgus monkey Fc γ R extracellular domain genes was performed by cloning cDNA of each Fc γ R from cynomolgus monkeys using methods known to those skilled in the art. The amino acid sequence of the constructed Fc γ R extracellular domain is shown in the sequence listing: (SEQ ID NO 210 is human Fc γ RIIaR, SEQ ID NO 211 is human Fc γ RIIaH, SEQ ID NO 214 is human Fc γ RIIb, SEQ ID NO 217 is human Fc γ RIIIaF, SEQ ID NO 218 is human Fc γ RIIIaV, SEQ ID NO 220 is cynomolgus monkey Fc γ RIIa1, SEQ ID NO 221 is cynomolgus monkey Fc γ RIIa2, SEQ ID NO 222 is cynomolgus monkey Fc γ RIIa3, SEQ ID NO 223 is cynomolgus monkey Fc γ RIIb, and SEQ ID NO 224 is cynomolgus monkey Fc γ RIIIaS). A His tag was then added to its C-terminus and each gene obtained was inserted into an expression vector designed for mammalian cell expression. The expression vector was introduced into FreeStyle293 cells (Invitrogen) derived from human embryonic kidney cells to express the target protein. After cultivation, the resulting culture supernatant was filtered and purified in principle by the following four steps. Cation exchange chromatography using SP Sepharose FF was performed as the first step, affinity chromatography against His tag (HisTrap HP) as the second step, gel filtration column chromatography (Superdex200) as the third step, and sterile filtration as the fourth step. The concentration of the purified Protein was determined using a spectrophotometer to measure the absorbance of the purified Protein at 280nm and using the extinction coefficient calculated by the PACE method (Protein Science 4:2411-2423 (1995)).

Kinetic analysis of the interaction between these antibodies and Fc γ R was performed using BIACORE (registered trademark) T200 or BIACORE (registered trademark) 4000(GE Healthcare). HBS-EP + (GE Healthcare) was used as a running buffer, and the measurement temperature was set to 25 ℃. Chips prepared by immobilizing protein A, protein A/G or mouse anti-human IgG kappa light chain (BD Biosciences) on Series S Sensor Chip CM4 or CM5(GE Healthcare) by amine coupling were used. The antibody of interest was captured on the chip to interact with each Fc γ R that had been diluted with running buffer, and the binding to the antibody was measured. After the measurement, the captured antibody on the chip was washed away by allowing to react with 10mM glycine-HCl, pH1.5 and 25mM NaOH to regenerate the chip and reuse it. Sensorgrams obtained as measurement results were analyzed by a 1:1Langmuir binding model using BIACORE (registered trademark) Evaluation Software to calculate an association rate constant ka (L/mol/s) and an dissociation rate constant KD (1/s), and a dissociation constant KD (mol/L) was calculated from these values. Because MS1032LO06-MY009 binds weakly to human Fc γ RIIaH, human Fc γ RIIIaV and cynomolgus monkey Fc γ RIIIaS, kinetic parameters such as KD cannot be calculated by the above mentioned analytical methods. Regarding the interaction, KD values were calculated using the following 1:1 binding model described in BIACORE (registered trademark) T100 Software Handbook BR1006-48 Edition AE.

The behaviour of the interacting molecule according to the 1:1 binding model on BIACORE (registered trade mark) can be described by equation 1: req ═ cx R max/(KD + C) + RI, where Req is a plot of steady-state binding level versus analyte concentration, C is concentration, RI is bulk refractive index (bulk reactive index) distribution in the sample, and Rmax is the analyte binding capacity of the surface. When rearranging this equation, KD can be expressed as equation 2: KD ═ Cx Rmax/(Req-RI) -C. KD may be calculated by substituting the values of Rmax, RI, and C into equation 1 or equation 2. From the current measurement conditions, RI ═ 0 and C ═ 2 μ Mol/L can be used. Further, Rmax values obtained when a sensorgram obtained as a result of analyzing the interaction of each Fc γ R with IgG1 was fully fitted using a 1:1Langmuir binding model were divided by the amount of captured SG1, multiplied by the amount of captured MY009, and the obtained values were used as Rmax. This calculation is based on the assumption that the limit amount of each Fc γ R that can be bound by SG1 remains unchanged for all variants prepared by introducing mutations into SG1, and Rmax at the time of measurement is proportional to the amount of antibody bound on the chip at the time of measurement. Req is defined as the amount of binding observed for each Fc γ R to each variant on the sensor chip when measured.

Table 14 shows the results of kinetic analysis of human and cynomolgus monkey Fc γ R by SG1 and MY 009. KD values filled in gray bins were calculated using [ equation 2] because their affinity was too weak to be correctly determined by kinetic analysis. KD fold values were calculated by dividing the KD value of SG1 for each Fc γ R by the value of the variant KD.

As shown in table 14, the human Fc γ RIIb enhanced variant MY009 does not show enhanced binding to cynomolgus monkey Fc γ RIIb but shows enhanced binding to human Fc γ RIIb. Its affinity for cynomolgus monkey Fc γ RIIb was reduced by 0.4 fold compared to SG1, indicating that the human Fc γ RIIb enhancing variant MY009 has no cross-reactivity to cynomolgus monkey Fc γ R.

Development of novel Fc variants showing enhanced binding to both human and cynomolgus monkey Fc γ RIIb

Ideally, the novel Fc variants should selectively have enhanced binding to both human and cynomolgus monkey Fc γ RIIb and reduced binding to other active Fc γ rs. However, since the putative Fc binding residues in cynomolgus Fc γ RIIb are identical to any allotype of cynomolgus Fc γ RIIa (fig. 23), it is theoretically not possible to achieve a selective enhancement of cynomolgus Fc γ RIIb binding relative to cynomolgus Fc γ RIIa. Thus, the novel Fc variants should selectively have enhanced binding to human Fc γ RIIb, cynomolgus monkey Fc γ RIIb and cynomolgus monkey Fc γ RIIa.

To obtain novel Fc variants showing selectively enhanced binding to both human and cynomolgus monkey Fc γ RIIb, the results of extensive mutagenesis studies performed in WO 2012/115241 were used. In extensive mutagenesis studies, extensive mutations were introduced at all positions in the Fc γ R binding region of IgG1 antibodies, and binding to each Fc γ R was analyzed comprehensively, as shown in the following procedure.

[ Table 14]

The variable region of the glypican 3 antibody comprising the CDRs of GpH7, which is the anti-glypican 3 antibody with improved plasma kinetics disclosed in WO 2009/041062, was used as the antibody heavy chain variable region (GpH 7: SEQ ID NO: 225). Similarly, for the antibody light chain, GpL16-k0(SEQ ID NO:226) of the glypican 3 antibody with improved plasma kinetics disclosed in WO 2009/041062 was used. In addition, B3(SEQ ID NO:228) was used as an antibody H chain constant region, and the K439E mutation in B3 was introduced into G1d (SEQ ID NO:227) prepared by removing the C-terminal Gly and Lys of IgG 1. This heavy chain, made by fusing GpH7 and B3, is referred to as GpH7-B3(VH, SEQ ID NO: 225; CH, SEQ ID NO: 228).

With respect to GpH7-B3, the amino acid residues believed to be involved in Fc γ R binding and the surrounding amino acid residues (positions 234 to 239, 265 to 271, 295, 296, 298, 300, and 324 to 337, according to EU numbering) were each replaced with 18 amino acid residues (excluding the original amino acid residue and Cys). These Fc variants are referred to as B3 variants. The B3 variants were expressed and the binding of protein-a purified antibodies to each of the Fc γ rs (Fc γ RIIa type H, Fc γ RIIa type R, Fc γ RIIb, and Fc γ RIIIaF) was evaluated comprehensively as follows. Analysis of the interaction between each of the altered antibodies and Fc γ receptor prepared as mentioned above was performed using BIACORE (registered trademark) T100(GE Healthcare), BIACORE (registered trademark) T200(GE Healthcare), BIACORE (registered trademark) a100, or BIACORE (registered trademark) 4000. HBS-EP + (GE Healthcare) was used as a running buffer, and the measurement temperature was set to 25 ℃. Chips prepared by fixing protein a (thermo scientific), protein a/g (thermo scientific), or protein L (ACTIGEN or BioVision) to Series S sensor chips CM5 or CM4(GE Healthcare) by amine coupling methods were used. After capturing the antibody of interest onto these sensor chips, the amount of binding to the antibody was measured by allowing Fc γ receptor interaction diluted with running buffer. However, since the amount of bound Fc γ receptor depends on the amount of captured antibody, the amount of bound Fc γ receptor is divided by the amount of each captured antibody to obtain corrected values, and these values are compared. In addition, the captured on the chip antibody with 10mM glycine-HCl, pH1.5 washing, and the chip regeneration and reuse. The value of Fc γ R binding amount of each B3 variant was divided by the value of Fc γ R binding amount of the parent B3 antibody (an antibody having the sequence of naturally occurring human IgG1 at positions 234 to 239, 265 to 271, 295, 296, 298, 300, and 324 to 337 according to EU numbering). The value obtained by multiplying this value by 100 was used as an indication of the relative Fc γ R-binding activity of each variant.

Table 15 shows the binding characteristics of promising substitutions selected based on the following criteria (more than 40% for human Fc γ RIIb, less than 100% for human Fc γ RIIaR and Fc γ RIIaH, less than 10% for human Fc γ RIIIaF compared to the parent B3 antibody).

[ Table 15]

Selected substitutions binding characteristics to human Fc γ R

In the next step, the cross-reactivity of these substitutions to cynomolgus monkey Fc γ R was evaluated. These 16 mutations were introduced into M103205H795-SG 1. The variants were expressed using M103202L889-SK1 as light chain and analyzed for affinity for cynomolgus monkey Fc γ RIIa1, IIa2, IIa3, IIb, IIIaS.

Table 16 shows the binding characteristics of these 16 variants to cynomolgus monkey Fc γ R. The value of the amount of bound Fc γ R is obtained by dividing the amount of bound Fc γ R by the amount of each captured antibody. This value was further normalized with the value of SG1 used as 100.

[ Table 16]

Binding characteristics of selected substitutions to cynomolgus monkey Fc gamma R

Of the 16 Fc variants selected, only MY001 retained 85% of binding to cynomolgus monkey Fc γ RIIb compared to SG 1. Furthermore, MY001 showed reduced binding to cynomolgus monkey Fc γ RIIIaS while its binding to cynomolgus monkey Fc γ RIIa1, IIa2 and IIa3 was maintained. Thus, the G236N mutation is the only substitution that shows similar binding characteristics to both human and cynomolgus monkey Fc γ R.

Although the G236N substitution showed cross-reactivity to both human and cynomolgus monkey Fc γ R, its affinity for Fc γ RIIb was lower than SG1 (75% for human Fc γ RIIb and 85% for cynomolgus monkey Fc γ RIIb, respectively). To increase its affinity for Fc γ RIIb, additional substitutions were evaluated. Specifically, substitutions showing enhanced binding to human Fc γ RIIb, attenuated binding to human Fc γ RIIIa and enhanced selectivity to human Fc γ RIIb relative to human Fc γ RIIa were selected based on the results of extensive mutagenesis studies (table 17).

[ Table 17]

Additional selected substitutions binding characteristics to human Fc γ R

Among these substitutions, L234W and L234Y were selected because of increased selectivity for human Fc γ RIIb relative to human Fc γ RIIa. And G236A, G236S, a330R, a330K and P331E were selected because of the diminished binding to human fcyriiia. All other substitutions were selected for enhanced binding to human Fc γ RIIb. The selected substitutions were evaluated for cross-reactivity to cynomolgus monkey Fc γ R. In addition, three substitutions, P396M, V264I and E233D, were also evaluated. The substitutions were introduced into M103205H795-SG1 and the variants were expressed using M103202L889-SK1 as the light chain.

Table 18 shows kinetic analysis of selected substitutions, including G236N, on both human and cynomolgus monkey Fc γ R. Specifically, the substitutions were introduced into M103205H795-SG 1. The variants were expressed using M103202L889-SK1 as the light chain and analyzed for affinity for cynomolgus monkey Fc γ RIIa1, IIa2, IIa3, IIb, IIIaS.

KD values filled in the gray grid were calculated using [ equation 2] because their affinity was too weak to be correctly determined by kinetic analysis. The values for KD fold were obtained by dividing the KD value of SG1 for each Fc γ R by the KD value of the variant.

G236N shows comparable binding affinity to SG1 for cynomolgus monkey Fc γ RIIa1, cynomolgus monkey Fc γ RIIa2, cynomolgus monkey Fc γ RIIa3 and cynomolgus monkey Fc γ RIIb. Although the affinity for human Fc γ RIIb decreased to 0.6 fold, the affinity for human Fc γ RIIaH and human Fc γ RIIaR decreased to 0.2 fold and 0.1 fold, respectively, indicating that this substitution is more selective for human Fc γ RIIb than for human Fc γ RIIa. Furthermore, it has 0.03-fold and 0.04-fold affinity for cynomolgus monkey Fc γ RIIIaS and human Fc γ RIIIaV, respectively, which is advantageous for eliminating ADCC activity. Based on this result, G236N showed nearly ideal cross-reactivity to human and cynomolgus monkey Fc γ R (although its affinity to both human and cynomolgus monkey Fc γ RIIb should be enhanced).

In additional substitutions evaluated, S298L, G236A, P331E, E233D, K334Y, K334M, L235W, S239V, K334I, L234W, K328T, Q295L, K334V, K326T, P396M, I332D, H268E, P271G, S267A and H268D show enhanced binding to both human and cynomolgus Fc γ RIIb. Specifically, H268D showed the most pronounced effect (7-fold for human Fc γ RIIb and 5.3-fold for cynomolgus monkey Fc γ RIIb). With respect to the attenuation of Fc γ RIIIa binding, G236S, a330K and G236D showed an affinity less than 0.5 times that of SG 1.

To enhance the affinity of MY001 to both human and cynomolgus monkey Fc γ RIIb, combinations of substitutions listed in table 18 were evaluated. Specifically, the substitutions were introduced into M103205H795-SG 1. The variants were expressed and analyzed for affinity using M103202L889-SK1 as the light chain. Table 19 shows the results of kinetic analysis for human and cynomolgus monkey Fc γ R. KD values filled in the gray boxes were calculated using [ equation 2] because their affinity was too weak to be correctly determined by kinetic analysis. The values for KD fold were obtained by dividing the KD value of SG1 for each Fc γ R by the KD value of the variant for each Fc γ R.

[ Table 18]

[ Table 19]

All variants inhibited affinity for human Fc γ RIIIaV (less than 0.26 fold compared to SG 1) and cynomolgus monkey Fc γ RIIIaS (less than 0.42 fold compared to SG 1). Furthermore, all variants (except MY047, MY051 and MY 141) successfully showed enhanced binding to both human and cynomolgus monkey Fc γ RIIb compared to MY001 and their human Fc γ RIIa binding remained less than 2-fold compared to SG 1. Wherein MY201, MY210, MY206, MY144, MY103, MY212, MY105, MY205, MY109, MY107, MY209, MY101, MY518, MY198 and MY197 show more than 4-fold enhanced binding to both human and cynomolgus monkey Fc γ RIIb compared to SG 1. Most importantly, MY205, MY209, MY198 and MY197 showed more than 7-fold enhanced binding to both human and cynomolgus monkey Fc γ RIIb.

Substitution at position 396 in the CH3 domain was shown in WO2014030728 to enhance affinity for human Fc γ RIIb. Extensive mutagenesis was introduced into position 396 of M103205H795-MY052 containing a G236N/H268E/P396M substitution. The resulting variants were expressed using M103202L889-SK1 as the light chain and evaluated for affinity for human and cynomolgus monkey Fc γ R (table 20). The values for KD fold were obtained by dividing the KD value of SG1 for each Fc γ R by the KD value of the variant for each Fc γ R.

Among the substitutions tested, P396I, P396K and P396L retained more than 5-fold binding to human fcyriib compared to SG1, and only P396L substitution enhanced affinity to human fcyriib compared to MY 052. Regarding binding to cynomolgus monkey Fc γ RIIb, parent MY052 shows the highest affinity, which is increased 3.9-fold over SG 1.

Since G236N shows ideal cross-reactivity to human and cynomolgus monkey Fc γ R, other substitutions at position 236 that were not evaluated in the previous examples were tested. Specifically, Asn in M103205H795-MY201 was substituted with Met, His, Val, Gln, Leu, Thr, and Ile. The resulting variants were expressed using M103202L889-SK1 as the light chain and evaluated for affinity for human and cynomolgus monkey Fc γ R (table 21). KD values filled in the gray grid were calculated using [ equation 2] because their affinity was too weak to be correctly determined by kinetic analysis. The values for KD fold were obtained by dividing the KD value of SG1 for each Fc γ R by the KD value of the variant for each Fc γ R.

[ Table 20]

[ Table 21]

[ Table 22]

While all variants showed reduced affinity to both human and cynomolgus Fc γ RIIb compared to the parent MY201, MY265, prepared by substituting Asn with Thr at position 236 of MY201, maintained enhanced binding compared to SG1, 2.1-fold for human and 3.1-fold for cynomolgus Fc γ RIIb, respectively. MY265 also retains attenuated binding to both human and cynomolgus monkey Fc γ RIIIa. Although the affinity for human Fc γ RIIaH and Fc γ RIIaR was enhanced by more than 2.5 fold compared to SG1, G236T at position 236 is second preferred.

To improve selectivity and affinity of MY265 for human Fc γ RIIb, extensive mutagenesis studies were performed in positions 231 and 232. Specifically, positions 231 and 232 of M103205H795-MY265 were substituted with 18 amino acid residues excluding the original amino acid and Cys. The resulting variants were expressed using M103202L889-SK1 as the light chain and evaluated for affinity for human and cynomolgus monkey Fc γ R (table 22). The values for KD fold were obtained by dividing the KD value of SG1 for each Fc γ R by the KD value of the variant for each Fc γ R.

Regarding affinity for Fc γ RIIb, the addition of a231T, a231M, a231V, a231G, a231F, a231I, a231L, a231W, P232V, P232Y, P232F, P232M, P232I, P232W, P232L improves binding to both human and cynomolgus monkey Fc γ RIIb compared to the parent MY 265. Most importantly, the addition of a231T and a231G attenuated human Fc γ RIIaR binding compared to MY 265.

Combinations of permutations that were not evaluated in the previous embodiment are evaluated. The permutations tested and shown to be promising were introduced combinatorially into M103205H795-SG 1. The resulting variants were expressed using M103202L889-SK1 as the light chain and evaluated for their affinity for human and cynomolgus monkey Fc γ R. Table 23 shows the results, and the KD values filled in the gray grid were calculated using [ equation 2] because their affinity was too weak to be correctly determined by kinetic analysis. The values for KD fold were obtained by dividing the KD value of SG1 for each Fc γ R by the KD value of the variant for each Fc γ R. The values of MY209 selected as promising variants in table 19 are again shown for comparison.

In the variants tested, only MY213 showed higher binding affinity to both human and cynomolgus monkey Fc γ RIIb compared to MY 209. However, MY213 has a higher affinity for human Fc γ RIIaH and human Fc γ RIIaR than MY 209.

[ Table 23A ]

[ Table 23B ]

[ Table 24]

Example 28

Evaluation of muscle growth inhibitory factor in fully human Fc γ R transgenic mice using Fc γ RIIb enhanced Fc variants Cleaning of

The Fc γ RIIb-enhanced Fc variants constructed in example 27 were evaluated for their effect on muscle growth inhibitory factor clearance in mice in which all murine Fc γ rs had been deleted and the human Fc γ R encoded as a transgene had been inserted into the mouse genome (proc. natl. acad. sci.,2012,109,6181). In this mouse, all mouse Fc γ rs were replaced with human Fc γ rs so that the affinity for human Fc γ RIIb enhanced effect on soluble antigen clearance can be assessed in mice. In addition, substitutions that increase pI were evaluated in conjunction with Fc γ RIIb-enhanced Fc variants constructed in example 27.

Preparation and characterization of test variants

The 8 antibodies tested and their binding characteristics are summarized in table 24. The heavy chain, MS103205H795-PK2, was prepared by introducing pI increasing substitutions (S400R/D413K) into MS103205H795-MY 101. MS103205H795-MY351, MS103205H795-MY344, MS103205H795-MY335 were prepared by introducing another pI raising substitution (Q311R/D413K) into MS103205H795-MY201, MS103205H795-MY205, MS103205H795-MY265, respectively. All MS1032LO06 variants were expressed with M103202L889-SK1 as the light chain according to the method shown in example 27 and their affinity to human and cynomolgus monkey Fc γ R was evaluated using the method in example 27.

Based on SPR analysis outlined in table 24, it was confirmed that substitutions that increase pI did not affect Fc γ R binding of Fc variants with enhanced Fc γ RIIb. MY101 and PK2 prepared by introducing S400R/D413K into MY101 showed 9-fold and 8-fold enhancement of human Fc γ RIIb binding, respectively. The affinity of MY101 and PK2 for human Fc γ RIIa remained comparable to SG 1. It shows almost the same binding characteristics for other human and cynomolgus monkey Fc γ rs. Similarly, MY351 showed similar binding characteristics as parent MY201, MY344 showed similar binding characteristics as parent MY205, and MY335 showed similar binding characteristics as parent MY265, respectively (table 21). These results indicate that neither substitution to increase pI, S400R/D413K or Q311R/D413K, affected the affinity for human and cynomolgus monkey Fc γ R.

PK studies in fully human Fc γ R transgenic mice

In vivo testing using fully human Fc γ R transgenic mice

The elimination of myostatin and anti-latent myostatin antibodies was evaluated in vivo after co-administration of anti-latent myostatin antibodies and human latent myostatin in fully human Fc γ R transgenic mice (fig. 24 and 25). The anti-latent myostatin antibody (0.3mg/ml) and latent myostatin (0.05mg/ml) were administered in a single dose of 10ml/kg into the tail vein. anti-CD 4 antibody (1mg/ml) was administered into the tail vein three times (once every 10 days) at a dose of 10ml/kg to inhibit anti-drug antibodies. Blood was collected at 5 minutes, 15 minutes, 1 hour, 4 hours, 7 hours, 1 day, 2 days, 7 days, 14 days, 21 days and 28 days post-administration. The collected blood was immediately centrifuged at 15,000rpm at 4 ℃ for 5 minutes to separate plasma. The separated plasma was stored below-20 ℃ until measured. The anti-latent myostatin antibodies used were MS1032LO06-SG1, MS1032LO06-MY101, MS1032LO06-PK2, MS1032LO06-MY201, MS1032LO06-MY351, MS1032LO06-MY205, MS1032LO06-MY344 and MS1032LO06-MY 335.

Measurement of total myostatin concentration in plasma by Electrochemiluminescence (ECL)

The concentration of total myostatin in the plasma of mice was measured by ECL. Plates immobilized with anti-mature myostatin antibody were prepared by distributing anti-mature myostatin antibody RK35(WO 2009058346) on a Multi-ARRAY 96-well plate (Meso Scale Discovery) and incubating overnight at 4 ℃. Mature myostatin calibration curve samples and mouse plasma samples diluted 4-fold or more were prepared. The sample was mixed in an acidic solution (0.2M glycine-HCl, pH2.5) to dissociate mature myostatin from its binding protein (e.g., propeptide). Subsequently, the sample was added to the plate on which the anti-mature myostatin antibody was immobilized, and allowed to bind at room temperature for 1 hour, followed by washing. Next, BIOTIN TAG-labeled anti-mature myostatin antibody RK22(WO 2009/058346) was added and the plates were incubated at room temperature for 1 hour, followed by washing. Next, SULFO TAG-labeled streptavidin (Meso Scale Discovery) was added and the plate was incubated at room temperature for 1 hour, followed by washing. Read Buffer T (x4) (Meso Scale Discovery) was immediately added to the plate and the signal detected by SECTOR Imager 2400(Meso Scale Discovery). Mature myostatin concentrations were calculated based on the response of the calibration curves using the analytical software SOFTmax PRO (Molecular Devices). The time course of total myostatin concentration in plasma after intravenous administration of anti-latent myostatin antibody and latent myostatin measured by this method is shown in figure 24.

Measurement of anti-latent muscle in plasma by high performance liquid chromatography-electrospray tandem mass spectrometry (LC/ESI-MS/MS) Concentration of meat growth inhibitory factor antibody

The anti-latent myostatin antibody concentration in the plasma of mice was measured by LC/ESI-MS/MS. In mouse plasma, the concentrations of the calibration standards were 1.5625, 3.125, 6.25, 12.5, 25, 50, 100 and 200. mu.g/mL. Mu.l of calibration standard and plasma sample were added to 50. mu.l of Ab-Capture Mag (ProteNova) and allowed to incubate at room temperature for 2 hours. Thereafter, the magnetic beads were recovered from the sample and washed twice with 0.2mL of 10mmol/L PBS (containing 0.05% Tween 20). Subsequently, the beads were washed with 10mmol/L PBS to ensure removal of Tween 20. After washing, the magnetic beads were suspended in 25. mu.l of 7.5mol/L urea, 8mmol/L dithiothreitol and 1. mu.g/mL lysozyme (egg white) in 50mmol/L ammonium bicarbonate and the suspended sample was incubated for 45 min at 56 ℃. Then, 2. mu.l of 500mmol/L iodoacetamide was added and the sample was incubated at 37 ℃ for 30 minutes in the dark. Next, Lysyl Endopeptidase digestion was performed by adding 150. mu.l of 0.67. mu.g/mL in 50mmol/L ammonium bicarbonate for biochemical Lysyl Endopeptidase (Wako) and incubating the sample at 37 ℃ for 3 hours. Subsequently, pancreatin digestion was performed by adding 10. mu.l of 10. mu.g/mL sequencing grade modified trypsin (Promega) in 50mmol/L ammonium bicarbonate. The sample was allowed to digest overnight at 37 ℃ with mixing and quenched by the addition of 5 μ l of 10% trifluoroacetic acid. Analysis by LC/ESI-MS/MS was performed on 50. mu.l of the digested sample. LC/ESI-MS/MS was performed using a Xevo TQ-S triple quadrupole instrument (Waters) equipped with 2D I-class UPLC (Waters). The anti-latent myostatin antibody specific peptide YAFGQGTK and lysozyme specific peptide GTDVQAWIR as internal standards were monitored by Selected Response Monitoring (SRM). SRM transition anti-latent myostatin antibodies are [ M +2H ]2+ (M/z 436.2) to y8 ions (M/z 637.3), and [ M +2H ]2+ (M/z 523.3) to y8 ions (M/z 545.3) for lysozyme. The peak areas plotted against concentration were used to construct an internal calibration curve by weighted (1/x or 1/x2) linear regression. Concentrations in mouse plasma were calculated from calibration curves using analytical software Masslynx ver.4.1 (Waters). The time course of the concentration after intravenous administration of the anti-latent myostatin antibody and the latent myostatin antibody in plasma measured by this method is shown in fig. 25.

Effect of pI and Fc Gamma R binding on in vivo concentration of myostatin

Following administration of MS1032LO06-SG1, the plasma total myostatin concentration at 7 hours was reduced by a factor of 5 compared to the plasma total myostatin concentration at 5 minutes. In contrast, after administration of MS1032LO06-MY101, MS1032LO06-MY201 and MS1032LO06-MY205, the plasma total myostatin concentration at 7 hours was reduced by 28-200 times compared to the plasma total myostatin concentration at 5 minutes. Furthermore, the plasma total myostatin concentration at 7 hours was decreased 361-fold 419-fold after administration of MS1032LO06-PK2, MS1032LO06-MY351, MS1032LO06-MY344 and MS1032LO06-MY335 compared to the plasma total myostatin concentration at 5 minutes. On the other hand, the concentration difference of each antibody at each sampling point was approximately within 2-fold compared to MS1032LO06-SG1, and the pI variants did not improve the elimination of antibodies from plasma. Both the human Fc γ RIIb binding enhancing antibody and the pI increasing substitutions increased the elimination of myostatin, but not the antibody.

The high pI variants have a more positive charge in plasma. Because this positive charge interacts with the negative charge on the cell surface, the antigen-antibody immune complex of the high pI variant becomes closer to the cell surface, resulting in increased cellular uptake of the antigen-antibody immune complex of the high pI variant.

Example 29

Assessment of muscle growth inhibitory factor clearance in monkeys using Fc γ RIIb enhanced Fc variants

The effect of enhanced binding of the Fc variants developed in example 27 to cynomolgus monkey Fc γ RIIb on myostatin clearance was evaluated in cynomolgus monkeys. And also to evaluate the combined effect with substitutions that increase pI.

Preparation and characterization of test variants

The 14 antibodies tested and their binding characteristics are summarized in table 25. Fc variants with enhanced binding to FcRn at acidic pH are reported to increase the in vivo half-life of the antibody (J.biol.chem.2006281: 23514-23524 (2006); nat.Biotechnol.28:157-159(2010), Clin Pharm. & Thera.89(2):283-290 (2011)). To increase antibody half-life without binding to rheumatoid factor, we combined these substitutions with Fc variants with enhanced Fc γ RIIb and increased pI.

Heavy chains, MS103205H795-SG1012, MS103205H795-SG1029, MS103205H795-SG1031, MS103205H795-SG1033, MS103205H795-SG1034 are prepared by introducing an N434A substitution into MS103205H795-MY101, MS103205H795-MY344, MS103205H795-MY351, MS103205H795-MY201, MS103205H795-MY335, respectively. MS103205H795-SG1016 was prepared by introducing pI raising substitutions (Q311R/D399K) into MS103205H795-SG 1012. MS103240H795-SG1071 and MS103240H795-SG1079 were prepared by introducing M428L/N434A/Y436T/Q438R/S440E substitutions and N434A/Q438R/S440E substitutions, respectively, into MS103240H795-MY344 (MS103240H 795: VH, SEQ ID NO; 92).

MS103240H795-SG1074 and MS103240H795-SG1077 were prepared by introducing pI-raising substitutions Q311R/P343R and M428L/N434A/Y436T/Q438R/S440E into MS103240H795-MY209 and MS103240H795-MY518, respectively. MS103240H795-SG1080 and MS103240H795-SG1081 were prepared by introducing pI-raising substitutions Q311R/P343R and N434A/Q438R/S440E into MS103240H795-MY209 and MS103240H795-MY518, respectively. MS103240H795-SG1071 was prepared by introducing pI raising substitutions Q311R/D413K and M428L/N434A/Y436T/Q438R/S440E into MS103240H795-MY 205. MS103240H795-SG1079 was prepared by introducing the pI raising substitutions Q311R/D413K and N434A/Q438R/S440E into MS103240H795-MY 205. These MS1032LO06 and MS1032LO19 variants were expressed as light chains using M103202L889-SK1 and M103202L1045-SK1(M103202L 1045: VL, SEQ ID NO:97), respectively, and their affinities to human and cynomolgus monkey Fc γ R were evaluated using the methods in example 27, according to the methods shown in example 34. In this example, the KD fold values for each Fc variant were calculated by dividing the KD value of parent SG1 for each Fc γ R by the KD value of the variant for each Fc γ R. For example, KD multiplier values for MS1032LO06-SG1012 and MS1032LO19-SG1071 are calculated by dividing the KD values for MS1032LO06-SG1 and MS1032LO19-SG1 by the KD values for MS1032LO06-SG1012 and MS1032LO19-SG1071, respectively.

[ Table 25]

The SPR analysis results are summarized in table 25. Among these variants, SG1012, SG1016, SG1074 and SG1080 showed the strongest affinity for human Fc γ RIIb, with 10-fold enhanced affinity for human Fc γ RIIb compared to SG 1.

29.2. PK Studies in monkeys

In vivo testing using cynomolgus monkeys

Accumulation of endogenous myostatin was assessed in vivo after administration of anti-latent myostatin antibody in 2-4 years old bundled monkeys (cynomolgus monkeys) (Shin Nippon biological Laboratories ltd., japan) from cambodia. The dosage level of 30mg/kg was injected into the cephalic vein of the forearm using a disposable syringe, extension tube, indwelling needle and infusion pump. The rate of administration was 30 minutes per body. Blood was collected before the start of administration and at 5 minutes, 7 hours and 1, 2, 3, 7, 14, 21, 28, 35, 42, 49 and 56 days after the end of administration or at 5 minutes and 2, 4 and 7 hours and 1, 2, 3, 7, 14, 21, 28, 35, 42, 49 and 56 days after the end of administration. Blood was drawn from the femoral vein using a syringe containing sodium heparin. The blood was immediately cooled on ice and plasma was obtained by centrifugation at 1700x g for 10 minutes at 4 ℃. Plasma samples were stored in a deep-freeze freezer (acceptable range: -70 ℃ or below) until measured. The anti-latent myostatin antibodies used were MS1032LO00-SG1, MS1032LO06-SG1012, MS1032LO06-SG1016, MS1032LO06-SG1029, MS1032LO06-SG1031, MS1032LO06-SG1033 and MS1032LO 35 06-SG1034 (herein, SG1012, SG1016, SG1029, SG1031, SG1033 and SG1034 are heavy chain constant regions constructed based on SG1 as described below).

For the anti-latent myostatin antibodies MS1032LO19-SG1079, MS1032LO19-SG1071, MS1032LO19-SG1080, MS1032LO19-SG1074, MS1032LO19-SG1081, and MS1032LO19-SG1077 (herein, SG1079, SG1071, SG1080, SG1074, SG1081 and SG1077 are heavy chain constant regions constructed based on SG1 as described below), 2mg/kg dose levels were administered into the cephalic or saphenous vein of the forearm using a disposable syringe and a indwelling needle. Blood was collected before the start of administration and at 5 minutes and 2, 4, and 7 hours and 1, 2, 3, 7, 14 days after the end of administration. Blood was treated as described above. The plasma samples were stored in a deep-freezing refrigerator (acceptable range: -70 ℃ or below) until measured.

Measurement of total myostatin concentration in plasma by Electrochemiluminescence (ECL)

Total myostatin concentrations in monkey plasma were measured by ECL as described in example 23. The time course of plasma total myostatin concentration after intravenous administration of anti-latent myostatin antibody measured by this method is shown in fig. 26.

Measurement of ADA in monkey plasma using Electrochemiluminescence (ECL)

Biotinylated drugs were coated on MULTI-ARRAY 96-well streptavidin plates (Meso Scale Discovery) and incubated in low cross buffer (Candor) at room temperature for 2 hours. Monkey plasma samples were diluted 20-fold in low cross buffer before addition to the plates. The samples were incubated overnight at 4 ℃. The next day, the plates were washed three times with wash buffer before adding the SULFO TAG-labeled anti-monkey IgG secondary antibody (Thermo Fisher Scientific). After one hour incubation at room temperature, the plates were washed three times with wash buffer. Read Buffer T (x4) (Meso Scale Discovery) was immediately added to the plate and the signal detected using SECTOR Imager 2400(Meso Scale Discovery).

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

In cynomolgus monkeys, administration of a pH independent antibody (MS1032LO00-SG1) resulted in at least a 60-fold increase in myostatin concentration from baseline at day 28. On day 28, the pH-dependent anti-latent myostatin antibodies MS1032LO06-SG1012 and MS1032LO06-SG1033 resulted in 3-fold and 8-fold increases from baseline, respectively. Strong clearance was mainly attributed to increased affinity for cynomolgus monkey Fc γ RIIb. On day 28, the pH-dependent anti-latent myostatin antibodies MS1032LO06-SG1029, MS1032LO06-SG1031, and MS1032LO06-SG1034 can clear antigen below baseline. The strong clearance of MS1032LO06-SG1029, MS1032LO06-SG1031 and MS1032LO06-SG1034 is due to increased non-specific uptake in cells due to increased positive charge clusters of the antibody and increased Fc γ R mediated cellular uptake due to enhanced binding to Fc γ R.

In cynomolgus monkeys, administration of the pH-dependent anti-latent myostatin antibodies MS1032LO19-SG1079, MS1032LO19-SG1071, MS1032LO19-SG1080, MS1032LO19-SG1074, MS1032LO19-SG1081 and MS1032LO19-SG1077 reduced the myostatin concentration below the detection limit (<0.25ng/mL) from day 1. On day 14, the concentration of myostatin increased above the detection limit for MS1032LO19-SG1079 and MS1032LO19-SG1071, while for MS1032LO19-SG1080, MS1032LO19-SG1074, MS1032LO19-SG1081 and MS1032LO19-SG1077, the concentration of myostatin remained below the detection limit. The weaker inhibition of MS1032LO19-SG1079 and MS1032LO19-SG1071 may be due to differences in pI mutations.

This data indicates that strong clearance of myostatin from plasma can be achieved by mutations that enhance binding to Fc γ RIIb or a combination of mutations that will increase the positive charge of the antibody and enhance binding to Fc γ RIIb. It is expected that strong clearance of myostatin in humans can be achieved by combining mutations that increase the positive charge of the antibody and enhance binding to Fc γ R.

Example 30

Substitutions with increased pI are screened to enhance clearance of myostatin.

To enhance clearance of myostatin, substitutions in the Fc portion of the antibody that increase pI were evaluated in this example. The method of adding an amino acid substitution to an antibody constant region to increase the pI is not particularly limited, but, for example, it may be performed by the method described in WO 2014/145159. As in the case of the variable region, the amino acid substitutions introduced into the constant region are preferably those which decrease the number of 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 may be introduced at any position in the antibody constant region, and may be a single amino acid substitution or a combination of multiple amino acid substitutions. Without particular limitation, the site for introducing an amino acid substitution is preferably a position at which an amino acid side chain can be exposed on the surface of an antibody molecule. Particularly preferred examples include methods of introducing a combination of multiple amino acid substitutions that can be exposed at positions on the surface of an antibody molecule. Alternatively, the preferred positions for the various amino acid substitutions introduced here are such that they are structurally close to one another. Further, without particular limitation, the plurality of amino acid substitutions introduced herein are preferably substitutions to positively charged amino acids, so that it is preferable that it results in a state in which a plurality of positive charges are present at structurally adjacent positions.

Preparation and characterization of test variants

The antibodies tested are summarized in table 26. The heavy chain, MS103205H795-SG141, was made by introducing the pI increasing substitutions Q311R/D399R into MS103205H795-SG 1. Other heavy chain variants were also prepared by introducing the corresponding substitutions shown in table 26 into MS103205H795-SG 1. All MS1032LO06 variants were expressed using M103202L889-SK1 as light chain according to the method shown in example 34.

Mouse Fc gamma RII-binding assay using Fc variants of BIACORE (registered trade Mark) with increased pI

With respect to the prepared Fc region variant-containing antibodies, binding assay between soluble mouse Fc γ RII and antigen-antibody complex was performed using BIACORE (registered trademark) T200(GE Healthcare). Soluble mouse Fc γ RII was prepared as His-tagged molecules by methods known to those skilled in the art. An appropriate amount of anti-His antibody was immobilized onto the sensor chip CM5(GE Healthcare) by amine coupling method using a His capture kit (GE Healthcare) to capture mouse Fc γ RII. Next, the antibody-antigen complex and running buffer (as reference solution) were injected and allowed to interact with the mouse Fc γ RII captured on the sensor chip. 20mM N- (2-acetamido) -2-aminoethanesulfonic acid, 150mM NaCl, 1.2mM CaCl ₂And 0.05% (w/v) Tween 20pH7.4 was used as a running buffer, and the corresponding buffer was also used to dilute the soluble mouse Fc γ RII. For regeneration of the sensor chip, 10mM glycine-HCl pH1.5 was used. All measurements were made at 25 ℃. Analysis was performed based on binding (RU) calculated from a sensorgram obtained by measurement, and a relative value was displayed (the amount of binding of SG1 was defined as 1.00). For calculating the parameters, BIACORE (registered trademark) T100Evaluation software (GE healthcare) was used.

The SPR analysis results are summarized in table 26. Some Fc variants were demonstrated to have enhanced affinity for mouse Fc γ RII immobilized on BIACORE (registered trademark) sensor chip.

Without being bound to a particular theory, the results may be explained as follows. BIACORE (registered trademark) sensor chips are known to be negatively charged, and the charged state can be considered to be similar to the cell membrane surface. More specifically, it was hypothesized that the affinity of the antigen-antibody complex to the mouse Fc γ RII immobilized on the negatively charged BIACORE (registered trademark) sensor chip resembles the manner in which the antigen-antibody complex binds to the mouse Fc γ RII present on the surface of a similarly negatively charged cell membrane.

[ Table 26]

Summary of Fc variants with increased pI

Name of heavy chain	Replacement of	Cellular uptake	biacore IC binding
				M103205H795·SG1		1.00	1.00
M103205H795·SG141	Q311R/D399R	4.42	1.64
				M103205H795·P1375m	Q311R//D413K	3.43	1.21
M103205H795·P1378m	S400R/D413K	3.86	1.23
				M103205H795·P1383m	Q311R/S400R/D413K	5.68	1.41
M103205H795·P1484m	Q311R/D312R	0.00	1.33
				M103205H795·P1485m	Q311R/N315R	1.88	1.29
M103205H795·P1486m	Q311R/N315K	1.52	1.20
				M103205H795·P1487m	Q311R/N384R	1.23	1.46
M103205H795·P1488m	Q311R/N384K	1.45	1.43
				M103205H795·P1489m	Q311R/E318R	1.68	1.24
M103205H795·P1490m	Q311R/E318K	1.33	1.28
				M103205H795·P1491m	Q311R//E333R	1.37	1.05
M103205H795·P1492m	Q311R/E333K	1.13	0.88
				M103205H795·P1493m	Q311R/T335R	0.99	1.10
M103205H795·P1494m	Q311R/T335K	1.12	1.14
				M103205H795·P1495m	Q311R/S337R	1.76	1.30
M103205H795·P1496m	Q311R/S337K	1.69	1.28
				M103205H795·P1497m	Q311R/Q342R	1.38	1.49
M103205H795·P1498m	Q311R/Q342K	1.56	1.38
				M103205H795·P1499m	Q311R/P343R	3.61	1.98
M103205H795·P1500m	Q311R/P343K	1.82	1.32
				M103205H795·P1501m	Q311R/D413R	2.40	1.82
M103205H795·P1522m	Q311R/H285R	2.13	1.24
				M103205H795·P1523m	Q311R/H285K	1.91	1.19
M103205H796·P1524m	Q311R/G341R	3.66	1.40
				M103205H795·P1525m	Q311R/G341K	3.55	1.31
M103205H795·P1526m	Q311R/G385R	1.20	1.04
				M103205H795·P1527m	Q311R/G355K	0.92	0.98
M103205H795·P1528m	Q311R/E388R	3.02	1.12
				M103235H795·P1529m	Q311R/E388K	2.88	1.08
M108205H795·P1530m	Q311R/N390R	3.12	1.09
				M103205H795·P1531m	Q311R/N390K	2.35	1.01
M103205H795·P1532m	Q311R/D401R	4.16	1.23
				M103205H795·P1533m	Q811R/D401K	3.52	1.18
M103205H795·P1534m	Q311R/G402R	3.12	1.12
				M103205H795·P1535m	Q311R/G402K	2.49	1.11
M103205H795·P1536m	Q311R/G420R	2.05	1.16
				M103205H795·P1537m	Q311R/V422R	2.77	1.32
M103205H795·P1538m	Q311R/V422K	1.84	1.11
				M103205H795·P1539m	Q311R/A431R	3.20	0.90
M103205H795·P1540m	D401R/D413K	7.64	1.73
				M103205H795·P1541m	D401K/D413K	7.17	1.57
M103205H795·P1542m	G402R/D413K	3.32	1.21
				M103205H795·P1543m	G402K/D413K	3.70	1.19
M103205H795·P1544m	Q311R/D401R/D413K	7.66	2.04
				M103205H795·P1545m	Q311R/D401K/D413K	7.73	1.80
M103205H795·P1546m	Q311R/G402R/D413K	5.10	1.41
				M103205H795·P1547m	Q311RG402K/D413K	4.85	1.33
M103205H795·P1548m	Q311R/G385R/D401R	2.30	1.37
				M103205H795·P1549m	Q311R/G385R/G402R	2.03	1.22
M103205H795·P1550m	Q311R/E338R/D401R	6.43	1.63
				M103205H795·P1551m	Q311R/E388R/G402R	2.91	1.33
M103205H795·P1552m	G385R/D401R/D413K	4.44	2.19
				M103205H795·P1553m	G385R/G402R/D413K	2.86	1.47
M103205H795·P1555m	E388R/G402R/D413K	3.71	2.01

Here, an antibody prepared by introducing a pI-increasing modification into an Fc region is an antibody in which the charge of the Fc region is more toward the positive side than before the modification is introduced. Thus, it is believed that the coulombic interaction between the Fc region (positive charge) and the sensor chip surface (negative charge) is enhanced by the pI-increasing amino acid modification. Furthermore, this effect is expected to similarly occur on the same negatively charged cell membrane surface; therefore, it is also expected to show an effect of accelerating the rate of uptake into cells in vivo.

In the Fc variant with increased pI compared to SG1 with two amino acid substitutions, the antigen-antibody complex formed by SG141, P1499m, P1501m and P1540m showed the strongest binding to human fcyriib. It is hypothesized that the amino acid substitutions of Q311R/D399R, Q311R/P343R, Q311R/D413R, and D401R/D413K have strong charge effects on binding to human fcyriib on the sensor chip.

Cellular uptake of Fc variants with increased pI

To assess intracellular uptake rates into cell lines expressing human Fc γ RIIb, the following assays were performed. An MDCK (Madin-Darby canine kidney) cell line constitutively expressing human Fc γ RIIb was prepared by a known method. These cells were used to assess intracellular uptake of antigen-antibody complexes.

Specifically, pHrodoRed (Life technologies) was used for labeling human latent muscle growth inhibitory factor (antigen) according to a determined protocol, and an antigen-antibody complex was formed in a culture solution having an antibody concentration of 10mg/mL and an antigen concentration of 2.5 mg/mL. The culture solution containing the antigen-antibody complex was added to the above-mentioned culture plate of MDCK cells constitutively expressing human Fc γ RIIb and incubated for one hour, and then the fluorescence intensity of the antigen taken into the cells was quantified using cell Analyzer 6000(GE healthcare). The amount of antigen taken up was expressed as a relative value with respect to SG1 value taken as 1.00.

The results of quantification of cellular uptake are summarized in table 26. Strong fluorescence from antigens in cells was observed in several Fc variants.

Without being bound to a particular theory, this result can be explained as follows.

The antigen and the antibody added to the cell culture solution form an antigen-antibody complex in the culture solution. The antigen-antibody complex binds to human Fc γ RIIb expressed on the cell membrane via the antibody Fc region and is taken into the cell in a receptor-dependent manner. The antibodies used in this experiment bind antigen in a pH-dependent manner; thus, the antibody can dissociate from the antigen in endosomes (acidic pH conditions) within the cell. Because the dissociated antigen is labeled with a pHrodoRed as described previously, it fluoresces in endosomes. Thus, it is believed that a strong fluorescence intensity within the cell indicates that faster or greater uptake of the antigen-antibody complex into the cell occurs.

In Fc variants with increased pI compared to SG1 with two amino acid substitutions, antigen-antibody complexes formed by SG141, P1375m, P1378m, P1499m, P1524m, P1525m, P1532m, P1533m, P1540m, P1541m and P1543m show stronger cellular uptake of antigen. It is hypothesized that the amino acid substitutions of Q311R/D399R, Q311R/D413K, S400R/D413K, Q311R/P343R, Q311R/G341R, Q311R/G341K, Q311R/D401R, Q311R/D401K, D401R/D413K, D401K/D413K and G402K/D413K have a strong charge effect on cellular uptake of the antigen-antibody complex.

PK studies in human FcRn transgenic mice

In vivo assay using human FcRn transgenic mice

After co-administration of anti-latent myostatin and latent myostatin antibodies to human FcRn transgenic mice (where mouse FcRn is replaced with human FcRn), depletion of myostatin and anti-latent myostatin antibodies was assessed in vivo. In experiment PK-2 (depicted in FIG. 27), anti-latent myostatin antibody (0.1mg/ml) and mouse latent myostatin (0.05mg/ml) were administered in a single dose of 10ml/kg into the tail vein. In experiment PK-4 (depicted in FIG. 28), anti-latent myostatin antibody (0.3mg/ml), human latent myostatin (0.05mg/ml) and human normal immunoglobulin (CSL Behring AG) (100mg/ml) were administered in a single dose of 10ml/kg into the tail vein. In experiment PK-2, blood was collected 5 minutes, 15 minutes, 1 hour, 4 hours, 7 hours, 1 day, 2 days, 7 days, 14 days, 21 days and 28 days after administration. In experiment PK-4, blood was collected 5 minutes, 1 hour, 4 hours, 7 hours, 1 day, 7 days, 14 days, 21 days and 30 days after administration. The collected blood was immediately centrifuged at 15,000rpm at 4 ℃ for 5 minutes to separate plasma. The separated plasma was stored below-20 ℃ until measured. The anti-latent myostatin antibodies used were MS1032LO06-SG1, MS1032LO06-P1375m, MS1032LO06-P1378m, MS1032LO06-P1383m in experiment PK-2, and MS1032LO06-P1375m, MS1032LO06-P149 1499m in experiment PK-4.

Measurement of total myostatin concentration in plasma by Electrochemiluminescence (ECL)

The concentration of total myostatin in mouse plasma was measured by ECL as described in example 28 (ravatch PK). The time course of total myostatin concentration in plasma after intravenous administration of anti-latent myostatin antibody and latent myostatin, as measured by this method, is shown in figures 27A and 28A.

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

The concentration of anti-latent myostatin antibody in mouse plasma was measured by ELISA in experiment PK-2. Anti-human IgG (gamma-chain specific) F (ab')2 antibody fragment (Sigma) was dispensed onto Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and allowed to stand overnight at 4 ℃ to prepare plates immobilized with anti-human IgG. Calibration curve samples with plasma concentrations of 2.5, 1.25, 0.625, 0.313, 0.156, 0.078 and 0.039 μ g/ml and mouse plasma samples diluted 100-fold or more were prepared. Then, the sample was dispensed onto a plate on which anti-human IgG was immobilized, and allowed to stand at room temperature for 1 hour. Subsequently, goat anti-human IgG (γ -chain specific) biotin conjugate (Southern Biotech) was added to react at room temperature for 1 hour. Then, streptavidin-PolyHRP 80 (Stereospeicic Detection Technologies) was added to react at room temperature for One hour, and a chromogenic reaction was performed using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as a Substrate. After the reaction was terminated with 1N sulfuric acid (Showa Chemical), the absorbance at 450nm was measured by a microplate reader. Concentrations in mouse plasma were calculated from absorbance calibration curves using the analytical software SOFTmax PRO (Molecular Devices). The time course of antibody concentration in plasma after intravenous administration of anti-latent myostatin antibody and latent myostatin measured by this method is shown in fig. 27B.

In experiment PK-4, byGyrolab Bioaffy^TMCd (gyros) the concentration of anti-latent myostatin antibodies in mouse plasma was measured. Biotinylated anti-human IgG Fc antibody was flowed over a streptavidin bead column within the microstructure of the CD. Calibration curve samples of human normal immunoglobulin (CSL Behring AG) at a plasma concentration of 5mg/mL and mouse latent myostatin at 200. mu.g/mL with peak (spiking) plasma concentrations of 0.5, 1, 2, 4, 8, 16 and 32. mu.g/mL, and mouse plasma samples of mouse latent myostatin at a peak plasma concentration of 200. mu.g/mL diluted 25-fold or more were prepared. The sample was then added to the CD and flowed over a bead column. Subsequently, Alexa-labeled goat anti-human IgG polyclonal antibody (BETHYL) was added to the CD and flowed over a bead column. Concentrations in mouse plasma were calculated from the response of the calibration curve using a Gyrolab analyzer Program. The time course of antibody concentration in plasma after intravenous administration of anti-latent myostatin antibody and latent myostatin measured by this method is shown in fig. 28B.

Effect of pI on in vivo concentration of myostatin

In experiment PK-2, the concentration of total myostatin at 7 hours was reduced 12-fold after administration of MS1032LO06-SG1 compared to the concentration of total myostatin at 5 minutes. In contrast, in experiment PK-2, the concentration of total myostatin at 7 hours was reduced by 26-67 times compared to the concentration of total myostatin at 5 minutes after administration of MS1032LO06-P1375m, MS1032LO06-P1378m and MS1032LO06-P1383 m. Although the concentration of MS1032LO06-P1375m was within a 2-fold difference at each sampling point compared to MS1032LO06-SG1 in experiment PK-2, the decrease in antibody concentrations of MS1032LO06-P1378m and MS1032LO06-P1383m was more than 3-fold compared to MS1032LO06-SG 1. pI variants including the amino acid substitutions Q311R/D413K were shown to enhance the elimination of myostatin from plasma without enhancing the elimination of antibodies from plasma.

In addition, total myostatin concentrations and antibody concentrations following administration of MS1032LO06-P1499m were evaluated in the presence of co-administered human normal immunoglobulin to mimic human plasma. In experiment PK-4, following administration of MS1032LO06-P1375m and MS1032LO06-P1499m, plasma total myostatin concentrations at 7 hours were reduced by 3.4 and 5.3 times compared to plasma total myostatin concentrations at 5 minutes, and total myostatin concentrations and antibody concentrations at each sample point in MS1032LO06-P1499m were within 1.5 times compared to MS1032LO06-P1375 m. pI variants including the amino acid substitutions Q311R/P343R were also shown to enhance the elimination of myostatin from plasma without enhancing the elimination of antibody from plasma.

The high pI variants have a more positive charge in plasma. Because this positive charge interacts with the negative charge on the cell surface, the antigen-antibody immune complex of the high pI variant is closer to the cell surface, which results in increased cellular uptake of the antigen-antibody immune complex of the high pI variant.

Example 31

pI enhanced substitutions in combination with Fc gamma RIIb enhanced Fc variants

The effect of Fc γ RIIb-enhanced Fc variants in combination with other pI-increasing substitutions on myostatin clearance was evaluated in human Fc γ RIIb transgenic mice. Human Fc γ RIIb transgenic mice were generated by standard techniques (see, e.g., J.Immunol.,2015 Oct 1; 195(7):3198-205) by microinjection of BAC (bacterial artificial chromosome) vectors containing all exons of the human FCGR2B gene into the pronuclei of C57BL/6N fertilized eggs. Mouse Fc γ RIIb deficient mice were generated using Zinc Finger Nucleases (ZFNs) designed to target exon 1. Humanized Fc γ RIIb mice were created by crossing human Fc γ RIIb transgenic mice with mouse Fc γ RIIb KO mice. The combined effect of affinity enhancement and pI enhancement on soluble antigen clearance of human Fc γ RIIb can be assessed using this mouse.

Preparation and characterization of the variants tested

The 7 antibodies tested and their binding characteristics are summarized in table 27. The heavy chain, MS103205H795-MY352, MS103205H795-PK55, MS103205H795-PK56, MS103205H795-PK57, were prepared by introducing the pI-increasing substitutions Q311R/D413R, Q311R/P343R, Q311R/P343R/D413R, Q311R/N384R/D413R into MS103205H795-MY201, respectively. All MS1032LO06 variants were expressed as light chains with M103202L889-SK1 according to the method shown in example 30 and their affinity for human and cynomolgus monkey Fc γ R was evaluated using the method in example 27.

[ Table 27]

Based on SPR analysis outlined in table 27, it was confirmed: substitutions that increase pI do not affect Fc γ R binding of Fc γ RIIb enhanced Fc variants. MY201 and MY351 prepared by introducing Q311R/D413K into MY201 showed 6-fold and 5-fold enhanced human Fc γ RIIb binding, respectively. It shows almost the same binding characteristics for other human and cynomolgus monkey Fc γ rs. Similarly, MY352, PK55, PK56 and PK57 showed similar binding characteristics as the parent MY201 (table 27). These results indicate that any substitution to increase pI does not affect affinity for human and cynomolgus monkey Fc γ R.

PK studies in human Fc γ RIIb transgenic mice

In vivo testing using human Fc γ RIIb transgenic mice

The elimination of myostatin and anti-latent myostatin antibodies was assessed in vivo after co-administration of anti-latent myostatin antibodies and human latent myostatin to human Fc γ RIIb transgenic mice (in which mouse Fc γ RII was replaced with human Fc γ RIIb). The anti-latent myostatin antibody (0.3mg/ml) and latent myostatin (0.05mg/ml) were administered in a single dose of 10ml/kg into the tail vein. Blood was collected at 5 minutes, 1 hour, 4 hours, 7 hours, 1 day, 7 days, 14 days, 21 days and 28 days after administration. The collected blood was immediately centrifuged at 15,000rpm at 4 ℃ for 5 minutes to separate plasma. The separated plasma was stored below-20 ℃ until measured. The anti-latent myostatin antibodies used were MS1032LO06-SG1, MS1032LO06-MY201, MS1032LO06-MY351, MS1032LO06-MY352, MS1032LO06-PK55, MS1032LO06-PK56 and MS1032LO06-PK 57.

Measurement of total myostatin concentration in plasma by electrochemiluminescence

The concentration of total myostatin in mouse plasma was measured by Electrochemiluminescence (ECL), as described in example 28. The time course of total myostatin concentration in plasma after intravenous administration of anti-latent myostatin antibody and latent myostatin, as measured by this method, is shown in figure 29.

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

The concentration of anti-latent myostatin antibody in mouse plasma was measured by ELISA. Anti-human IgG (gamma-chain specific) F (ab')2 antibody fragment (Sigma) was dispensed onto Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and allowed to stand overnight at 4 ℃ to prepare plates immobilized with anti-human IgG. Calibration curve samples with plasma concentrations of 2.5, 1.25, 0.625, 0.313, 0.156, 0.078 and 0.039 μ g/ml and mouse plasma samples diluted 100-fold or more were prepared. Then, the sample was dispensed onto a plate on which anti-human IgG was immobilized, and allowed to stand at room temperature for 1 hour. Subsequently, goat anti-human IgG (γ -chain specific) biotin conjugate (Southern Biotech) was added to react at room temperature for 1 hour. Then, streptavidin-PolyHRP 80 (Stereospeicic Detection Technologies) was added to react at room temperature for One hour, and a chromogenic reaction was performed using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as a Substrate. After the reaction was terminated with 1N sulfuric acid (Showa Chemical), the absorbance at 450nm was measured by a microplate reader. Concentrations in mouse plasma were calculated from absorbance calibration curves using the analytical software SOFTmax PRO (Molecular Devices). The time course of antibody concentration in plasma after intravenous administration of anti-latent myostatin antibody and latent myostatin measured by this method is shown in figure 30.

pI and Fc gamma R junctionEffect of the combination on the concentration of myostatin in vivo

Following administration of MS1032LO06-MY201, the plasma total myostatin concentration at 7 hours was reduced by 8-fold compared to the plasma total myostatin concentration at 5 minutes. In contrast, after administration of MS1032LO06-PK57, the plasma total myostatin concentration at 7 hours was reduced by 376-fold compared to the plasma total myostatin concentration at 5 minutes. Other high pI variants showed similar enhancement of elimination of myostatin from plasma. At day 28, the antibody concentration of the high pI variant was reduced 2-3 fold compared to the antibody concentration of MS1032LO06-SG1, and the pI variant showed slightly enhanced elimination of antibody from plasma.

The high pI variants have a more positive charge in plasma. Because this positive charge interacts with the negative charge on the cell surface, the antigen-antibody immune complex of the high pI variant is closer to the cell surface, which results in increased uptake of the antigen-antibody immune complex of the high pI variant by the cell.

Example 32

Development of human Fc gamma RIIb enhanced Fc variants

Human Fc γ RIIb enhanced Fc variants with anti-myostatin variable regions were constructed and evaluated for affinity for human Fc γ R. Specifically, human FcyRIIb enhanced Fc variants (VH, SEQ ID NO: 92; CH, SEQ ID NO:9) were constructed by introducing substitutions into MS103240H795-SG1 in combination with the T250V/T307P substitution. In addition, substitutions (Q311R/D413K or Q311R/P343R) were introduced. The variants were expressed using M103202L1045-SK1 as the light chain and analyzed for affinity to human fcyr according to the method shown in example 34. Table 28 shows the results of kinetic analysis for human and cynomolgus monkey Fc γ R. KD values filled in the gray grid were calculated using [ equation 2] because their affinity was too weak to be correctly determined by kinetic analysis.

All variants evaluated showed an increased affinity for human Fc γ RIIb and a reduced binding to human Fc γ RIIaR, Fc γ RIIaH, Fc γ RIIIaV, compared to SG 1. The affinity for human Fc γ RIIb was increased 4.1-fold to 30.5-fold compared to SG 1. The affinity for human Fc γ RIIaR is 0.02-fold to 0.22-fold. The affinities for human Fc γ RIIaH and human Fc γ RIIIaV were less than 0.04-fold and 0.012-fold, respectively. By comparing the affinities of MY009(P238D) and MY214(P238D/T250V/T307P), it was revealed that the substitutions (T250V/T307P) do not affect the binding characteristics to human fcyr. On the other hand, the two pairs of pI increasing substitutions (Q311R/D413K and Q311R/P343R) reduced binding affinity by about 0.5-fold compared to the variants not containing the pI increasing substitutions. For example, while TT14 showed 30.5-fold higher affinity for human Fc γ RIIb, it showed only 16.1-fold and 14.5-fold increases in combination with Q311R/D413K (TT33) and Q311R/P343R (TT32), respectively.

In addition, the substitutions used in example 29 to improve antibody half-life and reduce binding to rheumatoid factor were introduced into human Fc γ RIIb-enhanced Fc variants and their affinity for human Fc γ R was analyzed (table 29). In this SPR analysis, all data were obtained using mouse anti-human IgG kappa light chains for capturing the antibody of interest. Table 30 shows the results, and the KD values filled in the gray grid were calculated using [ equation 2] because their affinity was too weak to be correctly determined by kinetic analysis.

[ Table 28]

[ Table 29]

Amino acid sequence comprising the heavy chain constant region of a human Fc γ RIIb-enhanced Fc variant (shown as SEQ ID NO)

[ Table 30]

SG1 and TT33 were again evaluated in this measurement, with the capture method being different from that in table 28. As a result, the "KD fold" values of TT33 were consistent with those obtained using table 28 (16.1 fold in table 28, 15.7 fold in table 30 for human Fc γ RIIb). TT92 and TT93, which were constructed by introducing N434A/Q438R/S440E and M428L/N434A/Y436T/Q438R/S440E into TT33, showed comparable binding characteristics to TT 33. Similarly, TT90 and TT91 derived from TT32, TT72 and TT73 derived from TT31, TT70 and TT71 derived from TT30, TT68 and TT69 derived from TT21, and TT66 and TT67 derived from TT20 show binding characteristics comparable to those of the parent antibodies shown in table 28. These results indicate that substitutions used to improve antibody half-life and attenuate binding to rheumatoid factor do not affect the binding characteristics of human Fc γ RIIb-enhanced variants to human Fc γ R.

Example 33

Cellular imaging analysis of Fc γ RIIb enhanced Fc variants

To assess intracellular uptake of antigen-antibody complexes formed by the antibodies described in example 32, the cellular imaging assay described in example 30 was performed. In this example, to avoid signal saturation, antigen-antibody complexes were formed in a culture solution with an antibody concentration of 1.25mg/mL and an antigen concentration of 0.34 mg/mL.

The measurement results are shown in fig. 31. By increasing binding affinity to human Fc γ RIIb, cellular uptake of antigen-antibody complexes is increased. In the case of TT14, which enhanced binding affinity for human Fc γ RIIb by 30-fold compared to SG1, the intracellular antigen fluorescence intensity increased 7.5-fold over SG 1.

While the pI increasing substitutions (Q311R/D413K and Q311R/P343R) reduced the binding affinity for human fcyriib by about 0.5-fold compared to the variant not containing the pI increasing substitutions, cellular uptake of antigen-antibody complexes of the Fc variant with the pI increasing substitutions was increased compared to the Fc variant without the pI increasing substitutions. TT33 showed a 36-fold increase in intracellular antigen antibody uptake compared to SG1, whereas its parent TT14 showed a 7.5-fold increase in cellular uptake. Further, TT33 showed cellular uptake of intracellular antigen-antibody complexes comparable to SG1071, SG1074, SG1077, SG1079, SG1080 and SG1081, which showed strong clearance in cynomolgus monkeys. These results indicate that combining substitutions that increase pI with Fc γ RIIb enhanced Fc variants is a powerful tool for antigen clearance.

Example 34

Constructing an antibody expression vector;

and expression and purification of antibodies

Synthesis of the full-length gene of the nucleotide sequences encoding the H chain and L chain of the antibody variable region is carried out by a preparation method known to those skilled in the art using Assemble PCR or the like. Introduction of amino acid substitution is performed by a method known to those skilled in the art using PCR or the like. The obtained plasmid fragment was inserted into an animal cell expression vector, and an H-chain expression vector and an L-chain expression vector were prepared. The nucleotide sequence of the obtained expression vector is determined by methods known to those skilled in the art. The prepared plasmids were transiently introduced into HEK293H cell line (Invitrogen) or FreeStyle293 cells (Invitrogen) derived from human embryonic kidney cancer cells for antibody expression. The culture supernatant obtained was collected and then passed through 0.22 μm MILLEX (R) -GV filter (Millipore), or through 0.45 μm MILLEX (R) -GV filter (Millipore), to obtain a culture supernatant. The antibodies were purified from the culture supernatants obtained by methods known to the skilled person using rProtein a Sepharose Fast Flow (GE Healthcare) or Protein G Sepharose 4Fast Flow (GE Healthcare). For the concentration of the purified antibody, its absorbance at 280nm was measured using a spectrophotometer. From the obtained values, the extinction coefficient calculated by a method such as PACE was used to calculate the antibody concentration (Protein Sci.4:2411-2423 (1995)).

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims (8)

1. An isolated antibody that binds to latent myostatin, wherein the antibody comprises:

(i) HVR-H1 consisting of the amino acid sequence of SEQ ID NO:114,

HVR-H2 consisting of the amino acid sequence of SEQ ID NO:58,

HVR-H3 consisting of the amino acid sequence of SEQ ID NO:63,

HVR-L1, consisting of the amino acid sequence of SEQ ID NO:122,

HVR-L2 consisting of the amino acid sequence of SEQ ID NO:71, and

HVR-L3, consisting of the amino acid sequence of SEQ ID NO: 74; or

(ii) HVR-H1, consisting of the amino acid sequence of SEQ ID NO:114,

HVR-H2, consisting of the amino acid sequence of SEQ ID NO:116,

HVR-H3, consisting of the amino acid sequence of SEQ ID NO:121,

HVR-L1, consisting of the amino acid sequence of SEQ ID NO:122,

HVR-L2 consisting of the amino acid sequence of SEQ ID NO:71, and

HVR-L3, consisting of the amino acid sequence of SEQ ID NO: 74; or

(iii) HVR-H1, consisting of the amino acid sequence of SEQ ID NO:57,

HVR-H2, consisting of the amino acid sequence of SEQ ID NO:117,

HVR-H3, consisting of the amino acid sequence of SEQ ID NO:63,

HVR-L1, consisting of the amino acid sequence of SEQ ID NO:122,

HVR-L2 consisting of the amino acid sequence of SEQ ID NO:71, and

HVR-L3, consisting of the amino acid sequence of SEQ ID NO: 74; or

(iv) HVR-H1, consisting of the amino acid sequence of SEQ ID NO:57,

HVR-H2, consisting of the amino acid sequence of SEQ ID NO:118,

HVR-H3, consisting of the amino acid sequence of SEQ ID NO:63,

HVR-L1, consisting of the amino acid sequence of SEQ ID NO:122,

HVR-L2 consisting of the amino acid sequence of SEQ ID NO:71, and

HVR-L3, consisting of the amino acid sequence of SEQ ID NO: 74; or

(v) HVR-H1, consisting of the amino acid sequence of SEQ ID NO:57,

HVR-H2, consisting of the amino acid sequence of SEQ ID NO:119,

HVR-H3, consisting of the amino acid sequence of SEQ ID NO:63,

HVR-L1, consisting of the amino acid sequence of SEQ ID NO:122,

HVR-L2 consisting of the amino acid sequence of SEQ ID NO:71, and

HVR-L3, consisting of the amino acid sequence of SEQ ID NO: 74; or

(vi) HVR-H1 consisting of the amino acid sequence of SEQ ID NO:114,

HVR-H2 consisting of the amino acid sequence of SEQ ID NO:118,

HVR-H3 consisting of the amino acid sequence of SEQ ID NO:63,

HVR-L1 consisting of the amino acid sequence of SEQ ID NO:122,

HVR-L2 consisting of the amino acid sequence of SEQ ID NO:71, and

HVR-L3, consisting of the amino acid sequence of SEQ ID NO: 74; or

(vii) HVR-H1, consisting of the amino acid sequence of SEQ ID NO:114,

HVR-H2, consisting of the amino acid sequence of SEQ ID NO:58,

HVR-H3, consisting of the amino acid sequence of SEQ ID NO:63,

HVR-L1, consisting of the amino acid sequence of SEQ ID NO:123,

HVR-L2 consisting of the amino acid sequence of SEQ ID NO:71, and

HVR-L3, consisting of the amino acid sequence of SEQ ID NO: 74; or

(viii) HVR-H1, consisting of the amino acid sequence of SEQ ID NO:115,

HVR-H2, consisting of the amino acid sequence of SEQ ID NO:58,

HVR-H3, consisting of the amino acid sequence of SEQ ID NO:63,

HVR-L1, consisting of the amino acid sequence of SEQ ID NO:123,

HVR-L2 consisting of the amino acid sequence of SEQ ID NO:71, and

HVR-L3, consisting of the amino acid sequence of SEQ ID NO: 74; or

(ix) HVR-H1, consisting of the amino acid sequence of SEQ ID NO:114,

HVR-H2, consisting of the amino acid sequence of SEQ ID NO:58,

HVR-H3, consisting of the amino acid sequence of SEQ ID NO:63,

HVR-L1, consisting of the amino acid sequence of SEQ ID NO:124,

HVR-L2 consisting of the amino acid sequence of SEQ ID NO:125, and

HVR-L3, consisting of the amino acid sequence of SEQ ID NO: 74; or

(x) HVR-H1, consisting of the amino acid sequence of SEQ ID NO:115,

HVR-H2, consisting of the amino acid sequence of SEQ ID NO:120,

HVR-H3, consisting of the amino acid sequence of SEQ ID NO:63,

HVR-L1, consisting of the amino acid sequence of SEQ ID NO:123,

HVR-L2 consisting of the amino acid sequence of SEQ ID NO:71, and

HVR-L3, consisting of the amino acid sequence of SEQ ID NO: 74.

2. The antibody of any one of claims 1(i) to (vi) and (ix), further comprising: heavy chain variable domain framework FR1(H-FR1), said H-FR1 consisting of the amino acid sequence of SEQ ID NO: 132;

heavy chain variable domain framework FR2(H-FR2), said H-FR2 consisting of the amino acid sequence of SEQ ID NO: 135;

heavy chain variable domain framework FR3(H-FR3), said H-FR3 consisting of the amino acid sequence of SEQ ID NO: 137;

Heavy chain variable domain framework FR4(H-FR4), said H-FR4 consisting of the amino acid sequence of SEQ ID NO: 138;

light chain variable domain framework FR1(L-FR1), said L-FR1 consisting of the amino acid sequence of SEQ ID NO: 139;

light chain variable domain framework FR2(L-FR2), said L-FR2 consisting of the amino acid sequence of SEQ ID NO: 140;

light chain variable domain framework FR3(L-FR3), said L-FR3 consisting of the amino acid sequence of SEQ ID NO: 142;

light chain variable domain framework FR4(L-FR4), said L-FR4 consisting of the amino acid sequence of SEQ ID NO: 144; or

The antibody of claim 1(vii) or (x), further comprising

H-FR1 consisting of the amino acid sequence of SEQ ID NO. 133;

H-FR2 consisting of the amino acid sequence of SEQ ID NO: 136;

H-FR3 consisting of the amino acid sequence of SEQ ID NO: 137;

H-FR4 consisting of the amino acid sequence of SEQ ID NO: 138;

L-FR1 consisting of the amino acid sequence of SEQ ID NO: 139;

L-FR2 consisting of the amino acid sequence of SEQ ID NO: 140;

L-FR3 consisting of the amino acid sequence of SEQ ID NO: 142; and

L-FR4, consisting of the amino acid sequence of SEQ ID NO: 144; or

The antibody of claim 1(vii) or (viii), further comprising

H-FR1 consisting of the amino acid sequence of SEQ ID NO: 134;

H-FR2 consisting of the amino acid sequence of SEQ ID NO: 136;

H-FR3 consisting of the amino acid sequence of SEQ ID NO: 137;

H-FR4 consisting of the amino acid sequence of SEQ ID NO: 138;

L-FR1 consisting of the amino acid sequence of SEQ ID NO: 139;

L-FR2 consisting of the amino acid sequence of SEQ ID NO: 140;

L-FR3 consisting of the amino acid sequence of SEQ ID NO: 142; and

L-FR4, consisting of the amino acid sequence of SEQ ID NO: 144; or

The antibody of claim 1(vii), further comprising

H-FR1 consisting of the amino acid sequence of SEQ ID NO. 133;

H-FR2 consisting of the amino acid sequence of SEQ ID NO: 136;

H-FR3 consisting of the amino acid sequence of SEQ ID NO: 137;

H-FR4 consisting of the amino acid sequence of SEQ ID NO: 138;

L-FR1 consisting of the amino acid sequence of SEQ ID NO: 139;

L-FR2, consisting of the amino acid sequence of SEQ ID NO. 141;

L-FR3 consisting of the amino acid sequence of SEQ ID NO. 143; and

L-FR4, consisting of the amino acid sequence of SEQ ID NO: 144.

3. The antibody of claim 1, wherein the antibody comprises

(i) The VH sequence of SEQ ID NO 86 and the VL sequence of SEQ ID NO 96; or

(ii) The VH sequence of SEQ ID NO 86 and the VL sequence of SEQ ID NO 98; or

(iii) The VH sequence of SEQ ID NO 87 and the VL sequence of SEQ ID NO 96; or

(iv) The VH sequence of SEQ ID NO:88 and the VL sequence of SEQ ID NO: 96; or

(v) The VH sequence of SEQ ID NO. 89 and the VL sequence of SEQ ID NO. 96; or

(vi) The VH sequence of SEQ ID NO. 90 and the VL sequence of SEQ ID NO. 96; or

(vii) The VH sequence of SEQ ID NO 91 and the VL sequence of SEQ ID NO 96; or

(viii) The VH sequence of SEQ ID NO 92 and the VL sequence of SEQ ID NO 97; or

(ix) The VH sequence of SEQ ID NO 92 and the VL sequence of SEQ ID NO 99; or

(x) The VH sequence of SEQ ID NO 93 and the VL sequence of SEQ ID NO 97; or

(xi) The VH sequence of SEQ ID NO 94 and the VL sequence of SEQ ID NO 97; or

(xii) The VH sequence of SEQ ID NO. 95 and the VL sequence of SEQ ID NO. 97.

4. The antibody of claim 1, comprising a heavy chain constant region comprising the amino acid sequence of SEQ ID NO 352 and a light chain constant region comprising the amino acid sequence of SEQ ID NO 10.

5. A nucleic acid encoding the antibody of any one of claims 1 to 4.

6. A host cell comprising the nucleic acid of claim 5, wherein the host cell is not a plant cell.

7. A method of producing an antibody comprising culturing the host cell of claim 6 to produce the antibody.

8. A pharmaceutical formulation comprising the antibody of any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

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