Anti-myostatin antibodies and methods of use

The invention provides anti-myostatin antibodies and methods of using the same. In some embodiments, an isolated anti-myostatin antibody of the present invention binds to mature myostatin, and uptake of the antibody into cells is enhanced when complexed with the antigen. The invention also provides isolated nucleic acids encoding an anti-myostatin antibody of the present invention. The invention also provides host cells comprising a nucleic acid of the present invention. The invention also provides a method of producing an antibody comprising culturing a host cell of the present invention so that the antibody is produced. Anti-myostatin antibodies of the present invention may be for use as a medicament. Anti-myostatin antibodies of the present invention may be for use in treating a muscle wasting disease. Anti-myostatin antibodies of the present invention may be for use in increasing mass of muscle tissue. Anti-myostatin antibodies of the present invention may be for use in increasing strength of muscle tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase of PCT Application No. PCT/JP2016/088302, filed Dec. 22, 2016, which claims the benefit of Japanese Patent Application No. 2015-253346, filed Dec. 25, 2015, each of which is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing (Name: 6663 0076 sequence listing.txt; Size: 93.6 kilobytes; and Date of Creation: Jun. 19, 2018) filed with the application is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention relates to anti-myostatin antibodies and methods of using the same.

BACKGROUND ART

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

Like other members of the TGF-beta family, myostatin is synthesized as a large precursor protein containing an N-terminal propeptide domain, and a C-terminal domain considered as the active molecule (See, e.g., NPL 5; PTL 2). Two molecules of myostatin precursor are covalently linked via a single disulfide bond present in the C-terminal growth factor domain. Active mature myostatin (disulfide-bonded homodimer consisting of the C-terminal growth factor domain) is liberated from myostatin precursor through multiple steps of proteolytic processing. In the first step of the myostatin activation pathway, a peptide bond between the N-terminal propeptide domain and the C-terminal growth factor domain, Arg266-Asp267, is cleaved by a furin-type proprotein convertase in both chains of the homodimeric precursor. But the resulting two propeptides and one mature myostatin (disulfide-bonded homodimer consisting of the growth factor domains) remain associated, forming a noncovalent inactive complex, that is latent myostatin. Mature myostatin can then be liberated from latent myostatin through degradation of the propeptide. Members of the bone morphogenetic protein 1 (BMP-1) family of metalloproteinases cleave a single peptide bond within the propeptide, Arg98-Asp99, with concomitant release of the mature myostatin (See, e.g., NPL 6). Moreover, the latent myostatin can be activated in vitro by dissociating the complex with either acid or heat treatment as well (See, e.g., NPL 7).

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

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

Inhibition of myostatin expression leads to both muscle hypertrophy and hyperplasia (NPL 4). Myostatin negatively regulates muscle regeneration after injury and lack of myostatin in myostatin null mice results in accelerated muscle regeneration (See, e.g., NPL 9). Anti-myostatin (GDF-8) antibodies described in, e.g., PTL 3, PTL 4, PTL 5, PTL 6, PTL 7, PTL 8, PTL 9, PTL 10, and PTL 11 have been shown to bind to myostatin and inhibit myostatin activity in vitro and in vivo, including myostatin activity associated with the negative regulation of skeletal muscle mass. Myostatin-neutralizing antibodies increase body weight, skeletal muscle mass, and muscle size and strength in the skeletal muscle of wild type mice (See, e.g., NPL 10) and the mdx mice, a model for muscular dystrophy (See, e.g., NPL 11; NPL 12). However, there is a further need for improvements in efficacy and convenience of agents that bind myostatin and antagonize its activity in the art.

CITATION LIST

Patent Literature

Non Patent Literature

SUMMARY OF INVENTION

Technical Problem

An objective of the invention is to provide anti-myostatin antibodies and methods of using the same.

Solution to Problem

The invention provides anti-myostatin antibodies and methods of using the same.

In some embodiments, an isolated anti-myostatin antibody of the present invention binds to mature myostatin. In some embodiments, uptake of an isolated anti-myostatin antibody of the present invention into cells is enhanced when complexed with an antigen. In further embodiments, the uptake is caused by the interaction between Fc region of the antibody and Fc gamma R on the cells. In further embodiments, the antibody shows at least 2.5-fold higher uptake compared with a reference antibody which is identical to the antibody except that Fc region of the reference antibody has no Fc gamma R-binding activity. In some embodiments, an isolated anti-myostatin antibody of the present invention has an inhibitory activity against myostatin. In some embodiments, an isolated anti-myostatin antibody of the present invention binds to the same epitope as an antibody described in Table 2 or 3. In some embodiments, an isolated anti-myostatin antibody of the present invention competes for binding to myostatin with an antibody described in Table 2 or 3. In some embodiments, an isolated anti-myostatin antibody of the present invention binds to mature myostatin with higher affinity at neutral pH than at acidic pH.

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

In some embodiments, an isolated anti-myostatin antibody of the present invention comprises (a) HVR-H3 comprising the amino acid sequence GX1DNFGYSYX2DFNL, wherein X1is G or H, X2is I or H (SEQ ID NO: 86), (b) HVR-L3 comprising the amino acid sequence QTYDGISX1YGVA, wherein X1is S or H (SEQ ID NO: 88), and (c) HVR-H2 comprising the amino acid sequence IINIX1GX2TYYASWAX3G, wherein X1is S or E, X2is S or E, X3is K or E (SEQ ID NO: 85).

In some embodiments, an isolated anti-myostatin antibody of the present invention comprises (a) HVR-H1 comprising the amino acid sequence X1YVX2G, wherein X1is N or H, X2is M or K (SEQ ID NO: 84), (b) HVR-H2 comprising the amino acid sequence IINIX1GX2TYYASWAX3G, wherein X1is S or E, X2is S or E, X3is K or E (SEQ ID NO: 85), and (c) HVR-H3 comprising the amino acid sequence GX1DNFGYSYX2DFNL, wherein X1is G or H, X2is I or H (SEQ ID NO: 86). In further embodiments, the antibody comprises (a) HVR-L1 comprising the amino acid sequence QASX1SIX2X3X4LS, wherein X1is Q or E, X2is S or H, X3is N or H, X4is E or D (SEQ ID NO: 87); (b) HVR-L2 comprising the amino acid sequence LASTLAS (SEQ ID NO: 81); and (c) HVR-L3 comprising the amino acid sequence QTYDGISX1YGVA, wherein X1is S or H (SEQ ID NO: 88).

In some embodiments, an isolated anti-myostatin antibody of the present invention comprises (a) HVR-L1 comprising the amino acid sequence QASX1SIX2X3X4LS, wherein X1is Q or E, X2is S or H, X3is N or H, X4is E or D (SEQ ID NO: 87); (b) HVR-L2 comprising the amino acid sequence LASTLAS (SEQ ID NO: 81); and (c) HVR-L3 comprising the amino acid sequence QTYDGISX1YGVA, wherein X1is S or H (SEQ ID NO: 88).

In some embodiments, an isolated anti-myostatin antibody of the present invention comprises a heavy chain variable domain framework FR1 comprising the amino acid sequence of SEQ ID NO: 89; FR2 comprising the amino acid sequence of SEQ ID NO: 90; FR3 comprising the amino acid sequence of SEQ ID NO: 91; and FR4 comprising the amino acid sequence of SEQ ID NO: 92. In some embodiments, an isolated anti-myostatin antibody of the present invention comprises a light chain variable domain framework FR1 comprising the amino acid sequence of SEQ ID NO: 93; FR2 comprising the amino acid sequence of SEQ ID NO: 94; FR3 comprising the amino acid sequence of SEQ ID NO: 95; and FR4 comprising the amino acid sequence of SEQ ID NO: 96.

In some embodiments, an isolated anti-myostatin antibody of the present invention comprises (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 48-51; (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 52-55; or (c) a VH sequence as in (a) and a VL sequence as in (b). In further embodiments, the antibody comprises a VH sequence of any one of SEQ ID NOs: 48-51. In further embodiments, the antibody comprises a VL sequence of any one of SEQ ID NOs: 52-55.

The invention provides an antibody comprising a VH sequence of any one of SEQ ID NOs: 48-51 and a VL sequence of any one of SEQ ID NOs: 52-55.

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

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

Anti-myostatin antibodies of the present invention may be for use as a medicament. Anti-myostatin antibodies of the present invention may be for use in treating a muscle wasting disease. Anti-myostatin antibodies of the present invention may be for use in increasing mass of muscle tissue. Anti-myostatin antibodies of the present invention may be for use in increasing strength of muscle tissue.

Anti-myostatin antibodies of the present invention may be used in the manufacture of a medicament. In some embodiments, the medicament is for treatment of a muscle wasting disease. In some embodiments, the medicament is for increasing mass of muscle tissue. In some embodiments, the medicament is for increasing strength of muscle tissue.

The invention also provides a method of treating an individual having a muscle wasting disease. In some embodiments, the method comprises administering to the individual an effective amount of an anti-myostatin antibody of the present invention. The invention also provides a method of increasing mass of muscle tissue in an individual. In some embodiments, the method comprises administering to the individual an effective amount of an anti-myostatin antibody of the present invention to increase mass of muscle tissue. The invention also provides a method of increasing strength of muscle tissue in an individual. In some embodiments, the method comprises administering to the individual an effective amount of an anti-myostatin antibody of the present invention to increase strength of muscle tissue.

DESCRIPTION OF EMBODIMENTS

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

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

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

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

The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin,vincaalkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and the various antitumor or anticancer agents disclosed below.

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

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

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

The term “Fc receptor” or “FcR” also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and regulation of homeostasis of immunoglobulins. Methods of measuring binding to FcRn are known (see, e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004/92219 (Hinton et al.). Binding to human FcRn in vivo and serum half life of human FcRn high affinity binding polypeptides can be assayed, e.g., in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which the polypeptides with a variant Fc region are administered. WO 2000/42072 (Presta) describes antibody variants with improved or diminished binding to FcRs. See also, e.g., Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

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

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

The term “myostatin”, as used herein, refers to any native myostatin from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length”, unprocessed myostatin as well as any form of myostatin that results from processing in the 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 (promyostatin) is shown in SEQ ID NO: 1. The amino acid sequence of an exemplary C-terminal growth factor domain of human myostatin is shown in SEQ ID NO: 2. The amino acid sequence of an exemplary N-terminal propeptide domain of human myostatin is shown in SEQ ID NO: 97 or 100. Active mature myostatin is a disulfide-bonded homodimer consisting of two C-terminal growth factor domains. Inactive latent myostatin is a noncovalently-associated complex of two propeptides and the mature myostatin. The amino acid sequence of an exemplary cynomolgus monkey and murine myostatin (promyostatin) are shown in SEQ ID NO: 3 and 5, respectively. The amino acid sequence of an exemplary C-terminal growth factor domain of cynomolgus monkey and murine myostatin are shown in SEQ ID NO: 4 and 6, respectively. The amino acid sequence of an exemplary N-terminal propeptide domain of cynomolgus monkey and murine myostatin are shown in SEQ ID NO: 98 or 101, and 99 or 102, respectively. Amino acid residues 1-24 of SEQ ID NOs: 1, 3, 5, 100, 101, and 102 correspond to a signal sequence that is removed during processing in the cell and is thus missing from the exemplary amino acid sequence shown in SEQ ID NOs: 97, 98, and 99.

II. 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 to myostatin are provided. Antibodies of the invention are useful, e.g., for the diagnosis or treatment of a muscle wasting disease.

In one aspect, the invention provides isolated antibodies that bind to myostatin. In certain embodiments, an anti-myostatin antibody of the present invention binds to mature myostatin. Mature myostatin is a disulfide-bonded homodimer of a polypeptide having an amino acid sequence of, for example, SEQ ID NO: 2 in human, SEQ ID NO: 4 in cynomolgus monkey, and SEQ ID NO: 6 in mouse. In some embodiments, an anti-myostatin antibody of the present invention forms a complex with the antigen, myostatin (also described herein as an antigen-antibody complex or an immune complex). In a further embodiment, the antigen-antibody complex comprises at least two antibody molecules of the present invention. In a further embodiment, the antigen-antibody complex comprises at least two antigen molecules. In a further embodiment, the antigen-antibody complex comprises at least two myostatin mature form molecules.

In some embodiments, an anti-myostatin antibody of the present invention is taken up into cells. In another embodiments, an antigen-antibody complex formed by an anti-myostatin antibody of the present invention is taken up into cells. In further embodiments, uptake of an anti-myostatin antibody of the present invention into cells is enhanced when the antibody forms a complex with the antigen. In further embodiments, uptake of the antibody is enhanced when the antibody forms a complex with the antigen compared with when the antibody does not form a complex with the antigen. Enhanced uptake of an antigen-antibody complex into cells can lead to enhanced antigen clearance from plasma when the antibody is administered in a subject. In another embodiment, clearance of the antigen from plasma is enhanced when an anti-myostatin antibody of the present invention is administered in a subject.

In some embodiments, an anti-myostatin antibody of the present invention is taken up into cells through the interaction between an Fc region of the antibody and an Fc receptor on the surface of the cells. In certain embodiment, the Fc region of an anti-myostatin antibody of the present invention has an Fc receptor-binding activity. In further embodiments, the Fc receptor can be Fc gamma receptor (Fc gamma R), which includes, for example, Fc gamma RI including isoforms Fc gamma RIa, Fc gamma Rib, and Fc gamma RIc; Fc gamma RII including isoforms Fc gamma RIIa (including allotypes H131 (type H) and R131 (type R)), Fc gamma RIIb (including Fc gamma RIIb-1 and Fc gamma RIIb-2), and Fc gamma RIIc; and Fc gamma RIII including isoforms Fc gamma RIIIa (including allotypes V158 and F158), and Fc gamma RIIIb (including allotypes Fc gamma RIIIb-NA1 and Fc gamma RIIIb-NA2).

In another embodiment, an anti-myostatin antibody of the present invention shows higher uptake into cells when compared with an antibody which is identical to the anti-myostatin antibody except that the Fc region has no Fc gamma R-binding activity. In further embodiment, an anti-myostatin antibody of the present invention shows at least 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 20, 50, 100, 200, 500, or 1000 fold higher uptake into cells when compared with an antibody which is identical to the anti-myostatin antibody except that the Fc region has no Fc gamma R-binding activity. In another embodiment, when compared between two antibodies both of which are constructed by modifying an anti-myostatin antibody of the present invention, one of which is an antibody having an Fc region with Fc gamma R binding activity and the other of which is an antibody having an Fc region without Fc gamma R binding activity, the former antibody shows higher uptake into cells than the latter antibody. In certain embodiments, a modified antibody having a heavy chain constant region of G1m (SEQ ID NO: 7) or SG1 (SEQ ID NO: 64) can be used as an antibody having an Fc region with Fc gamma R binding activity. In certain embodiments, a modified antibody having a heavy chain constant region of F760 (SEQ ID NO: 68) can be used as an antibody having an Fc region without Fc gamma R binding activity. In further embodiments, the former antibody shows at least 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 20, 50, 100, 200, 500, or 1000 fold higher uptake into cells than the latter antibody.

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

When an antigen is a soluble protein, the binding of an antibody to the antigen can result in an extended half-life of the antigen in plasma (i.e., reduced clearance of the antigen from plasma), since the antibody can have a longer half-life in plasma than the antigen itself and may serve as a carrier for the antigen. This is due to the recycling of the antigen-antibody complex by FcRn through the endosomal pathway in cell (Roopenian and Akilesh (2007) Nat Rev Immunol 7(9): 715-725). However, an antibody with pH-dependent binding characteristics, which binds to its antigen in neutral extracellular environment while releasing the antigen into acidic endosomal compartments following its entry into cells, is expected to have superior properties in terms of antigen neutralization and clearance relative to its counterpart that binds in a pH-independent manner (Igawa et al (2010) Nature Biotechnol 28(11); 1203-1207; Devanaboyina et al (2013) mAbs 5(6): 851-859; International Patent Application Publication No: WO 2009/125825).

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

The binding properties of an antibody for a particular antigen may also be expressed in terms of the kd of the antibody. The kd of an antibody refers to the dissociation rate constant of the antibody with respect to a particular antigen and is expressed in terms of reciprocal seconds (i.e., sec−1). An increase in kd value signifies weaker binding of an antibody to its antigen. The present invention therefore includes antibodies that bind to myostatin with a higher kd value at acidic pH than at neutral pH. The present invention includes antibodies that bind to myostatin at acidic pH with a kd that is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more times greater than the kd of the antibody binding to myostatin at neutral pH. In another embodiment, the kd value of the antibody at neutral pH can be 10′ 1/s, 10−31/s, 1041/s, 10−51/s, 10−61/s, or less. In another embodiment, the kd value of the antibody at acidic pH can be 10−31/s, 10′ 1/s, 1041/s, or greater.

In certain instances, a “reduced binding to myostatin at acidic pH as compared to its binding at neutral pH” is expressed in terms of the ratio of the KD value of the antibody binding to myostatin at acidic pH to the KD value of the antibody binding to myostatin at neutral pH (or vice versa). For example, an antibody may be regarded as exhibiting “reduced binding to myostatin at acidic pH as compared to its binding at neutral pH”, for purposes of the present invention, if the antibody exhibits an acidic/neutral KD ratio of 2 or greater. In certain exemplary embodiments, the acidic/neutral KD ratio for an antibody of the present invention can be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or greater. In another embodiment, the KD value of the antibody at neutral pH can be 10−7M, 10−8M, 10−9m, 10−10M, 10−11M, 10−12M, or less. In another embodiment, the KD value of the antibody at acidic pH can be 10−9M, 10−8M, 10−7M, 10−6M, or greater.

In certain instances, a “reduced binding to myostatin at acidic pH as compared to its binding at neutral pH” is expressed in terms of the ratio of the kd value of the antibody binding to myostatin at acidic pH to the kd value of the antibody binding to myostatin at neutral pH (or vice versa). For example, an antibody may be regarded as exhibiting “reduced binding to myostatin at acidic pH as compared to its binding at neutral pH”, for purposes of the present invention, if the antibody exhibits an acidic/neutral kd ratio of 2 or greater. In certain exemplary embodiments, the acidic/neutral kd ratio for an antibody of the present invention can be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or greater. In another embodiment, the kd value of the antibody at neutral pH can be 10−21/s, 10−31/s, 10−41/s, 10−51/s, 10−61/s, or less. In another embodiment, the kd value of the antibody at acidic pH can be 10−31/s, 10−21/s, 10−11/s, or greater.

KD values, and kd values, as expressed herein, may be determined using a surface plasmon resonance-based biosensor to characterize antibody-antigen interactions. (See, e.g., Example 6, herein). KD values, and kd values can be determined at 25 degrees C. or 37 degrees C.

An anti-myostatin antibody of the present invention forms a large immune complex with antigen (myostatin). In this invention, a “large” immune complex (i.e. antigen-antibody complex) means an immune complex containing two or more antibody molecules and two or more antigen molecules. Myostatin can form a large immune complex when being bound by an appropriate antibody. Without being bound by a particular theory, this is possible because myostatin (including mature myostatin) exists as a homodimer containing two myostatin molecules (for example, human, cynomolgus monkey and mouse mature myostatin exists as a homodimer of a polypeptide comprising an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, respectively). Two molecules of an anti-myostatin antibody of the present invention may bind one each to the two myostatin molecules in the homodimer. Furthermore, because an antibody such as IgG is also a homodimer (or a heterotetramer) having two antigen binding sites, one antibody molecule may bind to two antigen molecules which may be in a single homodimer or in separate homodimers. As such, multiple myostatin molecules and multiple antibody molecules can be included in an immune complex formed by myostatin and an anti-myostatin antibody. A large immune complex containing two or more antibody molecules can bind to Fc receptors on a cell surface more strongly than an immune complex containing only one antibody molecule, because multiple interactions (avidity) between multiple Fc regions and Fc receptors caused by the former, large immune complex is larger than a single interaction (affinity) caused by the latter immune complex. Thus, such a large immune complex that can strongly bind to Fc receptors due to avidity effect through the multiple Fc regions in the complex could be efficiently taken up into cells expressing Fc receptors. In one embodiment, an anti-myostatin antibody of the present invention has two antigen-binding domains such as Fab, each of which binds to the same epitope on a myostatin molecule. In another embodiment, an anti-myostatin antibody of the present invention has two antigen-binding domains binding to different epitopes on a myostatin molecule, much like a bispecific antibody.

Furthermore, an antibody with pH-dependent binding characteristics is thought to have superior properties in terms of antigen neutralization and clearance relative to its counterpart that binds in a pH-independent manner (Igawa et al (2010) Nature Biotechnol 28(11); 1203-1207; Devanaboyina et al (2013) mAbs 5(6): 851-859; International Patent Application Publication No: WO 2009/125825). Therefore, an antibody having both properties mentioned above, that is, an antibody which forms a large immune complex containing two or more antibody molecules and which binds to an antigen in a pH-dependent manner, is expected to have even more superior properties for highly accelerated elimination of antigens from plasma (International Patent Application Publication No: WO 2013/081143).

In some embodiments, an anti-myostatin antibody of the present invention has an inhibitory activity against myostatin. In another embodiment, an anti-myostatin antibody of the present invention blocks myostatin signaling through myostatin receptor such as activin receptor type IIB (ActRIIB).

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

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 comprising the amino acid sequence of any one of SEQ ID NOs: 70-71, 84; (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 72-74, 85; (c) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 75-76, 86; (d) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 77-80, 87; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 81; and (f) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 82-83, 88.

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 comprising the amino acid sequence of any one of SEQ ID NOs: 70-71, 84; (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 72-74, 85; and (c) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 75-76, 86. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 75-76, 86. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 75-76, 86 and HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 82-83, 88. In a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 75-76, 86, HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 82-83, 88, and HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 72-74, 85. In a further embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 70-71, 84; (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 72-74, 85; and (c) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 75-76, 86.

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 comprising the amino acid sequence of any one of SEQ ID NOs: 77-80, 87; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 81; and (c) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 82-83, 88. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 77-80, 87; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 81; and (c) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 82-83, 88.

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 comprising the amino acid sequence of any one of SEQ ID NOs: 70-71, 84, (ii) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 72-74, 85, and (iii) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 75-76, 86; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 77-80, 87, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 81, and (iii) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 82-83, 88.

In another aspect, the invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 70-71, 84; (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 72-74, 85; (c) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 75-76, 86; (d) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 77-80, 87; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 81; and (f) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 82-83, 88.

All possible combinations of the above substitutions are encompassed by the consensus sequences of SEQ ID NOs: 84, 85, 86, 87, and 88 for HVR-H1, HVR-H2, HVR-H3, HVR-L1, and HVR-L3, respectively.

In any of the above embodiments, an anti-myostatin antibody is humanized. In one embodiment, an anti-myostatin antibody comprises HVRs as in any of the above embodiments, and further comprises an acceptor human framework, e.g. a human immunoglobulin framework or a human consensus framework. In another embodiment, an anti-myostatin antibody comprises HVRs as in any of the above embodiments, and further comprises a VH or VL comprising an FR sequence. In a further embodiment, the anti-myostatin antibody comprises the following heavy chain or light chain variable domain FR sequences: For the heavy chain variable domain, FR1 comprises the amino acid sequence of SEQ ID NO: 89, FR2 comprises the amino acid sequence of SEQ ID NO: 90, FR3 comprises the amino acid sequence of SEQ ID NO: 91, FR4 comprises the amino acid sequence of SEQ ID NO: 92. For the light chain variable domain, FR1 comprises the amino acid sequence of SEQ ID NO: 93, FR2 comprises the amino acid sequence of SEQ ID NO: 94, FR3 comprises the amino acid sequence of SEQ ID NO: 95, FR4 comprises the amino acid sequence of SEQ ID NO: 96.

In another aspect, an anti-myostatin antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 48-51. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence (such as the amino acid sequence of any one of SEQ ID NOs: 48-51), but an anti-myostatin antibody comprising that sequence retains the ability to bind to myostatin. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in any one of SEQ ID NOs: 48-51. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-myostatin antibody comprises the VH sequence in any one of SEQ ID NOs: 48-51, including post-translational modifications of that sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 70-71, 84, (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 72-74, 85, and (c) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 75-76, 86.

In another aspect, an anti-myostatin antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 52-55. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence (such as the amino acid sequence of any one of SEQ ID NOs: 52-55), but an anti-myostatin antibody comprising that sequence retains the ability to bind to myostatin. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in any one of SEQ ID NOs: 52-55. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-myostatin antibody comprises the VL sequence in any one of SEQ ID NOs: 52-55, including post-translational modifications of that sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 77-80, 87; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 81; and (c) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 82-83, 88.

In another aspect, an anti-myostatin antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in any one of SEQ ID NOs: 48-51 and any one of SEQ ID NOs: 52-55, respectively, including post-translational modifications of those sequences.

In certain embodiments, an anti-myostatin antibody of the present invention comprises a VH as in any of the embodiments provided above and a heavy chain constant region comprising the amino acid sequence of any one of SEQ ID NOs: 7, 64, and 68. In certain embodiments, an anti-myostatin antibody of the present invention comprises a VL as in any of the embodiments provided above and a light chain constant region comprising the amino acid sequence of any one of SEQ ID NOs: 9 and 65.

In one aspect, the invention provides an anti-myostatin antibody described in Table 2 or Table 3.

In a further aspect, the invention provides an antibody that binds to the same epitope as an anti-myostatin antibody provided herein. For example, in certain embodiments, an antibody is provided that binds to the same epitope as an antibody described in Table 2 or 3. In certain embodiments, an antibody is provided that binds to the same epitope as any one of the antibodies selected from the group of consisting of: MST0226, MST0796, MST0139, MST0182, MSLO00, MSLO01, MSLO02, MSLO03, and MSLO04 described in Example 2 or 6. In another aspect, the invention provides an antibody that competes for binding myostatin with an anti-myostatin antibody provided herein. For example, in certain embodiments, an antibody is provided that competes for binding myostatin with an antibody described in Table 2 or 3. In certain embodiments, an antibody is provided that competes for binding myostatin with any one of the antibodies selected from the group of consisting of: MST0226, MST0796, MST0139, MST0182, MSLO00, MSLO01, MSLO02, MSLO03, and MSLO04 described in Example 2 or 6. It is expected that the epitopes bound by the antibodies described above are located in conformationally appropriate positions to form a large antigen-antibody complex when bound by the antibodies. Therefore, not only the antibodies described above but also antibodies that bind to the same epitopes as them or antibodies that compete for binding myostatin with them would be useful in the present invention.

In a further aspect of the invention, an anti-myostatin antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized or human antibody. In one embodiment, an anti-myostatin antibody is an antibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)2fragment. In another embodiment, the antibody is a full length IgG antibody, e.g., an intact IgG1or IgG4 antibody or other antibody class or isotype as defined herein.

In a further aspect, an anti-myostatin antibody according to any of the above embodiments may incorporate any of the features, singly or in combination, as described in Sections 1-7 below.

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

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

3. Chimeric and Humanized Antibodies

Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

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

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

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

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

c) Fc Region Variants

B. Recombinant Methods and Compositions

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

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

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

1. Binding Assays and Other Assays

In one aspect, an antibody of the invention is tested for its antigen binding activity, e.g., by known methods such as ELISA, Western blot, BIAcore, etc.

In another aspect, competition assays may be used to identify an antibody that competes for binding to myostatin with any anti-myostatin antibody described herein. In certain embodiments, when such a competing antibody is present in excess, it blocks (e.g., reduces) the binding of a reference antibody to myostatin by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more. In some instances, binding is inhibited by at least 80%, 85%, 90%, 95%, or more. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear or a conformational epitope) that is bound by an anti-myostatin antibody described herein (e.g., an anti-myostatin antibody described in Table 2 or 3). Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, N.J.).

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

In another aspect, an antibody that binds to the same epitope as an anti-myostatin antibody provided herein or that competes for binding myostatin with an anti-myostatin antibody provided herein may be identified using sandwich assays. Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected. In a sandwich assay, the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three part complex. See David & Greene, U.S. Pat. No. 4,376,110. The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme. An antibody which simultaneously binds to myostatin with an anti-myostatin antibody provided herein can be determined to be an antibody that binds to a different epitope from the anti-myostatin antibody. Therefore, an antibody which does not simultaneously bind to myostatin with an anti-myostatin antibody provided herein can be determined to be an antibody that binds to the same epitope as the anti-myostatin antibody or that competes for binding myostatin with the anti-myostatin antibody.

In one aspect, the binding activity of an Fc region of an antibody towards an Fc receptor (e.g., Fc gamma R) can be measured by the Amplified Luminescent Proximity Homogeneous Assay (ALPHA), the BIACORE method which utilizes the surface plasmon resonance (SPR) phenomena, or such, in addition to ELISA or fluorescence activated cell sorting (FACS) (Proc Natl Acad Sci USA (2006) 103(11): 4005-4010). For example, in the BIACORE method, Fc receptors are subjected to interaction as an analyte with an antibody comprising an Fc region immobilized or captured onto the sensor chips using Protein A, Protein L, Protein A/G, Protein G, anti-lamda chain antibodies, anti-kappa chain antibodies, antigenic peptides, antigenic proteins, or such.

In one aspect, assays are provided for identifying anti-myostatin antibodies having biological activity. Biological activity may include, e.g., an inhibitory activity against myostatin. Antibodies having such biological activity in vivo and/or in vitro are also provided.

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

In certain embodiments, whether a test antibody has an inhibitory activity against myostatin is determined by detecting mature myostatin activity, for example, the activity of binding to a myostatin receptor, or the activity of mediating signal transduction in a cell expressing a myostatin receptor. Cells useful for such an assay can be those that express an endogenous myostatin receptor, for example, L6 myocytes, or can be those that are genetically modified, transiently or stably, to express a transgene encoding a myostatin receptor, for example, an activin receptor such as an activin type II receptor (See, for example, Thies et al (2001) Growth Factors 18(4): 251-259). Binding of myostatin to a myostatin receptor can be detected by using a receptor binding assay. Myostatin mediated signal transduction can be detected at any level in the signal transduction pathway, for example, by examining phosphorylation of a Smad polypeptide, examining expression of a myostatin regulated gene including a reporter gene, or measuring proliferation of a myostatin-dependent cell. Where a decreased mature myostatin activity is detected in the presence of (or following contact with) the test antibody, the test antibody is identified as an antibody that has an inhibitory activity against myostatin.

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

In certain embodiments, whether a test antibody is taken up into cells can be determined by cell imaging analysis. A fluorescence-labeled antibody is contacted with cells expressing an Fc receptor (e.g., Fc gamma R) in the absence and presence of antigen, and the resulting fluorescence intensity of the cells is measured using an image analyser. Cells useful for such an assay can be those that express an endogenous Fc receptor, or can be those that are genetically modified, transiently or stably, to express a transgene encoding an Fc receptor. Where an increased fluorescence intensity is detected in the presence of the antigen compared with in the absence of the antigen, it is determined that uptake of the test antibody into cells is enhanced when the test antibody is complexed with the antigen.

In another embodiment, uptake of an antibody into cells can be evaluated, for example by detecting formation of an immune complex (such as a “large” immune complex defined above) in vitro. In certain embodiments, formation of an immune complex is detected by a method such as size exclusion (gel filtration) chromatography, ultracentrifugation, light scattering, electron microscope, or mass spectrometry (Mol Immunol (2002) 39: 77-84, Mol Immunol (2009) 47: 357-364). These methods make use of the property that the size of an immune complex containing two or more antibodies is larger than that of an immune complex containing one antibody. Where a large difference is observed between the molecular sizes detected in the presence of the antigen and in the absence of the antigen, it is determined that uptake of the antibody into cells is enhanced when complexed with its antigen. In another embodiments, formation of an immune complex is detected by a binding assay to an Fc receptor (e.g., Fc gamma R) using such as ELISA, FACS, or SPR (surface plasmon resonance assay; for example, using Biacore) (J Biol Chem (2001) 276 (9): 6591-6604; J Immunol Methods (1982) 50: 109-114; J Immunol (2010) 184 (4): 1968-1976; mAbs (2009) 1(5): 491-504). These methods make use of the property that an immune complex containing two or more antibodies can bind to an Fc receptor more strongly than an immune complex containing only one antibody. Where a large difference is observed between the binding signals detected in the presence of the antigen and in the absence of the antigen, it is determined that uptake of the antibody into cells is enhanced when it is complexed with its antigen.

In another embodiment, uptake of an antibody into cells can be evaluated, for example by administering a test antibody to an animal (e.g., a mouse) and measuring the clearance of the antigen from plasma. Where an accelerated elimination of antigens from plasma is observed in a test antibody-administered animal compared to in a reference antibody-administered animal, it is determined that uptake of the test antibody into cells is enhanced when complexed with its antigen. As described above, an antibody which forms an immune complex containing two or more antibodies (and/or two or more antigens) is expected to accelerate the elimination of antigens from plasma. In certain embodiments, an antibody which does not form a large immune complex containing two or more antibodies can be used as a reference antibody. In certain embodiments, the difference between the two antibodies can be evaluated using a ratio of the plasma antigen concentration. For example, a large value of the ratio of (plasma antigen concentration measured in a reference antibody-administered animal)/(plasma antigen concentration measured in a test antibody-administered animal) indicates that the test antibody can accelerate elimination of antigens from plasma compared to the reference antibody and/or that uptake of the test antibody into cells is enhanced as compared to the reference antibody. In certain embodiments, such a large value of the ratio of (plasma antigen concentration measured in a reference antibody-administered animal)/(plasma antigen concentration measured in a test antibody-administered animal) can be at least 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 20, 50, 100, 200, 500, or 1000.

In another embodiment, uptake of an antibody into cells can be evaluated, for example by administering a test antibody to an animal (e.g., a mouse) and measuring the clearance of antigen from plasma. As described above, uptake of an immune complex into cells is expected to be caused through the interaction between an Fc region of the antibody and an Fc receptor (e.g., Fc gamma R) on the cells. Therefore, the extent of the cellular uptake of one test antibody can be evaluated by comparing antigen clearance from plasma caused by the test antibody and that caused by a reference antibody, the reference antibody being identical with the test antibody except that it has an Fc region with no Fc receptor (e.g., Fc gamma R) binding activity. In a certain embodiment, the extent of the cellular uptake of one test antibody can be evaluated by making two modified antibodies, one of which has an Fc region with Fc receptor (e.g., Fc gamma R) binding activity and the other of which has an Fc region without Fc receptor (e.g., Fc gamma R) binding activity, and comparing antigen clearance from plasma caused by the two antibodies. The difference in the antibody clearance reflects how large amounts of the test antibody complexed with its antigen are taken up into cells and cleared from plasma through an Fc receptor (e.g., Fc gamma R), and it is determined that the larger the difference is, the higher the uptake of the test antibody into cells is when it is complexed with its antigen. In certain embodiments, a modified antibody having a heavy chain constant region of G1m (SEQ ID NO: 7) or SG1 (SEQ ID NO: 64) can be used as an antibody which has an Fc region with Fc gamma R binding activity. In certain embodiments, a modified antibody having a heavy chain constant region of F760 (SEQ ID NO: 68) can be used as an antibody which has an Fc region without Fc gamma R binding activity. In certain embodiments, the difference between the two antibodies can be evaluated using a ratio of the plasma antigen concentration. For example, a large value of the ratio of (plasma antigen concentration measured in an animal to which the antibody without Fc gamma R binding activity is administered)/(plasma antigen concentration measured in an animal to which the antibody with Fc gamma R binding activity is administered) indicates that the uptake of the test antibody into cells is high. In certain embodiments, such a large value of the ratio of (plasma antigen concentration measured in an animal to which the antibody without Fc gamma R binding activity is administered)/(plasma antigen concentration measured in an animal to which the antibody with Fc gamma R binding activity is administered) can be at least 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 20, 50, 100, 200, 500, or 1000.

In those in vivo tests, an antibody can be administered via intradermal, intravenous, intravitreal, subcutaneous, intraperitoneal, parenteral or intramuscular injection. For example, an antibody can be administered via intravenous injection, as exemplified in Example 4. In certain embodiments, an antigen can be externally administered to an animal in addition to an antibody, either by co-injection with an antibody or by a separate steady-state infusion. For example, an antigen can be co-injected with an antibody, as exemplified in Example 4. In certain embodiments, plasma antigen concentration can be measured as free antigen concentration in plasma which means the concentration of the antigen not bound by an antibody in plasma, or total antigen concentration in plasma which means the sum of the concentrations of the antibody-bound antigen and the non-antibody-bound antigen in plasma (Pharm Res. 2006 January; 23 (1): 95-103). For example, plasma antigen concentration can be measured as total antigen concentration in plasma, as exemplified in Example 4. In certain embodiments, plasma antigen concentration can be measured at 15 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 7 days, 14 days, 28 days, 56 days, or 84 days after antibody administration. For example, plasma antigen concentration can be measured at 7 days after antibody administration, as exemplified in Example 4.

In certain embodiments, an anti-myostatin antibody of the present invention can be obtained using assays as described above for evaluating cellular uptake of an antibody into cells. For example, such an antibody can be obtained by preparing a group of anti-myostatin antibodies, performing assays as described above on the antibodies, and selecting an antibody whose uptake into cells is determined to be high when complexed with its antigen. In further embodiments, antibodies obtained by immunizing animals against myostatin or obtained by screening antibody libraries against myostatin can be used as a group of anti-myostatin antibodies.

In other embodiments, an anti-myostatin antibody of the present invention can be obtained using competition assays described above for identifying an antibody that competes for binding to myostatin. For example, such an antibody can be obtained by preparing a group of anti-myostatin antibodies, performing competition assays as described above on the antibodies, and selecting an antibody which competes for binding to myostatin with an anti-myostatin antibody described herein. Alternatively, an antibody which competes for binding to myostatin with an anti-myostatin antibody described in Table 2 or 3 can be selected. Alternatively, an antibody which competes for binding to myostatin with any one of the antibodies selected from the group of consisting of: MST0226, MST0796, MST0139, MST0182, MSLO00, MSLO01, MSLO02, MSLO03, and MSLO04 described in Examples 2 or 6 can be selected. In further embodiments, antibodies obtained by immunizing animals against myostatin or obtained by screening antibody libraries against myostatin can be used as a group of anti-myostatin antibodies.

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

In another embodiment, an immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes are available for the production of 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 comprise a radioactive atom for scintigraphic studies, for example tc99m or 1123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

E. Methods and Compositions for Diagnostics and Detection

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

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

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

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

G. Therapeutic Methods and Compositions

Any of the anti-myostatin antibodies provided herein may be used in therapeutic methods.

In one aspect, an anti-myostatin antibody for use as a medicament is provided. In further aspects, an anti-myostatin antibody for use in treating a muscle wasting disease is provided. In certain embodiments, an anti-myostatin antibody for use in a method of treatment is provided. In certain embodiments, the invention provides an anti-myostatin antibody for use in a method of treating an individual having a muscle wasting disease comprising administering to the individual an effective amount of the anti-myostatin antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In further embodiments, the invention provides an anti-myostatin antibody for use in increasing mass of muscle tissue. In certain embodiments, the invention provides an anti-myostatin antibody for use in a method of increasing mass of muscle tissue in an individual comprising administering to the individual an effective amount of the anti-myostatin antibody to increase mass of muscle tissue. In further embodiments, the invention provides an anti-myostatin antibody for use in increasing strength of muscle tissue. In certain embodiments, the invention provides an anti-myostatin antibody for use in a method of increasing strength of muscle tissue in an individual comprising administering to the individual an effective amount of the anti-myostatin antibody to increase strength of muscle tissue. An “individual” according to any of the above embodiments is preferably a human.

In further embodiments, the invention provides an anti-myostatin antibody for use in enhancing the clearance of myostatin from plasma. In certain embodiments, the invention provides an anti-myostatin antibody for use in a method of enhancing the clearance of myostatin from plasma in an individual comprising administering to the individual an effective amount of the anti-myostatin antibody to enhance the clearance of myostatin from plasma. In one embodiment, an anti-myostatin antibody which forms a large immune complex containing two or more antibody molecules enhances the clearance of myostatin from plasma, compared to an anty-myostatin antibody which does not form such a large immune complex. In another embodiment, an anti-myostatin antibody with pH-dependent binding characteristics enhances the clearance of myostatin from plasma, compared to an anti-myostatin antibody which does not have pH-dependent binding characteristics. In a further embodiment, an anti-myostatin antibody with a pH-dependent binding characteristic between binding at pH5.8 and pH7.4 enhances the clearance of myostatin from plasma, compared to an anti-myostatin antibody which does not have pH-dependent binding characteristics. In a further embodiment, an anti-myostatin antibody having both properties, that is, an antibody which forms a large immune complex containing two or more antibody molecules and which has pH-dependent binding characteristics, enhances the clearance of myostatin from plasma. An “individual” according to any of the above embodiments is preferably a human.

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

In a further embodiment, the medicament is for enhancing the clearance of myostatin from plasma. In a further embodiment, the medicament is for use in a method of enhancing the clearance of myostatin from plasma in an individual comprising administering to the individual an effective amount of the medicament to enhance the clearance of myostatin from plasma. An “individual” according to any of the above embodiments is preferably a human.

In a further aspect, the invention provides a method for treating a muscle wasting disease. In one embodiment, the method comprises administering to an individual having such a muscle wasting disease an effective amount of an anti-myostatin antibody. In one such 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 a further aspect, the invention provides a method for increasing mass of muscle tissue in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-myostatin antibody to increase mass of muscle tissue. In one embodiment, an “individual” is a human.

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

In a further embodiment, the invention provides a method for enhancing the 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 to enhance the clearance of myostatin from plasma. In one embodiment, an “individual” is a human.

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

In a further aspect, the pharmaceutical formulation is for treatment of a muscle wasting disease. In a further embodiment, the pharmaceutical formulation is for increasing mass of muscle tissue. In a further embodiment, the pharmaceutical formulation is for increasing strength of muscle tissue. In a further embodiment, the pharmaceutical formulation is for enhancing the 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.

It is believed that an anti-myostatin antibody which can form a large immune ecomplex, for example an immune complex containing two or more antibodies and two or more antigens, can be taken up into cells efficiently, and such enhanced uptake of an immune complex into cells can lead to enhanced antigen clearance from plasma, compared to a conventional anti-myostatin antibody which does not form a large immune complex. An anti-myostatin antibody which additionally has pH-dependent antigen binding characteristics would be able to further enhance antigen clearance from plasma, since such an antibody can bind to the antigen in neutral extracellular environment and release it into acidic endosomal compartments following the uptake of the antigen-antibody complex into cells.

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

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

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

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

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

H. Articles of Manufacture

It is understood that any of the above articles of manufacture may include an immunoconjugate of the invention in place of or in addition to an anti-myostatin antibody.

Expression and Purification of Human, Cynomolgus Monkey, and Mouse Myostatin Mature Form

Human latent myostatin (also described herein as myostatin latent form) (SEQ ID NO:1) was expressed transiently using FreeStyle293-F cell line (Thermo Fisher, Carlsbad, Calif., USA). Conditioned media containing expressed human myostatin latent form was acidified to pH 6.8 and diluted with ½ vol of milliQ water, followed by application to a Q-sepharose FF anion exchange column (GE healthcare, Uppsala, Sweden). The flow-through fraction was adjusted to pH 5.0 and applied to a SP-sepharose HP cation exchange column (GE healthcare, Uppsala, Sweden), and then eluted with a NaCl gradient. Fractions containing the human myostatin latent form were collected and subsequently subjected to a Superdex 200 gel filtration column (GE healthcare, Uppsala, Sweden) equilibrated with 1×PBS. Fractions containing the human myostatin latent form were then pooled and stored at −80 degrees C.

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

Expression and purification of myostatin latent and mature form from cynomolgus monkey (cynomolgus or cyno) (SEQ ID NOs: 3 and 4, respectively) and mouse (SEQ ID NOs: 5 and 6, respectively) were all done exactly the same way as the human counterpart.

The sequence homology of myostatin mature form among human, cynomolgus monkey and mouse are 100% identical, therefore in all the necessary experiments, regardless of species, SEQ ID NO: 2 was used as myostatin mature form (recombinant mature myostatin).

Identification of Anti-Mature Myostatin Antibody

Twelve to sixteen week old NZW rabbits were immunized intradermally with human mature myostatin, human latent myostatin or mature myostatin conjugated with KLH (50-100 micro g/dose/rabbit). This dose was repeated 4-5 times. One week after the final immunization, the spleen and blood from immunized rabbit was collected. Antigen-specific B-cells were stained with labelled antigen, sorted with FCM cell sorter (FACS aria III, BD), and plated in 96-well plates at one cell/well density together with 25,000 cells/well of EL4 cells (European Collection of Cell Cultures) and with rabbit T-cell conditioned medium diluted 20 times, and were cultured for 7-12 days. EL4 cells were treated with mitomycin C (Sigma) for 2 hours and washed 3 times in advance. The rabbit T-cell conditioned medium was prepared by culturing rabbit thymocytes in RPMI-1640 containing Phytohemagglutinin-M (Roche), phorbol 12-myristate 13-acetate (Sigma) and 2% FBS. After cultivation, B-cell culture supernatants were collected for further analysis and pellets were cryopreserved.

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

A total of 28,547 of B-cell lines were screened for binding to mature myostatin and/or human latent myostatin and 1154 lines were selected and designated MST0001-0254, 0288-0629, 0633-0676, 0760-0909, 0911-0931, and 1120-1462. RNA was purified from corresponding cell pellets by using ZR-96 Quick-RNA kits (ZYMO RESEARCH).

The DNA of their variable regions of the heavy and light chain were amplified by reverse transcription PCR and cloned into expression vectors with the heavy chain constant region G1m sequence (SEQ ID NO: 8 (the amino acid sequence is shown in SEQ ID NO: 7)) and with the light chain constant region k0MTC (SEQ ID NO: 10 (the amino acid sequence is shown in SEQ ID NO: 9)) or k0MC sequence (SEQ ID NO: 11 (the amino acid sequence is shown in SEQ ID NO: 9)), respectively. Recombinant antibodies were expressed transiently using the FreeStyle FS293-F cells and 293fectin (Life technologies), according to the manufacturer's instructions. Culture supernatant or recombinant antibodies were used for screening. Recombinant antibodies were purified with protein A (GE Healthcare) and eluted in D-PBS, TBS (Tris-buffered saline), or His buffer (20 mM Histidine, 150 mM NaCl, pH6.0). Size exclusion chromatography was further conducted to remove high molecular weight and/or low molecular weight component, if necessary. Several recombinant antibodies of which sequences are shown in Table 2 were selected for further experiments below.

Reporter gene assay was used to assess the biological activity of active myostatin in vitro. HEK-Blue™ TGF-beta cells (Invivogen), which express a Smad3/4-binding elements (SBE)-inducible SEAP (Secreted embryonic alkaline phosphatase) reporter genes, allow the detection of bioactive myostatin by monitoring the activation of the activin type 1 and type 2 receptors. Myostatin mature form stimulates the production of SEAP into cell supernatant by activating Smad3/4 signal through the binding to its receptor. The quantity of SEAP secreted is then assessed using QUANTIBlue™ (Invivogen).

HEK-Blue™ TGF-beta cells were maintained in DMEM medium (Gibco) supplemented with 10% fetal bovine serum, 50 micro g/mL streptomycin, 50 U/mL penicillin, 100 micro g/mL Normocin™, 30 micro g/mL of Blasticidin, 200 micro g/mL of HygroGold™ and 100 micro g/mL of Zeocin™. During functional assay, cells were changed to assay medium (DMEM with 0.1% bovine serum albumin, streptomycin, penicillin and Normocin™) and seeded to 96-well plate. Recombinant mature myostatin and anti-mature myostatin antibody were incubated at 37 degrees C. for 30 minutes. The sample mixtures were transferred to cells. After 20-hour incubation, cell supernatant was mixed with QUANTIBlue™ and the optical density at 620 nm was measured in a colorimetric plate reader.

41C1E4 and MY0029 were used as positive controls. Both 41C1E4 and MY029 are anti-mature myostatin antibodies and their sequences are described in U.S. Pat. No. 7,632,499 and WO2004037861, respectively.

As shown asFIG. 1, all anti-mature myostatin antibodies inhibited the secretion of SEAP. This indicates that the antibodies block the binding of mature myostatin to its receptor.

Comparison of Plasma Total Myostatin Concentration Between Antibodies with Fc Gamma R Binding and with Abolished Fc Gamma R Binding in Mice

In Vivo Test Using C.B-17 Scid Mice

The kinetics of total exogenous and endogenous myostatin was assessed in vivo upon administration of an anti-mature myostatin antibody and recombinant mature myostatin in C.B-17 scid mice (In Vivos, Singapore). An anti-mature myostatin antibody (0.6 mg/ml) and recombinant mature myostatin (0.05 mg/ml) was administered at a single dose of 10 ml/kg into the caudal vein. Blood was collected at 7 days after administration. The collected blood was centrifuged immediately at 14,000 rpm in 4 degrees C. for 10 minutes to separate the plasma. The separated plasma was stored at or below −80 degrees C. until measurement. The anti-mature myostatin antibodies used are those prepared based on MST0226, MST0796, MST0139, MST0182, 41C1E4 and MY0029 which are described above. To assess the effects of Fc gamma R binding on myostatin accumulation, two types of modified anti-mature myostatin antibodies were generated, one having an Fc region with Fc gamma R binding activity and the other having an Fc region without Fc gamma R binding activity (also described herein as silent Fc). Heavy chain constant regions G1m (amino acid sequence SEQ ID NO: 7, nucleotide sequence SEQ ID NO: 8) and SG1 (amino acid sequence SEQ ID NO: 64, nucleotide sequence SEQ ID NO: 66) described herein include an Fc region with Fc gamma R binding activity, and F760 (amino acid sequence SEQ ID NO: 68, nucleotide sequence SEQ ID NO: 69) include an Fc region without Fc gamma R binding activity. Binding affinity of G1m and SG1 against human Fc gamma Rs are comparable to that of natural human IgG1. On the other hand, binding affinity of F760 is abolished by amino acid modification in the Fc region.

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 antibody-immobilized plates were prepared by dispensing anti-mature myostatin antibody RK35 (as described in WO2009058346) onto a MULTI-ARRAY 96-well plate (Meso Scale Discovery) and incubated overnight at 4 degrees C. Mature myostatin calibration curve samples and mouse plasma samples diluted 40-fold or more were prepared. The samples were mixed in an acidic solution (0.2 M Glycine-HCl, pH 2.5) to dissociate mature myostatin from its binding protein (such as pro-peptide). Subsequently, the samples were added onto an anti-mature myostatin antibody-immobilized plate, and allowed to bind for 1 hour at room temperature before washing. Next, SULFO TAG labelled anti-mature myostatin antibody RK22 (as described in WO2009058346) was added and incubated for 1 hour at room temperature before washing. Read Buffer T (×4) (Meso Scale Discovery) was immediately added to the plate and signal was detected by SECTOR Imager 2400 (Meso Scale Discovery). The mature myostatin concentration was calculated based on the response of the calibration curve using the analytical software SOFTmax PRO (Molecular Devices). The ratio of total myostatin concentration in plasma at day 7 between F760 and G1 after intravenous administration measured by this method is shown inFIG. 2, as ratios of (plasma total myostatin concentration measured when the antibody having F760-type Fc region was administered)/(plasma total myostatin concentration measured when the antibody having G1-type Fc region was administered).

Effect of Fc Gamma R Binding on Myostatin Accumulation In Vivo

A 2.06 fold difference of plasma total myostatin concentration was observed between the 41C1E4-F760-administered group and the 41C1E4-G1-administered group, and a 1.92 fold difference of plasma total myostatin concentration was observed between the MY0029-F760-administered group and the MY0029-G1-administered group. In contrast, a 4.77, 2.56, 2.55, and 3.10 fold-difference of plasma total myostatin concentration was observed between MST0226-F760 and MST0226-G1, between MST0796-F760 and MST0796-G1, between MST0139-F760 and MST0139-G1, and between MST0182-F760 and MST0182-G1, respectively. Since mature myostatin is a dimeric protein, anti-mature myostatin antibodies are expected to form a multimeric, large immune complex which contains two or more Fc regions. Moreover, optimal size and form of immune complex can accelerate the uptake of immune complex into cell via Fc gamma R. Although 41C1E4 and MY0029 showed only 2 fold difference of plasma total myostatin concentration between their F760-type form and their G1-type form, MST0226, MST0796, MST0139 and MST0182 showed more than 2.5 fold difference of plasma total myostatin concentration between their F760-type form and their G1-type form. The result suggests that MST0226, MST0796, MST0139 and MST0182 have potential for faster uptake of immune complex compared to 41C1E4 and MY0029.

In Vivo Efficacy of Anti-Mature Myostatin Antibody on Muscle Mass

The in vivo efficacy of anti-mature myostatin antibodies 41C1E4 (as described in U.S. Pat. No. 7,632,499), MST0226-G1m, and MST0796-G1m was evaluated in mice. 41C1E4 was used as positive control in this study. To avoid potential immunomodulation due to mouse anti-human antibody response, in vivo studies were performed in immune-deficient Severe Combined Immunodeficient (SCID) mice. Five-week-old SCID (C.B-17 SCID) mice (Charles River Laboratories Japan, Inc. (Kanagawa, JAPAN)) were given intravenous administration of a monoclonal antibody at 2 mg/kg or 10 mg/kg, or vehicle (PBS) once per week for two weeks. On day 0, 4, 7 and 14, full body lean mass was assessed by nuclear magnetic resonance (NMR) (the minispec LF-50, Bruker Bio Spin (Kanagawa, JAPAN)). The animals were euthanized on day 14, and the gastrocnemius, quadriceps, plantaris, masseter, and soleus muscles were dissected and weighed. Each isolated muscle weight in antibody treatment group was standardized by the isolated muscle weight in the PBS treatment group. Statistical significance was determined by ANOVA, a Student's t-test and a Dunnett's test with JMP 9 software (SAS, Inc.). A p value of less than 0.05 was considered significant. The results are shown inFIGS. 3 and 4. Both antibodies MST0226-G1m and MST0796-G1m increased lean body mass measured by NMR and isolated muscle weight, compared with the PBS treatment group. This indicates that MST0226-G1m and MST0796-G1m have an ability to increase muscle in mice.

Generation of Humanized and pH-Dependent Anti-Mature Myostatin Antibody

Humanization was carried out on MST0226-G1m to generate a humanized antibody, MSLO00-SG1. The polynucleotides encoding the heavy and light chains were synthesized by GenScript Inc. and were cloned into expression vectors (See Table 3 for amino acid sequences and nucleotide sequences). MSLO00-SG1 was transiently expressed in FS293 cells and HEK Blue Assay was carried out as described above. As shown inFIG. 5, MSLO00-SG1 showed comparable inhibition activity to MST0226-G1m, hence, humanization was successfully completed.

To generate pH-dependent anti-mature myostatin antibodies, comprehensive mutagenesis was conducted on all CDRs of MSLO00-SG1. Each amino acid in the CDRs was individually substituted with any of 18 other amino acids except cysteine. Mutated variants were transiently expressed and evaluated by Biacore assay as described below.

pH dependent binding of MST0226 variants to human mature myostatin were determined at 37 degrees C. using Biacore T200 instrument (GE Healthcare). Biotinylated mature myostatin was immobilized onto streptavidin sensor chip (GE Healthcare). In order to assess pH dependent binding of MST0226 variants to mature myostatin, 100 nM of antibodies were injected over mature myostatin sensor surface at pH 7.4 (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2), 0.05% Tween 20, 0.005% NaN3), followed by dissociation at pH 7.4 and an additional dissociation phase at pH5.8. This is to assess the pH-dependent dissociation of antibody/antigen complexes formed at pH 7.4. The dissociation rate (kd) at both pH 7.4 and pH 5.8 buffer was determined by processing and fitting data using Scrubber 2.0 (BioLogic Software) curve fitting software. The ratio of (kd at pH5.8)/(kd at pH 7.4) gives indication of pH dependent binding, e.g. ratio>1 indicates pH dependent binding. The sensor surface was regenerated each cycle with 10 mM Glycine-HCl, pH 1.7.

After several cycles of mutagenesis and selections, four pH dependent variants: MSLO01-SG1, MSLO02-SG1, MSLO03-SG1, and MSLO04-SG1 were successfully generated. Amino acid and nucleotide sequences of the four variants are shown in Table 3. Amino acid sequences of their hypervariable regions (HVRs) are shown in Table 4. Results of Biacore assay and HEK Blue Assay are shown in Table 5 andFIG. 5. These pH dependent variants showed pH dependency under acidic condition with ratio of (kd at pH5.8)/(kd at pH 7.4)>17. As shown inFIG. 5, the pH dependent variants showed comparable or even stronger inhibition activity in HEK Blue Assay to MSLO00-SG1 (non-pH dependent antibody).

TABLE 3MST0226 variants and their DNA and amino acid sequences (shown as SEQ ID NOs)Variable regionConstant regionHeavyLightHeavyLightAntibody nameAbbreviationDNAProteinDNAProteinDNAProteinDNAProteinMS_M22601H-SG1/M22608L-SK1MSLO00-SG15648605266646765MS_M22601H1020-SG1/M22608L0744-SK1MSLO01-SG15749615366646765MS_M22601H1080-SG1/M22608L0837-SK1MSLO02-SG15850625466646765MS_M22601H1082-SG1/M22608L0837-SK1MSLO03-SG15951625466646765MS_M22601H1080-SG1/M22608L0846-SK1MSLO04-SG15850635566646765

Effect of pH Dependent Mature Myostatin Binding Against Plasma Total Myostatin Concentration in Mice

In Vivo Test Using C.B-17 Scid Mice

The kinetics of total exogenous and endogenous myostatin was assessed in vivo upon administration of an anti-mature myostatin antibody and recombinant mature myostatin in C.B-17 scid mice (In Vivos, Singapore) as described in Example 4. The anti-mature myostatin antibodies used are MSLO00-SG1, MSLO01-SG1, MSLO02-SG1, MSLO03-SG1, and MSLO04-SG1 which are described above. The four pH-dependent variants (MSLO01-SG1, MSLO02-SG1, MSLO03-SG1, and MSLO04-SG1) were compared with the non pH-dependent antibody MSLO00-SG1.

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 4. The lower limit of quantitation of assay was 2.44 ng/mL. When quantitative value was below the lower limit of quantitation, it was described as “BLQ” (below the limit of quantitation). The plasma total myostatin concentration at day 7 after intravenous administration of an antibody as measured by this method is shown inFIG. 6A. The ratio of total myostatin concentration in plasma at day 7 between F760 and G1 after intravenous administration measured by this method is shown inFIG. 6B. When quantitative value was below the lower limit of quantitation, 2.44 ng/ml was used to calculate the ratio of total myostatin concentration.

Effect of pH Dependent Binding to Myostatin Accumulation In Vivo

After administration of MSLO00-SG1, plasma total myostatin concentration at day 7 showed 25.49 ng/mL. In contrast, after administration of MSLO01-SG1, MSLO02-SG1, MSLO03-SG1, or MSLO04-SG1, plasma total myostatin concentration at day 7 showed BLQ. Administration of the pH-dependent anti-mature myostatin antibodies reduced total myostatin concentration over 10 fold compared to the non pH-dependent anti-mature myostatin antibody (FIG. 6A). A 2.98 fold difference of plasma total myostatin concentration was observed between the MSLO00-F760-administered group and the MSLO00-SG1-administered group, while a 7.49, 11.85, 10.02, and 11.92 fold difference of plasma total myostatin concentration was observed between MSLO01-F760 and MSLO01-SG1, between MSLO02-F760 and MSLO02-SG1, between MSLO03-F760 and =MSLO003-SG1, and between MSLO04-F760 and MSLO04-SG1, respectively. Administration of the pH-dependent anti-mature myostatin antibodies accelerated Fc gamma R-mediated elimination of mature myostatin from plasma, compared to the non pH-dependent anti-mature myostatin antibody (FIG. 6B).

In Vivo Efficacy of pH-Dependent Anti-Mature Myostatin Antibody

The in vivo efficacy of MSLO00-SG1 (non-pH dependent antibody) and MSLO03-SG1 (pH dependent antibody) were evaluated in mice as described in Example 5. Grip strength was measured with a grip strength test meter (e.g., GPM-100B, MELQUEST Ltd., (Toyama, JAPAN)). In this study, both antibodies were administered at different dose from 0.5 mg/kg to 10 mg/kg. Lean body mass was measured as an index of muscle increment, and grip strength was measured as an index of muscle function. The results are shown inFIGS. 7 and 8. Both antibodies increased lean body mass and grip strength dose dependently. When the increment of lean body mass and the improvement of grip strength of MSLO00-SG1 and MSLO03-SG1 were compared at the same dose, MSLO03-SG1 showed the superior efficacy in muscle increment and muscle function to MSLO00-SG1. This indicates that the reduction of mature myostatin concentration by pH dependent antibody leads to the increase in muscle mass and the improvement in muscle function at lower dose.