EA040534B1 - METHODS FOR INCREASING LESS BODY MASS USING AN ANTIBODY TO GDF8 OR ITS ANTIGEN-BINDING FRAGMENT AND RESISTANCE TRAINING - Google Patents

Description

Link to related applications

This application was filed April 15, 2016 as an International Patent Application under the PCT and this application claims priority under U.S. Provisional Application No. 62/147,853, filed April 15, 2015, U.S. Provisional Application No. 62/234899, filed September 30, 2015, and U.S. Provisional Application No. 62/261,528, filed December 1, 2015, the disclosures of which are incorporated herein in their entirety.

Sequence listing

This application includes a Sequence Listing in electronic format in a text file called 40848-0055WOU1_SeqList-text, which was created on April 15, 2016 and is 160 kilobytes (KB) in size. The content of the text file 40848-0055WOUl_SeqListtext is incorporated herein by reference.

State of the art

Decrease in skeletal muscle mass appears to play a significant pathological role in the development of a wide range of disorders associated with aging, frailty, and certain metabolic conditions. In the elderly, conditions such as sarcopenia and specific events such as hip fracture can be directly linked to significant loss of overall muscle mass. In both older and younger populations, recovery from immobilization and orthopedic surgery may be associated with a degree of acute muscular dystrophy associated with both lack of functional muscle loading and procedure-related atrophy. In addition, increasing or maintaining skeletal muscle mass may lead to the prevention of obesity as well as improvements in metabolism.

Myostatin, or growth and differentiation factor 8 (GDF8), is a soluble ligand of the TGF-β superfamily. It is a negative muscle growth regulator expressed predominantly in skeletal muscle, but there is evidence of low expression in other tissues such as the heart and adipose tissue at levels approximately 100 lower than that observed in skeletal muscle (McPherron Nature 387:83 1997, Sharma J Cell Phys 180:1, 1999, Lee Annrev Cell Dev Biol 20:61, 2004, Allen Pysiol Rev 88:257, 2008, Heineke Cir 121:419, 2010) . Mature myostatin is highly conserved across species, and inactivating myostatin gene mutations result in an overly muscular phenotype in many species, including mice, cattle, dogs, and humans. In contrast, overexpression of myostatin in mice (by injecting transfected CHO cells into the thigh of nude mice or generating striated muscle transgenic mice) caused a significant decrease in body weight due to a decrease in muscle fiber size (McPherron 1997, cited above, GrobetNat. Genet. 17: 71, 1997, Mosher PLOS Genet 3:e79, 2007, Schuelke NEJM 350:2682, 2004). While the myostatin null phenotype in mice demonstrates the importance of myostatin in controlling muscle size during development, hypertrophy can also be induced in adult muscle by inhibition of myostatin with neutralizing antibodies, decoy receptors, or other antagonists. However, the effects of myostatin inhibitors on functioning cardiac tissue can result in an undesirable side effect profile when these inhibitors are used to treat conditions such as sarcopenia and metabolic conditions.

Ways to increase strength and functionality with GDF8 inhibitors.

The present disclosure provides methods and compositions for use in the methods described herein. GDF8 inhibitors are useful for increasing lean muscle mass in a subject, for example, in combination with exercise. In some embodiments, the subject is a subject that does not have a disease or disorder that significantly limits the subject's ability to participate in resistance training. In embodiments, the disease or disorder is a disease or disorder in which a physician has recommended exercise restriction for the subject or in which exercise is contraindicated, such as uncontrolled diabetes mellitus, recent myocardial infarction, unstable heart conditions, acute heart failure, severe myocarditis, uncontrolled hypertension, valvular heart disease requiring surgery, and severe aortic stenosis.

In embodiments, a method for increasing lean body mass in a subject comprises providing the subject with an exercise regimen and administering a composition containing an effective amount of a GDF8 inhibitor, the effective amount being at least 400 mg. In embodiments, the exercise regimen includes, without limitation, resistance training, resistance training, yoga, aerobic exercise, and Pilates. In embodiments, the GDF8 inhibitor is an antibody or antigen-binding fragment that specifically binds GDF8. In embodiments, the antibody or antigen-binding fragment comprises a heavy chain CDR contained within a heavy chain variable region selected from the group consisting of SEQ ID

- 1 040534

NO: 2, SEQ ID NO: 18, SEQ ID NO: 34, SEQ ID NO: 50, SEQ ID NO: 66, SEQ ID NO: 82, SEQ ID NO: 98,

SEQ ID NO: 114, SEQ ID NO: 130, SEQ ID NO: 146, SEQ ID NO: 162, SEQ ID NO: 178, SEQ ID NO: 194,

SEQ ID NO: 210, SEQ ID NO: 226, SEQ ID NO: 242, SEQ ID NO: 258, SEQ ID NO: 274, SEQ ID NO: 290,

SEQ ID NO: 306, SEQ ID NO: 360, and SEQ ID NO: 376. In embodiments, the method further provides an antibody or antigen binding fragment that comprises light chain CDRs contained within light chain variable regions selected from the group consisting of SEQ ID NO: SEQ ID NO: 10, SEQ ID NO: 26, SEQ ID NO: 42, SEQ ID NO: 58, SEQ ID NO: 74, SEQ ID NO: 90, SEQ ID NO: 106, SEQ ID NO: 122 , SEQ ID NO: 138, SEQ ID NO: 154, SEQ ID NO: 170, SEQ ID NO: 186, SEQ ID NO: 202, SEQ ID NO: 218, SEQ ID NO: 234, SEQ ID NO: 250, SEQ ID NO: 266, SEQ ID NO: 282, SEQ ID NO: 298, SEQ ID NO: 314, SEQ ID NO: 322, SEQ ID NO: 368 and SEQ ID NO: 384.

In embodiments, the compositions are formulated to contain an effective amount of a GDF8 inhibitor to increase lean muscle mass. In embodiments, the effective amount is at least 0.1 mg/kg - about 10 g/kg, 1 mg/kg - about 1 g/kg, or 10-100 mg/kg. In embodiments, the composition is administered at least once a week, twice a week, three times a week, four times a week, or five times a week. In embodiments, the exercise regimen is followed for at least 12 weeks. In embodiments, the compositions are formulated for intravenous, subcutaneous, or oral administration.

Brief description of the figures

In FIG. 1(A) shows the results of a MAD (multiple incremental dose) study designed to evaluate the safety, tolerability, pharmacokinetics (PK), immunogenicity, and pharmacodynamic (PD) effects of REGN1033 (an anti-GDF8 antibody) administered subcutaneously (SC) to healthy volunteers aged 60 years and older. A total of 5 cohorts were studied with 12 subjects recruited into each cohort. Subjects received SC doses of REGN1033 (n=9) or placebo (n=14). The scheduled dosing regimens for REGN1033 were 100, 200 or 400 mg Q2W for a total of 6 doses per subject and 200 mg or 400 mg Q4W for a total of 3 doses per subject. % lean body mass was determined using dual energy X-ray absorptiometry (DEXA).

In FIG. 1(B) shows percentage change in lean muscle mass per study visit per group in subjects receiving placebo alone (third line from top), placebo plus resistance training (RT) (bottom line), 400 mg SC REGN1033 alone 6 doses total during the study period (second line from top) and 400 mg SC REGN1033 along with RT (upper line) for a total of 6 doses during the study period.

In FIG. 2 shows the percentage change in appendicular fat mass per study visit for each arm in subjects receiving placebo alone (third line from top), placebo plus resistance training (RT) (bottom line), 400 mg SC REGN1033 alone (second line, starting at from the upper line) for a total of 6 doses during the study period and 400 mg SC REGN1033 together with RT (upper line) for a total of 6 doses during the study period. Lines for placebo alone and 400 mg SC REGN1033 separately overlapped.

In FIG. 3(A) shows the percent change in thigh muscle mass, including intramuscular fat, per study visit per group in subjects receiving placebo alone (third line from top), placebo plus resistance training (RT) (bottom line), 400 mg SC REGN1033 alone for a total of 6 doses during the study period (second line from top) and 400 mg SC REGN1033 together with RT (upper line) for a total of 6 doses during the study period.

In FIG. 3(B) shows the percent change in thigh muscle mass, excluding intramuscular fat, per study visit per group in subjects receiving placebo alone (third line from top), placebo plus resistance training (RT) (bottom line), 400 mg SC REGN1033 alone for a total of 6 doses during the study period (second line from top) and 400 mg SC REGN1033 together with RT (upper line) for a total of 6 doses during the study period. The lines for placebo alone and placebo plus RT overlap.

In FIG. 4 shows the percentage change in gynoid fat per study visit per group in subjects receiving placebo alone (upper line), placebo plus resistance training (RT) (second line from top), 400 mg SC REGN1033 alone for a total of 6 doses during the study period (third line from top) and 400 mg SC REGN1033 along with RT for a total of 6 doses during the study period (bottom line). Lines for placebo together with RT and 400 mg SC REGN1033 overlapped separately.

In FIG. 5 shows the percentage change in bench press per study visit per group in subjects receiving placebo alone (lower line), placebo plus resistance training (RT) (third line from top), 400 mg SC REGN1033 alone for a total of 6 doses during the study period (second line from top) and 400 mg SC REGN1033 along with RT (upper line) for a total of 6 doses during the study period.

Detailed disclosure

Before describing the present methods, it should be understood that the present disclosure is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. In addition, it should be understood that the terminology used herein is provided solely for the purpose of describing specific embodiments and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

As used in the present specification and the appended claims, the singular forms include references to the plural forms, unless the context clearly dictates otherwise. Thus, for example, reference to a method includes one or more methods and/or steps of the type described herein and/or which will become apparent to those skilled in the art upon reading this disclosure.

Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as are generally understood by a person skilled in the art to which the present invention belongs.

As used herein, the term approximately when used with reference to a specific listed numerical value means that the value may vary from the listed value by no more than 1%. For example, the expression approximately 100 as used herein includes 99 and 101 and everything in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).

While any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, preferred methods and materials will be described below. All patents, applications and non-patent publications mentioned in the present specification are incorporated herein by reference in their entirety.

Definitions.

Human growth and differentiation factor-8, GDF8 and myostatin are used interchangeably to refer to the protein encoded by the nucleic acid sequence of SEQ ID NO: 338 and the protein characterized by the amino acid sequence of SEQ ID NO: 339 (propeptide) and SEQ ID NO: 340 (mature protein ).

As used herein, the term antibody is intended to refer to immunoglobulin molecules containing four polypeptide chains, two heavy (H) chains, and two light (L) chains linked together by disulfide bonds, as well as their multimers (e.g., IgM) . Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR, or VH) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2 and C _H 3. Each light chain contains a light chain variable region (abbreviated herein as LCVR, or VL) and a light chain constant region. The light chain constant region contains one domain (C _L 1). The VH and V _L regions can be further divided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with regions that are more conserved called framework regions (FRs). Each VH AND VL Consist of three CDRs and four FRs arranged from amino to carboxy in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. According to various embodiments of the present invention, the FR of an antibody to GDF8 (or an antigen-binding portion thereof) may be identical to human germline sequences, or may be naturally occurring or artificially modified. The amino acid consensus sequence can be determined based on a side-by-side analysis of two or more CDRs.

As used herein, the term antibody also includes antigen-binding fragments of whole antibody molecules. As used herein, the terms antigen-binding portion of an antibody, antigen-binding fragment of an antibody, and the like include any naturally occurring, enzymatically produced, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding antibody fragments can be generated, for example, from whole antibody molecules using any suitable standard techniques such as proteolytic cleavage or recombinant genetic engineering techniques, including manipulation and expression of DNA encoding the variable and optionally constant domains of an antibody. Such DNA is known and/or easily obtained from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to arrange one or more variable and/or constant domains in a suitable configuration, or to introduce codons, create cysteine residues, modify, add or remove amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv molecules

- 3 040534 (scFv); (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic an antibody's hypervariable region (eg, a complementarity-determining region (CDR)). Other engineered molecules, such as diabodies, tribodies, tetrabodies, and minibodies, are also included in the expression antigen-binding moiety used herein.

An antigen binding fragment of an antibody will typically contain at least one variable domain. The variable domain may be of any size or amino acid composition and will typically contain at least one CDR located adjacent to or in the same reading frame with one or more framework sequences. In antigen-binding fragments containing a V _H domain linked to a V _L domain, the VH and V _L domains can be arranged relative to each other in any suitable order. For example, the variable region may be dimeric and contain VH-VH, VH-VL, or VL-VL dimers. Alternatively, an antigen-binding fragment of an antibody may comprise a VH or VL monomeric domain.

In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary variable and constant domain configurations that can be found within an antigen-binding fragment of an antibody of the present invention include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) Vh-Ch1-Ch2-Ch3; (vi) Vh-Ch2-Ch3; (vii) Vh-Cl; (viii) Vl-Ch1; (ix) Vl-Ch2; (x) Vl-Ch3; (xi) Vl-Ch1-Ch2; (xii) V _l -C _h 1-C _h 2-C _h 3; (xiii) V _L -C _H 2 -C _H 3 and (xiv) V _L -C _L . In any configuration of the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may either be directly linked to each other or may be linked through a full or partial hinge or linker region. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that result in a flexible or semi-flexible connection between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present invention may comprise a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with each other and/or with one or more VH or VL monomeric domains (e.g. , via disulfide(s) bond(s)).

As with whole antibody molecules, antigen binding fragments can be monospecific or multispecific (eg, bispecific). A multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, with each variable domain being able to specifically bind to a different antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of an antigen-binding fragment of an antibody of the present invention using routine techniques available in the art.

Antibodies of the present invention may function through complement dependent cytotoxicity (CDC) or antibody dependent cell related cytotoxicity (ADCC). Complement dependent cytotoxicity (CDC) refers to the lysis of antigen expressing cells with an antibody of the present invention in the presence of complement. Antibody-dependent cell-mediated cytotoxicity (ADCC) refers to a cell-mediated response in which non-specific cytotoxic cells that express Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize the bound antibody on the cell -target and thereby lead to lysis of the target cell. CDC and ADCC can be measured using assays that are well known and available in the art. (See, for example, US Pat. Nos. 5,500,362 and 5,821,337 and Clynes et al., Proc. Natl. Acad. Sci. (USA) 95:652656 (1998)).

The term specifically binds or the like means that an antibody, or antigen-binding fragment thereof, forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding can be characterized by a dissociation constant of 1x10'6 M or less. Methods for determining whether two molecules are specifically bound are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, an antibody that specifically binds human GDF8 as used in the context of the present invention includes antibodies that bind human GDF8 or a portion thereof (e.g., a peptide comprising at least 6 contiguous amino acids of SEQ ID NO: 340) to KD, less than about 1000 nM, less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM,

less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM , less than about 2 nM, less than about 1 nM, or less than about 0.5 nM, as measured in a surface plasmon resonance assay. (See, for example, example 3 in this document). An isolated antibody that specifically binds human GDF8 may, however, be cross-reactive with other antigens, such as GDF8 molecules from other species. The term high affinity antibody refers to those antibodies that are capable of binding to GDF8 with a dissociation constant (KD) of about 10' ⁸ M or less, about 10' ⁹ M or less, about 10' ¹⁰ M or less, about 10' ¹¹ M or less or about 10' ¹² M or less, as measured by surface plasmon resonance, such as BIACORE™ or ELISA, to determine the affinity in solution.

By the term slow dissociation rate or Koff is meant an antibody that dissociates from GDF8 with a rate constant of 1x10'3 s ^-1 or less, preferably 1x10' ⁴ s' ¹ or less as determined by surface plasmon resonance, for example BIACORE™.

A neutralizing or blocking antibody is meant to refer to an antibody whose binding to GDF8 results in inhibition of the biological activity of GDF8. This inhibition of the biological activity of GDF8 can be assessed by measuring one or more indicators of the biological activity of GDF8. These indicators of the biological activity of GDF8 can be assessed using one or more of certain standard in vitro or in vivo assays known in the present technical field.

As used herein, the term anti-GDF8 antibody also includes multispecific antigen-binding molecules (eg, bispecific antibodies), wherein at least one binding domain (eg, binding arm) of the multispecific antigen-binding molecule specifically binds GDF8.

Exemplary anti-GDF8 antibodies that can be used in the context of the present invention include, for example, the fully human anti-GDF8 antibody H4H1657N2 (Regeneron/Sanofi) (for example, an anti-GDF8 antibody containing heavy and light chain variable regions with the amino acid sequences of SEQ ID NO : 360 and SEQ ID NO: 368, respectively, as presented in US patent No. 8840894). Other GDF8 antagonists that may be used in the context of the methods of the present invention include anti-GDF8 antibodies (e.g., antibody designated 2_112_1 (ATCC Depository PTA-6574)), as shown in US Pat. No. 7,807,159, anti-GDF8 antibodies ( e.g., 12A5-5) as shown in US Pat. No. 8,999,343 and US Patent Publication No. 2013/0209489, anti-GDF8 antibodies (e.g., 10B3H8L5 and 10B3H8L5-Fc deactivated) as shown in US Pat. Publication No. 2013/0142788, anti-GDF8 antibody stamulumab/MYO-29 as shown in, for example, US Pat. U.S. Patent No. 7,731,961 Anti-GDF8 antibodies (e.g. RK35) as presented in U.S. Patent No. 8,496,934 Anti-GDF8 antibodies (e.g., OGD1.0.0) as presented in US Pat. patent No. 1773041 B1, antit ate to GDF8 (e.g., 41C1E4) as presented in US Pat. all of the aforementioned patents and patent application publications are incorporated herein by reference in their entirety.

Fully human anti-GDF8 antibodies disclosed herein may contain one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from public antibody sequence databases. The present invention provides antibodies and antigen-binding fragments thereof which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are back-mutated to the corresponding residue(s). ) germline or up to a conservative amino acid substitution (whether or not naturally occurring) of the corresponding germline residue(s) (such sequence changes are referred to herein as germline backmutations). One skilled in the art, starting from the heavy and light chain variable region sequences disclosed herein, can easily generate numerous antibodies and antigen binding fragments that contain one or more single germline backmutations, or combinations thereof. According to

- 5 040534 certain embodiments, all framework residues and/or CDR residues within the VH and/or V _L domains are backmutated to the germline sequence. In other embodiments, only certain residues are backmutated to a germline sequence, such as only mutated residues occurring within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only mutated residues occurring within CDR1, CDR2, or CDR3. Moreover, the antibodies of the present invention may contain any combination of two or more germline backmutations within the framework and/or CDR regions, ie. wherein certain individual residues are backmutated to the germline sequence while retaining certain other residues that differ from the germline sequence. Once antibodies and antigen-binding fragments have been generated that contain one or more germline backmutations, they can be readily assayed for one or more of the desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (depending on specific case), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained by this general method are provided by the present invention.

The present invention also provides antibodies to GDF8 containing variants of any of the amino acid sequences of HCVR, LCVR, and/or CDR disclosed herein, characterized by one or more conservative substitutions. For example, the present invention provides anti-GDF8 antibodies comprising the amino acid sequences of HCVR, LCVR, and/or CDR, e.g., 10 or less, 8 or less, 6 or less, 4 or less, etc. conservative amino acid substitutions with respect to any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein. In one embodiment, the antibody comprises an HCVR comprising an amino acid sequence selected from SEQ ID NOs: 360 and 376 with 8 or fewer conservative amino acid substitutions. In another embodiment, the antibody comprises an HCVR comprising an amino acid sequence selected from SEQ ID NOs: 360 and 376 with 6 or fewer conservative amino acid substitutions. In another embodiment, the antibody comprises an HCVR comprising an amino acid sequence selected from SEQ ID NOs: 360 and 376 with 4 or fewer conservative amino acid substitutions. In another embodiment, the antibody comprises an HCVR comprising an amino acid sequence selected from SEQ ID NOs: 360 and 376 with 2 or fewer conservative amino acid substitutions. In one embodiment, the antibody comprises an LCVR comprising an amino acid sequence selected from SEQ ID NOs: 368 and 384 with 8 or fewer conservative amino acid substitutions. In another embodiment, the antibody comprises an LCVR comprising an amino acid sequence selected from SEQ ID NOs: 368 and 384 with 6 or fewer conservative amino acid substitutions. In another embodiment, the antibody comprises an LCVR comprising an amino acid sequence selected from SEQ ID NOs: 368 and 384 with 4 or fewer conservative amino acid substitutions. In another embodiment, the antibody comprises an LCVR comprising an amino acid sequence selected from SEQ ID NOs: 368 and 384 with 2 or fewer conservative amino acid substitutions.

In certain embodiments, an antibody or antibody fragment of the present invention can be conjugated to a therapeutic moiety (immunoconjugate), such as a cytotoxin, a chemotherapeutic drug, and an immunosuppressant or radioactive isotope.

As used herein, the term isolated antibody means an antibody that has been identified and isolated and/or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, tissue, or cell, in which the antibody exists naturally or is naturally produced, is an isolated antibody for the purposes of the present invention. An isolated antibody also includes an antibody in situ within a recombinant cell, as well as an antibody that has been subjected to at least one purification or isolation step. In certain embodiments, the isolated antibody may be substantially free of other cellular material and/or chemicals.

As used herein, the term surface plasmon resonance refers to an optical phenomenon that allows real-time analysis of biospecific interactions by detecting changes in protein concentrations within a biosensor array, for example, using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

As used herein, the term K _D is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

- 6 040534

The term epitope includes any determinant, preferably a polypeptide determinant, capable of specific binding to an immunoglobulin or T cell receptor. In certain embodiments, epitope determinants include reactive surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural characteristics and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. In certain embodiments, an antibody can be said to specifically bind an antigen if it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. For example, an antibody can be said to specifically bind an antigen if the K _D is less than or equal to 10 ^-8 M, less than or equal to 10 ^-9 M, or less than or equal to 10 ^-10 M.

A protein or polypeptide is substantially pure, substantially homogeneous, or substantially purified if at least about 60-75% of the sample exhibits one species of the polypeptide. The polypeptide or protein may be monomeric or multimeric. Essentially, a pure polypeptide or protein will typically contain approximately 50, 60, 70, 80, or 90% wt./wt. the protein sample will typically be about 95% and preferably over 99% pure. The purity and homogeneity of a protein can be determined by a number of methods well known in the art, such as polyacrylamide gel electrophoresis of a protein sample followed by visualization of a single polypeptide band by staining the gel with a dye well known in the art. For certain purposes, higher resolution can be achieved using HPLC or other means well known in the art for purification.

As used herein, the term polypeptide analog or variant refers to a polypeptide that consists of a segment of at least 25 amino acids that has significant identity with respect to a portion of the amino acid sequence and that has at least one of the following properties: (1) specific binding to GDF8 under suitable binding conditions or (2) the ability to block the biological activity of GDF8. Typically, polypeptide analogs or variants contain a conservative amino acid substitution (or insertion or deletion) relative to the naturally occurring sequence. Analogues are typically at least 20 amino acids long, at least 50, 60, 70, 80, 90, 100, 150, or 200 amino acids long or longer, and can often be the same length as a full-length naturally occurring polypeptide.

Preferred amino acid substitutions are those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) change binding affinity for nascent protein complexes, (4) change binding affinities, and (4) impart or modify other physico- chemical or functional properties of such analogues. Analogues may include various mutations in the sequence other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) can be made in a naturally occurring sequence (preferably in a portion of the polypeptide outside the domain(s) forming(their) intermolecular contacts). A conservative amino acid substitution should not significantly alter the structural characteristics of the original sequence (eg, the substituting amino acid should not tend to break the helix that occurs in the original sequence or disrupt other types of secondary structure that characterizes the original sequence). Examples of secondary and tertiary polypeptide structures accepted in the art are described in Proteins, Structures and Molecular Principles (Creighton 1984 W.H. Freeman and Company, New York; Introduction to Protein Structure (Branden & Tooze, eds., 1991, Garland Publishing, NY); and Thornton et al 1991 Nature 354:105, which are incorporated herein by reference.

Non-peptide analogues are widely used in the pharmaceutical industry as drugs with properties similar to those of the matrix peptide. These types of non-peptide compound are referred to as peptide mimetics or peptidomimetics (see, for example, Fauchere (1986) J. Adv. Drug Res. 15:29; and Evans et al. (1987) J. Med. Chem. 30:1229, which are included herein by reference.Systematic substitution of one or more amino acids of the consensus sequence with a D-amino acid of the same type (for example, D-lysine instead of Llysine) can also be used to create more stable peptides.In addition, peptides with limited conformational freedom containing a consensus sequence, or a substantially identical variant of the consensus sequence, can be obtained using methods known in the present technical field (Rizo et al. (1992) Ann. Rev. Biochem. 61:387, incorporated herein by reference), for example , by adding internal cysteine residues capable of forming intramolecular disulfide bridges that cyclize the peptide.

When applied to polypeptides, the term significant identity or substantially identical

- 7 040534 true means that two peptide sequences when optimally aligned, for example, using the GAP or BESTFIT programs using default deletion opening penalties, have at least about 80% sequence identity, at least about 90%, at least at least about 95%, at least about 98%, or at least about 99% sequence identity. Preferably, positions of residues that are not identical are distinguished by conservative amino acid substitutions. A conservative amino acid substitution is one in which an amino acid residue is replaced by another amino acid residue containing a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). In general, a conservative amino acid substitution will not significantly alter the functional properties of a protein. In the case where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity can be adjusted upward to allow for the conservative nature of the substitution. Methods for implementing said regulation are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference. Examples of groups of amino acids that contain side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) main side chains: lysine, arginine and histidine; and 6) the sulfur-containing side chains are cysteine and methionine. Preferred groups of conservative amino acid substitutions are valineleucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparaginglutamine. Alternatively, a conservative substitution is any change characterized by a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-45, incorporated herein by reference. Moderately conservative substitution is any change characterized by a non-negative value in the PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software determines similar sequence matches using similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG contains programs such as Gap and Bestfit that can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species, or between a wild-type protein and its mutein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA using the default parameters, or the recommended parameters, of the program in GCG version 6.1. FASTA (eg, FASTA2 and FASTA3) provides alignments and percent sequence identity of best match regions between query and search sequences (Pearson (2000), formerly). Another preferred algorithm for comparing a sequence according to the present invention with a database containing a large number of sequences from different organisms is the BLAST computer program, especially blastp or tblastn, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389 402, each of which is incorporated herein by reference.

The length of polypeptide sequences compared for homology will typically be at least about 16 amino acid residues, at least about 20 residues, at least about 24 residues, at least about 28 residues, or at least about 35 residues. When searching a database containing sequences from a large number of different organisms, it is preferable to compare amino acid sequences.

The term effective amount is the concentration or amount of an antibody or antigen-binding fragment of an antibody that results in the achievement of a specific stated goal. An effective amount of an anti-GDF8 antibody or antigen-binding antibody fragment thereof can be empirically determined. Moreover, a therapeutically effective amount is the concentration or amount of an anti-GDF8 antibody, or antigen-binding fragment thereof, that is effective to achieve the claimed therapeutic effect. This amount can also be determined empirically.

As used herein, the term healthy subject refers to a subject that is not characterized by the presence of a disease or disorder that significantly limits the subject's ability to participate in resistance training. In embodiments, the disease or disorder is a disease or disorder in which a physician has recommended exercise restriction for the subject or in which exercise is contraindicated, such as uncontrolled diabetes mellitus, recent myocardial infarction, unstable heart conditions, acute heart failure, severe myocarditis, uncontrolled hypertension, valvular heart disease requiring surgery, and severe aortic stenosis.

As used herein, the term resistance training refers to a set of physical exercises that causes a muscle to contract against external resistance. External resistance includes, for example, a weight, an expander, a kettlebell, or the subject's own body weight.

As used herein, the term exercise regimen refers to an exercise plan. In embodiments, the exercise regimen includes exercise such as resistance training, resistance training, aerobic training, walking, interval training, yoga, and combinations thereof.

aspects of disclosure.

The disclosure provides compositions, kits, and methods for using GDF8 inhibitors to increase lean body mass. In embodiments, the GDF8 inhibitor is an antibody, or an antigen-binding fragment thereof, that specifically binds GDF8.

Obtaining human antibodies.

Methods for making monoclonal antibodies, including fully human monoclonal antibodies, are known in the art. Any such known methods can be used in the context of the present invention to obtain human antibodies that specifically bind to GDF8.

Using VELOCIMMUNE™ technology or any other known method for producing monoclonal antibodies, high affinity chimeric anti-GDF8 antibodies characterized by a human variable region and a mouse constant region are first isolated. As in the experimental section below, the characteristics of the antibodies are determined and the antibodies are selected for the desired characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with the desired human constant region to generate a fully human antibody of the present invention, eg wild-type or modified IgG1 or IgG4. While the constant region chosen may vary according to the particular application, the characteristics of high affinity antigen binding and target specificity are inherent in the variable region.

In general, the antibodies of the present invention have very high affinities, typically having a KD of about 10 ^-12 to about 10 ^-9 M, as measured by antigen binding, either immobilized on a solid phase or in a liquid phase. Mouse constant regions are replaced with the desired human constant regions to generate fully human antibodies of the present invention, e.g., wild-type IgG1 (SEQ ID NO: 335) or wild-type IgG4 (SEQ ID NO: 336), or modified IgG1 or IgG4 (e.g., SEQ ID NO: 337). While the constant region chosen may vary according to the particular application, the characteristics of high affinity antigen binding and target specificity are inherent in the variable region.

Antibodies or antigen-binding fragments specific for GDF8.

The present invention provides anti-GDF8 antibodies and antigen-binding antibody fragments that bind specific epitopes of human GDF8 (SEQ ID NO: 340) and are capable of blocking the biological activity of GDF8. In one embodiment, the antibody or antigen-binding fragment thereof binds within an epitope containing amino acid residues 1-109; 1-54; 1-44; 1-34; 1-24 and 1-14. In another embodiment, the antibody or antigen-binding fragment thereof binds within an epitope containing amino acid residues 65-72; 35-109; 45-109; 55-109; 65-109; 75-109; 85-109; 92-109 or 95-109. In another embodiment, the antibody or antigen-binding fragment thereof binds within an epitope containing amino acid residue 48-72; 48-69; 48-65; 52-72; 52-65 or 56-65. In specific embodiments, the antibody, or antigen-binding fragment thereof, may bind within 2 or more epitopes.

In addition, the present invention provides antibodies and antigen-binding fragments thereof that bind wild-type mature GDF8 (SEQ ID NO: 340) but do not bind isolated peptides characterized by less than the full amino acid sequence of SEQ ID NO: 340. For example, according to the present invention Anti-GDF8 antibodies are provided that bind wild-type mature GDF8 (SEQ ID NO: 340) but do not bind isolated peptides consisting of 10-40 contiguous amino acids of SEQ ID NO: 340. The present invention also provides antibodies to GDF8 that do not bind any linear epitopes within mature wild-type GDF8. In certain embodiments, anti-GDF8 antibodies bind mature human wild-type GDF8 comprising SEQ ID NO: 340, but do not bind

- 9 040534 one or more isolated GDF8 peptides characterized by an amino acid sequence selected from the group consisting of amino acids 1-14, 1-18, 17-42, 48-65, 48-69, 48-72, 52-65, 5272 , 56-65, 56-72, 65-72, 73-90, 75-105, and 91-105 in SEQ ID NO: 340. In certain embodiments, anti-GDF8 antibodies do not bind any of the aforementioned GDF8 peptides. Methods for determining whether a given antibody is capable of binding a particular GDF8 peptide are known to those skilled in the art.

The present invention also provides isolated human antibodies, or antigen-binding fragments thereof, that specifically bind to mature human wild-type GDF8 (e.g., a protein or polypeptide comprising SEQ ID NO: 340), but do not bind to a GDF8 chimeric construct in which certain amino acids of GDF8 replaced by the corresponding amino acid(s) sequence(s) from a non-identical but related protein, such as TGFP-1. In one example, the chimeric construct is a GDF8/TGFP-1 chimera in which amino acids 48-72 of mature GDF8 are replaced with the corresponding TGFP-1 amino acid sequence (eg, amino acids 49-76 of TGFP-1). An example of one such chimera is provided by SEQ ID NO: 352. Thus, in certain embodiments, the antibodies of the present invention specifically bind to mature human wild-type GDF8 (SEQ III NO: 340), but do not bind to the GDF8/TGFP-1 chimeric construct according to SEQ ID NO: 352 indicating that the epitope to which such antibodies bind includes or encompasses amino acids located within residues 48-72 of SEQ ID NO: 340. Blocking bioassays can also be used to indirectly ascertain whether that the antibody binds mature human wild-type GDF8 (SEQ ID NO: 340) and does not bind the GDF8/TGFP-1 chimeric construct, e.g., the construct according to SEQ ID NO: 352. For example, an antibody that blocks the bioactivity of mature wild-type GDF8 human, but does not block the bioactivity of chimeric GDF8/TGFP1, binds to the portion of GDF8 that is substituted with the corresponding TGFP-1 sequence in the chimeric construct.

Similarly, the present invention also provides isolated human antibodies, or antigen-binding fragments thereof, that block mature wild-type GDF8 mediated activity in a bioassay but do not block the activity of a chimeric GDF8 construct (e.g., a GDF8/TGFP-1 chimera in which amino acids 48-72 of mature GDF8 substituted with the corresponding TGFP-1 amino acid sequence (eg amino acids 49-76 of TGFP-1).

The present invention provides antibodies to GDF8 that bind to the same epitope as any of the specific illustrative antibodies described herein. Similarly, the present invention also provides antibodies to GDF8 that cross-compete for binding to GDF8 or a fragment of GDF8 with any of the specific illustrative antibodies described herein.

Whether an antibody binds to the same epitope as a reference anti-GDF8 antibody or competes for binding with a reference anti-GDF8 antibody can be readily determined using routine methods known in the art. For example, to determine if an antibody of interest binds to the same epitope as a reference anti-GDF8 antibody of the present invention, the reference antibody is allowed to bind to a GDF8 protein or peptide under saturating conditions. Next, the ability of the test antibody to bind to the GDF8 molecule is evaluated. If the test antibody is able to bind to GDF8 after binding saturation with the anti-GDF8 reference antibody, it can be concluded that the test antibody binds to an epitope different from the reference anti-GDF8 antibody. On the other hand, if the test antibody is unable to bind to the GDF8 molecule after binding saturation with the anti-GDF8 reference antibody, the test antibody can bind to the same epitope as the epitope bound by the anti-GDF8 reference antibody of the present invention. Additional routine experiments (e.g., peptide mutation and binding assays) can then be performed to confirm that the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference antibody, or that steric blocking (or other phenomenon) is responsible. no observable binding. Experiments of this kind can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to certain embodiments of the present invention, two antibodies bind to the same (or overlapping) epitope if, for example, a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits binding of the other by at least 50 %, but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, for example, Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, two antibodies are considered to bind to the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other. Two antibodies are considered to have overlapping epitopes if only a subset of amino acid mutations that reduce or eliminate the binding of one antibody reduces or eliminates the binding of the other.

- 10 040534

To determine if an antibody competes for binding with a reference anti-GDF8 antibody, the above binding procedure is performed in two orientations: the first orientation allows the reference antibody to bind to the GDF8 molecule under saturating conditions, followed by evaluation of the binding of the test antibody to the GDF8 molecule. In the second orientation, the test antibody is allowed to bind to the GDF8 molecule under saturating conditions, followed by evaluation of the binding of the reference antibody to the GDF8 molecule. If in both orientations only the first (saturating) antibody is able to bind to the GDF8 molecule, it is concluded that the test antibody and the reference antibody compete for binding to GDF8. One skilled in the art will appreciate that an antibody that competes for binding with a reference antibody may not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding to an overlapping or contiguous epitope.

The present invention provides human or humanized antibodies and antigen-binding fragments of human or humanized antibodies that specifically bind human growth and differentiation factor 8 (GDF8). These antibodies are characterized by binding to GDF8 with high affinity and the ability to neutralize the activity of GDF8. Antibodies may be full-length (eg, an IgG1 or IgG4 antibody) or may contain only an antigen-binding portion (eg, a Fab fragment, F(ab')2, or scFv) and may be modified to affect functionality, such as to eliminate residual effector functions ( Reddy et al (2000) J Immunol 164:1925-1933).

In one embodiment, an antibody of the present invention comprises a heavy chain variable region (HCVR) amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 34, SEQ ID NO: 50, SEQ ID NO: 66, SEQ ID NO: 82, SEQ ID NO: 98, SEQ ID NO: 114, SEQ ID NO: 130, SEQ ID NO: 146, SEQ ID NO: 162, SEQ ID NO: 178, SEQ ID NO : 194, SEQ ID NO: 210, SEQ ID NO: 226, SEQ ID NO: 242, SEQ ID NO: 258, SEQ ID NO: 274, SEQ ID NO: 290, SEQ ID NO: 306, SEQ ID NO: 360 and SEQ ID NO: 376 or a sequence substantially identical to them.

In one embodiment, an antibody of the present invention comprises a light chain variable region (LCVR) amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 26, SEQ ID NO: 42, SEQ ID NO: 58, SEQ ID NO: 74, SEQ ID NO: 90, SEQ ID NO: 106, SEQ ID NO: 122, SEQ ID NO: 138, SEQ ID NO: 154, SEQ ID NO: 170, SEQ ID NO: 186, SEQ ID NO : 202, SEQ ID NO: 218, SEQ ID NO: 234, SEQ ID NO: 250, SEQ ID NO: 266, SEQ ID NO: 282, SEQ ID NO: 298, SEQ ID NO: 314, SEQ ID NO: 322 , SEQ ID NO: 368 and SEQ ID NO: 384 or substantially identical to them.

In one embodiment, an antibody of the present invention comprises an HCVR amino acid sequence and an LCVR amino acid sequence, wherein the sequences of the HCVR/LCVR pair are selected from the group consisting of SEQ ID NO: 2/10, SEQ ID NO: 18/26, SEQ ID NO: 34 /42, SEQ ID NO: 50/58, SEQ ID NO: 66/74, SEQ ID NO: 82/90, SEQ ID NO: 98/106, SEQ ID NO: 114/122, SEQ ID NO: 130/138 , SEQ ID NO: 146/154, SEQ ID NO: 162/170, SEQ ID NO: 178/186, SEQ ID NO: 194/202, SEQ ID NO: 210/218, SEQ ID NO: 226/234, SEQ ID NO: 242/250, SEQ ID NO: 258/266, SEQ ID NO: 274/282, SEQ ID NO: 290/298, SEQ ID NO: 306/314, SEQ ID NO: 114/322, SEQ DNO: 360/368 and SEQ ID NO: 376/384.

The present invention also provides a human or humanized antibody or antigen-binding antibody fragment comprising the amino acid sequence of a heavy chain complementarity determining region 3 (HCDR3) and the amino acid sequence of a light chain CDR3 (LCDR3), wherein HCDR3 is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 40, SEQ ID NO: 56, SEQ ID NO: 72, SEQ ID NO: 88, SEQ ID NO: 104, SEQ ID NO: 120, SEQ ID NO: 136, SEQ ID NO: 152, SEQ ID NO: 168, SEQ ID NO: 184, SEQ ID NO: 200, SEQ ID NO: 216, SEQ ID NO: 232, SEQ ID NO: 248, SEQ ID NO: 264, SEQ ID NO: 280, SEQ ID NO: 296, SEQ ID NO: 312, SEQ ID NO: 366, and SEQ ID NO: 382, or a sequence substantially identical to them, and the LCDR3 amino acid sequence is selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 32, SEQ ID NO: 48, SEQ ID NO: 64, SEQ ID NO: 80, SEQ ID NO: 96, SEQ ID NO: 112, SEQ ID NO: 128, SEQ ID NO: 144, SEQ ID NO: 160, SEQ ID NO: 176, SEQ ID N O: 192, SEQ ID NO: 208, SEQ ID NO: 224, SEQ ID NO: 240, SEQ ID NO: 256, SEQ ID NO: 272, SEQ ID NO: 288, SEQ ID NO: 304, SEQ ID NO: 320, SEQ ID NO: 328, SEQ ID NO: 374 and SEQ ID NO: 390, or essentially. their identical sequence. In another embodiment, the antibody or fragment thereof comprises an HCDR3/LCDR3 amino acid sequence pair selected from the group consisting of SEQ ID NO: 8/16, SEQ ID NO: 24/32, SEQ ID NO: 40/48, SEQ ID NO: 56/64, SEQ ID NO: 72/80, SEQ ID NO: 88/96, SEQ ID NO: 104/112, SEQ ID NO: 120/128, SEQ ID NO: 136/144, SEQ ID NO: 152/ 160, SEQ ID NO: 168/176, SEQ ID NO: 184/192, SEQ ID NO: 200/208, SEQ ID NO: 216/224, SEQ ID NO: 232/240, SEQ ID NO: 248/256, SEQ ID NO: 264/272, SEQ ID NO: 280/288, SEQ ID NO: 296/304, SEQ ID NO: 312/320, SEQ ID NO: 120/328, SEQ ID NO: 366/374 and SEQ ID NO: 382/390.

- 11 040534

In a related embodiment, the antibody or fragment thereof further comprises the amino acid sequences of a heavy chain CDR1 (HCDR1) and a heavy chain CDR2 (HCDR2), and the amino acid sequences of a light chain CDR1 (LCDR1) and a light chain CDR2 (LCDR2), wherein the amino acid sequence of HCDR1 is selected from the group, consisting of SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 36, SEQ ID NO: 52, SEQ ID NO: 68, SEQ ID NO: 84, SEQ ID NO: 100, SEQ ID NO: 116, SEQ ID NO: 132, SEQ ID NO: 148, SEQ ID NO: 164, SEQ ID NO: 180, SEQ ID NO: 196, SEQ ID NO: 212, SEQ ID NO: 228, SEQ ID NO: 244, SEQ ID NO: 260, SEQ ID NO: 276, SEQ ID NO: 292, SEQ ID NO: 308, SEQ ID NO: 362 and SEQ ID NO: 378 or substantially identical to them; the amino acid sequence of HCDR2 is selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 38, SEQ ID NO: 54, SEQ ID NO: 70, SEQ ID NO: 86, SEQ ID NO: 102 , SEQ ID NO: 118, SEQ ID NO: 134, SEQ ID NO: 150, SEQ ID NO: 166, SEQ ID NO: 182, SEQ ID NO: 198, SEQ ID NO: 214, SEQ ID NO: 230, SEQ ID NO: 246, SEQ ID NO: 262, SEQ ID NO: 278, SEQ ID NO: 294, SEQ ID NO: 310, SEQ ID NO: 364 and SEQ ID NO: 380, or a sequence substantially identical to them; the amino acid sequence of LCDR1 is selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 108 , SEQ ID NO: 124, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 172, SEQ ID NO: 188, SEQ ID NO: 204, SEQ ID NO: 220, SEQ ID NO: 236, SEQ ID NO: 252, SEQ ID NO: 268, SEQ ID NO: 284, SEQ ID NO: 300, SEQ ID NO: 316, SEQ ID NO: 324, SEQ ID NO: 370 and SEQ ID NO: 386 or, essentially , sequence identical to them; and the LCDR2 amino acid sequence is selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 30, SEQ ID NO: 46, SEQ ID NO: 62, SEQ ID NO: 78, SEQ ID NO: 94, SEQ ID NO: 110, SEQ ID NO: 126, SEQ

ID NO: 142, SEQ ID NO: 158, SEQ ID NO: 174, SEQ ID NO: 190, SEQ ID NO: 206, SEQ ID NO: 222, SEQ

ID NO: 238, SEQ ID NO: 254, SEQ ID NO: 270, SEQ ID NO: 286, SEQ ID NO: 302, SEQ ID NO: 318, SEQ

ID NO: 326, SEQ ID NO: 372 and SEQ ID NO: 388 or substantially identical to them. According to another embodiment, HCDR1, HCDR2 and HCDR3 are selected from the group consisting of SEQ ID NO: 36/38/40, SEQ ID NO: 116/118/120, SEQ ID NO: 228/230/232, SEQ ID NO: 362 /364/366 and SEQ ID NO: 378/380/382; and LCDR1, LCDR2 and LCDR3 are selected from the group consisting of SEQ ID NO: 44/46/48, SEQ ID NO: 124/126/128, SEQ ID NO: 236/238/240, SEQ ID NO: 370/372/ 374 and SEQ ID NO: 386/388/390. In another embodiment, the heavy and light chain CDRs are selected from the group consisting of SEQ ID NO: 36/38/40/44/46/48 (e.g., 21-E5), SEQ ID NO: 116/118/120/124/ 126/128 (e.g. 8D12), SEQ ID NO: 228/230/232/236/238/240 (e.g. 1A2), SEQ ID NO: 362/364/366/370/372/374 (e.g. H4H1657N2) and SEQ ID NO: 378/380/382/386/388/390 (eg H4H1669P). In a related embodiment, the present invention provides an antibody or antigen-binding antibody fragment that specifically binds GDF8, wherein the antibody or fragment comprises heavy and light chain CDR domains contained within heavy and light chain variable domain sequences selected from the group consisting of SEQ ID NO: 2/10, SEQ ID NO: 18/26, SEQ ID NO: 34/42, SEQ ID NO: 50/58, SEQ ID NO: 66/74, SEQ ID NO: 82/90, SEQ ID NO: 98/106, SEQ ID NO: 114/122, SEQ ID NO: 130/138, SEQ ID NO: 146/154, SEQ ID NO: 162/170, SEQ ID NO: 178/186, SEQ ID NO: 194/ 202, SEQ ID NO: 210/218, SEQ ID NO: 226/234, SEQ ID NO: 242/250, SEQ ID NO: 258/266, SEQ ID NO: 274/282, SEQ ID NO: 290/298, SEQ ID NO: 306/314, SEQ ID NO: 114/322, SEQ ID NO: 360/368 and SEQ ID NO: 376/384.

Methods and techniques for identifying CDRs within the HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein. Illustrative conventional approaches that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the position of structural loop regions, and the AbM definition is a combination between the Kabat and Chothia approaches. See, for example, Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al, J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. sci. USA 86:9268-9272 (1989). Public databases are also available to identify CDR sequences within an antibody.

The present invention also provides nucleic acid molecules encoding antibodies or antigen-binding fragments of the present invention. Recombinant expression vectors carrying antibody-encoding nucleic acids of the present invention and host cells into which such vectors have been introduced are also contemplated by the present invention, as well as methods for producing antibodies of the present invention by culturing host cells of the present invention.

In one embodiment, an antibody of the present invention comprises a CDR contained within an HCVR, or an HCVR encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 33, SEQ ID NO : 49, SEQ ID NO: 65, SEQ ID NO: 81, SEQ ID NO: 97, SEQ ID NO: 113, SEQ ID NO: 129, SEQ ID NO: 145, SEQ ID

- 12 040534

NO: 161, SEQ ID NO: 177, SEQ ID NO: 193, SEQ ID NO: 209, SEQ ID NO: 225, SEQ ID NO: 241, SEQ ID

NO: 257, SEQ ID NO: 273, SEQ ID NO: 289, SEQ ID NO: 305, SEQ ID NO: 359, and SEQ ID NO: 375, or a substantially similar sequence having at least 95% homology to to them.

In one embodiment, an antibody of the present invention comprises a CDR contained within an LCVR, or an LCVR encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 25, SEQ ID NO: 41, SEQ ID NO : 57, SEQ ID NO: 73, SEQ ID NO: 89, SEQ ID NO: 105, SEQ ID NO: 121, SEQ ID NO: 137, SEQ ID NO: 153, SEQ ID NO: 169, SEQ ID NO: 185 , SEQ ID NO: 201, SEQ ID NO: 217, SEQ ID NO: 233, SEQ ID NO: 249, SEQ ID NO: 265, SEQ ID NO: 281, SEQ ID NO: 297, SEQ ID NO: 313, SEQ ID NO: 321, SEQ ID NO: 367 and SEQ ID NO: 383 or a substantially similar sequence with at least 95% homology thereto.

The present invention also provides a fully human or humanized antibody or antibody fragment that binds GDF8 with an affinity (expressed as a dissociation constant, K _D ) of about 1 nM or less as measured by a surface plasmon resonance assay (e.g., BIACORE™). In certain embodiments, an antibody of the present invention exhibits a K _D of about 700 pM or less; about 500 pM or less; about 320 pM or less; about 160 pM or less; about 100 pM or less; about 50 pM or less; about 10 pM or less; or about 5 pM or less.

In one embodiment, the present invention provides a fully human or humanized monoclonal antibody (mAb) that specifically binds and inhibits human GDF8 and exhibits an IC ₅₀ of less than or equal to about 10 nM; about 5 nM or less; about 3 nM or less; about 2 nM or less; about 1 nM or less; about 500 pM or less, or about 200 pM or less, as measured by the GDF8 inducible luciferase assay. As shown in the experimental section below, some of the anti-GDF8 antibodies of the present invention block the activity of closely related proteins such as GDF11 with a much higher IC ₅₀ than GDF8 in the luciferase bioassay. In one embodiment, the present invention provides an antibody or antigen-binding antibody fragment that exhibits at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least at least about 500 times, at least about 1000 times, or at least about 1500 times the IC ₅₀ value for blocking GDF11 activity against GDF8.

The present invention provides anti-GDF8 antibodies having a modified glycosylation profile. In some applications, a modification can be used to remove unwanted glycosylation sites, or an antibody that lacks a fucose moiety present on the oligosaccharide chain, for example, to increase the function of antibody-dependent cell-mediated cytotoxicity (ADCC) (see Shield et al. (2002) JBC 277 :26733). In other applications, modification of galactosylation can be performed to modify complement dependent cytotoxicity (CDC).

The present invention provides antibodies to GDF8 that bind specific epitopes of GDF8 and are capable of blocking the biological activity of GDF8. In a first embodiment, an antibody of the present invention binds an epitope of the mature GDF8 protein (SEQ ID NO: 340) within amino acids from about 1 to about 109; from about 1 to about 54; from about 1 to about 44; from about 1 to about 34; from about 1 to about 24; and from about 1 to about 14. In a second embodiment, an antibody of the present invention binds one or more epitopes of a mature GDF8 protein (SEQ ID NO: 340) ranging from amino acids from about 35 to about 109; from about 45 to about 109; from about 55 to about 109; from about 65 to about 109; from about 75 to about 109; from about 85 to about 109; about 92 to about 109, or about 95 to about 109. In a third embodiment, the antibody or antigen-binding antibody fragment binds within an epitope of the mature human GDF8 protein from about amino acid residue 48 to about 72; from about 48 to about 69; about 48 to about 65; about 52 to about 72; about 52 to about 65, or about 56 to about 65.

In certain embodiments, anti-GDF8 antibodies bind to human GDF8 but not to GDF8 from other species. Alternatively, anti-GDF8 antibodies of the present invention, in certain embodiments, bind to human GDF8 and to GDF8 from one or more non-human species. For example, the anti-GDF8 antibodies of the present invention may bind to human GDF8 and may or may not bind, as the case may be, to one or more murine, rat, guinea pig, hamster, gerbil, porcine, cat, dog, rabbit, goats, sheep, cows, horses, camels, cynomolgus monkeys, monkeys, rhesus monkeys or chimpanzees.

The present invention provides a human or humanized anti-GDF8 monoclonal antibody conjugated to a therapeutic moiety (immunoconjugate), such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant, or a radioactive isotope. Cytotoxic agents include any agent that is detrimental to cells. Examples of suitable cytotoxic agents and chemotherapeutic agents for the formation of immunoconjugates are known in the art (see, for example, international patent publication WO 05/103081, which is specifically incorporated herein by reference).

The antibodies of the present invention may be monospecific, bispecific or multispecific.

Multispecific antibodies may be specific for different epitopes of the same target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. Anti-GDF8 antibodies of the present invention can be coupled or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or antibody fragment can be operably linked (e.g., by chemical linkage, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to form a bispecific or multispecific antibody with a second binding specificity. For example, the present invention provides for bispecific antibodies wherein one immunoglobulin arm is specific for human GDF8 or a fragment thereof and the other immunoglobulin arm is specific for a second therapeutic target or conjugated to a therapeutic moiety.

An exemplary bispecific antibody format that can be used in the context of the present invention includes the use of an immunoglobulin (Ig) first CH3 domain and a second Ig C _H 3 domain, wherein the first and second Ig C _H 3 domains differ from each other by at least one amino acid, and moreover, at least a difference in one amino acid reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody in which there is no difference in amino acids. In one embodiment, the first Ig CH3 domain binds protein A, and the second Ig CH3 domain contains a mutation that reduces or abolishes protein A binding, such as an H95R modification (according to IMGT exon numbering; H435R according to EU numbering). The second CH3 may further comprise the modification Y96F (as per IMGT; Y436F as per EU). Additional modifications that can be found within the second CH3 include D16E, L18M, N44S, K52N, V57M and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M and V422I according to EU) in the case of IgG1 antibodies; N44S, K52N and V82I (IMGT; N384S, K392N and V422I according to EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (as per IMGT; Q355R, N384s, K392N, V397M, R409K, E419Q and V422I as per EU) for IgG4 antibodies. Variations on the bispecific antibody format described above are contemplated within the scope of the present invention.

Anti-GDF8 antibodies and antibody fragments of the present invention include proteins having amino acid sequences that differ from the amino acid sequences of the disclosed antibodies, but which retain the ability to bind human GDF8. Such variant antibodies and antibody fragments contain one or more additions, deletions, or substitutions of amino acids compared to the original sequence, but exhibit biological activity that is substantially equivalent to that of the described antibodies. Similarly, the GDF8 antibody-encoding DNA sequences of the present invention include sequences that contain one or more nucleotide additions, deletions, or substitutions compared to the disclosed sequence, but which encode an anti-GDF8 antibody or antibody fragment that is substantially bioequivalent to the antibody. to GDF8 or an antibody fragment of the present invention. Examples of such variant amino acid and DNA sequences are discussed above.

Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption does not show a significant difference when administered at the same molar dose under similar experimental conditions, either single dose or multiple doses. . Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in their extent of absorption but not in their rate of absorption and may still be considered bioequivalent because such differences in absorption rates are intentional and reflective.

- 14 040534 on the label, are not essential to achieve effective concentrations of the drug in the body, for example, with long-term use and are considered medically insignificant for a particular study medicinal product.

In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity, and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if the patient can switch one or more times between the comparator drug and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity or reduced efficacy, compared to continuous therapy without such switching. .

In one embodiment, two antigen-binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.

Bioequivalence can be demonstrated using in vivo and in vitro methods. Measures of bioequivalence include, for example, (a) an in vivo study in humans or other mammals, in which the concentration of an antibody or its metabolites is measured in blood, plasma, serum, or other body fluid as a function of time; (b) an in vitro study that has been correlated with in vivo human bioavailability data and is reasonably predictive of in vivo human bioavailability data; (c) an in vivo study in humans or other mammals, in which the corresponding acute pharmacological effect of an antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical trial that establishes the safety, efficacy, or bioavailability or bioequivalence of an antibody.

Bioequivalent variants of antibodies to GDF8 according to the present invention can be constructed by, for example, making various substitutions of residues or sequences, or deletion of terminal or internal residues or sequences that are not necessary for biological activity. For example, cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other contexts, bioequivalent antibodies may include anti-GDF8 antibody variants containing amino acid changes that modify the glycosylation characteristics of the antibodies, such as mutations that abrogate or remove glycosylation.

Therapeutic administration and formulations.

The present invention provides therapeutic compositions comprising antibodies or antigen-binding fragments thereof according to the present invention. The administration of the therapeutic compositions of the present invention is carried out in conjunction with suitable carriers, excipients, and other agents that are included in the formulations to provide improved transport, delivery, tolerability, and the like.

Numerous acceptable formulations can be found in the pharmacological reference book known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. Said formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipid containing lipid (cationic or anionic) vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in- water and water-in-oil, carbovax emulsions (polyethylene glycols of various molecular weights), semi-solid gels and semi-solid mixtures containing carbovax. Any of the above mixtures may be acceptable in the treatments and therapies of the present invention provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and acceptable for the route of administration. See also Powell et al. Compendium of excipients for parenteral formulations PDA (1998) J. Pharm. sci. Technol. 52:238-311.

The dose may vary depending on the age and size of the subject to be administered, the target disease, conditions, route of administration, and the like. When an antibody of the present invention is used to treat various conditions and diseases associated with GDF8 in adults, it is preferred to administer the antibody of the present invention, typically at a single dose of about 0.01 to about 20 mg/kg body weight, from about 0.1 to about 10 mg/kg body weight, or about 0.1 to about 5 mg/kg body weight. Alternatively, the antibody, or antigen-binding fragment thereof, may be administered to a healthy subject in combination with an exercise regimen. In embodiments, the healthy subject experiences age-related lean muscle loss and/or postoperative muscle wasting. In embodiments, the effective amount for a subject is at least 400 mg, or about 36 mg/kg (assuming 70 kg is the average adult weight).

Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. In other parenteral and oral administration, the antibody may be administered at a dose corresponding to the dose presented above. If the condition is particularly severe, the dose may be increased according to the condition up to an amount that causes significant side effects, if any. In embodiments, the subject receives at least two or more subcutaneous doses. In embodiments, subjects receive multiple doses at intervals such as once a week, every two weeks, every three weeks, every four weeks, every five weeks, and once every six weeks.

Various delivery systems are known and can be used to administer the pharmaceutical composition of the present invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al. (1987) J Biol Chem 262:4429-4432). Routes of administration include, without limitation, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous membranes (eg, oral mucosa, rectal and intestinal mucosa, etc.) and may be administered with other biologically active agents. The administration may be systemic or local.

The pharmaceutical composition can also be delivered in a vesicle, in particular a liposome (see Langer (1990) Science 249:1527-1533).

The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen-shaped delivery device easily finds applications in the delivery of a pharmaceutical composition according to the present invention. Such a pen-style delivery device may be reusable or disposable. A reusable pen delivery device typically uses a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been introduced and the cartridge is empty, the empty cartridge can be easily removed and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. The disposable pen delivery device does not have a replaceable cartridge. Instead, the pen-shaped disposable delivery device comes pre-filled with a pharmaceutical composition contained in a reservoir within the device. Once the reservoir runs out of pharmaceutical composition, the entire device is discarded.

Various reusable pen and auto-injector delivery devices find use in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, without limitation, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen , HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), syringe - the BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™ and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to name but a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of the pharmaceutical composition of the present invention include, but are not limited to, the SOLOSTAR™ pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk) and KWIKPEN™ (Eli Lilly), SURECLICK™ autoinjector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.) and HUMIRA™ pen (Abbott Labs, Abbott Park IL), to name but a few.

In certain situations, the pharmaceutical composition can be delivered in a controlled release system, for example using a pump or polymeric materials. According to another embodiment, the controlled release system can be placed close to the target of the composition, thus ensuring that only a fraction of the systemic dose is needed.

Examples of a composition for oral administration include solid or liquid dosage forms, in particular tablets (including dragees and coated tablets), pills, granules, powder preparations, capsules (including soft capsules), syrup, emulsions, suspensions and etc. Such a composition is produced by conventional methods and contains a carrier, diluent or excipient commonly used in the field of pharmaceutical preparations. Examples of a carrier or tablet excipient are lactose, starch, sucrose, magnesium stearate and the like.

Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, and the like. These injectable preparations can be obtained using well-known methods. For example, injectable preparations can be made by dissolving, suspending, or emulsifying the antibody or its co

- 16 040534 whether as described above, in a sterile aqueous medium or in an oily medium normally used for injection. As the aqueous injection medium, there are, for example, physiological saline solution, isotonic solution containing glucose and other excipients, etc., which can be used in combination with a suitable solubilizing agent such as alcohol (for example, ethanol), polyhydric alcohol (eg propylene glycol, polyethylene glycol), non-ionic surfactant [eg polysorbate 80, HCO-50 adduct (polyoxyethylene (50 mol) hydrogenated castor oil)], etc. As the oil medium, for example, sesame oil, soybean oil, etc. are used, which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus obtained is preferably filled into a suitable ampoule.

Preferably, the oral or parenteral pharmaceutical compositions described above are formulated in single dose dosage forms suitable for adjusting the dosage of the active ingredients. Such single dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, and the like. The amount of the aforementioned antibody contained therein is typically about 5-500 mg per dosage form in a single dose; especially in the form of an injection, it is preferred that the aforementioned antibody be present in an amount of about 5-500 mg and about 10-400 mg for other dosage forms.

Therapeutic applications of antibodies.

The antibodies of the present invention are useful, inter alia, for increasing lean muscle mass in a subject. In some embodiments, a method for increasing lean muscle mass comprises providing an exercise regimen for the subject and administering a composition containing an effective amount of a GDF8 inhibitor. In embodiments, the subject is a subject who has not been prescribed exercise restriction by a physician and/or who does not have a condition or disorder for which exercise is contraindicated. In embodiments, the subject is characterized by a loss of lean muscle mass. In other embodiments, the subject is at least 50 years old, 55 years old, or 60 years old or older.

In embodiments, the antibodies of the present invention are useful, inter alia, for the treatment, prevention and/or amelioration of any disease or disorder associated with GDF8 activity. More specifically, the antibodies of the present invention are useful in the treatment of any condition or lesion that can be improved by increasing muscle strength/power and/or muscle mass and/or muscle function in an individual, or by favorably altering metabolism (metabolism of carbohydrates, lipids and proteins). ) by blocking GDF8 activity. Illustrative diseases, disorders, and conditions that may be treated with the anti-GDF8 antibodies of the present invention include, without limitation, the following: sarcopenia, cachexia (whether idiopathic or secondary to other conditions, such as cancer, chronic renal failure, or chronic obstructive pulmonary disease), muscle injury, muscle wasting, and muscle atrophy, such as muscle atrophy or muscle wasting, caused by or associated with lack of functional muscle loading, immobilization, bed rest, trauma, medical treatment, or surgery (eg, hip fracture , hip replacement, knee replacement, etc.) or the need for mechanical ventilation. Additional disorders that can be treated with the anti-GDF8 antibodies of the present invention include, but are not limited to, sIBM (sporadic inclusion myositis).

According to the present invention, therapeutic administration regimens are contemplated which involve administering an anti-GDF8 antibody of the present invention in combination with at least one additional therapeutically active ingredient. Non-limiting examples of such additional therapeutically active ingredients include other GDF8 antagonists (e.g., small molecule GDF8 inhibitors or other anti-GDF8 antibodies or GDF8-binding molecules), growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and cytotoxic/cytostatic agents. Additional(e) therapeutically active(e) component(s) can be entered before, simultaneously with or after the introduction of antibodies to GDF8 according to the present invention.

According to a related embodiment, the method comprises the combination of administering the antibody, or antigen-binding fragment thereof, to a subject who exercises on a regular basis, such as weight-bearing exercise. In embodiments, the method includes providing an exercise regimen, such as a regimen that stimulates an increase in lean muscle mass. In embodiments, the exercise regimen includes one or more of the following: resistance training, strength training, Pilates, aerobic exercise, weight training, and

- 17 040534 yoga. In a particular embodiment, the exercise regimen includes resistance training and/or resistance training. In embodiments, the exercise regimen is provided in writing and/or by way of illustration, on a computer-readable medium, video, or in an exercise room.

In embodiments, resistance training includes training any muscle in the body including, but not limited to, non-dominant palm, dominant palm, leg, arm, back, abdominal, quadriceps, calf, biceps, triceps, shoulder muscle, gluteal muscle and/or chest muscle. Resistance training may include the use of weights, elastic bands, exercise equipment, and/or the subject's own weight. Physical exercises may include bench press, leg press, arm curls, leg curls, wrist presses, abs, calf presses, biceps curls, triceps curls, elbows, side elbows, and/or walking on stairs.

In an embodiment, the exercise regimen provides for at least one exercise program per muscle group, more than one, two, or three exercise programs in specific embodiments. In embodiments, a set of physical exercises includes at least eight repetitions or more.

The subject follows the prescribed exercise regimen at least once a week, twice a week, three times a week, four times a week, or five times a week. In embodiments, the exercise regimen is followed for at least 12 weeks.

In embodiments, the subject complies with a set of physical exercises at a minimum intensity level, such as 50% or less of the maximum weight that can be lifted for a particular exercise (1-RM). In embodiments, the intensity is gradually increased over the course of the exercise regimen. In embodiments, the 1-RM for each exercise (bench press, leg press, leg curl, and arm curl) can be measured in the exercise room on equipment used for physical fitness at various time intervals such as baseline measurement, 4 week and 8 weeks. In embodiments, the intensity is increased by about 10% 1-RM per week. In embodiments, the intensity is at least 50% and maintained at about 65-90% of maximum.

In embodiments, the method further comprises monitoring resistance training performed by the subject, comprising providing a device that measures training and/or providing a log or computer program to track repetitions, duration, intensity, and frequency of resistance training. In embodiments, the instrument includes an accelerometer, a dynamometer, a linear positioning device, and an actigraph.

In embodiments, the method comprises administering a GDF8 inhibitor in an amount effective to increase lean muscle mass without causing adverse myocardial and/or cardiac function side effects. As previously discussed, GDF8 inhibitors may be associated with cardiac hypertrophy. However, according to embodiments, the dose and mode of administration is chosen to minimize any effects on the myocardium and/or cardiac function. In embodiments, markers such as creatinine kinase, troponin, and the like are monitored during treatment. In embodiments, treatment is stopped if cardiac hypertrophy is observed and/or if cardiac markers indicative of cardiac injury increase by at least 20%.

Examples

The following examples are set forth to provide those skilled in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention and are not intended to limit the scope of what the present inventors consider their invention. Efforts have been made to ensure accuracy with respect to the numerical values used (eg amounts, temperatures, etc.), but some experimental errors and deviations must be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

Example 1 Preparation of human antibodies to human GDF8.

Anti-human GDF8 antibodies were prepared as described in US Pat. No. 8,840,894. An exemplary GDF8 inhibitor used in the following example is an anti-human GDF8 antibody designated H4H1657N2 (also referred to as REGN1033). H4H1657N2 is characterized by the following amino acid sequence characteristics: heavy chain variable region (HCVR) containing SEQ ID NO: 360; light chain variable domain (LCVR) containing SEQ ID

- 18 040534

NO: 368. The REGN1033 CDRs contain the heavy chain complementarity determining region 1 (HCDR1) comprising SEQ ID NO: 362; HCDR2 containing SEQ ID NO: 364; HCDR3 containing

SEQ ID NO: 366; light chain complementarity determining region 1 (LCDR1) containing SEQ

ID NO: 370; LCDR2 containing SEQ ID NO: 372; and LCDR3 containing SEQ ID NO: 374.

Example 2 Clinical trial on the safety and biological effect of anti-GDF8 antibody with and without exercise.

A randomized, double-blind, placebo-controlled, multicenter, parallel group study was conducted on the effects of repeated doses of subcutaneous treatment with REGN1033 on safety, body composition and muscle volume, muscle strength, and stair-walking function in 120 healthy male and female subjects. with a sedentary lifestyle, whose age was 60 years and older. A 2x2 factorial design was used; up to 120 subjects (4 groups of 30 subjects each) were to be randomized 1:1:1:1 into groups: placebo alone, REGN1033 (400mg SC Q2W x 6 doses) alone, placebo + resistance training (RT) or REGN1033+RT. Randomization was stratified by gender and by study location. RT consisted of low-intensity resistance training in the gym at 50% 1-rep max (1-RM) twice a week for a total of 12 weeks with at least one gradual exercise adjustment during the study.

The primary aim of the study was to evaluate the effect of REGN1033, with and without exercise, on total lean body mass as measured by dual energy X-ray absorptiometry (DEXA). Secondary objectives include assessments of effects on safety and tolerability; effects on appendicular lean mass and fat mass with DEXA, effects on hip muscle volume measured by MRI, upper and lower body strength with 1-rep max methods, maximal hand press power, and stair walking power .

Selection of patients.

The target population for this study is 120 healthy sedentary male and female subjects aged 60 years and over.

The inclusion criteria were as follows:

1. Men and women 60 years of age or older, with no significant health problems or conditions;

2. Sexually active men who agree to use contraceptives and not donate sperm during the study and for 4 months after the study;

3. Women in need of clinical confirmation of postmenopausal status (at least 12 months since last menstrual period, confirmed by postmenopausal FSH levels >20 mIU/mL or surgically neutered);

4. Body mass index (BMI) from 19 to 35 kg/m ² inclusive;

5. No condition that could limit participation in instructor-supervised resistance training based on PAR-Q (Physical Activity Readiness Questionnaire);

6. Sedentary lifestyle, defined by a score of <125 on the CHAMPS-18 physical activity questionnaire in the past 3 months;

7. Agreeing to maintain the current diet and exercise programs described for the study and not to initiate any new nutrition/weight management programs;

8. Consent and ability to attend all clinic appointments and complete all study-related procedures;

9. Submit complete survey-related questionnaires; And

10. Submit an Informed Consent Form (ICF).

Exclusion criteria from the study include those subjects who have been hospitalized or who have had major surgery, who are characterized by the presence of osteoarthritis, rheumatological disease, or orthopedic disorders that limit joint range of motion or ability to exercise; which are characterized by the presence of gastrointestinal disorders, chronic kidney disease, malignant tumor, lung disease, heart disease, bronchial asthma, stroke with residual paresis, paralysis, multiple sclerosis, Parkinson's disease, cognitive impairment, psychiatric conditions that require acute or chronic therapeutic intervention ( eg, major depressive disorder, bipolar disorder, panic disorder, schizophrenia), current or previous use of any drug known to affect muscle mass or muscle activity, within 6 months, and unable to undergo an MRI of the hips .

The introduction of drugs.

REGN1033 was supplied as a lyophilized drug product and administered subcutaneously in the present study. Each vial of lyophilized REGN1033 was aseptically diluted to a final concentration of 100 mg/mL. Placebo corresponding to REGN1033,

- 19 040534 received in the same composition as REGN1033, without the addition of REGN1033. The placebo volume was the same at each dose level. Subjects received 400 mg REGN1033 or placebo (combined with RT resistance training for subjects in groups 3 and 4) every 2 weeks for a total of 6 doses of SC in the abdomen. A specific quadrant of the abdomen is documented.

Research plan.

The present study was characterized by a screening period of 28 days (day -28 day -1, screening/pre-treatment), a drug treatment period (day 1 - day 71), and a 10-week follow-up period after the last dose. Baseline measures of mass, strength and function of stair walking and electrocardiography were obtained during the screening period (Day -14 - Day -1). Baselines for DEXA and MRI were obtained between day -7±3 and day -1. For all other parameters, baseline values were obtained on day 1 (baseline). The total duration of the study from the first dose was approximately 20 weeks.

Subjects were screened on day -28 - day -1 and eligible subjects were randomized to 1 of 4 groups on day 1: placebo; placebo and RT; REGN1033; REGN1033 and RT. Subject eligibility was determined using standard screening procedures, as well as screening for cognitive impairment using the Mini Mental Status Assessment (MMSE) questionnaire and screening for sedentary lifestyle using the CHAMPS 18 physical activity questionnaire (Community Health Activities Model Program). for Seniors 18).

Not later than 2 weeks prior to dosing (Day -14 to Day -1), all subjects were introduced to exercise and measurements of muscle strength and function: leg press, bench press, leg curl, arm curl, dynamic hand strength, and stair walking without load and with load (8 steps). Baseline upper and lower body strength (defined by 1-rep max [1RM] for bench press and leg press), maximal dynamic hand strength, and unloaded and loaded stair-walking strength were determined for all subjects within 2 weeks ( day -14 - day -1) before dosing. For each strength/function measurement, 2 two tests were performed on subjects in all 4 treatment groups at 2 separate visits within 2 weeks prior to the first dose of study drug to ensure familiarity and education on testing procedures. The average value of the specified 2 measurements during the specified 2 testing sessions will be used as the initial value.

1-RM for each RT exercise (bench press, leg press, leg curl, and arm curl) was measured in the exercise room on equipment used for physical training at baseline, at week 4 and at week 8, and used for load calculation for RT training during weeks 1-4, weeks 5-8 and weeks 9-12. Physical training was performed twice a week during the 12-week treatment period. At least 1 day separated each exercise session. Within 10 days prior to dosing (day -7±3 days), all subjects were subjected to whole body DEXA to determine total and regional fat mass and appendicular lean mass. Subjects also underwent MRI of both thighs to determine muscle volume and SC and intramuscular fat. The obtained values served as a baseline for the indicated parameters.

On Day 1 and every 2 weeks thereafter, subjects received a total of 6 SC doses of study drug (400 mg REGN1033 or placebo) administered to the abdomen as 2 injections of 2 ml each at the injection site. Subjects were monitored for 30 minutes for vital signs and to collect adverse events (AEs), including the occurrence of reactions at the injection site. Accelerometry was used to monitor physical activity during the study.

Subjects in Groups 3 and 4 attended instructor-supervised low-intensity resistance training of major upper and lower limb major muscle groups using resistance training equipment twice a week for 12 weeks. All exercise in the workout was performed at a relatively low intensity of 50% 1-RM for each exercise.

All subjects completing the study underwent laboratory testing and safety assessments at weeks 2, 4, 6, 8, and 10 on days 15, 29, 43, 57, and 71 (all with ±3 days between visits) if subsequent dose of study drug. Subjects were followed up for 10 weeks after the last dose (Day 71) and returned to the clinic for laboratory and safety evaluations at Weeks 12, 14, 16, and 20 (Days 85±3, 99±3, 113±3 and 141±3 [until the end of the last study visit]). Performance measures (DEXA, MRI, muscle strength and physical functioning) were obtained at screening and at week 12 (day 85±3). DEXA and muscle strength (double leg press, bench press and maximum brush press) and physical function (stair walking strength) were measured at weeks 6 and 20. Electrocardiograms were obtained on day 1, at 4 weeks, 8 weeks, 12 weeks and at the end

- 20 040534 studies. Echocardiograms were performed at week 12 (day 85±3 days).

All subjects were instructed to wear the GT3X Actigraph (set at 30 Hz) on their wrist for the first 2 weeks of the study, from visit on day 1 to day 15, and for another 14-day period from day 71 to day 85 (excluding periods of resistance training in the gym and during sleep). Subjects returned the device to the study site for data download (Day 15 and Day 85). Time spent at various levels of activity was determined based on an activity threshold analysis using predetermined detection limits. Estimates of metabolic levels and energy expenditure were determined using pre-established algorithms (Actigraph Corp).

Sample analysis and statistics.

The primary efficiency variable, i.e. the percent change in total lean tissue mass by dual energy x-ray absorptiometry (DEXA) from baseline to week 12 was analyzed using a repeated measures mixed effects model (MMRM) approach. Secondary efficacy variables were analyzed in a similar manner. The model included factors (fixed effects) for treatment (with 4 levels R1033 + RT, R1033 no RT, placebo + RT, and placebo no RT), baseline (gender), visit, baseline, and treatment-visit relationship as covariates . The primary endpoint was compared between REGN1033 without RT and placebo without RT. The same comparison was made between REGN1033 with RT and placebo with RT. In addition, the effect of exercise was studied.

Results.

In all 4 treatment groups, 93.8-96.6% of randomized subjects completed their visit at week 12. Only one subject, in the placebo treatment group alone, who discontinued the study due to an adverse event. The % of subjects in each of the placebo (PLC), placebo + RT (PLC + RT), REGN1033 (R1033), and REGN1033 + RT (R1033+RT) groups who received all 6 doses of study drug was 87.5% , 93.1%, 84.4% and 90.6% respectively. There was no imbalance in all groups in adherence to either treatment or RT. Demographics were balanced among the 4 groups, except that the placebo group had an increased percentage (18.8%) of black subjects; and the placebo + RT group had an increased percentage (75.9%) and the REGN1033 group had a decreased percentage (56.3%) of subjects over 65 years of age. Baseline values for DEXA, MPT, muscle strength and physical functioning for each group were not significantly different from each other (data not shown).

The primary aim of the study was to evaluate the effect of REGN1033, with and without exercise, on total lean body mass as measured by dual energy X-ray absorptiometry (DEXA). Secondary objectives include assessments of effects on safety and tolerability; effects on appendicular lean mass and fat mass with DEXA, effects on hip muscle volume measured by MRI, upper and lower body strength with methods using 1-rep max, maximum hand press strength, and stair walking strength. In addition, the potential metabolic effects of REGN1033 on HbA1C and HOMA-IR were investigated.

The primary efficiency variable, i.e. the percentage change in total lean tissue mass by dual energy x-ray absorptiometry (DEXA) from baseline to week 12 was analyzed using a repeated measures mixed effects model (MMRM) approach. The model included factors (fixed effects) for treatment (with 4 levels R1033 + RT, R1033 no RT, placebo + RT, and placebo no RT), baseline (gender), visit, baseline, and treatment-visit relationship as covariates . The primary endpoint was compared between REGN1033 without RT and placebo without RT. The same comparison was made between REGN1033 with RT and placebo with RT. In addition, the effect of exercise was studied. Secondary efficacy endpoints with continuous treatment outcome were analyzed in a manner similar to that of the primary efficacy endpoint.

Doses and dosing regimens for the present study were selected based on the results of other studies.

In the completed SAD study, 76 healthy volunteers received REGN1033 at doses up to 10 mg/kg IV and 400 mg SC. Among them, an elderly cohort of 8 healthy volunteers aged 65-85 years received 6 mg/kg IV of study drug. All doses were well tolerated; did not observe any clinically significant signals of safety issues (data not shown).

Another study was designed to evaluate the safety, tolerability, pharmacokinetics (PK), immunogenicity and pharmacodynamic (PD) effects of REGN1033 administered by SC in healthy volunteers aged 60 years and older. A total of 5 cohorts were studied with 12 subjects recruited into each cohort. Subjects received SC doses of REGN1033 (n=9) or placebo (n=3). The scheduled dosing regimens for REGN1033 were 100, 200 or 400 mg Q2W for a total of 6 doses per subject and 200 mg or 400 mg Q4W for a total of 3 doses per subject. The test results shown in FIG. 1A indicate that these doses of REGN1033 are well tolerated and are associated with an increase in lean tissue mass as detected by DEXA.

From these studies, the PK and total PD data of GDF8 indicate that REGN1033 can reach concentrations associated with target lesion saturation over the entire dose range for dose levels of 400 mg Q4W and 400 mg Q2W. At the dose level of 100 mg Q4W, the effects of REGN1033 are expected to be clearly less than those achieved at higher doses. PK and PD data from phase 1 studies indicate that this reduced dose will not reach concentrations associated with target damage over the entire dose range in all individuals.

The results for the outcome for the primary efficacy variable are shown in Table 1. 1 and in FIG. 1B.

Table 1

Week 12 REGN alone vs. with RVO separately Week 12 REGN + RT vs. with PBO+RT Week 12 REGN combo in comparison with RVO comb. Week 12 RBO + RT vs. with RVO Week 12 REGN + RT vs. with REGN Model MMRM effect REGN MMRM model of the effect of exercise The MMRM model of the relationship between REGN and exercise Generalized view of primary effectiveness Percent change in total fat-free mass as measured by dual energy X-ray absorptiometry (DEXA) from baseline 1.66% cf. from 1.79% (p=0.8640) 3.10% cf. s 0.25% (p<0.0001) 2.39% cf. from 0.79% (p=0.0048) -0.25% cf. from 1.79% (p=0.0088) 3.10% cf. from 1.79% (p=0.0609) (p=0.0008) (p=0.6333) (p=0.0124)

Without exercise, REGN1033 (REGN) treatment did not increase total lean body mass measured by DEXA compared to placebo (PBO) treatment (placebo-adjusted change -0.13% p=0.8640, baseline to 12 weeks). With exercise, treatment with REGN1033 (REGN+RT) significantly increased total lean body mass measured by DEXA from baseline to week 12 compared with treatment with placebo (PBO+RT) (placebo-adjusted increase of 3.34 %, p<0.0001). See table. 1 and FIG. 1B. The percentage change in the placebo + RT group is significantly lower than in the placebo group alone: -0.25% compared to 1.79% (p = 0.0088), but the REGN1033 + RT group is numerically larger than the REGN1033 group alone : 3.10% vs. 1.66% (p=0.0609).

The MMRM factor model also shows a significant effect of treatment with REGN1033 on the percentage change in total lean body mass with DEXA from baseline to week 12 (P=0.0008), which was maintained at week 20 (P=0.0004). In addition, MMRM also shows an association effect (p=0.0124) between exercise and treatment.

In subjects randomized to receive progressive resistance exercise, treatment with REGN1033 (REGN+RT) significantly increased DEXA-measured appendicular lean body mass from baseline to week 12 compared with placebo treatment (PBO+RT). ) (unweighted marginal mean difference vs. placebo 4.34%, P=0.001). As observed in the analysis of total lean body mass, the percentage change in appendicular lean body mass in the placebo + RT group was significantly lower than in the placebo group alone: 0.08% versus 2.25% (p = 0.0200), but this indicator in the REGN1033+ RT group was significantly higher than in the REGN1033 group alone: 4.42% compared to 2.30% (p=0.0217). The MMRM factor model also shows a significant treatment effect.

- 22 040534 (0.0008) and the relationship between treatment and exercise at week 12 (p=0.0040), which persisted at week 20. See table. 2 and FIG. 2.

table 2

Week 12 REGN alone vs. srvo separately Week 12 REGN + RT vs. with PBO+RT Week 12 REGN combo in comparison with RVO comb. Week 12 RBO + RT vs. with RVO Week 12 REGN + RT vs. with REGN Model MMRM effect REGN MMRM model of the effect of exercise The MMRM model of the relationship between REGN and exercise Percent change in appendicular mass as measured by DEXA 2.30% cf. from 2.25% d=0.04% (p=0.9617) 4.42% cf. c 0.08% d=4.34% (p<0.0001) 3.37% cf. from 1.19% d=2.18% (p=0.0015) 0.08% ref. from 2.25% (p=0.0200) 4.42% cf. from 2.30% (p=0.0217) (p=0.0008) (p=0.9728) (p=0.0040)

Treatment with REGN1033, with or without exercise, resulted in statistically significant increases in MRI thigh muscle volume. See fig. 3. Placebo-adjusted effects on MRI thigh muscle volume (excluding intramuscular fat and vascularity) at week 12 were +3.7%, p=0.0006 without exercise (REGN alone) and +4.5%, p < 0.0001 with exercise (REGN+RT). There is a trend towards increased thigh muscle, including intramuscular fat and vascular volume, in the REGN1033 group alone compared to placebo alone, and an increase in thigh muscle, including intramuscular fat and vascular volume, in the REGN1033 + RT group compared to placebo group +RT. At week 12, the unweighted marginal mean of the percentage (%) change from baseline in thigh muscle volume, including intramuscular fat and vascular volume (original scale), for placebo alone, placebo + RT, REGN1033 alone, and REGN1033 + RT is -0 .03%, -0.434%, 2.076% and 3.405%, respectively. In subjects randomized to no exercise, treatment with REGN1033 significantly increased thigh muscle, including intramuscular fat and vascular volume, as measured by MRI from baseline to week 12 compared with placebo treatment (unweighted marginal mean difference compared with placebo 2.11%%, P=0.0139). In subjects randomized to receive progressive resistance training exercise, treatment with REGN1033 significantly increased thigh muscle, including intramuscular fat and vascular volume, as measured by MRI from baseline to week 12 compared with treatment with placebo (unweighted marginal mean difference compared to placebo 3.84%, p<0.0001).

The MMRM factor model also shows a significant effect of treatment (<0.0001) but no effect of exercise (p=0.4453). The relationship between treatment and exercise at week 12 is not significant (p=0.1651). An increase in lean body mass as measured by DEXA was observed in the placebo group, which was not accompanied by an increase in muscle volume as measured by MRI. The reason for this discrepancy is unknown.

There was a trend towards an increase in gynoid fat mass in the placebo group alone at week 12 compared to other treatment groups and a decrease in gynoid fat mass in the REGN1033 + RT group. At week 12, the unweighted marginal mean percentage (%) change from baseline in the placebo alone, placebo + RT, REGN1033 alone, and REGN1033 + RT groups is 2.42%, -0.49%, -0.27%, and -2 .17% respectively. See fig. 4. Factor model MMRM shows a trend of significant effects of treatment (p=0.0824) and exercise (0.0531) on gynoid fat mass, but no relationship between exercise and treatment (p=0.8774).

Four measurements of muscle strength were also obtained. Equipment was standardized across all study sites for 1-RM bench press and leg press measurements, but not for arm curl and leg curl measurements. Moreover, only a subset of subjects were measured for arm flexion and leg flexion.

A trend towards increased leg press strength was observed in all groups. At week 12, the unweighted marginal mean percentage (%) change from baseline in the placebo alone, placebo + RT, REGN1033 alone, and REGN1033 + RT groups is 4.7%, 8.9%, 6.7%, and 9.7% respectively. The MMRM factor model showed no significant treatment effect (p=0.8389), but a borderline effect of exercise (p=0.0541) on the leg press. The effect of the relationship between exercise and treatment was not significant (p=0.8029).

There was a trend towards increased bench press strength in all groups. The REGN1033 group alone increased more than the placebo group alone, and the REGN1033 + RT group also increased more than the placebo + RT group. See fig. 5. At week 12, the unweighted marginal mean percentage (%) change from baseline in the placebo alone, placebo + RT, REGN1033 alone, and REGN1033 + RT groups are 2.2%, 10.5%, 10.4%, and 15 .3% respectively. In subjects randomized not to receive progressive resistance training exercise, treatment with REGN1033 increased bench press strength from baseline to week 12 compared with placebo treatment (unweighted marginal mean difference versus placebo 8.2% , P=0.0524). In subjects randomized to receive progressive resistance training exercise, treatment with REGN1033 numerically increased bench press from baseline to week 12 compared to placebo treatment, but this was not significant (unweighted marginal mean difference vs. placebo 5.3%, P=0.2316). The MMRM factor model showed significant treatment effects (p=0.0525) and a statistically significant effect of exercise (0.0272) on bench press and no relationship effect (p=0.5089) between exercise and treatment.

There was a trend towards increased leg flexion strength in all groups up to 12 weeks. At week 12, the unweighted marginal mean percentage (%) change from baseline in the placebo alone, placebo + RT, REGN1033 alone, and REGN1033 + RT groups was 5.3%, 16.3%, 10.4%, and 9.7% respectively. The change in leg flexion strength in the REGN1033 group alone was 5.1% higher than in the placebo group alone, however, in the REGN1033 + RT group was 6.6% lower than in the placebo + RT group. The percentage change in the placebo + RT group was significantly higher than in the placebo group alone: 16.3% compared to 5.30% (p = 0.0050), however, in the REGN1033 + RT group it was not higher than in the REGN1033 group alone: 9.79% vs. 10.4% (p=0.8388). The MMRM factor model showed no significant association effect (p=0.0229) between exercise and treatment.

There was a trend towards increased arm flexion strength in all groups (data not shown). At week 12, the unweighted marginal mean percentage (%) change from baseline in the placebo alone, placebo + RT, REGN1033 alone, and REGN1033 + RT groups is 8.1%, 21.7%, 12.4%, and 15.7% respectively. In subjects randomized not to receive progressive resistance training, treatment with REGN1033 increased arm flexion strength from baseline to week 12 compared with placebo, but this difference was not statistically significant (unweighted marginal mean difference vs. placebo 4.3%, P=0.4589).

In subjects randomized to receive progressive resistance training exercise, treatment with REGN1033 increased less arm flexion strength from baseline to week 12 compared to placebo, but this was not significant (unweighted marginal mean difference vs. placebo - 6.0%, P=0.3121). The MMRM factor model showed no significant effect of treatment with REGN1033 (p=0.8958), borderline effects of exercise (p=0.053), and no significant effect of association (p=0.1318) between exercise and treatment.

There was a trend towards an increase in dominant hand press strength in the groups treated with REGN1033 (data not shown). At week 12, the unweighted marginal mean percentage (%) change from baseline in the placebo alone, placebo + RT, REGN1033 alone, and REGN1033 + RT groups was 1.8%, 1.2%, 4.8%, and 5.0% respectively. In subjects randomized not to receive progressive resistance training exercise, treatment with REGN1033 increased dominant hand pressing strength from baseline to week 12 compared with placebo, but this was not significant (unweighted marginal mean difference vs. placebo 3.0%, P=0.4180). In subjects randomized to receive progressive resistance training exercise, treatment with REGN1033 increased dominant hand pressing strength from baseline to week 12 more than placebo, but this was not significant (unweighted marginal mean difference vs. placebo 3.8%, P=0.3198). The MMRM factorial model shows a statistically significant effect of treatment with REGN1033 (p=0.0407), but no effect of exercise (p=0.7033) or relationship between exercise and treatment (p=0.8283).

There was a trend towards increased non-dominant hand press strength in the REGN1033 groups but not in the placebo groups (data not shown). At week 12, the unweighted marginal mean percentage (%) change from baseline in the placebo alone, placebo + RT, REGN1033 alone, and REGN1033 + RT groups were 1.8%, -0.8%, 7.0%, and 4.2 % respectively. In subjects randomized not to receive progressive resistance training, treatment with REGN1033 increased non-dominant hand pressing strength more from baseline to week 12 compared to placebo, but this was not significant (unweighted marginal mean difference vs. placebo 5.1%,

- 24 040534

P=0.1432). In subjects randomized to receive progressive resistance training exercise, treatment with REGN1033 increased non-dominant hand pressing strength more from baseline to week 12 compared to placebo, but this was not significant (unweighted marginal mean difference vs. placebo 5.0%, P=0.1708). The MMRM factorial model showed a statistically significant effect of treatment with REGN1033 (p=0.0039) and a marginal effect of exercise (p=0.0581) on non-dominant hand press strength. There was no relationship effect between exercise and treatment (p=0.6224).

There was a trend towards increased stair walking strength with a load in all groups (data not shown). At week 12, the unweighted marginal mean percentage (%) change from baseline in the placebo alone, placebo + RT, REGN1033 alone, and REGN1033 + RT groups was 5.6%, 12.9%, 12.2%, and 15.0% respectively. In subjects randomized not to receive progressive resistance training exercise, treatment with REGN1033 increased resistance stair walking strength from baseline to week 12 more than placebo, but this was not significant (unweighted marginal mean difference vs. with placebo 6.6%, P=0.1303). In subjects randomized to receive progressive resistance training exercise, treatment with REGN1033 increased resistance stair walking strength from baseline to week 12 more than placebo, but this was not significant (unweighted marginal mean difference vs. with placebo 2.2%, P=0.6368). The MMRM factor model did not show a statistically significant effect of treatment (p=0.3612) or relationship between exercise and treatment (p=0.8043), but did show a statistically significant effect of exercise (p=0.0151) when walking stairs with load.

There was a trend towards increased unstressed stair walking strength in all groups. At week 12, the unweighted marginal mean percentage (%) change from baseline in the placebo alone, placebo + RT, REGN1033 alone, and REGN1033 + RT groups were 6.1%, 12.5%, 5.4%, and 9.8 % respectively. There was no difference between the REGN1033 and placebo groups. The MMRM factor model did not show a statistically significant effect of treatment (p=0.4670) or relationship between treatment and exercise (p=0.4180), but showed a statistically significant effect of exercise (p=0.0295). The percentage change in the placebo + RT group was higher than in the placebo group alone 12.5% compared to 6.1% (p=0.0900), and higher in the REGN1033 + RT group than in the REGN1033 group alone: 9. 8% versus 5.4% (p=0.2253), however these differences are not statistically significant.

Treatment with REGN1033 and resistance training (RT) were generally well tolerated in the study. The results of treatment-related adverse events (TEA) are shown in table. 3.

Table 3

Placebo REGN1033

Separately (N=32) RT (N=29) 400 mg (N=32) 400 mg+ RT (N=32) TEAE number 151 103 129 98 Number of serious TEAEs 1 1 3 0 Subjects with at least one TEAE Subjects with at least one 26 (81.3%) 27 (93.1%) 27 (84.4%) 28 (87.5%) drug-related TEAE Subjects with at least one serious 10(31.3%) 6 (20.7%) 14 (43.8%) 10(31.3%) TEAE Subjects with TEAE resulting from 1 (3.1%) 1 (3.4%) 2 (6.3%) 0 discontinuation of the study 3 (9.4%) 1 (3.4%) 1 (3.1%) 0

medicinal product

Subjects with TEAE that resulted in death 0 0 0 0

There were no reported deaths in the present study, and a total of 5 serious TEAS occurred in 4 subjects, including a foot fracture in 1 subject in the placebo group alone; cholecystitis in 1 subject in the placebo + RT group, stress cardiomyopathy in 1 subject

- 25 040534 that in the REGN1033 group and hypokalemia and hypotension in 1 subject in the REGN1033 group. There were no reports of serious TEAS in the REGN1033 + RT group. Subjects in the placebo group separately recorded more TEAEs and treatment-related TEAEs compared to the other groups. The number and percentage of subjects experiencing at least one TEAE were similar across all treatment groups: 26 (81.3%) in the placebo alone group, 27 (93.1%) in the placebo + RT group, 27 (84.4 %) in the REGN1033 group and 28 (87.5%) in the REGN1033 + RT group, respectively. A total of 5 subjects (3 and 1 in the placebo and placebo + RT groups, compared with 1 and 0 subjects in the REGN1033 and REGN1033 + RT groups, respectively) discontinued drug treatment due to TEAE.

In the Potentially Clinically Significant Value (PCSV) categories of vital signs, ECG, and hematology, there was no evidence of an unbalanced higher signaling frequency for PCSV in the REGN1033 treatment groups. Numerically more subjects with chemical PCSV were observed in the REGN1033 combined group than were observed in the placebo combined group. Elevated creatine kinase values >3-fold from the upper limit of normal were observed in 2 (6.3%), 1 (3.4%), 2 (6.3%), and 4 (12.5%) subjects in placebo group alone, placebo + RT, REGN1033 and REGN1033 + RT, respectively. Creatine kinase values >10-fold higher than the upper limit of normal were not observed (Table 14.3.4.2.3). More subjects in the REGN1033-treated groups (7 [10.9%]) had >5% weight gain than those in the placebo-treated groups (3, [4.9%], Table 14.3.5.1. 2).

A review of echocardiogram parameters related to heart structure and function did not reveal any signal of cardiac hypertrophy or any other detrimental effects on heart function. Treatment with REGN1033 had no effect on left ventricular ejection fraction, LV wall thickness, or ventricular septal thickness. There were no reported increases in LV mass index or LV mass index in either REGN1033 or placebo treated treatment groups (data not shown).

Conclusions.

Together with exercise, REGN1033 treatment significantly increased total lean body mass in healthy subjects as measured by DEXA from baseline to week 12 compared with placebo treatment (3.1% vs -0.2%, < 0.0001). Without exercise, REGN1033 treatment did not significantly increase total lean body mass as measured by DEXA compared to placebo treatment.

For secondary efficacy endpoints, the results of appendicular lean body mass with DEXA are consistent with the results observed for total lean body mass with DEXA. Treatment with REGN1033, with or without exercise, resulted in statistically significant increases in MRI thigh muscle volume, excluding intramuscular fat and vascularity, at week 12 compared with placebo treatment (placebo-adjusted increase was 3.7% , P=0.0006 without exercise and 4.5%, P<0.0001 with exercise).

Resistance exercise (RT) in the present study was well tolerated in this elderly population. Significant effects of exercise were demonstrated using the MMRM model on most strength/functionality measurements. This level of RT exercise did not result in a significant increase in lean mass as measured by DEXA or muscle volume as measured by MRI. Treatment with REGN1033 also tended to have effects on some of the strength and function endpoints investigated in the present study. Positive effects were observed in the following functions: bench press strength, dynamic hand strength and stair walking with a load.

In general, 400 mg Q2W REGN1033 SC was generally well tolerated in the present trial. The number of patients reporting TEAE was comparable across all treatment groups. The TEAE review found no significant safety signal. Echocardiogram studies have shown no deleterious effects on heart structure or function.

Example 3 Clinical Trial Protocol for Anti-GDF8 Antibody Treatment of Sarcopenic Subjects with and without Exercise.

Conducted a randomized, double-blind, placebo-controlled, multicenter phase 2 study of the safety and efficacy of 3-month SC treatment with REGN1033 in patients with sarcopenia. 250 patients were recruited into the study, in 4 treatment groups. Eligible patients were male and female patients with sarcopenia and associated mobility impairment, aged 70 years or older, with a median age of 78 years. Patients were randomized 1:1:1:1 to receive placebo SC every 2 weeks (Q2W) for a total of 6 doses, REGN1033 at a dose of 300 mg SC Q2W for a total of 6 doses, REGN1033 at a dose of 300 mg SC every 4 weeks (Q4W) for a total of 3 administration (alternating placebo administration one week later), and REGN1033 at a dose of 100 mg SC Q4W

- 26 040534 3 injections in total (alternating placebo injections every other week). The study was characterized by a screening/pre-treatment period (day -28 - day -1), a 12-week treatment period (day 1 - day 85), and

An 8-week follow-up period (through day 141).

Screening procedures and pre-treatment procedures (day -28 - day -1).

A sequential screening process occurred over 3 visits, with pre-eligibility determined at 1 visit and pre-treatment procedures performed at 2 and 3 visits. Where possible at the study sites, Visit 1 and Visit 2 were conducted at the same time; if so, Visit 2 procedures were conducted no later than 21 days from the first dose of study drug. Preliminary eligibility was determined at visit 1 using standard screening procedures, as well as gait speed over 4 m [4 M] and a Mini Mental Status Assessment (MMSE) score. Patients meeting pre-qualification criteria were returned to the clinic at Visit 2 and Visit 3 for pre-treatment initial procedures and measurements.

Procedures included standard safety and laboratory assessments, DEXA scans, echocardiograms, strength measurements (leg press, bench press, and dynamic hand strength), and functional measurements (stair walking, Brief Physical Performance Test Battery [SPPB], 4 m gait speed and 6-minute walk test [6MWT]).

Treatment period and study drug administration (Day 1 - Day 85).

Beginning on day 1, patients were randomized to receive either REGN1033 or a matching placebo. The doses were as follows:

300 mg SC Q2W 6 injections in total,

300mg SC Q4W 3 total doses (alternating placebo every other week to maintain blinding)

100mg SC Q4W total 3 doses (alternating placebo every other week to maintain blind format), matched placebo SC Q2W total 6 doses.

Injections were administered into the abdomen. Patients were observed for 30 minutes for vital signs and to collect adverse events (AEs), including the occurrence of reactions at the injection site. Efficacy and safety procedures were performed and patient-reported end results (PRO) were recorded. Blood samples were collected for pharmacokinetics (PK), anti-drug antibodies (ADA) and study. All blood samples were collected after an overnight fast and before dosing.

Follow-up (day 86 - day 141).

Follow-up visits were made on day 141, 8 weeks after the end of the treatment visit on day 85.

end points.

The primary endpoint in the study was the percentage change in total lean body mass as measured by DEXA from baseline to week 12. Secondary endpoints were TEAE from baseline to end of study, change from baseline in appendicular lean mass with DEXA, maximum leg press strength, 1-rep max (1-RM), maximum bench press strength (1-RM) , gait speed over 4m, SPPB and parts of the SPPB scale, distance walked in the 6MWT test, regional and total fat mass as measured by DEXA, and hand press force as measured by a hand dynamometer.

Procedures and assessments.

The safety and tolerability of REGN1033 was assessed by vital signs, electrocardiogram (ECG), echocardiogram, adverse events (AE), and clinical laboratory studies. Patients were asked to monitor and report all AEs experienced from the time they signed the informed consent until the end of the study visit.

Efficacy was assessed using DEXA, strength measurements (leg press, bench press and dynamic hand strength) and functional measurements (stair walking, SPPB, 4m gait speed and 6MWT). Other measurements used were accelerometry and PRO (10-item Physical Functioning Form [PF-10], Fatigue Scale in Functional Evaluation of Chronic Disease Therapy [FACIT], Health Assessment Inventory: Disability Index [HAQ-DI], short form Nutrient Status Mini-Questionnaire [MNA-SF] and Rapid Physical Activity Assessment [RAPA]).

Results.

95% of randomized patients completed the study. As shown below in Table. 4, in each of the three dosing regimens studied, treatment with REGN1033 significantly increased total lean body mass from baseline to week 12 compared to placebo; average times-

- 27 040534 Differences from placebo were 1.7% (p=0.008), 1.8% (p=0.004) and 2.3% (p<0.001) for REGN1033 100 mg SC Q4W, 300 mg Q4W and 300 mg Q2W respectively, which corresponded to increases in lean tissue mass of 0.7, 0.8 and 1.0 kg. Appendicular lean mass also increased significantly in patients treated with REGN1033: placebo-adjusted changes ranged from 2.3-2.8%. Measured with DEXA, total fat mass, android fat mass and gynoid fat mass showed numerical reductions due to treatment with REGN1033. Treatment with REGN1033 resulted in directionally large mean changes from baseline in various measures of strength and function relative to placebo.

Table 4

REGN 100 mg Q4W (n=62) vs. RVO (n=65) REGN 300 mg Q4W (n=64) vs. RVO (n=65) REGN 300 mg Q2W (n=59) vs. RVO (n=65) Primary Efficacy Endpoint: DEXA - Percent change from baseline to week 12 Percent Change total lean tissue mass as measured by dual energy x-ray absorptiometry (DEXA) from baseline 1.191% vs -0.474% d= 1.664% (p=0.0077) 1.308% _vs -0.474% d= 1.781% (p=0.0043) 1.816% _vs -0.474% d=2.289% (p=0.0004) Summary view of secondary efficacy endpoints: DEXA - Percent change from baseline to week 12 Percent change in appendicular mass lean tissue (g) as measured by dexa 2.162% over -0.249% d=2.412% (p=0.0026) 2.033% vs -0.249% d=2.282% (p=0.0043) 2.502% vs -0.249% d=2.751% (p=0.0008) Percent change total fat mass (g) as measured by dexa -0.077% vs -0.076% d=-0.001% (p=0.9993) -2.666% vs. -0.076% d=-2.590% (p=0.0099) -0.947% vs. -0.076% d=-0.871% (p=0.3945) Percent change android fat mass (g) as measured by DEXA -0.496% vs. 2.501% d=-2.998% (p=0.0587) -3.164% vs. 2.501% d=-5.665% (p=0.0004) -2.209% vs. 2.501% d=-4.710% (p=0.0038) Percent change from baseline in gynoid fat mass (g) as measured by DEXA 0.022% vs. 0.156% d=-0.135% (p=0.9050) -2.608% compared to 0.156% d=-2.751% (p=0.0152) -2.185% vs. 0.156% d=-2.341% (p=0.0434)

REGN1033 was generally safe and well tolerated. The frequency of adverse events was similar in all treatment groups. The percentage of subjects experiencing at least one SAE was also similar across all treatment groups (7.7% in the placebo group compared to 7.4% in the REGN1033 treated groups). There was no discernible SAE distribution profile. There were no clinically significant trends observed for laboratory tests, vital signs, ECG, and echocardiograms.

Conclusions.

Treatment with REGN1033 significantly increased total lean and appendicular lean mass in patients with sarcopenia and was well tolerated.

The present invention is not limited in its scope to the specific embodiments described herein. Moreover, various modifications according to the present invention in addition to those described herein will become apparent to those skilled in the art from the above description and the accompanying figures. It is intended that such modifications fall within the scope of the appended claims. All publications referenced in this document are incorporated herein by reference.

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Claims (14)

CLAIM 1. A method for increasing lean body mass in a human subject, comprising: a) providing an exercise regimen for the subject, wherein the exercise regimen includes progressive resistance training, and b) administering a composition comprising an effective amount of a GDF8 inhibitor, wherein the GDF8 inhibitor is an anti-human GDF8 antibody or antigen-binding fragment thereof that specifically binds GDF8, and wherein the antibody or fragment thereof contains the three heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3, containing the amino acid sequences of SEQ ID NO: 362/364/366, respectively; and three light chain complementarity determining regions LCDR1, LCDR2, LCDR3 containing the amino acid sequences of SEQ ID NO: 370/372/374, respectively. 2. A method of treating a disease, disorder or condition associated with GDF8 activity and which can be improved by increasing lean body mass in a human subject, wherein the disease or disorder is selected from the group consisting of sarcopenia, cachexia, muscle injury, muscle wasting, muscular atrophy and sporadic inclusion myositis (sIBM), including: a) providing an exercise regimen for the subject, wherein the exercise regimen includes progressive resistance training, and b) administering to a subject in need thereof a composition comprising an effective amount of a growth and differentiation factor 8 (GDF8) inhibitor, wherein the GDF8 inhibitor is an anti-human GDF8 antibody or antigen-binding fragment thereof that specifically binds GDF8, and wherein the antibody or fragment thereof contains three complementarity-determining regions of the heavy chain HCDR1, HCDR2, HCDR3, containing the amino acid sequences of SEQ ID NO: 362/364/366, respectively; and three light chain complementarity determining regions LCDR1, LCDR2, LCDR3 containing the amino acid sequences of SEQ ID NO: 370/372/374, respectively. 3. The method of claim 2, wherein the cachexia is either idiopathic or secondary to other conditions selected from the group consisting of cancer, chronic renal failure, or chronic obstructive pulmonary disease. 4. The method of claim 2, wherein the muscle atrophy or muscle wasting is caused by or associated with lack of functional muscle loading, immobilization, bed rest, trauma, medical treatment, or surgery. 5. The method according to any one of claims 1 to 4, wherein the effective amount is at least 400 mg. 6. The method according to any one of claims 1-5, wherein the effective amount comprises a dosage regimen selected from the group consisting of a single dose of 0.01-20 mg/kg body weight, 0.1-10 mg /kg of body weight or 0.1-5 mg/kg of body weight. 7. The method according to any one of claims 1 to 6, wherein resistance training includes a set of physical exercises for training muscles selected from the group consisting of muscles of the dominant hand, non-dominant hand, leg, arm, back muscles, abdominal muscles, shoulder muscles, gluteal muscles, chest muscles and their combinations. 8. The method according to any one of claims 1 to 7, wherein administration of the GDF8 inhibitor in an amount effective to increase lean body mass does not result in adverse side effects on the myocardium and/or cardiac function. 9. The method according to any one of claims 1 to 8, wherein the composition is administered at least once every six weeks, once every five weeks, once every four weeks, once every three weeks, once every two weeks, once a week, twice once a week, three times a week, four times a week or five times a week. 10. The method according to any one of claims 1 to 9, wherein the composition is formulated for intravenous or subcutaneous administration. 11. The method according to any one of claims 1 to 10, wherein the antibody or antigen-binding fragment comprises the heavy chain variable region (HCVR) amino acid sequence of SEQ ID NO: 360 and the light chain variable region (LCVR) amino acid sequence of SEQ ID NO: 368. 12. The method according to any one of claims 1-11, wherein the antibody, or antigen-binding fragment thereof, binds an epitope of the mature GD8 protein of SEQ ID NO: 340 within amino acid residues 48-72. 13. The method of any one of claims 1 to 12, wherein the antibody or antigen-binding fragment thereof is a multispecific antibody or antigen-binding fragment thereof that can bind 2 or more GDF8 epitopes. 14. The method of any one of claims 1 to 13, wherein the anti-GDF8 antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof. -