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  Pharmaceutical Patents  

 

Title:  Anti-myostatin antibodies
United States Patent: 
8,063,188
Issued: 
November 22, 2011

Inventors: 
Sayers; Robert Owen (Encinitas, CA), Huang; Lihua (Carmel, IN)
Assignee: 
Eli Lilly and Company (Indianapolis, IN)
Appl. No.: 
12/376,390
Filed: 
August 23, 2007
PCT Filed: 
August 23, 2007
PCT No.: 
PCT/US2007/076604
371(c)(1),(2),(4) Date:  February 04, 2009
PCT Pub. No.: 
WO2008/030706
PCT Pub. Date: 
March 13, 2008


 

Pharm Bus Intell & Healthcare Studies


Abstract

Monoclonal anti-myostatin antibodies that preferentially bind myostatin over GDF-11, have strong binding affinity to myostatin and are resistant to chemical degradation. The antibodies of the invention are useful for increasing muscle mass, increasing bone density, or for the treatment or prevention of various disorders in mammalian and avian species.

Description of the Invention

SUMMARY OF THE INVENTION

Antibodies of the invention preferentially bind myostatin over GDF-11, i.e., they are significantly less reactive with GDF-11 than with myostatin. An antibody of the invention binds myostatin at least about 2, 3, 5, 10, 20, 22, or 25-times greater than it binds GDF-11 as measured by a technique in the art, e.g., by competition ELISA, or by BIACORE or KINEXA assay to demonstrate higher affinity (i.e., lower K.sub.D) of the antibody to GDF-8 than GDF-11. Most preferably, antibodies of the invention do not bind GDF-11 above background levels in the binding assay used.

The present invention encompasses an anti-myostatin monoclonal antibody that preferentially binds myostatin over growth differentiation factor-11 (GDF-11), wherein said antibody binds myostatin with an affinity no greater than about 3.times.10.sup.-8 M and wherein at least 95% of the monoclonal antibody is not cleaved when present for one year at 4.degree. C., six months at 25.degree. C., two months at 37.degree. C. or four weeks at 40.degree. C. in an antibody solution. An exemplary antibody solution comprises 1 mg/mL of an antibody of the invention, 10 mM phosphate, pH 7.4, and 150 mM NaCl. Preferably, antibodies of the invention are further characterized by having an IC.sub.50 of less than 25 nM, in the in vitro myostatin/SBE reporter assay described in Example 4 herein.

In one embodiment, an antibody of the invention is resistant to chemical degradation, i.e., 100%, 99%, 98%, 97%, 96%, or 95% of the antibodies are not cleaved, when present in an antibody solution for a time and temperature selected from the group consisting of: one year at 4.degree. C., six months at 25.degree. C., two months at 37.degree. C., and four weeks at 40.degree. C. in an antibody solution. A preferred time and temperature is four weeks at 40.degree. C.

In one embodiment, antibodies of the invention are further characterized by binding myostatin within the domain spanning amino acids 40-64 [ANYCSGECEFVFLQKYPHTHLVHQA (SEQ ID NO: 29) for human], 43-57 [CSGECEFVFLQKYPH (SEQ ID NO: 30) for human] or 45-59 [GECEFVFLQKYPHTH (SEQ ID NO: 31) for human] of mature myostatin. In another embodiment, antibodies of the invention are further characterized by binding a polypeptide consisting of amino acids 40-64, 43-57 or 45-59 of mature myostatin.

In one embodiment, an antibody of the invention comprises a heavy chain variable region (HCVR) with a sequence as shown in SEQ ID NO: 11 and a light chain variable region (LCVR) with a sequence selected from the group consisting of SEQ ID NO: 4, 5, 6, and 7. In another embodiment, a monoclonal antibody of the invention comprises a HCVR with a sequence as shown in SEQ ID NO: 12 and a LCVR with a sequence selected from the group consisting of SEQ ID NO: 9 and 10.

In one embodiment, an antibody of the invention comprises a HCVR and a LCVR, wherein said HCVR comprises a peptide at CDRH1 with the sequence as shown in SEQ ID NO: 23, a peptide at CDRH2 with the sequence as shown in SEQ ID NO: 25, and a peptide at CDRH3 with the sequence as shown in SEQ ID NO: 27, and wherein said LCVR comprises a peptide at CDRL1 with the sequence as shown in SEQ ID NO: 13, a peptide at CDRL2 with the sequence as shown in SEQ ID NO: 14, and a peptide at CDRL3 with the sequence selected from the group consisting of SEQ ID NO:16, 17, 18 and 19.

In another embodiment, an antibody of the invention comprises a HCVR and a LCVR, wherein said HCVR comprises a peptide at CDRH1 with the sequence as shown in SEQ ID NO: 24, a peptide at CDRH2 with the sequence as shown in SEQ ID NO: 26, and a peptide at CDRH3 with the sequence as shown in SEQ ID NO: 28, and wherein said LCVR comprises a peptide at CDRL1 with the sequence as shown in SEQ ID NO: 13, a peptide at CDRL2 with the sequence as shown in SEQ ID NO: 14, and a peptide at CDRL3 with the sequence selected from the group consisting of SEQ ID NO: 21, and 22.

In one embodiment, an antibody of the invention further comprises a constant region, wherein said constant region originates from the human genome or the genome of an animal selected from the group consisting of domestic animals, sports animals and food-source animals. In a more preferred embodiment, an antibody of the invention comprises a light chain with an amino acid sequence as shown in SEQ ID NO: 32 and a heavy chain with an amino acid sequence as shown in SEQ ID NO: 33.

In another embodiment, the invention provides a composition (e.g., a pharmaceutical composition) comprising an antibody of the invention. The composition of the invention may further comprise a pharmaceutically acceptable carrier. In said composition, the antibody of the invention is the active ingredient. Preferably the composition comprises a homogeneous or substantially homogeneous population of an anti-myostatin antibody of the invention. The composition for therapeutic or prophylactic use is sterile, may be lyophilized, and is preferably supplied with an appropriate diluent.

The invention provides a method of inhibiting at least one myostatin biological activity in an animal, preferably a mammalian or avian species, preferably a human, in need thereof, comprising administering a therapeutically effective amount or prophylactically effective amount of an anti-myostatin monoclonal antibody of the invention to said mammalian or avian species. The invention further provides a method of enhancing muscle mass or treating or preventing a disease or disorder or condition ameliorated by neutralizing or antagonizing a myostatin bioactivity that comprises administering to a patient (e.g., a human) in need of such treatment or prevention a therapeutically or prophylactically effective amount of an antibody of the invention.

The invention embodies an antibody of the invention for use in therapy.

The invention embodies the use of an antibody of the invention for the preparation of a medicament for the treatment of muscle wasting, frailty, age-related sarcopenia, disuse atrophy and cachexia.

The invention embodies the use of an antibody of the invention for the preparation of a medicament for the prevention of muscle wasting, frailty, age-related sarcopenia, disuse atrophy and cachexia.

The invention embodies the method of treating muscle wasting, frailty, age-related sarcopenia, disuse atrophy and cachexia in a mammal, preferably a human, in need thereof by administering a therapeutically effective amount of an antibody of the invention.

The invention embodies the method of preventing muscle wasting, frailty, age-related sarcopenia, disuse atrophy and cachexia in a mammal, preferably a human, in need thereof by administering a prophylactically effective amount of an antibody of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Antibody Characterization

The invention presents an antibody that preferentially binds myostatin over GDF 11 and is more stable than the parent antibody (i.e., more resistant to chemical degradation than the parent antibody) while maintaining, or improving, the binding affinity for myostatin as exhibited by the parent antibody. In one embodiment, a labile Asn residue in CDRL3 of the parent antibody, Mab C12 or Mab 510C2 as shown in FIG. 2 (see Original Patent), is substituted with a different amino acid.

Preferred amino acid substitutions in an antibody of the invention are those which: (1) reduce susceptibility to spontaneous chemical degradation, i.e., cleavage or deamidation, and (2) maintain the antigen-antibody binding affinity as exhibited by the parent antibody. In one preferred embodiment, the Asn residue in the CDRL3 of Mab C12 is replaced with H or R. In another preferred embodiment, the Asn residue in the CDRL3 of Mab 510C2 is replaced with H, S, T, or A.

In one embodiment, an antibody of the invention is resistant to spontaneous chemical degradation, i.e., 100%, 99%, 98%, 97%, 96%, or 95% of the antibodies are not cleaved when present in an antibody solution for a time and temperature selected from the group consisting of: one year at 4.degree. C., six months at 25.degree. C., two months at 37.degree. C., and four weeks at 40.degree. C. in an antibody solution. An antibody solution is any solution suitable for a pharmaceutical composition comprising an antibody. An exemplary antibody solution comprises 1 mg/mL of the antibody, 10 mM phosphate, pH 7.4 and 150 mM NaCl.

In one embodiment, antibodies of the invention are further characterized by having a strong binding affinity (K.sub.D) for myostatin, i.e., less than about 3.times.10.sup.-8 M, 1.times.10.sup.-8M or 1.times.10.sup.-9M, preferably less than about 9.times.10.sup.-10 M, 8.7.times.10.sup.-10 M or more preferably, less than about 8.times.10.sup.-11 M. Alternatively, the antibodies of the invention are characterized by a K.sub.D for myostatin of no greater than about 3.times.10.sup.-8M, 1.times.10.sup.-8M, 1.times.10.sup.-9M or 9.times.10.sup.-10 M, more preferably no greater than about 8.7.times.10.sup.-10 M and most preferably no greater than about 8.times.10.sup.-11 M. The binding affinity of an antibody of the invention is similar to or better than that of its parent antibody.

Preferably, antibodies of the invention characterized by a strong binding affinity as described above also have an IC.sub.50 of less than 25 nM, 20 nM, 16 nM, 14 nM, 10 nM, 9 nM, 6 nM, or 5.2 nM in the in vitro myostatin/SBE reporter assay as set forth in Example 4 herein. Preferably the IC.sub.50 of an antibody of the invention is similar to or better than that of its parent antibody. All antibodies of the invention are significantly less reactive with GDF-11 than with myostatin, i.e., they preferentially bind myostatin over GDF-11.

Antibodies of the invention are preferably chimeric, humanized or human antibodies or antigen-binding portions thereof and preferably bind to myostatin within the region of the mature form of myostatin spanning amino acids 40-64 or more preferably within the region of the mature form of myostatin spanning amino acids 43-57 and/or 45-59. Furthermore, antibodies of the invention neutralize a myostatin biological activity in vivo or in vitro. Specific binding of anti-myostatin monoclonal antibodies of the invention allows the antibodies of the invention to be used as therapeutics or prophylactics for myostatin-associated conditions, diseases or disorders, i.e., conditions, diseases or disorders which benefit from lowering myostatin levels or antagonizing or inhibiting a myostatin biological activity. Further, antibodies of the invention may be used to diagnose or monitor conditions, diseases or disorders which benefit from an altered level or bioactivity of myostatin or to determine the level of myostatin in a sample.

The anti-myostatin monoclonal antibodies of the invention bind an antigenic epitope discovered to be localized within amino acids 40-64 (SEQ ID NO: 1 for human) of mature myostatin preferably within amino acids 43-57 and/or 45-59 of mature myostatin. Furthermore, a myostatin immunogenic epitope of the invention is localized within amino acids 40-64 of mature myostatin (SEQ ID NO: 1 for human), preferably within amino acids 43-57 and/or 45-59 of mature myostatin of any mammalian or avian species. An immunogenic epitope of the invention is also contemplated to be an antigenic epitope. Additionally, residues of myostatin outside of the amino acids 40-64 may affect the conformational structure of the antigenic domain and thereby alter binding of an antibody of the invention to the antigenic epitope.

Single chain antibodies, and chimeric, humanized antibodies, as well as chimeric or CDR-grafted single chain antibodies, and the like, comprising portions derived from different species, are also encompassed by the present invention and the term "antibody" or "modified antibody." The various portions of these antibodies can be joined together chemically by conventional techniques, synthetically, or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein.

In addition, functional portions of antibodies, including antigen-binding portions of chimeric, humanized, human or single chain antibodies, can also be produced. Functional portions of the foregoing antibodies retain at least one antigen-binding function and/or biological function or bioactivity of the full-length antibody from which they are derived. Preferred functional portions retain an antigen-binding function of a corresponding full-length antibody (e.g., the ability to bind a mammalian mature form of myostatin). Particularly preferred functional portions or fragments retain the ability to inhibit one or more functions or bioactivities characteristic of a mammalian mature myostatin, such as a binding activity, a signaling activity, and/or stimulation of a cellular response. For example, in one embodiment, a functional portion or fragment can inhibit the interaction of mature myostatin with one or more of its ligands and/or can inhibit one or more receptor-mediated functions.

Antibody portions or fragments capable of binding to mature myostatin or a portion thereof (preferably within amino acids 40-64, 43-57 and/or 45-59 of mature myostatin), include, but are not limited to, Fv, Fab, Fab' and F(ab').sub.2 fragments and are encompassed by the invention. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For instance, papain or pepsin cleavage can generate Fab or F(ab').sub.2 fragments, respectively. The smallest antigen-binding fragment is the Fv, which consists of the HCVR and the LCVR domains. The Fab fragment consists of the HCVR-CH1 and LCVR-CL domains covalently linked by a disulfide bond between the constant regions. To overcome the tendency of non-covalently linked HCVR and LCVR domains in the Fv to dissociate when co-expressed in a host cell, a so-called single chain (sc) Fv fragment (scFv) can be constructed, in which a flexible and adequately long polypeptide links either the C-terminus of the HCVR to the N-terminus of the LCVR or the C-terminus of the LCVR to the N-terminus of the HCVR. A commonly used linker is a 15-residue (Gly.sub.4Ser).sub.3 peptide, but other linkers are also known in the art. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons has been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab').sub.2 heavy chain portion can be designed to include DNA sequences encoding the CH.sub.1 domain and hinge region of the heavy chain.

Identification of Labile Asn Residue(s)

Many methods are available to detect and quantify the spontaneous modifications of labile Asn residues in a protein, e.g., antibody. Deamidation, a modification resulting in the conversion of an asparagine residue to a mixture of isoaspartate and aspartate which may provide a signal for protein degradation, introduces negative charge and changes the protein mass (NH.sub.2 vs OH, .DELTA.=1 Da) and hydrophobicity. Separation techniques including electronic and chromatographic methods, such as, IEF, cIEF, urea gel electrophoresis, reversed-phase PHLC, ion exchange PHLC, and hydrophilic interaction, may be used to separate or isolate deamidated or cleaved forms of an antibody, or a protein or a polypeptide. Ion-exchange chromatography is widely used to isolate deamidated proteins. The location and extent of the spontaneous modifications of an antibody, or a protein, or a polypeptide may be further characterized by liquid-chromatography/mass spectrometry (LC/MS) and N-terminal sequencing.

Substitution of Labile Asn Residue(s) or Other Modification of Antibodies

To eliminate, e.g., by amino acid substitution, a labile Asn residue in an antibody, or other protein, the labile Asn residue may be substituted with a single amino acid. It is desirable that the amino acid substitution does not alter (i.e., negatively), or minimally alters (e.g., 10%, 5%, 4%, 3%, 2% or less) the antibody:antigen binding affinity. It is further desirable that the amino acid substitution does not alter (i.e., negatively), or minimally alters antibody neutralization, epitope specificity and the ability of the antibody to preferentially bind myostatin over GDF-11. An ELISA may be used to determine the effects of the individual amino acid substitutions on the binding affinity of an antibody of the invention to myostatin or an antigenic epitope thereof and the ELISA value compared to that of its parent antibody (e.g., C12 or 510C2 antibody) binding to the same antigen. Alternatively, a BIACORE.RTM. or KINEXA.RTM. assay may be used to measure binding affinity of an antibody.

Antibodies of the invention may be further mutagenized (or mutagenized prior to elimination of the Asn residue whose presence contributes to the parent antibody instability), e.g., within the CDR domain(s) to create a variant antibody with an optimized property of interest, e.g., binding affinity, IC.sub.50, specificity, etc. An antibody of the invention generated by amino acid substitution is preferred and has at least one amino acid residue of the parent antibody molecule removed and a different residue inserted in its place. The sites of greatest interest for such substitutional mutagenesis include the CDR regions, but FR alterations are also contemplated.

A convenient way for generating substitutional variants is affinity maturation using phage display. Briefly, several CDR region sites are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity, specificity, IC.sub.50) as herein disclosed. In order to identify candidate CDR region sites for modification, alanine scanning mutagens can be performed to identify CDR region residues contributing significantly to antigen binding. Alternatively, or in addition, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and myostatin. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein or known in the art. Alternatively, or in addition, random mutagenesis may be performed on one or more CDR sequences at one or more residue positions, either while the CDR is operably linked to the variable region or while the CDR is independent of other variable region sequence and then the altered CDR returned to a variable region using recombinant DNA technology. Once such variant antibodies are generated and expressed, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.

Antibody Expression

The present invention is also directed to cell lines that express an anti-myostatin monoclonal antibody of the invention or portion thereof. Creation and isolation of cell lines producing a monoclonal antibody of the invention can be accomplished using standard techniques known in the art. Preferred cell lines include COS, CHO, SP2/0, NS0 and yeast (available from public repositories such as ATCC, American Type Culture Collection, Manassas, Va.).

A wide variety of host expression systems can be used to express an antibody of the present invention including prokaryotic (bacterial) and eukaryotic expression systems (such as yeast, baculovirus, plant, mammalian and other animal cells, transgenic animals, and hybridoma cells), as well as phage display expression systems. An example of a suitable bacterial expression vector is pUC119 and a suitable eukaryotic expression vector is a modified pcDNA3.1 vector with a weakened DHFR selection system. Other antibody expression systems are also known in the art and are contemplated herein.

An antibody of the invention can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. To express an antibody recombinantly, a host cell is transformed, transduced, infected or the like with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and/or heavy chains of the antibody such that the light and/or heavy chains are expressed in the host cell. The heavy chain and the light chain may be expressed independently from different promoters to which they are operably linked in one vector or, alternatively, the heavy chain and the light chain may be expressed independently from different promoters to which they are operably linked in two vectors--one expressing the heavy chain and one expressing the light chain. Optionally the heavy chain and light chain may be expressed in different host cells. Preferably, the recombinant antibodies are secreted into the medium in which the host cells are cultured, from which the antibodies can be recovered or purified. Standard recombinant DNA methodologies are used to obtain antibody heavy and light chain genes, incorporate these genes into recombinant expression vectors, and introduce the vectors into host cells.

An isolated DNA encoding a HCVR region can be converted to a full-length heavy chain gene by operably linking the HCVR-encoding DNA to another DNA molecule encoding heavy chain constant regions (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art. See, e.g., Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991). DNA fragments encompassing these regions can be obtained e.g., by standard PCR amplification. The heavy chain constant region can be of any type, (e.g., IgG, IgA, IgE, IgM or IgD), class (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3 and IgG.sub.a) or subclass constant region and any allotypic variant thereof as described in Kabat (supra). Alternatively, the antigen binding portion can be a Fab fragment, Fab' fragment, F(ab').sub.2 fragment, Fd, or a single chain Fv fragment (scFv). For a Fab fragment heavy chain gene, the HCVR-encoding DNA may be operably linked to another DNA molecule encoding only a heavy chain CH1 constant region.

An isolated DNA encoding a LCVR region may be converted to a full-length light chain gene (as well as to a Fab light chain gene) by operably linking the LCVR-encoding DNA to another DNA molecule encoding a light chain constant region, CL. The sequences of human light chain constant region genes are known in the art. See, e.g., Kabat, supra. DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

To create an scFv gene, the HCVR- and LCVR-encoding DNA fragments are operably linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly.sub.4-Ser).sub.3, such that the HCVR and LCVR sequences can be expressed as a contiguous single-chain protein, with the LCVR and HCVR regions joined by the flexible linker.

To express an antibody of the invention, a DNA encoding a partial or full-length light and/or heavy chain, obtained as described above, is inserted into an expression vector such that the gene is operably linked to transcriptional and translational control sequences. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors or, more typically, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods. Additionally, the recombinant expression vector can encode a signal peptide that facilitates secretion of the anti-myostatin monoclonal antibody light and/or heavy chain from a host cell. The anti-myostatin monoclonal antibody light and/or heavy chain gene can be cloned into the vector such that the signal peptide is operably linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide.

In addition to the antibody heavy and/or light chain gene(s), a recombinant expression vector of the invention carries regulatory sequences that control the expression of the antibody chain gene(s) in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals), as needed, that control the transcription or translation of the antibody chain gene(s). The design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma virus.

In addition to the antibody heavy and/or light chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and one or more selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced. For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR-minus host cells with methotrexate selection/amplification), the neo gene (for G418 selection), and glutamine synthetase (GS) in a GS-negative cell line (such as NS0) for selection/amplification.

For expression of the light and/or heavy chains, the expression vector(s) encoding the heavy and/or light chains is introduced into a host cell by standard techniques e.g., electroporation, calcium phosphate precipitation, DEAE-dextran transfection, transduction, infection and the like. Although it is theoretically possible to express the antibodies of the invention in either prokaryotic or eukaryotic host cells, eukaryotic cells are preferred, and most preferably mammalian host cells, because such cells, are more likely to assemble and secrete a properly folded and immunologically active antibody. Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary (CHO cells), NS0 myeloma cells, COS cells, and SP2/0 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the host cell and/or the culture medium using standard purification methods.

Host cells can also be used to produce portions, or fragments, of intact antibodies, e.g., Fab fragments or scFv molecules by techniques that are conventional. It will be understood by a skilled artisan that variations on the above procedure are within the scope of the present invention. For example, it may be desirable to transfect a host cell with DNA encoding either the light chain or the heavy chain of an antibody of this invention. Recombinant DNA technology may also be used to remove some or all the DNA encoding either or both of the light and heavy chains that is not necessary for binding to myostatin. The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the invention.

In a preferred system for recombinant expression of an antibody of the invention, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into CHO cells by e.g., calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operably linked to enhancer/promoter regulatory elements to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformant host cells are cultured to allow for expression of the antibody heavy and light chains and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the antibody from the culture medium. Antibodies, or antigen-binding portions thereof, of the invention can be expressed in an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see, e.g., Taylor, et al., Nucleic Acids Res. 20:6287-95, 1992).

Uses

Antibodies of the present invention are useful in therapeutic, prophylactic, diagnostic and research applications as described herein. An antibody of the invention may be used to diagnose a disorder or disease associated with the expression of human myostatin. In a similar manner, the antibody of the invention can be used in an assay to monitor myostatin levels in a subject being treated for a myostatin-associated condition.

Therapeutic Uses for the Antibody

Myostatin plays a role in muscle development and a number of related disorders or diseases. In adults, myostatin mRNA is primarily detected in skeletal muscle although lower concentrations are also found in adipose tissue and cardiac tissue (Sharma, M., et al, J. Cell Physiol. 180:1, 1999). Myostatin knockout mice have two- to three-fold greater muscle mass than their wild type littermates. The increased muscle mass is the result of fiber hypertrophy and hyperplasia (McPherron, A., et al. Nature 387:83-90, 1997 and Zhu, X. et al., FEBS Letters 474:71). In addition, the myostatin knockout mice accumulate less fat than their wild type littermates but otherwise appear normal and healthy. Myostatin has also been recently shown to be an important regulator of adipogenesis (Rebbapragada, A., et al., Mol. and Cell. Bio. 23:7230-7242, 2003). Additionally, bone structure and content has been recently studied in myostatin deficient mice (Hamrick M. W., et al., J. Orthopaedic Research 21:1025, 2003; Hamrick, M. W., et al., Calcif Tissue Int 71:63, 2002.

Therefore, a composition comprising an anti-myostatin monoclonal antibody of the invention may be used to increase muscle mass, increase bone density, decrease muscle wasting, or may be useful for the treatment or prevention of conditions wherein the presence of myostatin causes or contributes to undesirable pathological effects or decrease of myostatin levels has a therapeutic benefit in mammals, preferably humans. Preferably a composition comprising an anti-myostatin monoclonal antibody of the invention may be used to increase muscle mass.

Preferably an antibody of the invention may be used in the treatment or prevention of muscle wasting, muscle injury, surgery, repair of damaged muscle, frailty, age-related sarcopenia, disuse atrophy, osteoporosis, osteoarthritis, ligament growth and repair, obesity, suppression of body fat accumulation, obesity, muscular dystrophy of any type, critical care myopathy, alcoholic myopathy, cachexia (e.g., cancer-related or HIV-induced, or resulting from COPD, chronic lung disease, recovery from sepsis, renal failure, liver failure, cardiac failure or disease), metabolic syndrome, post-burn muscle wasting, and Type II diabetes. More preferably, an antibody of the invention may be used in the treatment or prevention of muscle wasting, frailty, age-related sarcopenia, disuse atrophy and cachexia. Most preferably, an antibody of the invention may be used in the treatment or prevention of disuse atrophy or cachexia.

Disuse atrophy may result from numerous causes or incidents including any disorder or disease or state which leads to prolonged immobility or disuse or bed rest including, but not limited to, solid organ transplant, joint replacement, stroke, spinal cord injury, recovery from severe burn, sedentary chronic hemodialysis, post-sepsis recovery and exposure to microgravity. Since myostatin is highly conserved in sequence and function across species, the antibodies of the invention may be used to increase muscle mass, increase bone density or treat or prevent conditions in non-human mammals or avian species [e.g., domestic animals (e.g., canine and feline), sports animals (e.g., equine), food-source animals (e.g., bovine, porcine and ovine), avian species (e.g., chicken, turkey, other game birds or poultry)] wherein the presence of myostatin causes or contributes to undesirable pathological effects or decrease of myostatin levels has a therapeutic benefit.

The use of an anti-myostatin monoclonal antibody of the present invention for treating or preventing of at least one of the aforementioned disorders in which myostatin activity is detrimental or which benefits for decreased levels of bioactive myostatin is contemplated herein. Additionally, the use of an anti-myostatin monoclonal antibody of the present invention for use in the manufacture of a medicament for the treatment of at least one of the aforementioned disorders is contemplated.

As used herein, the terms "treatment", "treating", and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be therapeutic in terms of a partial or complete cure for a disease and/or adverse affect attributable to the disease. "Treatment", as used herein, includes administration of a compound of the present invention for treatment of a disease or condition in a mammal, particularly in a human, and includes: (a) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease or disorder or alleviating symptoms or complications thereof. Dosage regimens may be adjusted to provide the optimum desired response.

As used herein, the term, "preventing" refers to completely or partially preventing a disease or symptom thereof. "Prevention" as used herein, includes administration of a compound of the present invention for prevention the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it.

Composition

An antibody of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. The compounds of the invention may be administered alone or in combination with a pharmaceutically acceptable carrier, diluent, and/or excipients, in single or multiple doses. The compositions for administration are designed to be appropriate for the selected mode of administration, and pharmaceutically acceptable diluents, carrier, and/or excipients such as dispersing agents, buffers, surfactants, preservatives, solubilizing agents, isotonicity agents, stabilizing agents and the like are used as appropriate. Said compositions are designed in accordance with conventional techniques as in e.g., Remington, The Science and Practice of Pharmacy, 19.sup.th Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa. 1995 which provides a compendium of formulation techniques as are generally known to practitioners.

A composition of the invention preferably is a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody of the invention. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the antibody may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effect of the antibody, are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount.

A therapeutically-effective or prophylactically-effective amount is at least the minimal dose, but less than a toxic dose, of an active agent which is necessary to impart therapeutic benefit to a subject. Stated another way, a therapeutically-effective amount of an antibody of the invention is an amount which in mammals, preferably humans, increases muscle mass, increases bone density, or treats conditions wherein the presence of myostatin causes or contributes to undesirable pathological effects or decrease in myostatin levels results in a beneficial therapeutic effect in a mammal, preferably a human, including, but not limited to, muscle wasting, muscle injury, surgery frailty, age-related sarcopenia, disuse atrophy, osteoporosis, osteoarthritis, ligament growth and repair, obesity, suppression of body fat accumulation, muscular dystrophy of any type, critical care myopathy, cachexia (e.g., cancer-related or HIV-induced, or resulting from COPD, renal failure, liver failure, cardiac failure or disease), metabolic syndrome and Type II diabetes. Disuse atrophy may result from numerous causes or incidents including any disorder or disease or state which leads to prolonged immobility or disuse or bed rest including, but not limited to, solid organ transplant, joint replacement, stroke, spinal cord injury, recovery from severe burn, sedentary chronic hemodialysis, post-sepsis recovery and exposure to microgravity.

The route of administration of an antibody of the present invention is parenteral. Preferably, the antibodies of the invention can be incorporated into a pharmaceutical composition suitable for parenteral administration. The term parenteral as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, or intraperitoneal administration. Peripheral systemic delivery by intravenous or intraperitoneal or subcutaneous injection is preferred. Suitable vehicles for such injections are straightforward in the art.

The composition typically must be sterile and stable under the conditions of manufacture and storage in the container provided, including e.g., a sealed vial or syringe. Therefore, compositions may be sterile filtered after making the formulation, or otherwise made microbiologically acceptable. A typical composition for intravenous infusion could have a volume from 20-1000 mL of fluid, such as sterile Ringer's solution, physiological saline, dextrose solution and Hank's solution and a therapeutically effective dose, (e.g., 1 to 1000 mg) of antibody concentration. Dose may vary depending on the type and severity of the disease. As is well known in the medical arts, dosages for any one subject depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. A typical dose can be, for example, in the range of 1 to 1000 mg; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. A typical dosing regimen may occur daily, weekly, biweekly or monthly. A typical parenteral dosage regimen can be about 10 .mu.g/kg to about 20 mg/kg of total body weight, preferably from about 20 .mu.g/kg to about 10 mg/kg. Progress may be monitored by periodic assessment. For repeated administrations, depending on the condition, the treatment may be repeated until a desired suppression of disease symptoms occurs or desired prevention of disease symptoms occurs. However, other dosage regimens may be useful and are not excluded herefrom.

These suggested amounts of antibody are subject to a great deal of therapeutic discretion. The key factor in selecting an appropriate dose and scheduling is the result obtained. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the antibody, the particular type of antibody, the method of administration, the scheduling of administration, and other factors known to medical practitioners.

Therapeutic agents of the invention may be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use. Lyophilization and reconstitution can lead to varying degrees of antibody activity loss. Dosages may have to be adjusted to compensate. Generally, pH between 6 and 8 is preferred.

Articles of Manufacture.

In another embodiment of the invention, an article of manufacture containing materials useful for the treatment or prevention of the disorders or conditions described above is provided. The article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition of the invention which is effective for preventing or treating the disorder or condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition is an anti-myostatin antibody of the invention. The label on, or associated with, the container indicates that the composition is used for treating the condition of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
 

Claim 1 of 14 Claims

1. An anti-myostatin monoclonal antibody comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein said HCVR comprises: a) a peptide at CDRH1 with a sequence as shown in SEQ ID NO: 23, b) a peptide at CDRH2 with a sequence as shown in SEQ ID NO: 25, c) a peptide at CDRH3 with a sequence as shown in SEQ ID NO: 27, and wherein said LCVR comprises: a) a peptide at CDRL1 with a sequence as shown in SEQ ID NO: 13, b) a peptide at CDRL2 with a sequence as shown in SEQ ID NO: 14, and c) a peptide at CDRL3 with a sequence selected from the group consisting of SEQ ID NO: 16, 17, 18 and 19.

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