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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




PCT 

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification ? : 

G01N 33/50, 33/68, C07K 14/51, 14/475, 
7/08, 7/06, A01K 67/027, C12N 9/00, 
15/11 



A2 



(11) International Publication Number: 
(43) International Publication Date: 



WO 00/43781 

27 July 2000 (27.07.00) 



(21) International Application Number: PCT/USOO/01552 

(22) International Filing Date: 21 January 2000 (21.01.00) 



(30) Priority Data: 

60/116,639 
60/138,363 



2 1 January 1 999 (21 .01 .99) US 
10 June 1999(10.06.99) US 



(63) Related by Continuation (CON) or Continuation-in-Part 
(CIP) to Earlier Applications 

US 60/1 16,639 (CIP) 

Filed on 21 January 1999 (21.01.99) 

US 60/138,363 (CIP) 

Filed on 10 June 1999 (10.06.99) 



(71) Applicant (for alt designated States except US): METAMOR- 
PHIX, INC. [US/US]; 1450 South Rolling Road, Baltimore, 
MD 21227 (US). 

(72) Inventors; and 

(75) Inventors/Applicants (for US only): TOPOUZIS, Stavros 
[GR/US]; Apartment C305, 3821 14th Avenue, W., Seattle, 
WA 98119 (US). WRIGHT, Jill, F. [US/USJ; 131 Paden 
Ct., Forest Hill, MD 21050 (US). RATOVITSKI, Tamara 



[IL/US]; 6509 Hazel Thicket Terrace, Columbia, MD 
21044 (US). LIANG, Li-Fang [US/US]; 6645 Hunter 
Road, Elkridge, MD 21075 (US). BRADY, James, L., Jr. 
[US/US]; Apartment 821. 4977 Battery Lane, Bethesda, 
MD 20814 (US). SINHA, Debasish [IN/US]; 18022 
Fertile Meadow Court, Gaithersburg, MD 20877 (US). 
Y AS WEN-CORKER Y, Linda [US/US]; 1606 Dublin 
Drive, Silver Spring, MD 20902 (US). 

(74) Agents: MANDRAGOURAS, Amy, E. et al.; Lahive & 
Cockfield, LLP, 28 State Street, Boston, MA 02109 (US). 



(81) Designated States: AE, AL, AM, AT, AU, AZ, BA, BB, BG, 
BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, 
ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, 
KE, KG, KP, KR, KZ, LC. LK, LR, LS, LT, LU, LV, MA, 
MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, 
SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, 
US, UZ, VN, YU, ZA, ZW, ARIPO patent (GH, GM, KE, 
LS, MW, SD, SL, SZ, TZ, UG, ZW), Eurasian patent (AM, 
AZ, BY, KG, KZ, MD, RU, TJ. TM), European patent (AT, 
BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, 
MC, NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM, 
GA, GN, GW, ML, MR, NE, SN, TD, TG). 



Published 

Without international search report and to be republished 
upon receipt of that report. 



(54) Title: GROWTH DIFFERENTIATION FACTOR INHIBITORS AND USES THEREFOR 



OEAVAGESfTE. 



WKSOF-8 



RAG 



PROOOMAM 



MATURE GDf -8 



RAG 



M-GDF-8 



GOF-6 



PRECURSOR 



B 



CMV 



SIGNAL 
PEPTIDE 



RAG 



PRO-DOMAIN 



(57) Abstract 



Inhibitors of GDF proteins, such as GDF-8 or GDF-11, are disclosed. Also disclosed are methods for identifying and using the 
inhibitors, for example, to generate transgenic animals and to treat a variety of diseases. 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT. 



AL 


Albania 


ES 


Spain 


LS 


Lesotho 


SI 


Slovenia 


AM 


Armenia 


FI 


Finland 


LT 


Lithuania 


SK 


Slovakia 


AT 


Austria 


FR 


France 


L4J 


Luxembourg 


SN 


Senegal 


AU 


Australia 


GA 


Gabon 


LV 


Latvia 


sz 


Swaziland 


AZ 


Azerbaijan 


GB 


United Kingdom 


MC 


Monaco 


TD 


Chad 


BA 


Bosnia and Herzegovina 


GE 


Georgia 


MD 


Republic of Moldova 


TG 


Togo 


BB 


Barbados 


GH 


Ghana 


MG 


Madagascar 


TJ 


Tajikistan 


BE 


Belgium 


GN 


Guinea 


MK 


The former Yugoslav 


TM 


Turkmenistan 


BF 


Burkina Faso 


GR 


Greece 




Republic of Macedonia 


TR 


Turkey 


BG 


Bulgaria 


HU 


Hungary 


ML 


Mali 


TT 


Trinidad and Tobago 


Bj 


Benin 


IE 


Ireland 


MN 


Mongolia 


UA 


Ukraine 


BR 


Brazil 


IL 


Israel 


MR 


Mauritania 


UG 


Uganda 


BY 


Belarus 


IS 


Iceland 


MW 


Malawi 


US 


United States of America 


CA 


Canada 


IT 


Italy 


MX 


Mexico 


uz 


Uzbekistan 


CF 


Central African Republic 


JP 


Japan 


NE 


Niger 


VN 


Viet Nam 


CC 


Congo 


KE 


Kenya 


NL 


Netherlands 


YU 


Yugoslavia 


CM 


Switzerland 


KG 


Kyrgyzstan 


NO 


Norway 


ZW 


Zimbabwe 


CI 


C6te d'lvoire 


KP 


Democratic People's 


NZ 


New Zealand 






CM 


Cameroon 




Republic of Korea 


PL 


Poland 






CN 


China 


KR 


Republic of Korea 


PT 


Portugal 






CU 


Cuba 


KZ 


Kazakstan 


RO 


Romania 






CZ 


Czech Republic 


LC 


Saint Lucia 


RU 


Russian Federation 






DE 


Germany 


U 


Liechtenstein 


SD 


Sudan 






DK 


Denmark 


LK 


Sri Lanka 


SE 


Sweden 






EE 


Estonia 


LR 


Liberia 


SG 


Singapore 







WO 00/43781 PCt/USOO/01552 

- 1 - 

GROWTH DIFFERENTIATION FACTOR INHIBITORS AND USES 

THEREFOR 

Related Applications 

5 This application claims priority to U.S. provisional Application No. 60/1 16,639, 

filed on January 21, 1999, and U.S. provisional Application No. 60/138,363, filed on 
June 10, 1999, incorporated herein in their entirety by this reference. 

Field of the Invention 

10 The invention relates to inhibitors of GDF proteins, such as GDF-8 and GDF-1 1 

proteins, as well as methods of identifying these inhibitors and methods of using them. 

Background of the Invention 

Growth and Differentiation Factor-8 (GDF-8), also known as Myostatin, is a 

1 5 member of the Transforming Growth Factor-beta (TGF-jJ) superfamily of structurally- 
related growth factors, all of which are endowed with physiologically important growth- 
regulatory and morphogenetic properties (D.M. Kingsley et al, (1994) Genes Dev > 8, 
133-46; P.A. Hoodless et al. (1998) Curr. Topics Microbiol Immunol, 228, 235-72). 
Members of the TGF-P superfamily signal through a heteromeric protein kinase receptor 

20 complex. The family also includes Bone Morphogenetic Proteins (BMPs), Activins. 
lnhibins. Mullerian Inhibiting Substance, Glial-Derived Neurotrophic Factor, and a still 
growing number of Growth and Differentiation Factors (GDFs). Myostatin itself is 
highly expressed specifically in the developing and adult skeletal muscle and to a much 
lesser extent in fat. Myostatin seems to be implicated in a number of physiological 

25 processes, the best established of which is its ability to regulate skeletal muscle mass, 
hence its name "Myostatin". 

Myostatin "knock-out" mice generated by gene targeting develop normally, but 
have twice the normal skeletal muscle mass (A.C. McPherronst al. (1997) Nature, 387, 
83-90). The identification of two breeds ofdouble-muscled cattle, Belgian Blue and 

30 Piedmontese, and of the hypermuscular Compt mutant mouse, whose phenotypes are 
due to mutations of the Myostatin gene, suggests that Myostatin has the same 



WO 00/43781 PCT/US00/01SS2 

-2- 

developmental role in these two species (A.C. McPherron et al. (1997) PNAS, 94, 
12457-61; R. Kambaduret al. (1997) Genome Res., 7, 910-15; G. Szaboet al. (1998) 
Mammalian Genome \ 9, 671-2). Sequence data from various species indicate the GDF-8 
is highly conserved among vertebrates, suggesting that GDF-8 may have the same role 
5 across species (McPherron and Lee, (1997) PNAS, 94, 12457-61). 

In agreement with the above, recent studies have shown that HlV-infection- 
associated muscle wasting in humans is accompanied by increases in Myostatin protein 
expression (N.F. Gonzalez-Cadavid et al. (1998) PNAS, 95, 14938-43). Overall, this 
evidence strongly supports a pivotal role of GDF-8 (or Myostatin) in the control of 
10 skeletal muscle mass. 

Summary of the Invention 

The present invention is based, at least in part, on the identification of GDF-8 
inhibitory activity in media obtained from cells expressing GDF-8. Accordingly, in one 

15 aspect, the invention features a method for identifying a GDF inhibitor, e.g., a GDF-8 
inhibitor, such as a GDF polypeptide, which inhibits GDF, e.g., GDF-8, activity. In one 
embodiment, the inhibitor does not itself possess GDF, e.g., GDF-8 activity (e.g., the 
inhibitor is a GDF polypeptide which does not itself possess GDF activity). The method 
includes obtaining medium in which cells producing a GDF polypeptide have been 

20 cultured and testing the components of the medium for the ability to inhibit GDF 

activity, thereby identifying a GDF inhibitor. In one embodiment, the method includes 
performing chromatography on the medium before the medium is tested for the ability to 
inhibit GDF activity. In another embodiment, the method includes performing 
electrophoresis, e.g., preparative non-reducing or reducing SDS-PAGE, on fractions 

25 obtained from the chromatography and recovering the fractions, ^.g., by electrocution. 
In other preferred embodiments, the cells expressing a GDF polypeptide are 
cells, e.g., CHO cells, which are transfected with a plasmid containing an insert 
encoding a GDF polypeptide. In still other preferred embodiments, the cells produce a 
GDF polypeptide, e.g., GDF-8, endogenously. Such cells include, for example, the 

30 rhabdomyosarcoma line RD (ATCC, CCL-1 36) and QM7 muscle myoblast (ATCC, 
CRL-1962). 



WO 00/43781 PCT/US00/01 552 

-3- 

In another aspect, the invention features a method for identifying a GDF 
inhibitor, which includes preparing polypeptide fragments of a GDF polypeptide and 
testing the fragments for the ability to inhibit GDF activity, thereby identifying a GDF 
inhibitor. The polypeptide fragments can be prepared by digesting a GDF polypeptide, 
5 e.g., a native GDF polypeptide or a recombinantly produced GDF polypeptide, or they 
can be recombinantly or synthetically synthesized. For example, the GDF polypeptide 
can be digested using a protease, including but not limited to trypsin, thermolysin, 
chymotrypsin, pepsin, or any other known protease. The polypeptides can then be 
isolated before they are tested for the ability to inhibit GDF activity. 
1 0 Prior to preparing the peptide fragments, the peptides also can be selected to 

elicit neutralization of endogenous GDF via an immune response. The testing of the 
polypeptide fragments can be performed by screening the polypeptide fragments for the 
ability to elicit an immune response resulting in the generation of GDF inhibitory 
antibodies. 

1 5 GDF polypeptide inhibitors can be any length sufficient to inhibit GDF activity. 

Typically, the polypeptides range from 5-10, 10-25, 25-40 or 40 or more amino acids in 
length. In certain preferred embodiments, the GDF polypeptide fragments are at least 5, 
10, 15, 20, 25, 30, 35, 40, or 45 amino acids in length. 

In another aspect, the invention features a GDF inhibitor, e.g., an inhibitor 

20 which may or may not possess GDF activity, which has one or more of the following 
characteristics: it can be isolated from medium in which cells (e.g., CHO) stably 
transfected with an expression plasmid containing an insert encoding GDF-8 or GDF-1 1 
have been isolated by column chromatography; it retains activity after heating at 100°C 
for up to 10 minutes; it retains or does not retain activity after reduction; and it retains 

25 activity after treatment with 6M Urea. In one embodiment, the invention features a 
GDF-8 inhibitor having a molecular weight of less than 70 kDa in a reducing and 
denaturing gel. 



WO 00/43781 PCT/US00/01552 

-4- 

In another aspect, the invention features a GDF inhibitor, e.g., a GDF 
polypeptide, identified by the methods described herein. 

In another aspect, the invention features a GDF inhibitor comprising the pro- 
domain of a GDF polypeptide, or a portion thereof. In preferred embodiments, the pro- 
5 domain of a GDF polypeptide or a portion thereof is glycosylated. 

In another aspect, the invention features a GDF inhibitor comprising a variant of 
a GDF polypeptide. In one embodiment, the GDF polypeptide variant is a cysteine 
variant. In another embodiment, the GDF polypeptide variant is a pro-domain variant. 
In yet another embodiment, the GDF polypeptide variant is a post-translational 
1 0 modification variant. In yet a further embodiment, the GDF polypeptide variant is a 
cleavage site variant. 

In another aspect, the invention features a GDF inhibitor comprising an isolated 
nucleic acid molecule which binds to and/or cleaves the RNA transcripts produced by 
genes encoding a GDF polypeptide. In certain embodiments, the nucleic acid is a 
1 5 ribozyme comprising the nucleotide sequence of any one of SEQ ID NOs: 1 -4. In other 
embodiments, the nucleic acid is an antisense molecule comprising the nucleotide 
sequence of any one of SEQ ID NOs: 5-24. 

In another aspect, the invention features an assay for measuring GDF activity, 
which can be used to identify GDF inhibitors. Suitable bioassays for testing inhibition 
20 og GDF activity include but are not limited to assays which detect the activity of 
muscle-specific enzymes, such as creatine kinase; assays which detect adipocyte 
differentiation, such as differentiation of 3T3-L1 pre-adipocytes; DNA replication 
assays; and transcription-based assays. 

In yet another aspect, the invention features a method for testing GDF inhibitors 
25 in a cell system, using a protein secretion-based assay. 

In a further aspect, the invention features transgenic animals in which expression 
of genes that encode the GDF inhibitors of the invention interfere with GDF polypeptide 
processing, GDF polypeptide secretion, and/or GDF polypeptide biological activity. 
In other aspects, the invention features methods of using GDF inhibitors of the 
30 invention, for example, to treat a variety of diseases. 



WO 00/43781 PCT/US00/015S2 

-5- 

Other features and advantages of the invention will be apparent from the 
following detailed description, and from the claims. 

Brief Description of the Drawings 

5 Figure 1 shows an ion exchange chromatogram (HQ column) for fractions A and 

B which was measured at 230 nm. 

Figure 2 shows an ion exchange chromatogram (SP column) of fraction A 
measured at 215 nm. The inset shows the gradient used in the elution where buffer A 
was 20 mM NaPhos pH 5.0 and buffer B was 20 mM NaPhos pH 5.0/2 M NaCl. 
10 Figure 3 shows an ion exchange chromatogram (SP column) of fraction B 

measured at 21 5 nm. The inset shows the gradient used in the elution where buffer A 
was 20 mM NaPhos pH 5.0 and buffer B was 20 mM NaPhos pH 5.0/2 M NaCl. 

Figure 4 shows a chromatogram of the reverse phase chromatography (C4 
column) for fraction A which elutes between 21-23 minutes. The gradient used for the 
15 elution is also shown where buffer A was 0.1% TFA and buffer B was 0.1% TFA in 
80% acetonitrile. 

Figure 5 shows a chromatogram of the reverse phase chromatography (C4 
column) for fraction B which elutes at 27-29 minutes. The gradient used for the elution 
is also shown where buffer A was 0. 1 % TFA and buffer B was 0.085% TFA in 80% 
20 acetonitrile. 

Figures 6A and B are graphs showing that fraction A inhibits the effect of GDF-8 
on cell proliferation. Figure 6A shows the effect of fraction A on DNA synthesis in G8 
myoblasts as measured by BrdU incorporation. Figure 6B shows the effect of fraction A 
on DNA synthesis in CCL-64 mink lung epithelial cells as measured by PH]-TdR 
25 incorporation. 

Figures 7 A and B are graphs showing the GDF-8-inhibitory activity of Fraction 
A monitored by transcription-based assays. Luciferase expression is derived from the 
reporter plasmid p(CAGA)]2~MLP {Figures 1 A and 7B). 

Figure 8 is a graph showing the specificity of the inhibitory effect of Fraction A 
30 for GDF-8. 



WO 00/43781 PCT/US00/01552 

-6- 

Figure 9 is a graph showing the GDF-8-inhibitory activity of Fraction B 
monitored by transcription-based assays. 

Figure 10 is a graph showing the specificity of the inhibitory effect of Fraction B 
for GDF-8. 

5 Figure 77 is a depiction of an amino acid sequence alignment of murine, rat, 

human, baboon, bovine, porcine, ovine, chicken, turkey, and zebrafish GDF-8. 

Figure 12 is a schematic representation of various GDF-8 constructs. Figure 12A 
shows the construct for the wild-type, full-length GDF-8. GDF-8 is processed in cells 
generating the mature GDF-8 and the remainder pro-peptide. Figure 12B shows the 

10 uncleavable mutant with the replaced cleavage site. This mutant is secreted as a 
precursor molecule. Figure 12C shows the pro-domain of GDF-8, expressed 
independently. 

Figure 13 shows the nucleotide and amino acid sequence of mouse GDF-8. The 
mutations introduced to generate GDF-8 variants are indicated. The cleavage site is 
15 boxed. The beginning and the end of the pro-domain after the disposal of the signal 
sequence are marked by triangles. The predicted site of N-linked glycosylation is 
underlined. 

Figure 14 is graph showing that the pro-region of GDF-8 can inhibit the activity 
of mature GDF-8. 

20 Figure 15 is a graph, showing the specificity of the inhibitory effect of pro- 

domain of GDF-8 for GDF-8. 

Figure 76 is a graph, showing dose-dependent inhibition of GDF-8 activity by 
the pro-domain. 

Figure 1 7 shows an alignment of the predicted amino acid sequence of human 
25 GDF-1 1 (top lines) and human GDF-8 (bottom lines). Vertical lines indicate identities. 
Dots represent gaps introduced in order to maximize the alignment. Numbers represent 
amino acid positions relative to the N-terminus. The putative proteolytic processing 
sites are shown by the open box. The conserved cysteine residues on the C-terminal 
region are shown by the shaded boxes. 
30 Figure 18 shows ribozyme target sites in the mouse GDF-8 mRNA sequence. 

Inverted arrows indicate the four ribozyme cleavage sites that were chosen in the mouse 



WO 00/43781 PCT/USO0/01552 

-7- 

GDF-8 mRNA sequence. Underlined nucleotides indicate regions that are 
complementary to sequences that flank the catalytic domain in each ribozyme. 
Translation initiation and termination codons for the GDF-8 protein are enclosed in 
boxes. 

5 Figure 19 shows the nucleotide sequences of four mouse GDF-8 ribozymes. The 

sequence of each ribozyme is shown underneath the mouse GDF-8 mRNA sequence to 
which it is complementary. Inverted arrows indicate target sites for ribozyme cleavage 
in the GDF-8 sequence. 

Figure 20 shows the nucleotide sequence of the DNA cassette containing the 

1 0 four tandemly arrayed ribozymes shown in Figure 19 and the inverted repeats. The 
sequences of the four ribozymes (SEQ ID NOs: 1-4) are highlighted and the inverted 
repeats are indicated by arrows underneath the sequence. 

Figure 21 shows selected ribozyme expression constructs. A ribozyme cassette 
with inverted repeats was ligated into three different plasmids. pGDF8R-l contains 2.8 

1 5 kb upstream of the mouse GDF-8 translation start site, exonl , intron 1 , and part of exon 
2 from the mouse GDF-8 gene ligated upstream of the ribozyme cassette. The 
polyadenylation signal from the SV40 large T antigen gene was ligated onto the other 
side of the ribozyme cassette. pMLCR-1 contains -1 500 bp upstream of the 
transcription start site for the rat myosin light chain 1 gene ligated upstream of the 

20 ribozyme cassette and a ~ 900 bp fragment containing the rat myosin light chain gene 
3'enhancer ligated downstream of the ribozyme cassette. pCMVR-1 contains 
approximately 500 bp of the human cytomegalovirus major immediate early gene 
promoter ligated upstream of the ribozyme cassette. Rib 1 , Rib 2, Rib 3, and Rib 4 
denote the regions of DNA encoding the four different ribozymes; blocks with inverted 

25 arrows correspond to the inverted repeats. 

Figure 22 shows the sequences of twenty oli gonucleotides (SEQ ID NOs: 5-24) 
complementary to the human GDF-8 cDNA sequence in the regions shown. Circled 
numbers beneath the oligonucleotide sequences indicate the 5* ends of the respective 
oligonucleotides. 



WO 00/43781 PCT/US00/01SS2 

-8- 

Figure 23 is a graph showing the results from an electrospray/ionization mass 
spectrometry analysis performed to further characterize Fraction B. The mass spec 
spectrum showed three peaks separated by 600 Da with the major component of 
molecular mass of 29472.0 Da. 
5 Figure 24 is a graph showing the effect of chemical modifications of the pro- 

domain of GDF-8 on its ability to inhibit the activity of mature GDF-8. 

Figure 25 is a graph showing the effect of the GDF-8 pro-domain purified from 
Fraction B on GDF-8 induction of a reporter plasmid in A204 cells. Figure 25 shows the 
results obtained using reporter plasmid p(CAGA) I2 -MLP. 
10 Figure 26 is a graph showing the effect of GDF-8 and TGF-P, on myogenic 

differentiation of C2C12 and chick primary myoblasts, as determined by a creatine 
kinase-based assay. 

Figure 27 is a graph showing the effects of GDF-8 on glucose uptake in 3T3-L1 
adipocytes. The data indicate that GDF-8 inhibits glucose uptake in 3T3-L1 cells in 
1 5 response to insulin 

Figure 28 is a graph showing that in vitro deglycosylation of Fraction B results in 
the loss of its inhibitory activity. 

Figure 29 is a graph showing the results from a transcription-based reporter 
activation assay performed to assess the biological activity of the GDF-8 complexes 
20 produced by QM-7 myoblasts. 

Figures 30A-B are graphs showing the results from a transcription-based reporter 
activation assay performed to assess the biological activity of supernatants from m- 
calpain treated QM-7 cells (transfected with either the WT-GDF-8-F construct or a 
control). 

25 Figure 31 is a graph showing the effect of TGF(i and GDF-8 on the induction of 

a luciferase reporter plasmid in CCL-64 cells. 

Figure 32 is a graph showing the effect of TGFp and GDF-8 on the induction of a 
luciferase reporter plasmid in RIB cells. 

Figure 33 is a graph showing the rescue of RIB cell responsiveness to TGF£ and 
30 GDF-8 by the re-introduction of the ALK-5 type I receptor into Rl B cells. 



WO 00/43781 



-9- 



PCT/US00/015S2 



Figure 34 is a graph showing the effect of TGFp and GDF-8 on the induction of a 
luciferase reporter plasmid in DR26 cells. 

Figure 35 is a graph showing the effect of the ActRIIB KR expression vector on 
the responsiveness of DR26 cells to GDF-8. 

5 

Detailed Description 

The present invention provides compositions and methods for inhibiting Growth 
Differentiation Factor, (GDF) proteins, as well as methods for identifying such 
inhibitors. 

10 In particular, GDF inhibitors of the present invention can be identified using a 

variety of screening methods which test peptides from GDF proteins, such as GDF-8 and 
GDF-1 1, or medium from cells producing GDF proteins, for GDF inhibitory activity. In 
one screening method, fragments of GDF proteins (i.e., peptides which comprise any 
portion of a GDF protein which is less than the whole, full length protein) are prepared 

1 5 and tested for GDF inhibitory activity. In a preferred embodiment, the fragments are 
derived from the Pro-region of the GDF protein, e.g., from the N-terminus of the pro- 
region (pro-domain) of the protein. For example, the fragments can be derived from the 
region of the pro-domain that is upstream of Arg 99 in GDF-8 (see Figure 11). The 
peptides can be selected by, for example, the ability to elicit antibodies against GDF 

20 proteins which inhibit GDF activity. Alternatively, GDF inhibitors can be identified and 
isolated directly from media of cells producing GDF proteins, as described in detail 
below. For example, in the studies described herein, two chromatography fractions, 
Fractions A and B, were isolated from media of CHO -cells expressing GDF-8. 

As used herein, the terms "GDF polypeptide" and "GDF protein" include 

25 members of the Transforming Growth Factor-beta (TGF-p) superfamily of structurally- 
related growth factors, all of which are endowed with physiologically important growth- 
regulatory and morphogenetic properties. This family of related growth factors is 
described in, for example, D.M. Kingsley et al. (1994) Genes Dev., 8, 133-46; P.A. 
Hoodless et al. (1998) Curr. Topics Microbiol Immunol, 228, 235-72, the contents of 



WO 00/43781 



- 10- 



PCT/US00/01552 



which are incorporated hrein by reference. Members of the TGF-P superfamily signal 
through a heteromeric protein kinase receptor complex. 

Accordingly, the terms "GDF polypeptide" and "GDF protein, as used herein, 
refer to proteins within the TGF-P superfamily, including Bone Morphogenetic Proteins 
5 (BMPs), Activins, lnhibins, Mullerian Inhibiting Substance, Glial-Derived Neurotrophic 
Factor, and a still growing number of Growth and Differentiation Factors (GDFs), 
including GDF-8 (Myostatin) and GDF-1 1.. 

As used herein, the term "GDF inhibitor" includes any agent capable of 
inhibiting activity, expression, processing, and/or secretion of a GDF protein including 
10 but not limited to peptides, peptidomimetics, ribozymes, anti-sense oligonucleotides, or 
small molecules which specifically inhibit the action of GDF proteins. The GDF 
inhibitor may possess GDF activity or, preferably is a GDF inhibitor which does not 
possess GDF activity. 

As used herein, the terms "GDF-8 inhibitor" and "GDF-1 1 inhibitor" include any 
15 agent capable of inhibiting GDF-8 or GDF-1 1 activity, expression, processing, and/or 
secretion including but not limited to peptides, peptidomimetics, ribozymes, anti-sense 
oligonucleotides, or small molecules which specifically inhibit the action of GDF-8 or 
GDF-1 1 while, preferably, leaving intact the activity of TGF-P or Activin or other 
members of the TGF-p superfamily. The GDF-8 or GDF-1 1 inhibitor may possess 
20 GDF-8 or GDF-1 1 activity or, preferably is a GDF-8 or GDF-1 1 inhibitor which does 
not possess GDF-8 or GDF-1 1 activity. 

As used herein, the term "GDF-8 or GDF-1 1 activity" includes any activity 
mediated by GDF-8 or GDF-1 1 . For example. GDF-8 is known to inhibit fibroblast 
differentiation to adipocytes, modulate the production of muscle-specific enzymes, e.g., 
25 creatine kinase, and modulate uptake glucose by cells, and stimulate myoblast cell 
proliferation. Accordingly, GDF-8 or GDF-1 1 inhibitors x:an be identified by, for 
example, testing GDF-8 or GDF-1 1 activity, as measured by the ability of GDF-8 or 
GDF-1 1 to interfere with the differentiation process of 3T3-L1 pre-adipocytes 
(fibroblasts) to adipocytes, the ability to modulate the activity of muscle-specific 



WO 00/43781 PCT/USOO/01552 

- 11 - 

enzymes, e.g., creatine kinase, the ability to modulate glucose uptake by cells, or the 
ability to stimulate myoblast cell proliferation. 

As used herein, the term "bioassay" includes any assay designed to identify a 
GDF inhibitor. The assay can be an in vitro or an in vivo assay suitable for identifying 
5 whether a GDF inhibitor can inhibit one or more of the biological functions of a GDF 
protein. Examples of suitable bioassays include DNA replication assays, transcription- 
based assays, creatine kinase assays, assays based on the differentiation of 3T3-L1 pre- 
adipocytes, assays based on glucose uptake in 3T3-L1 adipocytes, and immunological 
assays. 

10 

Various aspects of the present invention are described in further detail in the 
following subsections. To illustrate the invention, these subsections are directed to 
inhibtors of GDF-8 and GDF- 1 1 (two highly homologous GDF proteins). However, the 
the invention (e.g.. the following description and assays) can be applied to make and use 
1 5 inhibitors for any GDF protein and, therefore, should not be construed as limited to 
GDF-8 and GDF-11. 

/. PROTEIN INHIBITORS 

20 A. GDF-8 AND GDF-1 1 PEPTIDE INHIBITORS 

L Identification of GDF-8 or GDF-1 1 Inhibitors From Media in which Cells 
Expressing GDF-8 or GDF-1 1 Have been Cultured 

25 In one embodiment, the invention provides a method which involves obtaining 

medium in which cells producing GDF-8 or GDF-1 1 have been cultured; and testing the 
medium for the ability to inhibit GDF-8 or GDF-1 1 activity, thereby identifying a GDF- 
8 or GDF-11 inhibitor. 

The medium from which the GDF-8 or GDF-1 1 inhibitor is identified can 

30 contain cells which are transfected with a plasmid containing an insert encoding GDF-8 
or GDF-1 1 . Alternatively, the medium from which the GDF-8 or GDF-1 1 inhibitor is 



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identified can contain cells which produce GDF-8 or GDF-1 1 endogenously, i.e., native 
GDF-8 or GDF-1 1 As used herein, the term "native protein" includes a protein recovered 
from a source occurring in nature. 

The cell producing GDF-8 or GDF-1 1 can be any prokaryotic or eukaryotic cell. 
5 For example, the GDF-8 or GDF-1 1 protein can be expressed in bacterial cells such as 
E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells 
(CHO) or COS cells). Other suitable host cells are readily known to those skilled in the 
art. 

The plasmid containing an insert encoding GDF-8 or GDF-1 1 can be introduced 
1 0 into prokaryotic or eukaryotic cells via conventional transformation or transfection 
techniques. As used herein, the terms "transformation" and "transfection" are intended 
to refer to a variety of art-recognized techniques for introducing foreign nucleic acid 
(e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co- 
precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. 
15 Suitable methods for transforming or transfecting host cells can be found in Sambrook, 
et al. {Molecular Cloning: A Laboratory Manual 2nd, ed, Cold Spring Harbor 
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and 
other laboratory manuals. 

For stable transfection of mammalian cells, it is known that, depending upon the 
20 expression vector and transfection technique used, only a small fraction of cells may 
integrate the foreign DNA into their genome. In order to identify and select these 
integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is 
generally introduced into the host cells along with the gene of interest. Preferred 
selectable markers include those which confer resistance to drugs, such as G41 8, 
25 hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be 
introduced into a host cell on the same vector as that encoding the GDF-8 or GDF-1 1 
protein or can be introduced on a separate vector. Cells stably transfected with the 
introduced nucleic acid can be identified by drug selection (e.g., cells that have 
incorporated the selectable marker gene will survive, while the other cells die). 



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As used herein, the term "GDF-8" includes all known forms of GDF-8 including 
but not limited to human GDF-8, bovine GDF-8, chicken GDF-8, murine GDF-8, rat 
GDF-8, porcine GDF-8, ovine GDF-8, turkey GDF-8, baboon GDF-8, and fish GDF-8. 
These molecules are described in McPherron A. C. et al. (1997) Proc. Natl Acad. ScL 
5 94:12457-12461, the contents of which are incorporated herein by reference. The amino 
acid sequences for these proteins are shown in Figure 1L 

As used herein, the term "GDF-1 1 " includes all known forms of GDF-1 1 
including but not limited to human GDF-1 1 , bovine GDF-1 1, chicken GDF-1 1 , murine 
GDF-1 1 , rat GDF-1 1 , porcine GDF-1 1 , ovine GDF-1 1 , turkey GDF-1 1 , baboon GDF- 
10 1 1 , and fish GDF-1 1 . These molecules are described in, for example, U.S. Patent 

5,871,935 and in L.W. Gamer et al (1999) Developmental Biology, 208, 222-232, the 
contents of which are incorporated herein by reference. An alignment of the amino acid 
sequences of the GDF-8 and the GDF-1 1 proteins is shown in Figure 1 7. 

GDF-8 or GDF-1 1 inhibitors can be identified and isolated from media of cells 
15 expressing GDF-8 or GDF-1 1 using techniques known in the art for purifying peptides 
or proteins including ion-exchange chromatography, reverse-phase chromatography, gel 
filtration chromatography, ultrafiltration, electrophoresis, and immunoaffmity 
purification with antibodies specific for the GDF-8 or GDF-1 1 inhibitor, or a portion 
thereof. In one embodiment, the media obtained from cultures of cells which express 
20 GDF-8 or GDF-1 1 are subjected to high performance liquid chromatography (HPLC) as 
described in the Examples section. 

The samples obtained can then be tested for GDF-8 or GDF-1 1 inhibitory 
activity as described below. 

25 2. Identification of Peptides From GDF-8 or GDF-1 1 Which Inhibit GDF-8 or 

GDF-1 1 Activity 

In another aspect of the invention, GDF-8 or GDF-1 1 inhibitors are identified by 
screening fragments of GDF-8 or GDF-1 1 for inhibitory activity. GDF-8 or GDF-1 1 
30 fragments can be produced by a variety of art known techniques. For example, specific 
oligopeptides (approximately 10-25 amino acids-long) spanning the GDF-8 or GDF-1 1 



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sequence can be synthesized (e.g., chemically or recombinantly) and tested for their 
ability to inhibit GDF-8 or GDF-1 1 , for example, using the assays described herein. The 
GDF-8 or GDF-1 1 peptide fragments can be synthesized using standard techniques such 
as those described in Bodansky, M. Principles of Peptide Synthesis, Springer Verlag, - 
5 Berlin (1993) and Grant, G.A (ed.). Synthetic Peptides: A User's Guide, W.H. Freeman 
and Company, New York (1992). Automated peptide synthesizers are commercially 
available (e.g., Advanced ChemTech Model 396; Milligen/ Biosearch 9600). 

Alternatively, GDF-8 or GDF-1 1 fragments can be produced by digestion of 
native or recombinantly produced GDF-8 or GDF-1 1 by, for example, using a protease, 

10 e.g., trypsin, thermolysin. chymotrypsin, or pepsin. Computer analysis (using 
commercially available software, e.g. MacVector, Omega, PCGene, Molecular 
Simulation. Inc.) can be used to identify proteolytic cleavage sites. 

GDF-8 or GDF-1 1 peptides of the invention are preferably isolated. As used 
herein, an "isolated" or "purified" protein or biologically active peptide thereof is 

1 5 substantially free of cellular material or other contaminating proteins from the cell or 
tissue source from which the GDF-8 or GDF-1 1 protein or peptide is derived, or 
substantially free from chemical precursors or other chemicals when chemically 
synthesized. The language "substantially free of cellular material" includes preparations 
of GDF-8 or GDF-1 1 protein or peptide thereof in which the protein or peptide thereof 

20 is separated from cellular components of the cells from which it is isolated or 
recombinantly produced. In one embodiment, the language "substantially free of 
cellular material" includes preparations of GDF-8 or GDF-1 1 protein or peptide thereof 
having less than about 30% (by dry weight) of non-GDF-8 or GDF-1 1 protein or peptide 
thereof (also referred to herein as a "contaminating protein"), more preferably less than 

25 about 20% of non-GDF-8 or GDF-1 1 protein or peptide thereof, still more preferably 
less than about 1 0% of non-GDF-8 or GDF-1 1 protein or peptide thereof, and most 
preferably less than about 5% non-GDF-8 or GDF-1 1 protein or peptide thereof. When 
the GDF-8 or GDF-1 1 protein or biologically active portion thereof is recombinantly 
produced, it is also preferably substantially free of culture medium, i.e., culture medium 

30 represents less than about 20%, more preferably less than about 10%, and most 
preferably less than about 5% of the volume of the protein preparation. 



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A two-step method can be used to produce and isolate such proteolytically 
cleaved GDF-8 or GDF-1 1 peptides. The first step involves enzymatic digestion of the 
GDF-8 or GDF-1 1 protein. GDF-8 or GDF-1 1 can be produced either as a dimer from 
CHO cell conditioned media, as a monomer in E.coli or yeast, or isolated from cells 

5 which naturally produce GDF-8 or GDF-1 1 . Following purification of GDF-8 or GDF- 
1 1 monomers or dimers by, for example, HPLC chromatography, their enzymatic 
digestion is performed as described infra. The amino acids cleaved during the digestion 
depend on the specific protease used in the experiment as is known in the art. For 
example, if the protease of choice were trypsin, the cleavage sites would be amino acids 

10 arginine and lysine. The GDF-8 or GDF-1 1 protein can be digested using one or more 
of such proteases. 

After the digestion, the second step involves the isolation of peptide fractions 
generated by the protein digestion. This can be accomplished by, for example, high 
resolution peptide separation as described infra. Once the fractions have been isolated, 
1 5 their GDF-8 or GDF-1 1 inhibitory activity can be tested for by an appropriate bioassay, 
as described below. 

The proteolytic or synthetic GDF-8 or GDF-1 1 fragments can comprise as many 
amino acid residues as are necessary to inhibit, e.g., partially or completely. GDF-8 or 
GDF-1 1 function, and preferably comprise at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 

20 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length. 

In one embodiment, peptides are selected which do not contain a sufficient 
number of T cell epitopes to induce T cell mediated immune responses and/or which 
contain a sufficient number of B cell epitopes to elicit antibodies when administered to a 
mammal. Preferred GDF-8 or GDF-1 1 peptide inhibitors do not contain a sufficient 

25 number of T cell epitopes to induce T-cell mediated {e.g., cytokine) responses. 

However, B cell epitopes may be desirable and can be selected for by, for example, 
testing the peptide's ability to elicit an antibody response, as discussed below. 

T cell epitopes within GDF-8 or GDF-1 1 fragments can be identified using a 
number of well known techniques. For example, T cell epitopes can be predicted using 

30 algorithms (see e.g., Rothbard, J. and Taylor, W.R. (1988) EMBOJ. 7:93-100; 

Berzofsky, J.A. (1 989) Philos Trans R. Soc. Lond 323:535-544). Preferably, human T 



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cell epitopes within a GDF-8 or GDF-1 1 protein can be predicted using known HLA 
class II binding specific amino acid residues. One algorithm for predicting peptides 
having T cell stimulating activity which has been used with success is reported in 
Rothbard, 1st Forum in Virology, Annals of the Pasteur Institute, pp 518-526 
5 (December, 1 986), Rothbard and Taylor, ( 1 988) Embo J. 7:93-1 00 and EP 0 304 279. 
These documents report defining a general T cell pattern (algorithm), its statistical 
significance and its correlation with known epitopes as well as its successful use in 
predicting previously unidentified T cell epitopes of various protein antigens and 
autoantigens. The general pattern for a T cell epitope as reported in the above- 

1 0 mentioned documents appears to contain a linear pattern composed of a charged amino 
acid residue or glycine followed by two hydrophobic residues. Other algorithms that 
have been used to predict T cell epitopes of previously undefined proteins include an 
algorithm reported by Margalit et al., (1987) J. Immunol, 138:2213-2229, which is 
based on an amphipathic helix model. 

1 5 Other methods for identifying T cell epitopes involve screening GDF-8 or GDF- 

1 1 inhibitory peptides of the invention for human T cell stimulating activity. This can 
be accomplished using one or more of several different assays. For example, in vitro, T 
cell stimulatory activity can be assayed by contacting a peptide of the invention with an 
antigen presenting cell which presents appropriate MHC molecules in a T cell culture. 

20 Presentation of a GDF-8 or GDF-1 1 inhibitory peptide of the invention in association 
with appropriate MHC molecules to T cells, in conjunction with the necessary co- 
stimulation can have the effect of transmitting a signal to the T cell that induces the 
production of increased levels of cytokines, particularly of interleukin-2 and interleukin- 
4. The culture supernatant can be obtained and assayed for interleukin-2 or other known 

25 cytokines. For example, any one of several conventional assays for interleukin-2 can be 
employed, such as the assay described in Proc. Natl Acad. Sci USA, 86:1333 (1989) the 
entire contents of which are incorporated herein by reference. A kit for an assay for the 
production of interferon is also available from Genzyme Corporation (Cambridge, MA). 
A common assay for T cell proliferation entails measuring tritiated thymidine 

30 incorporation. The proliferation of T cells can be measured in vitro by determining the 
amount of ^H-labeled thymidine incorporated into the replicating DNA of cultured 



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cells. Therefore, the rate of DNA synthesis and, in turn, the rate of cell division can be 
quantified. 

Other preferred fragments of GDF-8 or GDF-1 1 that are located on the surface of 
the protein, e.g., hydrophilic regions, as well as regions with high antigenicity or 

5 fragments with high surface probability scores can be identified using computer analysis 
programs well known to those of skill in the art (Hopp and Wood, (1983), Mol. 
Immunol., 20, 483-9, Kyte and Doolittle, (1982), J. Mol. Biol., 157, 105-32, Corrigan 
and Huang, (1 982), Comput. Programs Biomed, 3, 1 63-8). 

Still other preferred fragments of GDF-8 or GDF-1 1 to be tested for GDF-8 or 

10 GDF-1 1 inhibitory activity include one or more B-cell epitopes. Such peptides can be 
identified by immunizing a mammal with the peptide, either alone or combined with or 
linked to an adjuvant (e.g., a hapten), and testing sera from the immunized animal for 
anti-GDF-8 or GDF-1 1 antibodies. Preferred peptides generate anti-GDF-8 or GDF-1 1 
antibodies which inhibit GDF-8 or GDF-1 1 activity. For example, sera from immunized 

1 5 animals can be tested for GDF-8 or GDF-1 1 inhibitory activity using any of the GDF-8 
or GDF-1 1 bioassays described herein. 

A proteolytic or synthetic GDF-8 or GDF-1 1 fragment (alone or linked to a 
hapten) can be used to immunize a suitable subject, (e.g., rabbit, goat, mouse or other 
mammal or vertebrate). For example, the methods described in U.S. Patent Nos. 

20 5,422,1 10; 5,837,268; 5,708,155; 5,723,129;and 5,849,531, can be used (the contents of 
these patents are incorporated herein by reference). The immunogenic preparation can 
further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar 
immunostimulatory agent. Immunization of a suitable subject with an immunogenic 
proteolytic or synthetic GDF-8 or GDF-1 1 fragment preparation induces a polyclonal 

25 anti-GDF-8 or GDF-1 1 antibody response. The anti-GDF-8 or GDF-1 1 antibody titer in 
the immunized subject can be monitored over time by standard techniques, such as with 
an enzyme linked immunosorbent assay (ELISA) using immobilized GDF-8 or GDF-1 1 . 
Subsequently, the sera from the immunized subjects can be tested for their GDF-8 or 
GDF-1 1 inhibitory activity using any of the bioassays described herein. 



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The antibody molecules directed against GDF-8 or GDF-1 1 can be isolated from 
the mammal (e.g., from the blood) and further purified by well known techniques, such 
as protein A chromatography to obtain the IgG fraction. At an appropriate time after 
immunization, e.g., when the anti-GDF-8 or GDF-1 1 antibody titers are highest, 

5 antibody-producing cells can be obtained from the subject and used to prepare e.g., 
monoclonal antibodies by standard techniques, such as the hybridoma technique 
originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, 
Brown et al. (1981) J. Immunol 127:539-46; Brown et al. (1980) 1 Biol Chem 
.255:4980-83; Yeh et al. (1 976) Proc. Natl Acad Sci. USA 76:2927-3 1 ; and Yeh et al. 

1 0 (1 982) Int. J. Cancer 29:269-75), the more recent human B cell hybridoma technique 
(Kozbor et al. (1 983) Immunol Today 4:72), the EBV-hybridoma technique (Cole et al. 
(1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or 
trioma techniques. The technology for producing monoclonal antibody hybridomas is 
well known (see generally R. H. Kenneth, in Monoclonal Antibodies: A New Dimension 

1 5 In Biological Analyses. Plenum Publishing Corp., New York, New York (1 980); E. A. 
Lerner (1981) Yale J. Biol Med, 54:387-402; M. L. Gefter et al. (1977) Somatic Cell 
Genet. 3:231-36). Briefly, an immortal cell line (typically a myeloma) is fused to 
lymphocytes (typically splenocytes) from a mammal immunized with a GDF-8 or GDF- 
1 1 immunogen as described above, and the culture supernatants of the resulting 

20 hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody 
that binds GDF-8 or GDF-1 1 . 

Any of the many well known protocols used for fusing lymphocytes and 
immortalized cell lines can be applied for the purpose of generating an anti-GDF-8 or 
GDF-1 1 monoclonal antibody (see, e.g., G. Galfre et al. (1977) Nature 266:55052; 

25 Gefter et al. Somatic Cell Genet., cited supra; Lerner, Yale J. Biol Med, cited supra\ 
Kenneth, Monoclonal Antibodies, cited supra). Moreover, the ordinarily skilled worker 
will appreciate that there are many variations of such methods which also would be 
useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the 
same mammalian species as the lymphocytes. For example, murine hybridomas can be 

30 made by fusing lymphocytes from a mouse immunized with an immunogenic 

preparation of the present invention with an immortalized mouse cell line. Preferred 



WO 00/43781 PCT/US00/01552 

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immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium 
containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a 
number of myeloma cell lines can be used as a fusion partner according to standard 
techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/0-Agl 4 myeloma lines. 

5 These myeloma lines are available from ATCC. Typically, HAT-sensitive mouse 
myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG"). 
Hybridoma cells resulting from the fusion are then selected using HAT medium, which 
kills unfused and unproductively fused myeloma cells (unfused splenocytes die after 
several days because they are not transformed). Hybridoma cells producing a 

1 0 monoclonal antibody of the invention are detected by screening the hybridoma culture 
supernatants for antibodies that bind GDF-8 or GDF-1 1, e.g., using a standard EL1SA 
assay. The antibodies can then be tested for GDF-8 or GDF-1 1 inhibitory activity using, 
for example, the assays described herein. 

In another aspect of the invention, GDF-8 peptide inhibitors comprise all or a 

1 5 portion of the GDF-8 pro-domain. The pro-domain of GDF-8 or a portion thereof can 
be generated using various expression systems (e.g., CHO, baculovirus and the like). 
The expressed pro-domain of GDF-8 can be purified by, for example, using the method 
described in Bottinger et. al. (1996) PNAS 93:5877-5882, or any other art recognized 
method for purifying peptides. Alternatively, the pro-domain can be tagged with, for 

20 example, FLAG or 6-His, as described in the Examples below. In addition, the pro- 
domain of GDF-8 or a portion thereof can be generated by cleavage, e.g., chemical 
cleavage, of the native GDF-8. Moreover, the pro-domain of GDF-8 or a portion thereof 
can be chemically synthesized using art known techniques described herein. 
In a specific embodiments, the GDF-8 inhibitor is a peptide, e.g., a 

25 pentacosapeptide, that includes (e.g. spans) the C-terminus of mature GDF-8. 
Preferably, the GDF-8 peptide is about 25 amino acids in length and comprises or 
consists essentially of a sequence selected from: ANYCSGECEFVFLQKYPHTHLVH 
(SEQ ID NO:25), KIPAMVVDRCGCS (SEQ ID NO:29), and/or 
LSKLRLETAPN1SKDVIRQLLP (SEQ ID NO:30). In another embodiment, the GDF-8 

30 inhibitor has all or a portion of the above-identified sequence and a length of about, 20- 
25, 25-30, 30-35, 35-40, or 40-45 amino acid residues. GDF-8 peptide inhibitors which 



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are based on these peptides can then be tested for GDF-8 or GDF-1 1 inhibitory activity 
using the assays described herein. 

In another embodiment, GDF-8 and GDF-1 1 inhibitors of the present invention 
are peptides, peptidomimetics, or small molecules which inhibit the release of the 
5 mature GDF-8 protein from the GDF-8/pro-domain complex, and/or which stabilize 
the GDF-8/pro-domain complex. Such GDF-8 and GDF-1 1 inhibitors include cysteine 
protease inhibitors, e.g., m-calpain inhibitors. 

In yet another embodiment, GDF-8 and GDF-1 1 inhibitors of the present 
invention are soluble GDF-8 or GDF-1 1 receptors, e.g., soluble fragments of GDF-8 or 
1 0 GDF- 1 1 receptors which compete (with GDF-8 or GDF- 1 1 ) for binding to GDF-8 or 
GDF-1 1 receptors. Such soluble GDF-8 or GDF-1 1 receptors are described herein. 

B. GDF-8 AND GDF-1 1 PROTEIN VARIANTS WHICH INHIBIT GDF-8 
AND GDF-1 1 ACTIVITY 

1 5 In another embodiment, the GDF-8 and GDF-1 1 inhibitors of the present 

invention are GDF-8 or GDF-1 1 protein variants which do not possess detectable GDF-8 
activity. GDF-8 or GDF-1 1 variants of the invention may contain one or more 
conservative or non-conservative amino acid substitutions, deletions, insertions, or 
premature truncations of the amino acid sequence of wild type GDF-8 or GDF-1 1 . 

20 Alternatively, the variants may contain one or more conservative or non-conservative 
amino acid substitutions, insertions or deletions in critical residues or critical regions for 
activity of the GDF-8 or GDF-1 1 protein. The resulting GDF-8 or GDF-1 1 protein 
variants interfere with, for example, GDF-8 or GDF-1 1 processing, secretion or/and 
biological activity, thereby serving as a GDF-8 or GDF-1 1 inhibitor. 

25 A "conservative amino acid substitution" is one in which the amino acid residue 

is replaced with an amino acid residue having a similar side chain (e.g., charge, size 
etc.). A "non-conservative amino acid substitution" is one in which the amino acid 
residue is replaced with an amino acid residue having a side chain which is not similar 
(e.g., change, size etc.). Families of amino acid residues having similar side chains have 

30 been defined in the art. These families include amino acids with basic ^ide chains (e.g., 
lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), 



WO 00/43781 PCT/USOO/01552 

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uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, 
tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, 
proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., 
threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, 
5 tryptophan, histidine). 

1. Cleavage Site Variants 

In one embodiment, GDF-8 and GDF-1 1 inhibitors of the invention are dominant 
negative mutants of GDF-8 and GDF-1 1 proteins. Growth factors belonging to the 

10 TGF-p superfamily, such as GDF-8 and GDF-1 1, form homodimers and heterodimers 
by the dimerization of two precursor molecules. This interaction is necessary for the 
secretion of these factors. Activation of the ligand dimer requires cleavage of the 
carboxy-terminal mature peptide from the amino-terminal precursor remainder and 
occurs under correct physiological conditions. Thus, mutations in the conserved 

15 cleavage sequences, required for activation, can result in the synthesis of non-functional 
GDF-8 and GDF-1 1 molecules. Moreover, overexpression of these mutated precursor 
molecules can lead to heterodimer formation of the mutated GDF-8 and GDF- 1 1 
polypeptides with endogenous unmodified monomers in competitive fashion. When 
GDF-8 and GDF-1 1 mutants are expressed at high levels, most of the endogenous dimer 

20 will be titrated out by heterodimer formation, thus, completely blocking the secretion of 
the active growth factor, e.g., GDF-8 and/or GDF-1 1, in a specific manner. GDF-8 and 
GDF-1 1 dominant negative mutants, e.g., the cleavage site mutant, can be prepared as 
described in the Examples below using the techniques described in, for example, Lopez 
et al (1992) Mol Cell Biol., 12, 1674-1679; Wittbrodt and Rosa, (1994; Genes & Dev., 

25 8, 1448-1462; Suzuki et al (1997) Dev. Biol, 189, 1 12-122; and Hawley et al (1995) 
Genes & Dev., 9, 2923-2935, the contents of which are incorporated herein by reference. 

2. Cysteine Variants 

In another embodiment, GDF-8 and GDF-1 1 inhibitors of the present invention 
30 are GDF-8 and GDF-1 1 proteins containing point mutations at one or more critical 
cysteine residues involved in, for example, the intramolecular cysteine bridges which 



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maintain the tertiary structure of TGF-p family members such as GDF-8 and GDF-1 1. 
For example, cysteine residues which are conserved among all members of the TGF-P 
superfamily, e.g., the cysteine residue at position 313 of the GDF-8 polypeptide, may be 
non-conservatively replaced with an amino acid residue which does not have a similar 
5 side chain (as described above). The cysteine mutants of the present invention can be 
prepared as described in the Examples section below using the techniques described in, 
for example, McPherron and Lee (1997) Nature, 387, 83-90 and Kambadur et al (1997) 
Genome Res., 7, 910-15, the contents of which are incorporated herein by reference. 

10 3. Pro-domain Variants 

In another embodiment, GDF-8 and GDF-1 1 inhibitors of the present invention 
are modified forms of the GDF-8 or GDF-1 1 polypeptides which include all or a portion 
of the pro-domain of the GDF-8 or GDF-1 1 polypeptide. Inhibitors comprising the 
pro-domain of GDF-8 and GDF-1 1 proteins, referred to herein as "Pro-GDF-8" and 

1 5 "Pro-GDF-1 1", respectively, can be prepared as described in the previous sub-section 
and in the Examples section below using the techniques described in, for example, 
Bottinger et. al: (1996) PNAS, 93, 5877-5882 and Gentry and Nash (1990) Biochemistry 
29, 6851-6857, the contents of which are incorporated herein by reference. "Pro-GDF- 
8'' and "Pro-GDF-1 1" inhibitors of the present invention can be used to inhibit GDF-8 or 

20 GDF-1 1 activity in the same species from which the pro-domain was derived or in a 
different species (e.g., the mouse pro-GDF-8 can be used to inhibit human GDF-8 
activity). In a preferred ambodiment, the inhibitor (e.g.. Pro-GDF-8) comprises the N- 
terminus of the pro-domain of GDF-8 (e.g., comprises a region of the pro-domain that is 
upstream of Arg 99 (see Figure 11)). 

25 

4. Post-Translational Modification Variants 

In a further embodiment, GDF-8 and GDF-1 1 inhibitors of the present invention 
include GDF-8 or GDF-1 1 proteins or peptides which do not include post-translational 
modifications necessary for activity of the GDF-8 or GDF-1 1 protein or peptide (e.g., 
30 which include abberrant post-transational modifications). As used herein, the term 

"aberrant" includes a post-translational modification which deviates from the wild type 



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post-translational modification of the GDF-8 or the GDF-1 1 polypeptides. Aberrant 
post-translational modifications include increased or decreased post-translational 
modifications, as well as post-translational modifications which do not follow the wild 
type pattern of GDF-8 or GDF-1 1 polypeptide post-translational modifications. As used 

5 herein, the term "post-translational modifications" includes any modification that the 
GDF-8 or the GDF-1 1 polypeptide undergoes after it has been translated (e.g., after 
peptide bond formation has occurred). Examples of post-translational modifications 
include glycosylation, acylation, limited proteolysis, phosphorylation, and 
isoprenylation. Post-translational modifications play an important role in protein 

1 0 processing, secretion and biological activity. 

In a preferred embodiment, the GDF-8 and GDF-1 1 inhibitors of the present 
invention have aberrant glycosylation. For example, certain GDF-8 inhibitors of the 
present invention contain a mutation at the predicted N-linked glycosylation site within 
the GDF-8 pro-domain (see Figure J 3). 

15 

II. GDF-8 AND GDF-1 J NUCLEIC ACID INHIBITORS 



A. RIBOZYMES 

In still another embodiment, GDF-8 and GDF-1 1 inhibitors of the invention are 
20 ribozymes directed against GDF-8 or GDF-1 1 gene transcripts. Ribozymes are catalytic 
RNA molecules with ribonuclease activity which are capable of cleaving a single- 
stranded nucleic acid, such as an mRNA, to which they hybridize based on having a 
complementary region. Thus, ribozymes (e.g., hammerhead ribozymes described in 
Haselhoff and Gerlach (1988) Nature 334:585-591) of the invention can be used to 
25 catalytically cleave GDF-8 or GDF-1 1 mRNA transcripts, thereby inhibiting translation 
of GDF-8 or GDF-1 1 mRNA. A ribozyme having specificity for a GDF-8- or GDF-1 1- 
encoding nucleic acid can be designed based upon the nucleotide sequence of a GDF-8 
or GDF-1 1 cDNA such as those described in McPherron A. C. et al. (1997) Proc. Natl 
Acad. Set 94:12457-12461 and U.S. Application Serial No. 08/706958, the contents of 
30 which are incorporated herein by reference. For example, a derivative of a Tetrahymena 
L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is 



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complementary to the nucleotide sequence to be cleaved in a GDF-8- or GDF-1 1- 
encoding mRNA (see, e.g., Cech et al. U.S. Patent No. 4,987,071; and Cech et al. U.S. 
Patent No. 5,1 16,742). Alternatively, GDF-8 or GDF-1 1 mRNA can be used to select a 
catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules 

5 (see, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261 : 141 1-1418). 

Alternatively, GDF-8 or GDF-1 1 inhibitor ribozymes can comprise a nucleotide 
sequence complementary to one or more of the regulatory regions of GDF-8 or GDF-1 1 
genes (e.g., the GDF-8 or GDF-1 1 promoter and/or enhancers) to form triple helical 
structures that prevent transcription of the GDF-8 or GDF-1 1 gene in target cells. (The 

1 0 techniques for this are described in, for example, Helene, C. (1 991 ) Anticancer Drug 
Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N.Y. Acad Sci. 660:27-36; and Maher, 
LJ. (\992) Bioassays 14(12):807-15). 

In a particular embodiment, GDF-8 and GDF-1 1 inhibitors of the invention are 
ribozymes comprising one of the four nucleotide sequences set forth in SEQ ID NOs: 1- 

1 5 4. GDF-8 and GDF-1 1 inhibitors of the invention also include ribozymes having a 

nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or more identical to 
the nucleotide sequence set forth in SEQ ID NOs:l -4 which inhibit expression of GDF-8 
or GDF-1 1 . To determine the percent identity of two nucleic acid sequences, the 
sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in 

20 one or both of a first and a second nucleic acid sequence for optimal alignment and non- 
homologous sequences can be disregarded for comparison purposes). In a preferred 
embodiment, the length of a reference sequence aligned for comparison purposes is at 
least 30%, preferably at least 40%, more preferably at least 50%, even more preferably 
at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the 

25 reference sequence. The nucleotides at corresponding nucleotide positions are then 
compared. When a position in the first sequence is occupied by the same nucleotide as 
the corresponding position in the second sequence, then the molecules are identical at 
that position (as used herein nucleic acid "identity" is equivalent to nucleic acid 
"homology"). The percent identity between the two sequences is a function of the 

30 number of identical positions shared by the sequences, taking into account the number of 



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gaps, and the length of each gap, which need to be introduced for optimal alignment of 
the two sequences. 

The comparison of sequences and determination of percent identity between two 
sequences can be accomplished using a mathematical algorithm. In a preferred 
5 embodiment the percent identity between two nucleotide sequences is determined using 
the GAP program in the GCG software package (available at http://www.gcg.com), 
using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length 
weight of 1 , 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two 
nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller 
10 (CABIOS, 4:1 1-17 (1989)) which has been incorporated into the ALIGN program 
(version 2.0), using a PAM1 20 weight residue table, a gap length penalty of 12 and a 
gap penalty of 4. 

B. GDF-8 AND GDF-1 1 ANTISENSE OLIGONUCLEOTIDES 
15 In another aspect, the invention provides GDF-8 or GDF-1 1 inhibitors in the 

form of isolated antisense nucleic acid molecules. An "antisense" nucleic acid 
comprises a nucleotide sequence which is complementary to a "sense" nucleic acid 
encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA 
molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic 
20 acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acids of the 
invention can be complementary to an entire GDF-8 or GDF-1 1 coding strand, or to a 
portion thereof. In one embodiment, the GDF-8 or GDF-1 1 antisense nucleic acid is 
antisense to the coding region of a nucleotide sequence encoding GDF-8 or GDF-1 1 . 
The term "coding region" refers to the region of the nucleotide sequence comprising 
25 codons which are translated into amino acid residues. In another embodiment, the 
antisense nucleic acid is antisense to a noncoding region of a nucleotide sequence 
encoding GDF-8 or GDF-1 1 . The term "noncoding region" refers to 5' and 3' sequences 
which flank the coding region that are not translated into amino acids (i.e.. also referred 
to as 5 ! and 3' untranslated regions). 



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Based on the known nucleotide sequences encoding GDF-8 and GDF-1 1, 
antisense nucleic acids of the invention can be designed according to the rules of Watson 
and Crick base pairing. The antisense nucleic acid molecule can be complementary to 
the entire coding region of GDF-8 or GDF-1 1 mRNA, but typically is an oligonucleotide 

5 which is antisense to only a portion of the coding or noncoding region of GDF-8 or GDF- 
1 1 mKN A. For example, the antisense oligonucleotide can be complementary to the 
region surrounding the translation start site of GDF-8 or GDF-1 1 mRNA. Suitable 
antisense oligonucleotides are, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 
nucleotides in- length. Such antisense GDF-8 or GDF-1 1 inhibitors of the invention can 

1 0 be constructed using chemical synthesis and enzymatic ligation reactions in accordance 
with procedures known in the art. For example, an antisense nucleic acid (e.g.. an 
antisense oligonucleotide) can be chemically synthesized using naturally occurring 
nucleotides or variously modified nucleotides designed to increase the biological stability 
of the molecules or to increase the physical stability of the duplex formed between the 

15 antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine 
substituted nucleotides can be used. Examples of modified nucleotides which can be 
used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5- 
chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- 
(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- 

20 carboxymethylaminomethyluracil, dihydrouracil, beta-Dngalactosylqueosine, inosine, 
N6-isopentenyladenine, 1 -methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- 
methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- 
methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta- 
D-mannosylqueosine, S'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio- 

25 N6-isopentenyladenine 5 uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, 

queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, 
uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouraciL 3- 
(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, 
the antisense nucleic acid can be produced biologically using an expression vector into 

30 which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA 



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transcribed from the inserted nucleic acid will be of an antisense orientation to a target 
nucleic acid of interest, described further in the following subsection). 

The antisense GDF-8 or GDF-1 1 inhibitors of the invention are typically 
administered to a subject or generated in situ such that they hybridize with or bind to 

5 cellular mRNA and/or genomic DNA encoding a GDF-8 or GDF-1 1 polypeptide to 
thereby inhibit expression of the polypeptide, e.g., by inhibiting transcription and/or 
translation. The hybridization can be by conventional nucleotide complementarity to 
form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule 
which binds to DNA duplexes, through specific interactions in the major groove of the 

1 0 double helix. An example of a route of administration of antisense nucleic acid 
molecules of the invention include direct injection at a tissue site. Alternatively, 
antisense nucleic acid molecules can be modified to target selected cells and then 
administered systemically. For example, for systemic administration, antisense GDF-8 
or GDF-1 1 molecules can be modified such that they specifically bind to receptors or 

15 antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid 
molecules to peptides or antibodies which bind to cell surface receptors or antigens. The 
antisense GDF-8 or GDF-1 1 nucleic acid molecules can also be delivered to cells using 
art known vectors (e.g., expression vectors which are transcribed as an anti-sense mRNA 
directed against a GDF-8 or GDF-1 1 mRNA). To achieve sufficient intracellular 

20 concentrations of the antisense GDF-8 or GDF-1 1 molecules, vectorconstructs in which 
the antisense GDF-8 or GDF-1 1 nucleic acid molecule is placed under the control of a 
strong promoter, e.g., pol II or pol III promoter, are preferred. 

In yet another embodiment, antisense GDF-8 or GDF-1 1 inhibitors of the 
invention include a-anomeric nucleic acid molecules. An a-anomeric nucleic acid 

25 molecule forms specific double-stranded hybrids with complementary RNA in which, 
contrary to the usual p-units, the strands run parallel to each other (Gaultier et al. (19B7) 
Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also 
comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131- 
6148) or a chimeric RNA-DNA analogue <Inoue et al. (1987) FEES Lett. 215:327-330). 



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In a particularly preferred embodiment, GDF-8 and GDF-1 1 inhibitors of the 
invention are antisense oligonucleotides comprising one of the twenty nucleotide 
sequences set forth in SEQ ID NOs:5-24. 

5 III GDF-8 OR GDF-U ASSAYS TO TEST FOR INHIBITION 

GDF-8 or GDF-1 1 inhibitors of the present invention can be identified using a 
variety of appropriate assays which test for inhibition of GDF-8 or GDF-1 1 activity. 
Preferably, the inhibition is specific, i.e., the GDF-8 inhibitor can specifically inhibit the 

1 0 GDF-8 protein without affecting the activity of other proteins and the GDF-1 1 inhibitor 
can specifically inhibit the GDF-1 1 protein without affecting the activity of other 
proteins. In certain embodiments, the GDF-8 inhibitor is also able to inhibit GDF-1 1 
activity and the GDF-1 1 inhibitor is also able to inhibit GDF-8 activity. 

As used herein, the term "assay" includes any assay designed to identify a GDF- 

15 8 or GDF-1 1 inhibitor. The assay can be an in vitro or an in vivo assay suitable for 

identifying whether the GDF-8 or GDF-1 1 inhibitor effects, -e.g., downmodulates, one or 
more of the biological functions of GDF-8 or GDF-1 1 . Examples of suitable assays 
include DNA replication assays, transcription-based assays, creatine kinase assays, 
assays based on the differentiation of 3T3-L1 pre-adipocytes, assays based on glucose 

20 uptake control in 3T3-L1 adipocytes, and immunological assays, all as described in 
subsection II below and in the following Examples. 



Creatine Kinase Bioassay for the Identification of GDF-8 or GDF-1 1 Inhibitors 

GDF-8 and GDF-1 1 modulate the protein levels, and therefore the activity, of a 

25 muscle-specific enzyme, creatine kinase. This effect of GDF-8 or GDF-1 1 can be used 
to screen for potential GDF-8 or GDF-1 1 inhibitors. Specifically, a<reatine kinase 
assay can be performed in a myoblast, e.g., the mouse skeletal myoblast cell line CI CI 2 
or primary chick myoblasts isolated from Day 1 1 chick embryos. To test for the 
inhibitors' ability to modulate the activity of creatine kinase, cells are typically grown in 

30 48-well trays in serum-containing medium that maintains them undifferentiated. When 
a 70% confluence has been reached, medium is switched to 1% serum, thus allowing 



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differentiation and creatine kinase expression. At the time of the switch, the potential 
GDF-8 or GDF-1 1 -inhibitory fraction is added to some wells, followed some time later 
by GDF-8 or GDF-1 1 itself Cells are returned to the incubator for an additional two to 
three day period. In the end, cells are lysed and creatine kinase activity is measured in 
5 the lysates using a commercially available kit (available by Sigma, St Louis ; MO). 

Assay Based on the Differentiation of 3T3-L1 Pre-adipocytes 

GDF-8 and GDF-1 1 interfere with the differentiation process of 3T3-L1 pre- 
adipocytes (fibroblasts) to adipocytes. This effect of GDF-8 or GDF-1 1 can be used to 

10 screen for potential GDF-8 or GDF-1 1 inhibitors. Specifically, the bioassay of the 
invention can be performed in the following manner. 3T3-L1 pre-adipocytes are 
allowed to reach confluence, and subsequently differentiation can be achieved by 
successive replacements of their serum-containing DMEM media as follows: DMEM + 
serum + methylisobutylxanthine + dexamethasone + insulin for 2 days, DMEM + serum 

1 5 + insulin for 2 additional days, DMEM + serum for 3 additional days (Spiegelman et a/., 
(1993) J. Biol Chem. 268, 6823-6826). GDF-8 and potential inhibitors can be added at 
the onset of differentiation and re-supplied at each additional medium change. The 
degree of adipocyte differentiation can be assessed by various ways, e.g., visually by 
estimation of the content of fat droplets in the pre-adipocytes or, by quantitation of the 

20 glycerol/triglyceride content of cell lysates, using the Triglyceride (Gro-Trinder) kit 
from Sigma (St Louis, MO) according to the manufacturer's instructions. 

Assay Based on Glucose Uptake Control in 3T3-L1 Adipocytes 

GDF-8 and GDF-1 1 modulate glucose uptake control in adipocytes. This effect 

25 of GDF-8 or GDF- 1 1 can be used to screen for potential GDF-8 or GDF-1 1 inhibitors. 
Specifically, 3T3-L1 pre-adipocytes can be induced to fully differentiate following the 
protocol described above. Subsequently, a glucose transport assay is performed. 
Briefly, following differentiation, media are changed to serum-free DMEM, and cells are 
treated with the GDF-8 or GDF-1 1 inhibitor and GDF-8 or GDF-1 1 for a variable period 

30 of time ranging from 2 hours to three days. Subsequently, cells are switched for four 
hours to low glucose, serum-free DMEM (without the factors and inhibitors). Then. 



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cells are washed twice with Ringer/Krebs solution and insulin (0.01 to 1 ^M) is added to 
them for 5 to 20 minutes. Insulin is washed twice and tritiated (radioactive) deoxy- 
glucose (NEN Dupont) is added at 1 (iCi/ml final concentration in Ringer buffer. 
Glucose uptake is left to proceed for 5 to 1 0 minutes, at which time cells are washed 
5 with excess volume of Krebs, solubilized, and radioactive glucose uptake is determined 
by scintillation counting of the cell lysates. 

DNA Replication Assay 

GDF-8 and GDF-1 1 inhibitors of the invention can be tested using a DNA 

10 replication assay. This bioassay is a cell-based, growth assay and can be performed in 
any cell type responsive to GDF-8 or GDF-1 1 . In this type of assay DNA replication 
and, thus, proliferation is accurately measured by the incorporation of Bromo-deoxy- 
Uridine (BrdU) or tritiated thymidine ([ 3 H]-TdR) label into the DNA. This bioassay can 
be perfomed using several cell lines including, but not limited to, G8 mouse skeletal 

1 5 myoblasts, C2C12 mouse myoblasts and the mink lung epithelial cell line, CCL-64, and 
its mutant derivatives. 

Briefly, cells are plated using the appropriate culture conditions. For example, 
G8 myoblasts are plated in 96-well culture plates coated with 0.1% gelatin (Sigma), and 
CCL-64 cells are plated in un-coated plates. Cells are plated in serum-free DMEM 

20 containing 0.1% w/v BSA at lOxlO 3 cells per well. The following day, media are 

removed and replaced with fresh media. The relevant inhibitory fraction is added, and 
cells are returned to the incubator for 60 minutes before growth factors (GDF-8, Activin, 
and the like) are applied to them. In another embodiment, the inhibitory fraction can be 
mixed with the growth factor for 30 minutes to 2 hours at room temperature, to allow for 

25 inhibitor-ligand interactions, and thereafter the mixture can be applied onto the cells. In 
G8 myoblasts, the appropriate label is added 24 hours after the test factors, and 
incorporation is allowed to proceed for an additional 24 hours. In CCL-64 cells, the 
appropriate label is added 12 hours after the test factors, and incorporation is allowed to 
proceed for an additional 4 hours. When BrdU is used as the label, cells are finally fixed 

30 and processed according to the manufacturer's instructions manual using a commercially 
available kit (Boehringer-Manheim). The incorporated BrdU is determined 



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colorimetrically. When [ 3 H]-thymidine (NEN-Dupont) is used as a label, cells are 
washed twice with 10% trichloroacetic acid and radioactivity is extracted with G.3N 
NaOH and determined by scintillation counting. 

5 Transcription-Based Assay 

In another embodiment, GDF-8 or GDF-1 1 inhibitors of the present invention 
can be identified using a transcription-based assay in any GDF-8- or GDF-1 1 -responsive 
cell line including, but not limited to G8 myoblasts, C2C12 myoblasts, CCL-64 mink 
lung epithelial cells, and A204 human rhabdomyosarcoma cells. 
1 0 Artificial reporter plasmids which respond to GDF-8 or GDF-1 1 in this type of 

assay can be used. For example. p3TP-Lux (described in, for example, Wrana et al 
( 1 992) Cell, 71,1 003- 1 4) and p(CAGA) 12 -MLP (described in, for example, Dennter et 
al (1998) EMBO J., 17, 3091-3100) can be used in this assay. In both p3TP-Lux and 
p(CAGA) 12 -MLP, luciferase gene transcription (and thus activity) is driven by artificial 
1 5 minimal promoters which respond to members of the TGF-{5 family. Therefore, 

luciferase activity in cell lysates correlates linearly with the degree of stimulation of the 
cells by the applied growth factors. 

Briefly, cells are plated in 48-well plates in the appropriate media (e.g., DMEM 
for CCL-64 and McCoy's medium for A204) supplemented with 10% fetal bovine 
20 serum, antibiotics and L-Glutamine. Upon reaching 80% confluence, celis are 
transfected using FuGENE-6 (Boehringer-Manheim) to facilitate plasmid uptake, 
according to the manufacturer's instructions. Cells are transiently transfected with a 
cocktail of two plasmids; pSV-P-gal plasmid to monitor transfection efficiency, and 
either of the two luciferase reporter plasmids. After overnight incubation with the 
25 transfection reagents, cells are washed twice with the appropriate serum-fre^ medium 
containing 0.1% BSA. The putative inhibitory fraction is then added and-cells are 
returned to the incubator for 60 minutes. At the -end of this period, the following factors 
can be added: human recombinant Myostatin/GDF-8, produced from GDF-8-expr-essing 
CHO cell conditioned medium, human TGF-p j (R&D Biosystems, Minneapolis) and 
30 concentrated conditioned medium from CHO cells expressing Activin pA. 



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ln another embodiment, the putative inhibitor is mixed with GDF-8, GDF-1 1 , or 
other ligand for 30 minutes to 2 hours at room temperature and the mixture is added onto 
the recipient cells. Cells are lysed after a 6 hour incubation and luciferase and P- 
galactosidase activity can be determined in the same sample using the Dual-Light 
5 Luciferase Assay kit from Tropix Inc. Activity is expressed in Relative Luciferase units 
(RLU), i.e. luciferase activity corrected for transfection efficiency that is given by the 
corresponding values of P-galactosidase activity measured in the same sample. 

Protein Secretion Based Assays 

1 0 In another embodiment, GDF-8 or GDF-1 1 inhibitors of the present invention 

can be identified using a protein secretion based assay. Briefly, the constructs encoding 
the GDF-8 or GDF-1 1 inhibitors (e.g., the dominant negative mutants, cysteine mutants, 
pro-domain variants and post-translational modification variants) can be introduced or 
co-introduced in muscle cells lines such as QM7 and RD, with a wild-type GDF-8 or 

1 5 GDF-1 1 producing construct. Subsequently, the ability of the co-transfected cells to 
produce and secrete mature GDF-8 or GDF-1 1 can be tested by, for example, a western 
blot analysis. GDF-8 or GDF-1 1 inhibitors which inhibit production and/or secretion of 
mature GDF-8 or GDF-1 1 can then be selected. 

20 IV. USES OF GDF-8 OR GDF-1 1 INHIBITORS 

In another aspect, the invention provides<iDF-8 or GDF-1 1 inhibitors identified 
by the methods described herein, as well as compositions, e.g., pharmaceutical 
compositions, and methods of using the inhibitors. The GDF-8 or GDF-1 1 inhibitors 

25 identified by the methods described herein can be used to modulate GDF-8 or GDF-1 1 
expression or activity for therapeutic purposes. In an exemplary embodiment, a cell is 
contacted with a GDF-8 or GDF-1 1 inhibitor that modulates one or more of the activities 
of GDF-8 or GDF-1 1 protein activity associated with the cell. The contacting can be 
performed in vitro (e.g., by culturing the cell with the GDF-8 or GDF-1 1 inhibitor), in 

30 vivo (e.g., by administering the GDF-8 or GDF-1 1 inhibitor to a subject), or in ovo (e.g., 
by injecting the GDF-8 or GDF-1 1 inhibitor into eggs). The ovo injection can be 



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performed as described herein using the techniques of, for example, H. Kocamis et al 
(1998) Poult ScL 77, 1913-1919; or P.A. Johnston et al, (1997) Poult. ScL 76, 165-178; 
C.E. Dean et al. (1993) Growth Dev. Aging 57, 59-72. 

As such, the present invention further provides methods of treating an individual 

5 afflicted with a disease or disorder characterized by aberrant expression or activity of a 
GDF-8 or GDF-1 1 protein or nucleic acid molecule, e.g., a muscle-associated disorder. 
Inhibition of GDF-8 or GDF-1 1 activity is desirable in situations in which GDF-8 or 
GDF-1 1 is abnormally upregulated and/or in which decreased GDF-8 or GDF-1 1 
activity is likely to have a beneficial effect. Examples of disorders which can be treated 

10 using the GDF-8 or GDF-1 1 inhibitors of the invention include muscle-associated 
disorders such as cancer, muscular dystrophy, spinal cord injury, traumatic injury, 
congestive obstructive pulmonary disease, AIDS or cachexia. In addition, the GDF-8 or 
GDF-1 1 inhibitors of the invention can be used to treat obesity and related disorders, or 
disorders related to abnormal proliferation of adipocytes. 

1 5 In preferred embodiments, the GDF-8 or GDF-1 1 inhibitors of the invention are 

used to treat diabetes, obesity, and disorders related to obesity. For example, GDF-8 or 
GDF-1 1 inhibitors of the invention can be used to modulate glucose transport in a 
subject, e.g., by increasing the activity of the glucose transporter GLUT4 in the subject. 
The GDF-8 or GDF-1 1 inhibitors of the invention can further be used to decrease 

20 GDF-8 or GDF-1 1 activity in a subject. As used herein, the term "subject" includes any 
animal which expresses X3DF-8 or GDF-1 1 , preferably a mammal. In a preferred 
embodiment, the subject is a vertebrate. In an even more preferred embodiment, the 
vertebrate is a chicken, a turkey, a pig, a cow, a mouse, a rat, a rabbit, a^goat, a fish, or a 
human. For example, the GDF-8 or <}DF-1 1 inhibitors of the invention can be used to 

25 increase muscle mass in a subject, e.g., a chicken. 

The GDF-8 or GDF-1 1 inhibitors of the invention can be incorporated into 
pharmaceutical compositions suitable for administration. Such -compositions typically 
comprise the GDF-8. or GDF-1 1 inhibitor and a pharmaceutical^ acceptable carrier. As 
used herein the language "pharmaceutical^ acceptable carrier" is intended to include 

30 any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, 
isotonic and absorption delaying agents, and the like, compatible with pharmaceutical 



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administration. The use of such media and agents for pharmaceutical^ active 
substances is well known in the art. Except insofar as any conventional media or agent 
is incompatible with the GDF-8 or GDF-1 1 inhibitor, use thereof in the compositions is 
contemplated. Supplementary active compounds can also be incorporated into the 
5 compositions. 

A pharmaceutical composition of the invention is formulated to be compatible 
with its intended route of administration. Examples of routes of administration include 
parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), 
transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions 

10 used for parenteral, intradermal, or subcutaneous application can include the following 
components: a sterile diluent such as water for injection, saline solution, fixed oils, 
polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; 
antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as 
ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic 

1 5 acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of 
tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, 
such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be 
enclosed in ampoules, disposable syringes or multiple dose vials made of glass or 
plastic. 

20 Pharmaceutical compositions suitable for injectable use include sterile aqueous 

solutions (where water soluble) or dispersions and sterile powders for the 
extemporaneous preparation of sterile injectable solutions or dispersion. For 
intravenous administration, suitable carriers include physiological saline, bacteriostatic 
water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In 

25 all cases, the composition must be sterile and should be fluid to the extent that easy 
syringability exists. It must be stable under the conditions of manufacture and storage 
and must be preserved against the contaminating action of microorganisms such as 
bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for 
example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid 

30 polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity 
can be maintained, for example, by the use of a coating such as lecithin, by the 



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maintenance of the required particle size in the case of dispersion and by the use of 
surfactants. Prevention of the action of microorganisms can be achieved by various 
antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol- 
ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include 

5 isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium 
chloride in the composition. Prolonged absorption of the injectable compositions can be 
brought about by including in the composition an agent which delays absorption, for 
example, aluminum monostearate and gelatin. 

Sterile injectable solutions can be prepared by incorporating the GDF-8 or GDF- 

10 11 inhibitor in the required amount in an appropriate solvent with one or a combination 
of ingredients enumerated above, as required, followed by filtered sterilization. 
Generally, dispersions are prepared by incorporating the GDF-8 or GDF-1 1 inhibitor 
into a sterile vehicle which contains a basic dispersion medium and the required other 
ingredients from those enumerated above. In the case of sterile powders for the 

15 preparation of sterile injectable solutions, the preferred methods of preparation are 
vacuum drying and freeze-drying which yields a powder of the active ingredient plus 
any additional desired ingredient from a previously sterile-filtered solution thereof. 

Oral compositions generally include an inert diluent or an edible carrier. They 
can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral 

20 therapeutic administration, the GDF-8 or GDF-1 1 inhibitor can be incorporated with 
excipients and used in the form of tablets, troches, or capsules. Oral compositions can 
also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in 
the fluid carrier is applied orally and swished and expectorated or swallowed. 
Pharmaceutical ly compatible binding agents, and/or adjuvant materialscan be included 

25 as part of the composition. The tablets, pills, capsules, troches and the like can contain 
any of the following ingredients, or compounds of a similar nature: a binder such as 
microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or 
lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant 
such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a 

30 sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, 
methyl salicylate, or orange flavoring. 



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For administration by inhalation, the compounds are delivered in the form of an 
aerosol spray from pressured container or dispenser which contains a suitable propellant, 
e.g., a gas such as carbon dioxide, or a nebulizer. 

Systemic administration can also be by transmucosal or transdermal means. For 
5 transmucosal or transdermal administration, penetrants appropriate to the barrier to be 
permeated are used in the formulation. Such penetrants are generally known in the art, 
and include, for example, for transmucosal administration, detergents, bile salts, and 
fusidic acid derivatives. Transmucosal administration can be accomplished through the 
use of nasal sprays or suppositories. For transdermal administration, the active 
10 compounds are formulated into ointments, salves, gels, or creams as generally known in 
the art. 

The GDF-8 or GDF-1 1 inhibitor can also be prepared in the form of 
suppositories (e.g., with conventional suppository bases such as cocoa butter and other 
glycerides) or retention enemas for rectal delivery. 

15 In one embodiment, the GDF-8 or GDF-1 1 inhibitors are prepared with carriers 

that will protect the compound against rapid elimination from the body, such as a 
controlled release formulation, including implants and microencapsulated delivery 
systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl 
acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic 

20 acid. Methods for preparation of such formulations will be apparent to those skilled in 
the art. The materials can also be obtained commercially from Alza Corporation and 
Nova Pharmaceuticals, Inc. Liposomal suspensions {including liposomes targeted to 
infected cells with monoclonal antibodies to viral antigens) can also be used as 
pharmaceutically acceptable carriers. These can be prepared according to methods 

25 known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,81 1 . 
It is especially advantageous to formulate oral or parenteral compositions in 
dosage unit form for ease of administration and uniformity of dosage. Dosage unit form 
as used herein refers to physically discrete units suited as unitary dosages for the subject 
to be treated; each unit containing a predetermined quantity of active compound 

30 calculated to produce the desired therapeutic effect in association with the required 

pharmaceutical carrier. The specification for the dosage unit forms of the invention are 



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dictated by and directly dependent on the unique characteristics of the active compound 
and the particular therapeutic effect to be achieved, and the limitations inherent in the art 
of compounding such an active compound for the treatment of individuals. 

Toxicity and therapeutic efficacy of such compounds can be determined by 
5 standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for 
determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose 
therapeutically effective in 50% of the population). The dose ratio between toxic and 
therapeutic effects is the therapeutic index and it can be expressed as the ratio 
LD50/ED50. GDF-8 or GDF-1 1 inhibitors which exhibit large therapeutic indices are 

10 preferred. While GDF-8 or GDF-1 1 inhibitors that exhibit toxic side effects may be 
used, care should be taken to design a delivery system that targets such GDF-8 or GDF- 
1 1 inhibitors to the site of affected tissue in order to minimize potential damage to 
uninfected cells and, thereby, reduce side effects. 

The data obtained from the cell culture assays and animal studies can be used in 

1 5 formulating a range of dosage for use in humans. The dosage of such compounds lies 
preferably within a range of circulating concentrations that include the ED50 with little 
or no toxicity. The dosage may vary within this range depending upon the dosage form 
employed and the route of administration utilized. For any GDF-8 or GDF-1 1 inhibitor 
used in the method of the invention, the therapeutically effective dose can be estimated 

20 initially from cell culture assays. A dose may be formulated in animal models to 
achieve a circulating plasma concentration range that includes the IC50 (i.e., the 
concentration of the test GDF-8 or GDF-1 1 inhibitor which achieves a half-maximal 
inhibition of symptoms) as determined in cell culture. Such information can be used to 
more accurately determine useful doses in humans. Levels in plasma may be measured, 

25 for example, by high performance liquid chromatography. 

The pharmaceutical compositions can be included in a container, pack, or 
dispenser together with instructions for administration. 

The GDF-8 and GDF-1 1 inhibitors of the present invention, e.g., ribozymes or 
antisense inhibitors, can further be inserted into vectors and used in gene therapy. Gene 

30 therapy vectors can be delivered to a subject by, for example, intravenous injection, 
local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g.. 



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Chen et al. (\994)Proc. Nail. Acad Sci USA 91:3054-3057). The pharmaceutical 
preparation of the gene therapy vector can include the gene therapy vector in an 
acceptable diluent, or can comprise a slow release matrix in which the gene delivery 
vehicle is imbedded. Alternatively, where the complete gene delivery vector can be 
5 produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical 
preparation can include one or more cells which produce the gene delivery system. 

Vectors suitable for use in gene therapy are known in the art. For example, 
adeno virus-derived vectors can be used. The genome of an adenovirus can be 
manipulated such that it encodes and expresses a gene product of interest but is 

10 inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See for 
example Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 
252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral vectors 
derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g., 
Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art. Recombinant 

1 5 adenoviruses can be advantageous in certain circumstances in that they are not capable 
of infecting nondividing cells. Furthermore, the virus particle is relatively stable and 
amenable to purification and concentration, and as above, can be modified so as to affect 
the spectrum of infectivity. Additionally, introduced adenoviral DNA (and foreign DNA 
contained therein) is not integrated into the genome of a host cell but remains episomal, 

20 thereby avoiding potential problems that can occur as a result of insertional mutagenesis 
in situations where introduced DNA becomes integrated into the host genome (e.g., 
retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign 
DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al. 
cited supra; Haj-Ahmand and Graham (1986) J. Virol. 57:267). Most replication- 

25 defective adenoviral vectors currently in use and therefore favored by the present 

invention are deleted for all or parts of the viral El and E3 genes but retain as much as 
80 % of the adenoviral genetic material (see, e.g., Jones et al. (1979) Cell 16:683; 
Berkner et al., supra; and Graham et al. in Methods in Molecular Biology , E.J. Murray, 
Ed. (Humana, Clifton, NJ, 1991) vol. 7. pp. 109-127). Expression of the gene of interest 

30 comprised in the nucleic acid molecule can be under control of, for example, the El A 



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promoter, the major late promoter (MLP) and associated leader sequences, the E3 
promoter, or exogenously added promoter sequences. 

Yet another viral vector system useful for delivery of the GDF-8 and GDF-1 1 
inhibitors of the invention is the adeno-associated virus (AAV). Adeno-associated virus 

5 is a naturally occurring defective virus that requires another virus, such as an adenovirus 
or a herpes virus, as a helper virus for efficient replication and a productive life cycle. 
(For a review see Muzyczka et al. Curr. Topics in Micro, and Immunol (1 992) 1 58:97- 
129). Adeno-associated virusses exhibit a high frequency of stable integration (see for 
example Flotte et al. (1992) Am, J. Respir. Cell Mol Biol 7:349-356; Samulski et al. 

10 (1989) J. Virol 63:3822-3828; and McLaughlin et al. (1989) J. Virol 62:1963-1973). 
Vectors containing as few as 300 base pairs of AAV can be packaged and can integrate. 
Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as that 
described in Tratschin et al. (1985) Mol Cell Biol 5:3251-3260 can be used to 
introduce DNA into T cells. A variety of nucleic acids have been introduced into 

1 5 different cell types using AAV vectors (see for example Hermonat et al. (1 984) Proc. 
Natl Acad Set USA 81 -.6466-6470; Tratschin et al. (1985) Mol Cell Biol 4:2072- 
2081; Wondisford et al. (1988) Mol Endocrinol 2:32-39; Tratschin et al. (1984) J. 
Virol 51:611-619;andFlotteetal.(1993)J. Biol Chem. 268:3781-3790). Otherviral 
vector systems that may be useful for delivery of the GDF-8 and GDF-1 1 inhibitors of 

20 the invention are derived from herpes virus, vaccinia virus, and several RNA viruses. 

The GDF-8 and GDF-1 1 inhibitors of the present invention can further be used 
to generate transgenic animals in which the GDF-8 and GDF-1 1 inhibitors interfere with 
GDF-8 or GDF-1 1 processing, GDF-8 or GDF-1 1 secretion, and/or GDF-8 or GDF-1 1 
biological activity. As used herein, a "transgenic animal" is a non-human animal, 

25 preferably a mammal, more preferably a rodent such as a -rat or mouse, in which one or 
more of the cells of the animal includes a transgene. Other examples of transgenic 
animals include non-human primates, sheep, dogs, cows, goats, chickens, turkeys, 
amphibians, fish, and the like. A transgene is exogenous DNA which is integrated into 
the genome of a cell from which a transgenic animal develops and which remains in the 

30 genome of the mature animal, thereby directing the expression of an encoded gene 
product in one or more cell types or tissues of the transgenic animal. 



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A transgenic animal can be created by introducing GDF-8 or GDF-1 1 inhibitor- 
encoding nucleic acid or GDF-8- or GDF-1 1 -encoding nucleic acid into the male 
pronucleus of a fertilized oocyte, e.g., by microinjection, retroviral infection, and 
allowing the oocyte to develop in a pseudopregnant female foster animal. Intronic 
5 sequences and polyadenylation signals can also be included in the transgene to increase 
the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) 
can be operably linked to the GDF-8 or GDF-1 1 transgene to direct expression of the 
GDF-8 or GDF-1 1 inhibitor to particular cells. Methods for generating transgenic 
animals via embryo manipulation and microinjection, particularly animals such as mice, 

10 have become conventional in the art and are described, for example, in U.S. Patent Nos. 
4,736,866 and 4,870,009, both by Leder et al., U.S. Patent No. 4,873,1 91 by Wagner et 
al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory 
Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of 
other transgenic animals. A transgenic founder animal can be identified based upon the 

1 5 presence of the GDF-8 or GDF-1 1 inhibitor transgene in its genome and/or expression of 
the GDF-8 or GDF-1 1 inhibitor mRNA in tissues or cells of the animals. A transgenic 
founder animal can then be used to breed additional animals carrying the transgene. 
Moreover, transgenic animals carrying a transgene encoding the GDF-8 or GDF-1 1 
inhibitor can further be bred to other transgenic animals carrying other transgenes. 

20 Clones of GDF-8 or GDF-1 1 inhibitor transgenic animals can also be produced 

according, for examples, to the methods described in Wilmut, 1. et al. (1997) Nature 
385:810-813 and PCT International Publication Nos. WO 97/07668 and WO 97/07669. 
In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and 
induced to exit the growth cycle and enter G 0 phase. The quiescent cell can then be 

25 fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal 
of the same species from which the quiescent cell is isolated. The reconstructed oocyte 
is then cultured such that it develops to morula or blastocyte and then transferred io 
pseudopregnant female foster animal. The offspring borne of this female foster animal 
will be a clone of the animal from which the c«ll 5 e.g., the somatic cell, is isolated. 

30 In preferred embodiments, the GDF-8 or GDF-1 1 inhibitor transgenic animals 

can be selected such that the GDF-8 or GDF-1 1 function is only partially inhibitted. 



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This invention is further illustrated by the following examples which should not 
be construed as limiting. The entire contents of all references, patents and published 
patent applications cited throughout this application, as well as the Sequence Listing and 
the Figures are incorporated herein by reference. 

5 

EXAMPLES 

Materials and Methods 

The materials and methods used in the following Examples are first described. 

10 1 . Non-reducing SDS-PAGE and electroblotting 

The Novex NuPAGE protocol was used (Novex). The reducing agent from both 
sample and running buffers was omitted. 

2. Gel elution 

1 5 The BioRad mini gel elutor protocol, with the elution buffer (25 mM Tris, pH 

8.3 and 6 M urea), was used. 

3. Acetone precipitation 

Removal of excess SDS is necessary in order to successfully perform tryptic 
20 digestion. Therefore, ice-cold acetone was added to the SDS-containing sample to 

obtain final v/v of 20%. The solution was kept below -20°C for 20 minutes. The SDS- 
free protein was then recovered by centrifugation at maximum speed (1 3,000x g) for 15 
minutes at 4°C. The supernatant was then removed without disturbing the pellet. The 
solution was not discared until the presence of protein in the pellet has been confirmed 
25 by SDS-PAGE. A high percentage of recovery (>90% per run) is usually achieved, but 
in some cases, the optimal concentration of acetone required for this process can be 
protein dependent, and careful monitoring should be performed. 

4. Solubilization of SDS-free protein 

30 The SDS-free protein sample was air-dried for a few minutes to evaporate most 

of the acetone (never letting the pellet dry out). To solublize the sample, either solid 



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urea or a 1 0M urea solution was added to the wet pellet. Due to the large volume 
dilution in subsequent tryptic digestions, the amount of volume used for solubilization 
was kept to a minimum (<5|il) in order to avoid losing protein in the sample. 

5 5. Tryptic digestion 

A sufficient amount of digestion buffer (100 mM ammonium bicarbonate, pH 
7.8 to 8.0, no adjustment of pH is needed) was added to the sample (final concentration 
of urea should be kept below 0.1M). After adding trypsin (at l^g/ml in ImM HC1) in a 
w/w ratio of 1 (enzyme) to 20 (substrate), digestion was initiated by incubating at 37°C. 
10 After overnight incubation (12-16 hours), the reaction was quenched by adding PMSF 
(stock solution prepared in ethanol) to a final concentration of 1 mM. 

6. High resolution peptide separation 

A PMSF-treated tryptic sample solution was divided into two equal parts before 
15 it was subjected to HPLC separation using the conditions described below. One part 
was treated with reducing agent (TCEP/ 50 mM final concentration), and the other part 
is kept untreated (water is added to adjust volume). The samples were incubated at 
60°C for 30 minutes before acidifying with 1 0% TFA. In the case that an alternative 
sample treatment is used, the possibility of fragment loss due to aggregation should also 
20 be considered. For example, solid guanidine.HCl can be added to the PMSF-treated 
tryptic sample solution to a final concentration of 4 M. Subsequently, the protocol 
described above can be used. 

Column: CI 8 RP column <2.1x 250mm). 
25 Solvents: A: 0.1% TFA in water, B: 0.1% TFA in 80% acetonitrile. 
Flow Rate: 0.2ml/min. 

Gradient: 0%B to 1 00%B in 60 to 90 minutes. 
Detector: 215nm 

Optimization of the gradient is necessary in order to obtain the maximum resolution 
30 regarding disulfide containing peaks. 



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7. Secondary digestion 

Selection of proteases is highly sequence-dependent. When target sequences are 
not known, suitable proteases (which are less specific than trypsin) can be used in 
accordance with methods well known in the art, e.g., themnolysin, chymotrypsin, pepsin, 
5 or any other commercially available protease. Since the target substrates of these 
proteases are quite broad and non-specific, they often can cleave the tryptic fragment 
and generate smaller fragments. Proteases that are more specific than those mentioned 
can also be used to generate larger fragments. Secondary digestion is preferably 
performed using a procedure similar to the trypsin digestion described above, in 
10 paragraph 5 except for differences in buffer and in pH levels. 



8. Deglycosylation of the inhibitor 

To solublize the SDS-free sample (5 to 10 ^ig), a minimum amount of 
hydrogenated Triton X-100 and 50 mM of ammoniun bicarbonate buffer (pH 7.8 to 8:0) 

15 can be added in a small increment of 2 to 3^1 at a time. The final volume should be less 
than 30 and the detergent concentration should be below 0.5% (to prevent 
denaturation of the enzyme). One unit of peptide N-glycosidase F (PNGase F) is then 
added to the reaction mixture. The sample is incubated at 37°C for 16 hours before 
acidifying with 1 0% TFA. The deglycosylated protein is further purified by C4 

20 RPHPLC, and any inhibitory activity is monitored by an appropriate bioassay. 

Deglycosylation can also be performed using an N-Glycosidase F Deglycosylation Kit 
(Boehringer-Mannheim). 

9. Site-directed mutagenesis and vector construction 

25 The mutated GDF-8 cDN A that encodes a protein with a replaced cleavage ^ite 

(RSRR) was generated using the overlapping PCR technique. The pair of overlapping 
inside primers, incorporating the mutation of the cleavage site<RSSR to NAQT), was 
used to amplify 5' and 3 f ends of mouse GDF-8 cDNAs in the first round of PCR. Then, 
PCR products were combined and used as a template with the outside primers to 

30 generate the full-length mutated GDF-8 cDNA. referred to as dominant-negative mutant 



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(Mut-GDF-8). The PCR fragment was ligated into the PCR 2.1 vector (Invitrogen) and 
sequenced to confirm the incorporation of the mutation. The cDNA encoding Mut 
-GDF-8 was then inserted into the FLAG-CMV-5a vector (Sigma) to produce an 
in-frame fusion with the FLAG epitope at the 3' end. The FLAG epitope can be used for 
5 detection and purification of the unprocessed GDF-8 protein. This vector contains 

cytomegalovirus promoter sequences, which result in high level expression in eukaryotic 
cells. 

The expression plasmid containing the pro-domain of GDF-8 was generated 
through the same method. The partial mouse cDNA, encoding the pro-domain of 

1 0 GDF-8 (residues 1 -266) ? which is referred to herein as "Pro-GDF-8'\ was generated by 
PCR-based mutagenesis. The Asp-267 codon (GAC) was changed to a STOP codon 
(TGA). The resulting cDNA was ligated into the PCR 2.1 vector, sequenced, and 
inserted into FLAG-CMV-5a as described herein. The wild-type (WT) mouse GDF-8 
cDNA was also subcloned in the same vector to generate the full-length precursor 

1 5 protein. The resulting construct is referred to as WTGDF-8. 

10. Transfection, poly acrv lam ide gel electrophoresis, and immunoblotting 

The expression constructs were introduced into QM-7 quail myoblast cells by 
transient transfection using FuGene reagent (Boehringer Mannheim). Cells were plated 

20 on 60 mm dishes 24 hours before transfection and were 70-80% confluent at the time of 
transfection. The efficiency of transfection, monitored by a reporter construct 
containing P-galactosidase, was about 70-80%. Transfected cells were maintained in 
Opti-MEM (Gibco-Life Sciences), and conditioned media were collected 48 hours after 
transfection and concentrated in Centricon columns (Amicon) by about 50-fold. 

25 Expression of the various forms of recombinant GDF-8 proteins in QM-7 cells was 
confirmed by SDS-PAGE and immunoblotting with the anti-FLAG M2 specific 
antibody (Sigma). Proteins reactive with the anti-FLAG antibody were detected by a 
chemiluminescent technique according to the manufacturer's instructions (ECL, 
Amersham). 



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11. Protein purification 

Affinity chromatography using an affinity gel coupled to a specific monoclonal 
antibody directed against the FLAG epitope (Sigma) was used to purify the 
WT-GDF-8-FLAG, the MutGDF-8-FLAG, and the Pro-GDF-8-FLAG from conditioned 
5 media of QM-7 cells according to the manufacturer's instructions. The bound proteins 
were eluted in 0.1 M glycine (pH 3.5) and quantitated in silver-stained SDS-PAGE gels 
(Novex). 

12. Metabolic labeling and immunoprecipitation 

1 0 Transfected QM-7 cells were maintained in Opti-MEM for 48 hours and then 

labeled for 3 hours in cysteine-free DMEM containing 200 pCi/ml of [ 35 S] cysteine 
(Amersham). Cells were washed twice and incubated in Opti-MEM for indicated time 
periods. The conditioned media were then collected, and the cells were lysed in 500 |il 
of lysis buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCI, 5 mM EDTA, 1% SDS, 

1 5 0.25% deoxycholate, 0.25% NP-40, protease inhibitors cocktail {Boehringer 
Mannheim]) and spun for 1 0 minutes. For immunoprecipitations, either 1 ml of 
conditioned medium or 250 |il of lysate (diluted 1 :2 with 10 mM Tris-HCl [pH 7.5], 
1 50 mM NaCI, 5 mM EDTA), were incubated overnight at 4°C with anti-FLAG M2 
affinity gel (Sigma). The immunoprecipitates were r-esuspended in 2X Laemmli loading 

20 buffer containing 2% mercaptoethanol, heated for 10 minutes at 40°C and 
electrophoresed in 14% SDS-PAGE gels. Gels were permeated with Amplify 
(Amersham), dried, and exposed to Kodak film at -70°C. 

13. Identification of GDF-8 -inhibitory activity/Transcription-based assay 

25 This type of bioassay is transcription-based and was performed in two different 

cell lines. A204 human rhabdomyosarcoma cells were used to monitor the efficacy of 
the inhibition because they are highly responsive to Myostatin. To further characterize 
the specificity of the inhibition, both A204 cells and the well-characterized mink lung 
epithelial cell line CCL-64 were used. The advantages ofCCL-64 over A204 are 

30 two-fold: first, in contrast to A204-cells, CCL-64 respond to TGF-p, and second, 



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CCL-64 cells have been used for years as a model cell line to elucidate the molecular 
mechanisms of action of TGF-p superfamily members. The artificial reporter plasmid 
p(CAGA) I2 -MLP (described in Dennler et ah (1998) EMBO J., 17, 3091-3100) was used 
in this assay. Luciferase gene transcription and, thus, activity is driven by an artificial 

5 minimal promoter which is induced by the members of the TGF-p family. Therefore, 
luciferase activity in cell lysates correlates linearly with the degree of stimulation of the 
cells by the applied growth factors. 

Both cell types were plated in 48-well plates in their respective media (DMEM 
for CCL64, McCoy's medium for A204) supplemented with 10% fetal bovine serum, 

10 antibiotics and L-Glutamine. Upon reaching 80% confluence, cells were transfected 
using FuGENE-6 to facilitate plasmid uptake (Boehringer-Mannheim) according to the 
manufacturer's instructions. Cells were transiently transfected with a cocktail of two 
plasmids, pSV-P-gal plasmid to monitor transfection efficiency and luciferase reporter 
plasmid. The reporter plasmid p(CAGA) I2 -MLP is described in Dennler at al. (1998) 

15 supra. After overnight incubation with the transfection reagents, ceils were washed 
twice with the appropriate serum-free medium containing 0.1 % BSA. The inhibitory 
fraction was then added and cells were returned to the incubator for 60 minutes. At the 
end of this period, the following factors were added to the cells: recombinant human 
Myostatin produced from Myostatin-expressing CHO cell conditioned medium, human 

20 TGF-P, and concentrated conditioned medium from CHO cells expressing activin, and 
PA. Cells were lysed after a 6 hour incubation and luciferase and #-galactosidase 
activities were determined in the same samples using the Dual-Light Luciferase Assay 
kit (Tropix, Inc.). Activity is expressed in Relative Luciferase units (RLU), i.e. 
luciferase activity corrected for transfection efficiency that is given by the corresponding 

25 values of P-galactosidase activity measured in the same sample. 

To chemically reduce the inhibitor sample, it was diluted by adding an excess of 
50 mM Na 2 P0 4 , pH 7.0, urea, and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) 
were added to the solution at final concentrations of 6 M and 10 mM, respectively, and 
the solution was incubated at 37°C for 30 minutes. To quench the reduction, 

30 iodoacetamide was added to a final concentration of 10 mM, and the solution was 



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incubated at room temperature for 30 minutes. The sample was then acidified with 10 % 
TFA and the buffer exchanged to 0. 1 % TFA. 

EXAMPLE J: Production and Purification of Fractions A and B 

5 

Fractions A and B were derived separately, each from an independent batch of 
CHO-conditioned medium, using identical purification protocols. The conditioned 
media were collected from CHO cells stably transfected with the expression plasmid 
G8P-1/0.1 , which contains an insert encoding human GDF-8. CHO cells were cultured 

1 0 in alpha medium supplemented with 0.1 mM methotrexate and 1 mg/ml G41 8 
(Geneticin, GIBCO-Life Sciences). 

To obtain Fractions A and B, CHO-conditioned media were passed through three 
different HPLC columns. The first column was an ion exchange column (HQ from 
Pharmacia) where a fraction of the media components was associated with the flow- 

15 through. Figure 1 shows a representative chromatogram measured at 230 nm. The broad 
peak represents the HQ flow-through and the sharp peak represents the bound material. 
Subsequently, the pH of the HQ flow-through was adjusted to pH 5.0 with 6 N HC1, 
which allowed certain components of the HQ flow-through material to bind to an SP ion 
exchange column (Pharmacia). Using a shallow gradient, SP-bound material that was 

20 eluted between 6-9 minutes was collected to obtain Fraction A from one batch of CHO 
conditioned media and SP-bound material that was eluted between 14-19 minutes was 
collected to obtain Fraction B from another batch of CHO conditioned media. The 
gradients and the chromatograms measured at 21 5 nm for Fractions A and B are 
depicted in Figures 2 and 3, respectively. The SP material that eluted at the above times 

25 was desalted using 20 mM NaPhosphate, pH 5.0. Each SP material was then injected 
into a C4 column using a shallow acetonitrile gradient. The C4 fractions were collected 
and the acetonitrile was removed using a speed vac. The gradient and the chromatogram 
measured at 21 5 nm for the C4 runs that produced Fractions A (eluted between 2 1-23 
minutes) and B (eluted at 27 minutes) are depicted in Figures 4 and 5, respectively. C4 

30 fractions were stored in 0.1% trifluoroacetic acid (TFA) and were tested for GDF-8- 
inhibitory activity as described below. 



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To denature samples, urea to a final concentration of 6 M was added, and the 
sample was incubated at 70°C for 10 minutes, followed by chilling on ice. To reduce 
Fractions A and B, the TFA in fractions A and B was diluted by adding an excess of 50 
mM NaPhosphate, pH 7.0. Urea and tris(2-carboxyethyl)phosphine hydrochloride 

5 (TCEP) were added to the solution at a final concentration of 2 M and 1 0 mM, 
respectively. The solution was incubated at 37°C for 30 minutes. To quench the 
reduction, iodoacetamide was added to a final concentration of 10 mM and the solution 
was incubated at room temperature for 30 minutes. The sample was then acidified with 
10 % TFA and the buffer exchanged to 0.1 % TFA. Samples from a separate, blank C4 

10 run (no input protein) that eluted at 22.5 or 27 minutes were collected and treated in 
parallel with Fractions A and B, to be used as appropriate buffer controls in the 
bioassays. 

EXAMPLE 2: Identification of GDF-8-inhibitory Activity in 

] 5 Fractions A and B 

Two independent types of bioassays were developed to test for the inhibitory 
potential of the C4 fractions obtained from original CHO-conditioned media. 

20 DNA replication assay 

The first bioassay was a growth assay performed in G8 mouse skeletal myoblasts 
and in the mink lung epithelial cell line, CCL-64. In this type of assay DNA replication 
and thus proliferation is accurately measured by the incorporation of Bromo-deoxy- 
Uridine (BrdU) or tritiated thymidine label into the DNA. 

25 Briefly, G8 myoblasts were plated in 96- well culture plates coated with 0.1% 

gelatin (Sigma), while CCL-64 were plated in uncoated plates. Both cell types were 
plated in serum-free DMEM containing 0.1% w/v BSA at 10xl0 3 cells per well. The 
following day, media were removed and replaced with fresh media. The relevant 
inhibitory fraction was added, and cells were returned to the incubator for 60 minutes 

30 before growth factors were applied to them. BrdU label at 1 : 1,000 final dilution 

(Boehringer-Manheim) was added to G8 myoblasts 24 hours after addition of the test 



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factors, and incorporation was allowed to proceed for an additional 24 hours. G8 
myoblasts were then fixed and processed according to the manufacturer's instructions 
manual using a commercially available kit (Boehringer-Manheim). The incorporated 
BrdU was determined colorimetrically. [^HJ-thymidine (NEN-Dupont) was added to 
5 CCL-64 epithelial cells 12 hours after addition of test factors, for a total period of 4 
hours. CCL-64 cells were washed twice with 1 0% trichloroacetic acid and radioactivity 
was extracted with 0.3N NaOH and determined by scintillation counting. 

Transcription-based Assay 

1 0 The second type of bioassay was transcription-based and was performed in two 

different cell lines. A204 human rhabdomyosarcoma cells were used to monitor the 
efficacy of the inhibition, because they are particularly responsive to GDF-8. To further 
characterize the specificity of the inhibition, both A204 cells and the well-characterized 
mink lung epithelial cell line CCL-64 were used. The advantages of CCL-64 over A204 

1 5 are two-fold: first, in contrast to A204 cells, CCL-64 respond to TGF-{5, and second, 
CCL-64 cells have been used for years as a model cell line to elucidate the molecular 
mechanisms of action of TGF-p superfamily members. Two artificial reporter plasmids 
were used in this assay: p3TP-Lux (Wrana et aL (1992) Cell, 71, 1003-14) and 
p(CAGA) 12 -MLP (Dennler et aL, (1998) EMBOJ., 17, 3091-3100). In both, luciferase 

20 gene transcription (and thus activity) is driven by artificial minimal promoters which 
respond to members of TGF-P family members. Therefore, luciferase activity in cell 
lysates correlates linearly with the degree of stimulation of the cells by the applied 
growth factors. 

Briefly, both cell types were plated in 48-well plates in their respective media 
25 (DMEM for CCL-64, McCoy's medium for A204) supplemented with 10% fetal bovine 
serum, antibiotics and L-Glutamine. Upon reaching 80% confluence, cells were 
transfected using FuGENE-6 to facilitate plasmid uptake (Boehringer-Mannheim), 
according to the manufacturer's instructions. Cells were transiently transfected with a 
cocktail of two plasmids; pS V-p-gal plasmid (Promega) to monitor transfection 
30 efficiency, and either of the two luciferase reporter plasmids. The first reporter, p3TP- 



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lux, is described in Wrana et al., 1 992, supra. The second, p(CAGA)]2-MLP ? is 
described in detail in Dennler at al., 1998, supra. After overnight incubation with the 
transfection reagents, cells were washed twice with the appropriate serum-free medium 
containing 0.1% BSA. The inhibitory fraction was then added and cells were returned to 
5 the incubator for 60 minutes. At the end of this period, the following factors were added 
to the cells: human recombinant GDF-8-expressing CHOcell conditioned medium, 
human TGF-Pj (R&D Biosystems, Minneapolis) and concentrated conditioned medium 
from CHO cells expressing Activin PA (Genetics Institute, Cambridge, MA). Medium 
from mock-transfected cells was devoid of activity. 
1 0 Cells were lysed after a 6 hour incubation and luciferase and P-galactosidase 

activity were determined in the same sample using the Dual-Light Luciferase Assay kit 
from Tropix Inc. Activity is expressed in Relative Luciferase Units (RLU), i.e. 
luciferase activity corrected for transfection efficiency that is given by the corresponding 
values of P-galactosidase activity measured in the same sample. 

15 

Inhibitory Effects of Fraction A (C4 column fraction eluted at 22.5 min) 

I. Effect on proliferation of G8 myoblasts and of CCL-64 mink lung epithelial cells 

20 GDF-8 by itself at 100 ng/ml increased G8 myoblast proliferation 6-fold, as 

measured by increased incorporation of BrdU label in DNA {Figure 6A). The buffer 
vehicle used to resuspend fractions derived from the C4 column was devoid of activity 
on its own in this assay, and failed to substantially influence the effect of lOOng/ml of 
GDF-8 (Figure 6A). 

25 Of the various fractions that were obtained from the C4 column, a bioactive peak 

that eluted at 22.5 minutes was identified, called Fraction A. Fraction A by itself had 
no effect on G8 myoblast proliferation. However, it was able to almost abolish the 
positive effect of GDF-8 on cell proliferation {Figure 6A). Similar GDF-8-inhibitory 
effects of Fraction A were observed when tritiated thymidine was used instead of BrdU 

30 as label, and when the effects of GDF-8 and Fraction A were tested in another cell type, 



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CCL-64 cells (Figure 6B). In contrast to its effect on myogenic cell lines, GDF-8 
inhibits cell proliferation in the CCL-64 epithelial cell line, an effect that is identical to 
that of TGF-p. This growth inhibition of GDF-8 was reversed when CCL-64 cells were 
pretreated for 60 minutes with an excess weight/weight Fraction A prior to addition of 
5 GDF-8 (Figure 6B). In sum, regardless of the cell line, the type (positive or negative) 
proliferative effect of GDF-8 and the method of measuring cell proliferation, Fraction A 
consistently displayed clear antagonistic activity versus GDF-8. 

II. Effects on transcription from minimal promoters/Sensitivity to denaturing and 
10 reduction/Specificity of Action 

As shown seen in Figure 7A, 1-hour preincubation with Fraction A abolished 
the effect of 3 or 10 ng/ml of GDF-8 on p(CAGA)]2-MLP luciferase gene transcription 
in A204 rhabdomyosarcoma cells. This effect was also observed using the second, 
1 5 independent reporter plasmid p3TP-Lux in the same cells. 

Reduction of Fraction A using two different reducing agents. DTT and 
TCEP/iodoacetamide did not affect the inhibitory potential of Fraction A, regardless of 
the reporter plasmid used, indicating that the active moiety within this fraction is not 
sensitive to strong reducing agents (Figure 7A). The fact that these conditions are 
20 indeed capable of effecting reduction was supported independently by showing, for 
example, that they can abolish the activity of another reduction-sensitive fraction. 
Fraction B (see Figure 9). 

Denaturing of Fraction A by either heat (1*00°C for 10 minutes, then quick 
chilling on ice) or by 6M urea and heat did not affect its inhibitory activity either 
25 (Figure IB). 

The issue of specificity was addressed in CCL-64 cells using the p(CAGA)]2- 
MLP reporter. The effects of Fraction A on three different members of the TGF-P 
family: TGF-P itself, GDF-8, and Activin, were compared. 

As shown in Figure 8, Fraction A (or its vehicle) did not modulate basal 
30 (background) luciferase activity by itself, nor did it appreciably alter the effect of TGF-p 



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or Activin. In contrast, it still showed strong inhibitory activity against GDF-8. This 
piece of evidence indicates that Fraction A can clearly discriminate between structurally 
related factors that share a significant degree of homology (-40% at the amino acid 
level). 

5 

Inhibitory Effects of Fraction B (C4 column fraction eluted at 27 min) 

I. Effects of Fraction B on transcription from minimal promoters/Sensitivity to 
denaturing and reduction/Specificity of Action 

10 

A second, independently-derived inhibitory fraction was also identified from the 
processing of a separate batch of CHO-conditioned media. This novel inhibitor fraction 
had a retention time of 27 minutes in a C4 column. This fraction was tested in the 
transcription-based bioassay, under the same conditions as Fraction A. Similar to 

1 5 Fraction A, Fraction B was able to functionally inhibit the effect of GDF-8 on luciferase 
activity in A204 rhabdomyosarcoma cells {Figure 9). 

Further analysis indicated that, similar to the activity in Fraction A, the 
inhibitory activity within Fraction B was insensitive to strong denaturing conditions (6M 
urea plus heat). In contrast to Fraction A however, TCEP reduction of the Fraction B 

20 sample abolished its inhibitory activity {Figure 9). 

To test the specificity of this novel C4 fraction, an additional series of 
experiments was performed in CCL-64 cells using the p(CAGA)j2-MLP reporter. The 
results obtained indicate that Fraction B resembles Fraction A in its ability to 
specifically inhibit the action of GDF-8, while leaving intact the activity of TGF-f} or 

25 Activin {Figure 10). 

EXAMPLE 3: Biochemical Characterization of Fractions A and B 

As part of an effort to better understand the composition of Fractions A and B, a 
30 silver stain was run with Fractions A and B under both reduced and non-reduoed 

conditions. The methods used for reducing Fraction A are described above. Fraction A 



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in solution comprises several components whether it is in a reduced or non-reduced 
state, ranging in weight from very low (co-migrating with the dye front) to 200 kDa. 
However, when the sample is reduced new bands appear at lower molecular weight and 
some of the higher molecular weight bands disappear. 
5 Fraction B was reduced using the methodology and reducing agents supplied by 

Novex. The results reveal that there are two major species and several minor species in 
the non-reduced state of Fraction B. In the reduced state, a new band around 12 kDa is 
present giving a total of 3 major components with several minor components present as 
well. 

10 

EXAMPLE 4: Identification and Characterization of the GDF-8 Inhibitor in 

Fractions A and B 

A four-step strategy to identify and characterize the inhibitor in Fractions A and 
1 5 B can be performed as follows: 

I. Identification of the components of Fractions A and B that inhibit GDF-8 
activity 

20 Several distinct species are present in Fractions A and B. Therefore, it is 

necessary to further process these Fractions into their individual components in order to 
unambiguously identify the active moiety or moieties (e.g. the component(s) which 
inhibit GDF-8). This process can be done, as described in Example 5 below, by 
performing a preparative non-reducing SDS-PAGE, and by subsequently recovering the 

25 separated components by electrocution. Components possessing GDF-8 inhibitory 
activity can then be identified using bioassays described herein. 

After successfully identifying one or more GDF-8 inhibitory -components, the 
specificity of these components can be further studied, as described below in Example 5. 
For example, several molecules that possess structural and biological activity -similar to 

30 GDF-8 included in this study, such as Activin, BMPs, and TGF-p can be used to test for 



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GDF-8-specific inhibitory activity. The purified GDF-8 inhibitor can be tested for the 
ability to antagonize GDF-8 effects in a dose-dependent and GDF-8-specific fashion. 



II. Characterizing GDF-8 Inhibitors 

5 

As described in Example 5 below, one or more of a combination of N-terminal 
sequencing and mass spectrometry analyses can be used to characterize the inhibitor. In 
N-terminal sequencing analysis, at least 1 0 to 1 5 jig of either Fraction can be loaded 
onto non-reducing SDS-PAGE. Proteins are then separated and electroblotted onto a 

1 0 PVDF membrane. After staining with Coomassie blue, the protein band corresponding 
to the inhibitor is cut out for sequencing analysis. The number of residues observed at 
each sequencing cycle correspond directly with the number of different components in 
the sample, and the ratio of intensity among the various signals at each cycle is used to 
estimate the proportion of each different component. 

1 5 The precise molecular weights of the inhibitor and its components can be 

determined by electrospray/ionization mass spectrometry (ESI/MS) in the absence and 
presence of a reducing agent. The mass spectrometry information, combined with the 
expected molecular weights calculated from the known primary sequences, can be used 
for two purposes: first, to either confirm or refine the composition of the inhibitor 

20 through N-terminal sequencing analysis, and second, to evaluate the integrity of the 
inhibitor regarding potential C-terminal truncation, internal cleavage, or post- 
translational modification, in which N-terminal sequencing analysis is unable to provide 
the answer. 

25 III. Characterizing Disulfide Patterns of GDF-8 Inhibitors 

Experiments which characterize disulfide patterns are particularly relevant for 
the characterization of fractions, such as Fraction having inhibitory activity which is 
sensitive to reduction and which, therefore, contain disulfide bonds. Successful 
30 characterization of disulfide bonds is dependent on the consistency of both sample 

preparation and high resolution peptide mapping. The disuifide-containing fragments are 



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identified by comparing HPLC chromatograms between Tris(2-carboxyethyl) phosphine 
(TCEP)-treated (reduced) and non TCEP-treated samples. 

Excepting the early region (non-retentate) of the chromatogram, the 
disappearance of a peak in the chromatogram of the TCEP-treated sample suggests a 
5 disulfide-containing fragment. The corresponding fraction from a non TCEP-treated 
sample is analyzed both by N-terminal sequencing and by matrix-assisted laser 
desorption/ionization and time-of-flight mass spectrometry (MALDI-TOF/MS) 
analyses. In the case where a single disulfide bond is present, two amino acid residues 
are evidenced with comparable signal intensity, for each sequencing cycle. 

10 The preliminary characterization of the inhibitory Fractions A and B, as well as 

their presence in the conditioned media of GDF-8-expressing CHO cells, is compatible 
with the notion that they are GDF-8-related (e.g. contain peptides of GDF-8 which 
inhibit GDF-8 activity). If this is supported by the sequencing efforts described above, 
then by comparison with the known primary sequence of GDF-8, the identity of 

15 disulfide-1 inked peptides, and more importantly, the disulfide position within the GDF-8 
sequence is easily determined. The MALDI-TOF/MS analysis is used primarily for the 
purpose of confirmation. 

In the case of either multiple sequences or multiple disulfide bonds being 
involved, a secondary digestion by a protease (the selection of the protease being 

20 completely sequence-dependent) is needed to generate analyzable fragments. Another 
run of peptide mapping can then be performed as described in the materials and methods 
section above. 

IV. Identification of Post-translational Modifications GDF-8 Inhibitors 

25 

One of the most common forms of mammalian protein modification is 
glycosylation, which in some instances can substantially affect the bioactivity of the 
protein. Therefore, it is important to investigate whether glycosylation of GDF-8 
inhibitory peptides modulates the peptide's inhibitory activity. 



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To do so, the inhibitory species can be deglycosylated as described in the 
materials and methods section above and as illustrated in the following Example. The 
correlation between structure (glycosylation) and functional activity (inhibition) can be 
either confirmed or ruled out by the bioassays for GDF-8 inhibition described herein. 

5 

EXAMPLE 5: Identification and Characterization of the GDF-8 Inhibitor in 

Fraction B 

The individual component in Fraction B responsible for the inhibition of GDF-8 
10 was determined by running a preparative non-reducing SDS-Page gel and separating the 
components by electroelution. It was determined that a peptide having a molecular 
weight of approximately 36 kDa is responsible for the inhibition of GDF-8. The 
specificity of the GDF-8 inhibition was also shown as described below. 

The first step in the characterization of the inhibitor was the determination of the 
15 precise molecular weight by electrospray/ionization mass spectrometry. The mass spec 
spectrum showed three peaks separated by 600 Da with the major component of 
molecular mass of 29,472.0 Da (M-Scan, Inc.) (Figure 23). The difference in the 
molecular weights from mass spec and the SDS-Page could have been due to the gel and 
the molecular weight marker. 
20 The next step was to determine the amino acid sequence of the inhibitor by pulsed 

phase N-terminal sequencing with five cycles (determines the first five amino acids). 
Table 1 shows the major and minor amino acids detected with the major amino acids 
being greater than 90% present. The sequence (NENSE) (SEQ ID NO:26) corresponds 
to a sequence found in the pro-domain of GDF-8. 



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TABLE I 



Cycle No. 


Major PTH-AA Detected 


Minor PTH-AA Detected 


1 


Asn 




2 


Glu 


Pro,Ala,Phe 


3 


Asn 


Gin, Val, Lys 


4 


Ser 


Asp,Lys 


5 


Glu 


Leu 



The next step was to determine whether the inhibitor from Fraction B (i.e., the 
pro-domain of GDF-8) referred to as "pro-GDF-8" is effected by post-translational 

5 modification. The most common form of modification in mammalian proteins is 
glycosylation. In the pro-domain of GDF-8 there is only one asparagine for 
glycosylation. To determine if glycosylation of the asparagine is needed to maintain the 
inhibitory activity the GDF-8 pro-domain, the pro-GDF-8 inhibitor from Fraction B was 
deglycosylated using an N-Glycosidase F Deglycosylation kit (Boehringer Mannheim). 

10 The enzyme, denaturing reagents and salts were removed by passing the deglycosylated 
protein through a C4 column. A transcription-based bioassay (using the (CAGA) 12 -MLP 
construct) was used to determine the inhibitory activity of the glycosylated and 
deglycosylated protein. The results showed that the GDF-8 pro-domain must be 
glycosylated in order to inhibit GDF-8. (Figure 28) 

15 Overall, the foregoing studies demonstrated that the GDF-8 peptide inhibitor 

isolate from Fraction B is the entire pro-domain of GDF-8 in glycosylated form, and that 
glycosylation (e.g., production of the peptide in cells that can glycosylate the peptide) is 
necessary for inhibitory activity. 

20 Characterization of the Fraction A Inhibitor Using a DNA Replication Assay 

As shown in Figure 6A, GDF-8 itself at 100 ng/ml increases G8 myoblast 
proliferation 6-fold, as measured by increased incorporation of BrdU label in DNA. The 
buffer vehicle used to resuspend pro-GDF-8 was devoid of activity on its own in this 
assay, and failed to substantially influence the effect of 100 ng/ml of GDF-8. However, 



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the pro-GDF-8 was able to almost abolish the positive effect of GDF-8 on cell 
proliferation. 

Similar GDF-8-inhibitory effects of the pro-GDF-8 were observed when tritiated 
thymidine was used instead of BrdU as label, and the assay was performed in another 
5 cell type, CCL-64 cells (Figure 6B). In contrast to its effect on myogenic cell lines, 
GDF-8 inhibits cell proliferation in the CCL-64 epithelial cell line, an effect that is 
identical to that of TGF-p. This growth inhibition of GDF-8 was reversed when CCL- 
64 cells were pretreated for 60 minutes with a 6-fold excess weight/weight of pro-GDF- 
8 prior to addition of the mature GDF-8 protein (Figure 6B). 
1 0 In conclusion, regardless of the cell line, the type (positive or negative) effect of 

GDF-8 on proliferation, and the label used to measure DNA replication, this modified 
growth assay accurately reflects the activity of GDF-8 and, can be used to determine the 
GDF-8-inhibitory potential of GDF-8 antagonists, such as pro-GDF-8. 

15 Characterization of the GDF-8 Inhibitor (Fraction B) Using a Transcription-based Assay 
As shown in Figure 7A, reporter gene construct p(CAGA) I2 -MLP respond in a 
dose-dependent fashion to GDF-8, with substantial induction detected as early as 6 
hours after application of the growth factor. However, 1-hour pre-treatment of cells with 
the pro-domain of GDF-8 abolished the effect of 3 or 10 ng/ml of mature GDF-8 on 

20 p(CAGA)i 2 -MLP luciferase gene transcription in A204 rhabdomyosarcoma cells (Figure 
25). This effect was also observed using the second, independent reporter plasmid 
p3TP-Lux in the same cells. 

This type of transcription-based assay is more sensitive to the effect of GDF-8 
(compare GDF-8 concentrations in Figures 7 A and IB to those in Figures 6A and 6B). 

25 This type of assay also determines specificity of inhibition, for example, in CCL-64 cells 
using the p(CAGA) 12 -MLP reporter. Specifically, the effects of pro-GDF-8 on three 
different members of the TGF-p family: TGF-p itself, GDF-8 and Activin, were 
compared. As seen in Figure 8, pro-GDF-8 (or its vehicle) did not modulate basal 
(background) luciferase activity by itself, nor did it appreciably alter the effect of TGF-P 

30 or Activin. In contrast, it still showed strong inhibitory activity against GDF-8. 



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In conclusion, the above-described transcription based bioassay is specifically 
optimized to allow for sensitive monitoring of GDF-8 activity and for determining the 
inhibitory potential of putative GDF-8 antagonists, regardless of their mechanism of 
action (e.g., whether they act by binding and inactivating GDF-8 or by blocking the 
5 cognate receptors). 

Characterization of the GDF-8 Inhibitor Using a Creatine Kinase Assay 

As shown in Figure 26, treatment of chick primary myoblasts or C2C12 
myoblasts with GDF-8 for 72 hours at the onset of differentiation, reduced the activity 

10 of creatine kinase in cell lysates. This was also evident by comparison of cell 

morphology in the presence and absence of GDF-8. In the latter case, a lot fewer tubes 
had formed in the dish. TGF-p, had a similar effect to that of GDF-8 in C2C12 cells, 
but not in chick primary myoblasts (Figure 26). Therefore, the creatine kinase assay can . 
be used to identify and characterize GDF-8 inhibitors, such as pro-GDF-8, by 

15 determining their ability to inhibit the reduction in creatine kinase activity by GDF-8. 

Characterization of the GDF-8 Inhibitor Using an Assay Based on the Differentiation of 
3T3-L1 Pre-adipocytes 

Following a specific protocol, 3T3-L1 pre-adipocytes can differentiate into 

20 adipocytes, displaying the full range of molecular markers and morphological 
characteristics typical of adipocytes in vivo. As the results of this experiment 
demonstrate, treatment of 3T3-L1 cells with GDF-8 at the onset of differentiation 
process resulted in a severe blockade of differentiation. This was assessed using two 
independent criteria. The effect of GDF-8 is evident by the near-absence of refractile 

25 cells that contain lipid droplets. At the mRNA level, the expression of the adipocyte- 
specific gene GLUT4 is blocked in GDF-8-treated cells. Two cytokines that have 
previously been implicated in the control of adipocyte metabolism, namely TNF-a and 
TGF-p,, can mimick the effect of GDF-8. In addition, the -degree of differentiation -can 
be addressed by a variety of immunological, biochemical and molecular biological 

30 methods, as mentioned in the Materials and Methods section. Changes in adipocity in 



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mouse and cattle that lack functional GDF-8 have been described. Therefore, this assay 
provides a physiologically important measure of GDF-8 activity in vivo. Accordingly, 
the effect of GDF-8 inhibitors on GDF-8 blockade of adipocyte differentiation can be 
used to evaluate such inhibitors. 

5 

Characterization of the GDF-8 Inhibitor Using a Glucose uptake assay in 3T3-L1 
Adipocytes 

In differentiated adipocytes, insulin stimulates glucose transport through the Glut- 
4 transporter in a dose-dependent fashion (Figure 27). This effect can be blocked in a 
10 dose-dependent manner by treatment of the cells for 72 hours with GDF-8 (Figure 27). 
Importantly, this assay offers an in vitro correlate of GDF-8 activity of in vivo body fat 
metabolic functions. 

EXAMPLE 6: Generation of GDF-8 Inhibitors Derived From the GDF-8 

1 5 Pro-domain 

To produce GDF-8 inhibitors comprising all or a portion of the pro-domain of 
the GDF-8 protein, a partial mouse cDNA encoding the pro-domain (residues 1-266 
shown in Figure 13) of GDF-8 (also referred to as "pro-GDF-8"), was generated by 

20 PCR-based mutagenesis. The PGR fragment was subcloned in the TA vector 

(Invitrogen), and sequenced to confirm the incorporation of the mutation. The cDNA 
encoding pro-GDF-8 was inserted in the FLAG-CMV-5a vector (Sigma) to produce an 
in-frame fusion with the FLAG epitope at the 3' end, used for detection and purification 
of the pro-GDF-8 protein (Figure 12C). This vector contains cytomegalovirus promoter 

25 sequences, which result in high-level expression in eucaryotic cells. The construct 
expressing the wild type GDF-8 (WT-GDF-8) was generated using the same vector 
(Figure 7 2 A). 

An un-cleavable full-length GDF-8 mutant was expressed using the same 
method, by replacing the predicted cleavage site at the boundaries between the pro- 
30 domain and the mature protein (Figures 1 3B and 14). 



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The resulting three expression constructs were introduced in QM-7 quail 
myoblast cells by transient transfection using FuGENE reagent (Boehringer Mannheim). 
The efficiency of transfection, monitored by a reporter construct containing P-gal, was 
about 70-80%. Affinity chromatography using an affinity gel coupled to a specific 

5 monoclonal antibody directed against the FLAG epitope (Sigma) was used to purify the 
recombinant proteins, that is the Mut-GDF-FLAG, and the pro-GDF-8-FLAG from 
conditioned medium of transfected QM-7 cells. The bound proteins were eluted in 0.1 
glycine (pH 3.5), and quantitated by SDS-PAGE, followed by Silver Staining (Novex). 
The estimated concentration of the purified pro-GDF-8 was about 20 ng/ml. 

1 0 The eluted proteins were also analyzed by immunoblot with the AntiFlag 

antibody. The Mut-GDF-8 and pro-GDF-8 proteins were properly recovered from the 
column, and the immunoreative proteins of the expected molecular weights were present 
in the recovered fractions, to be used in the transcription-based bioassay. 

To test whether the pro-region of GDF-8 (pro-GDF-8) can interfere with the 

15 effects of mature GDF-8, column-purified pro-GDF-8 was pre-incubated with a specific 
amount of human recombinant GDF-8 for 30 minutes at room temperature. In parallel, 
comparable amounts of BSA, glycine buffer, or full-length uncteavable-GDF-8 (Mut- 
GDF-8) were added to separate tubes containing GDF-8, to be used as controls. Two 
different dilutions of pro-GDF-8 were chosen, to achieve 30-fold and 10-fold 

20 weight/weight excess compared to GDF-8. Following pre-incubation, the mixture was 
transferred onto A204 cells, transfected with either p(CAGA), 2 -MLP or p3TP-Lux 
reporter plasmids mixed with pSV-P-gal plasmid. Six hours later cells were lysed and 
luciferase and P-galactosidase activity were determined in the lysates. The final 
concentrations of the buffers and column-purified proteins in each well were 10 ng/ml 

25 for GDF-8 and 300 ng/ml and 1 ,000 ng/ml for pro-GDF-8 and 1 ,000 ng/ml for the un- 
cleavable full-length GDF-8. 

As seen in Figure 16 the column-purified pro domain of GDF-8 was able to 
totally suppress the induction of luciferase activity by GDF-8. This induction was not 
influenced when prior to its addition to the cells GDF-8 had been pre-incubated with 

30 either BSA, glycine buffer (used for column elution), or full length, uncleavable GDF-8 



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protein (Mut-GDF-8). This effect was dose-dependent (Figure 16) and specific for 
GDF-8, since pro-GDF-8 did not affect the induction of luciferase activity by TGF-pi or 
by activin (Figure 75). 

Similar to fractions A and B ? the effect of denaturing and reduction on the 
5 inhibitory activity of pro-GDF-8 was examined. As seen in Figure 24, pro-GDF-8 
retained its inhibitory activity after denaturing with 6M urea. However, reduction of 
pro-GDF-8 resulted in the complete loss of inhibition. 

To study the effect of the pro-domain on secretion of wild-type GDF-8, the 
conditioned media from QM-7 cells co-transfected with Pro-GDF-8 and WT-F-GDF-8 

10 expression plasmids were analyzed. Western blotting with anti-FLAG specific 

antibodies under reducing conditions detected major immunoreactive proteins migrating 
at 38 kDa in the conditioned media from cells transfected with Pro-GDF-8. This is 
consistent with the predicted size for the GDF-8 pro-domain. An additional polypeptide 
immunoreactive with the FLAG antibody, was also identified. This polypeptide has a 

1 5 molecular weight of approximately 25 kDa. 

To determine the identity of the polypeptides represented by the 38 KDa and 25 
kDa bands, the polypeptides were purified by affinity chromatography and the 
corresponding bands from the Coomassie-stained gel were subjected to N-terminal 
sequencing. The N-terminal sequence of the 25 kt> polypeptide was DDSSD (SEQ ID 

20 NO:27). demonstrating that this 25 KDa polypeptide is the product of an alternative 

cleavage at a specific site (Arg 99). (The same cleavage site was also identified for both 
wild type and cleavage site mutant GDF-8 precursors). 

Dimers of the pro-domain migrating at 80 kDa, as well as monomeric forms of 
the pro-domain were also present on the non-reducing gel. 

25 Co-transfection of the Pro-GDF-8 construct with the WT-F-GDF-8 construct 

resulted in a significant decrease in the amount of secreted mature GDF-8. 

Since two FLAG-immunoreactive proteins were -detected in the conditioned 
media of QM-7 cells transfected with Pro-GDF-8, it was important to determine which 
of these polypeptides possessed the inhibitory activity wer GDF-8. This was achieved 

30 by elution of the proteins from denaturing SDS-PAGE. Individual polypeptides were 
then tested for their ability to act as GDF-8 inhibitors in a reporter activation bioassay, 



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as described above. The results demonstrate that only the higher molecular weight 
polypeptide (migrating at 38 kDa) representing the full-length pro-domain possessed 
inhibitory activity, whereas the polypeptide migrating at 25 kDa, a product of an 
alternative cleavage at Arg 99, was inactive. This indicates that the inhibitory (or GDF- 
5 8-binding) domain is located at the N-terminus of the pro-domain, upstream of Arg 99. 
Thus, GDF-8 inhibitors of small molecular size may be designed based on the sequence 
of the pro-domain upstream of Arg 99 (see Figure 13). 

Overall, the foregoing studies demonstrate that the GDF-8 pro-domain is able to 
specifically inhibit the biological activity of GDF-8 in vitro in a transcription-based 
10 assay and that it affects the secretion of the mature GDF-8 in the conditioned media of 
cultured cells, when co-expressed with the wild type GDF-8 protein. 

EXAMPLE 7: Generation Of Transgenic Mice Overexpressing The GDF-8 

Pro-Domain 

15 

To confirm the results described above in Example 6, in vivo, a DNA construct 
for muscle-specific expression of the GDF-8 pro-domain in mice was generated. 
Briefly, the partial mouse cDNA encoding the pro-domain (residues 1-266 shown in 
Figure 13) was fused with the FLAG epitope at the C-terminus, and inserted in the 

20 pMEX-NMCS2 expression vector downstream of the rat Myosin Light Chain 1 

promoter. This vector was shown to facilitate fast fiber specific expression in skeletal 
muscle of transgenic mice (Neville, C. et ah (1996) Dev. Genetics 19: 157). 

Transgenic mice were generated by standard pronuclei microinjections. 
Offspring were screened for transgene integration by tail PCR with specific primers to 

25 the FLAG epitope. Fourteen germ-line-integrated founders were established. The 
Northern blotting analysis of muscle tissues with GDF-8 specific probes identified 3 
lines with high level of transgene expression in skeletal muscle. The expression of the 
transgene (the GDF-8 pro-domain) exceeded the levels of endogenous GDF-8 by 3-10 
fold. 



30 



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EXAMPLE 8: GDF-8 Is Secreted Within A Latent Complex With Its 
Pro-Domain Which Is Activated By Acidification 



Upon characterization of media from QM-7 cells transfected with WT GDF-8, an 
5 additional 38 kDa "double" band was detected. To determine the identity of this 

polypeptide the FLAG-tagged proteins were purified by affinity chromatography and the 
bands from the Coomassie-stained gel were subjected to N-terminal sequencing. The N- 
terminal sequence of the 38 kDa "double" band polypeptide was DDSSD (SEQ ID 
NO:27), indicating that it is the product of an alternative cleavage at a specific site (Arg 
10 99) . The sequence analysis of this "double" band migrating at 38 kDa revealed also the 
polypeptide with an amino-terminal sequence GPVDLNE (SEQ ID NO:28), which is 
identical to that of the GDF-8 precursor protein. This band represents the pro-domain, 
lacking the signal peptide, which is co-purified with the FLAG-tagged GDF-8 precursor 
and mature fragment. This polypeptide, not recognized by an anti-FLAG M2 specific 
15 antibody, co-migrates with the independently expressed pro-domain. Since the FLAG 
epitope is located at the C -terminus of the WT-GDF-8 protein, the GDF-8 pro-domain 
could only bind to the anti-FLAG affinity gel if it is indeed associated with the mature 
GDF-8. 

To further characterize the secreted GDF-8 complexes, cell media from QM-7 
20 cells transfected with WT-GDF-8-F was size-fractionated using Microcon centrifugal 
filters (with a molecular weight cut-off of 50K). The retentate contained almost all 
GDF-8 related immunoreactive proteins, further supporting the presence of the high 
molecular weight complexes, which migrated at 60-120kDa under non-reduced 
conditions. Upon reduction, the mature GDF-8 fragments, migrating at 15kDa, as well 
25 as unprocessed forms of GDF-8 were detected. No mature GDF-8 dimer was found in 
the flow-through fraction. These data confirm that mature GDF-8 is present in the cell 
media within a high molecular weight complex. 

To assess the biological activity of the GDF-8 complexes produced by QM-7 
myoblasts, a transcription-based reporter activation assay was employed (see Figure 29). 
30 The crude cell media did not exhibit any GDF-8-like biological activity. In contrast, 
purified human mature GDF-8 efficiently induced luciferase activity from the reporter 



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plasmid p(CAGA) 12 -MLP (described above). Unlike crude cell media, the affinity 
purified GDF-8 complexes possessed activity. This limited activation occurred, most 
likely, from chemical acidification resulting from elution with glycine buffer (pH 3.5). 
Further purification of affinity purified complexes through an HPLC C4 column (upon 

5 acidification with TFA) resulted in the identification of a more active fraction eluting at 
approximately 18 minutes. This fraction (fraction #18) evoked the highest response in 
the transcription-based bioassay and is mainly composed of the mature GDF-8 migrating 
at!5 kDa on the reduced SDS-PAGE. Importantly, fraction #18 contains far less 
amounts of unprocessed GDF-8, than the starting material (Figure 29, lane 1) or fraction 

10 # 20 (Figure 29, lane 4). 

Thus, it is possible that high molecular weight GDF-8-related proteins, which are 
secreted by QM-7 cells and co-purified (by affinity chromatography) with the mature 
GDF-8, can inhibit its biological activity. The aforementioned results suggest that the 
mature GDF-8 is secreted by QM-7 cells within a latent complex, containing the pro- 

1 5 domain. This is consistent with the ability of the purified pro-domain to inhibit the 
activity of GDF-8 in a transcription-based assay (described above). The underlying 
mechanism may be the association of the mature GDF-8 with its pro-domain, preventing 
its interaction with the signaling receptor. 

20 EXAMPLE 9: Activation Of The Latent GDF-8 Complex By Calpain 

M-calpain is a ubiquitiously expressed calcium-dependent member of the calpain 
family of cysteine proteases. In these experiments, the ability of m-calpain to activate 
the GDF-8 complex was investigated. First, the ability of m-calpain to cleave GDF-8 

25 proteins in vitro (i.e. , in the test tube) was studied. Briefly, QM-7 conditioned media 
containing WT-GDF-8-F or Pro-GDF-8-F proteins were treated with 0, 0.1, and 1 U/ml 
of calpain at 37 °C for an overnight period. The results demonstrated that m-calpain can 
specifically cleave the pro-domain of GDF-8, generating additional lower molecular 
weight species. Notably, the full-length precursor of wild type GDF-8 is also a substrate 

30 for m-calpain. Treatment with a higher concentration of calpain (lU/ml) resulted in a 
complete depletion of the full-length wild type GDF-8 protein. Only trace amounts of 



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proteolytic products of this cleavage were detected. Importantly, the mature GDF-8 was 
not degraded by m-calpain. In the presence of QM-7 cells, m-calpain also cleaved the 
pro-domain, as evidenced by the appearance of lower molecular weight bands on the 
SDS-PAGE. 

5 Since m-calpain was able to digest the GDF-8 pro-domain, it was hypothesized 

that m-calpain might activate the latent GDF-8 complex. To verify ths hypothesis, 
supernatants from QM-7 cells, transfected with WT-GDF-8-F construct or mock- 
transfected cells, were treated with 0 or lU/ml of m-calpain for 2 hours at 37° C, 
followed by affinity purification of GDF-8 complexes on the anti-FLAG column 

1 0 (described above). The purified proteins were tested for activity in the transcription- 
based bioassay described herein. Luciferase activity was 2.5-fold higher after the 
incubation of GDF-8 complexes with m-calpain, compared to the untreated control 
(Figure 30A). In agreement with its inability to digest the mature factor, m-calpain did 
not inhibit the activity of mature human recombinant GDF-8 (hrGDF-8) (Figure SOB). 

1 5 However, pre-incubation of the pro-domain with m-calpain drastically reduced the pro- 
domain's ability to inhibit mature GDF-8 (Figure 30B). These results demonstrate that 
m-calpain can release active GDF-8 from the latent complex by cleaving the pro- 
domain. 

The aforementioned data show that activation of GDF-8 and the release of the 
20 mature GDF-8 protein from the GDF-8/pro-domain complex targets for inhibiting GDF 
function. For example, the stability of the 

pro-domain can be increased to, thereby, prevent its potential cleavage by cellular 
proteases. This will, in turn, lead to the stabilization of the GDF-8/pro-domain complex, 
thus, preventing the release of active GDF-8. 

25 

EXAMPLE 10: Production And Characterization Of a GDF-8 Cleavage-site 

Mutant (Dominant-Negative Mutant) 

A GDF-8 cleavage-site mutant (dominant-negative mutant) was generated and 
30 tested for GDF-8 inhibition as follows: 



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Briefly, a construct expressing an un-cleavable full-length GDF-8 mutant 
(Mut-GDF-8) was generated by replacing the predicted cleavage site at the boundaries 
between the pro-domain and the mature protein. After removal of the signal peptide, the 
predicted protein comprises the pro-domain followed by the C -terminal mature region of 

5 GDF-8 {Figure 72B). Unlike wild-type GDF-8, the un-cleavable mutant can not be 
cleaved to generate the mature GDF-8 protein and is, therefore, not biologically active. 
The wild-type (WT) mouse GDF-8 cDNA was also subcloned in the same vector to 
generate the full-length precursor protein tagged with the FLAG epitope at the 
C-terminal. The resulting construct is referred to as WT-F-GDF-8 (Figure 72A). The 

10 unmodified (not tagged) GDF-8 cDNA subcloned in the CMV-based expression vector 
is referred to as WT-GDF-8. 

Both expression constructs were introduced into QM-7 quail myoblast cells by 
transient transfection, and GDF-8 proteins were immunoprecipitated from conditioned 
media with anti-FLAG affinity gel. The immunoprecipitates were further analyzed by 

1 5 SDS-PAGE, followed by detection with an anti-FLAG specific antibody as described in 
Methods. The wild-type (WT-F-GDF-8) was expressed and processed properly in 
QM-7 cells. Under reducing conditions two immunoreactive bands were detected, 
representing the full-length precursor (45-50kDa) and the mature GDF-8 (15kDa). The 
sizes of these proteins are consistent with ones previously reported for GDF-8. The 

20 remainder (pro-domain) is not detectable, as it lacks the FLAG epitope. No 

immunoreactive proteins were detected in conditioned media from cells transfected with 
the control empty vector. Transfection of the un-cleavable mutant MutGDF-8 resulted, 
as expected, in the generation of a major immunoreactive species, corresponding to 
precursor molecules, as well as some amounts of GDF-8 related proteins migrating at 38 

25 kDa. . 

To test the ability of the cleavage site mutant to act as a dominant-negative 
inhibitor of the wild type GDF-8, QM-7 cells were co-transfected with different ratios of 
the Mut-GDF-8-F and WT-GDF-8-F constructs. The results demonstrated that the 
cleavage site mutant inhibits the secretion of the mature X3DF-8 in a -dose-dependent 
30 manner. To confirm that the cleavage site mutant also inhibits the secretion of non - 
tagged wild-type GDF-8, Mut-GDF-8-F and WT-GDF-8 constructs were co-introduced 



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into QM-7 cells, and antibodies to GDF-8 were used to detect expressed proteins. The 
results were similar to those with the FLAG-tagged GDF-8. 

The specificity of the inhibition was examined using recombinant BMP-2, a 
member of the TGF-p family sharing 41% homology with GDF-8 within the mature 

5 region. The expression constructs for both BMP-2 and Mut-GDF-8-F were co- 
introduced into QM-7 cells, and secreted proteins were detected with an anti-BMP-2 
antibody. The data demonstrated that the mutant GDF-8 did not affect the secretion and 
processing of mature BMP-2. Thus, inhibitory action of Mut-GDF-8 is selective with 
regard to other members of the TGF-P protein family. 

10 To address the issue of stability of the dominant-negative mutant and in an 

attempt to reveal the mechanism of the inhibition transfected QM-7 cells were grown in 
the presence of 200jaCi/ml of [ 35 S] cysteine for 2 hours and chased at indicated time 
periods. The conditioned media were collected and cell lysates were prepared for further 
analysis. All FLAG-tagged proteins were immunoprecipitated with anti-FLAGM2 

1 5 affinity gel and fractionated on SDS-PAGE. Data from this experiment demonstrates 
that the precursor form of WT-F-GDF-8 and the full-length Mut-GDF-8 proteins 
initially accumulate inside the cells. Three forms of GDF-8 were detected in the 
supernatants of the cells transfected with the WT-F-GDF-8: the precursor, the mature 
GDF-8 and the pro domain. No intracellular accumulation of the processed forms of 

20 GDF-8 was detected, both the C-terminal mature GDF-8 and the pro-domain were 

secreted immediately after cleavage and were detected in the supernatants as early as 3 
hours after synthesis. Processed forms of GDF-8 were stable in the conditioned media 
for at least 24 hours. No additional accumulation of the processed forms of GDF-8 was 
observed after incubation of conditioned medium for 24 hours at 37° C. This indicates 

25 that the presence of the cells seem to be essential for processing. However, the 

possibility exists that some cleavage can occur outside of the cells upon availability of 
the specific protease. The unprocessed forms of WT-F-GDF-8 and the full-length Mut- 
GDF-8 were also efficiently secreted, but some intracellular accumulation was observed 
as well. 



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Upon co-transfection of the WT-F-GDF-8 together with the Mut-GDF-8, a pool 
of the mature C-terminal fragments, accumulating inside the cells was observed. The 
same phenomenon was observed when the Mut-GDF-8 was introduced into QM-7 stable 
cell lines constitutively expressing WT-F- GDF-8. Two of the positive clones were 

5 transiently transfected with the Mut-GDF-8 construct, labeled as above, and cell lysates 
were analyzed 24 hours later. As the data indicate, only in the cells co-transfected with 
the mutant GDF-8 there was a detectable cell-associated pool of the mature C-terminal 
fragment. These data confirm the hypothesized mechanism of the dominant-negative 
inhibition of secretion of GDF-8 by the un-cleavable mutant, i.e., formation of 

1 0 heterodimers (or other complexes) between the wild-type and mutant proteins which 
interfere with the normal secretion of this growth factor. 

Pro-GDF-8 interacts and inhibits the activity of mature GDF-8 only when it is 
produced as such, and not when it is embedded in a larger protein, as is the case with the 
un-cleavable GDF-8 (Mut-GDF-8), which contains the GDF-8 pro-domain sequence. 

1 5 The studies described above show that pro-GDF-8 can act as an antagonist of 

endogenous mature GDF-8 either when the pro-GDF-8 is administered to a subject in 
protein form or when it is expressed from a suitable vector (i.e., gene therapy-type 
approach), since its target is the secreted mature, processed GDF-8. In contrast, the 
mode action of the Mut-GDF-8 is intracellular, during GDF-8 synthesis, resulting in the 

20 intracelluar formation of un-cleavable heterodimers. As a consequence, Mut-GDF-8 can 
inhibit GDF-8 function when it is expressed intracellularly. 

EXAMPLE 11: Production And Testing Of a GDF-8 Cysteine Mutant 

25 A GDF-8 cysteine mutant was produced by introducing a point mutation into the 

GDF-8 cDNA using a PCR- based approach as described for the other GDF-8 mutants 
The mutated GDF-8 sequences were inserted in the FLAG-CMVoa expression vector to 
produce an in-frame fusion with the FLAG epitope. The resulting construct 
(C-Mut-GDF-8) was introduced alone into QM7 cells, or cotransfected with 

30 WT-F-GDF-8 to test the effect of the mutant on the production and accumulation of the 
mature GDF-8. 



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Since immunoreactive proteins could not be detected in the conditioned media of 
cells transfected with C-Mut-GDF-8 by Western blotting, metabolic labeling of these 
cells was performed after transfection. As the experiments demonstrated, the 
C-Mut-GDF-8 protein was produced and properly processed in QM-7 cells, but it was 
5 secreted in much lower amounts than the wild-type GDF-8. Most of the full-length 
precursor form accumulated inside the cells, while both mature and unprocessed forms 
were secreted in the conditioned media. Thus, the mutation of one of the cysteines in 
the mature region of GDF-8 can still be processed and secreted. However, 
co-transfection experiments demonstrated that the cysteine mutant has the ability to 
1 0 inhibit secretion of the wild-type mature factor, when it is introduced into QM-7 cells 
together with the WT GDF-8. 

EXAMPLE 12: Analysis Of The Role OfN-Linked Glycosylation Of 

GDF-8 Variants 

15 

Glycosylation is one of the major forms of post-translational modifications of 
proteins. The carbohydrate structures of glycoproteins play important roles in protein 
processing, secretion and biological activity. The GDF-8 precursor contains one 
predicted site of N-linked glycosylation at Asn 72 within its pro-domain. To determine 

20 if this glycosylation site is used, the purified recombinant GDF-8 proteins were first 
treated with N-glycosydase F. This enzyme is able to release all common classes of N- 
glycans from the protein backbone. WT-F-GDF-8, Mut-GDF-8 and Pro-GDF-8 proteins 
were purified using anti FLAG affinity chromatography. The protein samples were 
denatured, incubated with N-glycosidase F in the test tube, and subjected to the SDS- 

25 PAGE in reducing conditions. As the results of this experiment demonstrated, the 

enzymatic removal in vitro of the carbohydrate structures results in the shift to a lower 
apparent molecular weight for precursor forms of the wild type and mutant <3DF-8. The 
same shift was observed for the pro domain of GDF-8, expressed independently. 
Notably, there was no change in the mobility of the mature form of GDF-8, consistent 

30 with the absence of putative glycosylation sites within this region. 



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To examine the role of glycosylation in GDF-8 processing and secretion, the 
glycosylation inhibitor tunicamycin was used. This nucleoside antibiotic prevents the 
formation of the dolichol intermediate, necessary for oligosaccharide addition to the 
nascent polypeptide chain. The QM-7 quail myoblast cells were transiently transfected 
5 with the constructs expressing wild type GDF-8, or GDF-8 variants (the cleavage site 
mutant and the pro-domain). All three proteins were fused to a C-terminal FLAG 
epitope. Cells were treated with 2 jig/ml of tunicamycin 16 hours after transfection. 
Conditioned media and cell lysates were analyzed 24 hours later by Western blotting or 
immunoprecipitation, followed by detection of the proteins with anti-FLAG antibody. 

10 The results from this experiment demonstrate that tunicamycin appears to block the 
secretory exit of all three forms of GDF-8. An increase in the cell-associated non- 
glycosylated precursor form of GDF-8 and its pro-domain which migrate faster than the 
glycosylated species was observed. The data suggest that GDF-8 is a glycoprotein and 
its glycosylation is essential for normal processing and secretion. Treatment of Fraction 

1 5 B (containing the pro-domain) with N-glycosidase F eliminated the inhibitory activity of 
the pro-domain (Figure 36), thus providing further support that N-linked glycosylation is 
important for the inhibitory activity of the pro-domain. 

EXAMPLE 13: Effect Of The Human GDF-8 Pro-Domain From Fraction B 
20 And The Mouse GDF-8 Pro-Domain On Chicken Growth 

And Muscle Development 

Since GDF-8 "knock-out" mice generated by gene targeting develop normally, but 
have twice the normal skeletal muscle mass, it was hypothesized that introduction of the 

25 GDF-8 pro-domain during development may result in an increase in skeletal muscle 

mass in treated animals. To test this hypothesis and the efficacy of in ovo administration 
of the GDF-8 pro-domain to enhance skeletal muscle development, purified GDF-8 pro- 
domain (human from Fraction B, described above, or mouse from Example 6) was 
injected into fertilized eggs from Cobb and Ross chickens. Fertilized eggs were injected 

30 on Day 0, 1 ? 2, 3, 1 1 , 12, 1 3. 14. 1 5, 1 8, and 20 as described herein using the techniques 
described in, for example, H. Kocamis el al (1998) Poult. Sci. 77, 1913-1919. Live, 



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carcass, breast, and legs weights were obtained at the end of the 42-day grow-out period. 
Table II shows the percentage increase in live, breast, and leg weights for treated male 
and female birds (Cobb eggs, injection days Day 13 and Day 20) over control birds 
(uninjected). 



5 

Table II 



Cobb 


Live Weight 


Breast 


Leg 


Male (Day 13) 


11.8 


18.0 


18.6 


Female (Day 1 3) 


7.7 


9.7 


13.8 










Male (Day 20) 


3.4 


13.8 


7.4 


Female (Day 20) 


10.4 


17.4 


13.8 



Table III shows the percentage increase in live, carcass, breast, and leg weights for 
treated male and female birds (Ross eggs, injection day Day 1 5) oyer control birds 
10 (buffer alone). 



Table 111 



Ross 


Live Weight 


Carcass 


Breast 


Leg 


Male (Day 15) 


4.2 


5.5 


3.3 


6.7 


Female (Day 15) 


15.8 


18.0 


20.8 


9.2 



These results demonstrate that in ovo administration of the<3DF-8 pro-domain 
1 5 results in an increase in skeletal muscle mass for treated birds. 



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EXAMPLE 14: Identification Of GDF-8 Receptors That Can Be Used As 

GDF-8 Inhibitors 



Binding of [ 125 -I1-GDF-8 to receptors expressed in COS-7 cells 
5 To identify known (cloned) type II receptors that can bind GDF-8, expression 

constructs encoding three different serine-threonine receptors, or an empty vector 
(control) were introduced by transient transfection into COS-7 cells (50% confluent) 
using FuGENE 6 (Boehringer Mannheim). The type II receptors used were the TGF-p 
type II receptor, the Activin type IIB receptor (the B2 splice variant isoform), and the 
10 BMP type II receptor. Binding assays were performed 48 hours later, using [ I25 -I]- 
labeled GDF-8 as the ligand, followed by two types of analysis. First, an analysis was 
performed which involves cell lysis and quantitative determination of GDF-8 binding to 
the surface of the cells transfected by the various constructs, using scintillation counting. 
The second type of analysis was confirmatory, involving the visualization of GDF-8 
1 5 binding to the surface of COS-1 cells and was performed as described in, for example, 
Lin el a/.(1992) Cell 68, 775-85. 



Functional assays based on GDF-8-induced transcription from reporter constructs 

The transcription-based bioassay was performed in two different cell lines, the 

20 A204 human rhabdomyosarcoma cell line and the mink lung epithelial cell line CCL-64. 
Two artificial reporter plasmids were used in this assay: p3TP-Lux and p(CAGA) 12 - 
MLP (described above). In both, luciferase -gene transcription (and thus activity) is 
driven by artificial minimal promoters that respond to members of TGF-P family 
members. Therefore, luciferase activity in cell lysates correlates linearly with the degree 

25 of stimulation of the cells by the applied growth factors. 

Both cell types were plated in 48-well plates in their respective media (DMEM 
for CCL-64, McCoy's medium for A204) supplemented with 10% fetal bovine serum, 
antibiotics and L-Glutamine. Upon reaching 80% confluence, cells were transfected 
using FuGENE-6 to facilitate plasmid uptake (Boehringer-Mannheim), according to the 

30 manufacturer's instructions. Cells were transiently transfected with a cocktail of the 



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following plasmids; the receptor expression plasmids pCMV-ANpRII K-R (encoding the 
mutated, dominant-negative type II TGF-p receptor), pCMV-ANActR!IB2 K-R 
(encoding the mutated, dominant-negative type II Activin receptor) and the reporter 
plasmids pSV-Prgal (used to monitor transfection efficiency) and either of the two 
5 luciferase reporter plasmids. After overnight incubation with the transfection reagents, 
cells were washed twice with the appropriate serum-free medium containing 0.1% BSA. 
Human recombinant GDF-8 was then added to the cells. Cells were lysed after a 6 hour 
incubation and Luciferase and P-galactosidase activity were determined in the same 
sample using the Dual-Light Luciferase Assay kit (Tropix Inc.) Activity is expressed in 
10 Relative Luciferase units (RLU), i.e., luciferase activity corrected for transfection 

efficiency that is given by the corresponding values of P-galactosidase activity measured 
in the same sample. 

Functional identification of the TGF-B receptor Alk-5 as the type 1 receptor for GDF-8. 

1 5 The mink lung epithelial cell line CCL-64 is highly responsive to TGF-p. The 

cell line RIB, derived from CCL-64 by chemical mutagenesis, is unresponsive to TGF- 
p. It has been established that its unresponsiveness is due to lack of functional TPRJ 
receptors. As a definitive proof, upon re-introduction of T{JRI in this cell line, by 
transfection with a plasmid encoding this receptor, TGF-P responses can be fully 

20 restored (Wrana et aL, Cell, (1992), 71, 1003-14). 

Similarly to TGF-P, GDF-8 also elicits strong responses in the wild-type cell line 
CCL-64 (Figure 31), but is inactive in the RIB mutant (Figure 31). However, GDF-8 
responsiveness can be fully recovered when RIB cells are transfected with a plasmid 
encoding TpRI, thus resulting in full functional rescue (Figure 33). The foregoing data 

25 indicate that TpRI is a functional GDF-8 type I receptor. 

Identification of ActRIIB2 as a type II receptor for GDF-8. 

Another CCL-64-derived mutant cell line, DR26, lacks the TGF-P type II 
receptor and is therefore unresponsive to TGF-p. In contrast to the RIB cell line, DR26 



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cells fully retain their responsiveness to GDF-8, indicating that the TGF-P type II 
receptor is dispensable for GDF-8 activity, and some other type II receptor subunit must 
mediate GDF-8 responses (Figure 34). 

* To find out whether any of the already known (cloned) type 11 serine-threonine 

5 kinase receptor subunits can serve as a GDF-8 receptor, a variety of expression 
constructs encoding type II receptors were transfected into COS-7 cells. Cells were 
subsequently tested for [ 125 -I]-GDF-8 binding. In agreement with the functional analysis 
described in the preceding paragraph, no specific GDF-8 binding was detected to either 
TGF-P or BMP type II receptors. In contrast, the Activin type II receptor ActRIIB2 was 

10 identified as a GDF-8-binding receptor. This was confirmed by the increased binding of 
[ ,2 M]-GDF-8 to the surface of COS-7 cells transfected with ActWIB, while cells 
transfected with a control vector displayed no specific binding. 

The functional significance of the binding data was further demonstrated by 
using dominant-negative (AN) forms of ActRIIB2. Dominant-negative receptor 

1 5 isoforms were derived by point mutation of a specific amino acid residue in each 

receptor construct (K to R mutation) in the intracellular domain region of the receptors. 
These mutations do not interfere with the ability of the type II subunits to be properly 
expressed in the cell membrane, to bind ligand or to form heteromers with the respective 
type I subunits. However, these mutations eliminate the receptors' kinase activity and 

20 thus prevent their ability to relay functional signals after ligand binding (Wrana et 
al.(1992) Cell 71, 1003-14). 

These dominant-negative receptor isoforms were introduced in CCL*64 mink 
lung epithelial cells or in A204 human rhabdomyosarcoma cells that are highly 
responsive to GDF-8. As expected from the binding experiment tiata and the functional 

25 data obtained in RIB cells, the AN form of the TGF-p type U receptor did not affect 
GDF-8 responses, thus, confirming that it is dispensable in this respect. In contrast, 
introduction of the AN form of ActRlIB2 in both^cell backgrounds resulted in an 
inhibition of GDF-8 responses in DR26 cells (Figure 35) and A204 cells transfected 
with the reporter construct, presumably by out-competing endogenous ActRlIB2 

30 receptors (i.e. by acting in a dominant-negative fashion). 



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The combination of the binding and functional data demonstrates that ActRIlB2 
can serve as a functional GDF-8 receptor. In all, the above studies demonstrate that the 
heteromeric complex of TpRI and ActRIIB2 is a functional receptor combination that 
mediates GDF-8 responses. Of note, this particular combination of type I-type II 
5 receptor units has not been described as a functional transducer of responses of any other 
member of the TGF-p growth factor family. It is likely that the TpR]-ActRIIB2 
heteromer can be activated only by GDF-8 and by mokcules highly homologous to 
GDF-8. The active (mature) region of GDF-1 1 bears more than 90% homology to that 
of GDF-8, suggesting that these two closely-related factors share similar binding and 
1 0 signaling properties. Therefore, the reagents described herein as well as their 
applications can be used for both GDF-8 aad GDF-1 1 biological research and 
diagnostics. 

Methods for using GDF-8 receptors 

1 5 The identification of GDF-8 receptors allows the construction of "receptor 

probes". The utility of such "receptor probes" is directly related to their ability to 
recognize and bind GDF-8, GDF-8-like molecules (e.g. GDF-1 1) and presumably other, 
structurally related or unrelated molecules or peptides, whether these are chemically 
synthesized or naturally occurring. These, in virtue of their interaction with a GDF-8 

20 "receptor probe", are potential GDF-8 inhibitors. 

Overall, the " receptor probes" can be utilized in a number of ways: (1) in the 
form of a soluble receptor as GDF-8 protein traps in vivo by directly binding and 
neutralizing GDF-8, (2) as modified ELISA systems for GDF-8 detection and 
quantitation in biological and other fluids, for use to diagnose muscle wasting in humans 

25 or (3) as a screening reagent to identify GDF-8 inhibitors/antagonists at the reoeptor 
level. 

The generation of GDF-8 "receptor probes" may involve the generation of GDF- 
8 receptor fusion proteins (using techniques described in, for example,George et al. 
(1999) Proc. Natl Acad: ScL USA 96: 12719-12724. In brief, the receptor fusion 
30 proteins are a heteromeric complex of two fusion proteins: a GDF-8 type 1 receptor 
extracellular domain fused to, e.g., an immunoglobuiinG, (IgG,) Fc fragment, and a 



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GDF-8 type II receptor extracellular domain fused to. e.g., an immunoglobulinG, (IgG^ 
Fc fragment (or any other fusion proteins that contain the extracellular domain of GDF-8 
type I receptor fused to a protein domain X and a GDF-8 type II receptor extracelluar 
domain fused to a protein domain Y, whereby protein domain X and protein domain Y 
5 can interact to form heteromeric complex of the two fusion proteins). The construction 
of fusion receptors is not limited to using the Fc fragment; proteins or portions of 
proteins, both mammalian and bacterial that have the property of forming dimers or 
heterodimers can be utilized in this approach (e.g. basic helix-loop-helix domains). 
Methods for preparing such fusion receptor proteins are described in, for example, 
1 0 Davis el aL (1 994) Science 266: 8 1 6-8 1 9 or US Patent No.5,844,099, the contents of 
which are incorporated herein by reference. 

The following steps are necessary for developing the heterodimeric complex or 
"receptor probe". 

15 Creation of receptor/Fc hybrids 

DNA encoding receptor/Fc fusion proteins is generated through multiple rounds 
of PCR. Based on similar experiments conducted with receptors for other TGF-P 
family members, DNA encoding the amino-terminal 160 amino acids of the receptor is 
amplified from a cDNA clone. The 3' PCR primer overlaps with the 5' sequence of the 

20 human IgGl sequence that is to be included in the fusion gene. Likewise, DNA 

encoding the Fc region of the human IgGl gene (Pro 100 - Lys 330) is amplified from 
human genomic DNA using a 5' primer that overlaps with the receptor sequence. The 
receptor and IgGl PCR products are then combined and used as overlapping templates 
to generate an amplification product that represents a fusion of the sequences encoding 

25 the receptor ligand binding domain and the IgGl Fc region. The final PCR product is 
ligated into the TA cloning vector (Invitrogen, San Diego, CA) and then subcloned into 
pCMV5 (Sigma) to produce a -construct thatean express the fusion protein in transfected 
eukaryotic cells. The constructs are tested in CHO eells and in NSO mouse myeloma 
cells. 



WO 00/43781 PCT/US00/01552 

-78- 

Receptor/Fc hybrid proteins are purified from the conditioned media of 
transfected cells using a Protein A Sepharose column (Pharmacia, Piscataway, NJ). The 
fusion proteins are purified in the form of homodimers. Heterodimers are created by 
reducing the homodimers under conditions that favor the disruption of inter-chain 

5 disulfide bonds without affecting intra-chain disulfides. The two different types of 
monomers are mixed in equimolar amounts and oxidized to form a mixture of homo- 
and heterodimers. The homodimers can be separated then from the heterodimers by 
HPLC. Alternatively, one of the fusion proteins may be tagged with a string of carboxy- 
terminal histidine residues which allows purification of the recombinant proteins by 

1 0 nickel-chelate chromatography. The three types of dimeric proteins are then separated 
by elution of the bound proteins using increasing amounts of imidazole. Homodimers 
lacking the histidine tag are eluted at the lowest concentrations of imidazole; 
heterodimers with only one tagged protein are eluted at intermediate concentrations; and 
homodimers in which both subunits have histidine tags are eluted only at the highest 

1 5 concentrations of imidazole. 

The receptor/Fc heterodimer preparations are analyzed by polyacrylamide gel 
electorphoresis and silver staining for the presence of contaminating proteins. If 
necessary, additional purification steps are performed. The purified heterodimers are 
also tested to ensure that they can bind GDF-8, as described below, and they are tested 

20 as antagonists in one of the bioassays described herein. 

Development of a "ligand trap" assay for quantitation of GDF-8 protein 

The GDF-8 quantitation system is a modified sandwich EL1SA that incorporates 

competitive and capture techniques and that uses heterodimers of receptor/Fc hybrid 
25 proteins as capture reagents in place of a capture antibody. The following steps 

comprise a general protocol that may be used with the GDF-8 "ligand trap" assay. 

ELISA plates are coated with receptor/Fc protein heterodimers and the unattached 

receptor/Fc proteins are washed off. The proteins are fixed and the plates are washed 

again. The protein samples are combined with GDF-8 standards at different -dilutions, 
30 the anti-GDF-8 antibody is added, and the plate is incubated at 37°C. The 

protein/antibody mixtures are added to coated plates, the unattached ligands are washed 



WO 00/43781 



-79- 



PCT/USOO/015S2 



off under stringent conditions, a secondary antibody coupled to horse radish peroxidase 
(HRP) is added, and the unbound secondary antibody is washed off. Subsequently, 
OPD substrate is added and the optical densities are measured using a microplate reader. 
Finally, the level of GDF-8 in a sample is determined using the absorbance value 
5 approach. 

Equivalents 

Those skilled in the art will recognize, or be able to ascertain using no more than 
routine experimentation, many equivalents to the specific embodiments of the invention 
10 described herein. Such equivalents are intended to be encompassed by the following 
claims. 



WO 00/43781 



-80- 



PCT/US00/01S52 



Claims: 

1 . A method for identifying an inhibitor of a GDF protein, comprising: 

(a) obtaining medium in which cells producing a GDF protein have been 

5 cultured; 

(b) testing the medium for the ability to inhibit GDF activity, thereby 
identifying a GDF inhibitor. 

2. The method of claim 1 , further comprising performing chromatography 
10 on said medium before said medium is tested for the ability to inhibit GDF activity. 

3. The method of claim 1, further comprising performing electrophoresis on 
fractions obtained from said chromatography. 

15 4. The method of claim 1 . wherein said cells are transfected with a plasmid 

containing an insert encoding a GDF protein. 

5. The method of claim 4 ? wherein the cells are CHO cells. 

20 6. The method of claim 1 , wherein the cells produce a GDF protein 

endogenously. 

7. The method of claim 2, wherein the chromatography is ion exchange and 
reverse phase chromatography. 

25 

8. The method of claim 3. wherein the electrophoresis is selected from the 
group consisting of preparative non-reducing SDS-PAGE and preparative reducing 
SDS-PAGE. 



30 9. 
protein. 



The method of claim 1 , wherein the GDF protein is a human GDF 



WO 00/43781 



PCT/USOO/01552 



-81 - 



1 0. The method of claim 1 , wherein the GDF protein is selected from the 
group consisting of bovine GDF-8 or GDF-1 1, chicken GDF-8 or GDF-1 1, murine 
GDF-8 or GDF-1 1 , rat GDF-8 or GDF-1 1 , porcine GDF-8 or GDF-1 L ovine GDF-8 or 

5 GDF- 1 1 , turkey GDF-8 or GDF- 1 1 , and baboon GDF-8 or GDF- 1 1 . 

1 1 . The method of claim 1 , wherein the testing detects the activity of a 
muscle-specific enzyme. 

10 12. The method of claim 1 1 , wherein the muscle-specific enzyme is creatine 

kinase. 

13. The method of claim 1 , wherein the testing detects adipocyte 
differentiation. 

14. The method of claim 1 3, wherein the differentiation of 3T3-L1 pre- 
adipocytes is detected. 

1 5. The method of claim 1 , wherein the testing is performed using a 
20 transcription-based assay. 

1 6. The method of claim 1 , wherein the GDF inhibitor is a GDF polypeptide. 

1 7. The method of claim 1 , wherein the GDF inhibitor comprises the pro- 
25 domain of a GDF protein, or a portion of the pro domain. 

1 8. A method for identifying an inhibitor of a GDF protein, comprising: 

(a) preparing fragments of a GDF protein; 

(b) testing the fragments for the ability to inhibit GDF activity, thereby 
30 identifying a GDF inhibitor. 



WO 00/43781 PCTAJSO0/O1S52 

-82- 

19. The method of claim 1 8, wherein said fragments are prepared by 
digesting a GDF protein. 

20. The method of claim 1 8, wherein the fragments are synthetically 
5 synthesized. 

21 . The method of claim 1 8, further comprising isolating the fragments 
before they are tested for the ability to inhibit GDF activity. 

10 22. The method of claim 1 8, further comprising selecting for fragments 

which do not induce a T cell mediated response. 

23. The method of claim 1 8, further comprising selecting for fragments 
having an amino acid sequence which will elicit an immune response. 

15 

24. The method of claim 1 8, wherein the testing comprises screening said 
fragments for the ability to elicit an immune response resulting in the generation of GDF 
inhibitory antibodies. 

20 25. The method of claim 1 8, wherein the GDF protein is recombinantly 

produced. 

26. The method of claim 1 8, wherein the GDF protein is a native GDF 
protein. 

25 

27. The method of claim 1 9, wherein the GDF protein is digested by the use 
of a protease. 



30 



28. The method of claim 27, wherein the protease is-selected from the group 
consisting of trypsin, thermolysin, chymotrypsin, and pepsin. 



PCT/US00/01552 

-83- 

The method of claim 18, wherein the fragments are 10-25 amino acids in 

30. The method of claim 1 8, wherein the fragments are 25-40 amino acids in 

5 length. 

31. A GDF-8 or GDF-1 1 inhibitor which can be isolated from medium in 
which CHO cells stably transfected with an expression plasmid containing an insert 
encoding human GDF-8 or GDF-1 1 have been isolated by ion exchange 

10 chromatography, which retains activity after heating at 100°C for up to 10 minutes, 

which retains activity after reduction, and which retains activity after treatment with 6M 
Urea. 

32. The GDF-8 or GDF-1 1 inhibitor of claim 3 1 , which has a molecular 
1 5 weight of less than about 70 kDa. 

33. A GDF-8 or GDF-1 1 inhibitor which can be isolated from medium in 
which CHO cells stably transfected with an expression plasmid containing an insert 
encoding human GDF-8 or GDF-1 1 have been isolated by ion exchange 

20 chromatography, which retains activity after heating at 100°C for up to 10 minutes, 

which looses activity after reduction, and which retains activity after treatment with 6M 
Urea. 

34. The GDF-8 or GDF-1 1 inhibitor ofclaim 33, which has a molecular 
25 weight of less than about 70 kDa. 

35. The GDF-8 or GDF-1 1 inhibitor of any one of claims 3 1 or 33, wherein 
the inhibitor does not possess GDF-8 or GDF-1 1 activity. 



WO 00/43781 



29. 

length. 



30 



36. 



A GDF inhibitor identified by the method of any one of claims 1 or 1 7. 



WO 00/43781 PCT/US00/01552 

-84- 

37. The GDF inhibitor of claim 36, wherein the inhibitor is a GDF protein. 

38. A GDF protein or peptide which inhibits GDF activity. 

5 39. The protein or peptide of claim 38, wherein the protein or peptide does 

not itself exhibit GDF activity. 

40. The protein or peptide of claim 38, wherein the protein or peptide is 
synthetically produced. 

10 

41 . The protein or peptide of claim 38, wherein the protein or peptide is 
derived from a native GDF polypeptide. 

42. The protein or peptide of claim 38 produced recombinantly. 

15 

43. A GDF inhibitor comprising the pro-domain of a GDF protein or a 
portion of the pro domain. 

44. The GDF inhibitor of claim 43, wherein the inhibitor is glycosylated. 

20 

45. An isolated nucleic acid comprising a nucleotide sequence selected from 
the group consisting of SEQ ID NO:l, 2, 3, and 4 which inhibits GDF expression when 
transfected in a cell. 

25 46. An isolated nucleic acid -comprising a nucleotide sequence selected from 

the group consisting of SEQ ID NOs:5-24 which inhibits GDF -expression when 
transfected in a cell. 

47. A GDF inhibitor comprising a variant of a GDF protein. 

30 



WO 00/43781 



PCJ7USOO/01552 



-85- 

48. The GDF inhibitor of claim 47, wherein the GDF protein variant is a 
cysteine variant. 



49. The GDF inhibitor of claim 47. wherein the GDF protein variant is a pro- 
5 domain variant. 

50. The GDF inhibitor of claim 47, wherein the GDF protein variant is a 
post-translational modification variant. 

10 51 . An isolated polypeptide comprising an amino acid sequence selected 

from the group consisting of SEQ ID NOs:25, 29, or 30, wherein said isolated 
polypeptide inhibits GDF activity in a cell. 

52. A non-human animal which expresses a GDF inhibitor. 

15 

53. The non-human animal of claim 52, wherein the GDF inhibitor 
comprises the pro-domain of a GDF protein. 

54. The non-human animal of claim 53, wherein the GDF protein is GDF-8 
20 orGDF-11. 

55. The non-human animal of claim 55, wherein the animal is a chicken. 



WO 00/43781 



2/28 



PCTAJS00/01552 



00 




i i i : — i 1 1 — : 1 

5.00 10.00 15.00 20.00 25.00 30.00 35.00 



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3/28 



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Fig. 4 



SUBSTITUTE SHEET (RULE 26) 



WO 00/43781 PCT/US00/015S2 

4/28 




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SUBSTITUTE SHEET (RULE 26) 



WO 00/43781 



PCT/US00/015S2 



5/28 



0.35 -| 
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£ 0.20- 



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lOOng/ml 



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lOOng/ml 



Fig. 6B 



SUBSTITUTE SHEET (RULE 26) 



WO 00/43781 PCT/US00/O1SS2 

6/28 



D 



2000-1 



1600- 



1200- 



800- 



400- 



□ NO GDF-8 
Ea 3ng/ml GDF-8 
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BUFFER OR 

FRACTION A NONE 
AT600ng/ml 

TREATMENT OF none 
BUFFER/FRACTION A NUNt 



BUFFER FRACTION A BUFFER FRACTION A 
NONE NONE TCEP TCEP 

Fig.7A 



SUBSTITUTE SHEET (RULE 26) 



WO 00/43781 PCT/US00/015S2 

7/28 



140 -i 



to 

Z 
3 



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100- 



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CONTROL TRACTION A UREA-TREATED 
AT 300ng/ml FRACTION A 
AT300ng/ml 



Fig. 7B 



SUBSTITUTE SHEET (RULE 26) 



WO 00/43781 



8/28 



PCT/USOO/01552 



3 



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SUBSTITUTE SHEET (RULE 26) 



WO 00/43781 



9/28 



PCT7US00/01552 




70-i 



60- 



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VTA TGF-pl 0.3ng/ml 
hrt3DF-810ng/ml 
HI CHO-ACT 1:1,000 




BSA 
CONTROL 



FRACTION 6 
ATSOOng/ml 



Fig. 10 



SUBSTITUTE SHEET (RULE 26) 



WO 00/43781 



10/28 



PCT/US00/015S2 



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11/28 




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WO 00/43781 PCT/US00/01552 

12/28 & 

ATGATGCAAMACTGCAAATGTATGTTTATATTTACCTGTTCATGCT 
MMOKLQMYVYIYLFMLIA AG 

OCAGTGGATCTAAATGAGCGCAGTGAGAGAGAAGAAAAT 
PVDLNEGSERE ENVEKEGLC 

AATGCATGTGCGTGGAGACAAAACACGAGGTACTC^ 
NACAWRQNTRYSRIE'AIKIQ 

ATCCTCAGTAAGCTGCGGCTQGAMCAGCTCCTAACATCAGCAAAGATGCTATAAtaACAA 
ILSKLRLETAPNISKDAIRQ 

CTTCTGCCAAGAGQGOCTOCACTCOGGGAACTGATCGATCACT 
L LPRAP PLRELIDQYDVQRD 

GACAGCAGTGATGGCTCTTTGGAAGATGAC 
DS SDGSLED DDYHAT TETI I 

AOCATC&TACAGAGTCTGACT7TCTAATGCAA 
TMPTESDFLMQADGKPKC CF 

TTTAAATTTAGCTCTAAAATACAGTACAACAAAGrAGTAAAAGOCCAACrGrGGATATAT 
FKFS SKIQYNKV VKAQLWIY 

CTGAGA<X03TCAAGACTCCTACAAC^^ 
LRPVKTPT TVFVOILRLIKP 

ATGAAAGAOSGTTACAAGGTATACTGGAATOCGATCT 
MKDGTRYTGIRSLKLDMSPG 

ACTGCTATTTGGCAGAGTATTGATGTGAAGAC^^ 
TGIWQSIDVKTVLQNWLKQP 

GAATXAACTTAGGCATTGAAATCMAGCTTTGGATGAGAATGG 
ESNLGIEIKALD€NGHDLAV 



Mut-GDF-8 



AOCTTCOCAGGAOCAGGAGAAGATGGGCTGAATCOCTTTTTAGAAGTCAA<3^ 
TFPGPGEDGL FLEVKVTO 

< I AA TGCCC A G A CA 



ACAOCCAAG^CWmi<^ A QAGACTTTGGGCTTGACTGD^ 
T P K R S 



R Dl FGLDCDEHST-ES 



OGGTGCTGCCGCTAOCOCCTCACGGTCGATTTTGA 

RC CRYPLTVDFEAFGWDWI I 

A ► C-Mut-GDF-8 

GCA0CCAAAAGATATAAG3GCAATTACTGCTCAGGAGAGfrcn"GAATTTCT I I 1 I ACAA 

A PKRYKANYCSGECEFV T 

AAATATCEGCATACTCATCTTCTGCACCAAGC^^ 
KYPHTHLVHQ. ANPRGSAGPC 

TGCACTCCGACAAAAATGTTCTOCCATTAATATG^ 
CTPTKMSPINMLYFNGKEQI 

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Fig. 13 



SUBSTITUTE SHEET (RULE 26) 



WO 00/43781 



PCT/US00/01S52 



13/28 



20 n 



15- 



10- 





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EZZJGDF-8 10ng/ml 




BSA 
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GLYCINE UN 
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FLAG FLAG 

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GDF-8 AN-GDF-8 
PRO- UN- 
DOMAIN CLEAVABLE 
<@V.20 @1:20 
~300ng/ml) (~300ng/ml) 



Fig. 15 



SUBSTITUTE SHEET (RULE 26) 



WO 00/43781 



14/28 



PCT/USO0/O1SS2 




1000 



Fig. 16 



SUBSTITUTE SHEET (RULE 26) 



WO 00/43781 



15/28 



PCT/US00/01552 



1 MVLAAPLLLGFLLLALELRPRGEAAEGPAAAAAAAAAAAAAGVGGERSSR 50 

I I III 

1 MQKLQLCVYIYLFML . . . IVAGPVDLNENSE 28 

51 PAPSVAPEPDGCPVCVWRQHSRELRLESIKSQILSKLRLKEAPNISREVV 100 

I I I I III I I II Mill II I Mill I 

29 QKENVEKE.GLCNACTWRQNTKSSRIEAIKIQILSKLRLETAPN1SKDVI 77 
101 KQLLPKAPPLQQILDLHDFQGDALQPEDFLEEDEYHATTETVISMAQETD 150 

I Ml 1 1 III I III II I 1 1 Mill I I II 

78 RQLLPKAPPLRELIDQYDVQRDD . SSDGSLEDDDYHATTETIITMPTESD 126 
151 PAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRL 2 00 

I II I II I II I II Mill Mill III Mill 

127 FLMQVDGKPKCCFFKFSSKIQYNKWKAQLWIYLRPVETPTTVFVQILRL 17 6 
2 01 KPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQ 250 

II I Mill I Mill I II I I 

177 ikpmkdgt rytg i r s lk ldmnp gtg i wq s i dvkt vl qnwlkq 218 

251 pqsnwgieinafdpsgtdlavtslgpgaeglhpfmelrvlentk^rIJni. 3 00 

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219 PESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPRRSRRDF 268 



301 GLDiDEHSSESR^RYPLTVDFEAFGWDWIIAPKRYKANYgSGQpEYMFM 350 

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269 GLD.eDEHSTESRGeRYPLTVDFEAFGWDWIIAPKRYKANY l GSGE€EFVFL 318 



351 qkyphthlvqqanprgsagpMtptkmspinmlyfndkqqiiygkipgmv 400 
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Fig. 17 



SUBSTITUTE SHEET (RULE 26) 



WO 00/43781 



PCT/US00/01SS2 



16/28 

1 GTCTCTCGGA CGGTACATGC ACTAATATTT CACTTGGCAT TACTCAAAAG CAAAAAGAAG 
61 AAATAAGAAC AAGGGAAAAA AAAAGATTGT GCTGATTTTT AA AlATGk TGC AAAAACTGCA 



241 ACAAAACACG AGGTACTCCA GAATAGAAGC CATAAAAATT CAAATCCTCA GTAAGCTGCG 
301 CCTGGAAACA GCTCCTAACA TCAGCAAAGA TGCTATAAGA CAACTTCTGC CAAGAGCGCC 
361 TCCACTCCGG GAACTGATCG ATCAGTACGA CGTCCAGAGG GATGACAGCA GTGATGGCTC 
421 TTTGGAAGAT GACGATTATC ACGCTACCAC GGAAACAATC ATTACCATGC CTACAGAGTC 
481 TGACTTTCTA ATGCAAGCGG JVTGGCAAGCC CAAATGTTGC TTTTTTAAAT TTAGCTCTAA 
541 AATACAGTAC AACAAAGTAG T^AAGCCCA AC TGTGGATA TATCTCAGAC CCGTCAAGAC 
601 TCCTACAACA GTGTTTGTGC AAATCCTGAG ACTCATCAAA CCCATGAAAG ACGGTACAAG 
661 GTATACTGGA ATCCGATCTC TGAAACTTGA CATGAGCCCA GGCACTGGTA T TTGGCAGAG 
72 1 t^TTGATGTG AA GACAGTGT TGCAAAATTG GCTCAAACAG CCTGAATCCA ACTTAGGCAT 
781 TGAAATCAAA GCTTTGGATG AGAATGGCCA TGATCTTGCT GTAACCTTCC CAGGACCAGG 
841 AGAAGATGGG CTGAATCCCT TTTTAGAAGT CAAGGTGACA GACACACCCA AGAGGTCCCG 
901 GAGAGACTTT GGGCTTGACT GCGATGAGCA CTCCACGGAA TCCCGGTGCT GCCGCTACCC 
961 CCTCACGGTC GATTTTGAAG CGTTTGGATG GGACTGGATT ATCGCACCCA AAAGATATAA 
1021 GGCCAATTAC TGCTCAGGAG AGTGTGAATT TGTGTTTTTA CAAAAATATC CGCATACTCA 
1081 TCTTGTGCAC CAAGCAAACC CCAGAGGCTC AGCAGGCCCT TGCTGCACTC CGACAAAAAT 
1141 GTCTCCCATT AATATGCTAT ATTTTAATGG CAAAGAACAA ATAATATATG GGAAAATTCC 
1201 AGCCATGGTA GTAGACCGCT GTGGGTGCTC A lTSgk CTTTG CATTAGGTTA GAAACTTCCC 
1261 AAGTCATGGA AGGTCTTCCC CTCAATTTCG AAACTGTGAA TTCAAGCACC ACAGGCTGTA 
1321 GGCCTTGAGT ATGCTCTAGT AACGTAAGCA CAAGCTACAG TGTATGAACT AAAAGAGAGA 
1381 ATAGATGCAA TGGTTGGCAT TCAACCACCA AAATAAACCA TACTATAGGA TGTTGTAT-GA 
1441 TTTCCAGAGT TTTTGAAATA GATGGAGATC AAATTACATT TATGTCCATA TATGTATATT 
1501 ACAACTACAA TCTAGGCAAG GAAGTGAGAG CACATCTTGT GGTCTGCTGA GTTAGGAGGG 
1561 TATGATTAAA AGGTAAAGTC TTATTTCCTA ACAGTTTCAC TTAATATTTA CAGAAGAATC 
1621 TATATGTAGC CTTTGTAAAG TGTAGGATTG TTATCATTTA AAAACATCAT GTACACTTAT 
1681 ATTTGTATTG TATACTTGGT AAGATAAAAT TCCACAAAGT AGGAATGGGG CCTCACATAC 
1741 ACATTGCCAT TCCTATTATA ATTGGACAAT CCACCACGGT GCTAATGCAG TGCTGAATGG 
1801 CTCCTACTGG ACCTCTCGAT AGAACACTCT ACAAAGTACG AGTCTCTCTC TCCCTTCCAG 
1861 GTGCATCTCC ACACACACAG CACTAAGTGT TCAATGCATT TTCTTTAAGG AAAGAAGAAT 
1921 CTTTTTTTCT AGAGGTCAAC TTTCAGTCAA CTCTAGCACA GCGGGAGTGA CTGCTGCATC 
1981 TTAAAAGGCA GCCAAACAGT ATTCATTTTT TAATCTAAAT TTCAAAATCA CTGTCTGCCT 
2 041 TTATCACATG GCAATTTTGT GGTAAAATAA TGGAAATGAC TGGTTCTATC AATATTGTAT 




121 AATGtATGT T TAT ATTTACC TGTTCATGCT GATTGCTGCT GGCCCAGTGG ATCTAAATGA 
181 GGGCAGTGAG AGAGAAGAAA ATGTGGAAAA AGAGGGGCTG TGTAATGCAT GTGCGTGGAG 



Fig. 



18 



SUBSTITUTE SHEET (RULE 26) 



WO 00/43781 PCMJSOO/01552 

17/28 



r 



5' CTGCAAATGTATGTTTATATT 3' 

3' GACGTTTACA ACAAATATAA 5' 
A C T 

A 6 A 
G T 

C GA G 
AT 

G C 

G C 

A T 



5' GGGGCTGTGTAATGCATGTGC 3' 

3' CCCCGACACA TACGTACACG 5' 
A C T 

A <3 A 
G T 

C GA € 

A T 

G C 

G C 

A T 



GDF-8 Ribozyme 1 GDF-8 Ribozyme 2 



5' ACAAAGTAGTAAAAGCCCAAC 3' 

3' TGTTTCATCA TTTCGGGTTG 5' 
A C T 

A G A 
G T 

C GA G 

A T 

G C 

G C 

A T 



5 ' TTGGCAGAGTATTGATGTGAA 

3' AACCGTCTCA AACTACACTT 
A C T 
A G A 
G T 
C GA G 
A T 
'G C 
G C 
A T 



3 
5 



GDF-8 Ribozyme 3 GDF-8 Ribozyme 4 



Fig. 19 



SUBSTITUTE SHEET (RULE 26) 



WO 00/43781 PCT/USOO/015S2 

18/28 



ten 

< 

o 
o 

Eh 
U 

u 
Eh 

8 

Eh 
O 
C5 
H 
Eh 
Eh 
Eh 
Eh 
Eh 
Eh 
< 
U 
U 
Eh 
U 
U 

i 



3 

C9 
O 

8 

u 
o 

u 
< 

Eh 
U 

S 

Eh 
O 
Eh 

S 



EH 

O 
O 



U 

Eh 
O 
O 
U 
O 
O 
O 
U 
CD 
U 
< 

S 

o 
u 
u 



O 
CM 



% 3 



LL 



SUBSTITUTE SHEET (RULE 26) 



WO 00/43781 PCT/USOO/01552 

19/28 




SUBSTITUTE SHEET {RULE 26) 



WO 00/43781 20 / 28 PCT/US00/01552 

MQKLQLCV'y I YLF ML I VAGP 
ATGCAAAAACTGCAACTCTGTGTTTATATTTACCTGTTTATGCTGATTGTTGCTGGTCCA 
ACGT TTTTGACGTTGAGA^ AAATATAAATGGACAAAT^ 

VDLNENSEQKENVEKEGLCN 
GTGGATCTAAATGAGAACAGTGAGCAAAAAGAAAATGTGGAAAAAGAGGGGCTGTGTAAT 
CTCTTGTCACTCGTTTTTC^ 

ACTWRQNTKSSRIEAIKIQI 
GCATGTACTTGGAGACAAAACACTAAATCTTCAAGAATAGAAGCCATTAAGATACAAATC 
TACATGAACCTCTGTTTTGT ATGTTTAG 

© 

LSK LRLETAPNISKDVI RQL 
CTCAGTAAACTTCGTCTGGAAACAGCTCCTAACATCAGCAAAGATGTTATAAGACAACTT 
GAGTCATTTGA^ TCTACAATATTCTGTTAAA 

LPKAPPLRELIDQYDVQRDD 
TTACCCAAAGCTCCTCCACTCCGGGAACTGATTGATCAGTATGATGTCCAGAGGGATGAC 
A ACTAGTCATACTACAGGTCT 
© © 

SSDGSLEDDDYH ATTET I IT 
AGCAGCGATGGCTCTTTGGAAGATGACGATTATCACGCTACAACGGAAACAATCATTACC 

TACTGCTAATAGTGCGATGT 
® 

MPTESDFLMQVDGKPKCCFF 
ATGCCTACAGAGTCTGATTTTCTAATGCAAGTGGATGGAAAACCCAAATGTTGCTTCTTT 
AGACTAAAAGATTACGTTC^ ' 

KFS S K I Q YNKVVKAQLW I Y L 
AAATTTAGCTCTAAAATACAATACAATAAAGTAGTAAAGGCCCAACTATGGATATATTTG 

ATATAAAC 

RPVETPTTVFVQI LRLI KPM 
AGACCCGTCGAGACTCCTACAACAGTGTTTGTGCAAATCCTGAGACTCATCAAACCTATG 
TCTGGGCAGCTCTGAGGATGTTGTCACAAACA TCTGAGTAGTTTGGATAC 

© <0> 

KDGTRYTGIRSLKLDMNP GT 
AAAGACGGTACAAGGTATACTGGAATCCGATCTCTGAAACTTGACATGAACCCAGGCACT 
TXTCTGCCATGTTCCATATGACL AGACTTTGAACTGTACTTfiGGTCCGTGA 

© © t3) 

G I WQS I DVKTVLQN-WL.KQPE 
GGTATTTGGCAGAGCATTGATGTGAAGACAGTGTTGCAAAATTGGCTCAAACAACCTGAA 
CCATAAACCGTC ACTTCTGTCACAACGTTTTAACCGAGTTTGTTGGACTT 

<0> © 

SNL (a I E I KALDENGH DLAVT 
TCCAACTTAGGCATTGAAATAAAAGCTTTAGATGAGAATGGTCATGATCTTGCTGTAACC 
AG TAACTTTATTTTCCAAATCIACTCTTACCAGTACTAGAAC 

© <L5) *© 

FPG PGEDGLNPFLEV KVTDT 
TTCCCAGGACCAGGAGAAGATGGGCTGAATCCGTTTTTAGAGGTCAAGGTAACAGACACA 
GGGTCCTGGTCCTCTTCTAC 
<§) 

PKRSRRDFGLDCDEHSTES R 
CCAAAAAGATCCAGAAGGGATTTTGGTCTTGACTGTGATGAGCACTCAACAGAATCACGA 

CCRYPLTVDF EAFGWDWI IA 
TGCTGTCGTTACCCTCTAACTGTGGATTTTGAAGCTTTTGGATGGGATTGGATTATCGCT 



Fig. 22 



SUBSTITUTE SHEET (RULE 26) 



WO 00/43781 



PCTAJSO0/01SS2 



21/28 




■n MASS 



28000 28500 29000 29S00 30000 30500 31000 

Fig. 23 



SUBSTITUTE SHEET (RULE 26) 



WO 00/43781 



PCT/US00/O1S52 



22/28 



to 

< 

at 



u 

LU 
> 

LU 



45 
40- 
35 - 
30 
25 
20 
15 
10 
5 -I 



1 



CD BSA CONTROL 
EZa hrGDF-8 lOng/ml 



GDF-8 BUFFER UNMODIFIED UREA-TREATED REDUCED 

PRE- FOR PRO-GDF-8 PRO-GDF-8 PRO-GDF-8 

INCUBATED DENATURING AT300ng/ml AT 300nq/ml AT 300ng/ml 
WITH: OR REDUCTION y 



Fig. 24 



200 

180- 

160- 

140- 

120- 

| 100H 
80 
60- 
40- 
20- 
0 



1 




BSA 
CONTROL 



i 



□ NO GDF-8 
E2 3ng/ml DGF-8 
10ng/mlOGF-8 



BUFFER 



Fig. 25 



t3DF-8 PRO-DOMAIN 
600ng/ml 



SUBSTITUTE SHEET (RULE 26) 



WO 00/43781 



PCT/US00/01S52 



23/28 



30n 



25- 



> 

1X1 

2 



O 
2 



20- 



15- 



LLI <f 

2 uj 
lli <r 

u a. 



10- 



5 - 



0 



(Zj C2C12 myoblasts 

E2 chick primary myoblasts 





CONTROL hrGDF-8 TGF-61 
NO FACTORS (300hg/ml) (lOng/ml) 

72HOURS-/+ FACTORS 
*:p<0.05 



Fig. 26 




-i r — 1 i—i 

1 10 100 1000 

INSULIN friM) 



Fig. 27 



SUBSTITUTE SHEET (RULE 26) 



PCT/US00/01S52 



24/28 



1000^ 
900- 
800- 
700- 
600-i 
500- 
400- 
300- 
200- 
100- 
0- 



□ BSA 

E23 lOng/ml GDF-8 

E3 lOng/ml GDF-8+C0NTR0L BUFFER 

m 10ng/ml GDF-8+100ng/ml FRACTION B 




II 




UN-TREATED (NAIVE) BUFFER OR FRACTION B 
BUFFER OR FRACTION B TREATED WITH 

N-GLYCOSIDASE F 



Rg. 28 



30-, 





1 




P 




1 




BUFFER "GDF-8 GDF-8 18 19 20 21 
CM PURIF. C4 FRACTIONS 



BEFORE C4 

Rg.29 

SUBSTITUTE SHEET (RULE 26) 



WO 00/43781 PCT/US00/01SS2 

25/28 



lO-i 




CONTROL WT-CDF-8-F WT-GDF-8-F 

+CALPAIN 



Fig. 30A 



300 -, 



250 
200 

o <t 

< CD 

$1 150 

Oc u. 

5 9 loo - 



50- 



0 

hrGDF-8 
CALPAIN 
mrPRO-GDF-8 



i 



i 



+ - + 
+ + 



+ + 



Fig. 30B 



i 



+ + 
+ 

+ + 



SUBSTITUTE SHEET (RULE 26) 



WO 00/43781 



PCT/USOO/01552 



2000 

1800 

g 1600 

| 1400 

£ 1200 

^ 1000 

2 800 

lu 600 
cc 

O 400 
O 

200 



26/28 



— O- TGF-beta 10ng/ml 
■-O- GDF-8 300ng/ml 




-5 



~~ i 1 1 1 1 — 

0 5 10 15 20 
HOURS POST TREATMENT 



— i — 
25 



30 



Fig. 31 



2000 1 
1800- 
t 1600 
2 1400 

g 1200 
it 

O 1000 
—I 

Q 800 

UJ 

§ 600 H 
§ 400H 

o 

200H 



— o- TGPbeta 10ng/ml 
— □- GDF8 300ng/ml 



=3 



-i 1 1 1 r— 

0 5 10 15 20 

HOURS POST TREATMENT 



25 



I 

0 



Fig. 32 



SUBSTITUTE SHEET (RULE 26) 



WO 00/43781 PCMJSOO/01552 

27/28 



5000 -, 



z 



u 

3 



4000- 



3000- 



£ 2000- 



1000- 



I I BSA CONTROL 
(22 GDF-8 30ng/ml 
^ TGF-Pi lOng/ml 




tI 



1 




NOPLASMID 



pBK-CMV 
EMPTY 
VECTOR 



X 




pBK-Alk-5 pBK-Tsk 7L 



Rg.33 



SUBSTITUTE SHEET (RULE 26) 



WO 00/43781 



PCT/US00/01SS2 



28/28 



z 

UJ 

w 
< 

cc 

111 
LL 

o 

3 



o 

UJ 

cc 
cc 
o 
o 



2000 -, 
1800 
1600- 
1400- 
1200- 
1000- 
800- 
600- 
400 
200 -I 
0 



O 
cc 



8 
S 

o 
a 

o 



3 

DC 



12n 



10- 



8- 



6- 



Q 
Q 



2- 



— O- TGF-beta 10ng/ml 
--D- GDF8 300ng/ml 



i 1 1 1 — 

' 5 10 15 20 

HOURS POST TREATMENT 



25 30 



Fig. 34 



-O- pBK-EMPTY VECTOR 
--D-pCMV5ActRIIBKR 
--&-pCMV5flRIIKR 




0.1 



1 10 
hrGDF-8 (ng/ml) 

Fig. 35 



100 



SUBSTITUTE SHEET (RULE 26) 



WO 00/43781 

- 1 

SEQUENCE LISTING 

<110> METAMORPHIX, INC. 

<120> GROWTH DIFFERENTIATION FACTOR INHIBITORS AND USES 
THEREFOR 

<130> MTN-024PC 

<140> v 
<141> 

<150> 60/116, 639 
<151> 1999-01-21 

<150> 60/138,363 
<151> 1999-06-10 

<160> 30 

<170> Patentln Ver. 2.0 

<210> 1 
<211> 42 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Synthetic 
Sequence 

<400> 1 

aatataaaca ctgatgagtc cgtgaggacg aaacatttgc ag 42 

<210> 2 
<211> 42 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Synthetic 
Sequence 

<'4 00> 2 

gcacatgcat ctgatgagtc cgtgaggacg aaacacagcc cc 42 

<210> 3 
<211> 42 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Synthetic 
Sequence 

<400> 3 

gttgggcttt ctgatgagtc cgtgaggacg aaactacttt gt 42 



PCT/US00/01552 



<210> 4 



WO 00/43781 

-2 - ■ 

<211> 42 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Synthetic 
Sequence 

<400> 4 

ttcacatcaa ctgatgagtc cgtgaggacg aaactctgcc aa 

<210> 5 
<211> 20 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Synthetic 
Sequence 

<400> 5 

acgtttttga cgttgagaca 

<210> 6 
<211> 20 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Synthetic 
Sequence 

<400> 6 

aaatataaat ggacaaatac 

<210> 7 
<211> 20 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Synthetic 
Sequence 

<400> 7 

ctcttgtcac tcgtttttct 

<210> 8 
<211> 20 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Synthetic 
Sequence 

<400> 8 

tacatgaacc tctgttttgt 



WO 00/43781 



PCT/US00/0r5S2 



<210> 9 
<211> 20 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial 
Sequence 

<400> 9 

atgtttagga gtcatttgaa 

<210> 10 
<211> 20 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial 
Sequence 

<400> 10 

tctacaatat tctgttaaaa 

<210> 11 
<211> 20 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial 
Sequence 

<400> 11 

actagtcata ctacaggtct 

<210> 12 
<211> 20 
<212> DNA 

<213> Artificial Sequence 
<220>. 

<223> Description of Artificial 
Sequence 

<400> 12 

tactgctaat agtgcgatgt 

<210> 13 
<211> 20 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial 
Sequence 



Sequence: Synthetic 



20 



Sequence: Synthetic 



20 



Sequence: Synthetic 



20 



Sequence : Synthetic 



20 



Sequence: Synthetic 



<400> 13 

agactaaaag attacqttca 



20 



WO 00/43781 



-4- 



<210> 14 
<211> 20 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Synthetic 
Sequence 

<400> 14 

atataaactc tgggcagctc 

<210> 15 
<211> 20 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Synthetic 
Sequence 

<400> 15 

tgaggatgtt gtcacaaaca 

<210> 16 

<211> 20 

<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Synthetic 
Sequence 

<400> 16 

tctgagtagt ttggatactt 

<210> 17 
<211> 20 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223>" Description of Artificial Sequence: Synthetic 
Sequence 

<400> 17 

tctgccatgt tccatatgac 

<210> 18 
<211> 19 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Synthetic 
Sequence 

<400> 18 



WO 00/43781 



-5- 



agactttgaa ctgtacttg 

<210> 19 
<211> 21 . 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Synthetic 
Sequence 

<400> 19 

ggtccgtgac cataaaccgt c 

<210> 20 
<211> 20 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Synthetic 
Sequence 

<400> 20 

acttctgtca caacgtttta 

<210> 21 
<211> 20 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Synthetic 
Sequence 

<400> 21 

accgagtttg ttggacttag 

<210> 22 
<211> 20 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Synthetic 
Sequence 

<400> 22 

taactttatt ttcgaaatct 

<210> 23 
<211> 20 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Synthetic 
Sequence 



VVO 00/43781 



-6- 



PCT/USOO/01552 



<400> 23 

actcttacca gtactagaac 

<210> 24 
<211> 20 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Synthetic 
Sequence 

<400> 24 

gggtcctggt cctcttctac 

<210> 25 
<211> 23 
<212> PRT 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Synthetic 
Sequence 

<400> 25 

Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val Phe Leu Gin Lys Tyr 
1 5 10 15 

Pro His Thr His Leu Val His 
20 



<210> 26 
<211> 5 
<212> PRT 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Synthetic 
Sequence 

<400> 26 

Asn Glu Asn Ser Glu 
1 5 



<210> 27 
<211> 5 
<212> PRT 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Synthetic 
Sequence 

<400> 27 

Asp Asp Ser Ser Asp 

' 1 5 



WO 00/43781 



-7- 



PCT/US00/01552 



<210> 28 
<211> 7 
<212> PRT 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Synthetic 
Sequence 

<400> 28 

Gly Pro Val Asp Leu Asn Glu 
1 5 



<210> 29 
<211> 13 
<212> PRT 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Synthetic 
Sequence 

<400> 29 

Lys lie Pro Ala Met Val Val Asp Arg Cys Gly Cys Ser 
1 5 10 



<210> 30 
<211> 22 
<212> PRT 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Synthetic 
Sequence 

<400> 30 

Leu Ser Lys Leu Arg Leu Glu Thr Ala Pro Asn lie Ser Lys Asp Val 
15 10 15 

lie Arg Gin Leu Leu Pro 
20 



(J2) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(19) World Intellectual Property Organization 
International Bureau 

(43) International Publication Date 
27 July 2000 (27.07.2000) 




(10) International Publication Number 

PCT WO 00/43781 A3 



(51) Internationa! Patent Classification 7 : G01N 33/50, 
33/68, C07K 14/51, 14/475, 7/08, 7/06, A01K 67/027, 
CI 2N 9/00, 15/11 



(71) Applicant (for all designated Stales except US): META- 
MORPHIX, INC. [US/US]; 1450 South Rolling Road, 
Baltimore, MD 2 1 227 (US). 



(21) International Application Number: PCIYUSOO/01552 

(22) International Filing Date: 21 January 2000 (21.01.2000) 

(25) Filing Language: English 

(26) Publication Language: English 

(30) Priority Data: 

60/116,639 21 January 1999(21.01.1999) US 

60/138363 10 June 1999 (10.06.1999) US 

(63) Related by continuation (CON) or continuation-in-part 
(C1P) to earlier applications: 

US 60/11 6,639 (CIP) 

Filed on 21 January 1999 (21.01.1999) 

US 60/138,363 (CIP) 

Filed on 10 June 1999 (10.06.1999) 



(72) Inventors; and 

(75) Inventors/Applicants (for US only): TOPOUZIS, 
Stavros IGR/US]; Apartment C305, 3821 14th Avenue, 
W., Seattle, WA 981 19 (US). WRIGHT, Jill, F. fUS/US]; 
131 Paden Ct., Forest Hill, MD 21050 (US). RATOVIT- 
SKI, Tamara [IL/US]; 6509 Hazel Thicket Terrace, 
Columbia, MD 21044 (US). LIANG, Li-Fang [US/USJ; 
6645 Hunter Road, Elkridge, MD 21075 (US). . BRADY, 
James, L„ Jr. [US/US]; Apartment 821, 4977 Battery 
Lane, Bethesda, MD 20814 (US). SIN HA, Debasish 
fIN/US]; 18022 Ferule Meadow Court, Gaithersburg, MD 
20877 (US). YASWEN-CORKERV, Linda fUS/US]; 
1606 Dublin Drive, Silver Spring, MD 20902 (US). 

(74) Agents: MANDRAGOURAS, Amy, E. et al.; Lahive & 
Cockfield, LLP, 28 State Street, Boston, MA 02109 (US). 

(81) Designated States (national): AE, AL, AM, AT, AU, AZ, 
BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, 

I Continued on next page] 



(54) Title: GROWTH DIFFERENTIATION FACTOR INHIBITORS AND USES THEREFOR 



CMV 



SIGNAL 
PEPTIDE 



CLEAVAGE SITE 



FLAG 



WH30F-8 



PRO-DOMAIN 




MATURE GDF-8 



CMV 



SIGNAL 
PEPTIDE 



M-GDF-8 



< 

00 

o 
o 

O 



CMV 



SIGNAL 
PEPTIDE 



PRO-GDF-8 



mmmmm 



FLAG 



GDF-8 



B 



PRO-DOMAIN 

C 



PRECURSOR 



FLAG 



(57) Abstract: Inhibitors of GDF proteins, such as GDF-8 orGDF-11, are disclosed. Also disclosed are methods for identifying 
and using the inhibitors, for example, to generate transgenic animals and to treat a variety of diseases. 



wo 00/43781 A3 i inn iiiiiii ii mill inn iiH i it in mil [mi mil mil mi miiu iih hi j iih 



Published: 

— With international search report. 



DM, EE, ES, Fl, GB, GD, GE, GH, GM, HR, HU, ID, IL, 
IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, 
LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, 
RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, IT, TZ, UA, 
UG, US, UZ, VN, YU, ZA, ZW. 

(84) Designated States (regional): ARIPO patent (GH, GM, 
KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), Eurasian patent 
(AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European patent 
(AT. BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IX LU, 
MC NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CL CM, 
GA, GN, GW, ML, MR, NE, SN, TD, TG). 



(88) Date of publication of the international search report: 

1 February 2001 



For two-letter codes and other abbreviations, refer to the "Guid- 
ance Notes on Codes and Abbreviations" appearing at the begin- 
ning of each regular issue of the PCT Gazette. 



INTERNATIONAL SEARCH REPORT 



Interna .a I Application No 

PCT/US 00/01552 



A. CLASSIFICATION OF SUBJECT MATTER 

IPC 7 G01N33/50 G01N33/68 C07K14/51 
C07K7/06 A01K67/027 C12N9/00 



C07K14/475 C07K7/08 
C12N15/11 



According to International Patent Classification (IPC) or to both national classification and IPC 



B. FIELDS SEARCHED 



Minimum documentation searched (classification system followed by classification symbols) 

IPC 7 G01N 



Documentation searched other than minimum documentation to the extent that such documents ere included in the fields searched 



Electronic data base consulted during the international search (name of data base and, where practical, search terms used) 

EPO-Internal, UP! Data, PAJ, BIOSIS, MEDLINE, STRAND 



C. DOCUMENTS CONSIDERED TO BE RELEVANT 



Category ° Citation ot document, with indication, where appropriate, of the relevant passages 



Relevant to claim No. 



WO 98 35019 A (UNIV JOHNS HOPKINS MED) 
13 August 1998 (1998-08-13) 



page 19, line 3 - line 12; claims 1,3,9,16 

page 25, line 7 -page 26, line 24; claims 
42-48 

W0 98 33887 A (UNIV JOHNS HOPKINS MED) 
6 August 1998 (1998-08-06) 



the whole document 



-/-- 



18-21, 

23-28, 

36-50, 

52-55 

1-17, 

31-35 



18-21 , 

23-28, 

36-50, 

52-55 

1-17, 

31-35 



m 



Further documents are listed in the continuation of box C. 



ID 



Patent family members are listed in annex. 



° Special categories of cited documents : 

"A" document defining the general state of the an which is not 

considered to be of particular relevance 
k E" eartier document but published on or after the international 

filing date 

V document which may throw doubts on priority daim(s) or 
which is cited to establish the publication date of another 
citation or other special reason (as specified) 

"O" document referring to an oral disclosure, use, exhibition or 
other means 

*P' document published prior to the international filing date but 
later than the priority date claimed 



"T" later document published after the international filing date 
or priority date and not in conflict with the application but 
cited to understand the principle or theory underlying the 
invention 

"X" document of particular relevance; the claimed invention 
cannot be considered novel or cannot be considered to 
involve an inventive slep when the document is taken alone 

*Y" document of particular relevance; the claimed invention 
cannot be considered to involve an inventive step when the 
document is combined with one or more other such docu- 
ments, such combination being obvious to a person skilled 
in the art. 

*&* document member of the same patent family 



Date of the actual completion of the international search 



18 September 2000 



Date of mailing of the international search report 



\ V AO. 



Name and mailing address of the ISA 

European Patent Office. P.B. 5818 Patenttaan 2 
NL - 2280 HV Rtjswijk 
Tel. (+31-70) 340-2040. Tx. 31 651 epo nl, 
Fax: (+31-70) 340-3016 



Authorized officer 



Gundlach, B 



Form PCTASA/210 (second sheet) (July 1992) 



page I of 3 



INTERNATIONAL SEARCH REPORT 



Interni al Application No 



PCT/US 00/01552 



4 



C.(Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT 


Category c 


Citation ot document, wrth indication, where appropriate* ot the relevant passages 


Relevant to claim No. 


X 


WO 94 21681 A (UNIV JOHNS HOPKINS MED ;LEE 
SE JIN (US)- MCPHERRON ALEXANDRA C (US) 
29 September 1994 (1994-09-29) 
the whole document 


45,46 


X 


WO 96 01845 A (UNIV JOHNS HOPKINS MED ; LEE 
SE JIN (USV MCPHERRON AL FXANDRA C (US) 
25 January 1996 (1996-01-25) 
the whole document 


45,46 


P X 

r , a 


WO 99 06559 A (MCPHFRRON Al FXANDRA -IFF SF 
JIN (US); UNIV JOHNS HOPKINS MED (US)) 
11 February 1999 (1999-02-11) 
the whole document 


lR-?7 

lO L 1 , 

31-44, 
47-55 


P,X 


W0 99 42573 A (BI0STAR INC) 
26 August 1999 (1999-08-26) 
the whole document 


18-55 


A 


MCPHERRON, A.C. ET AL.: "Double muscling 

in cattle due to mutations in the 

myostatin gene" 

PR0C. NATL. ACAD. SCI. USA, 

vol. 94, November 1997 (1997-11), pages 

12457-12461, XP002085801 

abstract 


1-55 


X 


W0 98 24925 A (PENN STATE UNIVERSITY 

; SCHMIDT MICHAEL G (US); UNIV SOUTH 

CAROLINA) 11 June 1998 (1998-06-11) 

abstract; example 1 

& DATABASE GCG_GENESEQ_D 'Online! 

Accession no. V41859, 

26 October 1998 (1998-10-26) 

& DATABASE GCG_GENESEQ_D 'Online! 

Accession no. V41871, 

26 October 1998 (1998-10-26) 

abstract 

& DATABASE GCG GfNFSFO D 'flnlinpi 

Accession no. V41862, 

26 October 1998 (1998-10-26) 

abstract 

& DATABASE GCG GENESEO D 'Online* 

U UniriwnJL VJ w ^ Ilk w Lu U \Jl\ I 1 1 1 V * 

Accession no. V41872, 

26 October 1998 (1998-10-26) 

abstract 


45 


X 


W0 97 17433 A (UNIV 'SOUTH -CAROLINA) 
15 May 1997 (1997-05-15) 
abstract 


45 


X 


US 5 574 143 A (JENNINGS PHILIP A ET AL) 
12 November 1996 (1996-11-12) 
abstract 

-/-- 


45 



Form PCT71SA/210 (continuation of second sheet) (July 1982) 



page 2 of 3 



INTERNATIONAL SEARCH REPORT 



Interna. al Application No 

PCT/US 00/01552 



C.(Continuetion) DOCUMENTS CONSIDERED TO BE RELEVANT 



Category c Citation d document, with indication .where appropriate, of the relevant passages 



Relevant to claim No. 



p,x 



US 5 616 466 A (CANTOR GLENN H 
1 April 1997 (1997-04-01) 
abstract 



ET AL) 



W0 98 48809 A (FR0ELAND STIG ;HANSS0N 
VIDAR (NO); TASKEN KJETIL (NO); AUKRUST 
PAA) 5 November 1998 (1998-11-05) 
abstract 

WO 99 02667 A (GEORGES MICHEL ;GR0BET LUC 
(BE); UNIV LIEGE (BE); PONCELET D0MINIQ) 
21 January 1999 (1999-01-21) 
abstract; claim 86 

W0 96 28555 A (UNIV JEFFERSON ;GI0RDAN0 
ANTONIO (US)) 

19 September 1996 (1996-09-19) 
abstract 



W0 97 11162 A (ENDORECHERCHE INC 

FERNAND (CA); LUU THE VAN (CA)) 

27 March 1997 (1997-03-27) 
abstract 



;LABR1E 



45 



45 



46 



46 



46 



Form PCT/ISA/210 (continuation of second sheet) (July 1992) 



pa$e 3 of 3 



INTERNATIONAL SEARCH REPORT 



International application No. 

PCT/US 00/01552 



Box I Observations where certain claims were found unsearchable (Continuation of item 1 of first sheet) 



This international Search Report has not been established in respect ol certain claims under Article I7(2)(a) for the following reasons: 



because they relate to parts of the International Application that do not comply with the prescribed requirements to such 
an extent that no meaningful International Search can be carried out specifically: 



3. | I Claims Nos.: 

because they are dependent claims and are not dratted in accordance with the second and third sentences of Rule 6.' 



Box II Observations where unity of invention is lacking (Continuation of item 2 of first sheet) 



This International Searching Authority found multiple inventions in this international application, as follows: 



see additional sheet 



1 . FT"! As all required additional search tees were timely paid by the applicant, this International Search Report covers all 
I-*-* searchable claims. 



2. | | As all searchable claims could be searched without effort justifying an additional fee, this Authority did not invite payment 

of any additional fee. 



3. | | As only some of the required additional search tees were timely paid by the applicant, this International Search Report 
' — ' covers only those claims tor which fees were paid, specifically claims Nos.: 



4. | { No required additional search tees were timely paid by the applicant. Consequently, this International Search Report is 
restricted to the invention first mentioned in the claims; it is covered by claims Nos.: 




because they relate to subject matter not required to be searched by this Authority, namely: 




Remark on Protest 





No protest accompanied the payment of additional search fees. 



Form PCT/ISA/210 Continuation of first sheet (1 )) {July 1 998) 



International Application No. PCTAJS 00 /)1552 

FURTHER INFORMATION CONTINUED FROM PCT/ISA/ 210 

This International Searching Authority found multiple (groups of) 
inventions in this international application, as follows: 

1. Claims: 1-44, 47-55 (fully) 

Competitive inhibitors of GDF-8/11 protein 

2. Claim : 45 (fully) 

Ribozymes as inhibitors of GDF-8/11 expression (Seq. ID Nos 
1-4) 

3. Claim : 46 (partially) 

Anti-sense nucleotides as inhibitors of GDF-8/11 expression 
(Seq. ID Nos. 5-14) 

4. Claim : 46 (partially) 

Anti-sense nucleotides as inhibitors of GDF-8/11 expression 
(Seq. ID Nos. 15-24) 



INTERNATIONAL SEARCH REPORT 

Information on patent family members 



Interns* al Application No 

PCT/US 00/01552 



Patent document 


Publication 




Patent family 


Publication 


cited in search report 


date 




member(s) 


date 


WO 9835019 A 


13-08-1998 


US 


6008434 A 


28-12-1999 






AU 


6271498 A 


26-08-1998 



W0 9833887 A 06-08-1998 US 5994618 A 30-11-1999 

AU 6274298 A 25-08-1998 



W0 9421681 A 29-09-1994 CA 2157577 A 29-09-1994 

EP 0690873 A 10-01-1996 

JP 9507829 T 12-08-1997 

US 6096506 A 01-08-2000 

US 5827733 A 27-10-1998 



W0 9601845 A 25-01-1996 CA 2194660 A 25-01-1996 

EP 0776337 A 04-06-1997 

JP 10502811 T 17-03-1998 

US 5914234 A 22-06-1999 

US 6008434 A 28-12-1999 



WO 9906559 
W0 9942573 
W0 9824925 



A 
A 
A 



11-02-1999 
26-08-1999 



AU 8666398 A 



22-02-1999 



AU 2507399 A 



11-06-1998 



US 
AU 
EP 
AU 
CA 
CN 
EP 



5824519 
5688698 
0943005 
7727296 
2236998 
1207769 
0866852 



JP 2000500967 



W0 



9717433 



06-09-1999 



20-10-1998 
29-06-1998 
22-09-1999 

29- 05-1997 
15-05-1997 
10-02-1999 

30- 09-1998 
02-02-2000 
15-05-1997 



W0 9717433 



15-05-1997 



US 5824519 A 

AU 7727296 A 

CA 2236998 A 

CN 1207769 A 

EP 0866852 A 

JP 2000500967 T 

WO 9824925 A 



20-10-1998 

29- 05-1997 
15-05-1997 
ifl-02-1999 

30- 09-1998 
02-02-2000 
11-06-1998 



US 5574143 A 12-11-1996 



Form PCT/lSA/210 (patsm family annexHJuty 1892) 



US 


5494814 


A 


27-02-1996 


us 


5254678 


A 


19-10-1993 


AT 


115999 


T 


15-01-1995 


AU 


632993 


B 


21-01-1993 


AU 


2800789 


A 


19-07-1989 


WO 


8905852 


A 


29-06-1989 


6G 


51160 


A 


15-02-1993 


CN 


1033838 


A 


12-07-1989 


DE 


3852539 


0 


02-02-1995 


DE 


3852539 


T 


■04-05-1995 


DK 


143390 


A 


14-08-1990 


EP 


0321201 


A 


21-^06-1989 


EP 


0640688 


A 


01-03-1995 


ES 


2065919 


T 


01-03-1*95 


FI 


104562 


B 


29-02-2000 


•GR 


3015374 


T 


30-06-1995 


HI) 


54407 


A 


28-02-1991 


HU 


9500362 


A 


28-09-1995 



page 1 of 2 



INTERNATIONAL SEARCH REPORT 



Information on patent family members 



Interna at Application No 

PCT/US 00/01552 



Patent document 
cited in search report 



Publication 
date 



Patent family 
member(s) 



Publication 
date 



US 5574143 



IL 
IN 
JP 
JP 
KR 
MC 
NO 
NO 
NZ 
RO 
SG 
US 
US 
US 
US 
US 
US 
ZA 



88683 
171821 



3046318 B 
3502638 T 
9710758 B 
2115 A 
902661 
20000369 
227332 
114469 
52270 
5543508 
5747335 
5589580 
5766942 
5840874 
5707835 
8809352 



27-11 

23- 01 

29- 05 
20-06 

30- 06 

05- 07 
14-08 
14-08 

27- 08 

30- 04' 

28- 09 

06- 08 
05-05 

31- 12 
16-06 

24- 11 
13-01 
27-12' 



-1995 
-1993 
-2000 
-1991 
-1997 
1991 
-1990 
-1990 
-1991 
-1999 
1998 
-1996 
1998 
-1996 
-1998 
-1998 
-1998 
-1989 



US 5616466 



01-04-1997 



NONE 



WO 


9848809 


A 


05- 


-11- 


1998 


AU 


7086598 A 


24-11-1998 














EP 


1024809 A 


09-08-2000 














NO 


995269 A 


13-12-1999 


wo 


9902667 


A 


21- 


-01- 


1999 


US 


6103466 A 


15-08-2000 














AU 


8457198 A 


08-02-1999 














EP 


1002068 A 


24-05-2000 


wo 


9628555 


A 


19- 


-09- 


1996 


US 


5674748 A 


07-10-1997 














CA 


2220830 A 


19-09-1996 














EP 


0815240 A 


07-01-1998 














JP 


11511646 T 


12-10-1999 



WO 9711162 A 27-03-1997 AU 6867896 A 09-04-1997 

CA 2232500 A 27-03-1997 

EP 0856048 A 05-08-1998 

JP 11511329 T 05-10-1999 



Form PCT/lSA/210 (pet em family annex) (July 1992) 



page 2 of 2 



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