Skip to main content

Full text of "USPTO Patents Application 09804625"

See other formats


WORLD INTBLUECTUAL PROPERTY ORGANaAnON 
International Bureau 




PCX 

INTERNATIONAL APPUCATION PUBUSHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent ClassificatifHi ^ : 

C07K 14/52, 14/495, C12N 15/19, 15/63, 
5/10, 1/21, 1/15 



Al 



(11) International Publication Numbo*: WO 96/01845 

(43) International PublicaUon Date: 25 January 1996 (25.01.96) 



(21) Intermtional Application Number: PCT/US95/08543 

(22) International Filing Date: 7 July 1995 (07.07.95) 



(30) Priority Data: 

08/272.763 



8 July 1994 (08.07.94) 



US 



(71) Applicant (for all designated States except US): THE JOHNS 

HOPKINS UNIVERSITY SCHOOL OF MEDICINE 
[US/US]; 720 Rutland Avenue. Baltimore, MD 21205 (US). 

(72) Inventors; and 

(75) Inventors/Applicants (for US only): LEE. Se-Jin [US/USJ; 
6711 Chokcbeny Road. Baltimore. MD 21209 (US). 
McPHERRON, Alexandra. C. [US/US]; 3905 Keswick 
Road. Baltimore. MD 21211 (US). 

(74) Agents: HAILE. Lisa. A. et al.; Fish & Richardson P.C., Suite 
1400, 4225 Executive Square, La Jolla, CA 92037 (US). 



(81) Designated States: CA. JP, US. European patent (AT. BE. CH. 
DE. DK. ES. FR. GB. GR. IE. IT. LU. MC. NL. PT, SE). 



Published 

With international search report. 



(54) Title: GROWTH DIFFERENTIATION FACTOR- 11 
(57) Abstract 



Growth differentiation factor-ll (GDF-11) is disclosed along with its polynucleotide sequence and amino acid sequence. Also 
disclosed are diagnostic and therapeutic methods of using the GDF-11 polypeptide and polynucleotide sequences. 



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. 



AT 


Austria 


GB 


United Kingdom 


MR 


Mauritania 


AU 


Australia 


GE 


Georgia 


MW 


Malawi 


BB 


Barbados 


GN 


Guinea 


NE 


Niger 


BE 


Belgium 


GR 


Greece 


NL 


Netheriands 


BF 


Burkina Faso 


HU 


Hungary 


NO 


Norway 


BG 


Bulgaria 


IE 


Ireland 


NZ 


New Zealand 


BJ 


Benin 


IT 


Italy 


PL 


Poland 


BR 


Brazil 


JP 


Japan 


PT 


Portugal 


BY 


Belarus 


K£ 


Kenya 


RO 


Romania 


CA 


Canada 


KG 


Kyrgystan 


RU 


Russian Federation 


CF 


Central African Republic 


KP 


Democratic People's Republic 


SD 


Sudan 


CG 


Congo 




of Korea 


SE 


Sweden 


CH 


Switzerlaml 


KR 


Republic of Korea 


SI 


Slovenia 


CI 


Caie d'lvoirc 


KZ 


Kazakhstan 


SK 


Slovakia 


CM 


Cameroon 


U 


Liechtenstein 


SN 


Senegal 


CN 


China 


LK 


Sri Lanka 


TD 


Chad 


cs 


Czechoslovakia 


LU 


Luxembourg 


TG 


Togo 


C2 


Czech Republic 


LV 


Latvia 


TJ 


Tajikistan 


DE 


Germany 


MC 


Monaco 


TT 


Trinidad and Tobago 


DK 


Denmark 


MD 


Republic of Moldova 


UA 


Ukraine 


ES 


Spain 


MG 


Madagascar 


US 


United States of America 


n 


Finland 


ML 


Mali 


U2 


Uzbekistan 


FR 


France 


MN 


Mongolia 


VN 


Viet Nam 


GA 


Gabon 











wo 96/01845 



PCTAJS95/08543 



GROWTH DIFFERENTIATION FACTOR-11 

BACKGROUND OF THE INVENTION 

1 , Field of the Invention 

The invention relates generally to growth factors and specifically to a new mennber of the 
5 transfonming growth factor beta (TGF-p) superfamily. which Is denoted, growth 
differentiation factor-1 1 (GDF-11). 

2. Description of Related Art 

The transfomning growth factor p (TGF-p) superfamily encompasses a group of 
structurally-related proteins which affect a wide range of differentiation processes during 

10 embryonic development. The family Includes, Mullerian inhibiting substance (MIS), 
which is required for normal male sex development (Behringer, et al.. Nature, 345:167, 
iggO), Drosophila decapentaplegic (DPP) gene product, which is required for dorsal- 
ventral axis formation and morphogenesis of the imaginal disks (Padgett, et ai, Nature, 
325:81-84, 1987), the Xenopus Vg-1 gene product, which localizes to the vegetal pole 

15 of eggs ((Weeks, et ai, Cell, 51:861-867, 1987). the activins (Mason, et aL, Biochem, 
Biophys. Res, Commun., 135:957-964, 1986), which can induce the fonmation of 
mesoderm and anterior structures in Xenopus embryos (Thomsen. et aL, Cell, 63:485, 
1990), and the bone morphogenetic proteins (BMPs, osteogenin, OP-1) which can 
induce de novo cartilage and bone formation <Sampath, et aL, J. BioL Chem,, 265:13198, 

20 1990). The TGF-Ps can influence a variety of differentiation processes, including 
adipogenesis. myogenesis, chondrogenesis, hematopoiesis, and epithelial cell 
differentiation (for review, see Massague. Ce// 49:437, 1987). 

The proteins of the TGF-p family are initially synthesized as a \arge precursor protein 
which subsequently undergoes proteolytic cleavage at a cluster of basic residues 



wo 96/01845 



PCTAJS95«)8S43 



-2- 

approximately 110-140 amino acids from the C-temiinus. The C-temiinal regions, or 
mature regions, of the proteins are all structurally related and the different family 
members can be classified Into distinct subgroups based on the extent of their homology. 
Although the homologies within particular subgroups range from 70% to 90% amino acid 

5 sequence identity, the homologies between subgroups are significantly lovirer, generally 
ranging from only 20% to 50%. In each case, the active species appears to be a 
disulfide-linked dimer of C-temninal fragments. Studies have showm that when the pro- 
region of a member of the TGF-P family is coexpressed with a mature region of another 
member of the TGF-p family, intracellular dimerization and secretion of biologically active 

10 homodimers occur (Gray. A., and Maston, A.. Science, 242:1328, 1990). Additional 
studies by Hammonds, et al., {Molec. Endocrin. 5:149. 1991) showed that the use of the 
BMP-2 pro-region combined with the BMP-4 mature region led to dramatically improved 
expression of mature BMP-4. For most of the family members that have been studied, 
the homodimeric species has been found to be biologically active, but for other family 

15 members, like the inhibins (Ling, et aL. Nature, 321:779, 1986) and the TGF-Ps 
(Cheifetz, et al., Cell, 48:409, 1987), heterodimers have also been detected, and these 
appear to have different biological properties than the respective homodimers. 

Identification of new factors that are tissue-specific in their expression pattern wnll provide 
a greater understanding of that tissue's development and function. 



wo 96/01845 



-3- 



PCT/US95/08543 



SUMMARY OF THE INVENTION 

The present invention provides a cell growth and differentiation factor. GbF-11, a 
polynudeotide sequence which encodes the factor, and antibodies which are bind to the 
factor. This factor appears to relate to various cell proliferative disorders, especially 
5 those involving muscle, neural, and uterine cells, as well as disorders related to the 
function of the immune system. 

Thus, in one embodiment, the invention provides a method for detecting a cell 
proliferative disorder of muscle, neural, uterine, spleen, or thymus origin and which is 
associated vwth GDF-1 1. In another embodiment, the invention provides a method for 
1 0 treating a cell proliferative or immunologic disorder by suppressing or enhancing GDF-1 1 
activity. 



wo 96/01845 



PCr/US95/08543 



-4- 

BRIEF DESCRIPTION OF THE DRAWINGS 

FIGURE 1 shows the nucleotide and predicted amino acid sequences of murine 
(FIGURE 1a) and human (FIGURE 1b) GDF-11. The putative proteolytic processing 
sites are shown by the shaded boxes. In the human sequence, the potential N-linked 
5 glycosylation signal is shown by the open box, and the consensus polyadenylation signal 
is underlined; the poly A tail is not shown. 

FIGURE 2 shows Northern blots of RNA prepared from adult (FIGURE 2a) or fetal and 
neonatal (FIGURE 2b) tissues probed with a murine GDF-1 1 probe. 

FIGURE 3 shows amino acid homologies among different nr>embers of the TGF-P 
10 superfamily. Numbers represent percent amino acid identities between each pair 
calculated from the first conserved cysteine to the Oterminus. Boxes represent 
homologies among highly-related members within particular subgroups. 

FIGURE 4 shows an alignment of the predicted amino acid sequences of human GDF-1 1 
(top lines) with human GDF-8 (bottom lines). Vertical lines indicate identities. Dots 
15 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 shovwi by the open box. The conserved cysteine residues on the C-terminal region 
are shown by the shaded boxes. 

FIGURE 5 shows the expression of GDF-11 in mammalian <:ells. Conditioned nrwdium 
20 prepared from Chinese hamster ovary cells transfected with a hybrid <3DF-8/GDF-1 1 
gene (see text) cloned into the MSXND expression vector in either the antisehse (lane 
1) or sense (lane 2) orientation was dialyzed, lyophilized, and subjected to Western 
analysis using antibodies directed against the C-temiinal portion of <30F--8 protein. 
Arrows at right indicate the putative unprocessed (pro-GDF-e/GDF-ll) or processed 
25 GDF-1 1 proteins. Numbers at left indicate mobilities of molecular weight standards. 



wo 96/01845 



-5- 



PCTAJS95/08543 



FIGURE 6 shows the chromosomal mapping of human GDF-11. DNA samples prepared 
from human/rodent somatic cell lines were subjected to PGR, electrophoresed on 
agarose gels, blotted, and probed. The human chromosome contained in each of the 
hybrid cell lines is identified at the top of each of the first 24 lanes (1-22, X. and Y). In 
5 the lanes designated CHO, M, and H, the starting DNA template was total genomic DNA 
from hamster, mouse, and human sources, respectively. In the lane marked B1, no 
template DNA was used. Numbers at left indicate the mobilities of DNA standards. 

FIGURE 7 shows the FISH localization of GDF-11. Metaphase chromosomes derived 
from peripheral blood lymphocytes were hybridized with digoxigenin-labelled human 
1 0 GDF-1 1 probe (a) or a mbcture of human GDF-1 1 genomic and chromosome 12-speclf(c 
centromere probes (b) and analyzed as described in the text. A schematic showing the 
location of GDF-1 1 at position 12q13 is shown in panel (c). 

FIGURE 8 shows the nucleotide and deduced amino acid sequence of murine GDF-8. 



wo 96/01845 



PCT/US9S/08543 



DETAILED DESCRIPTION OF THE INVENTION 

The present invention provides a growth and differentiation factor, GDF-11, and a 
polynucleotide sequence encoding GDF-11. GDF-11 is expressed at highest levels in 
muscle, brain, uterus, spleen, and thymus and at lower levels in other tissues. In one 
5 embodiment, the invention provides a method for detection of a cell proliferative or 
immunologic disorder of muscle, neural, uterine, spleen, or thymus origin which is 
associated with GDF-11 expression or function. In another embodiment, the invention 
provides a method for treating a cell proliferative or immunologic disorder by using an 
agent which suppresses or enhances GDF-1 1 activity. 

1 0 The TGF-P superfamily consists of multifunctional polypeptides that control proliferation, 
differentiation, and other functions in many cell types. Many of the peptides have 
regulatory, both positive and negative, effects on other peptide growth factors. The 
structural homology between the GDF-11 protein of this invention and the members of 
the TGF-3 family, indicates that GDF-11 is a new member of the family of growth and 

15 differentiation factors. Based on the known activities of many of the other members, it 
can be expected that GDF-11 will also possess biological activities that will make it 
useful as a diagnostic and therapeutic reagent. 

Certain members of this superfamily have expression pattems or possess activities that 
relate to the function of the nervous system. For example, one family member, namely 

20 GDNF, has been shown to be a potent neurotrophic factor that can promote the survival 
of dopaminergic neurons (Lin, et aL, Science, 260:1130). Another family member, 
namely dorsalin-1, is capable of promoting the differentiation of neural crest cells (Easier, 
etal., Cell, 73:687, 1993). The inhibins and activins have been shown to be expressed 
in the brain (Meunier, et at., Proc. Natl Acad, Sc/., USA, 85:247, 1988; Sawchenko, et 

25 aL, Nature, 334:615, 1988), and activin has been shown to be capable of functioning as 
a nerve cell survival molecule (Schubert, ef a/., Nature, 344:868. 1990). Another family 
member, namely GDF-1, is nervous system-specific in its expression pattern -(Lee, Proc, 



wo 96/01845 



PCTAJS95/08543 



-7- 

Natl Acad Sa, USA, 88:4250, 1991), and certain other family members, such as Vgr-1 
(Lyons. etaL, Proc. Natl Acad Sc/., USA, 86:4554. 1989; Jones, ef at, Development, 
111:581. 1991), OP-1 (Ozkaynak. etaL, J. BioL Chem,, 267:25220. 1992). and BMP-4 
(Jones, et al, Development, 111:531. 1991). are also known to be expressed in the 
5 nen^ous system. The expression of GDF-1 1 In brain and muscle suggests that GDF-1 1 
may also possess activities that relate to the function of the nervous system. In 
particular, it is known, for example, that skeletal muscle produces a factor or factors that 
promote the survival of motor neurons (Brown. Trends NeuroscL, 7:10, 1984). The 
known neurotrophic activities of other members of this family and the expression of <3DF- 

10 1 1 in muscle suggest that one activity of GDF-1 1 may be as a trophic factor for motor 
neurons; indeed. GDF-1 1 is highly related to GDF-8. which is virtually muscle-specific 
in its expression pattern. Altematively. GDF-1 1 may have neurotrophic activities for 
other neuronal populations. Hence, GDF-1 1 may have in vitro and in vivo applications 
in the treatment of neurodegenerative diseases, such as amyotrophic lateral sclerosis. 

15 or in maintaining ceils or tissues in culture prior to transplantation. 

GDF-1 1 may also have applications in treating disease processes involving muscle, such 
as in musculodegenerative diseases or in tissue repair due to trauma. In this regard, 
many other members of the TGF-P family are also important mediators of tissue repair. 
TGF-p has been shown to have marked effects on the formation of collagen and to 

20 cause a striking angiogenic response in the newbom mouse (Roberts, et al., Proc. Natl. 
Acad. Sci.. USA 83:4167, 1986). TGF-p has also been shown to inhibit the 
differentiation of myoblasts in culture (Massague, et al, Proc. Nati Acad. Sci, USA 
83:8206, 1986). Moreover, because myoblast cells may be used as a vehicle for 
delivering genes to muscle for gene therapy, the properties of GDF-1 1 could be exploited 

25 for maintaining cells prior to transplantation or for enhancing the efficiency of the fusion 
process. 

GDF-1 1 may also have applications in the treatment of immunologic disorders. In 
particular, TGF-P has been shown to have a wide range of immunoregulatory activities. 



wo 96/01845 



PCTAJS95/08S43 



including potent suppressive effects on B and T cell proliferation and function (for review, 
see Palladino. etaL, Ann. N,Y. Acad, ScL, 593:181. 1990). The expression of GDF-11 
in spleen and thymus suggests that GDF-11 may possess similar activities and therefore, 
may be used as an anti-inflammatory agent or as a treatment for disorders related to 
5 abnormal proliferation or function of lymphocytes. 

The term "substantially pure" as used herein refers to GDF-1 1 which is substantially free 
of other proteins, lipids, carbohydrates or other materials with which it is naturally 
associated. One skilled in the art can purify GDF-11 using standard techniques for 
protein purification. The substantially pure polypeptide will yield a single major band on 
10 a non-reducing polyacrylamide gel. The purity of the GDF-11 polypeptide can also be 
determined by amino-terminal amino acid sequence analysis. GDF-11 polypeptide 
includes functional fragments of the polypeptide, as long as the activity of GDF-11 
remains. Smaller peptides containing the biological activity of GDF-11 are included in 
the invention. 

15 The invention provides polynucleotides encoding the GDF-11 protein. These 
polynucleotides include DNA, cDNA and RNA sequences which encode GDF-11. It is 
understood that all polynucleotides encoding all or a portion of GDF-1 1 are also included 
herein, as long as they encode a polypeptide with GDF-1 1 activity. Such polynucleotides 
include naturally occuning. synthetic, and intentionally manipulated polynucleotides. For 

20 example. GDF-11 polynucleotide may be subjected to site-directed mutagenesis. The 
polynucleotide sequence for GDF-11 also includes antisense sequences. The 
polynucleotides of the invention include sequences that are degenerate as a result of the 
genetic code. There are 20 natural amino acids, most of which are specified by more 
than one codon. Therefore, all degenerate nucleotide sequences are included in the 

25 invention as long as the amino acid sequence of GDF-11 polypeptide encoded by the 
nucleotide sequence is functionally unchanged. 



wo 96/01845 



PCTAJS95/08S43 



-9- 

Specifically disclosed herein is a DNA sequence containing the human GDF-11 gene. 
The sequence contains an open reading franne encoding a polypeptide 407 amino acids 
in length. The sequence contains a putative RXXR proteolytic cleavage site at amino 
acids 295-298. Cleavage of the precursor at this site would generate an active C- 
5 terminal fragment 109 amino acids in length with a predicted molecular weight of 
approximately 12.500 kD. Also disclosed herein is a partial murine genomic sequence. 
Preferably, the human GDF-11 nucleotide sequence is SEQ ID NO:1 and the mouse 
nucleotide sequence is SEQ ID NO:3. 

The polynucleotide encoding GDF-11 includes SEQ ID NO:1 and 3, as well as nucleic 
acid sequences complementary to SEQ ID NO's:1 and 3. A complementary sequence 
may include an antisense nucleotide. When the sequence is RNA, the deoxynucleotides 
A, G, C, and T of SEQ ID NO:1 and 3 are replaced by ribonucleotides A, G, C. and U. 
respectively. Also included in the invention are fragments of the above-described nucleic 
acid sequences that are at least 15 bases in length, which is sufficient to permit the 
fragment to selectively hybridize to DNA that encodes the protein of SEQ ID NO: 2 or 4 
under physiological conditions. 

The Otenminal region of GDF-11 following the putative proteolytic processing site shows 
significant homology to the known members of the TGF-P superfamily. The GDF-11 
sequence contains most of the residues that are highly conserved in other family 
20 members (see FIGURE 1). Like the TGF-Ps and inhibin Ps. GDF-11 contains an extra 
pair of cysteine residues in addition to the 7 cysteines found in virtually all other family 
members. Among the known family members, GDF-11 is most homologous to GDF-iB 
(92% sequence identity) (see FIGURE 3). 

Minor modifications of the recombinant GDF-1 1 primary amino acid sequence may result 
25 in proteins which have substantially equivalent activity as compared to the GDF-11 
polypeptide described herein. Such modifications may be deliberate, as by site-directed 
mutagenesis, or may be spontaneous. All of the polypeptides produced by these 



10 



15 



wo 96/01845 



PCT/US95/08543 



-10- 

modifications are included herein as long as the biological activity of GDF-11 still exists. 
Further, deletion of one or more amino acids can also result in a modification of the 
stmdure of the resultant molecule without significantly altering its biological, activity. This 
can lead to the development of a smaller active molecule which would have broader 
5 utility. For example, one can remove amino or carboxy temninal amino acids which are 
not required for GDF-1 1 biological activity. 

The nucleotide sequence encoding the GDF-1 1 polypeptide of the invention includes the 
disclosed sequence and conservative variations thereof. The term "conservative 
variation" as used herein denotes the replacement of an amino acid residue by another. 

1 0 biologically similar residue. Examples of conservative variations include the substitution 
of one hydrophobic residue such as isoleucine. valine, leucine or methionine for another, 
or the substitution of one polar residue for another, such as the substitution of arginine 
for lysine, glutamic for aspartic acid, or glutamine for asparagine. and the like. The tenm 
"conservative variation" also includes the use of a substituted amino acid in place of an 

15 unsubstituted parent amino acid provided that antibodies raised to the substituted 
polypeptide also immunoreact with the unsubstituted polypeptide. 

DMA sequences of the invention can be obtained by several methods. For example, the 
ONA can be isolated using hybridization techniques which are well known in the art. 
These include, but are not limited to: 1) hybridization of genomic or cDNA libraries with 
20 probes to detect homologous nucleotide sequences, 2) polymerase chain reaction (PCR) 
on genomic DNA or cDNA using primers capable of annealing to the DNA sequence of 
interest, and 3) antibody screening of expression libraries to detect cloned DNA 
fragments with shared structural features. 

Preferably the GDF-11 polynucleotide of the invention is derived from a mammalian 
25 organism, and most preferably from a mouse, rat, or human. Screening procedures 
which rely on nucleic acid hybridization make it possible to isolate any gene sequer>ce 
from any organism, provided the appropriate probe is available. Oligonucleotide probes, 



wo 96/01845 



-11- 



PCTAJS95/08543 



which correspond to a part of the sequence encoding the protein in question, can be 
synthesized chemically. This requires that short, oligopeptide stretches of amino acid 
sequence must be known. The DNA sequence encoding the protein can be deduced 
from the genetic code, however, the degeneracy of the code must be taken into account. 
5 It is possible to perform a mixed addition reaction when the sequence is degenerate. 
This includes a heterogeneous mixture of denatured double-stranded DNA. For such 
screening, hybridization is preferably perfomned on either single-stranded DNA or 
denatured double-stranded DNA. Hybridization is particularly useful in the detection of 
cDNA clones derived from sources where an extremely low amount of mRNA sequences 

10 relating to the polypeptide of interest are present. In other words, by using stringent 
hybridization conditions directed to avoid non-specific binding, it is possible, for example, 
to allow the autoradiographic visualization of a specific cDNA clone by the hybridization 
of the target DNA to that single probe in the mixture which is its complete complement 
(Wallace, et al, NucL Acid Res., 9:879. 1981; Maniatis. et al, Molecular Cloning: A 

1 5 Laboratory Manual, Cold Spring Harbor, N.Y. 1989). 

The development of specific DNA sequences encoding GDF-1 1 can also be obtained by: 
1) isolation of double-stranded DNA sequences from the genomic DNA; 2) chemical 
manufacture of a DNA sequence to provide the necessary codons for the polypeptide 
of interest; and 3) in vitro synthesis of a double-stranded DNA sequence by reverse 
20 transcription of mRNA isolated from a eukaryotic donor cell. In the latter case, a double- 
stranded DNA complement of mRNA is eventually fonmed which is generally referred to 
ascDNA. 

Of the three above-noted methods for developing specific DNA sequences for use in 
recombinant procedures, the isolation of genomic DNA isolates is the least common. 
25 This is especially true when it is desirable to obtain the microbial expression of 
mammalian polypeptides due to the presence of introns. 



wo 96/01845 



PCr/US95/08543 



-12- 

The synthesis of DNA sequences is frequently the method of choice when the entire 
sequence of amino acid residues of the desired polypeptide product is known. When the 
entire sequence of amino acid residues of the desired polypeptide is not known, the 
direct synthesis of DNA sequences is not possible and the method of choice is the 
5 synthesis of cDNA sequences. Among the standard procedures for isolating cDNA 
sequences of interest is the formation of plasmid- or phage-carrying cDNA libraries which 
are derived from reverse transcription of mRNA which is abundant in donor cells that 
have a high level of genetic expression. When used in combination with polymerase 
chain reaction technology, even rare expression products can be cloned. In those cases 

1 0 where significant portions of the amino acid sequence of the polypeptide are known, the 
production of labeled single or double-stranded DNA or RNA probe sequences 
duplicating a sequence putatively present in the target cDNA may be employed in 
DNA/DNA hybridization procedures which are carried out on cloned copies of the cDNA 
which have been denatured into a single-stranded form (Jay, ef aL, Nucl. Acid Res., 

15 11:2325,1983). 

A cDNA expression library, such as lambda gtl 1, can be screened indirectly for GDF-1 1 
peptides having at least one epitope, using antibodies specific for GDF-1 1. Such 
antibodies can be either polyctonally or monoclonally derived and used to detect 
expression product indicative of the presence of GDF-1 1 cDNA. 

20 DNA sequences encoding GDF-1 1 can be expressed in vitro by DNA transfer into a 
suitable host cell. "Host cells" are cells in which a vector can be propagated and its DNA 
expressed. The term also includes any progeny of the subject host cell. It is understood 
that all progeny may not be identical to the parental cell since there may be mutations 
that occur during replication. However, such progeny are included when the term "host 

25 cell" is used. Methods of stable transfer, meaning that the foreign DNA is continuously 
maintained in the host, are known in the art. 



wo 96/01845 



PCTAJS9S/08543 



-13- 

In the present invention, the GDF-11 polynucleotide sequences may be inserted into a 
recombinant expression vector. The term "recombinant expression vector" refers to a 
plasmid. vims or other vehicle known in the art that has been manipulated by insertion 
or incorporation of the GDF-1 1 genetic sequences. Such expression vectors contain a 
5 promoter sequence which facilitates the efficient transcription of the inserted genetic 
sequence of the host. The expression vector typically contains an origin of replication, 
a promoter, as well as specific genes which allow phenotypic selection of the 
transformed cells. Vectors suitable for use in the present invention include, but are not 
limited to the T7-based expression vector for expression in bacteria (Rosenberg, ei ai, 
1 0 Gene, 56:125, 1987). the pMSXND expression vector for expression in mammalian cells 
(Lee and Nathans, J. Bioi Chem,, 263:3521 1988) and baculovims-derived vectors for 
expression in insect cells. The DNA segment can be present in the vector operably 
linked to regulatory elements, for example, a promoter (e.g.. T7, metallothionein I. or 
polyhedrin promoters). 

1 5 Polynucleotide sequences encoding GDF-1 1 can be expressed in either prokaryotes or 
eukaryotes. Hosts can include microbial, yeast, insect and mammalian organisms. 
Methods of expressing DNA sequences having eukaryotic or viral sequences in 
prokaryotes are well known in the art. Biologically functional viral and plasmid DNA 
vectors capable of expression and replication in a host are known in the art. Such 

20 vectors are used to incorporate DNA sequences of the invention. Preferably, the mature 
G-terminal region of GDF-1 1 is expressed from a DNA clone containing the entire coding 
sequence of GDF-11. Alternatively, the C-terminal portion of GDF-11 can be expressed 
as a fusion protein with the pro- region of another member of the TGF-P family or co- 
expressed with another pro- region (see for example, Hammonds, et ai, Molec, 

25 Endocrin. 5:149, 1991; Gray. A., and Mason, A., Science, 247:1328. 1990). 

Transformation of a host cell wnth recombinant DNA may be carried out by conventional 
techniques as are well known to those skilled in the art. Where the host is prokaryotic, 
such as £ CO//, competent cells which are capable of DNA uptake -can be prepared from 



wo 96/01845 



PCTAJS95/08543 



-14- 

cells harvested after exponential growth phase and subsequently treated by the CaCIs 
method using procedures well known in the art. Alternatively, MgCl2 or RbCI can be 
used. Transformation can also be performed after forming a protoplast of the host cell 
if desired. 

5 When the host is a eukaryote, such methods of transfection of DNA as calcium 
phosphate co-precipitates, conventional mechanical procedures such as microinjection, 
electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be 
used. Eukaryotic cells can also be cotransformed with DNA sequences encoding the 
GDF-1 1 of the invention, and a second foreign DNA molecule encoding a selectable 
10 phenotype, such as the herpes simplex thymidine kinase gene. Another method is to 
use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, 
to transiently infect or transfomn eukaryotic cells and express the protein, (see for 
example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982), 

Isolation and purification of microbial expressed polypeptide, or fragments thereof, 
15 provided by the invention, may be carried out by conventional means including 
preparative chromatography and immunological separations involving monoclonal or 
polyclonal antibodies. 

The GDF-1 1 polypeptides of the invention can also be used to produce antibodies which 
are immunoreactive or bind to epitopes of the GDF-11 polypeptides. Antibody which 
20 consists essentially of pooled monoclonal antibodies with different epitopic specificities, 
as well as distinct monoclonal antibody preparations are provided. Monoclonal 
antibodies are made from antigen containing fragments of the protein by methods well 
known in the art (Kohier, et a!., Nature, 256:495. 1975; Current Protocols ir) Molecular 
Biology, Ausubel, ef a/., ed., 1989). 

25 The term "antibody" as used in this invention includes intact molecules as well as 
fragments thereof, sucrfi as Fab, F(ay)2, and Fv which are capable of binding the epitopic 



wo 96/01845 PCT/US95/08543 

-15- 

determinant. These antibody fragments retain some ability to selectively bind with its 
antigen or receptor and are defined as follows: 

(1) Fab, the fragment which contains a monovalent antigen-binding fragment of an 
antibody molecule can be produced by digestion of whole antibody with the 

5 enzyme papain to yield an intact light chain and a portion of one heavy chain; 

(2) Fab', the fragment of an antibody molecule can be obtained by treating whole 
antibody with pepsin, followed by reduction, to yield an intact light chain and a 
portion of the heavy chain; two Fab* fragments are obtained per antibody 
molecule; 

10 (3) (Fab')2. the fragment of the antibody that can be obtained by treating whole 
antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a 
dimer of two Fab' fragments held together by two disulfide bonds; 

(4) Fv. defined as a genetically engineered fragment containing the variable region 
of the light chain and the variable region of the heavy chain expressed as two 

15 chains; and 

(5) Single chain antibody ("SCA"), defined as a genetically engineered molecule 
containing the variable region of the light chain, the variable region of the heavy 
chain, linked by a suitable polypeptide linker as a genetically fused single chain 
molecule. 



20 



Methods of making these fragments are known in the art. <See for example, Harlow and 
Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York 
(1988). incorporated herein by reference). 



wo 96/01845 



PCrAJS9S/08543 



-16- 

As used in this invention, the tenn "epitope" means any antigenic detemninant on an 
antigen to which the paratope of an antibody binds. Epitopic determinants usually 
consist of chemically active surface groupings of molecules such as amino acids or 
sugar side chains and usually have specific three dimensional structural characteristics. 
5 as well as specific charge characteristics. 

Antibodies which bind to the GDF-11 polypeptide of the invention can be prepared using 
an intact polypeptide or fragments containing small peptides of interest as the 
immunizing antigen. The polypeptide or a peptide used to immunize an animal can be 
derived from translated cDNA or chemical synthesis w^ich can be conjugated to a carrier 
10 protein, if desired. Such commonly used earners which are chemically coupled to the 
peptide include keyhole limpet hemocyanin (KLH), thyroglobulin. bovine serum albumin 
(BSA). and tetanus toxoid. The coupled peptide is then used to immunize the animal 
(e.g., a mouse, a rat, or a rabbit). 

If desired, polyclonal or monoclonal antibodies can be further purified, for example, by 
15 biriding to and elution from a matrix to which the polypeptide or a peptide to wrhich the 
antibodies were raised is bound. Those of skill in the art will know of various techniques 
common in the immunology arts for purification and/or cor>centration of polyclonal 
antibodies, as well as monoclonal antibodies <See for example, Coligan, et aL, Unit 9, 
Current Protocols in Immunology, Wiley Interscience, 1991, incorporated by reference). 

20 It is also possible to use the anti-idiotype technology to produce monoclonal antibodies 
which mimic an epitope. For example, an anti-idiotypic monoclonal antibody made to a 
first monoclonal antibody will have a binding domain in the hypervariable region which 
is the "image" of the epitope bound by the first monoclonal antibody. 

The term "cell-proliferative disorder" denotes malignant as well as non-malignant cell 
25 populations which often appear to differ from the surrounding tissue both morphologteally 
and genotypically. Malignant cells (i.e. cancer) develop as a result of a multistep 



wo 96/01845 



PCr/US95/08S43 



-17- 

process. The GDF-11 polynucleotide that is an antisense molecule is useful In treating 
malignancies of the various organ systems, particularly, for example, cells in muscle, 
uterus, spleen, thymus, or neural tissue. Essentially, any disorder which is etiologicalty 
linked to altered expression of GDF-11 could be considered susceptible to treatment with 
5 a GDF-11 suppressing reagent. One such disorder is a malignant cell proliferative 
disorder, for example. 

The invention provides a method for detecting a cell proliferative disorder of muscle, 
uterine or neural tissue, for example, which comprises contacting an anti-GDF-11 
antibody with a cell suspected of having a GDF-11 associated disorder and detecting 

1 0 binding to the antibody. The antibody reactive with GDF-1 1 is labeled with a compound 
which allows detection of binding to GDF-1 1. For purposes of the invention, an antibody 
specific for GDF-11 polypeptide may be used to detect the level of GDF-11 in biological 
fluids and tissues. Any specimen containing a detectable amount of antigen can be 
used. A preferred sample of this invention is muscle, utenjs, spleen, thymus, or neural 

15 tissue. The level of GDF-11 in the suspect cell can be compared with the level in a 
nomnal cell to determine whether the subject has a GDF-1 1-associated cell proliferative 
disorder. Preferably the subject is human. 

The antibodies of the invention can be used in any subject in which it is desirable to 
administer in vitro or in vivo immunodiagnosis or immunotherapy. The antibodies of the 

20 invention are suited for use. for example, in immunoassays in which they can be utilized 
in liquid phase or bound to a solid phase carrier. In addition, the antibodies in these 
immunoassays can be detectably labeled in various ways. Examples of types of 
immunoassays which can utilize antibodies of the invention are competitive and non- 
competitive immunoassays in either a direct or indirect format. Examples of such 

25 immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) 
assay. Detection of the antigens using the antibodies of the invention can be done 
utilizing immunoassays which are run in either the forward, reverse, or simultaneous 
modes, including immunohistochemical assays on physiological samples. Those of skill 



wo 96/01845 



PCTAJS95/08543 



"18- 

in the art will know, or can readily discern, other immunoassay formats without undue 
experimentation. 

The antibodies of the Invention can be bound to many different carriers and used to 
detect the presence of an antigen comprising the polypeptide of the invention. Examples 
5 of well-known earners include glass, polystyrene, polypropylene, polyethylene, dextran, 
nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and 
magnetite. The nature of the carrier can be either soluble or insoluble for purposes of 
the invention. Those skilled in the art will know of other suitable carriers for binding 
antibodies, or will be able to ascertain such, using routine experimentation. 

10 There are many different labels and methods of labeling known to those of ordinary skill 
in the art. Examples of the types of labels which can be used in the present invention 
include enzymes, radioisotopes, fluorescent compounds. coHoidal metals, 
chemiluminescent compounds, phosphorescent compounds, and bioluminescent 
compounds. Those of ordinary skill in the art will know of other suitable labels for 

1 5 binding to the antibody, or will be able to ascertain such, using routine experimentation. 

Another technique which may also result in greater sensitivity consists of coupling the 
antibodies to low molecular weight haptens. These haptens can then be specifically 
detected by means of a second reaction. For example, it is common to use such 
haptens as biotin, which reacts with avidin, or dinitrophenyl, puridoxal. and fluorescein, 
20 which can react with specific antihapten antibodies. 

In using the monoclonal antibodies of the invention for the in vivo detection of antigen, 
the detectably labeled antibody is given a dose which is diagnostically effective. The 
term "diagnostically effective" means that the amount of detectably labeled monoclonal 
antibody is administered in sufficient quantity to enable detection of the site having the 
25 antigen comprising a polypeptide of the invention for which the monoclonal antibodies 
are specific. 



wo 96/01845 



PCTAJS95/08543 



-19- 

The concentration of detectably labeled monoclonal antibody which is administered 
should be sufficient such that the binding to those cells having the polypeptide is 
detectable compared to the background. Further, it is desirable that the detectably 
labeled monoclonal antibody be rapidly cleared from the circulatory system in order to 
5 give the best target-to-bacl^ground signal ratio. 

As a rule, the dosage of detectably labeled monoclonal antibody for in vivo diagnosis will 
vary depending on such factors as age, sex, and extent of disease of the individual. 
Such dosages may vary, for example, depending on whether multiple injections are 
given, antigenic burden, and other factors known to those of skill in the art. 

1 0 For in vivo diagnostic imaging, the type of detection instrument available is a major factor 
in selecting a given radioisotope. The radioisotope chosen must have a type of decay 
which is detectable for a given type of instrument. Still another important factor in 
selecting a radioisotope for in vivo diagnosis is that deleterious radiation with respect to 
the host is minimized. Ideally, a radioisotope used for in vivo imaging will lack a particle 

1 5 emission, but produce a large number of photons in the 140-250 keV range, which may 
readily be detected by conventional gamma cameras. 

For in vivo diagnosis radioisotopes may be bound to immunoglobulin either directly or 
indirectly by using an intermediate functional group. Intermediate functional groups 
which often are used to bind radioisotopes which exist as metallic ions to 
20 immunoglobulins are the bifunctional chelating agents such as 
diethylenetriaminepentacetic acid (DTPA) and ethylenediaminetetraacetic acid (EDTA) 
and similar molecules. Typical examples of metallic ions which can be bound to the 
monoclonal antibodies of the invention are "^In, ®'Ru, ^<3a, ^Ga, ^^As, ®^Zr, and ^°^TI. 

The monoclonal antibodies of the Invention can also be labeled with a paramagnetic 
25 isotope for purposes of in vivo diagnosis, as in magnetic resonance imaging (MRI) or 
electron spin resonance (ESR). In general, any conventional method for visualizing 



wo 96/01845 PCTAJS95/08543 

-20. 

diagnostic imaging can be utilized. Usually gamma and positron emitting radioisotopes 
are used for camera imaging and paramagnetic isotopes for MRI. Elements which are 
particularly useful in such techniques include ^^^Gd, ^Mn, ^^Dy. '^Cr, and ^Fe. 

The monoclonal antibodies of the Invention can be used in vitro and in vivo to monitor 
5 the course of amelioration of a GDF-11 -associated disease in a subject. Thus, for 
example, by measuring the increase or decrease in the number of cells expressing 
antigen comprising a polypeptide of the invention or changes in the concentration of 
such antigen present in various body fluids, it v^ould be possible to determine whether 
a particular therapeutic regimen aimed at ameliorating the GDF-1 1 -associated disease 
10 is effective. The term "ameliorate" denotes a lessening of the detrimental effect of the 
GDF-1 1 -associated disease in the subject receiving therapy. 

The present invention identifies a nucleotide sequence that can be expressed in an 
altered manner as compared to expression in a normal cell, therefore it is possible to 
design appropriate therapeutic or diagnostic techniques directed to this sequence. Thus, 

1 5 where a cell-proliferative disorder is associated with the expression of GDF-1 1 , nucleic 
acid sequences that interfere with GDF-11 expression at the translational level can be 
used. This approach utilizes, for example, antisense nucleic acid and ribozymes to block 
translation of a specific GDF-11 mRNA. either by masking that mRNA with an antisense 
nucleic acid or by cleaving it with a ribozyme. Such disorders include neurodegenerative 

20 diseases, for example. 

Antisense nucleic acids are DNA or RNA molecules that are complementary to at least 
a portion of a specific mRNA molecule (Weintraub, Scientific American, 262:40. 1990). 
In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a 
double-stranded molecule. The antisense nucleic acids interfere with the translation of 
25 the mRNA. since the cell will not translate a mRNA that is double-stranded. Antisense 
oligomers of about 15 nucleotides are preferred, since they are easily synthesized and 
are less likely to cause problems than larger molecules when introduced into the target 



wo 96/01845 



PCTAJS9S/08543 



-21- 

GDF-11 -producing cell. The use of antisense methods to inhibit the in vitro translation 
of genes is well knovym in the art (Marcus-Sakura, Anal.Biochem,, 172:289, 1988). 

Ribozymes are RNA molecules possessing the ability to specifically deave other single- 
stranded RNA in a manner analogous to DNA restriction endonucleases. Through the 
5 modification of nucleotide sequences which encode these RNAs, it is possible to 
engineer molecules that recognize specific nucleotide sequences in an RNA molecule 
and cleave it (Cech, JAmerMed, Assn., 260:3030. 1988). A major advantage of this 
approach is that, because they are sequence-specific, only mRNAs with particular 
sequences are inactivated. 

10 There are two basic types of ribozymes namely, tetratiymena-lype (Hasselhoff, Nature, 
334:585, 1988) and "hammerhead"-type. TetrahymenaAype ribozymes recognize 
sequences which are four bases in length, while "hammerhead'-type ribozymes 
recognize base sequences 11-18 bases in length. The longer the recognition sequence, 
the greater the likelihood that the sequence will occur exclusively in the target mRNA 

15 species. Consequently, hammertiead-type ribozymes are preferable to tetrahynwna- 
type ribozymes for inactivating a specific mRNA species and 18-based recognition 
sequences are preferable to shorter recognition sequences. 

The present invention also provides gene therapy for the treatment of cell proliferative 
or immunologic disorders which are mediated by GDF-11 protein. Such therapy would 
20 achieve its therapeutic effect by introduction of the GDF-1 1 antisense polynucleotide into 
cells having the proliferative disorder. Delivery of antisense GDF-1 1 polynucleotide -can 
be achieved using a recombinant expression vector such as a chimeric virus or a 
colloidal dispersion system. Especially preferred for therapeutic delivery of antisense 
sequences is the use of targeted liposomes. 

25 Various viral vectors which can be utilized for gene therapy as taught herein include 
adenovirus, herpes virus, vaccinia, or, preferably, an RNA virus such as a retrovirus. 



wo 96/01845 



PCT/US95/08543 



-22- 

Preferably, the retroviral vector is a derivative of a murine or avian retrovirus. Examples 
of retroviral vectors in which a single foreign gene can be inserted include, but are not 
limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus 
(HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). 
5 A number of additional retroviral vectors can incorporate multiple genes. All of these 
vectors can transfer or incorporate a gene for a selectable marker so that transduced 
cells can be identified and generated. By inserting a GDF-1 1 sequence of interest into 
the viral vector, along with another gene which encodes the ligand for a receptor on a 
specific target ceil, for example, the vector is now target specific. Retroviral vectons can 

10 be made target specific by attaching, for example, a sugar, a glycolipid, or a protein. 
Prefen-ed targeting is accomplished by using an antibody to target the retroviral vector. 
Those of skill in the art will know o?, or can readily ascertain without undue experimenta- 
tion, specific polynucleotide sequences which can be inserted into the retroviral genome 
or attached to a viral envelope to allow target specific delivery of the retroviral vector 

15 containing the GDF-11 antisense polynucleotide. 

Since recombinant retroviruses are defective, they require assistance in order to produce 
infectious vector particles. This assistance can be provided, for example, by using 
helper cell lines that contain plasmids encoding all of the structural genes of the 
retrovirus under the control of regulatory sequences within the LTR. These plasmids 

20 are missing a nucleotide sequence which enables the packaging mechanism to 
recognize an RNA transcript for encapsidation. Helper cell lines which have deletions 
of the packaging signal include, but are not limited to ^2, PA317 and PA12, for example. 
These cell lines produce empty virions, since no genome is packaged. If a retroviral 
vector is introduced into such cells in which the packaging signal is intact, but the 

25 stnjctural genes are replaced by other genes of interest, the vector can be packaged and 
vector virion produced. 

Alternatively, NIH 3T3 or other tissue culture cells can be directly transfected with 
plasmids encoding the retroviral stmctural genes gag, po/ and env, by conventional 



wo 96701845 



PCTAJS95/08543 



-23- 

calcium phosphate transfedion. These cells are then transfected with the vector plasmid 
containing the genes of interest. The resulting cells release the retroviral vector into the 
culture medium. 

Another targeted delivery system for GDF-11 antisense polynucleotides is a colloidal 

5 dispersion system. Colloidal dispersion systems include macromolecule complexes, 
nanocapsules, microspheres, beads, and lipid-based systems including oiNn-water 
emulsions, micelles, mixed micelles, and liposomes. The prefenred colloidal system of 
this invention is a liposome. Liposomes are artificial membrane vesicles which are useful 
as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles 

10 (LUV), which range in size from 0.2-4.0 pim can encapsulate a substantial percentage 
of an aqueous buffer containing large macromolecuies. RNA, ONA and intact virions can 
be encapsulated within the aqueous interior and be delivered to cells in a biologically 
active form (Fraley. et al„ Trends Biochem, Sc/.. 6:77, 1981). In addition to mammalian 
cells, liposomes have been used for delivery of polynucleotides in plant, yeast and 

1 5 bacterial cells. In order for a liposome to be an efficient gene transfer vehicle, the 
following characteristics should be present: (1) encapsulation of the genes of interest at 
high efficiency while not compromising their biological activity; <2) preferential and 
substantial binding to a target cell in comparison to non-target cells; (3) delivery of the 
aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) 

20 accurate and effective expression of genetic information (Mannino, et al., Biotechniques, 
6:682. 1988). 

The composition of the liposome is usually a combination of phospholipids, particularly 
high-phase-transition-temperature phospholipids, usually in combination with steroids, 
especially cholesterol. Other phospholipids or other lipids may also be used. The 
25 physical characteristics of liposomes depend on pH, ionic strength, and the presence of 
divalent cations. 



wo 96/01845 



PCTAJS95/08543 



-24. 

Examples of lipids useful in liposome production include phosphatidyl compounds, such 
as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine. phosphatidyletha- 
nolamine, sphingolipids, cerebrosides, and gangliosides. Particulariy useful are 
diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, 
5 particularly from 16-18 carbon atoms, and is saturated. Illustrative phospholipids include 
egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphos- 
phatidylchoiine. 

The targeting of liposomes can be classified based on anatomical and mechanistic 
factors. Anatomical classification is based on the level of selectivity, for example, organ- 
specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished 
based upon whether it is passive or active. Passive targeting utilizes the natural 
tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in 
organs which contain sinusoidal capillaries. Active targeting, on the other hand, involves 
alteration of the liposome by coupling the liposome to a specific ligand such as a 
monoclonal antibody, sugar, glycolipid. or protein, or by changing the composition or size 
of the liposome in order to achieve targeting to organs and cell types other than the 
naturally occurring sites of localization. 

The surface of the targeted delivery system may be modified in a variety of ways. In the 
case of a liposomal targeted delivery system, lipid groups can be incorporated into the 
20 lipid bilayer of the liposome in order to maintain the targeting ligand in stable association 
with the liposomal bilayer. Various linking groups can be used for joining the lipid chains 
to the targeting ligand. 

Due to the expression of GDF-11 in muscle, spleen, uterus, thymus, and neural tissue, 
there are a variety of applications using the polypeptide, polynucleotide, and antibodies 
25 of the invention, related to these tissues. Such applications include treatment of cell 
proliferative and immunologic disorders involving these and other tissues. In addition, 
GDF-1 1 may be useful in various gene therapy procedures. 



10 



15 



wo 96/01845 



PCT/US95y08543 



-25- 

The following examples are intended to illustrate but not limit the invention. While they 
are typical of those that might be used, other procedures known to those skilled in the 
art may alternatively be used. 



EXAMPLE 1 

5 IDENTIFICATION AND ISOLATION 

OF A NOVEL TGF-B FAMILY MEMBER 

To identify novel members of the TGF-p superfamily, a murine genomic library was 
screened at reduced stringency using a murine GDF-8 probe (FIGURE 8; nucleotides 
865-1234) spanning the region encoding the Otenninal portion of the GDF-8 precursor 

10 protein. Hybridization was carried out as described (Lee. Mol Endocrinol, 4:1034, 1990) 
at 65°C, and the final wash was camed out at the same temperature in a buffer 
containing 0.5 M NaCI. Among the hybridizing phage was one that could be 
distinguished from GDF-8-containing phage on the basis of its reduced hybridization 
intensity to the GDF-8 probe. Partial nucleotide sequence analysis of the genomic insert 

1 5 present in this weakly hybridizing phage showed that this clone contained a sequence 
highly related to but distinct from murine GDF-8. 

A partial nucleotide sequence of the genomic insert present in this phage is shown in 
FIGURE 1a. The sequence contained an open reading frame extending from 
nucleotides 198 to 575 that showed significant homology to the known members of the 
20 TGF-P superfamily (see below). Preceding this sequence was a 3' splice consensus 
sequence at precisely the same position as in the GDF-8 gene. This new TGF-P family 
member was given the designation GDF-11 (grov/th/differentiation factor-11). 



wo 96/01845 



PCTAJS95/08543 



-26- 

EXAMPLE 2 
EXPRESSION OF GDF-11 

To determine the expression pattern of GDF-11. RNA samples prepared from a variety 
of tissues were screened by Northem analysis. RNA isolation and Northem analysis 
5 were canied out as described previously (Lee, MoL Endocrinol., 4:1034, 1990) except 
that the hybridization was canied out in 5X SSPE, 10% dextran sulfate, 50% formamide. 
1% SDS, 200A^g/ml salmon DNA, and 0.1% each of bovine serum albumin, ficoll. and 
polyvinylpyn'olidone. Five micrograms of twice poly A-selected RNA prepared from each 
tissue (except for 2 day neonatal brain, for which only 3.3 ^9 RNA were used) were 

10 electrophoresed on formaldehyde gels, blotted, and probed with GDF-11. As shown in 
FIGURE 2, the GDF-11 probe detected two RNA species, approximately 4.2 and 3.2 kb 
in length, in adult thymus, brain, spleen, uterus, and muscle as well as in whole embryos 
isolated at day 12.5 or 18.5 and in brain samples taken at various stages of 
development. On longer exposures of these blots, lower levels of GDF-11 RNA could 

1 5 also be detected in a number of other tissues. 

EXAMPLE 3 

ISOLATION OF cDNA CLONES ENCODING GDF-11 

In order to isolate cDNA clones encoding GDF-11, a cDNA library was prepared in the 
lambda ZAP It vector (Stratagene) using RNA prepared from human adult spleen. From 

20 5 ug of tv^ce poly A-selected RNA prepared from human spleen, a cDNA library 
consisting of 21 million recombinant phage was constructed according to the instructions 
provided by Stratagene. The library was screened without amplification. Library 
screening and characterization of cDNA inserts were carried out as described previously 
(Lee, MoL Endocrinol., 4:1034, 1990). From this library, 23 hybridizing phage were 

25 obtained. 



wo 96/01845 



PCT/US95/08543 



-27- 

The entire nucleotide sequence of the clone extending furthest toward the 5* end of the 
gene was determined. The 1258 base pair sequence contained a single long open 
reading frame beginning from the 5' end of the clone and extending to a TAA stop codon. 
Because the open reading frame and the homology with GDF-8 (see below) extended 
5 to the very 5' end of the clone, it seemed likely that this clone was missing the coding 
sequence corresponding to the N-terminal portion of the GDF-1 1 precursor protein. In 
order to obtain the remaining portion of the GDF-11 sequence, several genomic clones 
were isolated by screening a human genomic library with the human GDF-1 1 cDNA 
probe. Partial sequence analysis of one of these genomic clones showed that this clone 

10 contained the GDF-11 gene. From this clone, the remaining GDF-11 coding sequence 
was obtained. FIGURE lb shows the predicted sequence of GDF-11 assembled from 
the genomic and cDNA sequences. Nucleotides 136 to 1393 represent the extent of the 
sequence obtained from a cDNA clone. Nucleotides 1 to 135 were obtained from a 
genomic clone. The sequence has been arbitrarily numbered beginning with a Sac II site 

15 present in the genomic clone, but the location of the mRNA start site is not known. The 
sequence contains a putative initiating methionine at nucleotide 54. Whether the 
sequence upstream of this methionine codon is all present in the mRNA is not known. 
Beginning with this methionine codon. the open reading frame extends for 407 amino 
adds." The sequence contains one potential N-linked glycosylation site at asparagine 94. 

20 The sequence contains a predicted RXXR proteolytic cleavage site at amino acids 295 
to 298. and cleavage of the precursor at this site would generate an active C-tenminal 
fragment 109 amino acids in length with a predicted molecular weight of approximately 
12,500 kD. In this region, the predicted murine and human GDF-11 amino acid 
sequences are 100% identical. The high degree of sequence conservation across 

25 species suggests that GDF-1 1 plays an important role in vivo. 

The C-terminal region following the predicted cleavage site contains all the hallmarks 
present in other TGF-P family members. GDF-1 1 contains most of the residues that are 
highly conserved in other family members, including the seven cysteine residues with 
their characteristic spacing. Like the TGF-p's, the inhibin P*s. and GDF-8. GDF-1 1 also 



wo 96/01845 



PCTAJS95/08543 



-28- 

contains two additional cysteine residues. In the case of TGF-p2. these additional 
cysteine residues are known to form an intramolecular disulfide bond (Daopin, et aL, 
Science, 257:369. 1992; Schlunegger and Grutter, Nature, 358:430. 1992). Ambulation 
of the amino acid sequence homologies between GDF-11 and the other TGF-P family 
5 members is shown in FIGURE 3. Numbers represent percent amino acid identities 
between each pair calculated from the first conserved cysteine to the Oterminus. Boxes 
represent homologies among highly-related members within particular subgroups, in this 
region, GDF-11 is most highly related to GDF-8 (92% sequence identity). 

An alignment of GDF-8 (SEQ ID NO:5) and GDF-11 (SEQ ID N0:6) amino acid 
10 sequences is shown in FIGURE 4. The two sequences contain potential N-linked 
glycosylation signals (NIS) and putative proteolytic processing sites (RSRR) at 
analogous positions. The two sequences are related not only in the C-terminal region 
following the putative cleavage site (90% amino acid sequence identity), but also in the 
pro-region of the molecules (45% amino acid sequence identity). 

15 EXAMPLE 4 

COMSTRUCTION OF A HYBRID GDF-8/GDF11 GENE 

In order to express GDF-11 protein, a hybrid gene was constructed in which the N- 
temiinal region of GDF-11 was replaced by the analogous region of GDF-8. Such hybrid 
constajcts have been used to produce biologically-active BMP-4 (Hammonds, et ai, MoL 

20 Endocrinol., 5:149, 1991) and Vg-1 (Thomsen and Melton, Cell, 74:433, 1993). In order 
to ensure that the GDF-11 protein produced from the hybrid construct would represent 
authentic GDF-11, the hybrid gene was constructed in such a manner that the fusion of 
the two gene fragments would occur precisely at the predicted deavage sites. In 
particular, an >*vall restriction site is present in both sequences at the location 

25 corresponding to the predicted proteolytic cleavage site. The N-terminal pro-region of 
GDF-8 up to this >Avall site was obtained by partial digestion of the clone with AvaW and 



wo 96/01845 



PCTAJS95/08543 



-29" 

fused to the C-terminal region of GDF-11 beginning at this AvaW site. The resulting 
hybrid constmct was then inserted into the pMSXND mammalian expression vector (Lee 
and Nathans, J. BioL Chem,, 263:3521) and transfected into Chinese hamster ovary 
cells. As shown in FIGURE 5, Western analysis of conditioned medium from G418- 
5 resistant cells using antibodies raised against the C-terminal portion of GDF-8 showed 
that these cells secreted GDF-11 protein into the medium and that at least some of the 
hybrid protein was proteolytically processed. Furthermore, these studies demonstrate 
that the antibodies directed against the C-terminal portion of GDF-8 will also react with 
GDF-11 protein. 



10 EXAMPLE 5 

CHROMOSOMAL LOCALIZATION OF GDF-11 

In order to map the chromosomal location of GDF-1 1 , DMA samples from human/rodent 
somatic cell hybrids (Drwinga, etaL, Genomics, 16:311-313, 1993; Dubois and Naylor. 
Genomics, 16:315-319, 1993) were analyzed by polymerase chain reaction followed by 

15 Southern blotting. Polymerase chain reaction was earned out using primer #101, 5- 
GAGTCCCGCTGCTGCCGATATCC-3\ (SEQ ID NO:7) and primer #102. 5- 
TAGAGCATGTTGATTGGGGACAT-3'. (SEQ ID NO:8) for 35 cycles at 94*^C for 2 
minutes, 58°C for 1 minutes, and 72X for 1 minute. These primers correspond to 
nucleotides 981 to 1003 and the reverse complement of nucleotides 1182 to 1204, 

20 respectively, in the human GDF-1 1 sequence. PCR products were electrophoresed on 
agarose gels, blotted, and probed with oligonucleotide #104, 5'- 
AAATATCCGCATACCCATTT-3'. (SEQ ID N0:9) which conresponds to a sequence 
internal to the region flanked by primer #101 and #102. Filters were hybridized in 6 X 
SSC, 1 X Denhardfs solution, 100 //g/ml yeast transfer RNA. and 0.05% sodium 

25 pyrophosphate at 50°C. 



wo 96/01845 



PCT/US95/08543 



-30- 

As shown in FIGURE 6, the human-specific probe detected a band of the predicted size 
(approximately 224 base pairs) in the positive control sample (total human genomic 
DNA) and in a single DNA sample from the human/rodent hybrid panel. This positive 
signal corresponds to human chromosome 12. The human chromosome contained in 
5 each of the hybrid cell lines is identified at the top of each of the first 24 lanes (1-22, X. 
and Y). In the lanes designated CHO. M, and H, the starling DNA template was total 
genomic DNA from hamster, mouse, and human sources, respectively. In the lane 
marked B1, no template DNA was used. Numbers at left indicate the mobilities of DNA 
standards. These data show that the human GDF-11 gene is located on chromosome 
10 12. 

In order to detemiine the more precise location of GDF-11 on chromosome 12, the GDF- 
11 gene was localized by florescence in situ hybridization (FISH). These FISH 
localization studies were carried out by contract to BIOS laboratories (New Haven, 
Connecticut). Purified DNA from a human GDF-11 genomic clone was labelled with 

15 digoxigenin dUTP by nick translation. Labelled probe was combined with sheared 
human DNA and hybridized to nonnal metaphase chromosomes derived from PHA 
stimulated peripheral blood lymphocytes in a solution containing 50% formamide. 10% 
dextran sulfate and 2xSSC. Specific hybridization signals were detected by incubating 
the hybridized slides in fluorescein-conjugated sheep antidigoxigenin antibodies. Slides 

20 were then counterstained with propidium iodide and analyzed. As shown in FIGURE 7a, 
this experiment resulted in the specific labelling of the proximal long arm of a group C 
chromosome, the size and morphology of which were consistent with chromosome 12. 
In order to confirm the identity of the specifically labelled chronriosome, a second 
experiment was conducted in which a chromosome 12- specific centromere probe was 

25 cohybridized with GDF-11. As shown in FIGURE 7b. this experiment clearly 
demonstrated that GDF-11 is located at a position which is 23% of the distance from the 
centromere to the telomere of the long arm of chromosome 12, an area which 
corresponds to band 12q13 (FIGURE 7c). A total of 85 metaphase cells were analyzed 
and 80 exhibited specific labelling. 



wo 96/01845 



PCTAJS95/08543 



-31- 

Although the invention has been described with reference to the presently preferred 
embodiment, It should be understood that various modifications can be made without 
departing from the spirit of the invention. Accordingly, the invention is limited only by 
the following claims. 



wo 96/01845 



PCTAJS95/08543 



-32- 

SEQX7EMCE LISTING 



(1) GENERAL INFORMATION: 

(i) APPLICANT: The Johns Hopkins University School of Medicine 
(ii) TITLE OF INVENTION: GROWTH DIFFERENTIATION FACTOR-11 
(iii) NUMBER OF SEQUENCES: 9 

(iv) CORRESPONDENCE ADDRESS: 

(A) ADDRESSEE: Fish & Richardson P.C. 

(B) STREET: 4225 Executive Square, Suite 1400 

(C) CITY: La Jolla 

(D) STATE: California 

(E) COUNTRY: US 

(F) ZIP: 92037 

(V) COMPUTER READABLE FORM: 

(A) MEDIUM TYPE: Floppy disk 

(B) COMPUTER: IBM PC compatible 

(C) OPERATING SYSTEM: PC-DOS /MS -DOS 

(D) SOFTWARE: Patentin Release #1.0, Version #1.25 

(vi) CURRENT APPLICATION DATA: 

(A) APPLICATION NUMBER: PCT/US95/ 

(B) FILING DATE: 07-JUL-1995 

(C) CLASSIFICATION: 

(viii) ATTORNEY /AGENT INFORMATION: 

(A) NAME: HAILE, PH.D., LISA A. 

(B) REGISTRATION NUMBER: 38,347 

(C) REFERENCE/DOCKET NUMBER: 07265/036WO1 

(ix) TELECOMMUNICATION INFORMATION: 

(A) TELEPHONE: 619/678-5070 

(B) TELEFAX: 619/678-5099 



wo 96/01845 



PCTAJS95/08543 



-33- 



(2) INFORMATION FOR SEQ ID N0:1: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1393 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 



(vii) IMMEDIATE SOURCE: 

(B) CLONE: HUMAN GDF-11 

10 (ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 54.. 1274 



(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1: 
CCGCGGGACT CCGGCGTCCC CGCCCCCCAG TCCTCCCTCC CCTCCCCTCC AGO ATG 56 

15 Met 

1 

GTG CTC GCG GCC CCG CTG CTG CTG GGC TTC CTG CTC CTC GCC CTG GAG 104 
Val Leu Ala Ala Pro Leu Leu Leu Gly Phe Leu Leu Leu Ala Leu Glu 
5 10 15 

20 CTG CGG CCC CGG GGG GAG GCG GCC GAG GGC CCC GCG GCG -GCG GCG GCG 152 

Leu Arg Pro Arg Gly Glu Ala Ala Glu Gly Pro Ala Ala Ala Ala Ala 
20 25 30 

GCG GCG GCG GCG GCG GCA GCG GCG GGG GTC GGG GGG GAG GGC TCC AGC 200 
Ala Ala Ala Ala Ala Ala Ala Ala Gly Val Gly Gly Glu Arg Ser Ser 
25 35 40 45 

CGG CCA GCC CCG TCC GTG GCG CCC GAG CCG GAC GGC TGC CCC GTG TGC 248 
Arg Pro Ala Pro Ser Val Ala Pro Glu Pro Asp Gly Cys Pre Val Cys 
50 55 60 65 

GTT TGG CGG CAG CAC AGC CGC GAG -CTG CGC CTA <;AG AGC ATC AAG TCG 296 
30 Val Trp Arg Gin His Ser Arg Glu Leu Arg Leu Glu Ser lie Lys Ser 

70 75 80 



wo 96/01845 



PCT/US95/08543 



-34- 



CAG ATC TTG AGC AAA CTG CGG CTC AAG GAG GCG CCC AAC ATC AGC CGC 344 
Gin lie Leu Ser Lys Leu Arg Leu Lys Glu Ala Pro Asn lie Ser Arg 
85 90 95 

GAG GTG GTG AAG CAG CTG CTG CCC AAG GCG CCG CCG CTG CAG CAG ATC 392 
5 Glu Val Val Lys Gin Leu Leu Pro Lys Ala Pro Pro Leu Gin Gin lie 

100 105 110 

CTG GAC CTA CAC GAC TTC CAG GGC GAC GCG CTG CAG CCC GAG GAC TTC 440 
Leu Asp Leu His Asp Phe Gin Gly Asp Ala Leu Gin Pro Glu Asp Phe 
115 120 125 

10 CTG GAG GAG GAC GAG TAC CAC GCC ACC ACC GAG ACC GTC ATT AGC ATG 488 

Leu Glu Glu Asp Glu Tyr His Ala Thr Thr Glu Thr Val lie Ser Met 
13C 135 140 145 

GCC CAG GAG ACG GAC CCA GCA GTA CAG ACA GAT GGC AGC CCT CTC TGC 536 
Ala Gin Glu Thr Asp Pro Ala Val Gin Thr Asp Gly Ser Pro Leu Cys 
15 150 155 160 

TGC CAT TTT CAC TTC AGC CCC AAG GTG ATG TTC ACA AAG GTA CTG AAG 584 
Cys His Phe His Phe Ser Pro Lys Val Met Phe Thr Lys Val Leu Lys 
165 170 175 

GCC CAG CTG TGG GTG TAC CTA CGG CCT GTA CCC CGC CCA GCC ACA GTC 632 
20 Ala Gin Leu Trp Val Tyr Leu Arg Pro Val Pro Arg Pro Ala Thr Val 
ISO 185 190 

TAC CTG CAG ATC TTG CGA CTA AAA CCC CTA ACT GGG GAA ^G ACC GCA 680 
Tyr Leu Gin lie Leu Arg Leu Lys Pro Leu Thr Gly Glu Gly Thr Ala 
195 200 205 

25 GGG GGA GGG GGC GGA GGC CGG CGT CAC ATC CGT ATC CGC TCA CTG AAG 728 

Gly Gly Gly Gly Gly Gly Arg Arg His He Arg He Arg Ser Leu Lys 
210 215 220 225 

AT7 GAG CTG CAC TCA CGC TCA GGC CAT TGG CAG AGC ATC GAC TTC AAG 776 
He Glu Leu His Ser Arg Ser Gly His Trp <31n Ser He Asp Phe Lys 
30 230 235 240 



CA-. GTG CTA CAC AGC TGG TTC CGC CAG CCA CAG AGC AAC TGG GGC ATC 
Gir. Val Leu His Ser Trp Phe Arg Gin Pro -Gin Ser Asn Trp Gly lie 
245 250 255 



824 



wo 96/01845 



PCTAJS95/08543 



.35- 



GAG ATC AAC GCC TTT GAT CCC AGT GGC ACA GAC CTG GCT GTC ACC TCC 872 
Glu He Asn Ala Phe Asp Pro Ser Gly Thr Asp Leu Ala Val Thr Ser 
260 265 270 

CTG GGG CCG GGA GCC GAG GGG CTG CAT CCA TTC ATG GAG CTT CGA GTC 920 
5 Leu Gly Pro Gly Ala Glu Gly Leu His Pro Phe Met Glu Leu Arg Val 
275 280 285 

CTA GAG AAC ACA AAA CGT TCC CGG CGG AAC CTG GGT CTG GAC TGC GAC 968 
Leu Glu Asn Thr Lys Arg Ser Arg Arg Asn Leu Gly Leu Asp Cys Asp 
290 295 300 305 

10 GAG CAC TCA AGC GAG TCC CGC TGC TGC CGA TAT CCC CTC ACA GTG GAC 1016 

Glu His Ser Ser Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val Asp 
310 315 320 

TTT GAG GCT TTC GGC TGG GAC TGG ATC ATC GCA CCT AAG CGC TAC AAG 1064 
Phe Glu Ala Phe Gly Trp Asp Trp He He Ala Pro Lys Arg Tyr Lys 
15 325 330 335 

GCC AAC TAC TGC TCC GGC CAG TGC GAG TAC ATG TTC ATG CAA AAA TAT 1112 
Ala Asn Tyr Cys Ser Gly Gin Cys Glu Tyr Met Phe Met Gin Lys Tyr 
340 345 350 

CCG CAT ACC CAT TTG GTG CAG CAG GCC AAT CCA AGA GGC TCT GCT GGG 1160 
20 Pro His Thr His Leu Val Gin Gin Ala Asn Pro Arg Gly Ser Ala Gly 

355 360 365 

CCC TGT TGT ACC CCC ACC AAG ATG TCC CCA ATC AAC ATG CTC TAC TTC 1208 
Pro Cys Cys Thr Pro Thr Lys Met Ser Pro He Asn Met Leu Tyr Phe 
370 375 380 . 385 

25 AAT GAC AAG CAG CAG ATT ATC TAC GGC AAG ATC CCT GGC ATG GTG GT<S 1256 

Asn Asp Lys Gin Gin He He Tyr Gly Lys He Pro Gly Met Val Val 
390 395 400 

GAT CGC TGT GGC TGC TCT TAAGTGGGTC ACTACAAGCT GCTGGAGCAA 1304 
Asp Arg Cys Gly Cys Ser 
30 405 

AGACTTGGTG GGTGGGTAAC TTAACCTCTT CACAGAGGAT AAAAAAT<3CT T-GTGAGTATG 1364 



ACAGAAGGGA ATAAACAGGC TTAAAGGGT 



1393 



wo 96/01845 



PCT/US95/08543 



-36- 



(2) INFORMATION FOR SEQ ID NO: 2: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 407 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 

Met Val Leu Ala Ala Pro Leu Leu Leu Gly Phe Leu Leu Leu Ala Leu 
1 5 10 15 

Glu Leu Arg Pro Arg Gly Glu Ala Ala Glu Gly Pro Ala Ala Ala Ala 
20 25 30 

Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Val Gly Gly Glu Arg Ser 
35 40 45 

Ser Arg Pro Ala Pro Ser Val Ala Pro Glu Pro Asp Gly Cys Pro Val 
50 55 60 

Cys Val Trp Arg Gin His Ser Arg Glu Leu Arg Leu Glu Ser lie Lys 
€5 70 75 80 

Ser Gin He Leu Ser Lys Leu Arg Leu Lys Glu Ala Pro Asn He Ser 
85 90 95 

Arg Glu Val Val Lys Gin Leu Leu Pro Lys Ala Pro Pro Leu Gin -Gin 
100 105 110 

He Leu Asp Leu His Asp Phe Gin Gly Asp Ala Leu Gin Pro Glu Asp 
115 120 125 

Phe Leu Glu Glu Asp Glu Tyr His Ala Thr Thr Glu Thr Val He Ser 
130 135 140 

Met Ala Gin Glu Thr Asp Pro Ala Val Gin Thr Asp Gly Ser Pro Leu 
145 150 155 160 

Cys Cys His Phe His Phe Ser Pro Lys Val Met Phe Thr Lys Val Leu 
165 170 175 



Lys Ala Gin Leu Trp Val Tyr Leu Arg Pro Val Pro Arg Pro Ala Thr 



wo 96/01845 



PCT/US95/08543 



-37. 



180 185 190 

Val Tyr Leu Gin lie Leu Arg Leu Lys Pro Leu Thr Gly Glu Gly Thr 
195 200 205 

Ala Gly Gly Gly Gly Gly Gly Arg Arg His lie Arg He Arg Ser Leu 
210 215 220 

Lys He Glu Leu His Ser Arg Ser Gly His Trp Gin Ser He Asp Phe 
225 230 235 240 

Lys Gin Val Leu His Ser Trp Phe Arg Gin Pro Gin Ser Asn Trp Gly 
245 250 255 

He Glu He Asn Ala Phe Asp Pro Ser Gly Thr Asp Leu Ala Val Thr 
260 265 270 

Ser Leu Gly Pro Gly, Ala Glu Gly Leu His Pro Phe Met Glu Leu Arg 
275 280 285 

Val Leu Glu Asn Thr Lys Arg Ser Arg Arg Asn Leu Gly Leu Asp Cys 
290 295 300 

Asp Glu His Ser Ser Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val 
305 310 315 320 

Asp Phe Glu Ala Phe Gly Trp Asp Trp He He Ala Pro Lys Arg Tyr 
325 330 335 

Lys Ala Asn Tyr Cys Ser Gly Gin Cys Glu Tyr Met Phe Met Gin Lys 
340 345 350 

Tyr Pro His Thr His Leu Val Gin Gin Ala Asn Pro Arg Gly Ser Ala 
355 360 365 

Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro He Asn Met Leu Tyr 
370 375 380 

Phe Asn Asp Lys Gin Gin He He Tyr Gly Lys He Pro Gly Met Val 
385 390 395 400 



Val Asp Arg Cys Gly Cys Ser 
405 



wo 96/01845 



PCTAJS95/08543 



-38" 



(2) INFORMATION FOR SEQ ID NO: 3: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 630 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 



(vii) IMMEDIATE SOURCE: 

(B) CLONE: MOUSE GDF-11 

10 (ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 198. .575 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: 

TCTAGATGTC AAGAGAAGTG GTCACAATGT CTGGGTGGGA GCCGTAAACA AGCCAAGAGG 60 

15 TTATGGTTTC TGGTCTGATG CTCCTGTTGA GATCAGGAAA TGTTCAGGAA ATCCCCTGTT 120 

GAGATGTAGG AAAGTAAGAG GTAAGAGACA TTGTTGAGGG TCATGTCACA TCTCTTTCCC 180 

CTCTCCCTGA CCCTCAG CAT CCT TTC ATG GAG CTT CGA GTC CTA GAG AAC 230 
His Pro Phe Met Glu Leu Arg Val Leu Glu Asn 
1 5 10 

20 ACG AAA AGG TCC CGG CGG AAC CTA GGC CTG GAC TGC GAT GAA CAC TCG 278 

Thr Lys Arg Ser Arg Arg Asn Leu Gly Leu Asp Cys Asp <31u His Ser 
15 20 25 

AGT GAG TCC CGC TGC TGC CGA TAT CCT CTC ACA GTG GAC TTT GAG GCT 326 
Ser Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala 
25 30 35 40 

TTT GGC TGG GAC TGG ATC ATC GCA CCT AAG CGC TAC AAG <3CC AAC TAC 374 
Phe Gly Trp Asp Trp lie lie Ala Pro Lys Arg Tyr Lys Ala Asn Tyr 
45 50 55 



30 



TGC TCC GGC CAG TGC GAA TAC ATG TTC ATG CAA AAG TAT CCA CAC ACC 
Cys Ser Gly Gin Cys Glu Tyr Met Phe Met Gin Lys Tyr Pro His Thr 



422 



wo 96/01845 



PCrAJS95/08543 



-39- 

60 65 70 75 

CAC TTG GTG CAA CAG GCC AAC CCA AGA GGC TCT GCT GGG CCC TGC TGC 
His- Leu Val Gin Gin Ala Asn Pro Arg Gly Ser Ala Gly Pro Cys Qys 
80 85 90 

ACC CCT ACC AAG ATG TCC CCA ATC AAC ATG CTC TAC TTC AAT GAC AAG 
Thr Pro Thr Lys Met Ser Pro lie Asn Met Leu Tyr Phe Asn Asp Lys 
95 100 105 

CAG CAG ATT ATC TAC GGC AAG ATC CCT GGC ATG GTG GTG GAT CGA TGT 
Gin Gin He He Tyr Gly Lys He Pro Gly Met Val Val Asp Arg Cys 
110 115 120 

GGC TGC TCC TAAGTTGTGG GCTACAGTGG ATGCCTCCCT CAGACCCTAC 
Gly Cys Ser 
125 

CCCAAGAACC CCAGC 



(2) INFORMATION FOR SEQ ID NO: 4: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 126 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 

His Pro Phe Met Glu Leu Arg Val Leu Glu Asn Thr Lys Arg Ser Arg 
1 5 10 15 

Arg Asn Leu Gly Leu Asp Cys Asp Glu His Ser Ser Glu Ser Arg Cys 
20 25 30 

Cys Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp 
35 40 45 



He He Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Gin Cys 
50 55 60 



wo 96/01845 



PCTAJS95/08543 



-40- 



Glu Tyr Met Phe Met Gin Lys Tyr Pro His Thr His Leu Val Gin Gin 
65 70 75 80 

Ala Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys Met 
85 90 95 

Ser Pro lie Asn Met Leu Tyr Phe Asn Asp Lys Gin Gin lie He Tyr 
100 105 110 

Gly Lys He Pro Gly Met val Val Asp Arg Cys Gly Cys Ser 
115 120 125 

(2) INFORMATION FOR SEQ ID NO: 5: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 375 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



(vii) IMMEDIATE SOURCE: 
(B) CLONE: GDF-8 

ax) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..375 



(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: 

Met Gin Lys Leu Gin Leu Cys Val Tyr He Tyr Leu Phe Met Leu He 
15 10 15 

Val Ala Gly Pro Val Asp Leu Asn Glu Asn Ser -Glu Gin Lys Glu Asn 
20 25 30 

Val Glu Lys Glu Gly Leu Cys Asn Ala Cys Thr Trp Arg -Gin Asn Thr 
35 40 45 



Lys Ser Ser Arg He Glu Ala He Lys He -Gin He Leu Ser Lys Leu 
50 55 60 



wo 96/01845 



PCTAJS95/08543 



-41- 



Arg Leu Glu Thr Ala Pro Asn lie Ser Lys Asp Val lie Arg Gin Leu 
65 70 75 80 

Leu Pro Lys Ala Pro Pro Leu Arg Glu Leu lie Asp Gin Tyr .Asp Val 
85 90 95 

Gin Arg Asp Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr His 
100 105 110 

Ala Thr Thr Glu Thr lie He Thr Met Pro Thr Glu Ser Asp Phe Leu 
115 120 125 

Met Gin Val Asp Gly Lys Pro Lys cys Cys Phe Phe Lys Phe Ser Ser 
130 - 135 140 

Lys He Gin Tyr Asn Lys Val Val Lys Ala Gin Leu Trp He Tyr Leu 
145 150 155 160 

Arg Pro Val Glu Thr Pro Thr Thr Val Phe Val Gin He Leu Arg Leu 
165 170 175 

He Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly He Arg Ser Leu 
180 185 190 

Lys Leu Asp Met Asn Pro Gly Thr Gly He Trp Gin Ser He Asp Val 
195 200 205 

Lys Thr Val Leu Gin Asn Trp Leu Lys Gin Pro Glu Ser Asn Leu Gly 
210 215 220 

He Glu He Lys Ala Leu Asp Glu Asn Gly His Asp Leu Ala Val Thr 
225 230 235 240 

Phe Pro Gly Pro Gly Glu Asp Gly Leu Asn Pro Phe l^eu -Glu Val Lys 
245 250 255 

Val Thr Asp Thr Pro Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp Cys 
260 265 270 

Asp Glu His Ser Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val 
275 280 285 



Asp Phe Glu Ala Phe Gly Trp Asp Trp He He Ala Pro Lys Arg Tyr 
290 295 300 



wo 96/01845 



PCTAJS95/08543 



-42- 



Lys Ala Asn Tyr Cys Ser Gly Glu 
305 310 

Tyr Pro His Thr His Leu Val His 
325 

Gly Pro Cys Cys Thr Pro Thr Lys 
340 

Phe Asn Gly Lys Glu Gin lie He 
355 360 

Val Asp Arg Cys Gly Cys Ser 
370 375 



Cys Glu Phe Val Phe Leu Gin Lys 
315 320 

Gin Ala Asn Pro Arg Gly jSer Ala 
330 335 

Met Ser Pro He Asn Met Leu Tyr 
345 350 

Tyr Gly Lys He Pro Ala Met Val 
365 



(2) INFORMATION FOR SEQ ID NO: 6: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 407 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: protein 



(via) IMMEDIATE SOURCE: 
(B) CLONE: GDF-11 

(ix) FEATURE: 

(A) .NAME/KEY: Protein 

(B) LOCATION: 1..407 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 

Met Val Leu Ala Ala Pro Leu Leu Leu Gly Phe Leu Leu Leu Ala Leu 
1 5 10 15 

Glu Leu Arg Pro Arg Gly Glu Ala Ala Glu Gly Pro Ala /Via Ala Ala 
20 25 30 



Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Val Gly Gly Glu Arg Ser 
35 40 45 



wo 96/01845 



PCTAJS95/08543 



-43- 



Ser Arg Pro Ala Pro Ser Val Ala Pro Glu Pro Asp Gly Cys Pro Val 
50 55 60 

Cys Val Trp Arg Gin His Ser Arg Glu Leu Arg Leu Glu Ser Jle Lys 
65 70 75 80 

ser Gin lie Leu Ser Lys Leu Arg Leu Lys Glu Ala Pro Asn lie Ser 
85 90 95 

Arg Glu Val Val Lys Gin Leu Leu Pro Lys Ala Pro Pro Leu Gin Gin 
100 105 110 

He Leu Asp Leu His Asp Phe Gin Gly Asp Ala Leu Gin Pro <31u Asp 
115 120 125 

Phe Leu Glu Glu Asp Glu Tyr His Ala Thr Thr Glu Thr Val He Ser 
130 135 140 

Met Ala Gin Glu Thr Asp Pro Ala Val Gin Thr Asp Gly Ser Pro Leu 
145 150 155 160 

Cys Cys His Phe His Phe Ser Pro Lys Val Met Phe Thr Lys Val Leu 
165 170 175 



Lys Ala Gin Leu Trp Val Tyr Leu 
180 

Val Tyr Leu Gin He Leu Arg Leu 
195 200 

Ala Gly Gly Gly Gly Gly Gly Arg 
210 215 

Lys He Glu Leu His Ser Arg Ser 
225 230 

Lys Gin Val Leu His Ser Trp Phe 
245 

He Glu He Asn Ala Phe Asp Pro 
260 



Arg Pro Val Pro Arg Pro Ala Thr 
185 190 

Lys Pro Leu Thr Gly Glu Gly Thr 
205 

Arg His He Arg He Arg Ser Leu 
220 

Gly His Trp Gin Ser He Asp Phe 
235 240 

Arg Gin Pro Gin Ser Asn Trp Gly 
250 255 

Ser Gly Thr Asp Leu Ala Val Thr 
265 270 



Ser Leu Gly Pro Gly Ala Glu Gly Leu His Pro Phe Met <;lu Leu Arg 
275 280 285 



wo 96/01845 



PCT/US95/08543 



-44- 



Val Leu Glu Asn Thr Lys Arg Ser Arg Arg Asn Leu Gly Leu Asp Cys 
290 295 300 

Asp Glu His Ser Ser Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val 
305 310 315 320 

Asp Phe Glu Ala Phe Gly Trp Asp Trp lie lie Ala Pro Lys Arg Tyr 
325 330 335 



Lys Ala Asn Tyr Cys Ser Gly Gin Cys Glu Tyr Met Phe Met Gin Lys 
340 345 350 

Tyr Pro His Thr His Leu Val Gin Gin Ala Asn Pro Arg Gly Ser Ala 
355 360 365 

Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro lie Asn Met Leu Tyr 
370 375 380 

Phe Asn Asp Lys Gin Gin He He Tyr Gly Lys He Pro Gly Met Val 
385 390 395 400 

Val Asp Arg Cys Gly Cys Ser 
405 

(2) INFORMATION FOR SEQ ID NO: 7: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 23 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 



(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 1. .23 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: 



GAGTCCCGCT GCTGCCGATA TCC 



wo 96/01845 



PCTAJS9S/08543 



-45- 



(2) INFORMATION FOR SEQ ID NO: 8: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 23 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 



(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 1..23 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: 
TAGAGCATGT TGATTGGGGA CAT 
(2) INFORMATION FOR SEQ ID NO: 9: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 20 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 



(ix) FEATURE: 

(A) N?iME/KEY: CDS 

(B) LOCATION: 1..20 

. (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: 
AAATATCCGC ATACCCATTT 



wo 96/01845 



PCT/US95/08543 



-46- 



CLAIMS 

1 . Substantially pure growth differentiation factor-1 1 (GDF-11). 

2. An isolated polynucleotide sequence encoding the GDF-11 polypeptide of claim 
1. 

3. The polynucleotide of claim 2, wherein the GDF-11 nucleotide sequence is 
selected from the group consisting of: 

a. SEQ ID NO:1 , wherein T can also be U; 

b. SEQ ID NO:3. wherein T can also be U; 

5 c. nucleic acid sequences complementary to SEQ ID NO:1; 

d. nucleic acid sequences complementary to SEQ ID NO:3; 

e. fragments of a. or c. that are at least 15 bases in length and that will 
selectively hybridize to DNA which encodes the GDF-11 protein of SEQ 
ID NO:2; and 

10 f. fragments of b. or d. that are at least 15 bases in length and that will 

selectively hybridize to DNA which encodes the GOF-1 1 protein of SEQ 
ID NO:4. 



4. The polynucleotide sequence of claim 2, wherein the polynucleotide is isolated 
from a mammalian cell. 



5. The polynucleotide of claim 4, wherein the mammalian cell is selected from the 
group consisting of mouse, rat» and human cell. 

6. An expression vector including the polynucleotide of claim 2. 

7. The vector of claim 6, wherein the vector is a plasmid. 



wo 96/01845 PCr/US95/08543 

-47- 

8. The vector of claim 6, wherein the vector is a virus. 

9. A host cell stably transformed with the vector of claim 6. 

10. The host cell of claim 9. wherein the cell is prokaryotic. 

1 1 . The host cell of claim 9. wherein the cell is eukaryotic. 

12. Antibodies that bind to the polypeptide of claim 1 or fragments thereof. 

13. The antibodies of claim 12, wherein the antibodies are polyclonal. 

14. The antibodies of claim 12, wherein the antibodies are monoclonal. 

15. A method of detecting a cell proliferative disorder comprising contacting the 
antibody of daim 12 with a specimen of a subject suspected of having a GDF-1 1 
associated disorder and detecting binding of the antibody. 

16. The method of claim 15, wherein the cell is a muscle cell. 

17. The method of claim 15. wherein the detecting is in vivo, 

18. The method of claim 17, wherein the antibody is detectably labeled. 

19. The method of claim 18. wherein the detectable label is selected from the group 
consisting of a radioisotope, a fluorescent compound, a bioluminescent 
compound and a chemlluminescent compound. 

20. The method of claim 15, v^rherein the detection is in vitro. 



wo 96/01845 PCT/US95/08543 

-48- 

21 . The method of claim 20, wherein the antibody is detectably labeled. 

22. The method of claim 21. wherein the label is selected from the group .consisting 
of a radioisotope, a fluorescent compound, a bioluminescent compound, a 
chemoluminescent compound and an enzyme. 

23. A method of treating a cell proliferative disorder associated with expression of 
GDF-11, comprising contacting the cells with a reagent which suppresses the 
GDF-11 activity. 

24. The method of claim 23, wherein the reagent is an anti-GOF-1 1 antibody. 

25. The method of claim 23, wherein the reagent is a GDF-11 antisense 
sequence. 

26. The method of claim 23, wherein the cell is a muscle cell. 

27. The method of claim 23, wherein the reagent which suppresses GDF-1 1 activity 
is introduced to a cell using a vector. 

28. The method of claim 27, wherein the vector Is a colloidal dispersion system. 

29. The method of claim 28, wherein the colloidal dispersion system is a liposome. 

30. The method of claim 29, wherein the liposome Is essentially target specific. 

31. The method of claim 30, wherein the liposome is anatomically targeted. 

32. The method of claim 31, wherein the liposome is mechanistically targeted. 



wo 96/01845 PCT/US95/08543 

33. The method of claim 32. wherein the mechanistic targeting is passive. 

34. The method of claim 32. wherein the mechanistic targeting is active.. 

35. The method of claim 34. wherein the liposome is actively targeted by coupling 
with a moiety selected from the group consisting of a sugar, a glycoiipid. and a 
protein. 

36. The method of claim 35, wherein the protein moiety is an antibody. 

37. The method of claim 36, wherein the vector is a virus. 

38. The method of claim 37, wherein the virus is an RNA virus. 

39. The method of claim 38, wherein the RNA virus is a retrovirus. 

40. The method of claim 39, wherein the retrovirus is essentially target specific. 

41. The method of claim 40, wherein a moiety for target specificity is encoded by a 
polynucleotide inserted into the retroviral genome. 

42. The method of claim 40. wherein a moiety for target specificity is selected from 
the group consisting of a sugar, a glycoiipid, and a protein. 

43. The method of claim 42, wherein the protein is an antibody. 



wo 96/01845 PCT/US95/08S43 

1/13 



1 TCTAGATCTCAMAGAAGTGGTCACAATCnC^ 60 

" TTATSUiXUt.lWlXriXa,TG C TCCTUri^AGATCAGgAAATCrrTCA^ 120 

°^i°ATGTAGGAAAGTAAGAGGTAA(»C»CAlT^^ 180 

181 CTCTCCCTCACCCTCACCATCCTrTCATCGAGCTICGACTC 240 

HPPMBLRVLEN TK li^^ii 

241 CCCSCCMAACCTACGCCTtMACltXGATGAACACTCGAGTGAGTCC^ 300 

BMBgi NLGLDCDERSSESRCCRY 

301 ATCCTCTCACAC?IX»ACTTTW«XK:TriTCCC^^ 3gO 

P LTVDPBAPGWOW IIAPKRY 
361 ACAAGGCCAACTACTOrTCaXKrCACTeCGAATACATSTTCATarAAAACTATCC^ ' 420 

KANY CSGQ CEYMPMC KYPHT 
421 CCCACTTOGTOaiACACX^CCAACCCAAGAGGCTCTCCTaXXCCTGC^^ 480 

HLVQQANPRGSAGPCCTPTK 
481 AGATCrrCCCCAATCAACATGCTCTACTTCAATCAaU^XrAGCAGATTAT^ 540 

MSPINMLYPNDKQQIIYCKI 
541 TCCCTGGCATGGTGGTOSATCGATGlXXKrrocrccrAAGTI^^ 600 

PGMVVDRC GCS* 
601 TCCCTCAGACCCTACCCCAAGAACCCCAGC 630 



FIG. la 



wo 96/01845 



2/13 



PCTAJS95/08543 



1 CCGCGGGACTCCGGCGTCCCCCCCCCCCAGTCCTCCCTCCCCTCCCCrCCAGCATCGTC 60 

M V L 

61 TCGCGGCCCCGCTGCrocrcCXXriTCC^ 120 

AAPLLLGFLL LALE LRPRGE 
121 AGGCGGCCGAGGGCCCCGCGGCGGCGGCGGCGGCGGCGGCGGCOGCGGCAGCGGCGGGGG 180 

A AEGPAAAAAAAAAAAAAGV 
181 TCGGGGGGGAGCGCTCCAGCCGGCCAGCCCCGTCCGTGGCGCCCGAGCOXJACGGCT^ 240 

G GERS SR PA PSVAPEPDGC P 
241 CCGTGTGCGTTTGGCGGCAGCACAGCCGCGAGCTGCGCCTAGAGAGCATC^^ 300 

VCVWRQHSRELR LES IRSQ I 
301 T C T TOA GCAAACnGCGGCTCAAGGAGGCGCC CAACATCAG CaSCGAGGTG^ 360 

LSKLRLKEAP ]N I S| R E V V K Q L 
361 TGCTGCCCAAGGCGCCGCCGCTGCAGCAGATCCIXXJACCTACACGACTTCCAGG^^ 420 

LPRAPPLQQILDLHDFQGOA 

421 cgcto:agcccgaggacttcctggaggaggacgagtaccacgccaccaccgagaccgt^ 480 

LQPEDFLE EDEYHATTE TVI 
481 rrAGCATGGCCCAGGAGACGGACCCAGCAGTACAGACAGAlTOCAGCCCTCT^^ 540 

SMAQETDPAVQTDGSPL CCH 
541 ATTTICACTTCAGCCCCAAGGTGATGTTCACAAAGGTACTG AAGGCCCAGCTG^ 600 

PHFS PKVMFTKVLKAQ L WVY 
601 ACCTACGGCCTGTACCCOXrCCAGCCACAGTCTACCTGCAGATC^^ 660 

LRPVPRP ATVYLQILRLKPL 
661 TAACTGGGGAAGGGACCGCAGGGGGAGGGGGC^AGGCCGGCGTCACATCCGTATCCCCT 720 

TGEGTAG G GGGGRRHIR IRS 
721 CACTGAAGATTGAGCTGCACTCACGCTCAGGCCATT^ 780 

LRIELKSRSGHWQSIDFKQV 
781 TGCTACACAGCTGGTTCCGCCAGCCACAGAGCAACTGGGGCATOT 840 

LH5W FRQPQSNWGIEINAF0 
841 ATCCCAGTGGCACAGACCTGGCTGTCACCTCCCTGGGGCCGGGAGCGGAGGOGCT^^ 900 

PSGTDLAVTSLGPGAE GLHP 
901 CATTCATGGAGCTTCGAGTCCTAGAGAACACAAA ACGTTCCCGG 960 

FMELRVLENTK ^^S^I^Ell N L G L O 
961 ACTGCGACGAGCACTCAAGGGAGTCCCGCrGCTGCCGATATCCCCTC^ 1020 
CDEH5SES RCCRYPLTVDFE 
1021 AGGCTTTCGGCIXXXSACTGGATCATCGCACCTAAGCGCTAC^^ 1080 

A FGWDWI1APKRYKANYCS<; 
1081 GCCAGTCCGAGTACATGITCATGCAAAAATATCCGCATACCC ATTTCG 1140 

QCEYMFMQKY PHTH LVQQAN 
1141 ATCCAAGAGGCTCTGCTGGGCCCTGTTGTACCCCCACCAAGATC 1200 

PRGSAGPCCTPTKMSPINML 
1201 TCTACTTCAATGACAAGCAGCAGATTATCTACGGCAAGATCCCT^ 1260 

YFNDRQQIIYGKI PGMVVD R 
1261 GCTGIXXOTGCTCrrAAGTGGGTCACTACAAGCT^^ 1320 
C G C S ♦ 

1321 TAACTTAACCIXrrTCACAGAGGATAAAAAATGCTTXr^ 1380 
1381 AGGCTTAAAGGGT 1393 



FIG. 1b 



wo 96/01845 



3/13 



PCTAJS95/08543 




FIG. 2a 



wo 96/01845 



4/13 



PCTAJS95/08543 




wo 96/01845 



PCT/US95/08543 

5/13 



TGF-fi3 




o 
fn 


<n 


n 


00 

m 


00 

ro 


ro 




in 
d 


d 


d 
ro 


ro 


in 
ro 


r> 


00 

<o 


vo 
n 


00 in 
ro d 




fS 

n 


00 
CN 


«-i 
m 


m 


u> 
n 


in 

ro 


r* 
ro 


ro 


in 
d 


o 
ro 


d 
ro 


ro 


ro 


n 


00 

ro 


in 
ro 


in 
m 


ro 
d 


TGF-fil 


fi 
m 


so 

n 


vo 


fO 

ro 


in 
n 


vo 

fO 


ro 


•V 
ro 


d 


m 
d 


d 
ro 


in 
ro 


TP 

ro 


in 

fO 


T^ TS« 

ro ro 


O 00 

ro d 


GCNF 


00 


CM 


CM 


C4 
CM 


CM 


CM 


xo 


VO 
«H 


o 
d 


o\ 

•H 




ro 
d 


d 
d 


d 


d 
d 


ro 
d 


d 


00 
fH 


Nodal 


in 
n 






PI 


in 


in 


ro 


<*> 

ro 


o 
m 


O 




d 


o 
TP 


ro 
TP 


TP 


o 


TP 


d 
d 




in 


in 
d 








vo 


d 


d 






r* 


d 


d 


fH 


d 




00 


in 
d 




r- 


n 


CN| 


o 


m 




CO 


o 


o 


in u> 


d 




TP 


ro 


fO 


d 


TP 

d 


Inhibin- a 


ro 
cs 


o 

CI 


in 

CI 


TP 

CM 


CM 


VO 
CM 


lO 

d 


m 
d 


d 


o\ 
d 


a\ 
d 


d 
d 


d 
d 


in 
d 


TP 

d 


TT 

d 


so 00 
d ^ 


HIS 




o 
n 


d 
n 


r» 

d 


U> 
CM 


in 
d 


fO 


o\ 
d 


d 


tn 


o 
<n 


d 


d 


TP 

d 


d 


d 


1^ o 
d o 
... «-l 


OP-2 




in 


in 
in 


o 
in 


CM 

in 


m 
in 


o 


ro 


d 


o 




in 
in 


in 
in 










BMP-5 




in 
in 


o 
m 


CM 

in 


in 


CM 

in 


d 


O 


f-« 

ro 


d 


ro 


\o 


a\ 
in 












OP-1 




M 

m 


o 
in 


in 


ro 
in 


ro 
in 


d 


o 


o 
ro 


d 


d 


o 
\o 


00 

in 












Vgr-1 




in 
in 


in 


in 


fO 

in 


d 
in 


in 




«H 

ro 






H 

\o 


o 

VO 













BMP-4 
BMP-2 

BMP-3 
GDF-10 

GDF-9 
GDF-11 

GDF-8 
GDF-7 
GDF-6 
GDF-5 
GDF-3/Vgr-2 
GDF-2 
GDF-1 




d 
I 

u 



tHdninvor*00f-ia>fHrodTp 
I t I I I I I I t I I I I 

ooaooooooaacsz 



C5^ S 
III 

c c c 

•H «r4 •H 

fH in i3 X) i3 fH 

I tH I d -H •H <d ft« 

OlOuxaiM cc^PQ 

>0(Q0XMIHHZ0 



tH d ro 

I I I 

^4 



wo 96/01845 



6/13 



PCT/US95/08543 



1 MVnLJUVFLLIX:nXUa£IJlPRG£AAH;PAAAAAAAAAAAAAGVGGERSSR 50 

I I I I I 

1 MQKLQLCVYIYLmL IVAGFVDLNENSC 28 



51 



29 



101 



78 



151 



127 



PAPSVAPEFIX^ZFVCVWRQHSRELRLESIKSQILSKLRLKEAPNISREVV 

I I I I III I I II iiiiiiii nil! I 

QKENVEKE . GLCNACTVRQm^SRIEAIRIQILSKLRUETAPNISKDVI 

KQLLPKAPPLQQILDLHDFQGDALQFEDFLEEDEYHATTETVISMAQETD 
lllllllll I I I I II I lllllll I I I i 

RQLLPKAPPLRELIDQYDVQRDD . SSDGSLEDDDYHATTETirmPTESD 



100 



77 



ISO 



126 



200 



PAVQTIX3SPIXCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRL 

I II lllllll II iiiii iiiii I It Hill 

FLMQVIXSKPKCCFPTCT'SSKIQYinCVVKAQLVaYIJlPVETPTTVFVQILRL 176 



201 RFLTCEGTAGCXSCXXSRRfilRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQ 250 

II I IIIII I Hill I H I I 

177 IKPMKD3T RYTGIRSUOJMNPGTGIWQSIDVKTVLQNWLKQ 218 



251 PQSNWGIEINAFDP5GTDLAVTSLGPGAEGLHPFMELRVLENTK RSRB »1L 

I 11 II II I I I I I 1 I I Hi Hill I IIIII 
219 PESNLGIEIKALDENGHDIAVTFPGPGEDGLNPFLEVKVTDTPF EISRP DF 



301 GLE I DEHSSESF Sl RYPLTVDFEAFGWDWIIAPKRYKAIW t SGC 



III! 



IHI HI 



II 1 II 1 1 1 1 II I II II II H H H H ] 



269 CLE S DEHSraSF SE RYPLTVDFEAFGWDWIIAPKRYKANY ? 3GE 



I I 




300 
268 
350 
318 



351 QKYPHTHLVQQANPRGSAGEiErPTKMSPINMLYFNDKQQIiyGKlPGMV 400 



ilHIIIil I IIIII II Hit 



IIHHHHHH I IIIIIIII H 



319 QKYPHTHLVHQANPRGSAGI^rPTKMSPINMLYFNGKEQIIYGKIPAMV 368 



401 vd: 
I I I 



407 

369 vD fjaaa s 375 



FIG. 4 



wo 96/01845 



7/13 



PCTAJS95/08543 




FIG. 5 



wo 96/01845 



8/13 



PCT/US95/08543 



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1920 21 22 X YCHOM H B1 



1018- 
506/517 - 




FIG. 6 



wo 96/01845 



9/13 



PCrAJS95/08543 




FIG. 7a 



SUD31 ilUTE. SHcE : (RjLi:2v; 



wo 96/01845 



10/13 



PCTAJS9S/08543 




FIG. 7b 



w L w j : 



2B! 



wo 96/01845 



11/13 



PCTAJS9S/08543 




12 

FIG. 7c 



wo 96/0184S 



12/13 



PCTAJS95/08S43 



GTCTCTCGGACGGTACATGCACTAATATTTCACTTGGCATTACTCAAAAGCAAAAAGAAG 60 
61 AAATAAGAACAACaAAAAAAAAAGATTGTGCTGATTTTTAAAATGATGCAAAAACTGCA 120 

M M Q K L Q' 

121 AATGTATGTTTATATTTACCTGTTCATGCTGATTGCTGCTGGCCCAGTGGATCTAAATGA 180 
MYVYIYLFMLIAAGPVOLNE 

181 GGGCAGTGAGAGAGAAGAAAATGTGGAAAAAGAGGGGCTGTGTAATGCATGTGCCTGGAG 240 
G S £ R E £ N V E K E G L C N A C A W R 

241 ACAAAACACGAGGTACTCCAGAATAGAAGCaTAAAAATTCAAATCCTCAGTAAGCTGCG 300 
QNTRYSRIEAIKIQILSKLR 

301 CCTGGAAACAGCTCCTAACATCAGCAAAGATGCTATAAGACAACTTCTGCCAAGAGCGCC 360 



L E T A P [N-:i:VS] KDAIRQLLPRAP 
351 TCCACTCCGGGAACTGATCGATCAGTACGACGTCCAGAGGGATGACAGCAGTGATGGCTC 420 

PLRELIDQYDVQRDOSSDGS 
421 TTTGGAAGATGACCATTATCACGCTACCACGGAAACAATCATTACCATGCCTACAGAGTC 480 

LEODDYHATTETIITMPTES 
481 TCACTTTCTAATGCAAGCGGATGGCAAGCCCAAATGTTGCTTTTTTAAATTTAGCTCTAA 540 

D F L M Q A D G K P K C C F F K F S S K 
541 AATACAGTACAACAAAGTAGTAAAAGCCCAACTGTGGATATATCTCAGACCCGTCAAGAC 600 

IQYNKVVKAQLWIYLRPVKT 
601 TCCTACAACAGTGTTTGTGCAAATCCTGAGACTCATCAAACCCATGAAAGmTACAAG 660 

PTTVFVQILRLIKPMKOGTR 
661 GTATACTGGAATCCGATCTCTGAAACTTGACATGAGCCCA(X;CACTGGTATTTGGCAGAG 720 

YTGIRSLKLDMSPGTGIWQS 
721 TATTGATGTGAAGACAGTGTTGCAAAATTGGCTCAAACAGCCTGAATCCAACTTAGGCAT 780 

IDVKTVLQNWLKQPESNLGI 
781 TGAAATCAAAGCTTTGGATGACAATGGCCATGATCTTGCTGTAACCTTCCCAGGACCAGG 840 

EIKALDENGHDLAVTFPGPC 
841 AGAAGATGGGCTGAATCCCTTTTTAGAAGTCAAGGTGACAGACACACCCAAGAGGTCTCG 900 

EDGLNPFLEVKVTDTP K iR S R 
901 GAGAGACTTTGGGCTTGACTGCGATGAGCACTCCACGGAATCCCGGTGCTGCCGCTACCC 960 

T|DFGLDCDEHSTESRCCRYP 
961 CCTCACGGTCGATTTTGAAGCCTTTGGATGGGACTCGATtATOJCACCCAAAAGATATAA 1020 

LTVOFEAFGWOWIIAPKRYK 
1021 GGCCAATTACTGCTCAGGAGAGTGTGAATTTGTGTTTTTACAAAAATATCCGCATACTCA 1080 

ANYCSGECEFVFLQKYPHTH 
1081 TCTTGTGCACCAAGCAAACCCCAGAGGCTCAGCAGGCCCTTGCTGCACTCCGACAAAAAT 1140 

LVHQANPRGSAOPCCTPTKM 
1141 GTCTCCCATTAATATGCTA7ATTTTAAT6GCAAAGAACAAATAATATATGGGAAAATTCC 1200 

SPINMLYFNGKEQIIYGKIP 
1201 AGCCATGGTAGTAGACCGCTGTGGGTGCTCATGAGCTTTCCATTAGGTTAGAAACTTCCC 1260 

AMVVDRCGCS* 



FIG. 8a 



wo 96/01845 



13/13 



PCTAJS95/08543 



1261 AAGTCATaJAAGGTCTTCCCCTCAATTTOJAAACTGTGMTTCMXIACCACAGGCTGTA 1320 

1321 GGCCTTGAGTATGCTCTAGTAACGTAAGCACAAGCTACAGTGTATGAACTAAAAGAGAGA 1380 

1381 ATAGATGCAATGGTTGGCATTCAACCACCAAAATAAACCATACTATAGGATGTTGTATGA 1440 

1441 TTTCCAGAGTTTTTGAAATAGATGGAGATCAAATTACATTTATGTaATATATGTATATT 1500 

1501 ACAACTACAATCTAGGCAAGGAAGTGAGAGCACATCTTGTGGTCTGCTGAGTTAGGAGGG 1560 

1561 TATGATTAAAAGGTAAAGTCTTATTTCCTAACAGTTTCACTTAATATTTACAGAAGAATC 1620 

1621 TA7ATGTAGCCTTTGTAAAGTGTAGGATTGTTATCATTTAAAAACATCATGTACACTTAT 1680 

1681 ATTTGTATTGTATACTTGGTAAGATAAAATTCCACAAAGTAGGAATGGGGCCTCACATAC 1740 

1741 ACATTGCCATTCCTATTATAATTGGACAATCCACCAaXJTGCTAATGCACTGCTGAATGG 1800 

1801 CTCCTACTGGACCTCTCGATAGAACACTCTACAAAGTACGAGTCTCTCTCTCCCTTCCAG 1860 

1861 GTGCATCTCaCACACACAGCACTAAGTGTTCAATGCATTTTCTTTAAGGAAAGAAGAAT 1920 

1921 CTTTTTTTCTACAGGTCAACTTTCAGTCAACTCTAGCACAGCGGGAGTGACTGCTGCATC 1980 

1981 TTAAAAGGCAGCCAAACAGTATTCATTTTTTAATCTAAATTTCAAAATCACTGTCTGCCT 2040 

2041 TTATCACATGGGAATTTTGTGGTAAAATAATGGAAATGACTGGTTCTATCAATATTGTAT 2100 

2101 AAAAGACTCTGAAACAATTACATTTATATAATATGTATACAATATTGTTTTGTAAATAAG 2160 

2161 TGTCTCCTTTTATATTTACTTTGGTATATTTTTACACTAATGAAATTTCAAATCATTAAA 2220 

2221 GTACAAAGACATGTCATGTATCACAAAAAAGGTGACTGCTTCTATTTCAGAGTGAA7TAG 2280 

2281 CAGATTCAATAGTGGTCTTAAAACTCTGTATGTTAAGATTAGAAGGTTATATTACAATCA 2340 

2341 ATTTATGTATTTTTTACATTATCAACTTATGGTTTCATGGTGGCTGTATCTATGAATGTG 2400 

2401 GCTCCCAGTCAAATTTCAATGCCCCACCATTTTAAAAATTACAAGCATTACTAAACATAC 2460 

2461 CAACATGTATCTAAAGAAATACAAATATGGTATCTCAATAACAGCTACTTTTTTATTTTA 2520 

2521 TAATTTGACAATGAATACATTTCTTTTATTTACTTCAGTTTTATAAATTGGAACTTTGTT 2580 

2581 TATCAAATGTATTGTACTCATAGCTAAATGAAATTATTTCTTACATAAAAATGTGTAGAA 2640 

2641 ACTATAAATTAAAGTGTTTTCACATTTTTGAAAGGC 2676 

FIG. 8b 



INTERNATIONAL SEARCH REPORT 



Inu.naiional application No. 
PCT/US95/08543 



A. CLASSIFICATION OF SUBJECT MATTER 
iPC(6) :C07K 14/52, 14/495; C12N 15/19. 15/63. 5/10, 1/21, 1/15 
US CL ;Plcasc Sec Extra Sheet. 

According to Inlcmalional Patent Classirication (IPC) or to both national classification and IPC 



B. FIELDS SEAKCnED 



Minimum documentation searched (classification system followed by classification symbols) 
U.S. : 530/350, 351. 395; 536/23.5, 24.31; 435/240.2. 252.3. 254.11, 320.1, 69.1, 69.5 



Documentation searched otlicr than minimum documentation to the extent that such documents arc included in the ficids searched 



Electronic data base consulted during llic international search (name of data base and, where practicable, search terms used) 
Please See Extra Sheet. 



C. DOCUMENTS CONSIDERED TO BE RELEVANT 



Category* 



Citation of document, with indication, where appropriate, of the relevant passages 



Relevant to claim No. 



Y.P 
A,P 



Proceedings of the National Academy of Sciences USA, 
Volume 90, issued July 1993, T. K. Sampath et al., 
"Drosophila transforming growth factor 0 superfamily 
proteins induce endochondral bone formation in mammals", 
pages 6004-6008. 

WO, A, 94/21681 (LEE ET AL.) 29 September 1994, 
especially Figs. 5a and 5b and Claim 3. 



1-11 



3 

1 , 2, 4-1 1 



I I Further documents arc listed in the continuation of Box C. Sec patent family annex. 



'A' 



special CBicf ories of cited docuinenb: 

document denning the genenl Btate of the ait which ia not coniklered 
Ij be of parttcubr relevance 

e&rticr dociuncDt published on or after the international filing date 

document which msy throw doubia on priority ckim(t) or which b 
cited to establish tlw publtcation dote of another ctUiioo or other 
apccial reason (as tpecificU) 

docimteot referring to an oral disclosure, use. exhibition or other 



document publttbed prior to the intemutional HUng date but bter than 
the priority date claimed 



*T* bter document published after the intcniatiooal Tding date or prioriiy 

date and not in connict with the application but cited to understand the 
principle or theory underlying the invention 

*X* document of particular relevance; the claimed inveolion cannot be 

considered novel or cannot be considered to invoNe wa ioveotivc step 
when the document is taken alone 

'Y* document of particular relevaoce; the claimed invention cannot t>c 

considered to involve an invcittive ttgp when the document is 
combined with one or more other such <tocumcntt. sudt combinattoo 
being obvious to a peraon skilled in the ait 

'St' document member of the same patent family 



Date of the actual completion of the international search 
17 AUGUST 1995 



Date of mailing of the international search report 

120CT1995 



Name and mailing address of the ISA/US 
Commissioner of Patents and Trademarks 

Box per 

Wnshtngion.D.C. 20231 
Facsimile No. (703) 305-3230 



Authorized officer 

DAVID L. FITZGERALD 
Telephone No, HPS) 308-0196 



Form PCT/ISA/210 (second shtfct)(July 1992)* 



INTERNATIONAL SEARCH REPORT 



Inu..»ationat application No. 
PCT/US95/08543 



Box I Observations wliere certain claims were found unsearchable (Continuntton of item 1 of first sheet) 



This international repoit has not been established in respect of certain claims under Article 17(2)(a) for the following reasons: 
Claims Nos.: 

because lliey relate to subject matter not required to be searched by this Authority, namely: 



Claims Nos.: 

because they relate to pans of the international application thai do not comply with the prescribed requirements to such 
an extent that no meaningful international search can be carried out. spectfically: 



Claims Nos.: 

because they arc dependent cbims and arc not drafted in accordance wih the second and third sentences of Rule 6.4(a). 



Hox 11 Observations where uuhy of invention is lacking (Continuation of item 2 of first sheet) 
Tliis International Searching Authority found multiple inventions in this international application, as follows: 
Please See Extra Sheet. 



1 . rn As all required additional search fees were timely paid by the applicant, this international search report covers all 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. [ I As only some of the required additional search fees were timely paid by the applicant, this international search report covers 

only those claims for which fees were paid, specifically claims Nos.: 



4. No required additional search fees were timely paid by the applicant. Consequently, this inlcmational search report is 

restricted to the invention first mentioned in the claims; it is covered by claims Nos.: 
1-11 



Remark on Protest The additional search fees were accompanied by the applicant's protest. 

I I No protest accompanied the payment of additional search fees. 



Form PCT/ISA/210 (continuation of first shecl(l))(Juiy 1992)* 



INTERNATIONAL SEARCH REPORT 



International application No. 
PCT/US95/08543 



A. CLASSIFICATION OF SUBJECT MATTER: 
USCL : 

530/350, 351, 395; 536^23.5» 24.31; 435/240.2. 252.3. 254.11. 320.1. 69.1. 69.5 

B. RELDS SEARCHED 

Electronic data bases consulted (Name of data base and where practicable terms used): 

Keyword databases: Medline, Biosis, SciScarch, Denvcnt WPI. USPTO-APS 

search terms: growth differentiation factor; TGF-^ (supcrjfamily 
Sequence databases: GcnBank/EMBL/DDBJ. GcneSeq, SwissProt, PIR 

BOX II. OBSERVATIONS WHERE UNITY OF INVENTION WAS LACKING 
This ISA found multiple inventions as follows: 

This application contains the following inventions or groups of inventions which arc not so linkoi as to form a single 
inventive concept under PCT Rule 13.1. In order for all inventions to be examined, the appropriate additional 
examination fees must be paid. 

I. Claims 1-11. directed to a GDF-11 protein, a nucleic acid encoding it. and corresponding vectors and transformed 
cells. 

II. Claims 12-22, directed to an antibody which binds to a GDF-11 polypeptide and an immunoassay using the same. 

III. Claims 23-43, directed to ilicrapcuiic mcUiods involving the suppression of GDF-11 activity. 

The inventions listed as Croups I-lII do not relate to a single inventive concept under PCT Rule 13.1 because, under 
PCT Rule 13.2, they lack the same or corresponding special technical features for the following reasons. 

The special technical feature of Group I which defines an advance over the art is the novel protein, GDF-11. Neither 
Group II nor Group III shares this special technical feature because each relates to product(s) which are materially 
unlike the products of Group I, and the inventions of these groups are not required to make or use the invention of that 
group. Since the GDF-U peptide is closely related to GDF-8 and other members of the TGF-i3 famUy. the antibodies 
of Group II may be alternatively made using, e.g.. GDF-8 as an antigen. The methods of Group Hi relate to the 
suppression of GDF-1 1 activity; they relate to methods and reagents which arc wholly independent of the GDF-1 1 
protein itself. Each of Groups II and III thus requires an advance over the art which is not dependent on the special 
technical feature embodied in the GDF-U protein of Group I. 

Groups II and III do not share a special technical feature. The special technical fealuic of Group 11 involves antibodies 
characterized by their ability to bind to GDF-11 and the exploitation of such binding in an analytical context. Group 111 
does not share this special technical feature because it relates to the suppression of GDF-11 activity rather than the 
detection of the protein. 

For the above reasons, this Authority considers that the inventions are not so linked by any special technical feature so 
as to form a single inventive concept within the meaning of PCT Rule 13.2. 



Form PCT/ISA/210 (extra shcet)(JuIy 1992)» 



This Page is Inserted by IFW Indexing and Scanning 
Operations and is not part of the Official Record 

BEST AVAILABLE IMAGES 

Defective images within this document are accurate representations of the original 
documents submitted by the appUcant. 

Defects in the images include but are not limited to the items checked: 

□ BLACK BORDERS 

□ IMAGE CUT OFF AT TOP, BOTTOM OR SIDES 
□TfADED TEXT OR DRAWING 

BLURRED OR ILLEGIBLE TEXT OR DRAWING 

□ SKEWED/SLANTED IMAGES 

□ COLOR OR BLACK AND WHITE PHOTOGRAPHS 

□ GRAY SCALE DOCUMENTS 

□ LINES OR MARKS ON ORIGINAL DOCUMENT 

□ REFERENCE(S) OR EXHIBIT(S) SUBMITTED ARE POOR QUALITY 

□ OTHER: ■ 

IMAGES ARE BEST AVAILABLE COPY. 
As rescanning these documents will not correct the image 
problems checked, please do not report these problems to 
the IFW Image Problem Mailbox.