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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




PCT 

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 6 : 
C07K 14/71, C07H 21700 



Al 



(11) Internationa] Publication Number: WO 95/10539 

(43) International Publication Date: 20 April 1995 (20.04.95) 



(21) International Application Number: PCT/US94/1 1440 

(22) Internationa] Filing Date: 7 October 1994 (07.10.94) 



(30) Priority Data: 

08/134,078 



8 October 1993 (08.10.93) 



US 



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

HOPKINS UNIVERSITY SCHOOL OF MEDICINE 
[US/US]; Suite 2-100, 2024 E. Monument Street, Balti- 
more, MD 21205 (US). 

(72) Inventors; and 

(75) Inventors/Applicants (for US only): LEE, Se-Jin [US/US]; 
6711 Chokeberry Road, Baltimore, MD 21209 (US). CUN- 
NINGHAM, Noreen [US/US]; 1400 Mimosa Lane, Silver 
Spring, MD 20904 (US). 

(74) Agents: WETHERELL, John, JL, Jr. ct al.; Spensley Horn 
Jubas & Lubitz, 5th floor, 1880 Century Park East, Los 
Angeles, CA 90067 (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-10 
(57) Abstract 



Growth differentiation factor- 10 (GDF-10) is disclosed along with its polynucleotide sequence and amino acid sequence. Also 
disclosed are diagnostic and therapeutic methods of using the GDF-10 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 


AD 


Australia 


GE 


Georgia 


MW 


Malawi 


BB 


Barbados 


GN 


Guinea 


NE 


Niger 


BE 


Belgium 


GR 


Greece 


NL 


Netherlands 


BP 


Burkina Paso 


HU 


Hungary 


NO 


Norway 


BG 


Bulgaria 


IE 


Ireland 


NZ 


New Zealand 


Bl 


Benin 


IT 


Italy 


PL 


Poland 


BR 


Brazil 


JP 


Japan 


FT 


Portugal 


BY 


Belarus 


KE 


Kenya 


RO 


Romania 


CA 


Canada 


KG 


Kyrgystan 


RU 


Russian Federation 


CF 


Centra) African Republic 


KP 


Democratic People's Republic 


SD 


Sudan 


CG 


Congo 




of Korea 


SE 


Sweden 


CH 


Switzerland 


KR 


Republic of Korea 


SI 


Slovenia 


CI 


Cote d'lwire 


KZ 


Kazakhstan 


SK 


Slovakia 


CM 


Cameroon 


U 


Liechtenstein 


SN 


Senegal 


CN 


China 


LK 


Sri Lanka 


TD 


Chad 


CS 


Czechoslovakia 


LU 


Luxembourg 


TG 


Togo 


CZ 


Czech Republic 


LV 


Latvia 


TJ 


Tajikistan 


DE 


Germany 


MC 


Monaco 


TT 


Trinidad and Tobago 


DK 


Denmark 


MD 


Republic of Moldova 


UA 


Ukraine 


E8 


Spain 


MG 


Madagascar 


US 


United States of America 


n 




ML 


Mali 


UZ 


Uzbekistan 


FR 


Prance 


MN 


Mongolia 


VN 


Viet Nam 


GA 


Gabon 











WO9S/10539 



PCTAJS94/11440 



" 1 ' 

GROWTH DIFFERENTIATION FACTOR-10 
BACKGROUND OF THE INVENTION 

t 

1. Field of the Invention 

The invention relates generally to growth factors and specifically to a 
5 new member of the transforming growth factor beta (TGF-/?) super- 
family, which is denoted, growth differentiation factor-10 (GDF-10). 

2. Description of Related Art 

The transforming growth factor ft (TGF-0) superfamily encompasses a 
group of structurally-related proteins which affect a wide range of 

10 differentiation processes during embryonic development. The family 
includes, Mullerian inhibiting substance (MIS), which is required for 
normal male sex development (Behringer, et a/., Nature, 345:167. 
1990), Drosophila decapentaplegic (DPP) gene product, which is 
required for dorsal-ventral axis formation and morphogenesis of the 

15 imaginal disks (Padgett, et ai, Nature, 325:81-84, 1987), the Xenopus 
Vg-1 gene product, which localizes to the vegetal pole of eggs ((Weeks, 
et ai, Cell, 51:861-867, 1987), the activins (Mason, et ai, Biochem, 
Biophys. Res. Commun., 135:957-964, 1986), which can induce the 
formation of mesoderm and anterior structures in Xenopus embryos 

20 (Thomsen, et ai, Cell, 63:485, 1990), and the bone morphogenetic 
proteins (BMPs, osteogenin, OP-1) which can induce <Je novo cartilage 
and bone formation (Sampath, era/., J. Biol. Chem., 265:13198. 1990). 
The TGF-/Ss can influence a variety of differentiation processes, 
including adipogenesis, myogenesis, chondrogenesis, hematopoiesis, 



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-2- 

and epithelial cell differentiation (for review, see Massague, Cell 49:437, 
1987). 

The proteins of the TGF-/? family are initially synthesized as a large 
precursor protein which subsequently undergoes proteolytic cleavage at 
5 a cluster of basic residues approximately 110-140 amino acids from the 
C-terminus. The C-terminai 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 

10 90% amino acid sequence identity, the homologies between subgroups 
are significantly lower, generally ranging from only 20% to 50%. In each 
case, the active species appears to be a disulfide-linked dimer of 
C-terminal fragments. For most of the family members that have been 
studied, the homodimeric species has been found to be biologically 

15 active, but for other family members, like the inhibins (Ling, et a/., 
Nature, 321:779, 1986) and the TGF-/?s (Cheifetz, et ai, Cell, £8:409, 
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 
20 pattern will provide a greater understanding of that tissue's development 
and function and allow development of effective diagnostic and 
therapeutic regimens. 



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-3- 

SUMMARY OF THE INVENTION 

The present invention provides a cell growth and differentiation factor, 
GDF-10, a polynucleotide sequence which encodes the factor, and 
antibodies which are immunoreactive with the factor. This factor 
5 appears to relate to various cell proliferative disorders, especially those 
involving those involving uterine, nerve, bone, and adipose tissue. 

Thus, in one embodiment, the invention provides a method for detecting 
a cell proliferative disorder of uterine, nerve, or fat origin and which is 
associated with GDF-1 0. In another embodiment, the invention provides 
10 a method for treating a cell proliferative disorder by suppressing or 
enhancing GDF-10 activity. 



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BRIEF DESCRIPTION OF THE DRAWINGS 

FIGURE 1 shows expression of GDF-10 mRNA in adult tissues. 

t 

FIGURE 2 shows nucleotide and predicted amino acid sequence murine 
GDF-10. Consensus N-glycosylation signals are denoted by plain 
5 boxes. 

FIGURE 3 shows the alignment of the C-terminal sequences of GDF-10 
with other members of the TGF-/? superfamily. The conserved cysteine 
residues are boxed. Dashes denote gaps introduced in order to 
maximize alignment. 

10 FIGURE 4 shows amino acid homologies with different members of the 
TGF-/? superfamily. Numbers represent percent amino acid identities 
between each pair calculated from the first conserved cysteine to the C- 
terminus. 

FIGURE 5 shows an alignment of the C-terminal sequences of human 
15 (top lines) and murine (bottom lines) GDF-10. 

FIGURE 6 shows an autoradiogram of labeled secreted proteins 
synthesized by 293 cells transfected with a pcDNAI vector into which 
the GDF-10 cDNA was inserted in either the antisense (lanes 1 and 2) 
or sense (lanes 3 and 4) orientation. 



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-5- 

DETAILED DESCRIPTION OF THE INVENTION 

The present invention provides a growth and differentiation factor, GDF- 
10 and a polynucleotide sequence encoding GDF-10. GDF-10 is 
expressed at highest levels in uterus and fat and at lower levels in other 
5 tissues, such as brain. In one embodiment, the invention provides a 
method for detection of a cell proliferative disorder of uterine, nerve, or 
fat origin which is associated with GDF-10 expression. In another 
embodiment, the invention provides a method for treating a cell 
proliferative disorder by using an agent which suppresses or enhances 
10 GDF-10 activity. 

The TGF-fi 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-10 protein of this invention and the 
members of the TGF-/? family, indicates that GDF-10 is a new member 
of the family of growth and differentiation factors. Based on the known 
activities of many of the other members, it can be expected that GDF-10 
will also possess biological activities that will make it useful as a 
diagnostic and therapeutic reagent. 

The expression of GDF-10 in uterine and fat tissue suggests a variety 
of applications using the polypeptide, polynucleotide, and antibodies of 
the invention, related to contraception, fertility, pregnancy, and cell 
proliferative diseases. Abnormally low levels of the factor my be 
25 indicative of impaired function in the uterus while abnormally high levels 
may be indicative of hypertrophy, hyperplasia, or ihe presence of 
ectopic tissue. Hence, GDF-10 my be useful in detecting not only 



15 



20 



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-6- 

primary and metastatic neoplasms of uterine origin but in detecting 
diseases such as endometriosis as well. In addition, GDF-10 may also 
be useful as an indicator of developmental anomalies in prenatal 
screening procedures. 

5 Several members of the TGF-/? superfamily possess activities 
suggesting possible applications for the treatment of cell proliferative 
disorders, such as cancer. In particular, TGF-/? has been shown to be 
potent growth inhibitor for a variety of cell types (Massague, Cell 
49:437, 1987). MIS has been shown to inhibit the growth of human 

10 endometrial carcinoma tumors in nude mice (Donahoe, et a/., Ann. 
Surg. 194:472. 1981), and inhibin a has been shown to suppress the 
development of tumors both in the ovary and in the testis (Matzuk, et 
al., Nature, 360:313, 1992). GDF-10 may have similar activity and may 
therefore be useful as an antiproliferative agent, such as for the 

15 treatment of endometrial cancer or endometriosis. 

Many of the members of the TGF-/? family are also important mediators 
of tissue repair. TGF-/? has been shown to have marked effects on the 
formation of collagen and causes of striking angiogenic response in the 
newborn mouse (Roberts, et al, Proc. Natl Acad. Sc/., USA 83:4167, 

20 1986). The BMP's can induce new bone growth and are effective for 
the treatment of fractures and other skeletal defects (Glowacki, et ai, 
Lancet, 1:959, 1981; Ferguson, et aL, Clin. Orthoped. Relat Res., 
227:265, 1988; Johnson, et aL, Clin Orthoped Relat Res., 230:257, 
1988). Based on the high degree of homology between GDF-10 and 

25 BMP-3, GDF-10 may have similar activities and may be useful in repair 
of tissue injury caused by trauma or burns for example. 



WO 95/10539 PCT/US94/11440 

-7- 

GDF-10 may play a role in regulation of the menstrual cycle or 
regulation of uterine function during pregnancy, and therefore, GDF-10, 
anti-GDF-10 antibodies, or antisense polynucleotides may be useful 
either in contraceptive regimens, in enhancing the success of in vitro 
5 fertilization procedures, or in preventing premature labor. 

Certain members of this superfamily have expression patterns or 
possess activities that relate to the function of the nervous system. For 
example, one family member, namely GDNF, has been shown to be a 
potent neurotrophic factor that can promote the survival of dopaminergic 

10 neurons (Lin, et al., Science, 260:1130). Another family member, 
namely dorsalin, is capable of promoting the differentiation of neural 
crest cells (Baster, et a/., Cell, 73:687). The inhibins and activins have 
been shown to be expressed in the brain (Meunier, et al., Proc. Nat'l 
Acad. Sci., USA, 85:247, 1988; Sawchenko, et al., Nature, 334:615. 

15 1988), and activin has been shown to be capable of functioning as a 
nerve cell survival molecule (Schubert, et al., Nature, 344:868. 1990). 
Another family member, namely GDF-1 , is nervous system-specific in 
its expression pattern (Lee, Padc. Nat'l Acad. Sci., USA, 88:4250, 1991), 
and certain other family members, such as Vgr-1 (Lyons, et al., Proc. 

20 Nafl Acad. Sci., USA, §6:4554, 1989; Jones et al., Development, 
111:581, 1991), OP-1 (Ozkaynak, et al., J. Biol. Chem., 26Z:25220, 
1992), and BMP-4 (Jones, etal., Development, 111:531, 1991), are also 
known to be expressed in the nervous system. By analogy GDF-10 
may have applications in the treatment of neurodegenerative diseases 

25 or in maintaining cells or tissues in culture prior to transplantation. 

The expression of GDF-10 in adipose tissue also raises the possibility 
of applications for GDF-10 in the treatment of obesity or of disorders 
related to abnormal proliferation of adipocytes. In this regard, TGF-0 



WO 95/10539 



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-8- 

has been shown to be a potent inhibitor of adipocyte differentiation in 
vitro (ignotz and Massague, Proc. Natl. Acad ScL, USA 82:8530, 1 985). 

The term "substantially pure" as used herein refers to GDF-10 which is 
5 substantially free of other proteins, lipids, carbohydrates or other 
materials with which it is naturally associated. One skilled in the art can 
purify GDF-10 using standard techniques for protein purification. The 
substantially pure polypeptide will yield a single major band on a non- 
reducing polyacrylamide gel. The purity of the GDF-10 polypeptide can 
10 also be determined by amino-terminal amino acid sequence analysis. 
GDF-10 polypeptide includes functional fragments of the polypeptide, as 
long as the activity of GDF-1 0 remains. Smaller peptides containing the 
biological activity of GDF-10 are included in the invention. 

The invention provides polynucleotides encoding the GDF-10 protein. 

15 These polynucleotides include DNA, cDNA and RNA sequences which 
encode GDF-10. it is understood that all polynucleotides encoding all 
or a portion of GDF-10 are also included herein, as long as they encode 
a polypeptide with GDF-10 activity. Such polynucleotides include 
naturally occurring, synthetic, and intentionally manipulated 

20 polynucleotides. For example, GDF-10 polynucleotide may be 
subjected to site-directed mutagenesis. The polynucleotide sequence 
for GDF-10 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 

25 specified by more than one codon. Therefore, all degenerate nucleotide 
sequences are included in the invention as long as the amino acid 
sequence of GDF-10 polypeptide encoded by the nucleotide sequence 
is functionally unchanged. 



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PCT/US94/11440 



Specifically disclosed herein is a cDNA sequence for GDF-10 which is 
2322 base pairs in length and contains an open reading frame 
beginning with a methionine codon at nucleotide 126. The encoded 
polypeptide is 476 amino acids in length with a molecular weight of 
5 about 52.5 kD, as determined by nucleotide sequence analysis. The 
GDF-10 sequence contains a core of hydrophobic amino acids near the 
N-terminus, suggestive of a signal sequence for secretion. GDF-10 
contains four potential N-glycosylation sites at asparagine residues 114, 
152, 277, and 467. GDF-10 contains several potential proteolytic 

10 processing sites. Cleavage most likely occurs following arginine 365, 
which would generate a mature fragment of GDF-10 predicted to be 111 
amino acids in length and have an unglycosylated molecular weight of 
about 12.6kD, as determined by nucleotide sequence analysis. One 
skilled in the art can modify, or partially or completely remove, the 

15 glycosyl groups from the GDF-10 protein using standard techniques. 
Therefore the functional protein or fragments thereof of the invention 
includes glycosylated, partially glycosylated and unglycosylated species 
of GDF-10. 

The C-terminal region of GDF-10 following the putative proteolytic 
20 processing site shows significant homology to the known members of 
the TGF-/? superfamily. The GDF-10 sequence contains most of the 
residues that are highly conserved in other family members. Among the 
known family mammalian TGF-0 family members, GDF-10 is most 
homologous to BMP-3 (83% sequence identity beginning with the first 
25 conserved cysteine residue). GDF-10 also shows significant homology 
to BMP-3 (approximately 30% sequence identity) in the pro-region of the 
molecule. Based on these sequence comparisons, GDF-10 and BMP-3 
appear to define a new subfamily within the larger superfamily. 



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-10- 

Minor modifications of the recombinant GDF-10 primary amino acid 
sequence may result in proteins which have substantially equivalent 
activity as compared to the GDF-1 0 polypeptide described herein. Such 
modifications may be deliberate, as by site-directed mutagenesis, or 
5 may be spontaneous. All of the polypeptides produced by these 
modifications are included herein as long as the biological activity of 
GDF-10 still exists. Further, deletion of one or more amino acids can 
also result in a modification of the structure of the resultant molecule 
without significantly altering its biological activity. This can lead to the 
10 development of a smaller active molecule which would have broader 
utility. For example, one can remove amino or carboxy terminal amino 
acids which, are not required for GDF-10 biological activity. 

The nucleotide sequence encoding the GDF-10 polypeptide of the 
invention includes the disclosed sequence and conservative variations 

15 thereof. The term "conservative variation" as used herein denotes the 
replacement of an amino acid residue by another, 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 

20 another, such as the substitution of arginine for lysine, glutamic for 
aspartic acid, or glutamine for asparagine, and the like. The term 
"conservative variation" also includes the use of a substituted amino 
acid in place of an unsubstituted parent amino acid provided that 
antibodies raised to the substituted polypeptide also immunoreact with 

25 the unsubstituted polypeptide. 



DNA sequences of the invention can be obtained by several methods. 
For example, the DNA can be isolated using hybridization techniques 
which are well known in the art. These include, but are not limited to: 



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1) hybridization of genomic or cDNA libraries with 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 
5 libraries to detect cloned DNA fragments with shared structural features. 

Preferably the GDF-10 polynucleotide of the invention is derived from 
a mammalian 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 sequence from any organism, 

10 provided the appropriate probe is available. Oligonucleotide probes, 
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 

15 code, however, the degeneracy of the code must be taken into account. 
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 
performed on either single-stranded DNA or denatured double-stranded 

20 DNA. Hybridization is particularly useful in the detection of cDNA clones 
derived from sources where an extremely low amount of mRNA 
sequences 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 

25 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 a/., Nuci Acid Res. t 9:879, 1981). 



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The development of specific DNA sequences encoding GDF-1 0 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 
5 synthesis of a double-stranded DNA sequence by reverse transcription 
of mRNA isolated from a eukaryotic donor cell. In the latter case, a 
double-stranded DNA complement of mRNA is eventually formed which 
is generally referred to as cDNA. 

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

The synthesis of DNA sequences is frequently the method of choice 
15 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 
synthesis of cDNA sequences. Among the standard procedures for 
20 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 
25 cloned. In those cases where significant portions of the amino acid se- 
quence 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 



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DNA/DNA hybridization procedures which are carried out on cloned 
copies of the cDNA which have been denatured into a single-stranded 
form (Jay, et ai, Nucl. Acid Res., 11:2325, 1983). 

A cDNA expression library, such as lambda gt11, can be screened 
5 indirectly for GDF-10 peptides having at least one epitope, using 
antibodies specific for GDF-10. Such antibodies can be either 
polyclonally or monoclonally derived and used to detect expression 
product indicative of the presence of GDF-10 cDNA. 

DNA sequences encoding GDF-10 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 cell" is used. Methods of stable transfer, meaning that the 
foreign DNA is continuously maintained in the host, are known in the art. 

In the present invention, the GDF-10 polynucleotide sequences may be 
inserted into a recombinant expression vector. The term "recombinant 
expression vector" refers to a plasmid, virus or other vehicle known in 
20 the art that has been manipulated by insertion or incorporation of the 
GDF-10 genetic sequences. Such expression vectors contain a 
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 
25 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, 



10 



15 



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-14- 

et a/., Gene, 56:125, 1987), the pMSXND expression vector for 
expression in mammalian cells (Lee and Nathans, J. Biol. Chem., 
263:3521, 1988) and baculovirus-derived vectors for expression in 
insect cells. The DNA segment can be present in the vector operably 
5 linked to regulatory elements, for example, a promoter (e.g., T7, 
metallothionein I, or polyhedrin promoters). 

Polynucleotide sequences encoding GDF-1 0 can be expressed in either 
prokaryotes or eukaryotes. Hosts can include microbial, yeast, insect 
and mammalian organisms. Methods of expressing DNA sequences 

10 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 
vectors are used to incorporate DNA sequences of the invention. 
Preferably, the mature C-terminal region of GDF-1 0 is expressed from 

15 a cDNA clone containing the entire coding sequence of GDF-1 0. 
Alternatively, the C-terminal portion of GDF-1 0 can be expressed as a 
fusion protein with the pro- region of another member of the TGF-/? 
family or co-expressed with another pro- region (see for example, 
Hammonds, era/., Molec. Endocrin. 5:149, 1991; Gray, A., and Mason, 

20 A., Science, 247:1328, 1990). 

Transformation of a host cell with 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 E. coii, competent cells which 
are capable of DNA uptake can be prepared from cells harvested after 
25 exponential growth phase and subsequently treated by the CaCI 2 
method using procedures well known in the art. Alternatively, MgCI 2 or 
RbCI can be used. Transformation can also be performed after forming 
a protoplast of the host cell if desired. 



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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 
5 be cotransformed with DNA sequences encoding the GDF-10 of the 
invention, and a second foreign DNA molecule encoding a selectable 
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 transform 
10 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, provided by the invention, may be carried out by 
conventional means including preparative chromatography and 
15 immunological separations involving monoclonal or polyclonal 
antibodies. 

The invention includes antibodies immunoreactive with GDF-10 
polypeptide or functional fragments thereof. Antibody which consists 
essentially of pooled monoclonal antibodies with different epitopic 

20 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 to those skilled in the 
art (Kohler, et a/., Nature, 256:495. 1975). The term antibody as used 
in this invention is meant to include intact molecules as well as 

25 fragments thereof, such as Fab and F(ab , ) 2 , which are capable of 
binding an epitopic determinant on GDF-10. 



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The term "cell-proliferative disorder" denotes malignant as well as non- 
malignant cell populations which often appear to differ from the 
surrounding tissue both morphologically and genotypically. The term 
"cell-proliferative disorder" also includes situations in which a normally 
5 occurring process could be enhanced or suppressed for clinical benefit; 
an example of such a process would be fracture healing. Malignant 
cells (i.e. cancer) develop as a result of a multistep process. The GDF- 
10 polynucleotide that is an antisense molecule is useful in treating 
malignancies of the various organ systems, particularly, for example, 
10 cells in uterine or adipose tissue. Essentially, any disorder which is 
etiologically linked to altered expression of GDF-10 could be considered 
susceptible to treatment with a GDF-10 suppressing reagent. One such 
disorder is a malignant cell proliferative disorder, for example. 

The invention provides a method for detecting a cell proliferative 
15 disorder of uterine or adipose tissue which comprises contacting an 
anti-GDF-10 antibody with a cell suspected of having a GDF-10 
associated disorder and detecting binding to the antibody. The antibody 
reactive with GDF-10 is labeled with a compound which allows detection 
of binding to GDF-10. For purposes of the invention, an antibody 
20 specific for GDF-10 polypeptide may be used to detect the level of 
GDF-10 in biological fluids and tissues. Any specimen containing a 
detectable amount of antigen can be used. A preferred sample of this 
invention is uterine or fat tissue. The level of GDF-10 in the suspect 
cell can be compared with the level in a normal cell to determine 
25 whether the subject has a GDF-1 0-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 



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immunotherapy. The antibodies of the 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 
5 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 immunoassays are the 
radioimmunoassay (RIA) and the sandwich (immunometric) assay. 
Detection of the antigens using the antibodies of the invention can be 
10 done utilizing immunoassays which are run in either the forward, 
reverse, orsimultaneous modes, including immunohistochemical assays 
on physiological samples. Those of skill in the art will know, or can 
readily discern, other immunoassay formats without undue 
experimentation. 

15 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 of well-known carriers include 
glass, polystyrene, polypropylene, polyethylene, dextran, nylon, 
amylases, natural and modified celluloses, polyacrylamides, agaroses 

20 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. 

There are many different labels and methods of labeling known to those 
25 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, colloidal metals, chemiluminescent compounds, 
phosphorescent compounds, and bioluminescent compounds. Those 



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of ordinary skill in the art will know of other suitable labels for binding 
to the antibody, or will be able to ascertain such, using routine 
experimentation. 

Another technique which may also result in greater sensitivity consists 
5 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, 
which can react with specific antihapten antibodies. 

10 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 

15 the antigen comprising a polypeptide of the invention for which the 
monoclonal antibodies are specific. 

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. 
20 Further, it is desirable that the detectably labeled monoclonal antibody 
be rapidly cleared from the circulatory system in order to give the best 
target-to-background 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 
25 extent of disease of the individual. Such dosages may vary, for 



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example, depending on whether multiple injections are given, antigenic 
burden, and other factors known to those of skill in the art. 

For /'n vivo diagnostic imaging, the type of detection instrument available 
is a major factor in selecting a given radioisotope. The radioisotope 
5 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 emission, but produce a large number of photons in the 140- 
10 250 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 

15 radioisotopes which exist as metallic ions to 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 111 ln, 97 Ru, ^Ga, "Ga, "As, 

20 89 Zr, and ^Tl. 

The monoclonal antibodies of the invention can also be labeled with a 
paramagnetic 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 diagnostic imaging can be 
25 utilized. Usually gamma and positron emitting radioisotopes are used 
for camera imaging and paramagnetic isotopes for MRI. Elements 



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which are particularly useful in such techniques include 157 Gd, 55 Mn, 
162 Dy, 52 Cr, and ^Fe. 

The monoclonal antibodies of the invention can be used in vitro and in 
vivo to monitor the course of amelioration of a GDF-1 0-associated 
5 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 would be possible to determine 
whether a particular therapeutic regimen aimed at ameliorating the 
10 GDF-1 0-associated disease is effective. The term "ameliorate" denotes 
a lessening of the detrimental effect of the GDF-1 0-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 

15 cell, therefore it is possible to design appropriate therapeutic or 
diagnostic techniques directed to this sequence. Thus, where a cell- 
proliferative disorder is associated with the expression of GDF-1 0, 
nucleic acid sequences that interfere with GDF-10 expression at the 
translational level can be used. This approach utilizes, for example, 

20 antisense nucleic acid and ribozymes to block translation of a specific 
GDF-10 mRNA, either by masking that mRNA with an antisense nucleic 
acid or by cleaving it with a ribozyme. 

Antisense nucleic acids are DNA or RNA molecules that are 
complementary to at least a portion of a specific mRNA molecule 
25 (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 



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the translation of 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 
5 GDF-10-producing cell. The use of antisense methods to inhibit the in 
vitro translation of genes is well known in the art (Marcus-Sakura, 
Anal.Biochem., 172:289, 1988). 

Ribozymes are RNA molecules possessing the ability to specifically 
cleave other single-stranded RNA in a manner analogous to DNA 

10 restriction endonucleases. Through the 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, JAmer.Med. Assn., 260:3030, 1988). A 
major advantage of this approach is that, because they are sequence- 

15 specific, only mRNAs with particular sequences are inactivated. 

There are two basic types of ribozymes namely, tetrahymena-type 
(Hasselhoff, Nature, 334:585. 1988) and "hammerhead"-type. 
Tetrahymena-type ribozymes recognize sequences which are four bases 
in length, while "hammerhead"-type ribozymes recognize base 

20 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 species. Consequently, hammerhead-type ribozymes are 
preferable to tetrahymena-type ribozymes for inactivating a specific 
mRNA species and 18-based recognition sequences are preferable to 

25 shorter recognition sequences. 

The present invention also provides gene therapy for the treatment of 
cell proliferative disorders which are mediated by GDF-10 protein. Such 



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therapy would achieve its therapeutic effect by introduction of the GDF- 
10 antisense polynucleotide into cells having the proliferative disorder. 
Delivery of antisense GDF-10 polynucleotide can be achieved using a 
recombinant expression vector such as a chimeric virus or a colloidal 
5 dispersion system. Especially preferred for therapeutic delivery of 
antisense sequences is the use of targeted liposomes. 

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. Preferably, the retroviral vector is a 

10 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). A number of additional retroviral vectors 

15 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-10 sequence of 
interest into the viral vector, along with another gene which encodes the 
ligand for a receptor on a specific target cell, for example, the vector is 

20 now target specific. Retroviral vectors can be made target specific by 
inserting, for example, a polynucleotide encoding a sugar, a glyco lipid, 
or a protein. Preferred targeting is accomplished by using ah antibody 
to target the retroviral vector. Those of skill in the art will know of, or 
can readily ascertain without undue experimentation, specific polynu- 

25 cleotide sequences which can be inserted into the retroviral genome to 
allow target specific delivery of the retroviral vector containing the GDF- 
10 antisense polynucleotide. 



L 



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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 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 
M*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 
structural 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 
15 transfected with plasmids encoding the retroviral structural genes gag, 
pol and env, by conventional calcium phosphate transfection. 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. 

20 Another targeted delivery system for GDF-10 antisense polynucleotides 
is a colloidal dispersion system. Colloidal dispersion systems include 
macromolecule complexes, nanocapsules, microspheres, beads, and 
lipid-based systems including oil-in-water emulsions, micelles, mixed 
micelles, and liposomes. The preferred colloidal system of this 

25 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 (LUV), which range in size from 
0.2-4.0 fjm can encapsulate a substantial percentage of an aqueous 



5 



10 



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buffer containing large macromolecules. RNA, DNA 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 
5 for delivery of polynucleotides in plant, yeast and 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 
10 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) 
accurate and effective expression of genetic information (Mannino, et 
aL, Biotechniques, 6:682, 1988). 

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

20 Examples of lipids useful in liposome production include phosphatidyl 
compounds, such as phosphatidylglycerol, phosphatidylcholine, 
phosphatidylserine, phosphatidylethanolamine, sphingolipids, 
cerebrosides, and gangliosides. Particularly useful are 
diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 

25 carbon atoms, particularly from 16-18 carbon atoms, and is saturated. 
Illustrative phospholipids include egg phosphatidylcholine, 
dipalmitoylphosphatidylcholine and distearoylphosphatidylchotine. 



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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 
5 whether it is passive or active. Passive targeting utilizes the natural 
tendency of liposomes to distribute to cells of the reticuloendothelial 
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 
10 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 
15 groups can be incorporated into the 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-10 primarily in uterine and adipose 
20 tissue, there are a variety of applications using the polypeptide, 
polynucleotide, and antibodies of the invention, related to these and 
other tissues. Such applications include treatment of cell proliferative 
disorders involving these and other tissues, including bone. In addition, 
GDF-10 may be useful in various gene therapy procedures. 

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. 



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EXAMPLE 1 

IDENTIFICATION AND ISOLATION OF A NOVEL 
TGF-fl FAMILY MEMBER 



To identify new members of the TGF-jff superfamily, degenerate 
5 oligonucleotides were designed which corresponded to two conserved 
regions among the known family members: one region downstream of 
the first conserved cysteine residue and the other region spanning the 
invariant cysteine residues near the C-terminus. These primers were 
used for polymerase chain reactions on lung and brain cDNA followed 
10 by subcloning the PCR products using restriction sites placed at the 5' 
ends of the primers, picking individual E. coli colonies carrying these 
subcloned inserts, and using a combination of random sequencing and 
hybridization analysis to eliminate know members of the superfamily. 

GDF-10 was identified from a mixture of PCR products obtained with the 
15 primers: 

NSC1: 5*- 

CCGGAATTCAA(G/A)GT(G/A/T/C)GA(T/C)TT(T/C)GC(G/An-/C)GA 
(T/C)AT(A/C/T)GG(G/A/T/C)TGG-3' 

NSC2: 5 - 

20 CCGGAATTC(A/G)CA(G/A/T/C)GC(A/G)CA<G/A)CT{T/C)TC<G/AAr/C) 
AC(G/A/T/C)GTCAT-3' 

NSC3: 5'- 

CCGGAATTC(A/G)CA{G/A/T/C)GC(AA3)CA(G/An'/C)GA(T/C)TC 
(G/A/T/C)AC(G/Afl7C)GTCAT-3' 

25 PCR using primers NSC1 with NSC2 or NSC1 with NSC3 was carried 
out with cDNA prepared from 0.25 /jg of lung or brain mRNA for 35 
cycles at 94°C for 1 min, 50°C for 2 min, and 72°C for 2 min. PCR 
products of approximately 300 base pairs were digested with Eco Rl, gel 
purified, and subcloned in the Bluescript vector (Stratagene, San Diego, 



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CA). DNA was prepared from bacterial colonies carrying individual 
subclones and sequenced. Of 11 clones that were sequenced, 9 
corresponded to BMP-3, and two represented a novel sequence, which 
was designated GDF-10. 

5 EXAMPLE 2 

EXPRESSION PATTERN AND SEQUENCE OF GDF-10 

To determine the expression pattern of GDF-10, RNA samples prepared 
from a variety of adult tissues were screened by Northern analysis. 2.5 
micrograms of twice polyA-selected RNA prepared from each tissue 
10 were electrophoresed on formaldehyde gels, blotted and probed with 
GDF-10. As shown In Figure 1, the GDF-10 probe detected an mRNA 
expressed at highest levels in uterus, fat, and brain. 

A murine uterus cDNA library consisting of 3 x 10 6 recombinant phage 
was constructed in lambda ZAP II and screened with a probe derived 

15 from the GDF-10 PCR product. The entire nucleotide sequence of the 
longest of 7 hybridizing clones is shown in Figure 2. Consensus N- 
glycosylation signals are denoted by plain boxes. Numbers indicate 
nucleotide position relative to the 5' end. The 2322 bp sequence 
contains a long open reading frame beginning with a methionine codon 

20 at nucleotide 126 and potentially encoding a protein 476 amino acids in 
length with a molecular weight of 52.5 kD. The predicted GDF-10 
amino acid sequence contains a hydrophobic N-terminal region, 
suggestive of a signal sequence for secretion, four potential N-linked 
glycosylation sites at asparagine residues 114, 152, 277, and 467 and 

25 a putative proteolytic processing site at amino acid 365. Cleavage of 
the GDF-10 precursor at this site would generate a mature GDF-10 



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protein 111 amino acids in length with a predicted unglycosylated 
molecular weight of 12.6 kD. 

The C-terminal region of GDF-10 following the putative proteolytic 
processing site shows significant homology to the known members of 
5 the TGF-/? superfamily (Figure 3). Figure 3 shows the alignment of the 
C-terminal sequences of GDF-10 with the corresponding regions of 
human GDF-1 (Lee, Proc. Natl. Acad. Sci. USA, 88:4250-4254, 1991), 
murine GDF-3 and GDF-9 (McPherron and Lee, J. Biol. Chem. 
268:3444. 1993), human BMP-2 and 4 (Wozney, et ai, Science, 

10 242:1528-1534, 1988), human Vgr-1 (Celeste, et ai, Proc. Natl. Acad. 
Sci. USA, 82:9843-9847, 1990), human OP-1 (Ozkaynak, era/., EMBO 
J., 9:2085-2093, 1990), human BMP-5 (Celeste, era/., Proc. Natl. Acad. 
Sci. USA, 82:9843-9847, 1990), human OP-2 (Ozkaynak, etai, J. Biol. 
Chem., 262:25220-25227, 1992), human BMP-3 (Wozney, et ai, 

1 5 Science, 242:1 528-1 534, 1 988), human MIS (Cate, et ai, Cell, 45:685- 
698, 1986), human inhibin alpha, 0A, and/?B (Mason, etai, Biochem, 
Biophys. Res. Commun., 135:957-964. 1986), murine nodal (Zhou, et 
ai, Nature, 361:543-547, 1993), human TGF-01 (Derynck, et ai, 
Nature, 316:701-705, 1 985), humanTGF-02 (deMartin, et ai, EMBO J., 

20 6:3673-3677, 1987), and human TGF-/J3 (ten Dijke, et ai, Proc. Natl. 
Acad. Sci. USA. 85:4715-4719. 1988). The conserved cysteine residues 
are boxed. Dashes denote gaps introduced in order to maximize the 
alignment. 

GDF-10 contains most of the residues that are highly conserved in other 
25 family members, including the seven cysteine residues with their 
characteristic spacing. 



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FIGURE 4 shows the amino acid homologies among the different 
members of the TGF-0 superfamily. Numbers represent percent amino 
acid identities calculated from the first conserved cysteine to the C- 
terminus. In this region, GDF-10 is most homologous to BMP-3 (83% 
5 sequence identity). 

EXAMPLE 3 
ISOLATION OF HUMAN GDF-10 

To isolate human GDF-10, a human uterus cDNA library consisting of 
16.2 x 10 6 recombinant phage was constructed in lambda ZAP II and 

10 screened with a murine GDF-10 probe. From this library, 20 hybridizing 
clones were isolated. Partial nucleotide sequence analysis of the 
longest clone showed that human and murine GDF-10 are highly 
homologous; the predicted amino acid sequences are 97% identical 
beginning with the first conserved cysteine residue following the 

15 predicted cleavage site (Figure 5). 

EXAMPLE 4 

SECRETION OF GDF-10 BY MAMMALIAN CELLS 

To determine whether GDF-10 is secreted by mammalian cells, the 
GDF-10 cDNA was cloned into the pcDNAI expression vector and 

20 transfected into 293 cells. Following DNA transfection, the cells were 
metabolically labeled with a mixture of [^SJ-cysteine and [ 35 S]- 
methionine, and labeled secreted proteins were analyzed by SDS- 
polyacrylamide gel electrophoresis. As shown in Figure 6, additional 
bands were detected in cells transfected with a sense GDF-10 construct 

25 compared to an antisense control construct. The presence of multiple 



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protein species most likely indicates that 293 cells are capable of 
proteolytically processing GDF-10. Hence, these data suggest that 
GDF-10 is secreted by these cells and that GDF-10 is cleaved, as 
predicted from the cDNA sequence. 

5 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. 



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SEQUENCE LISTING 

(1) GENERAL INFORMATION: 

(i) APPLICANT: THE JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE 
(ii) TITLE OF INVENTION: GROWTH DIFFERENTIATION FACTOR-10 
5 (iii) NUMBER OF SEQUENCES: 26 

(iv) CORRESPONDENCE ADDRESS: 

(A) ADDRESSEE: Spensley Horn Jubas & Lubitz 

(B) STREET: 1880 Century Park East, Suite 500 
10 (C) CITY: Los Angeles 

(D) STATE: California 

(E) COUNTRY: USA 

(F) ZIP: 90067 

(v) COMPUTER READABLE FORM: 
15 (A) MEDIUM TYPE: Floppy disk 

(B) COMPUTER: IBM PC compatible 

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

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

(vi) CURRENT APPLICATION DATA: 
20 (A) APPLICATION NUMBER: PCT 

(B) FILING DATE: 07 -OCT- 19 94 

(C) CLASSIFICATION: 

(viii) ATTORNEY /AGENT INFORMATION: 

(A) NAME: LISA A. HAILE, PH.D. 
25 (B) REGISTRATION NUMBER: P- 38 ,347 

(C) REFERENCE /DOCKET NUMBER: FD-3054 PCT 

(ix) . TELECOMMUNICATION INFORMATION: 

(A) TELEPHONE: (619) 455-5100 

(B) TELEFAX: (619) 455-5110 

30 (2) INFORMATION FOR SEQ ID NO:l: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH : 36 base pairs 

(B) TYPE: nucleic acid 



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(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 

t 

(vii) IMMEDIATE SOURCE: 

5 (B) CLONE: NSC1 

(ix) FEATURE: 

(A) NAME /KEY: CDS 

(B) LOCATION: 1..36 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: 
10 CCGGAATTCA ARGTNGAYTT YGCNGAYATH GGNTGG 36 

(2) INFORMATION FOR SEQ ID NO: 2: 

(i) SEQUENCE CHARACTERISTICS : 

(A) LENGTH: 33 base pairs 

(B) TYPE: nucleic acid 
15 (C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 



(vii) IMMEDIATE SOURCE: 
(B) CLONE: NSC2 

20 (ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 1..33 



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

CCGGAATTCR CANGCRCARC TYTCNACNGT CAT 33 

25 (2) INFORMATION FOR SEQ ID NO: 3: 

(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base pairs 



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(B) TYPE: nucleic acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 



(vii) IMMEDIATE SOURCE: 
(B) CLONE: NSC3 

(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 1..33 



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

CCGGAATTCR CANGCRCANG AYTCNACNGT CAT 33 

(2) INFORMATION FOR SEQ ID NO: 4: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 2322 base pairs 
15 (B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 



(vii) IMMEDIATE SOURCE: 
20 (B) CLONE: Murine GDF-10 

(ix) FEATURE: 

(A) NAME/KEY: CDS 



(B) LOCATION: 126.. 1553 



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

TGGGGTCATC CGGGCTGTCC GAGTCCCACA GGGACAACTC CAGCCGCGGA CGAGGTGCAC 60 

AGCCAACACT GAGCCCTCCT TGTCTGTTCT CCTGGGCTCA GACCCTTCAC CACCGTTACT 120 

CAGCC ATG GCT CCA GGT CCT GCT CGG ATC AGC TTG GGG TCC CAG CTG 167 
5 Met Ala Pro Gly Pro Ala Arg lie Ser Leu Gly Ser Gin Leu 

1 5 10 

CTG CCC ATG GTG CCG CTG CTC CTG CTG CTG CGG GGC GCA GGC TGC GGC 215 
Leu Pro Met Val Pro Leu Leu Leu Leu Leu Arg Gly Ala Gly Cys Gly 
15 20 25 30 

10 CAC AGG GGC CCC TCA TGG TCC TCA TTG CCC TCG GCA GCT GCC GGT CTG 263 

His Arg Gly Pro Ser Trp Ser Ser Leu Pro Ser Ala Ala Ala Gly Leu 
35 40 45 

CAG GGG GAC AGG GAC TCC CAG CAG TCA CCC GGG GAC GCA GCA GCC GCT 311 
Gin Gly Asp Arg Asp Ser Gin Gin Ser Pro Gly Asp Ala Ala Ala Ala 
15 50 55 60 

CTG GGC CCA GGC GCC CAG GAC ATG GTC <5CT ATC CAC ATG CTC AGG CTC 359 
Leu Gly Pro Gly Ala Gin Asp Met Val Ala lie His Met Leu Arg Leu 
65 70 75 

TAT GAG AAG TAC AAC CGA AGA GGT GCT CCA CCG GGA GGA GGC AAC ACC 407 
20 Tyr Glu Lys Tyr Asn Arg Arg Gly Ala Pro Pro Gly Gly Gly Asn Thr 

80 85 90 

GTC CGA AGC TTC CGT GCC CGG CTG GAA ATG ATC GAC CAA AAG CCT GTG 455 
Val Arg Ser Phe Arg Ala Arg Leu Glu Met lie Asp Gin Lys Pro Val 
95 100 105 110 

25 TAT TTC TTC AAC TTG ACT TCC ATG CAA GAC TCA GAA ATG ATC CTC ACA 503 

Tyr Phe Phe Asn Leu Thr Ser Met Gin Asp Ser Glu Met lie Leu Thr 
115 120 125 

GCC GCC TTC CAC TTC TAC TCA <3AA CCT CCA CGG TGG CCC CGG GCT GGT 551 
Ala Ala Phe His Phe Tyr Ser Glu Pro Pro Arg Trp Pro Arg Ma Gly 
30 130 135 140 

GAG GTA TTC TGC AAG CCC CGA GCT AAG AAC GCA TCC TGC C<3C CTC CTG 599 
Glu Val Phe Cys Lys Pro Arg Ala Lys Asn Ala Ser Cys Arg Leu Leu 
145 150 155 



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ACC CCA GGG CTG CCT GCA CGC TTG CAC CTA ATC TTC CGC AGT CTT TCC 647 
Thr Pro Gly Leu Pro Ala Arg Leu His Leu lie Phe Arg Ser Leu Ser 
160 165 170 

CAG AAC ACC GCC ACT CAG GGG CTG CTC CGC GGG GCC ATG GCC CTG ACG 695 
5 Gin Asn Thr Ala Thr Gin Gly Leu Leu Arg Gly Ala Met Ala Leu Thr 
175 180 185 190 

CCT CCA CCA CGT GGC CTG TGG CAG GCC AAG GAC ATC TCC TCA ATC ATC 743 
Pro Pro Pro Arg Gly Leu Trp Gin Ala Lys Asp lie Ser Ser lie He 
195 200 205 

10 AAG GCT GCC CGA AGG GAT GGA GAG CTG CTT CTC TCT GCT CAG CTG GAT 791 

Lys Ala Ala Arg Arg Asp Gly Glu Leu Leu Leu Ser Ala Gin Leu Asp 
210 215 220 

ACT GGG GAG AAG GAC CCC GGA GTG CCA CGG CCC AGT TCC CAC ATG CCC 839 
Thr Gly <31u Lys Asp Pro Gly Val Pro Arg Pro Ser Ser His Met Pro 
15 225 230 235 

TAT ATC CTT GTC TAC GCC AAT GAC CTG GCC ATC TCC GAA CCC AAC AGT 887 
Tyr He Leu Val Tyr Ala Asn Asp Leu Ala He Ser Glu Pro Asn Ser 
240 245 250 

GTA GCA GTG TCG CTA CAG AGA TAC GAC CCA TTT CCA GCT GGA GAC TTT 935 
20 Val Ala Val Ser Leu Gin Arg Tyr Asp Pro Phe Pro Ala Gly Asp Phe 
255 260 265 270 

GAG CCT .GGA GCA CCC CCC AAC AGC TCA GCT GAT CCC CGC GTG CGC AGG 983 
Glu Pro Gly Ala Ala Pro Asn Ser Ser Ala Asp Pro Arg Val Arg Arg 
275 280 285 

25 GCG GCT CAG GTG TCA AAA CCC CTG CAA GAC AAT GAA CTG CCG GGG CTG 1031 

Ala Ala Gin Val Ser Lys Pro Leu Gin Asp Asn Glu Leu Pro Gly Leu 
290 295 30 0 

GAT GAA AGA CCA GCG CCT GCC CTG CAT GCC CAG AAT TTC CAC AAG CAC 1079 
Asp Glu Arg Pro Ala Pro Ala Leu His Ala Gin Asn Phe His Lys His 
30 305 310 315 



GAG TTC TGG 
Glu Phe Trp 
320 



TCC 
Ser 



AGT CCT TTC GGG GCA CTG AAA CCC CGC ACG -GCG GGC 
Ser Pro Phe Arg Ala Leu Lys Pro Arg Thr Ala Arg 
325 330 



1127 



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AAA GAC CGC AAG AAG AAG GAC CAG GAC ACA TTC ACC GCC GCC TCC TCT 1175 
Lys Asp Arg Lys Lys Lys Asp Gin Asp Thr Phe Thr Ala Ala Ser Ser 
335 340 345 350 

CAG GTG CTG GAC TTT GAC GAG AAG ACG ATG CAG AAA GCC AGG AGG CGG 1223 
5 Gin Val Leu Asp Phe Asp Glu Lys Thr Met Gin Lys Ala Arg Arg Arg 

355 360 365 

CAG TGG GAT GAG CCC CGG GTC TGC TCC AGG AGG TAC CTG AAG GTG GAT 1271 
Gin Trp Asp Glu Pro Arg Val Cys Ser Arg Arg Tyr Leu Lys Val Asp 
370 375 380 

10 TTT GCA GAC ATC GGG TGG AAT GAA TGG ATC ATC TCT CCC AAA TCC TTT 1319 

Phe Ala Asp lie Gly Trp Asn Glu Trp lie lie Ser Pro Lys Ser Phe 
385 390 395 



GAC GCC TAC TAC TGT GCT GGG GCC TGC GAG TTC CCC ATG CCC AAG ATT 1367 
Asp Ala Tyr Tyr Cys Ala Gly Ala Cys Glu Phe Pro Met Pro Lys lie 
15 400 405 410 



GTC CGC CCA TCC AAC CAT GCC ACC 
Val Arg Pro Ser Asn His Ala Thr 
415 420 

GGC ATT GTC CCT GGC ATC CCA GAG 
20 Gly He Val Pro Gly He Pro Glu 

435 



ATC CAG AGC ATC GTC AGA GCT GTG 1415 
He Gin Ser He Val Arg Ala Val 
425 430 

CCA TGC TGT GTT CCA GAC AAG ATG 1463 
Pro Cys Cys Val Pro Asp Lys Met 
440 445 



AAC TCC CTT GGA GTC CTT TTC CTG GAT GAA AAT CGG AAT GCG GTT CTG 1511 
Asn Ser Leu Gly Val Leu Phe Leu Asp Glu Asn Arg Asn Ala Val Leu 
450 455 460 



25 AAG GTG TAC CCC AAT ATG TCC GTA GAG ACC TGT GCC TGT CGG IS 53 

Lys Val Tyr Pro Asn Met Ser Val Glu Thr Cys Ala Cys Arg 
465 470 475 



TAAGATGGCT TCAAGATAGA AGACAGACCT -GCTTCATCCC TGCCCTGCAG AGTGGCAATC 1613 
TTGGAGCCAG GGACTTGACT CGGGGAGGTT CCAGGTGCTA GACAGAGCTT ACAGGCAGCC 1673 
30 CTGCTGGGAC CAAGAAAGAT CTGCCCACCA CATCGCAATT CTTCAGTTCT TCCGTGCTGG 1733 



TGGTAGCTCT GTAAAGACGT GTTGAGTTCC TGGAAGAAAT CTGGAATTAA CTGTGGTCTG 1793 



CAATTTGCCC ATCATCCCTG CCCACACTTT TCAAGGCCTA GAAATAACGT GTGTCCTCAA 1853 



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-37- 

ATGTCAACTC CAGGCATTTG TCCTCTCAAA ACCTAGAAAG ACTATGCAAA TCTTGGGGTA 1913 
CTCCCCCCCC CCATGGCAGT TTAAATGCTG TTTTAAAACC CTCAGGCTGC ATTCTAGAAA 1973 
CAGGGCCTAA CCCATGGCAC GAGTGAGTAT TTTCTCTTAC GTTTCACTAC ACGTGCTTTT 2033 
ATACATGCAG TATGCACATG TAATCACGGT TGATTTCTTC TTTTAATATA TGTATTTCTA 2093 
5 TTTCAAAGCA AAACGGAGAG AGTCGATCCC ATCCCCTGCA GAGGTAATAA TGCAAGTTAG 2153 

GTGTGGGTTG TCTAAGCATG TGTATGGAAA TAATACATAC AGTAATATGC TGGAATACTA 2213 
AAAAAGTAAC CAAGATTTTA TATTTTTGTA AATTATACTT TGTATACTGT AGATTGTGAG 2273 
TGTTCTGTGT TTTTATGGAA AGCTAATAAA TTAAAGGTGC GGAGGTATC 2322 

(2) INFORMATION FOR SEQ ID NO: 5: 

10 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 476 amino acids 

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

(ii) MOLECULE TYPE: protein 

15 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: 

Met Ala Pro Gly Pro Ala Arg lie Ser Leu Gly Ser Gin Leu Leu Pro 
15 10 15 

Met Val Pro Leu Leu Leu Leu Leu Arg Gly Ala Gly Cys Gly His Arg 
20 25 30 

20 Gly Pro Ser Trp Ser Ser Leu Pro Ser Ala Ala Ala Gly Leu Gin Gly 
35 40 45 

Asp Arg Asp Ser Gin Gin Ser Pro Gly Asp Ala Ala Ala Ala Leu Gly 
50 55 60 

Pro Gly Ala Gin Asp Met Val Ala lie His Met Leu Arg Leu Tyr <31u 
25 65 70 75 80 

Lys Tyr Asn Arg Arg Gly Ala Pro Pro Gly Gly Gly Asn Thr Val Arg 
85 90 95 



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-38- 



Ser Phe Arg Ala Arg Leu Glu Met lie Asp Gin Lys Pro Val Tyr Phe 
100 105 110 

Phe Asn Leu Thr Ser Met Gin Asp Ser Glu Met lie Leu Thr Ala Ala 
115 120 125 

5 Phe His Phe Tyr Ser Glu Pro Pro Arg Trp Pro Arg Ala Gly Glu Val 

130 135 140 

Phe Cys Lys Pro Arg Ala Lys Asn Ala Ser Cys Arg Leu Leu Thr Pro 
145 150 155 160 

Gly Leu Pro Ala Arg Leu His Leu He Phe Arg Ser Leu Ser Gin Asn 
10 165 170 175 



Thr Ala Thr Gin Gly Leu Leu Arg 
180 

Pro Arg Gly Leu Trp Gin Ala Lys 
195 200 

15 Ala Arg Arg Asp Gly Glu Leu Leu 

210 215 

Glu Lys Asp Pro Gly Val Pro Arg 
225 230 

Leu Val Tyr Ala Asn Asp Leu Ala 
20 245 

Val Ser Leu Gin Arg Tyr Asp Pro 
260 



Gly Ala Met Ala Leu Thr Pro Pro 
185 190 

Asp He Ser Ser He He Lys Ala 
205 

Leu Ser Ala Gin Leu Asp Thr Gly 
220 

Pro Ser Ser His Met Pro Tyr He 
235 240 

He Ser Glu Pro Asn Ser Val Ala 
2S0 255 

Phe Pro Ala Gly Asp Phe Glu Pro 
265 270 



Gly Ala Ala Pro Asn Ser Ser Ala Asp Pro Arg Val Arg Arg Ala Ala 
275 280 285 

25 Gin Val Ser Lys Pro Leu Gin Asp Asn Glu Leu Pro Gly Leu Asp Glu 

290 295 300 

Arg Pro Ala Pro Ala Leu His Ala Gin Asn Phe His Lys His Glu Phe 
305 310 315 320 

Trp Ser Ser Pro Phe Arg Ala Leu Lys Pro Arg Thr Ala Arg Lys Asp 
30 325 330 335 



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Arg Lys Lys Lys Asp Gin Asp Thr Phe Thr Ala Ala Ser Ser Gin Val 
340 345 350 

Leu Asp Phe Asp Glu Lys Thr Met Gin Lys Ala Arg Arg Arg Gin Trp 
355 360 365 

5 Asp Glu Pro Arg Val Cys Ser Arg Arg Tyr Leu Lys Val Asp Phe Ala 
370 375 380 

Asp lie Gly Trp Asn Glu Trp lie lie Ser Pro Lys Ser Phe Asp Ala 
385 390 395 400 

> 

Tyr Tyr Cys Ala Gly Ala Cys Glu Phe Pro Met Pro Lys lie Val Arg 
10 405 410 415 

Pro Ser Asn His Ala Thr lie Gin Ser He Val Arg Ala Val Gly He 
420 425 430 

Val Pro Gly He Pro Glu Pro Cys Cys Val Pro Asp Lys Met Asn Ser 
435 440 445 

15 Leu Gly Val Leu Phe Leu Asp Glu Asn Arg Asn Ala Val Leu Lys Val 

450 455 460 

Tyr Pro Asn Met Ser Val Glu Thr Cys Ala Cys Arg 
465 470 475 

(2) INFORMATION FOR SEQ ID NO: 6: 

20 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 120 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 



25 (ii) MOLECULE TYPE: protein 



(vii) IMMEDIATE SOURCE: 
(B) CLONE: GDF-10 

(ix) FEATURE: 

(A) NAME/ KEY: Protein 
30 (B) LOCATION: 1..120 



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-40- 



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

Glu Lys Ser Met Gin Lys Ala Arg Arg Arg Gin Trp Asp Glu Pro Arg 
15 10 15 

Val Cys Ser Arg Arg Tyr Leu Lys Val Asp Phe Ala Asp lie Gly Trp 
5 20 25 30 

Asn Glu Trp lie lie Ser Pro Lys Ser Phe Asp Ala Tyr Tyr Cys Ala 
35 40 45 

Gly Ala Cys Glu Phe Pro Met Pro Lys lie Val Arg Pro Ser Asn His 
50 55 60 

10 Ala Thr He Gin Ser He Val Arg Ala Val Gly He Val Pro Gly He 

65 70 75 80 

Pro Glu Pro Cys Cys Val Pro Asp Lys Met Asn Ser Leu Gly Val Leu 
85 90 95 

Phe Leu Asp Glu Asn Arg Asn Ala Val Leu Lys Val Tyr Pro Asn Met 
15 100 105 110 

Ser Val Glu Thr Cys Ala Cys Arg 
115 120 

(2) INFORMATION FOR SEQ ID NO: 7: 

(i) SEQUENCE CHARACTERISTICS: 
20 (A) LENGTH: 123 amino acids 

(B) TYPE: amino acid 

(C) STRAND EDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



25 (vii) IMMEDIATE SOURCE: 

(B) CLONE: <5DF-1 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1. .123 



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-41- 



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

Arg Pro Arg Arg Asp Ala Glu Pro Val Leu Gly Gly Gly Pro Gly Gly 
15 10 15 

Ala Cys Arg Ala Arg Arg Leu Tyr Val Ser Phe Arg Glu Val Gly Trp 
5 20 25 30 

His Arg Trp Val lie Ala Pro Arg Gly Phe Leu Ala Asn Tyr Cys Gin 
35 40 45 

Gly Gin Cys Ala Leu Pro Val Ala Leu Ser Gly Ser Gly Gly Pro Pro 
50 55 60 

10 Ala Leu Asn His Ala Val Leu Arg Ala Leu Met His Ala Ala Ala Pro 

65 70 75 80 

Gly Ala Ala Asp Leu Pro Cys Cys Val Pro Ala Arg Leu Ser Pro lie 
85 90 95 

Ser Val Leu Phe Phe Asp Asn Ser Asp Asn Val Val Leu Arg Gin Tyr 
15 100 105 no 

Glu Asp Met Val Val Asp Glu Cys Gly Cys Arg 
115 120 

(2) INFORMATION FOR SEQ ID NO: 8: 

(i) SEQUENCE CHARACTERISTICS: 
20 (A) LENGTH: 118 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



25 (vii) IMMEDIATE SOURCE: 

(B) CLONE: GDF-3 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..118 



r 



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-42- 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: 

Arg Lys Arg Arg Ala Ala He Ser Val Pro Lys Gly Phe Cys Arg Asn 
x 5 10 15 

^ Phe Cys His Arg His Gin Leu Phe He Asn Phe Gin Asp Leu Gly Trp 

5 20 25 30 

His Lys Trp Val He Ala Pro Lys Gly Phe Met Ala Asn Tyr Cys His 
35 40 45 

Gly Glu Cys Pro Phe Ser Met Thr Thr Tyr Leu Asn Ser Ser Asn Tyr 
50 55 60 

10 Ala Phe Met Gin Ala Leu Met His Met Ala Asp Pro Lys Val Pro Lys 

65 70 75 80 

Ala Val Cys Val Pro Thr Lys Leu Ser Pro lie Ser Met Leu Tyr Gin 
85 90 95 

Asp Ser Asp Lys Asn Val He Leu Arg His Tyr Glu Asp Met Val Val 
15 100 105 110 

Asp Glu Cys Gly Cys Gly 
115 

(2) INFORMATION FOR SEQ ID NO: 9: 

(i) SEQUENCE CHARACTERISTICS: 
20 (A) LENGTH: 119 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



25 (vii) IMMEDIATE SOURCE: 

(B) CLONE: GDF-9 

(ix) FEATURE: 

(A) NAME /KEY: Protein 

(B) LOCATION: 1..119 



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-43- 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: 

Ser Phe Asn Leu Ser Glu Tyr Phe Lys Gin Phe Leu Phe Pro Gin Asn 
1 5 10 15 - 

Glu Cys Glu Leu His Asp Phe Arg Leu Ser Phe Ser Gin Leu Lys Trp 
5 20 25 30 

Asp Asn Trp lie Val Ala Pro His Arg Tyr Asn Pro Arg Tyr Cys Lys 
35 40 45 

Gly Asp Cys Pro Arg Ala Val Arg His Arg Tyr Gly Ser Pro Val His 
50 55 60 

10 Thr Met Val Gin Asn He He Tyr Glu Lys Leu Asp Pro Ser Val Pro 

65 70 75 80 

Arg Pro Ser Cys Val Pro Gly Lys Tyr Ser Pro Leu Ser Val Leu Thr 
85 90 95 

He Glu Pro Asp Gly Ser He Ala Tyr Lys Glu Tyr Glu Asp Met He 
15 100 105 110 

Ala Thr Arg Cys Thr Cys Arg 
115 

(2) INFORMATION FOR SEQ ID NO: 10: 

(i) SEQUENCE CHARACTERISTICS: 
20 (A) LENGTH: 118 amino acids 

(B) TYPE : amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



25 (vii) IMMEDIATE SOURCE: 

(B) CLONE: BMP -2 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..118 



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-44- 



<xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: 

Arg Glu Lys Arg Gin Ala Lys His Lys Gin Arg Lys Arg Leu Lys Ser 
15 10 15 

Ser Cys Lys Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp 
5 20 25 30 

Asn Asp Trp lie Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His 
35 40 45 

Gly Glu Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His 
50 55 60 

10 Ala He Val Gin Thr Leu Val Asn Ser Val Asn Ser Lys He Pro Lys 

65 70 75 80 

Ala Cys Cys Val Pro Thr Glu Leu Ser Ala He Ser Met Leu Tyr Leu 
85 90 95 

Asp Glu Asn Glu Lys Val Val Leu Lys Asn Tyr Gin Asp Met Val Val 
15 100 105 110 

Glu Gly Cys Gly Cys Arg 
115 

(2) INFORMATION FOR SEQ ID NO: 11: 

(i) SEQUENCE CHARACTERISTICS: 
20 (A) LENGTH: 116 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



25 (vii) IMMEDIATE SOURCE: 

(B) CLONE: BMP-4 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..118 



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-45- 



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

Lys Arg Ser Pro Lys His His Ser Gin Arg Ala Arg Lys Lys Asn Lys 
1 5 10 15 . 

Asn Cys Arg Arg His Ser Leu Tyr Val Asp Phe Ser Asp Val Gly Trp 
5 20 25 30 

Asn Asp Trp lie Val Ala Pro Pro Gly Tyr Gin Ala Phe Tyr Cys His 
35 40 45 

Gly Asp Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His 
50 55 60 

10 Ala He Val Gin Thr Leu Val Asn Ser Val Asn Ser Ser He Pro Lys 

65 70 75 80 

Ala Cys Cys Val Pro Thr Glu Leu Ser Ala He Ser Met Leu Tyr Leu 
85 90 95 

Asp Glu Tyr Asp Lys Val Val Leu Lys Asn Tyr Gin Glu Met Val Val 
15 100 105 110 

Glu Gly Cys Gly Cys Arg 
115 

(2) INFORMATION FOR SEQ ID NO: 12: 

(i) SEQUENCE CHARACTERISTICS: 
20 (A) LENGTH: 119 amino acids 

(B) TYPE: amino acid 
<C) STRANDEDNESS : single 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



25 (vii) IMMEDIATE SOURCE: 

(B) CLONE: Vgr-1 

(ix) FEATURE: 

(A) NAME /KEY: Protein 

(B) LOCATION: 1..119 



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-46- 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: 

Ser Arg Gly Ser Gly Ser Ser Asp Tyr Asn Gly Ser Glu Leu Lys Thr 
1 5 10 15 

Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Gin Asp Leu Gly Trp 
5 20 25 30 

Gin Asp Trp lie lie Ala Pro Lys Gly Tyr Ala Ala Asn Tyr Cys Asp 
35 40 45 

Gly Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His 
50 55 60 

10 Ala He Val Gin Thr Leu Val His Leu Met Asn Pro Glu Tyr Val Pro 

65 70 75 80 

Lys Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala He Ser Val Leu Tyr 
85 90 95 

Phe Asp Asp Asn Ser Asn Val He Leu Lys Lys Tyr Arg Asn Met Val 
15 100 105 no 

Val Arg Ala Cys Gly Cys His 
115 

(2) INFORMATION FOR SEQ ID NO: 13: 

(i) SEQUENCE CHARACTERISTICS: 
20 (A) LENGTH: 119 amino acids 

(B) TYPE : amino acid 

(C) STRANDEDNESS: single 
<D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



25 (vii) IMMEDIATE SOURCE: 

(B) CLONE: OP-1 

(ix) FEATURE: 

(A) NAME /KEY: Protein 

(B) LOCATION: 1..119 



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PCTYUS94/11440 



-47- 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: 

Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser Ser Asp Gin Arg Gin 
1 5 10 15 

£ Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp 

5 20 25 30 

Gin Asp Trp lie lie Ala Pro Glu Gly Tyr Ala Ala Tyr Tyr Cys <3lu 
35 40 45 

Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn Ala Thr Asn His 
50 55 60 

10 Ala He Val Gin Thr Leu Val His Phe He Asn Pro <31u Thr Val Pro 

65 70 75 80 

Lys Pro Cys Cys Ala Pro Thr Gin lieu Asn Ala He Ser Val Leu Tyr 
85 90 95 

Phe Asp Asp Ser Ser Asn Val He Leu Lys Lys Tyr Arg Asn Met Val 
15 100 105 110 

Val Arg Ala Cys Gly Cys His 
115 

(2) INFORMATION FOR SEQ ID NO: 14: 

(i) SEQUENCE CHARACTERISTICS: 
20 (A) LENGTH : 119 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



25 (vii) IMMEDIATE SOURCE : 

(B) CLONE: BMP- 5 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..119 



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-48- 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: 

Ser Arg Met Ser Ser Val Gly Asp Tyr Asn Thr Ser Glu Gin Lys Gin 
1 5 10 15 

Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp 
5 20 25 30 

Gin Asp Trp lie lie Ala Pro Glu Gly Tyr Ala Ala Phe Tyr Cys Asp 
35 40 45 

Gly Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His 
50 55 60 

10 Ala lie Val Gin Thr Leu Val His Leu Met Phe Pro Asp His Val Pro 

65 70 75 80 

Lys Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala lie Ser Val Leu Tyr 
85 90 95 

Phe Asp Asp Ser Ser Asn Val lie Leu Lys Lys Tyr Arg Asn Met Val 
15 100 105 110 

Val Arg Ser Cys Gly Cys His 
115 

(2) INFORMATION FOR SEQ ID NO: 15: 

(i) SEQUENCE CHARACTERISTICS: 
20 (A) LENGTH: 119 amino acids 

(6) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



25 (vii) IMMEDIATE SOURCE: 

(B) CLONE: OP-2 

(ix) FEATURE: 

(A) NAME /KEY: Protein 

(B) LOCATION: 1. .119 



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-49- 



<xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: 



Arg Leu Pro Gly lie Phe Asp Asp Val His Gly Ser His Gly Arg Gin 

1 5 10 15 

Val Cys Arg Arg His Glu Leu Tyr Val Ser Phe Gin Asp Leu Gly Trp 
5 20 25 30 



Leu Asp Trp Val He Ala Pro Gin Gly Tyr Ser Ala Tyr Tyr Cys Glu 
35 40 45 

Gly Glu Cys Ser Phe Pro Leu Asp Ser Cys Met Asn Ala Thr Asn His 
50 55 60 

10 Ala He Leu Gin Ser Leu Val His Leu Met Lys Pro Asn Ala Val Pro 

65 70 75 80 

Lys Ala Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr Ser Val Leu Tyr 
65 90 95 

Tyr Asp Ser Ser Asn Asn Val He Leu Arg Lys Ala Arg Asn Met Val 
15 100 105 110 

Val Lys Ala Cys Gly Cys His 
115 



(2) INFORMATION FOR SEQ ID NO: 16: 



(i) SEQUENCE CHARACTERISTICS : 
20 (A) LENGTH: 120 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



25 (vii) IMMEDIATE SOURCE: 

(B) CLONE: BMP-3 



( ix) FEATURE : 

(A) NAME/KEY: Protein 
<B) LOCATION: 1. .120 



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-50- 



<xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: 

Glu Gin Thr Leu Lys Lys Ala Arg Arg Lys Gin Trp He Glu Pro Arg 
15 10 15 

Asn Cys Ala Arg Arg Tyr Leu Lys Val Asp Phe Ala Asp He Gly Trp 
5 20 25 30 

Ser Glu Trp He He Ser Pro Lys Ser Phe Asp Ala Tyr Tyr Cys Ser 
35 40 45 

Gly Ala Cys Gin Phe Pro Met Pro Lys Ser Leu Lys Pro Ser Asn His 
50 55 60 

10 Ala Thr lie Gin Ser He Val Arg Ala Val Gly Val Val Pro Gly He 

65 70 75 80 

Pro Glu Pro Cys Cys Val Pro Glu Lys Met Ser Ser Leu Ser He Leu 
85 90 95 

Phe Phe Asp Glu Asn Lys Asn Val Val Leu Lys Val Tyr Pro Asn Met 
15 100 105 110 

Thr Val Glu Ser Cys Ala Cys Arg 
115 120 

(2) INFORMATION FOR SEQ ID NO: 17: 

(i) SEQUENCE CHARACTERISTICS: 
20 (A) LENGTH : 116 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



25 (vii) IMMEDIATE SOURCE: 

(8) CLONE: MIS 



(ix) 



FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..116 



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-51- 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: 

Gly Pro Gly Arg Ala Gin Arg Ser Ala Gly Ala Thr Ala Ala Asp Gly 
15 10 15 

Pro Cys Ala Leu Arg Glu Leu Ser Val Asp Leu Arg Ala Glu Arg Ser 
5 20 25 30 

Val Leu He Pro Glu Thr Tyr Gin Ala Asn Asn Cys Gin Gly Val Cys 
35 40 45 

Gly Trp Pro Gin Ser Asp Arg Asn Pro Arg Tyr Gly Asn His Val Val 
50 55 60 

10 Leu Leu Leu Lys Met Gin Ala Arg Gly Ala Ala Leu Ala Arg Pro Pro 

65 70 75 80 

Cys Cys Val Pro Thr Ala Tyr Ala Gly Lys Leu Leu He Ser Leu Ser 
85 90 95 

Glu Glu Arg He Ser Ala His His Val Pro Asn Met Val Ala Thr Glu 
15 100 105 no 

Cys Gly Cys Arg 
115 

(2) INFORMATION FOR SEQ ID NO: 18: 

(i) SEQUENCE CHARACTERISTICS: 
20 (A) LENGTH: 122 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii> MOLECULE TYPE: protein 



25 (vii) IMMEDIATE SOURCE: 

(B) CLONE: Inhibin-alpha 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..122 



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-52- 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: 

Ala Leu Arg Leu Leu Gin Arg Pro Pro Glu Glu Pro Ala Ala His Ala 
! 5 10 15 

Asn Cys His Arg Val Ala Leu Asn He Ser Phe Gin Glu Leu Gly Trp 
5 20 25 30 

Glu Arg Trp He Val Tyr Pro Pro Ser Phe He Phe His Tyr Cys His 
35 40 45 

Gly Gly Cys Gly* Leu His He Pro Pro Asn Leu Ser Leu Pro Val Pro 
50 55 60 

10 Gly Ala Pro Pro Thr Pro Ala Gin Pro Tyr Ser Leu Leu Pro Gly Ala 

65 70 75 80 

Gin Pro Cys Cys Ala Ala Leu Pro Gly Thr Met Arg Pro Leu His Val 
85 90 95 

Arg Thr Thr Ser Asp Gly Gly Tyr Ser Phe Lys Tyr Glu Thr Val Pro 
15 100 105 110 

Asn Leu Leu Thr Gin His Cys Ala Cys He 
115 120 

(2) INFORMATION FOR SEQ ID NO: 19: 

(i) SEQUENCE CHARACTERISTICS : 
20 (A) LENGTH: 121 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



25 (vii) IMMEDIATE SOURCE: 

(B) CLONE: Inhibin-beta-A 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1.-121 



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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: 

Arg Arg Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys Val Asn He Cys 
15 10 15 

Cys Lys Lys Gin Phe Phe Val Ser Phe Lys Asp He Gly Trp Asn Asp 
5 20 25 30 

Trp He He Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu 
35 40 45 

Cys Pro Ser His He Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His 
50 55 60 

10 Ser Thr Val He Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala 

65 70 75 80 

Asn Leu Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met 
85 90 95 

Leu Tyr Tyr Asp Asp Gly Gin Asn He He Lys Lys Asp He Gin Asn 
15 100 105 110 

Met He Val Glu Glu Cys Gly Cys Ser 
115 120 

(2) INFORMATION FOR SEQ ID NO: 20: 

(i) SEQUENCE CHARACTERISTICS: 
20 (A) LENGTH: 120 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



25 (vii) IMMEDIATE SOURCE: 

(B) CLONE: Inhibin-beta-B 

(ix) FEATURE: 

{A) NAME/KEY: Protein 
(B) LOCATION: 1. .120 



WO 95/10539 



PCT/US94/11440 



-54- 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: 

Arg He Arg Lys Arg Gly Leu Glu Cys Asp Gly Arg Thr Asn Leu Cys 
1 5 10 15 

Cys Arg Gin Gin Phe Phe He Asp Phe Arg Leu He Gly Trp Asn Asp 
5 20 25 30 

Trp He He Ala Pro Thr Gly Tyr Tyr Gly Asn Tyr Cys Glu Gly Ser 
35 40 45 

Cys Pro Ala Tyr Leu Ala Gly Val Pro Gly Ser Ala Ser Ser Phe His 
50 55 60 

10 Thr Ala Val Val Asn Gin Tyr Arg Met Arg Gly Leu Asn Pro Gly Thr 

65 70 75 80 

Val Asn Ser Cys Cys He Pro Thr Lys Leu Ser Thr Met Ser Met Leu 
85 90 95 

Tyr Phe Asp Asp Glu Tyr Asn He Val Lys Arg Asp Val Pro Asn Met 
15 100 105 110 

He Val Glu Glu Cys Gly Cys Ala 
115 120 

(2) INFORMATION FOR SEQ ID NO: 21: 

(i) SEQUENCE CHARACTERISTICS: 
20 (A) LENGTH: 118 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



25 (vii) IMMEDIATE SOURCE: 

{B) CLONE: Nodal 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..118 



WO 95/10539 



PCT/US94/11440 



-55- 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 

Gly Trp Gly Arg Arg Gin Arg Arg His His Leu Pro Asp Arg Ser Gin 
15 10 15 

Leu Cys Arg Arg Val Lys Phe Gin Val Asp Phe Asn Leu lie Gly Trp 
5 20 25 30 

Gly Ser Trp lie lie Tyr Pro Lys Gin Tyr Asn Ala Tyr Arg Cys Glu 
35 40 45 

Gly Glu Cys Pro Asn Pro Val Gly Glu Glu Phe His Pro Thr Asn His 
50 55 60 

10 Ala Tyr lie Gin Ser Leu Leu Lys Arg Tyr Gin Pro His Arg Val Pro 

€5 70 75 80 

Ser Thr Cys Cys Ala Pro Val Lys Thr Lys Pro Leu Ser Met Leu Tyr 
85 90 95 

Val Asp Asn Gly Arg Val Leu Leu Glu His His Lys Asp Met lie Val 
15 100 105 110 

Glu Glu Cys Gly Cys Leu 
115 

(2) INFORMATION FOR SEQ ID NO: 22: 

(i) SEQUENCE CHARACTERISTICS: 
20 (A) LENGTH: 114 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



25 (vii) IMMEDIATE SOURCE: 

(B) CLONE: TGF-beta-1 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..114 



WO 95/10539 



PCT/US94/11440 



-56- 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 

Arg Arg Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser Thr Glu Lys Asn 
15 10 15 

Cys Cys Val Arg Gin Leu Tyr lie Asp Phe Arg Lys Asp Leu Gly Trp 
5 20 25 30 

Lys Trp He His Glu Pro Lys Gly Tyr His Ala Asn Phe Cys Leu Gly 
35 40 45 

Pro Cys Pro Tyr He Trp Ser Leu Asp Thr Gin Tyr Ser Lys Val Leu 
50 55 60 

10 Ala Leu Tyr Asn Gin His Asn Pro Gly Ala Ser Ala Ala Pro Cys Cys 

65 70 75 80 

Val Pro Gin Ala Leu Glu Pro Leu Pro He Val Tyr Tyr Val Gly Arg 
85 90 95 

Lys Pro Lys Val Glu Gin Leu Ser Asn Met He Val Arg Ser Cys Lys 
15 100 105 110 

Cys Ser 



(2) INFORMATION FOR SEQ ID NO: 23: 

(i) SEQUENCE CHARACTERISTICS: 
20 (A) LENGTH: 114 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



25 (vii) IMMEDIATE SOURCE: 

(B) CLONE: TGF-beta-2 



(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..114 



WO 95/10539 



PCT/US94/11440 



-57- 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: 

Lys Arg Ala Leu Asp Ala Ala Tyr Cys Phe Arg Asn Val Gin Asp Asn 
15 10 15 

£ Cys Cys Leu Arg Pro Leu Tyr lie Asp Phe Lys Arg Asp Leu Gly Trp 

5 20 25 30 

Lys Trp lie His Glu Pro Lys Gly Tyr Asn Ala Asn Phe Cys Ala Gly 
35 40 45 

Ala Cys Pro Tyr Leu Trp Ser Ser Asp Thr Gin His Ser Arg Val Leu 
50 55 60 

10 Ser Leu Tyr Asn Thr lie Asn Pro Glu Ala Ser Ala Ser Pro Cys Cys 

65 70 75 80 

Val Ser Gin Asp Leu Glu Pro Leu Thr lie Leu Tyr Tyr lie Gly Lys 
85 90 95 

Thr Pro Lys He Glu Gin Leu Ser Asn Met He Val Lys Ser Cys Lys 
15 100 105 110 

Cys Ser 



(2) INFORMATION FOR SEQ ID NO: 24: 

(i) SEQUENCE CHARACTERISTICS: 
20 (A) LENGTH: 114 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



25 (vii) IMMEDIATE SOURCE: 

(B) CLONE: TGF-beta-3 

(ix) FEATURE : 

(A) NAME/KEY: Protein 
<B) LOCATION: 1..114 



WO 95/10539 



PCT/US94/11440 



-58- 



<xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: 

Lys Arg Ala Leu Asp Thr Asn Tyr Cys Phe Arg Asn Leu Glu Glu Asn 
15 10 15 

Cys Cys Val Arg Pro Leu Tyr lie Asp Phe Arg Gin Asp Leu Gly Trp 
5 20 25 30 

Lys Trp Val His Glu Pro Lys Gly Tyr Tyr Ala Asn Phe Cys Ser Gly 
35 40 45 

Pro Cys Pro Tyr Leu Arg Ser Ala Asp Thr Thr His Ser Thr Val Leu 
50 55 60 

10 Gly Leu Tyr Asn Thr Leu Asn Pro Glu Ala Ser Ala Ser Pro Cys Cys 

65 70 75 80 

Val Pro Gin Asp Leu Glu Pro Leu Thr lie Leu Tyr Tyr Val Gly Arg 
85 90 95 

Thr Pro Lys Val Glu Gin Leu Ser Asn Met Val Val Lys Ser Cys Lys 
15 100 105 110 

Cys Ser 



(2) INFORMATION FOR SEQ ID NO: 25: 

(i) SEQUENCE CHARACTERISTICS: 
20 (A) LENGTH: 115 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



25 (vii) IMMEDIATE SOURCE: 

(B) CLONE: Human GDF-10 

(ix) FEATURE: 

(A) NAME /KEY: Protein 

(B) LOCATION: 1..115 



WO 95/10539 



PCT/US94/11440 



-59- 



<xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 

Lys Ala Arg Arg Lys Gin Trp Asp Glu Pro Arg Val Cys Ser Arg Arg 
15 10 15 

Tyr Leu Lys Val Asp Phe Ala Asp lie Gly Trp Asn Glu Trp lie He 
5 20 25 30 

Ser Pro Lys Ser Phe Asp Ala Tyr Tyr Cys Ala Gly Ala Cys Glu Phe 
35 40 45 

Pro Met Pro Lys He Val Arg Pro Ser Asn His Ala Thr He Gin Ser 
50 55 60 

10 He Val Arg Ala Val Gly He He Pro Gly He Pro Glu Pro Cys Cys 

65 70 75 80 

Val Pro Asp Lys Met Asn Ser Leu Gly Val Leu Phe Leu Asp Glu Asn 
85 90 95 

Arg Asn Val Val Leu Lys Val Tyr Pro Asn Met Ser Val Asp Thr Cys 
15 100 105 110 

Ala Cys Arg 
115 

(2) INFORMATION FOR SEQ ID NO: 26: 

(i) SEQUENCE CHARACTERISTICS: 
20 (A) LENGTH: 115 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



25 (vii) IMMEDIATE SOURCE: 

(B) CLONE: Murine GDF-10 

(ix) FEATURE: 

(A) NAME /KEY: Protein 

(B) LOCATION: 1..115 



WO 95/10539 



PCT/US94/11440 



-60- 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: 

Lys Ala Arg Arg Lys Gin Trp Asp Glu Pro Arg Val Cys Ser Arg Arg 
15 10 15 

^ Tyr Leu Lys Val Asp Phe Ala Asp lie Gly Trp Asn Glu Trp lie lie 

5 20-25 30 

Ser Pro Lys Ser Phe Asp Ala Tyr Tyr Cys Ala Gly Ala Cys Glu Phe 
35 40 45 

Pro Met Pro Lys lie Val Arg Pro Ser Asn His Ala Thr He Gin Ser 
50 55 60 

10 He Val Arg Ala Val Gly He Val Pro Gly He Pro Glu Pro Cys Cys 

65 70 75 80 

Val Pro Asp Lys Met Asn Ser Leu Gly Val Leu Phe Leu Asp Glu Asn 
85 90 95 

Arg Asn Ala Val Leu Lys Val Tyr Pro Asn Met Ser Val Glu Thr Cys 
15 100 105 110 



Ala Cys Arg 
115 



WO 95/10539 PCT/US94/11440 

-61- 

CLAIMS 

1. Substantially pure growth differentiation factor-10 (GDF-10) 
and functional fragments thereof. 

2. An isolated polynucleotide sequence encoding the GDF-10 
polypeptide of claim 1 . 

3. The polynucleotide sequence of claim 2, wherein the polynu- 
cleotide is isolated from a mammalian cell. 

4. The polynucleotide of claim 3, wherein the mammalian cell is 
selected from the group consisting of mouse, rat, and human 
cell. 

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

6. The vector of claim 5, wherein the vector is a plasmid. 

7. The vector of claim 5, wherein the vector is a virus. 

8. A host cell stably transformed with the vector of claim 5. 

9. The host cell of claim 8, wherein the cell is prokaryotic. 

10. The host cell of claim 8, wherein the cell is eukaryotic. 

11. Antibodies reactive with the polypeptide of claim 1 or 
fragments thereof. 



WO 95/10539 



PCT/US94/11440 



-62- 

12. The antibodies of claim 11 , wherein the antibodies are polyclonal. 

13. The antibodies of claim 11, wherein the antibodies are 
monoclonal. 

14. A method of detecting a cell proliferative disorder comprising 
contacting the antibody of claim 11 with a specimen of a 
subject suspected of having a GDF-10 associated disorder 
and detecting binding of the antibody. 

15. The method of claim 14, wherein the cell is a uterine cell. 

16. The method of claim 14, wherein the cell is a fat cell. 

17. The method of claim 14, 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, a 
chemiluminescent compound, and an enzyme. 

20. The method of claim 14, wherein the detection is in vitro. 

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



WO 95/10539 



PCT/US94/11440 



-63- 

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-10, comprising contacting the cells 
with a reagent which suppresses the GDF-10 activity. 

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

25. The method of claim 23, wherein the reagent is a GDF-10 
antisense sequence. 

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

27. The method of claim 23, wherein the cell is a fat cell. 

28. The method of claim 23, wherein the reagent which 
suppresses GDF-10 activity is introduced to a cell using a 
vector. 

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

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



WO 95/10539 PCTAJS94/1 1440 

-64- 

31. The method of claim 30, wherein the liposome is essentially 
target specific. 

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

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

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

35. The method of claim 33, wherein the mechanistic targeting is 
active. 

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

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

38. The method of claim 28, wherein the vector is a virus. 

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

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

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



WO 95/10539 



PCT/US94/11440 



-65- 

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

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

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



WO 9S/10539 



PCT/US94/11440 



1/7 




SUBSTITUTE SHEET (RULE 26) 



WO 95/10539 



PCT/US94/11440 



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PCT/US94/1J440 



3/7 



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WO 95/10539 



PCT/US94/11440 



k/1 



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PCT/US94/11440 



5/7 




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SUBSTITUTE SHEET IflULE 26) 



WO 95/10539 



PCT/US94/11440 



6/7 





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FIG. 5 

SUBSTITUTE SHEET (RULE 20) 



WO 95/10539 



PCT/US94/11440 



7/7 



12 3 4 




FIG. 6 



SUBSTITUTE SHEET (RULE 26) 



INTERNATIONAL SEARCH REPORT 



International application No. 
PCT/US94/11440 



A. CLASSIFICATION OF SUBJECT MATTER 
IPC(6) :C07K 14/71; C07H 21/00 

US CL :530/399; 536/23.5; 435/69.1. 69.4, 320.1. 252.3 
According to International Patent Classification (IPC) or to both national classification and IPC 

B. FIELDS SEARCHED 

Minimum documentation searched (classification system followed by classification symbols) 
U.S. : 530/399; 536723.5; 435/69.1. 69.4, 320.1. 252.3 



Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched 



Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) 
GenBank, APS, Dialog 

search terms: GDF, endometriosis, uterine, pregnancy, cancer, malignancy 



DOCUMENTS CONSIDERED TO BE RELEVANT 



Category* 



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



Relevant to claim No. 



Proceedings of the National Academy of Sciences USA, 
Volume 88, issued May 1991, S. Lee, "Expression of 
growth/differentiation factor 1 in the nervous system: 
Conservation of a bicistronic structure", pages 4250-4254. 

Journal of Biological Chemistry, Volume 268, No. 5, issued 
15 February 1993, A.C. McPherron et al., "GDF-3 and GDF- 
9: Two members of the Transforming Growth Factor-/? 
Superfamily Containing a Novel Pattern of Cysteines", pages 
3444-3449. 



1-10 



1-10 



| x| Further documents are listed in the continuation of Box C. | | See patent family annex. 



Special categories of cited document!: 

documentdcftnmg the general state of the an which a Dot considered 
to be of particular relevance 

*E* earlier document published on or after the international filing date 

L* document which may throw doubta oo priority cUim(») or which m 

cited co establish the publication date of another citation or other 
special reason (as specified) 

O" document referring to an oral disclosure, use, exhibition or other 

P* document published prior to the mtematkma) filing dale but utter than 



*Y- 



later document published after the alternations! filing date or priority 
date and not in conflict with the application but cited to undenumd the 
principle or theory underlying the invention 

document of particular relevance; the claimed invention cannot be 
considered novel or cannot be considered to involve an inventive atep 
when the document is taken alone 

document of particular relevance; the claimed invention cannot be 
cons id era! to involve an inventive step when the document is 
rffmbhif^ with one or more other such documents, such combination 
being obvious to a person skilled in the art 

document member of the same patent family 



Date of the actual completion of the international search 
02 DECEMBER 1994 


Date of mailing of the international search report 

JAN 2 5 1996 


Name and mailing address of the ISA/US 
Commissioner of Patent* and Trademarks 
Box PCT 

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


Authorize* oflicc, j^ffa* j& 

SHELLY GUEST CERMAK ScS f 
Telephone No. (703)308-0196 



Form PCT/ISA/210 (second sheet)(Juiy 1992)* 



INTERNATIONAL SEARCH REPORT 



International application No. 
PCT/US94/11440 



C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT 


Category* 


L*iUUion oi aocumcni, wim inaicauon* wncrc appropriate, 01 ine rcicvani passages 


Relevant tn plnim No 


A 


Molecular Endocrinology, Volume 6, No. 11, issued 1992, CM. 
Jones et al., "Isolation of Vgr-2, a Novel Member of the 
Transforming Growth Factors-Related Gene Family", pages 
1961-1968. 


1-10 


A 


Molecular Endocrinology, Volume 4, No. 7, issued 1990, S. Lee, 
"Identification of a Novel Member (GDF-1) of the Transforming 
Growth Factor-0 Superfamily", pages 1034-1039. 


1-10 



Form PCT/ISA/210 (continuation of second sheci)(July 1992)* 



INTERNATIONAL SEARCH REPORT 



International application No. 
PCT/US94/ 11440 



Box I Observations where certain claims were found unsearchable (Continuation of item 1 of first sheet) 



This international report has not been established in respect of certain claims under Article 17(2) fa) for the following reasons: 

1. I I Claims Nos.: 

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



I ] Claims Nos.: 

because they relate to parts of the international application that do not comply with the prescribed requirements to such 
an extent that no meaningful international search can be carried out, specifically: 



3. Q Claims Nos.: 

because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a). 



Box II Observations where unity of invention is lacking (Continuation of item 2 of first sheet) 



This International Searching Authority found multiple inventions in this international application, as follows: 
Please See Extra Sheet. 



1 . | | 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. 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. | x| No required additional search fees were timely paid by the applicant. Consequently, this international search report i 
restricted to the invention first mentioned in the claims; it is covered by claims Nos.: 
1-10 . 



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

{ | No protest accompanied the payment of additional search fees. 



Form PCT/ISA/210 (continuation of first shect(l)KJuly 1992)* 



INTERNATIONAL SEARCH REPORT 



International application No. 
PCT/US94/ 11440 



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 are not so linked 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. 

Group I, claims 1-10, drawn to a GDF protein and the DNA encoding the GDF-10 protein. 

Group II, claims 11-44, drawn to an antibody and methods of using the antibody. 

The inventions listed as Groups I and II 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 inventions of Groups I and II are drawn to structurally distinct molecules, and although the antibody and GDF-10 
protein are related immunochemically, the inventions are considered independent and distinct because they are not so 
linked by a special technical feature within the meaning of PCT Rule 13.2 so as to form a single inventive concept. 



Form PCT/ISA/210 (extra sheet)(July 1992)* 



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