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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
Intemational Bureau 




PCX 

INTERNATIONAL APPLICATION PUBUSHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) Internationa] Patent Classification ^ : 
C07K 13/00, 15/28, C12N 15/18 



Al 



(11) Internationa] Pablication Number: 
(43) International Publication Date: 



WO 94/15965 

2] July 1994(21.07^) 



(21) International Application Niunber: PCT/US94/00666 

(22) Intemational Filmg Date: 12 January 1994 (12.01.94) 



(30) Priority Data: 

08/003.140 



12 January 1993 (12.01.93) 



US 



(60) Parent Application or Grant 
(63) Related by Continuation 
US 

Filed CD 



08/003,140 (OP) 
12 January 1993 (12.0153) 



(71) Applicant ifor all designated States except US): JOHNS HOP- 

KINS UNTVERSmr SCHOOL OF MEDICINE [US/US]; 
720 Rutland Avenue, Baltximwie, MD 21205 (US). 

(72) Inventors; and 

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

(74) Agents: WETHERELL, John, R., Jr. et al.; Spensley Horn 
Jubas & Lubiiz, 1880 Century Parte East, 5tb floor. 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, FT, SE), 



Publisbed 

With intemational search report 



(S4)TiUe: GROWTH DIFFERENTIATION FACrOR-3 
(57) Abstract 

Growth difTerentiation factor-3 (GDF-3) is disclosed along with its polynucleotide sequence and amino add sequence. Also disclosed 
are diagnostic and tiscrapeutic methods of using the GDF-3 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 intemational 
i^lications under the PCT. 



AT 


Auatrii 


GB 


UnitBd ttrngdfwn 


MR 


Maivitania 


AU 


Austnlu 


GE 


Georgia 


MW 


Malawi 


BB 


Barbados 


GN 


Ginoea 


NE 


Niger 


BE 


BdgUnn 


GR 


Greece 


NL 


Netberlandf 


BF 


Buridtts Fmo 


av 


Htmgiry 


NO 


N<nwiy 


EG 


Bolgsia 


IE 


Iitlaod 


NZ 


New Zealand 


BJ 


Bcoio 


rr 


Italy 


PL 


Polaod 


BR 


Bnzil 


JP 


lapao 


FT 


Portugal 


BY 


Belans 


K£ 


Keoya 


RO 


RomaQia 


CA 




KG 


KyigysiiD 


RU 


Rnssiao Federatioii 


CF 


Geotral AfticiD Republic 


KP 


Dconaitic People's RepabUc 


SD 


Sudan 


CG 


Congo 




of Korea 


SE 


Sweden 


CB 


Switzerland 


KR 


Repobtic of Koiea 


SI 


Slovtoia 


a 


COtetrivDiR 


KZ 


KaxakbstiD 


SK 


Slovakia 


CM 


CamaooD 


U 




SN 


Senegal 


CN 


Quu 


LK 


Sri Lanka 


TD 


Chad 


C5 


CzedunlovakU 


UJ 


Ltaemiboaig 


T6 


Togo 


CZ 


C^ecfa Reptiblic 


LV 


Latvia 


TJ 


Tajilostao 


DE 


Gennaiiy 




Mooaoo 


TT 


Thoidad and Tobago 


DK 




MD 


Repobfic of MddovB 


UA 


Ukiaine 


ES 


S|MiQ 


MG 


Madagascar 


US 


Uohed States of Amoica 


n 


Rnlifld 


ML 


Mali 


UZ 


Uzbekistan 


FR 


BraoDe 


MN 


MoQgolxa 


VN 


Viet Nam 


GA 


Gabon 











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-1- 



GROWTH DIFFERENTIATION FACTOR-3 
BACKGROUND OF THE INVENTION 

1. Field of the Invention 

The invention relates generally to growth factors and specifically to a new 
5 member of the transforming growth factor beta (IGF-p) superfamity, which is 
denoted, growth differentiation factor-3 (GDF-3). 

2. Description of Related Art 

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

10 processes during embryonic development. The family includes, Mullerian 
inhibiting substance (MIS), ^Nh\ch is required for normal male sex development 
(Behringer, et aL, Nature, 345:167, 1990), Drosophila decapentaplegic (DPP) 
gene product, which is required for dorsal-ventral axis formation and 
morphogenesis of the imaginal disks (Padgett, et aL, Nature, 325:81-84, 1987), 

15 the Xenopus Vg-1 gene product, which localizes to the vegetal pole of eggs 
({Weeks, eta!., Cell, 51:861-867, 1987), the activins (Mason, et aL, Biochem, 
Biophys, Res. Commun., 135:957-964, 1986). which can induce the formation 
of mesoderm and anterior structures in Xenopus embryos (Thomsen, et aL, 
Cell, 63:485, 1990), and the bone morphogenetic proteins <BMPs, osteogenin, 

20 OP-1) which can induce de novo cartilage and bone formation (Sanrpath, ef 
aL, J. BioL Chem., 265:13198, 1990). The TGF-)9S can influence a variety of 
differentiation processes, including adipogenesis. myogenesis, chondrogenesis, 
hematopoiesis, and epithelial cell differentiation (for review, see Massague. Cell 
49:437, 1987). 



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The proteins of the JGF-fi family are initially synthesized as a large precursor 
protein which subsequently undergoes proteolytic cleavage at a cluster of basic 
residues approximately 110-140 amino acids from the C-terminus; The C- 
terminal regions of the proteins are all structurally related and the different 
5 family members can be classified into distinct subgroups based on the extent 
of their homology. Although the homologies within particular subgroups range 
from 70% to 90% amino acid 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 

10 C-terminal fragments. For most of the family members that have been studied, 
the homodimeric species has been found to be biologically active, but for other 
family members, like the inhibins (Ling, ef a/., Nature, 221:779, 1986) and the 
TGF-^s (Cheifetz, ef a/.. Cell, 43:409, 1987), heterodimers have also been 
detected, and these appear to have different biological properties than the 

15 respective homodimers. 

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



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.3- 

SUMMARY OF THE INVENTION 

The present invention provides a cell growth and differentiation factor, GDF-3, 
a polynucleotide sequence which encodes the factor, and antibodies which are 
immunoreactive with the factor. This factor appears to relate to various cell 
5 proliferative disorders, especially those involving those involving hematopoietic 
and adipose tissue, as well as disorders related to the function of the immune 
system. 

Thus, in one embodiment, the invention provides a method for detecting a cell 
proliferative or immunologic disorder of bone marrow, spleen, thymus or fat 
10 origin and which is associated with GDF-3. In another embodiment, the 
invention provides a method of treating a cell proliferative or immunologic 
disorder associated with abnormal levels of expression of GDF-3, by 
suppressing or enhancing GDF-3 activity. 



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-4- 

BRIEF DESCRIPTION OF THE DRAWINGS 

FIGURE 1 shows expression of GDF-3 mRNA in adult tissues, 

FIGURE 2 shows nucleotide and predicted amino acid sequence of GDF-3. 
Consensus N-glycosylation signals are denoted by plain boxes. The putative 
5 tetrabasic processing sites are denoted by stippled boxes. The in-frame 
termination codons upstream of the putative initiating ATG and the consensus 
polyadenylation signals are underlined. The poly A tails are not shown. 
Numbers indicate nucleotide position relative to the 5' end. 

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

FIGURE 4 shows amino acid homologies among the different members of the 
TGF-^ superfamily. Numbers represent percent amino add identities between 
each pair calculated from the first conserved cysteine to the C-terminus. Boxes 
15 represent homologies among highly-related nf>embers within particular 
subgroups. 

FIGURE 5 shows the partial nucleotide and predicted amino acid sequences 
of human GDF-3. 



20 



FIGURE 6 shows Southern analysis of murine and human genomic DNA 
digested vwth Bam HI (B), Eco Rl (E). or Hind III (H) and probed with either 
mouse or human GDF-3. 



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DETAILED DESCRIPTION OF THE INVENTION 

The present invention provides a growth and differentiation factor, GDF-3 and 
a polynucleotide sequence encoding GDF-3. GDF-3 is expressed primarily in 
the bone marrow, spleen, thymus and adipose tissue and may have multiple 
5 regulatory roles in animals. In one embodiment, the invention provides a 
method for detection of a cell proliferative or immunologic disorder of the bone 
marrow, spleen, thymus or adipose tissue which is associated with GDF-3 
expression. In another embodiment, the invention provides a method for 
treating a cell proliferative or immunologic disorder associated with abnormal 
10 expression of GDF-3 by using an agent which suppresses or enhances GDF-3 
activity. 

The TGF-^ superfamily consists of multifunctionally 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 

15 growth factors. The structural homology between the GDF-3 protein of this 
invention and the members of the TGF-^ family, indicates that GDF-3 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-3 
will also possess biological activities that will make it useful as a diagnostic and 

20 therapeutic reagent. 

For example, TGF-^ has been shown to have a wide range of immuno- 
regulatory activities, including potent suppressive effects on B and T cell 
proliferation and function (for review, see Palladino, et aL, AnnMYAcad.ScL, 
§93:181, 1990). GDF-3 may also have similar activities and, therefore, may t>e 
25 useful as an anti-inflammatory agent or as a treatment for disorders related to 
abnormal proliferation of lymphocytes. In additran, both T<3F-/3 and activin have 
been postulated to play a role in hematopoiesis. Specifically, TGF-^ has been 



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PCT/US94/00666 



shown to be an inhibitor of the growth of early hematopoietic progenitor cells 
(for review, see Moore, B/ood 78:1, 1991); in this regard, GDF-3 may be useful 
for protecting hematopoietic stem cells during chemotherapy. In addition, 
activin has been shown to be expressed in the bone marrow and spleen 
5 (Shiozaki, et aL, Proc. NatL Acad. ScL USA, 89:1553, 1992) and to be capable 
of inducing erythroid differentiation (Murata, ef al., Proc. NatL Acad. ScLUSA, 
£5:2434, 1988), GDF-3 may possess a similar activity and may be useful for the 
treatment of diseases like thalassemias or sickle cell anemia. TGF-^ has also 
been shown to be a potent inhibitor of adipocyte differentiation in vitro (Ignotz 
10 and Massague, Proc.Natl.Acad.Sci.,USA 82:8530, 1985); in this regard, GDF-3 
may be useful for the treatment of obesity or of disorders related to abnormal 
proliferation of adipocytes. 

GDF-3 may also function as a growth stimulatory factor and therefore be useful 
for the survival of various cell populations in vitro. In particular, if GDF-3 plays 

15 a role in the stimulation of proliferation of hematopoietic stem cells, GDF-3 may 
have applications in chemotherapy, in bone marrow transplants or in the 
treatment of certain types of anemias. GDF-3 can be used to rapidly expand 
stem cell and progenitor cell populations in vitro, greatly reducing the amount 
of tissue required for transplantation. In addition, GDF-3 may be useful in 

20 maintaining stem cell populations prior to transplantation. Many other 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 a striking angiogenic response in the newborn mouse (Roberts, 
etal., Proc. Natl. Acad. Sci. USA, 83:4167, 1986). GDF-3 may also have similar 

25 activities and may be useful in repair of tissue injury caused by trauma or 
burns for example. 



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-7" 



PCT/US94/00666 



The term "substantially pure" as used herein refers to GDF-3 which is 
substantially free of other proteins, lipids, carbohydrates or other materials with 
which it is naturally associated. One skilled in the art can purify GDF-3 using 
standard techniques for protein purification. The substantially pure polypeptide 
5 will yield a single major band on a non-reducing polyacrylamide gel. The purity 
of the GDF-3 polypeptide can also be determined by amino-terminal amino 
acid sequence analysis. GDF-3 polypeptide includes functional fragments of 
the polypeptide, as long as the activity of GDF-3 remains. Smaller peptides 
containing the biological activity of GDF-3 are included in the invention. 

The invention provides polynucleotides encoding the GDF-3 protein. These 
polynucleotides include DNA, cDNA and RNA sequences which encode GDF-3. 
It is understood that all polynucleotides encoding all or a portion of GDF-3 are 
also included herein, as long as they encode a polypeptide with GDF-3 activity. 
Such polynucleotides include naturally occurring, synthetic, and intentionally 
manipulated polynucleotides. For example, GDF-3 polynucleotide may be 
subjected to site-directed mutagenesis. The polynucleotide sequence for GDF- 
3 also includes antisense sequences. The polynucleotides of the invention 
include sequences that are degenerate as a result of the genetic code. There 
are 20 natural amino acids, most of which are specified by more than one 
codon. Therefore, all degenerate nucleotide sequences are included in the 
invention as long as the amino acid sequer>ce of GDF-3 polypeptide encoded 
by the nucleotide sequence is functionally unchanged. 

Specifically disclosed herein is a cDNA sequence for GDF-3 which is 1 280 base 
pairs in length and contains an open reading frame beginning with a 
25 methionine codon at nucleotide 122. The encoded polypeptide is 366 amino 
acids in length with a molecular weight of about 41.5 kD, as determir>ed by 
nucleotide sequence analysis. Upstream of the putative initiating methionine 
is an in-frame termination codon beginning at nucleotide 77. The GDF-3 



15 



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sequence contains a core of hydrophobic amino acids near the N-terminus, 
suggestive of a signal sequence for secretion. GDF-3 contains two potential 
N-glycosylation sites at asparagine residues 113 and 308 and a putative 
tetrabasic proteolytic processing site (RKRR) at amino acids 249-252. 
5 Cleavage at this site would generate a mature fragment of GDF-3 predicted to 
be 114 amino acids in length and have an unglycosylated molecular weight of 
about 13.0 kD. as determined by nucleotide sequence analysis. One skilled 
in the art can modify, or partially or completely remove, the glycosyl groups 
from the GDF-3 protein using standard techniques. Therefore the functional 
10 protein or fragments thereof of the invention includes glycosylated, partially 
glycosylated and unglycosylated species of GDF-3. 

The C-terminal region of GDF-3 following the putative proteolytic processing 
site shows significant homology to the known members of the TGF-^ 
superfamily. The GDF-3 sequence contains most of the residues that are 

15 highly conserved in other family members (see Figure 3). However, the GDF-3 
sequence contains an altered pattern of cysteine residues in this C-terminal 
region. In particular, GDF-3 lacks one of the seven cysteine residues that are 
conserved in all other family members; that is, at amino acid position 330, 
where all other family members contain a cysteine residue, the GDF-3 

20 sequence contains a valine residue. In addition, GDF*3 contains an additional 
cysteine residue at position 262. ten amino acids following the predicted 
cleavage site. 

Among the known mammalian TGF-^ family members, GDF-3 is most 
homologous to Vgr-1 and BMP-2 (53% sequence kientity). GDF-3 is slightly 
25 more homologous to Xenopus Vg-1 (57% sequence kientity), but is unlikely to 
be the murine homolog of Vg-1 (for example. Vgr-1 and BMP-2 are as 
homologous to Vg-1 as GDF-3 is to Vg-1). However, GDF-3 does show 
homology to both GDF-1 and Vg-1 in the pro-region upstream of the putative 



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tetrabasic processing site (28% and 29%, respectively); this degree of 
sequence relatedness is comparable to that seen in the pro- regions between 
TGF-^1 and TGF-^2 (33%; de Martin, ef a/., EMBO J., g:3673, 1987). GDF-3 
is also similar to GDF-1 in the degree to which its sequence is diverged across 
5 species. As is the case for GDF-1, the sequence homology between murine 
and human GDF-3 appears to be only in the range of 80-85% amino acid 
identity. 

Minor modifications of the recombinant GDF-3 primary amino acid sequence 
may result in proteins which have substantially equivalent activity as compared 

10 to the GDF-3 polypeptide described herein. Such modifications may be 
deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the 
polypeptides produced by these modifications are included herein as long as 
the biological activity of GDF-3 still exists. Further, deletion of one or more 
amino acids can also result in a modification of the structure of the resultant 

15 molecule without significantly altering its biological activity. This can lead to the 
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-3 biological activity. 

The nucleotide sequence encoding the GDF-3 polypeptide of the invention 
20 includes the disclosed sequence and conservative variations thereof. The term 
"conservative variation" as used herein denotes the replacement of an amino 
acid residue by another, 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 
25 polar residue for another, such as the substitution of arginine for lysine, 
glutamic for aspartio acids, or glutamine for asparagine, and the like. The term 
"conservative variation" also includes the use of a substituted amino acid in 



wo 94/15965 PCTAJS94/00666 

-10- 

place of an unsubstituted parent amino acid provided that antibodies raised to 
the substituted polypeptide also immunoreact with the unsubstituted polypep>* 
tide. 

DNA sequences of the invention can be obtained by several methods. For 
5 example, the DNA can be isolated using hybridization techniques which are 
well known in the art. These include, but are not limited to: 1) hybridization of 
genomic or cDNA libraries with probes to detect homologous nucleotide 
sequences, 2) polymerase chain reaction (PGR) on genomic DNA or cDNA 
using primers capable of annealing to the DNA sequence of interest, and 3) 
1 0 antibody screening of expression libraries to detect cloned DNA fragments with 
shared structural features. 

Preferably the GDF-3 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 

15 to isolate any gene sequence from any organism, 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 

20 genetic code, however, the degeneracy of the code must be taken into 
account. It Is possible to perform a mixed addition reactfon when the 
sequence is degenerate. This includes a heterogeneous mixture of denatured 
double-stranded DNA. For such screening, hybrkJization is preferably 
performed on either single-stranded DNA or denatured double-stranded DNA. 

25 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. 



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for example, to allow the autoradiographic visualization of a specific cDNA 
clone by the hybridization of the target DNA to that single probe in the mixture 
which IS Its complete complement (Wallace, ef al., NucL Acid fles., 9:879, 
1981). 

5 The development of specific DNA sequences encoding GDF-3 can also be 
obtained by: 1) isolation of double-stranded DNA sequences from the genomic 
DNA; 2) chemical manufacture of a DNA sequence to provide the necessary 
codons for the polypeptide of interest; and 3) in vitro synthesis of a double- 
stranded DNA sequence by reverse transcription of mRNA isolated from a 
10 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 spedfic DNA sequences for 
use in recombinant procedures, the isolation of genomic DfsIA isolates is the 
least common. This is especially true when it is desirable to obtain the 
15 microbial expression of mammalian polypeptides due to the presence of 
introns. 

The synthesis of DNA sequences is frequently the method of choice when the 
entire sequence of amino acid residues of the desired polypeptide product is 
known. When the entire sequence of amino acid residues of the desired 

20 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 isolating cDNA sequences of interest is the 
formation of plasmid- or phage-carrying cDNA libraries whteh are derived from 
reverse transcription of mRNA which is abundant in donor cells that have a 

25 high level of genetic expression. When used in combination with polymerase 
chain reaction technology, even rare expression products can be cloned. In 
those cases where significant portions of the amino acid sequence of the 



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-12- 

polypeptide are known, the production of labeled single or double-stranded 
DNA or RNA probe sequences duplicating a sequence putatively present in the 
target cDNA may be employed in DNA/DNA hybridization procedures which are 
carried out on cloned copies of the cDNA which have been denatured into a 
5 single-stranded form (Jay, et aL, NucL Acid Res., 11:2325, 1983). 

A cDNA expression library, such as lambda gtll, can be screened indirectly 
for GDF-3 peptides having at least one epitope, using antibodies specific for 
GDF-3. Such antibodies can be either polyclonally or monoclonally derived 
and used to detect expression product indicative of the presence of GDF-3 
10 cDNA. 

DNA sequences encoding GDF-3 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 
15 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-3 polynucleotide sequences may be inserted 
20 into a recombinant expression vector. The term "recombinant expression 
vector" refers to a plasmid, ^nrus or other vehicle known in the art that has 
been manipulated by insertion or incorporation of the GDF-3 genetic sequenc- 
es. Such expression vectors contain a promoter sequence which facilitates the 
efficient transcription of the inserted genetic sequence of the host. The 
25 expression vector typically contains an origin of replication, a promoter, as well 
as specific genes which allow phenotypic selection of the transformed cells. 
Vectors suitable for use in the present invention include, but are not limited to 



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the T7-based expression vector for expression in bacteria (Rosenberg, et al., 
Gene, 5S: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 
5 can be present in the vector operably linked to regulatory elements, for 
example, a promoter (e.g,, T7, metallothionein I, or polyhedrin promoters). 

Polynucleotide sequences encoding GDF-3 can be expressed in either 
prokaryotes or eukaryotes. Hosts can include microbial, yeast, insect and 
mammalian organisms. Methods of expressing DNA sequences having 
10 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 incorp- 
orate DNA sequences of the invention. 

Transformation of a host cell with recombinant DNA may be carried out by 
15 conventional techniques as are well known to those skilled in the art. Where 
the host is prokaryotic, such as E. coli, competent cells which are capable of 
DNA uptake can be prepared from cells han^ested after exponential growth 
phase and subsequently treated by the CaClg method using procedures well 
known in the art. Alternatively, MgCl2 or RbCI can be used. Transformation 
20 can also be performed after forming a protoplast of the host cell if desired. 

When the host is a eukaryote, such methods of transfection of DNA as calcium 
phosphate co-precipitates, conventional mechanteal procedures such as 
microinjection, electroporation, insertion of a plasmid encased in liposomes, or 
virus vectors may be used. Eukaryotic cells can also be cotransformed with 
25 DNA sequences encoding the GDF-3 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, 



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PCT/US94/00666 



such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect 
or transform 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 
5 thereof, provided by the invention, may be carried out by conventional means 
including preparative chromatography and immunological separations involving 
monoclonal or polyclonal antibodies. 

The invention includes antibodies immunoreactive with GDF-3 polypeptide or 
functional fragments thereof. Antibody vyrhich consists essentially of pooled 

10 monoclonal antibodies with different epitopic specificities, as well as distinct 
monoclonal antibody preparations are provided. Monoclonal antibodies are 
made from antigen containing fragments of the protein by methods well known 
to those skilled in the art (Kohler, et aL, Nature, 25g:495, 1975). The terni 
antibody as used in this invention is meant to include intact molecules as well 

15 as fragments thereof, such as Fab and F(ab')2, which are capable of binding 
an epitopic determinant on GDF-3. 

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 GDF-3 polynucleotide that is an 

20 antisense molecule is useful In treating malignancies of the various organ 
systems, particularly, for example, cells in the bone marrow, spleen, thymus or 
adipose tissue. Essentially, any disorder whk:h is etiologrcaliy linked to altered 
expression of GDF-3 could be considered susceptible to treatment with a GDF- 
3 suppressing reagent. One such disorder of associated with bone marrow- 

25 derived cells is leukemia, for example. The term "immunobgic disorder*' refers 
to a disorder involving cells of the immune system, for example lymphocytes. 
Such immunologic disorders include disorders associated with the inflammatory 



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process for example. The immunologic disorder is not limited to an 
immunologic cell proliferative disorder. 

The invention provides a method for detecting a cell proliferative or 
immunologic disorder of the bone marrow, spleen, thymus or adipose tissue 
5 which comprises contacting an anti-GDF-3 antibody with a cell suspected of 
having a GDF-3 associated disorder and detecting binding to the antibody. 
The antibody reactive with GDF-3 is labeled with a compound which allows 
detection of binding to GDF-3. For purposes of the invention, an antibody 
specific for GDF-3 polypeptide may be used to detect the level of GDF-3 in 

10 biological fluids and tissues. Any specimen containing a detectable amount of 
antigen can be used. A preferred sample of this invention is tissue of bone 
marrow origin, specifically tissue containing hematopoietic stem or progenitor 
cells. The level of GDF-3 in the suspect cell can be compared with the level 
in a normal cell to determine whether the subject has a GDF-3-associated cell 

15 proliferative or immunologic disorder. Preferably the subject is human. 

The antibodies of the invention can be used in any subject in which it is 
desirable to administer in vitro or in vivo immunodiagnosis or immunotherapy. 
The antibodies of the invention are suited for use, for example, in immuno- 
assays in which they can be utilized in liquid phase or bound to a solid phase 

20 carrier. In addition, the antibodies in these immunoassays can be detectably 
labeled in various ways. Examples of types of immunoassays which can utilize 
antibodies of the invention are competitive and non-competitive immunoassays 
in either a direct or indirect format. Examples of such immunoassays ar« the 
radioimmunoassay (RIA) and the sandwich (immunometric) assay. Detection 

25 of the antigens using the antibodies of the invention can be done utilizing 
immunoassays which are run in either the forward, reverse, or simultaneous 
modes, including immunohistochemical assays on physiological samples. 
Those of skill in the art will know, or can readily discern, other immunoassay 
formats without undue experimentation. 



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-16- 

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 
5 celluloses, potyacrylamides, agaroses and magnetite. The nature of the carrier 
can be either soluble or insoluble for purposes of the invention. Those skilled 
in the art will know of other suitable carriers for binding antibodies, or will be 
able to ascertain such, using routine experimentation. 

There are many different labels and methods of labeling known to those of 
10 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 of ordinary skill in the art will know of 
other suitable labels for binding to the antibody, or will be able to ascertain 
15 such, using routine experimentation. 

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

In using the monoctonal 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 
25 detectably labeled monoclonal antibody is administered in sufficient quantity to 
enable detection of the site having the antigen comprising a polypeptide of the 
invention for which the morK>ctonal antibodies are specific. 



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The concentration of detectably labeled monoclonal antibody which is 
administered should be sufficient such that the binding to those cells having 
the polypeptide is detectable compared to the background. Further, it is 
desirable that the detectably labeled monoclonal antibody be rapidly cleared 
5 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 extent of 
disease of the individual. Such dosages may vary, for example, depending on 
10 whether multiple injections are given, antigenic burden, and other factors 
known to those of skill in the art. 

For in vivo diagnostic imaging, the type of detection instrument available is a 
major factor in selecting a given radioisotope. The radtoisotope chosen must 
have a type of decay which is detectable for a given type of instrument. Still 
15 another important factor in selecting a radioisotope for in vivo diagnosis is that 
deleterious radiation with respect to the host is minimized. Ideally, a radio- 
isotope used for in vivo imaging will lack a particle emissk)n, but produce a 
large number of photons in the 140-250 keV range, which may readily be 
detected by conventional gamma cameras. 

20 For in vivo diagnosis radioisotopes may be lx)und to immunoglobulin either 
directly or indirectly by using an intermediate functional group. Intermediate 
functional groups which often are used to bind radtoisotopes which exist as 
metallic ions to immunoglobulins are the bifunctional chelating agents such as 
diethylenetriaminepentacetic acid (DTPA) and ethylenediaminetetraacetic acid 

25 (EDTA) and similar molecules. Typical examples of metallic tons which can be 
bound to the monoclonal antibodies of the invention are ^^'in, ®^Ru. ^^Ga, ^Ga, 
^As, ^^r. and ^^Tl. 



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-18- 

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

The monoclonal antibodies of the invention can be used in vitro and in vivo to 
monitor the course of amelioration of a GDF-3-associated disease in a subject. 

10 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 GDF-3-associated disease is effective. The term "ameliorate" 

15 denotes a lessening of the detrimental effect of the GDF-3-associated disease 
in the subject receiving therapy. 

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

20 to this sequence. Thus, where a cell-proliferative or immunologic disorder is 
associated with the expression of GDF-3, nucleic acid sequences that interfere 
with GDF-3 expression at tiie translational level can be used. This approach 
utilizes, for example, antisense nucleic acid and ribozymes to block translation 
of a specific GDF-3 mRNA, either by masking that mRNA with an antisense 

25 nucleic acid or by cleaving it with a ribozyme. 



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Antisense nucleic acids are DNA or RNA molecules that are complementary to 
at least a portion of a specific mRNA molecule (Weintraub, Scientific American, 
262:40, 1990). In the cell, the antisense nucleic acids hybridize to the 
corresponding mRNA, forming a double-stranded molecule. The antisense 
5 nucleic acids interfere with 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 GDF- 
3-producing cell. The use of antisense methods to inhibit the in vitro 
10 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 restriction 
endonucleases. Through the modification of nucleotkie sequences which 
1 5 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-specific, only mRNAs with particular sequences are 
inactivated. 

20 There are two basic types of ribozymes namely. tetrahymena-\ype (Hasselhoff. 
Nature, 334:585, 1988) and "hammerhead"-type. 7e/raA?ymena-type ribozymes 
recognize sequences which are four bases in length, while "hammerhead'^-type 
ribozymes recognize base sequences 11-18 bases in length. The longer the 
recognition sequence, the greater the likelihood that the sequence will occur 

25 exclusively in the target mRNA species. Consequently, hammerhead-type 
ribozymes are preferable to tetrahymena-t^pe ribozymes for inactivating a 
specific mRNA species and 18-based recognition sequences are preferable to 
shorter recognition sequences. 



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-20- 

The present invention also provides gene therapy for the treatment of cell 
proliferative or immunologic disorders which are mediated by GDF-3 protein. 
Such therapy would achieve its therapeutic effect by introduction of the GDF-3 
antisense polynucleotide into cells having the proliferative disorder. Delivery of 
5 antisense GOF-3 polynucleotide can be achieved using a recombinant expres- 
sion vector such as a chimeric virus or a colloidal dispersion system. 
Especially preferred for therapeutic delivery of antisense sequences is the use 
of targeted liposomes. 

Various viral vectors which can be utilized for gene therapy as taught herein 

10 include adenovirus, herpes vims, vaccinia, or, preferably, an RNA virus such 
as a retrovirus. Preferably, the retroviral vector is a derivative of a murine or 
avian retrovirus. Examples of retroviral vectors in which a single foreign gene 
can be inserted include, but are not limited to: Moloney murine leukemia virus 
(MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor 

15 virus (MuMTV), and Rous Sarcoma Virus (RSV). A number of additional 
retroviral vectors can incorporate multiple genes. All of these vectors can 
transfer or incorporate a gene for a selectable marker so that transduced cells 
can be identified and generated. By inserting a GDF-3 sequence of interest 
into the viral vector, along with another gene which encodes the ligarKl for a 

20 receptor on a specific target cell, for example, the vector is now target specific. 
Retroviral vectors can be made target specific by inserting, for example, a 
polynucleotide encoding a sugar, a glycolipid, or a protein. Preferred targeting 
is accomplished by using an antibody to target the retroviral vector. Those of 
skill in the art will know of, or can readily ascertain without undue experimenta- 

25 tion, specific polynucleotide sequences which can be inserted into the retroviral 
genome to allow target specific delivery of the retroviral vector containing the 
GDF-3 antisense polynucleotide. 



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Since recombinant retroviruses are defective, tiiey require assistance in order 
to produce Infectious vector particles. This assistance can be provided, for 
example, by using helper ceil lines that contain plasmids encoding all of the 
structural genes of the retrovirus under the control of regulatory sequences 
5 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 *2, PA317 and PA12, for example. These cell 
lines produce empty virions, since no genome is packaged. If a retroviral 
10 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 transfected 
with plasmids encoding the retroviral structural genes gag, pot and env, by 
1 5 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. 

Another targeted delivery system for GDF-3 antisense polynucleotides is a 
colloidal dispersion system. Colloidal dispersion systems include macromole- 

20 cule complexes, nanocapsules, microspheres, beads, and lipid-based systems 
including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The 
preferred colloidal system of this invention is a liposome. Liposomes are 
artificial membrane vesicles which are useful as delivery vehicles in vitro and 
in vivo. It has been shown that large unilamellar vesicles <LUV), which range 

25 in size from 0.2-4.0 ^m can errcapsulate a substantial percentage of an 
aqueous 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 a!., Trends Biochem. Sc/., 6:77. 1981). In 



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-22- 

addition to mammalian cells, liposomes have been used 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 
5 compromising their biological activity; (2) preferential and substantial binding 
to a target cell in comparison to non-target cells; (3) delivery of the aqueous 
contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) 
accurate and effective expression of genetic information (Mannino, et al, 
Biotechniques. fi:682, 1988). 

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

15 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 carbon atoms, particularly from 16-18 carbon 

20 atoms, and is saturated. Illustrative phospholipids include egg phosphatidyl- 
choline, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine. 

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 orgapelle-specific. 
25 Mechanistic targeting can be distinguished based upon whether it is passive 
or active. Passive targeting utilizes the natural tendency of liposomes to 
distribute to cells of the reticulo-endothelial system (RES) In organs which 



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contain sinusoidal capillaries. Active targeting, on the other hand, involves 
alteration of the liposome by coupling the liposome to a specific ligand such 
as a monoclonal antibody, sugar, gtycolipid, or protein, or by changing the 
composition or size of the liposome in order to achieve targeting to organs and 
5 cell types other than the naturally occurring sites of localization. 

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

Due to the expression of GDF-3 in the bone marrow, spleen, thymus and 
adipose tissue, there are a variety of applications using the polypeptide, 
polynucleotide and antibodies of the invention, related to these tissues. GDF-3 
could play a role in regulation of the hematopoiesis and therefore could be 
15 useful in various transplantation procedures. In addition to applications for 
tissue transplantation, applications include treatment of cell proliferative and 
immunologic disorders. 

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 
20 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 a new member of the TGF-^ superfamily, degenerate 
5 oligonucleotides were designed which corresponded to two conserved regions 
among the known family members: one region spanning the two tryptophan 
residues conserved in all family members except MIS and the other region 
spanning the invariant cysteine residues near the C-terminus. These primers 
were used for polymerase chain reactions on mouse genomic DNA followed 
10 by subcloning the PGR 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 known members of the superfamily. 

GDF-3 was identified from a mixture of PGR products obtained with the primers 
15 SJL120: 5'-GGGGAATTGGA(A/G)GTIGGITGGGA(T/G)(A/G)GITGGG 
TIATIGGIGG-3' and 

SJL121 : 5'-GGGGAATTG{G/A)GAIGG(G/A)GA{T/G)TG(G/A)TGIAGIAGGAT(G/A) 

TG(T/G)TG(G/A)TA-3\ 
PGR using these primers was carried out with 2 mouse genomic DNA at 
20 94 *G for 1 min, 42*0 for 2 min, and 72'G for 3.5 min for 40 cycles, 

PGR products of approximately 280 bp were gel-purified, digested with Eco Rl, 
gel-purified again, and subcloned in the Bluescript vector (Stratagene, San 
Diego, GA). Bacterial colonies carrying individual sutx:lones were picked into 
96 well microtiter plates, and multiple replicas were prepared by plating the 



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cells onto nitrocellulose. The replicate filters were hybridized to probes 
representing known members of the family, and DNA was prepared from non- 
hybridizing colonies for sequence analysis. 

The primer combination of SJL120 and SJL121. encoding the amino acid 
5 sequences EVGWH{FVS)WV(I/M)AP and YEDMWDECGC respectively yielded 
one previously identified sequence GDF-1 and two novel sequences, one of 
which was designated GDF-3, among 80 subclones analyzed. 

RNA isolation and Northern analysis were carried out as described previously 
(Lee, SJ„ MoL Endocrinol., 4:1034, 1990) except that hybridization was carried 

10 out in 5x SSPE, 10% dextran sulfate, 50% formamide, 1% SDS, 200 ^g/ml 
salmon DNA and 0.1% each of bovine serum albumin, ficoll, and polyvinylpyr- 
rolidone. An oligo dT-primed cDNA library was prepared from 2.5 ^g of bone 
marrow poly A-selected RNA in the lambda ZAP II vector according to the 
instructions provided by Stratagene. The library was amplified once prior to 

15 screening. Filters were hybridized as described previously {Lee, S.J., Proc. 
Natl. Acad. ScL USA., 88:4250-4254, 1991). DNA sequencing of both strands 
was carried out using the dideoxy chain termination method {Sanger, ef a/., 
Proc. Natl. Acad. ScL, USA. 24:5463-5467. 1977) and a combination of the SI 
nuclease/exonuclease III strategy (Henikoff, S., Gene, 28:351-359, 1984) and 

20 synthetic oligonucleotide primers. 



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EXAMPLE 2 

EXPRESSION PATTERN AND SEQUENCE OF GbF-3 

To determine the expression pattern of GDF-3, RNA samples prepared from 
a variety of adult tissues were screened by Northern analysis. Five micrograms 
5 of twice polyA-selected RNA prepared from each tissue were electrophoresed 
on formaldehyde gels, blotted and probed with GDF-3. As shown in Figure 1 , 
the GDF-3 probe detected a 1.3 kb mRNA expressed in thymus, spleen, bone 
marrow and adipose tissue. 

10 A bone marrow cDNA library consisting of 1.8 x 10^ recombinant phage was 
constructed in lambda ZAP II and screened with a probe derived from the 
GDF-3 PGR product. The entire nucleotide sequence of the longest hybridizing 
clone is shown in Figure 2. Consensus N-glycosylation signals are denoted 
by plain boxes. The putative tetrabasic processing sites are denoted by 

15 stippled boxes. The in-frame termination codons upstream of the putative 
initiating ATG and the consensus polyadenylation signals are underlined. The 
poly A tails are not shown. Numbers indicate nucleotide position relative to the 
5* end. The 1280 bp sequence contains a long open reading frame beginning 
v^h a methionine codon at nucleotide 122 and potentially encoding a protein 

20 366 amino acids in length with a molecular weight of 41 .5 kD. Upstream of the 
putative initiating methionine is an in-frame termination codon beginning at 
nucleotide 77. The predicted GDF-3 amino acid sequence contains a 
hydrophobic N-terminal region, suggestive of a signal sequence for secretion, 
two potential N-linked glycosylation sites at asparagine residues 113 and 308, 

25 and a putative tetrabasic proteolytic processing site (RKRR) at amino acids 
249-252, Cleavage of the GDF-3 precursor at this site would generate a 
mature GDF-3 protein 114 amino acids in length with a predicted 
unglycosylated molecular weight of 13.0 kD. 



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The C-terminal region of GDF-3 following the putative proteolytic processing 
site shows significant homology to the known members of the TGF-^ 
superfamily (Figure 3). Figure 3 shows the alignment of the C-terminal 
sequences of GDF-3 with the corresponding regions of human GDF-1 (Lee, 
5 Proc. NatL Acad. ScL USA, 88:4250-4254, 1 991), Xenopus Vg-1 (Weeks, et aL, 
Cell. 51:861-867, 1987), human Vgr-1 (Celeste, ef a/., Proc. NatL Acad. ScL 
USA, fiZ:9843-9847, 1990), human OP-1 (Ozkaynak, et aL, EMBO J., 
a:2085-2093, 1990), human BMP-5 (Celeste, ef a/., Proc. NatL Acad. ScL USA, 
27:9843-9847, 1990), Drosophila 60A (Wharton, ef aL, Proc. NatL Acad. ScL 

10 USA, 88:9214-9218, 1991). human BMP-2 and 4 (Wozney, et aL, Science, 
242:1528-1534, 1988). Drosophila DPP (Padgett, et aL, Nature, 325:81-84, 
1987), human BMP-3 (Wozney, etaL, Science, 242:1528-1534, 1988). human 
MIS (Cate, ef aL, Cell, 45:685-698, 1986), human inhibin alpha, pA, and 
(Mason, et aL, Biochem, Biophys. Res. Commun., 135:957-964. 1986), human 

15 TGF-^1 (Derynck, et aL, Nature, 316:701-705, 1985), humanTGF-^2 (deMartin, 
et aL, EMBO J., 6:3673-3677. 1987), human TGF-^3 {ten Dijke, ef aL, Proc. 
NatL Acad. ScL USA, 85:4715-4719, 1988), chicken TGF-^4 (Jakowlew, ef aL, 
MoL EndocrinoL, 2:1186-1195, 1988). and Xenopus TGF-i95 (Kondaiah, etaL, 
J. BioL Chem., 265:1089-1093, 1990). The conserved cysteine residues are 

20 shaded. Dashes denote gaps introduced in order to maximize the alignment. 

Figure 4 shows the amino acid homologies among the 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. Boxes represent homologies among highly-related members within 
25 particular subgroups. 



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GDF-3 lacks the fourth cysteine residue of the seven cysteines that are 
conserved In every other family member. This cysteine residue is known in the 
case of TGF-^2to be the only cysteine involved in intermolecular disulfide bond 
formation in the mature dimer (Daopin. et aL, Science, 2§Z^369. 1992; 
5 Schlunegger and Grutter, Nature, 358:430, 1992). Therefore, GDF-3 may not 
form dimers or may form non-covalently-linked dimers In which the interaction 
between the subunits may be dynamic and subject to regulation. The GDF-3 
sequence contains an additional cysteine residue four amino acids upstream 
of the first conserved cysteine. The only family members known to contain 

10 additional cysteine residues are the TGF-^s and inhibin ^s, each of which 
contain two additional cysteine residues. In the case of TGF-^2, these 
additional cysteine residues are known to form an intramolecular disulfide bond 
(Daopin, supra). Because GDF-3 contains only a single additional cysteine 
residue, GDF-3 appears to be the only family member containing an unpaired 

1 5 cysteine. Alternatively, it is conceivable that GDF-3 does form a disulfide-linked 
dimer, either as a homodimer or as a heterodimer with another family member, 
and that this additional cysteine is involved in forming the intermolecular 
disulfide bond. Indeed, If the overall structure of GDF-3 is similar to that of 
TGF-^2, the location of this extra cysteine in the 'thumb" of the "hand" (Daopin, 

20 supra) would be consistent with such a role. 



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EXAMPLE 3 
ISOLATION OF HUMAN GDF>3 

Using the same primer pair described in Example 1 (primers SJL 120 and 121) 
with human genomic DNA, a PGR product was obtained that showed 

5 significant homology (approximately 82% amino acid identity) to GDF-3 (Figure 
5). Southern analysis of mouse and human genomic DMA was carried out in 
0.9 M sodium chloride, 50 mM sodium phosphate (pH 7.0), 10 mM EDTA, 10% 
dextran sulfate, 50% formamide, 1% SDS, 200 /ig/ml salmon testis DMA and 
0.1% each of bovine serum albumin, ficoll. and polyvinylpyrrolidone at 37* C, 

10 As shown in Figure 6. the same pattern of hybridizing bands was obtained 
whether the probe was derived from the mouse GDF-3 sequence or from the 
highly related human sequence. Therefore the data show that the human 
sequence shown in Figure 5 is the human GDF-3. 

Although the invention has been described with reference to the presently 
15 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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SUMMARY OF SEQUENCES 



SEQUENCE ID NO 1 is the nucleotide sequence of PGR primer, SJL120, for 
GDF-3. 

SEQUENCE ID NO 2 is the nucleotide sequence of PGR primer, SJL121, for 
5 GDF-3. 

SEQUENCE ID NO 3 is the amino acid sequence encoded by primer SJL120. 
SEQUENCE ID NO 4 is the amino acid sequence encoded by primer SJL121. 
SEQUENCE ID NO 5 is the nucleotide sequence and deduced amino acid 
sequence for murine GDF-3. 
10 SEQUENCE ID NO 6 is the deduced amino acid sequence for murine GDF-3. 
SEQUENCE ID NO 7 is the amino acid sequence of the C-terminal region of 
GDF-3. 

SEQUENCE ID NO 8 is the amino acid sequence of the C-terminal region of 
GDF-9. 

15 SEQUENCE ID NO 9 is the amino acid sequence of the C-terminal region of 
GDF-1. 

SEQUENCE ID NO 10 is the amino acid sequence of the C-terminal region of 
Vg-1. 

SEQUENCE ID NO 11 is the amino acid sequence of the C-terminal region of 
20 Vgr-1. 

SEQUENCE ID NO 12 is the amino acid sequence of the C-terminal region of 
OP-I. 

SEQUENCE ID NO 13 is the amino acid sequence of the C-terminal region of 
BMP-5. 

25 SEQUENCE ID NO 14 is the amino acid sequence of the C-terminal region of 
60A. 

SEQUENCE ID NO 15 is the amino acid sequence of the C-terminal regton of 
BMP-2. 



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SEQUENCE ID NO 16 is the amino acid sequence of the C-terminal region of 
BMP.4. 

SEQUENCE ID NO 17 is the amino acid sequence of the C-terminal region of 
DPP. 

5 SEQUENCE ID NO 18 is the amino acid sequence of the C-terminal region of 
BMP-3. 

SEQUENCE ID NO 19 is the amino acid sequence of the C-terminal region of 
MIS. 

SEQUENCE ID NO 20 is the amino acid sequence of the C-terminal region of 
10 Inhibin-Q. 

SEQUENCE ID NO 21 is the amino acid sequence of the C-terminal region of 
Inhibin-^A. 

SEQUENCE ID NO 22 is the amino acid sequence of the C-terminal region of 
Inhibin-^B. 

15 SEQUENCE ID NO 23 is the amino acid sequerrce of the C-terminal region of 
TGF-^1. 

SEQUENCE ID NO 24 is the amino acid sequence of the C-terminal region of 
TGF.^2. 

SEQUENCE ID NO 25 is the amino acid sequence of the C-terminal region of 
20 TGF-^3. 

SEQUENCE ID NO 26 is the amino acid sequence of the C-terminal region of 
TGF-^4. 

SEQUENCE ID NO 27 is the amino acid sequence of the C-terminal region 
ofTGF-^5. 

25 SEQUENCE ID NO 28 is the nucleotide sequence of human GDF-a 

SEQUENCE ID NO 29 is the deduced amino acid sequence of human GDF-3. 



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SEQUENCE LISTING 

(1) GENERAL INFORMATION: 

(i) APPLICANT: JOHNS HOPKINS UNIVERSITY 

5 TITLE OF INVENTION: GROWTH DIFFERENTIATION FACTOR- 3 

(iii) NUMBER OF SEQUENCES: 29 

(iv) CORRESPONDENCE ADDRESS: 

(A) ADDRESSEE: SPENSLEY HORN JUBAS & LUBITZ 
10 (B) STREET: 1880 CENTURY PARK EAST, FIFTH FLOOR 

(C) CITY: LOS ANGELES 

(D) STATE: CALIFORNIA 

(E) COUNTRY: US 

(F) ZIP: 90067 

15 (v) COMPUTER READABLE FORM: 

(A) MEDIUM TYPE: Floppy disk 

(B) COMPUTER: IBM PC compatible 

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

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

20 (vi) CURRENT APPLICATION DATA: 

(A) APPLICATION NUMBER: PCT 

(B) FILING DATE: 12-JAN-199A 

(C) CLASSIFICATION: 

(viii) ATTORNEY/AGENT INFORMATION: 
25 (A) NAME: WETHERELL, JR. Ph.D., JOHN R. 

(B) REGISTRATION NUMBER: 31,678 

(C) REFERENCE/DOCKET NUMBER: FD2279 PCT 

(ix) TELECOMMUNICATION INFORMATION: 
(A) TELEPHONE: (619) 455-5100 
30 (B) TELEFAX: (619) 455-5110 

(2) INFORMATION FOR SEQ ID N0:1: 



(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 41 base pairs 



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(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 



5 (vii) IMMEDIATE SOURCE: 

(B) CLONE: SJL120 

(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 1..41 

10 (D) OTHER INFORMATION: /note- "Where "R" Occurs, R - 

Adenine or Guanine; N - Inosine; Y - Thymine or 
Cytosine; M - Adenine or Cytosine." 



(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1: 
CCGGAATTCG ARGTNGGNTG GCAYMGNTGG GTNATNGCNC C 
15 (2) INFORMATION FOR SEQ ID NO: 2: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 42 base pairs 

(B) TYPE: nucleic acid 

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

(ii) MOLECULE TYPE: DNA (genomic) 



(vii) IMMEDIATE SOURCE: 
(B) CLONE: SJL121 



(ix) FEATURE: 
25 (A) NAME/KEY: CDS 

(B) LOCATION: 1,.42 

(D) OTHER INFORMATION: /note- "WHERE "R" OCCURS. R - 

GUANINE OR ADENINE; WHERE "N" OCCURS, N -= INOSINE; 
WHERE "Y" OCCURS, Y - THYMINE OR CYTOSINE." 



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PCT/US94/00666 



(xl) SEQUENCE DESCRIPTION: SEQ ID N0:2: 

CCGGAATTCR CANCCRCAYT CRTCNACNAC CATRTCYTCR TA 

(2) INFORMATION FOR SEQ ID NO: 3: 

(i) SEQUENCE CHARACTERISTICS: 
5 (A) LENGTH: 11 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 



10 (vii) IMMEDIATE SOURCE: 

(B) CLONE: SJL120 



(ix) FEATURE: 

(A) NAME/KEY: Peptide 

(B) LOCATION: l.,ll 

15 (D) OTHER INFORMATION: /note- "Where "Arg" Occurs, Arg - 

Arg or Ser; Where "lie" occurs, lie - lie or Met." 



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

Glu Val Gly Trp His Arg Trp Val He Ala Pro 
15 10 

20 (2) INFORMATION FOR SEQ ID NO: 4: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 11 amino acids 

(B) TYPE: amino acid 

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

(ii) MOLECULE TYPE: peptide 



(vii) IMMEDIATE SOURCE: 
(B) CLONE: SJL121 



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(ix) FEATURE: 

(A) NAME/KEY: Peptide 

(B) LOCATION: 1..11 



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

Tyr Glu Asp Met Val Val Asp Glu Cys Gly Cys 
1 5 10 



(2) INFORMATION FOR SEQ ID NO: 5: 

(1) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1280 base pairs 
10 (B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 



(vii) IMMEDIATE SOURCE: 
15 (B) CLONE: GDF-3 

(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 122.. 1219 



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

20 TGAGGGGCTG AGAAGAGAGC AATTCACACT TGATTAGCTC CCAGGCTCCT GAATTGAGCA 60 

GAGGAGGCTA GACCGCTGAG CTGCGCACCC CAGAGGCTGC TCTACCCTGG CTCAGACGAC 120 

C ATG CAG CCT TAT CAA CGG CTT CTG GCG CTT GGC TTC CTT CTC TTA 166 
Met Gin Pro Tyr Gin Arg Leu Leu Ala Leu Gly Phe Leu Leu Leu 
15 10 1*5 

25 ACC CTG CCC TGG GGC CAG ACA TCC GAG TTT CAA GAC TCT GAC CTT TTG 21A 
Thr Leu Pro Trp Gly Gin Thr Ser Glu Phe Gin Asp Ser Asp Leu Leu 
20 25 30 



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CAG TTT CTG GGA TTA GAG AAA GCG CCT TCA CCT CAC AGG TTC CAA CCT 
Gin Phe Leu Gly Leu Glu Lys Ala Pro Ser Pro His Arg Phe Gin Pro 
35 AO 45 



262 



GTG CCT CGC GTC TTA AGG AAA ATC ATC CGG GCT CGA GAA GCC GCT GCA 
Val Pro Arg Val Leu Arg Lys lie He Arg Ala Arg Glu Ala Ala Ala 
50 55 60 



310 



GCC ACT GCG GCC ICG CAG GAC TTA TGC TAG GTG AAG GAG CTG GGT GTT 
Ala Ser Gly Ala Ser Gin Asp Leu Cys Tyr Val Lys Glu Leu Gly Val 
65 70 75 



358 



10 CGT GGG AAC CTG CTT CAG CTT CTC CCA GAC CAG €GT TTT TTC CTT AAT 

Arg Gly Asn Leu Leu Gin Leu Leu Pro Asp Gin Gly Phe Phe Leu Asn 
80 85 90 95 



406 



15 



ACA CAG AAA CCT TTC CAA GAT GGC TCC TGT CTC CAG AAG GTC CTC TAT 
Thr Gin Lys Pro Phe Gin Asp Gly Ser Cys Leu Gin Lys Val Leu Tyr 
100 105 110 



A54 



TTT AAC TTG TCT GCC ATC AAA GAA AAG GCA AAG TTG ACC ATG GCC CAG 
Phe Asn Leu Ser Ala He Lys Glu Lys Ala Lys Leu Thr Met Ala Gin 
115 120 125 



502 



CTG ACT CTA GAC TTG GGG CCC AGG TCC TAG TAT AAC CTG CGA CCA GAG 
20 Leu Thr Leu Asp Leu Gly Pro Arg Ser Tyr Tyr Asn Leu Arg Pro Glu 
130 135 140 



550 



CTG GTG GTT GCT CTG TCT GTG GTT CAG GAC CGG GGC GTG T<3G GGG CGA 
Leu Val Val Ala Leu Ser Val Val Gin Asp Arg Gly Val Trp Gly Arg 
145 150 155 



598 



25 TCC CAC CCT AAG GTG GGC AGA TTG CTT TTT CTG CGG TCT GTC CCT GGG 

Ser His Pro Lys Val Gly Arg Leu Leu Phe Leu Arg Ser Val Pro Gly 
160 165 170 175 



646 



30 



CCT CAA GGT CAG CTC CAG TTC AAC CTG CAG GGT GCG CTT AAG GAT TGG 
Pro Gin Gly Gin Leu Gin Phe Asn Leu Gin Gly Ala Leu Lys Asp Trp 
180 185 190 



694 



AGC AGG AAC CGA CTG AAG AAT TTG GAC TTA CAC TTA GAG ATT TTG GTC 
Ser Ser Asn Arg Leu Lys Asn Leu Asp Leu His Leu Glu He Leu Val 
195 200 205 



742 



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PCT/US94/00666 



AAA GAG GAG AGA TAG TGC AGG GTA ACT GTC GAG CCC GAG AAC CCC TGT 790 
Lys Glu Asp Arg Tyr Ser Arg Val Thr Val Gin Pro Glu Asn Pro Cys 
210 215 220 

GAG CGG CTG CTC CGC TGT GTA GAT GGC TCG GTG CTG GTG GTA ACC GTC 838 
5 Asp Pro Leu Leu Arg Ser Leu His Ala Ser Leu Leu Val Val Thr Leu 
225 230 235 

AAT CCT AAA GAG TGT CAT GCT TCT TGC AGA AAA AGG AGG GCG GGC ATC 886 
Asn Pro Lys His Cys His Pro Ser Ser Arg Lys Arg Arg Ala Ala He 
240 245 250 255 

10 TCT GTC CCC AAG GGT TTC TGT AGG AAC TTC TGC CAC CGT CAT GAG CTG 934 

Ser Val Pro Lys Gly Phe Cys Arg Asn Phe Cys His Arg His Gin Leu 
260 265 270 

TTC ATC AAC TTC GAG GAC CTG GGT TGG CAC AAG TGG GTC ATC GCG CCT 982 
Phe He Asn Phe Gin Asp Leu Gly Trp His Lys Trp Val He Ala Pro 
15 275 280 285 

AAG GGG TTC ATG GCA AAT TAG TGT CAT GGA GAG TGC CCC TTC TCA ATG 1030 
Lys Gly Phe Met Ala Asn Tyr Cys His Gly Glu Cys Pro Phe Ser Met 
290 295 300 

ACC AGG TAT TTA AAT AGT TCG AAT TAT GCT TTC ATG CAG GGT CTG ATG 1078 
20 Thr Thr Tyr Leu Asn Ser Ser Asn Tyr Ala Phe Met Gin Ala Leu Met 
305 310 315 

CAT ATG GCT GAC CCC AAG GTC CCC AAG GCT GTC TGT GTC CCC ACC AAG 1126 
His Met Ala Asp Pro Lys Val Pro Lys Ala Val Cys Val Pro Thr Lys 
320 325 330 335 

25 CTC TCG CCC ATC TCG ATG GTC TAT CAG GAT AGT GAT AAG AAC GTC ATT 1174 

Leu Ser Pro He Ser Met Leu Tyr Gin Asp Ser Asp Lys Asn Val He 
340 345 350 

CTC GGA CAT TAT GAA GAC ATG GTA GTC GAT GAG TGT GGG TGT GGG 1219 
Leu Arg His Tyr Glu Asp Met Val Val Asp Glu Cys Gly Cys Gly 
30 355 360 365 

TAGTCTCGGG ACTAGGCTAG GAGTGTGCTT AGGGTAAATC CTTTAATAAA ACTACCACCC 1279 

C 1280 



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PCT/US94/00666 



(2) INFORMATION FOR SEQ ID NO: 6: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 366 amino acids 

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

(ii) MOLECULE TYPE: protein 

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

Met Gin Pro Tyr Gin Arg Leu Leu Ala Leu Gly Phe Leu Leu Leu Thr 
1 5 10 15 

10 Leu Pro Trp Gly Gin Thr Ser Glu Phe Gin Asp Ser Asp Leu Leu Gin 

20 25 30 

Phe Leu Gly Leu Glu Lys Ala Pro Ser Pro His Arg Phe Gin Pro Val 
35 40 45 

Pro Arg Val Leu Arg Lys He He Arg Ala Arg Glu Ala Ala Ala Ala 
15 50 55 60 

Ser Gly Ala Ser Gin Asp Leu Cys Tyr Val Lys Glu Leu Gly Val Arg 
65 70 75 80 

Gly Asn Leu Leu Gin Leu Leu Pro Asp Gin Gly Phe Phe Leu Asn Thr 
85 90 95 

20 Gin Lys Pro Phe Gin Asp Gly Ser Cys Leu Gin Lys Val Leu Tyr Phe 
100 105 110 

Asn Leu Ser Ala He Lys Glu Lys Ala Lys Leu Thr Met Ala Gin Leu 
115 120 125 

Thr Leu Asp Leu Gly Pro Arg Ser Tyr Tyr Asn Leu Arg Pro Glu Leu 
25 130 135 lAO 

Val Val Ala Leu Ser Val Val Gin Asp Arg Gly Val Trp Gly Arg Ser 
145 150 155 160 



His Pro Lys Val Gly Arg Leu Leu Phe Leu Arg Ser 
165 170 



Val Pro Gly Pro 
175 



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Gin Gly Gin Leu Gin Phe Asn Leu Gin Gly Ala Leu Lys Asp Trp Ser 
180 185 190 

Ser Asn Arg Leu Lys Asn Leu Asp Leu His Leu Glu lie Leu Val Lys 
195 200 205 

5 Glu Asp Arg Tyr Ser Arg Val Thr Val Gin Pro Glu Asn Pro Cys Asp 
210 215 220 

Pro Leu Leu Arg Ser Leu His Ala Ser Leu Leu Val Val Thr Leu Asn 
225 230 235 2A0 

Pro Lys His Cys His Pro Ser Ser Arg Lys Arg Arg Ala Ala lie Ser 
10 245 250 255 

Val Pro Lys Gly Phe Cys Arg Asn Phe Cys His Arg His Gin Leu Phe 
260 265 270 

He Asn Phe Gin Asp Leu Gly Trp His Lys Trp Val He Ala Pro Lys 
275 280 285 

15 Gly Phe Met Ala Asn Tyr Cys His Gly Glu Cys Pro Phe Ser Met Thr 
290 295 300 

Thr Tyr Leu Asn Ser Ser Asn Tyr Ala Phe Met Gin Ala Leu Met His 
305 310 315 320 

Met Ala Asp Pro Lys Val Pro Lys Ala Val Cys Val Pro Thr Lys Leu 
20 325 330 335 

Ser Pro He Ser Met Leu Tyr Gin Asp Ser Asp Lys Asn Val He Leu 
340 345 350 

Arg His Tyr Glu Asp Met Val Val Asp Glu Cys Gly Cys Gly 
355 360 365 

25 (2) INFORMATION FOR SEQ ID NO: 7: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 117 amino acids 

(B) TYPE: amino acid 

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



(ii) MOLECULE TYPE: protein 



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(vii) IMMEDIATE SOURCE: 
(B) CLONE: GDF-3 

(ix) FEATURE: 

(A) NAME/KEY: Protein 
5 (B) LOCATION: 1..117 



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



Lys Arg Arg Ala Ala He Ser Val Pro Lys Gly Phe Cys Arg Asn Phe 
1 5 10 15 

Cys His Arg His Gin Leu Phe He Asn Phe Gin Asp Leu Gly Trp His 
10 20 25 30 



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

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



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

65 70 75 80 

Val Cys Val Pro Thr Lys Leu Ser Pro He Ser Met Leu Tyr Gin Asp 
85 90 95 

Ser Asp Lys Asn Val He Leu Arg His Tyr Glu Asp Met Val Val Asp 
20 100 105 110 



Glu Cys Gly Cys Gly 
115 

(2) INFORMATION FOR SEQ ID NO: 8: 

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

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



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PCT/US94/00666 



(vil) IMMEDIATE SOURCE: 
(B) CLONE: GDF-9 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..118 



(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8: 

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

Cys Glu Leu His Asp Phe Arg Leu Ser Phe Ser Gin Leu Lys Trp Asp 
10 20 25 30 

Asn Trp lie Val Ala Pro His Arg Tyr Asn Pro Arg Tyr Cys Lys Gly 
35 40 A5 

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

15 Met Val Gin Asn lie lie Tyr Glu Lys Leu Asp Pro Ser Val Pro Arg 

65 70 75 80 

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

Glu Pro Asp Gly Ser lie Ala Tyr Lys Glu Tyr Glu Asp Met lie Ala 
20 100 105 110 

Thr Arg Cys Thr Cys Arg 
115 

(2) INFORMATION FOR SEQ ID NO: 9: 

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

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: protein 



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(vii) IMMEDIATE SOURCE: 
(B) CLONE: GDF-1 

(Ix) FEATURE: 

(A) NAME/KEY: Protein 
5 (B) LOCATION: 1..122 



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

Pro Arg Arg Asp Ala Glu Pro Val Leu Gly Gly Gly Pro Gly Gly Ala 
1 5 10 15 

Cys Arg Ala Arg Arg Leu Tyr Val Ser Phe Arg Glu Val Gly Trp His 
10 20 25 30 

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

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

15 Leu Asn His Ala Val Leu Arg Ala Leu Met His Ala Ala Ala Pro Gly 

65 70 75 80 

Ala Ala Asp Leu Pro Cys Cys Val Pro Ala Arg Leu Ser Pro He Ser 
85 90 95 

Val Leu Phe Phe Asp Asn Ser Asp Asn Val Val Leu Arg Gin Tyr Glu 
20 100 105 110 



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

(2) INFORMATION FOR SEQ ID NO: 10: 

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

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



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(vii) IMMEDIATE SOURCE: 
(B) CLONE: Vg-1 

(ix) FEATURE: 

(A) NAME/KEY: Protein 
5 (B) LOCATION: 1..118 



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

Arg Arg Lys Arg Ser Tyr Ser Lys Leu Pro Phe Thr Ala Ser Asn lie 
1 5 10 15 

Cys Lys Lys Arg His Leu Tyr Val Glu Phe Lys Asp Val Gly Trp Gin 
10 20 25 30 

Asn Trp Val lie Ala Pro Gin Gly Tyr Met Ala Asn Tyr Cys Tyr Gly 
35 40 A5 

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

15 lie Leu Gin Thr Leu Val His Ser He Glu Pro Glu Asp He Pro Leu 

65 70 75 80 

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

Asp Asn Asn Asp Asn Val Val Leu Arg His Tyr Glu Asn Met Ala Val 
20 100 105 110 

Asp Glu Cys €ly Cys Arg 
115 

(2) INFORMATION FOR SEQ ID NO: 11: 

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

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: protein 



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PCTAJS94/00666 



(vii) IMMEDIATE SOURCE: 
(B) CLONE: Vgr-1 

(ix) FEATURE: 

(A) NAME/KEY: Protein 
5 (B) LOCATION: 1..118 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: 

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

Cys Arg Lys His Glu Leu Tyr Val Ser Val Gin Asp Leu Gly Trp Gin 
10 20 25 30 

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

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

15 He Val Gin Thr Leu Val His Leu Met Asn Pro Glu Tyr Val Pro Lys 

65 70 75 80 

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

Asp Asp Asn Ser Asn Val He Leu Lys Lys Tyr Arg Asn Met Val Val 
20 100 105 110 

Arg Ala Cys Gly Cys His 
115 

(2) INFORMATION FOR SEQ ID NO: 12: 

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

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: protein 



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(vil) IMMEDIATE SOURCE: 
(B) CLONE: OP-1 



(ix) FEATURE: 

(A) NAME/KEY: Protein 
5 (B) LOCATION: 1..118 



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

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

Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gin 
10 20 25 30 

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

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

15 He Val Gin Thr Leu Val His Phe He Asn Pro Glu Thr Val Pro Lys 

65 70 75 80 

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

Asp Asp Ser Ser Asn Val He Leu Lys Lys Tyr Arg Asn Met Val Val 
20 100 105 110 



Arg Ala Cys Gly Cys His 
115 



(2) INFORMATION FOR SEQ ID NO: 13: 



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

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: protein 



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(vii) IMMEDIATE SOURCE: 
(B) CLONE: BMP- 5 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: l.,118 



(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13: 

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

Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gin 
10 20 25 30 

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

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

15 He Val Gin Thr Leu Val His Leu Met Phe Pro Asp His Val Pro Lys 

65 70 75 80 

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

Asp Asp Ser Ser Asn Val He Leu Lys Lys Tyr Arg Asn Met Val Val 
20 100 105 110 

Arg Ser Cys Gly Cys His 
115 

(2) INFORMATION FOR SEQ ID NO: 14: 

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

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: protein 



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(vii) IMMEDIATE SOURCE: 
(B) CLONE: 60A 

(ix) FEATURE: 

(A) NAME/KEY: Protein 
5 (B) LOCATION: 1..118 



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

Ser Pro Asn Asn Val Pro Leu Leu Glu Pro Met Glu Ser Thr Arg Ser 
15 10 15 

Cys Gin Met Gin Thr Leu Tyr He Asp Phe Lys Asp Leu Gly Trp His 
10 20 25 30 

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

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

15 He Val Gin Thr Leu Val His Leu Leu Glu Pro Lys Lys Val Pro Lys 

65 70 75 80 

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

Leu Asn Asp Glu Asn Val Asn Leu Lys Lys Tyr Arg Asn Met He Val 
20 100 105 110 

Lys Ser Cys Gly Cys His 
115 

(2) INFORMATION FOR SEQ ID NO: 15: 

(i) SEQUENCE CHARACTERISTICS: 
25 (A) LENGTH: 117 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



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(vli) IMMEDIATE SOURCE: 
(B) CLONE: BMP- 2 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..I17 



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

Glu Lys Arg Gin Ala Lys His Lys Gin Arg Lys Arg Leu Lys Ser Ser 
1 5 10 15 

Cys Lys Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn 
10 20 25 30 

Asp Trp He Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly 
35 AO 45 

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

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

65 70 75 80 

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

Glu Asn Glu Lys Val Val Leu Lys Asn Tyr Gin Asp Met Val Val Glu 
20 100 105 110 

Gly Cys Gly Cys Arg 
115 

(2) INFORMATION FOR SEQ ID NO: 16: 

(i) SEQUENCE CHARACTERISTICS: 
25 (A) LENGTH: 117 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: protein 



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(vii) IMMEDIATE SOURCE: 
(B) CLONE: BMP-4 

(ix) FEATURE: 

(A) NAME/KEY: Protein 
5 (B) LOCATION: 1..117 



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

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

Cys Arg Arg His Ser Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn 
10 20 25 30 

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

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

15 lie Val Gin Thr Leu Val Asn Ser Val Asn Ser Ser He Pro Lys Ala 

65 70 75 80 

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

Glu Tyr Asp Lys Val Val Leu Lys Asn Tyr Gin €lu Met Val Val Glu 
20 100 105 110 



Gly Cys Gly Cys Arg 
115 

(2) INFORMATION FOR SEQ ID N0:17: 

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

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(li) MOLECULE TYPE: protein 



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

(ix) FEATURE: 

(A) NAME/KEY: Protein 
6 (B) LOCATION: 1..118 



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

Lys Arg His Ala Arg Arg Pro Thr Arg Arg Lys Asn His Asp Asp Thr 
15 10 15 

Cys Arg Arg His Ser Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asp 
10 20 25 30 

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

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

15 Val Val Gin Thr Leu Val Asn Asn Met Asn Pro Gly Lys Val Pro Lys 

65 70 75 80 

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

Asn Asp Gin Ser Thr Val Val Leu Lys Asn Tyr Gin Glu Met Thr Val 
20 100 105 110 

Val Gly Cys Gly Cys Arg 
115 

(2) INFORMATION FOR SEQ ID NO: 18: 

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

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: protein 



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(vii) IMMEDIATE SOURCE: 
(B) CLONE: BMP- 3 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..119 



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

Gin Thr Leu Lys Lys Ala Arg Arg Lys Gin Trp lie Glu Pro Arg Asn 
1 5 10 15 

Cys Ala Arg Arg Tyr Leu Lys Val Asp Phe Ala Asp lie Gly Trp Ser 
10 20 25 30 

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

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

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

65 70 75 80 

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

Phe Asp Glu Asn Lys Asn Val Val Leu Lys Val Tyr Pro Asn Met Thr 
20 100 105 110 

Val Glu Ser Cys Ala Cys Arg 
115 

(2) INFORMATION FOR SEQ ID NO: 19: 

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

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



(ii) MOLECULE TYPE: protein 



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

(ix) FEATURE: 

(A) NAME/KEY: Protein 
5 (B) LOCATION: l.,115 



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

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

Cys Ala Leu Arg Glu Leu Ser Val Asp Leu Arg Ala Glu Arg Ser Val 
10 20 25 30 

Leu lie Pro Glu Thr Tyr Gin Ala Asn Asn Cys Gin Gly Val Cys Gly 
35 40 A5 

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

16 Leu Leu Lys Met Gin Ala Arg Gly Ala Ala Leu Ala Arg Pro Pro Cys 

65 70 75 80 

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

Glu Arg He Ser Ala His His Val Pro Asn Met Val Ala Thr Glu Cys 
20 100 105 110 

Gly Cys Arg 
115 

(2) INFORMATION FOR SEQ ID NO: 20: 

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

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: protein 



wo 94/15965 



-53- 



PCT/US94/00666 



(vil) IMMEDIATE SOURCE; 

(B) CLONE: Inhibin alpha 

(ix) FEATURE: 

(A) NAME/KEY: Protein 
5 (B) LOCATION: 1..121 



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

Leu Arg Leu Leu Gin Arg Pro Pro Glu Glu Pro Ala Ala His Ala Asn 
1 5 10 15 

Cys His Arg Val Ala Leu Asn He Ser Phe Gin Glu Leu Gly Trp Glu 
10 20 25 30 

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

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

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

65 70 75 80 

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

Thr Thr Ser Asp Gly Gly Tyr Ser Phe Lys Tyr -Glu Thr Val Pro Asn 
20 100 105 110 

Leu Leu Thr Gin His Cys Ala Cys He 
115 120 



(2) INFORMATION FOR SEQ ID NO: 21: 

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

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: protein 



wo 94/15965 



PCT/US94/00666 



-54- 



(vii) IMMEDIATE SOURCE: 

(B) CLONE: Inhibin beta A 

(ix) FEATURE: 

(A) NAME/KEY: Protein 
5 (B) LOCATION: l.,121 



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

Arg Arg Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys Val Asn lie Cys 
1 5 10 15 

Cys Lys Lys Gin Phe Phe Val Ser Phe Lys Asp lie Gly Trp Asn Asp 
10 20 25 30 

Trp lie lie Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu 
35 40 A5 

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

15 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 
20 100 105 110 

Met He Val Glu Glu Cys Gly Cys Ser 
115 120 

(2) INFORMATION FOR SEQ ID NO: 22: 

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

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: protein 



wo 94/15965 



PCTAJS94/00666 



-55- 



(vii) IMMEDIATE SOURCE: 

<B) CLONE: Inhibin beta B 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1,.120 



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

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

Cys Arg Gin Gin Pbe Pbe He Asp Phe Arg Leu He Gly Trp Asn Asp 
10 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 

15 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 
20 100 105 110 

He Val Glu Glu Cys Gly Cys Ala 
115 120 

(2) INFORMATION FOR SEQ ID NO: 23: 

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

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: protein 



wo 94/15965 



-56- 



PCT/US94/00666 



(vii) IMMEDIATE SOURCE: 

(B) CLONE: TGF-beta 1 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..114 



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

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 
10 20 25 30 

Lys Trp He His Glu Pro Lys Gly Tyr His Ala Asn Phe Cys Leu Gly 
35 AO A5 

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

15 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 
20 100 105 110 

Cys Ser 



(2) INFORMATION FOR SEQ ID N0:2A: 

(i) SEQUENCE CHARACTERISTICS: 
25 (A) LENGTH: HA amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



wo 94/15965 



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PCTAJS94/00666 



(vii) IMMEDIATE SOURCE: 

(B) CLONE: TGF-beta 2 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..11A 



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

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 
10 20 25 30 

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

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

15 Ser Leu Tyr Asn Thr He Asn Pro Glu Ala Ser Ala Ser Pro Cys Cys 

65 70 75 80 

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

Thr Pro Lys He Glu Gin Leu Ser Asn Met He Val Lys Ser Cys Lys 
20 100 105 110 



Cys Ser 



(2) INFORMATION FOR SEQ ID NO: 25: 

(i) SEQUENCE CHARACTERISTIGS : 
25 (A) LENGTH: 114 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: protein 



wo 94/15965 PCT/US94/00666 

-58- 



(vii) IMMEDIATE SOURCE: 

(B) CLONE: TGF-beta 3 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..11A 



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

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 
10 20 25 30 

Lys Trp Val His Glu Pro Lys Gly Tyr Tyr Ala Asn Phe Cys Ser Gly 
35 AO A5 

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

15 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 
20 100 105 110 

Cys Ser 



(2) INFORMATION FOR SEQ ID NO: 26: 

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

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: protein 



wo 94/15965 PCTAJS94/00666 

-59- 



(vil) IMMEDIATE SOURCE: 

(B) CLONE: TGF-beta h 



(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..116 



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

Arg Arg Asp Leu Asp Thr Asp Tyr Cys Phe Gly Pro Gly Thr Asp Glu 
15 10 15 

Lys Asn Cys Cys Val Arg Pro Leu Tyr lie Asp Phe Arg Lys Asp Leu 
10 20 25 30 

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

Met Gly Pro Cys Pro Tyr He Trp Ser Ala Asp Thr Gin Tyr Thr Lys 
50 55 60 

15 Val Leu Ala Leu Tyr Asn Gin His Asn Pro Gly Ala Ser Ala Ala Pro 

65 70 75 80 

Cys Cys Val Pro Gin Thr Leu Asp Pro Leu Pro He He Tyr Tyr Val 
85 90 95 

Gly Arg Asn Val Arg Val Glu Gin Leu Ser Asn Met Val Val Arg Ala 
20 100 105 110 

Cys Lys Cys Ser 
115 

(2) INFORMATION FOR SEQ ID NO: 27: 

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

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: protein 



wo 94/15965 



-60- 



PCT/US94/00666 



(vii) IMMEDIATE SOURCE: 

(B) CLONE: TGF-beta 5 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: 

Lys Arg Gly Val Gly Gin Glu Tyr Cys Phe Gly Asn Asn Gly Pro Asn 
15 10 15 

Cys Cys Val Lys Pro Leu Tyr lie Asn Phe Arg Lys Asp Leu Gly Trp 
10 20 25 30 

Lys Trp lie His Glu Pro Lys Gly Tyr Glu Ala Asn Tyr Cys Leu Gly 
35 40 A5 

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

15 Ser Leu Tyr Asn Gin Asn Asn Pro Gly Ala Ser He Ser Pro Cys Cys 

65 70 75 80 

Val Pro Asp Val Leu Glu Pro Leu Pro He He Tyr Tyr Val Gly Arg 
85 90 95 

Thr Ala Lys Val Glu Gin Leu Ser Asn Met Val Val Arg Ser Cys Asn 
20 100 105 110 

Cys Ser 



(2) INFORMATION FOR SEQ ID NO: 28: 

(i) SEQUENCE CHARACTERISTICS: 
25 (A) LENGTH: 201 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 



wo 94/15965 



-61- 



PCT/US94/00666 



(vii) IMMEDIATE SOURCE: 
(B) CLONE: GDF-3 

(ix) FEATURE: 

(A) NAME/KEY: CDS 
5 (B) LOCATION: 1..201 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: 

AAG GGG TTC ATG GCA AAT TAC TGC CAT GGA GAG TGT CCC TTC TCA CTG 48 
Lys Gly Phe Met Ala Asn Tyr Cys His Gly Glu Cys Pro Phe Ser Leu 
15 10 15 

10 ACC ATC TCT CTC AAC AGC TCC AAT TAT GCT TTC ATG CAA GCC CTG ATG 96 

Thr lie Ser Leu Asn Ser Ser Asn Tyr Ala Phe Met Gin Ala Leu Met 
20 25 30 

CAT GCC GTT GAC CCA GAG ATC CCC CAG GCT GTG TGT ATC CCC ACC AAG 146 
His Ala Val Asp Pro Glu lie Pro Gin Ala Val Cys lie Pro Thr Lys 
15 35 AO 45 

CTG TCT CCC ATT TCC ATG CTC TAC CAG GAC AAT AAT GAC AAT GTC ATT 192 
Leu Ser Pro lie Ser Met Leu Tyr Gin Asp Asn Asn Asp Asn Val lie 
50 55 60 

CTA CGA CAT 201 
20 Leu Arg His 

65 



(2) INFORMATION FOR SEQ ID NO: 29: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 67 amino acids 
25 (B) TYPE: amino acid 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: 

Lys Gly Phe Met Ala Asn Tyr Cys His Gly Glu Cys Pro Phe Ser Leu 
30 1 5 10 15 



wo 94/15965 



-62- 



PCT/US94/00666 



Thr He Ser Leu Asn Ser Ser Asn Tyr Ala Phe Met Cln Ala Leu Met 
20 25 30 

His Ala Val Asp Pro Glu He Pro Gin Ala Val Cys He Pro Thr Lys 
35 40 45 

5 Leu Ser Pro He Ser Met Leu Tyr Gin Asp Asn Asn Asp Asn Val He 
50 * 55 60 

Leu Arg His 
65 



wo 94/15965 PCT/US94/00666 

-63- 

CLAIMS 

1 . Substantially pure growth differentiation factor-3 (GDF-3) and functional 
fragments thereof. 

2. An isolated polynucleotide sequence encoding the GDF-3 polypeptide 
of claim 1 . 

3. The polynucleotide sequence of claim 2, wherein the polynucleotide 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. 

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

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



wo 94/15965 



PCT/US94/006d6 



-64- 

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-3 associated disorder and detecting binding 
of the antibody. 

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

16. The method of claim 14, wherein the cell proliferative disorder is 
leukemia. 

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 and a chemiluminescent compound. 

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

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

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



wo 94/15965 PCT/US94/00666 

-65- 

23. A method of treating a cell proliferative disorder associated with 
expression of GDF-3, comprising contacting the cells with a reagent 
which suppresses the GDF-3 activity. 

24. The method of claim 23, wherein the reagent is an anti-GDF-3 antibody. 

25. The method of claim 23, wherein the reagent is a GDF-3 antisense 
sequence. 

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

27. The method of claim 23, wherein the cell proliferative disorder is 
leukemia. 

28. The method of claim 23, wherein the reagent which suppresses GDF-3 
activity is introduced to a cell using a vector. 

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

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

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 32, wherein the liposome is mechanistically 
targeted. 



wo 94/15965 



PCT/US94/00666 



-66- 

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 37, 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. 

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 glycol^ld, and a 
protein. 



44. 



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



wo 94/15965 PCT/IIS94/00666 

1/9 



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SUBSTITUTE SHEET (RULE 26) 



wo 94/15965 



9/9 



PC.TAJS94/00666 



PROBE: MOUSE HUMAN 

DNA: MOUSE HUMAN MOUSE HUMAN 

BEHBEH B EH B EH 




FIG.6 



aJBSmUTE SHEET <RULE 26) 



INTERNATIONAL SEARCH REPORT 



111 national application No. 
PCT/US94/00666 



A. CLASSinCATION OF SUBJECT MATTER 
IPC(5) :C07K 13/00, 15/28; C12N 15/18 

US CL :530/399, 350; 536/23.5. 23.4; 435/6, 91.1, 69.1, 252.3, 320.1 
According to IntenuUional I^ateat 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. 350; 536/23.5. 23.4; 435/6, 91.1, 69.1. 252.3, 320.1 



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) 
APS, DIALOG. GENBANK, EMBL (search terms: GDF-3}« sequence search 



DOCUNfENTS CONSIDERED TO BE RELEVANT 



Category* 


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


Relevant to claim No. 


Y 


LI ET AL, "FUNDAMENTALS OF MOLECULAR EVOLUTION," 
published 1991 by SINAUER ASSOCIATES, INC (Mass.), 
pages 12-15, see entire document. 


1-44 


Y 


MOLECULAR ENDOCRINOLOGY. Volume 6, issued 1992, 
Jones et al., "Isolation of Vgr-2, a Novel Member of the 
Transforming Growth Factor-beta-Related Gene Family," 
pages 1961-1968, see figure 1. 


1-44 


Y 


US. A, 4.683,195 (MULLIS ET AL.) 28 JULY 1987, col. 15, 
lines 30-37 and col. 16, lines 35-46. 


1-44 



~x\ Fuither documents arc listed in the continuation of Box C. | | Sec patent family annex. 



* Special cacefora of deed docuoMatt: *T* 

'A* doctnnffoidcfipiag die gcoenl tttte of tfie ut wfaicb ii not oomidefcd 

to be put of puikiikr rckvaaoe 

*X" 

'E* oriiCT documeaipublkfaed oa or after the intcnitfioiMJ filiot dale 

'L* document whicfa amy tttrov doubli oo pfiority cluiii(s) or wfaidi ii 

cited to dtabliah die publicetioo date of aaMher citBtioo or other , 
qwctal reaaoe (aa qtecified) 

O* document referrug to ao oral diackiaure. um. exhibitioo or other 

P" rf*w ii m f f x i p^ibiiriiftd prior lo the jnir nf*******^ ! Wit th— i *a* 



later document pubtiihed after the inteniatiooal filing date or priority 
date and not in ooofBct widi the appUcatioD but cited to UDdentand die 
principle or theory uaderiyinc ^ invention 

document of paiticutar relevaaoe; the claimed inveotioo cannot be 
cooaiderad nowcl or canoot be cooaideml to involve an inventive step 
wfaen the doc u ment ii taken alooe 



document of pasticular relevance; die cbimed inveotioo < 
cooaidcred to involve an inventive itcp when the 
combined with one or more other aucfa documoita. nicb cnmbinatian 
bong obvioua to a petion akilled in the art 

documeot mcmSrr of die aame patent ftunily 



Date of the actual completion of the international search 
26 MARCH 1994 


Date of mailing of the international search report 

25 APR1994 


Name and mailing address of the ISAAJS 
Coininissiooer of Patents aixi Tndemariu 

Box per 

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


Authorized oHicer ^-n 

SheUy Guest Cermak^ T^^^M^ 
Telephone No. (703) 308-0196 ^ 



Form PCT/ISA/210 (second sheet)(July 1992)^ 



INTERNATIONAL SEARCH REPORT 



In. attonai application No. 
PCT/US94/00666 



C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT 

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to ctaim No. 



A MOLECULAR ENDOCRINOLOGY, Volume 4, issued 1990, 1-44 

Lee, "Identification of a Novel Member (GDF-1) of the 
Transfonning Growth Factor-beta Superfamily," pages 1034-1039. 

P,A THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 268, 1-44 
Number 5, issued 15 February 1993, McPherron et al., "GDF-3 
and GDF-9: Two New Members of the Transforming Growth 
Factor-beta Superfamily Containing a Novel Pattern of Cysteines," 
pages 3444-3449. 

A PROCEEDINGS OF THE NATIONAL ACADEMY OF 1-44 

SCIENCES, USA, Volume 88, issued May 1991, Lee et al., 
"Expression of growth/differentiation factor I in the nervous 
system: Conservation of a bicistronic structure", pages 4250-4254, 
see entire document. 



Form PCT/ISA/210 (continuation of second 8hcct)(July 



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