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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




PCT 

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 5 : 

A61K 37/24, 37/36, 39/00, C07K 3/00, 
13/00, 15/00, 17/00, C07H 17/00, C12Q 
1/00, G01N 33/53, C12N 5/00, 1/20, 15/00 



Al 



(11) Internationa) Publication Number: WO 95/01801 

(43) Internationa] Publication Date: 19 January 1995 (19.01 .95) 



(21) International Application Number: 

(22) Internationa] Filing Date: 



PCT/US94/07762 
8 July 1994 (08.07.94) 



(30) Priority Data: 
08/089300 



9 July 1993(09.0753) 



US 



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

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

(72) Inventors; and 

(75) Inventors/Applicants (for US only): LEE, Se-Jin [US/US]; 
6711 Cbokeberry Road, Baltimore, MD 21209 (US). 
HUYNH, Thanh [US/US]; 5100 South Bend Road, 
Baltimore, MD 21209 (US). 

(74) Agents: WETHERELL, John, R., Jr. et al.; Spensley Horn 
Jubas & Lubitz, 5th floor, 1880 Century Park East, Los 
Angeles, CA 90067 (US). 



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



Published 

With international search report 



(54) Title: GROWTH DIFFERENTIATION FACTOR-6 
(57) Abstract 



Growth differentiation factor-6 (GDF-6) is disclosed along with its polynucleotide sequence and amino acid sequence. Also disclosed 
are diagnostic and therapeutic methods of using the GDF-6 polypeptide and polynucleotide sequences. 



FOR THE PURPOSES OF INFORMATION ONLY 



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



AT 


Austria 


GB 


United Kingdom 


MR 


Mauritania 


AIT 


Australia 


GE 


Georgia 


MW 


Malawi 


BB 


Barbados 


GN 


Guinea 


NE 


Niger 


BE 


Belgium 


GR 


Greece 


NL 


Netherlands 


BF 


Burkina Ftao 


HU 


Hungary 


NO 


Norway 


BG 


Bulgaria 


IE 


Ireland 


NZ 


New Zealand 


Bl 


Benin 


IT 


Italy 


PL 


Poland 


BR 


Brazil 


JP 


Japan 


FT 


Portugal 


BY 


Belarus 


KE 


Kenya 


RO 


Romania 


CA 




KG 


Kyrgystan 


RU 


Russian Federation 


CF 


Central African Republic 


KP 


Democratic People*! Republic 


SD 


Sudan 


CG 


Congo 




of Korea 


SE 


Sweden 


CH 


Switzerland 


KR 


Republic of Korea 


SI 


Slovenia 


a 


Cote d'lvoirc 


KZ 


Kazakhstan 


SK 


Slovakia 


CM 


Carnrrooo 


U 




SN 


Senegal 


CN 


China 


LK 


Sri Lanka 


TD 


Chad 


CS 


Czechoslovakia 


LU 


Luxembourg 


TG 


Togo 


CZ 


Czech Republic 


tv 


Latvia 


TJ 


Tajikistan 


DE 


Germaoy 


MC 


Monaco 


TT 


Trinidad and Tobago 


DK 


Denmark. 


MD 


RepubBc of Moldova 


DA 


Ukraine 


ES 


Spain 


MG 


Madagascar 


US 


United States of America 


n 


Finland 


ML 


Mali 


VZ 


Uzbekistan 


FR 


France 


MN 


Mongolia 


VN 


VktNam 


GA 


Gabon 











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GROWTH DIFFERENTIATION FACTOR-6 
BACKGROUND OF THE INVENTION 

1 . Field of tfie Invention 

The invention relates generally to growth factors and specifically to a new 
5 member of the transforming growth factor beta (TGF-£) superfamily, which is 
denoted, growth differentiation factor-6 (GDF-6). 

2. Description of Related Art 

The transforming growth factor p (TGF-0) 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), which is required for normal male sex development 
(Behringer, ef a/., Nature, 345:167, 1990), Drosophila decapentaplegic (DPP) 
gene product, which is required for dorsal-ventral axis formation and 
morphogenesis of the imaginal disks (Padgett, ef a/., Nature, 325:81-84, 1987), 

15 the Xenopus Vg-1 gene product, which localizes to the vegetal pole of eggs 
((Weeks, et aL, Cell, §1:861-867, 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 (Sampath, ef 
aL, J. Biol. Chem., 265:13198, 1990). The TGF-^s 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 TGF-j3 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, or mature regions, of the proteins are all structurally related 

5 and the different family members can be classified into distinct subgroups 
based on the extent of their homology. Although the homologies within 
particular subgroups range from 70% to 90% amino acid 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- 

10 linked dimer of C-terminal fragments. Studies have shown that when the pro- 
region of a member of the TGF-0 family is coexpressed with a mature region 
of another member of the TGF-p family, intracellular dimerization and secretion 
of biologically active homodimers occur (Gray, A., and Maston, A., Science, 
247:1328, 1990). Additional studies by Hammonds, et a/., (Molec. Endocrin. 

15 5:149, 1991) showed that the use of the BMP-2 pro-region combined with the 
BMP-4 mature region led to dramatically improved expression of mature BMP- 
4. For most of the family members that have been studied, the homodimeric 
species has been found to be biologically active, but for other family members, 
like the inhibins (Ling, ef a/., Nature, 321:779, 1986) and the TGF-ps (Cheifetz, 

20 ef a/., Cell, 48:409, 1987), heterodimers have also been detected, and these 
appear to have different biological properties than the respective homodimers. 

Identification of new factors that are tissue-specific in their expression pattern 
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-6, 
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 placental tissue. 

Thus, in one embodiment, the invention provides a method for detecting a cell 
proliferative disorder of placental origin and which is associated with GDF-6. 
In another embodiment, the invention provides a method for treating a cell 
proliferative disorder by suppressing or enhancing GDF-6 activity. 



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BRIEF DESCRIPTION OF THE DRAWINGS 

FIGURE 1 shows expression of GDF-6 mRNA in placenta. The arrow denotes 
the position of the major mRNA species.. 

FIGURE 2 shows nucleotide and predicted amino acid sequence of murine 
5 GDF-6. The putative pentabasic processing site is boxed. 

FIGURE 3 shows the alignment of the C-terminal sequences of GDF-6 with 
other members of the TGF-0 superfamily. The conserved cysteine residues are 
boxed. Dashes denote gaps introduced in order to maximtee alignment. 

FIGURE 4 shows amino acid homologies among different members of the TGF- 
10 fi 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 particular 
subgroups. 



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DETAILED DESCRIPTION OF THE INVENTION 

The present invention provides a growth and differentiation factor, GDF-6 and 
a polynucleotide sequence encoding GDF-6. GDF-6 is expressed in placental 
tissue. In one embodiment, the invention provides a method for detection of 
5 a cell proliferative disorder of placental origin which is associated with GDF-6 
expression. In another embodiment, the invention provides a method for 
treating a cell proliferative disorder by using an agent which suppresses or 
enhances GDF-6 activity. 

The TGF-0 superfamily consists of multifunctional polypeptides that control 
1 0 proliferation, differentiation, and other functions in many cell types. Many of the 
peptides have regulatory, both positive and negative, effects on other peptide 
growth factors. The structural homology between the GDF-6 protein of this 
invention and the members of the TGF-p family, indicates that GDF-6 is a new 
member of the family of growth and differentiation factors. Based on the 
15 known activities of many of the other members, it can be expected that GDF-6 
will also possess biological activities that will make it useful as a diagnostic and 
therapeutic reagent. 

The expression of GDF-6 in the placenta suggests a variety of applications 
using the polypeptide, polynucleotide, and antibodies of the invention, related 

20 to pregnancy and cell proliferative diseases. Abnormally low levels of the factor 
may be indicative of impaired function in the placenta while abnormally high 
levels may be indicative of hypertrophy or hyperplasia. Hence, GDF-6 may be 
useful in detecting primary and metastic neoplasms of placental origin. In 
addition, GDF-6 may also be useful as an indicator of developmental anomalies 

25 in prenatal screening procedures. 



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-6- 

Several members of the TGF-0 superfamily possess activities suggesting 
possible applications for the treatment of cell proliferative disorders, such as 
cancer. In particular, TGF-0 has been shown to be potent growth inhibitor for 
a variety of cell types (Massague, Cell, 49:437, 1987). MIS has been shown 

5 to inhibit the growth of human endometrial carcinoma tumors in nude mice 
(Donahoe, et a/. f Ann. Surg., 194:472, 1981), and inhibin a has been shown to 
suppress the development of tumors both in the ovary and in the testis 
(Matzuk, et a/., Nature, 360:313, 1992) GDF-6 may have a simlar actiity and 
may therefore be useful as an antiproliferative agent, such as for the treatment 

10 choriocarcinoma. 

Many of the members of the TGF-£ family are also important mediators of 
tissue repair. TGF-£ has been shown to have marked effects on teh formation 
of collagen and causes of striking angiogenic response in teh newborn mouse 
(roberts, et al., Proc. Natl. acad. ScL, USA, 83:4167, 1986). The BMP's can 
15 induce new bone growth and are effective for the treatment of fractures and 
other skeletal defects (Glowacki, ef al., Lancet, 1:959, 1981; Ferguson, ef a/., 
Clin. Orthoped. Relat. Res.,, 221:265, 1988; Johnson, ef al., Clin Orthoped. 
Relat. Res., 230:257, 1988). GDF-6 may have simlar activities and may be 
useful in repair of tissue injury caused by trauma or burns for example. 

20 GDF-6 may play a role in the regulation of uterine function during pregmancy, 
and therefore, GDF-6, anti-GDF-6 antibodies, or antisense polynucleotides may 
be useful in preventing premature labor. 

The term "substantially pure" as used herein refers to GDF-6 which is 
substantially free of other proteins, lipids, carbohydrates or other materials with 
25 which it is naturally associated. One skilled in the art can purify GDF-6 using 
standard techniques for protein purification. The substantially pure polypeptide 
will yield a single major band on a non-reducing polyacrylamide gel. The purity 



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-7- 

of the GDF-6 polypeptide can also be determined by amino-terminal amino 
acid sequence analysis. GDF-6 polypeptide includes functional fragments of 
the polypeptide, as long as the activity of GDF-7 remains. Smaller peptides 
containing the biological activity of GDF-7 are included in the invention. 

5 The invention provides polynucleotides encoding the GDF-6 protein. These 
polynucleotides include DNA, cDNA and RNA sequences which encode GDF-6. 
It is understood that all polynucleotides encoding all or a portion of GDF-6 are 
also included herein, as long as they encode a polypeptide with GDF-6 activity. 
Such polynucleotides include naturally occurring, synthetic, and intentionally 

10 manipulated polynucleotides. For example, GDF-6 polynucleotide may be 
subjected to site-directed mutagenesis. The polynucleotide sequence for GDF- 
6 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 

15 codon. Therefore, all degenerate nucleotide sequences are included in tine 
invention as long as the amino acid sequence of GDF-6 polypeptide encoded 
by the nucleotide sequence is functionally unchanged. 

Specifically disclosed herein is a genomic DNA sequence containing a portion 
of the GDF-6 gene. The sequence contains an open reading frame 
20 corresponding to the predicted C-terminal region of the <3DF-6 precursor 
protein. The encoded polypeptide is predicted to contain a potential 
pentabasic proteolytic processing site. Cleavage of the precursor at this site 
would generate a mature biologically active C-terminal fragment of 120 amino 
acids with a predicted molecular weight of approximately 13,600. 

25 The C-terminal region of GDF-6 following the putative proteolytic processing 
site shows significant homology to the known members of the TGF-0 
superfamily. The GDF-6 sequence contains most of the residues that are 



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highly conserved in other family members (see Figure 3). Among the known 
family members, GDF-6 is most homologous to BMP-2 (57% sequence identity) 
(see Figure 4). 

Minor modifications of the recombinant GDF-6 primary amino acid sequence 
5 may result in proteins which have substantially equivalent activity as compared 
to the GDF-6 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-6 still exists. Further, deletion of one or more 
10 amino acids can also result in a modification of the structure of the resultant 
molecule without significantly altering its biological activity. This can lead to the 
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-6 biological activity. 

15 The nucleotide sequence encoding the GDF-6 polypeptide of the invention 
includes the disclosed sequence and conservative variations thereof. The term 
"conservative variation" as used herein denotes the replacement of an amino 
acid residue by another, biologically similar residue. Examples of conservative 
variations include the substitution of one hydrophobic residue such as 

20 isoleucine, valine, leucine or methionine for another, or the substitution of one 
polar residue for another, such as the substitution of arginine for lysine, 
glutamic for aspartic acid, or glutamine for asparagine, and the like. The term 
"conservative variation 1 ' also includes the use of a substituted amino acid in 
place of an unsubstituted parent amino acid provided that antibodies raised to 

25 the substituted polypeptide also immunoreact with the unsubstituted polypep- 
tide. 



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

Preferably the GDF-6 polynucleotide of the invention is derived from a 

10 mammalian organism, and most preferably from a mouse, rat, or human. 
Screening procedures which rely on nucleic acid hybridization make it possible 
to isolate any gene sequence from any organism, 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. 

15 This requires that short, oligopeptide stretches of amino acid sequence must 
be known. The DNA sequence encoding the protein can be deduced from the 
genetic code, however, the degeneracy of the code must be taken into 
account. It is possible to perform a mixed addition reaction when the 
sequence is degenerate. This includes a heterogeneous mixture of denatured 

20 double-stranded DNA. For such screening, hybridization is preferably 
performed on either single-stranded DNA or denatured double-stranded DNA. 
Hybridization is particularly useful in the detection of cDNA clones derived from 
sources where an extremely low amount of mRNA sequences relating to the 
polypeptide of interest are present. In other words, by using stringent 

25 hybridization conditions directed to avoid non-specific binding, it is possible, 
for example, to allow the autoradiographic visualization of a specific cDNA 
clone by the hybridization of the target DNA to that single probe in the mixture 
which is its complete complement (Wallace, ef a/., Nucl. Acid Res., §:879, 
1981). 



WO 95/01801 PCT/US94/07762 

-10- 

The development of specific DNA sequences encoding GDF-6 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- 
5 stranded DNA sequence by reverse transcription of mRNA isolated from a 
eukaryotic donor cell. In the latter case, a double-stranded DNA complement 
of mRNA is eventually formed which is generally referred to as cDNA. 

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

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

15 known. When the entire sequence of amino acid residues of the desired 
polypeptide is not known, the direct synthesis of DNA sequences is not 
possible and the method of choice is the synthesis of cDNA sequences. 
Among the standard procedures for isolating cDNA sequences of interest is the 
formation of plasmid- or phage-carrying cDNA libraries which are derived from 

20 reverse transcription of mRNA which is abundant in donor cells that have a 
high level of genetic expression. When used in combination with polymerase 
chain reaction technology, even rare expression products can be cloned. In 
those cases where significant portions of the amino acid sequence of the 
polypeptide are known, the production of labeled single or double-stranded 

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



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A cDNA expression library, such as lambda gt1 1 , can be screened indirectly 
for GDF-6 peptides having at least one epitope, using antibodies specific for 
GDF-6. Such antibodies can be either polyclonal^ or monoclonally derived 
and used to detect expression product indicative of the presence of GDF-6 
5 cDNA. 

DNA sequences encoding GDF-6 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 
10 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-6 polynucleotide sequences may be inserted 
15 into a recombinant expression vector. The term "recombinant expression 
vector" refers to a plasmid, virus or other vehicle known in the art that has 
been manipulated by insertion or incorporation of the GDF-6 genetic 
sequences. Such expression vectors contain a promoter sequence which 
facilitates the efficient transcription of the inserted genetic sequence of the host. 
20 The expression vector typically contains an origin of replication, a promoter, as 
well as specific genes which allow phenotypic selection of the transformed 
cells. Vectors suitable for use in the present invention include, but are not 
limited to the T7-based expression vector for expression in bacteria 
(Rosenberg, et a/., Gene, 56:125, 1987), the pMSXND expression vector for 
25 expression in mammalian cells (Lee and Nathans, J. Biol. Chem., 263:3521. 
1 988) and baculovirus-derived vectors for expression in insect cells. The DNA 
segment can be present in the vector operably linked to regulatory elements, 
for example, a promoter (e.g., T7, metallothionein I, or polyhedrin promoters). 



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-12- 

Polynucleotide sequences encoding GDF-6 can be expressed in either 
prokaryotes or eukaryotes. Hosts can include microbial, yeast, insect and 
mammalian organisms. Methods of expressing DNA sequences having 
eukaryotic or viral sequences in prokaryotes are well known in the art. 
5 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. Preferably, the mature C-terminal 
region of GDF-6 is expressed from a cDNA clone containing the entire coding 
sequence of GDF-6. Alternatively, the C-terminal portion of GDF-6 can be 
10 expressed as a fusion protein with the pro- region of another member of the 
TGF-£ family or co-expressed with another pro- region (see for example, 
Hammonds, et ai, Molec. Endocrin. 5:149, 1991; Gray, A., and Mason, A., 
Science, 247:1328, 1990). 

Transformation of a host cell with recombinant DNA may be carried out by 
conventional techniques as are well known to those skilled in the art. Where 
the host is prokaryotic, such as E. co//, competent cells which are capable of 
DNA uptake can be prepared from cells harvested after exponential growth 
phase and subsequently treated by the CaCI 2 method using procedures well 
known in the art. Alternatively, MgCI 2 or RbCI can be used. Transformation 
can also be performed after forming a protoplast of the host cell if desired. 

When the host is a eukaryote, such methods of transfection of DNA as calcium 
phosphate co-precipitates, conventional mechanical procedures such as 
microinjection, electroporation, insertion of a plasmid encased in liposomes, or 
virus vectors may be used. Eukaryotic cells can also be cotransformed with 
25 DNA sequences encoding the GDF-6 of the invention, and a second foreign 
DNA molecule encoding a selectable phenotype, such as the herpes simplex 
thymidine kinase gene. Another method is to use a eukaryotic viral vector, 
such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect 



15 



20 



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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 
thereof, provided by the invention, may be carried out by conventional means 
5 including preparative chromatography and immunological separations involving 
monoclonal or polyclonal antibodies. 

The invention includes antibodies immunoreactive with GDF-6 polypeptide or 
functional fragments thereof. Antibody which consists essentially of pooled 
monoclonal antibodies with different epitopic specificities, as well as distinct 

10 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, ef a/., Nature, 256:495, 1975). The term 
antibody as used in this invention is meant to include intact molecules as well 
as fragments thereof, such as Fab and F{ab') 2 . which are capable of binding 

15 an epitopic determinant on GDF-6. 

The term "cell-proliferative disorder 11 denotes malignant as well as non-malignant 
cell populations which often appear to differ from the surrounding tissue both 
morphologically and genotypically. Malignant cells (i.e. cancer) develop as a 
result of a multistep process. The GDF-6 polynucleotide that is an antisense 
20 molecule is useful in treating malignancies of the various organ systems, 
particularly, for example, cells in placental tissue. Essentially, any disorder 
which is etiologically linked to altered expression of GDF-6 could be considered 
susceptible to treatment with a GDF-6 suppressing reagent. One such 
disorder is a malignant cell proliferative disorder, for example. 



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-14- 

The invention provides a method for detecting a cell proliferative disorder of 
placental tissue which comprises contacting an anti-GDF-6 antibody with a cell 
suspected of having a GDF-6 associated disorder and detecting binding to the 
antibody. The antibody reactive with GDF-6 is labeled with a compound which 
allows detection of binding to GDF-6. For purposes of the invention, an 
antibody specific for GDF-6 polypeptide may be used to detect the level of 
GDF-6 in biological fluids and tissues. Any specimen containing a detectable 
amount of antigen can be used. A preferred sample of this invention is 
placental tissue. The level of GDF-6 in the suspect cell can be compared with 
the level in a normal cell to determine whether the subject has a GDF-6- 
associated cell proliferative disorder. Preferably the subject is human. 

The antibodies of the invention can be used in any subject in which it is 
desirable to administer in vitro or in vivo immunodiagnosis or immunotherapy. 
The antibodies of the 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 
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 are the 
radioimmunoassay (RIA) and the sandwich {immunometric) assay. Detection 
of the antigens using the antibodies of the invention can be done utilizing 
immunoassays which are run in either the forward, reverse, or simultaneous 
modes, including immunohistochemical assays on physiological samples. 
Those of skill in the art will know, or can readily discern, other immunoassay 
formats without undue experimentation. 

The antibodies of the invention can be bound to many different carriers and 
used to detect the presence of an antigen comprising the polypeptide of the 
invention. Examples of well-known carriers include glass, polystyrene, 



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-15- 

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

There are many different labels and methods of labeling known to those of 
ordinary skill in the art. Examples of the types of labels which can be used in 
the present invention include enzymes, radioisotopes, fluorescent compounds, 
colloidal metals, chemiluminescent compounds, phosphorescent compounds, 
10 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 
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 
15 then be specifically detected by means of a second reaction. For example, it 
is common to use such haptens as biotin, which reacts with avidin. or 
dinitrophenyl, puridoxal, and fluorescein, which can react with specific anti- 
hapten antibodies. 

In using the monoclonal antibodies of the invention for the in vivo detection of 
20 antigen, the detectably labeled antibody is given a dose which is diagnostically 
effective. The term "diagnostically effective" means that the amount of 
detectably labeled monoclonal antibody is administered in sufficient quantity to 
enable detection of the site having the antigen comprising a polypeptide of the 
invention for which the monoclonal 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 radioisotope 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 emission, 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 bound to immunoglobulin either 
directly or indirectly by using an intermediate functional group. Intermediate 
functional groups which often are used to bind radioisotopes which exist as 
metallic ions to immunoglobulins are the Afunctional chelating agents such as 
diethylenetriaminepentacetic acid (DTPA) and ethylenediaminetetraacetic acid 

25 (EDTA) and similar molecules. Typical examples of metallic ions which can be 
bound to the monoclonal antibodies of the invention are 111 In, 97 Ru, ^Ga, ^Ga, 
72 As, »Zr f and ^Tl. 



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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 157 Gd, ^Mn, 162 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-6-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-6-associated disease is effective. The term "ameliorate" 

15 denotes a lessening of the detrimental effect of the GDF-6-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-prol'rferative disorder is associated with 
the expression of GDF-6, nucleic acid sequences that interfere with GDF-6 
expression at the translational level can be used. This approach utilizes, for 
example, antisense nucleic acid and ribozymes to block translation of a specific 
GDF-6 mRNA, either by masking that mRNA with an antisense nucleic acid or 

25 by cleaving it with a ribozyme. 



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-18- 

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- 
6-producing cell. The use of antisense methods to inhibit the in vitro 
10 translation of genes is well known in the art (Marcus-Sakura, AnaLBiochem., 
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 nucleotide sequences which 
15 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-type (Hasselhoff, 
Nature, 334:585, 1988) and "hammerhead'-type. Tetrahymena-type ribozymes 
recognize sequences which are four bases in length, while "hammerhead'-type 
ribozymes recognize base 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-type ribozymes for inactivating a 
specific mRNA species and 18-based recognition sequences are preferable to 
shorter recognition sequences. 



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The present invention also provides gene therapy for the treatment of cell 
proliferative or immunologic disorders which are mediated by GDF-6 protein. 
Such therapy would achieve its therapeutic effect by introduction of the GDF-6 
antisense polynucleotide into cells having the proliferative disorder. Delivery of 
5 antisense GDF-6 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 virus, 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-6 sequence of interest 
into the viral vector, along with another gene which encodes the ligand 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 experiments- 

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-6 antisense polynucleotide. 



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

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, pol and env t by 
15 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-6 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 can encapsulate a substantial percentage of ah 
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, ef a/., Trends Biochem. Sci., g:77, 1981). In 



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-21- 

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, ef a/., 
Biotechniques, 6:682, 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. 

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 
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 prganelle-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 



15 



20 



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-22- 

contain sinusoidal capillaries. Active targeting, on the other hand, involves 
alteration of the liposome by coupling the liposome to a specific ligand such 
as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the 
composition or size of the liposome in order to achieve targeting to organs and 
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-6 in placenta! tissue, there are a variety of 
applications using the polypeptide, polynucleotide, and antibodies of the 
invention, related to this tissue. Such applications include treatment of cell 
proliferative disorders involving this tissue. In addition, GDF-6 may be useful 
15 in various gene therapy procedures. 

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



EXAMPLE 1 

20 IDENTIFICATION AND ISOLATION OF A NOVEL 

TGF-l FAMILY MEMBER 

To identify a new member of the TGF-p superfamily, degenerate 
oligonucleotides were designed which corresponded to two conserved regions 
among the known family members: one region spanning the two tryptophan 
25 residues conserved in all family members except MIS and the other region 



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spanning the invariant cysteine residues near the C-terminus. These primers 
were used for polymerase chain reactions on-mouse genomic DNA followed 
by subcloning the PCR products using restriction sites placed at the 5' ends 
of the primers, picking individual E coli colonies carrying these subcloned 
5 inserts, and using a combination of random sequencing and hybridization 
analysis to eliminate known members of the superfamily. 

GDF-6 was identified from a mixture of PCR products obtained with the primers 
SJL141 : 5'-CCGGMTTCGGITGG(G/C/A)A(G/A/T/C){A/G)A(T/C)TGG(A/G) 

(A/G)TI(T/G)CICC-3' (SEQ ID NO:1) 
10 SJL145:5 , -CCGGMTTC(G/A)CAI(G/C)C(G/A)CAIG(C/AHG/A/T/C)TC^ 

(T/C)CAT-3 > (SEQ ID NO:2) 

PCR using these primers was carried out with 2 mouse genomic DNA at 
94°C for 1 min, 50°C for 2 min ( and 72°C for 2 min for 40 cycles. 

PCR products of approximately 280 bp were gel-purified, digested with Eco Rl ( 
15 gel-purified again, and subcloned in the Bluescript vector (Stratagene, San 
Diego, CA). Bacterial colonies carrying individual subclones were picked into 
96 well microt'rter plates, and multiple replicas were prepared by plating the 
cells onto nitrocellulose. The replicate filters were hybridized to probes 
representing known members of the family, and DNA was prepared from non- 
20 hybridizing colonies for sequence analysis. 

The primer combination of SJL141 and SJL145, encoding the amino acid 
sequences GW(H/Q/N/K/D/E)(D/N)W(V/I/M)(V/I/M)(A/S)P (SEQ ID NO:3) and 
M(V/l/MyT/A)V(D/E)(A/S)C<G/A)C (SEQ ID NO:4) respectively, yielded four 
previously identified sequences <BMP-4, inhibin ^B, GDF-3 and GDF-5) and two 
25 novel sequences, which were designated GDF-6 and GDF-7 among 134 
subclones analyzed. 



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EXAMPLE 2 

EXPRESSION PATTERN AND SEQUENCE OF GPF-6 

To determine the expression pattern of GDF-6, RNA samples prepared from 
a variety of adult tissues were screened by Northern analysis. RNA isolation 
5 and Northern analysis were carried out as described previously (Lee, S.-J., 
Mol. Endocrinol., 4:1034, 1990) except that hybridization was carried out in 5X 
SSPE, 10% dextran sulfate, 50% formamide, 1% SDS, 200 pg/ml salmon DNA, 
and 0.1% each of bovine serum albumin, ficoll, and polyvinylpyrrolidone. Five 
micrograms of twice poly A-selected RNA were electrophoresed on 
10 formaldehyde gels, blotted, and probed with GDF-6. As shown in Figure 1 , the 
GDF-6 probe detected a single mRNA species expressed in placentas during 
late gestation. 

To obtain a larger segment of the GDF-6 gene, a mouse genomic library was 
screened with a probe derived from the GDF-6 PCR product. The partial 

15 sequence of a GDF-6 genomic clone is shown in Figure 2a. The sequence 
contains an open reading frame corresponding to the predicted C-terminal 
region of the GDF-6 precursor protein. The predicted GDF-6 sequence 
contains a potential proteolytic processing site, which is boxed. Cleavage of 
the precursor at this site would generate a mature C-terminal fragment 120 

20 amino acids in length with a predicted molecular weight of 13,600. 

The C-terminal region of GDF-6 following the putative proteolytic processing 
site shows significant homology to the known members of the TGF-0 
superfamily (Figure 3). Figure 3 shows the alignment of the C-terminal 
sequences of GDF-6 with the corresponding regions of human GDF-1 (Lee, 
25 Proc. Natl. Acad. ScL USA, 88:4250-4254, 1991), human BMP-2 and 4 
(Wozney, et al., Science, 242:1528-1534, 1988), human Vgr-1 (Celeste, et a/., 
Proc. Natl. Acad. ScL USA, 8Z:9843-9847, 1990), human OP-1 <Ozkaynak, et 



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aL, EMBO J., 9:2085-2093, 1990), human BMP-5 (Celeste, et aL, Proc. Natl. 
Acad. Sci. USA, £7:9843-9847, 1990), human BMP-3 (Wozney, etal., Science, 
242:1528-1534, 1988), human MIS (Cate, etaL, Cell, 45:685-698, 1986), human 
inhibin alpha, ^A, and pB (Mason, et aL, Biochem, Biophys. Res. Commun., 
5 135:957-964, 1986), human TGF-^1 (Derynck, ef aL, Nature, 316:701-705, 
1985), humanTGF-^2 (deMartin, ef aL, EMBO J., 6:3673-3677, 1987), and 
human TGF-^3 (ten Dijke, et aL, Proc. Natl. Acad. Sci. USA, 85:4715-4719, 
1988). The conserved cysteine residues are boxed. Dashes denote gaps 
introduced in order to maximize the alignment. 

10 GDF-6 contains most of the residues that are highly conserved in other family 
members, including the seven cysteine residues with their characteristic 
spacing. 

FIGURE 4 shows the amino acid homologies among the different members of 
the TGF-£ superfamily. Numbers represent percent amino acid identities 
15 between each pair calculated from the first conserved cysteine to the C- 
terminus. Boxes represent homologies among highly-related members within 
particular subgroups. In this region, GDF-6 is most homologous to BMP-2 
(57% sequence identity). 

Although the invention has been described with reference to the presently 
20 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 



SEQ 



ID 



NO: 



1 is the nucleotide sequence for the GDF-6 primer, SJL141. 



SEQ 



ID 



NO: 



2 is the nucleotide sequence for the GDF-6 primer, SJL145. 



SEQ 



ID 



NO: 



3 is the amino acid sequence for the primer, SJL141. 



5 



SEQ 



ID 



NO: 



4 is the amino acid sequence for primer SJL145. 



SEQ ID NO: 5 is the nucleotide and deduced amino acid sequence for GDF-6. 

SEQ ID NO: 6 is the deduced amino acid sequence for GDF-6. 

SEQ ID NO: 7 is the amino acid for the C-terminal sequence of GDF-6. 

SEQ ID NO: 8 is the amino acid for the C-terminal sequence of GDF-1 . 
10 SEQ ID NO: 9 is the amino acid for the C-terminal sequence of BMP-2. 

SEQ ID NO: 10 is the amino acid for the C-terminal sequence of BMP-4. 

SEQ ID NO: 1 1 is the amino acid for the C-terminal sequence of Vgr-1 . 

SEQ ID NO: 12 is the amino acid for the C-terminal sequence of OP-1. 

SEQ ID NO: 13 is the amino acid for the C-terminal sequence of BMP-5. 
15 SEQ ID NO: 14 is the amino acid for the C-terminal sequence of BMP-3. 



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-27- 

SEQ ID NO: 15 is the amino acid for the C-terminal sequence of MIS. 

SEQ ID NO: 16 is the amino acid for the C-terminal sequence of Inhibin-alpha. 

SEQ ID NO: 17 is the amino acid for the C-terminal sequence of Inhibin-beta- 
alpha. 

5 SEQ ID NO: 18 is the amino acid for the C-terminal sequence of Inhibin-beta- 
beta. 

SEQ ID NO: 19 is the amino acid for the C-terminal sequence of TGF-beta-1. 
SEQ ID NO: 20 is the amino acid for the C-terminal sequence of TGF-beta-2. 
SEQ ID NO: 21 is the amino acid for the C-terminal sequence of TGF-beta-3. 



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SEQUENCE LISTING 

(1) GENERAL INFORMATION: 

(i) APPLICANT: THE JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE 
(ii) TITLE OF INVENTION: GROWTH DIFFERENTIATION FACTOR- 6 
5 (iii) NUMBER OF SEQUENCES: 21 

(iv) CORRESPONDENCE ADDRESS: 

(A) ADDRESSEE: Spensley Horn Jubas & Lubitz 

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

(D) STATE: California 

(E) COUNTRY: USA 

(F) ZIP: 90067 

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

<B) COMPUTER: IBM PC compatible 

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

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

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

(B) FILING DATE: 08-JUL-1994 

(C) CLASSIFICATION : 

(viii) ATTORNEY/AGENT INFORMATION: 

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

(C) REFERENCE/DOCKET NUMBER: FD2349 

(ix) TELECOMMUNICATION INFORMATION: 

(A) TELEPHONE: (619) 455-5100 

(B) TELEFAX: (619) 455-5110 

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



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 35 base pairs 

(B) TYPE: nucleic acid 



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(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 



(vii) IMMEDIATE SOURCE: 
5 (B) CLONE: SJL141 



(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 1. .35 

(D) OTHER INFORMATION: /note= "V=guanine , cytosine or 
10 adenine; N«adenine f cytosine, guanine or thymine 

R«=adenine or guanine; Y=cytosine or thymine; 
K= thymine or guanine; B*=inosine" 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: 
CCGGAATTCG GBTGGVANRA YTGGRTBRTB KCBCC 
15 (2) INFORMATION FOR SEQ ID NO:2: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 33 base pairs 

(B) TYPE: nucleic acid 

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

(ii) MOLECULE TYPE: DNA (genomic) 



(vii) IMMEDIATE SOURCE: 
(B) CLONE: SJL145 

(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 1..29 

(D) OTHER INFORMATION: /note- "R-adenine or guanine; 
S«cytosine or guanine; M=adenine or cytosine; 
N=adenine, cytosine, guanine or thymine; 
Y^cytosine or thymine; B»inosine n 



25 



30 



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-30- 



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

CCGGAATTCR CABSCRCABG MNTCBACBRY CAT 

(2) INFORMATION FOR SEQ ID NO: 3: 

(i) SEQUENCE CHARACTERISTICS: 
5 (A) LENGTH: 9 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: peptide 



10 (vii) IMMEDIATE SOURCE: 

(B) CLONE: SJL141 



(ix) FEATURE: 

(A) NAME/KEY: Peptide 

(B) LOCATION: 1. .9 

15 (D) OTHER INFORMATION: 

or Glu; Asp-Asp 
Ala«Ala or Ser" 



/note*= n His=His, Gin, Asn, Lys, Asp 
or Asn; Val-Val, lie or Met; 



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

Gly Trp His Asp Trp Val Val Ala Pro 
20 1 5 

(2) INFORMATION FOR SEQ ID NO: 4: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 8 amino acids 

(B) TYPE: amino acid 

25 (C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: peptide 



(vii) IMMEDIATE SOURCE: 
(B) CLONE: SJL145 



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(ix) FEATURE: 

(A) NAME/KEY: Peptide 

(B) LOCATION: 1..8 

(D) OTHER INFORMATION: /note- "Val, position 1-Val, He, Met, 
Thr or Ala; Asp«Asp or Glu; Ala«Ala or Ser; Gly-Gly or 
Ala" 



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

Met Val Val Asp Ala Cys Gly Cys 
1 5 

10 (2) INFORMATION FOR SEQ ID NO: 5: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 530 base pairs 

(B) TYPE: nucleic acid 

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

(ii) MOLECULE TYPE: DNA (genomic) 



(vii) IMMEDIATE SOURCE: 
(B) CLONE: GDF-6 

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

(B) LOCATION: 126.. 527 



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

GCCCTGCTTG TAGTGTTCAC CAGATCGCAG CGCAAGAACC TGTTCACTGA GATGCATGAG 60 

CAGCTGGGCT CTGCAGAGGC TGCGGGAGCC GAGGGGTCAT GGCCAGCGCC GTCGGGCTCC 120 

25 CAGAC GCC GGG TCT TCG CTG CCC TCG CCC GGC CGC €GG CGG CGA GGC 167 

Ala Gly Ser Trp Leu Pro Ser Pro Gly Arg Arg Arg Arg Arg 
1 5 10 

ACC GCC TTC GCC AGC CGT CAC GGC AAG CGA CAT GGC AAG AAG TCC AGG 215 
Thr Ala Phe Ala Ser Arg His Gly Lys Arg His Gly Lys Lys Ser Arg 
30 15 20 25 30 



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CTG CGC TGC AGC AGA AAG CCT CTG CAC GTG AAT TTT AAG GAG TTA GGC 263 
Leu Arg Cys Ser Arg Lys Pro Leu His Val Asn Phe Lys Glu Leu Gly 
35 40 45 

TGG GAC GAC TGG ATT ATC GCG CCC CTA GAG TAC GAG GCC TAT CAC TGC 311 
5 Trp Asp Asp Trp lie lie Ala Pro Leu Glu Tyr Glu Ala Tyr His Cys 

50 55 60 

GAG GGC GTG TGC GAC TTT CCG CTG CGC TCG CAC CTT GAG CCC ACT AAC 359 
Glu Gly Val Cys Asp Phe Pro Leu Arg Ser His Leu Glu Pro Thr Asn 
65 70 75 

10 CAT GCC ATC ATT CAG ACG CTG ATG AAC TCC ATG GAC CCG GGC TCC ACC 407 

His Ala lie lie Gin Thr Leu Met Asn Ser Met Asp Pro Gly Ser Thr 
80 85 90 

CCG CCT AGC TGC TGC GTT CCC ACC AAA CTG ACT CCC ATT AGC ATC CTG 455 
Pro Pro Ser Cys Cys Val Pro Thr Lys Leu Thr Pro lie Ser lie Leu 
15 95 100 105 110 

TAC ATC GAC GCG GGC AAT AAT GTA GTC TAC AAG CAG TAT GAG GAC ATG 503 
Tyr lie Asp Ala Gly Asn Asn Val Val Tyr Lys Gin Tyr Glu Asp Met 
115 120 125 

GTG GTG GAG TCC TGC GGC TGT AGG TAG 530 
20 Val Val Glu Ser Cys Gly Cys Arg 
130 



(2) INFORMATION FOR SEQ ID NO: 6: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 134 amino acids 
25 (B) TYPE: amino acid 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

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

Ala Gly Ser Trp Leu Pro Ser Pro Gly Arg Arg Arg Arg Arg Thr Ala 
30 1 5 10 15 



Phe Ala Ser Arg His Gly Lys Arg His Gly Lys Lys Ser Arg Leu Arg 
20 25 30 



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Cys Ser Arg Lys Pro Leu His Val Asn Phe Lys Glu Leu Gly Trp Asp 
35 40 45 

Asp Trp lie lie Ala Pro Leu Glu Tyr Glu Ala Tyr His Cys Glu Gly 
50 55 60 

5 Val Cys Asp Phe Pro Leu Arg Ser His Leu Glu Pro Thr Asn His Ala 
65 70 75 80 

lie lie Gin Thr Leu Met Asn Ser Met Asp Pro Gly Ser Thr Pro Pro 
85 90 95 

Ser Cys Cys Val Pro Thr Lys Leu Thr Pro lie Ser He Leu Tyr He 
10 100 105 110 

Asp Ala Gly Asn Asn Val Val Tyr Lys Gin Tyr Glu Asp Met Val Val 
115 120 125 

Glu Ser Cys Gly Cys Arg 
130 

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

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 119 amino acids 

(B) TYPE: amino acid 

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

(ii) MOLECULE TYPE: protein 



(vii) IMMEDIATE SOURCE: 

(B) CLONE: GDF-6 (C-terminal) 

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

(B) LOCATION: 1..119 



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



Ala 
1 



Phe Ala Ser Arg His Gly Lys Arg His Gly Lys Lys Ser Arg Leu 
5 10 15 



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Arg Cys Ser Arg Lys Pro Leu His Val Asn Phe Lys Glu Leu Gly Trp 
20 ' 25 30 

Asp Asp Trp lie lie Ala Pro Leu Glu Tyr Glu Ala Tyr His Cys Glu 
35 AO 45 

5 Gly Val Cys Asp Phe Pro Leu Arg Ser His Leu Glu Pro Thr Asn His 

50 55 60 

Ala lie He Gin Thr Leu Met Asn Ser Met Asp Pro Gly Ser Thr Pro 
65 70 75 80 

Pro Ser Cys Cys Val Pro Thr Lys Leu Thr Pro He Ser He Leu Tyr 
10 85 90 95 

He Asp Ala Gly Asn Asn Val Val Tyr Lys Gin Tyr Glu Asp Met Val 
100 105 110 

Val Glu Ser Cys Gly Cys Arg 
115 

15 (2) INFORMATION FOR SEQ ID NO: 8: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 123 amino acids 

(B) TYPE: amino acid 

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

(ii) MOLECULE TYPE: protein 



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

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

(B) LOCATION: 1..123 



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



Arg 
1 



Pro Arg Arg Asp Ala Glu Pro Val Leu Gly Gly Gly Pro -Gly <31y 
5 10 15 



WO 95/01801 



PCT/US94/07762 



-35- 



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

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

5 Gly Gin Cys Ala Leu Pro Val Ala Leu Ser Gly Ser Gly Gly Pro Pro 

50 55 60 

Ala Leu Asn His Ala Val Leu Arg Ala Leu Met His Ala Ala Ala Pro 
65 70 75 80 

Gly Ala Ala Asp Leu Pro Cys Cys Val Pro Ala Arg Leu Ser Pro He 
10 85 90 95 

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

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

15 (2) INFORMATION FOR SEQ ID NO: 9: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 118 amino acids 

(B) TYPE: amino acid 

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

(ii) MOLECULE TYPE: protein 



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

(ix) FEATURE: 
25- (A) NAME/KEY: Protein 

(B) LOCATION : I. .118 



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



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



WO 95/01801 



PCT/US94/07762 



-36- 



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

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

5 Gly Glu Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His 

50 55 60 

Ala He Val Gin Thr Leu Val Asn Ser Val Asn Ser Lys He Pro Lys 
65 70 75 80 

Ala Cys Cys Val Pro Thr Glu Leu Ser Ala He Ser Met Leu Tyr Leu 
10 85 90 95 

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

Glu Gly Cys Gly Cys Arg 
115 

15 (2) INFORMATION FOR SEQ ID NO: 10: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 118 amino acids 

(B) TYPE: amino acid 

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

(ii) MOLECULE TYPE: protein 



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

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

(B) LOCATION: 1..118 



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



Lys 
1 



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



WO 95/01801 



PCT/US94/07762 



-37- 



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

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

5 Gly Asp Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His 

50 55 60 

Ala He Val Gin Thr Leu Val Asn Ser Val Asn Ser Ser He Pro Lys 
65 70 75 80 

Ala Cys Cys Val Pro Thr Clu Leu Ser Ala He Ser Met Leu Tyr Leu 
10 85 90 95 

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

Glu Gly Cys Gly Cys Arg 
115 

15 (2) INFORMATION FOR SEQ ID NO: 11: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 119 amino acids 

(B) TYPE: amino acid 

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

(ii) MOLECULE TYPE: protein 



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

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

(B) LOCATION: 1. .119 



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



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



WO 95/01801 



PCT/US94/07762 



-38- 



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

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

5 Gly Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His 

50 55 60 

Ala He Val Gin Thr Leu Val His Leu Met Asn Pro Glu Tyr Val Pro 
65 70 75 80 

Lys Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala He Ser Val Leu Tyr 
10 85 90 95 

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

Val Arg Ala Cys Gly Cys His 
115 

15 (2) INFORMATION FOR SEQ ID NO: 12: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 119 amino acids 

(B) TYPE: amino acid 

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

(ii) MOLECULE TYPE: protein 



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

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

(B) LOCATION: 1..119 



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



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



WO 95/01801 



PCTAJS94/07762 



-39- 



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

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

5 Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn Ala Thr Asn His 

50 55 60 

Ala lie Val Gin Thr Leu Val His Phe He Asn Pro Glu Thr Val Pro 
65 70 75 80 

Lys Pro Cys Cys Ala Pro Thr Gin Leu Asn Ala He Ser Val Leu Tyr 
10 85 90 95 

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

Val Arg Ala Cys Gly Cys His 
115 

15 (2) INFORMATION FOR SEQ ID NO: 13: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 119 amino acids 

(B) TYPE: amino acid 

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

(ii) MOLECULE TYPE: protein 



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

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

(B) LOCATION: 1..119 



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



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



WO 95/01801 



PCT/US94/07762 



-40- 



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

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

5 Gly Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His 

50 55 60 

Ala He Val Gin Thr Leu Val His Leu Met Phe Pro Asp His Val Pro 
65 70 75 80 

Lys Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala He Ser Val Leu Tyr 
10 85 90 95 

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

Val Arg Ser Cys Gly Cys His 
115 

15 (2) INFORMATION FOR SEQ ID NO: 14: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 120 amino acids 

(B) TYPE: amino acid 

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

(ii) MOLECULE TYPE: protein 



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

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

(B) LOCATION: 1..120 



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

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



WO 95/01801 



PCT/US94/07762 



-41- 



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

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

5 Gly Ala Cys Gin Phe Pro Met Pro Lys Ser Leu Lys Pro Ser Asn His 

50 55 60 

Ala Thr He Gin Ser He Val Arg Ala Val Gly Val Val Pro Gly He 
65 70 75 80 

Pro Glu Pro Cys Cys Val Pro Glu Lys Met Ser Ser Leu Ser He Leu 
10 85 90 95 

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

Thr Val Glu Ser Cys Ala Cys Arg 
115 120 

15 (2) INFORMATION FOR SEQ ID NO: 15: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 116 amino acids 

(B) TYPE: amino acid 

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

(ii) MOLECULE TYPE: protein 



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

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

(B) LOCATION: 1..116 



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

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



WO 95/01801 



PCT/US94/07762 



-42- 



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

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

5 Gly Trp Pro Gin Ser Asp Arg Asn Pro Arg Tyr Gly Asn His Val Val 

50 55 60 

Leu Leu Leu Lys Met Gin Ala Arg Gly Ala Ala Leu Ala Arg Pro Pro 
65 70 75 80 

Cys Cys Val Pro Thr Ala Tyr Ala Gly Lys Leu Leu lie Ser Leu Ser 
10 85 90 95 

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

Cys Gly Cys Arg 
115 

15 (2) INFORMATION FOR SEQ ID NO: 16: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 122 amino acids 

(B) TYPE: amino acid 

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

(ii) MOLECULE TYPE: protein 



(vii) IMMEDIATE SOURCE: 

(B) CLONE: Inhibin-alpha 

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

(B) LOCATION: 1..122 



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



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



WO 95/01801 



PCT/US94/07762 



-43- 



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

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

5 Gly Gly Cys Gly Leu His He Pro Pro Asn Leu Ser Leu Pro Val Pro 

50 55 60 

Gly Ala Pro Pro Thr Pro Ala Gin Pro Tyr Ser Leu Leu Pro Gly Ala 
65 70 75 80 

Gin Pro Cys Cys Ala Ala Leu Pro Gly Thr Met Arg Pro Leu His Val 
10 85 90 95 

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

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

15 (2) INFORMATION FOR SEQ ID NO: 17: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 122 amino acids 

(B) TYPE: amino acid 

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

(ii) MOLECULE TYPE: protein 



(vii) IMMEDIATE SOURCE: 

(B) CLONE: Inhibin -beta -alpha 

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

(B) LOCATION: 1. .122 



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



His 
1 



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



WO 95/01801 



PCT/US94/07762 



-44- 



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

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

5 Glu Cys Pro Ser His He Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe 

50 55 60 

His Ser Thr Val He Asn His Tyr Arg Met Arg Gly His Ser Pro Phe 
65 70 75 80 

Ala Asn Leu Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser 
10 85 90 95 

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

Asn Met He Val Glu Glu Cys Gly Cys Ser 
115 120 

15 (2) INFORMATION FOR SEQ ID NO: 18: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 121 amino acids 

(B) TYPE: amino acid 

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

(ii) MOLECULE TYPE: protein 



(vii) IMMEDIATE SOURCE: 

(B) CLONE: Inhibin-beta-beta 

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

(B) LOCATION: 1..121 



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

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



WO 95/01801 



PCT/US94/07762 



-45- 



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

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

5 Ser Cys Pro Ala Tyr Leu Ala Gly Val Pro Gly Ser Ala Ser Ser Phe 

50 55 60 

His Thr Ala Val Val Asn Gin Tyr Arg Met Arg Gly Leu Asn Pro Gly 
65 70 75 80 

Thr Val Asn Ser Cys Cys He Pro Thr Lys Leu Ser Thr Met Ser Met 
10 85 90 95 



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

Met He Val Glu Glu Cys Gly Cys Ala 
115 120 



15 (2) INFORMATION FOR SEQ ID NO: 19: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 115 amino acids 

(B) TYPE: amino acid 

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



(ii) MOLECULE TYPE: protein 



(vii) IMMEDIATE SOURCE: 

(B) CLONE: TGF-beta-1 

(ix) FEATURE: 
25. (A) NAME/KEY: Protein 

(B) LOCATION: 1..115 



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



His 
1 



Arg Arg Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser Thr Glu Lys 
5 10 15 



WO 95/01801 



PCT/US94/07762 



-46- 



Asn Cys Cys Val Arg Gin Leu Tyr He Asp Phe Arg Lys Asp Leu Gly 
20 25 30 

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

5 Gly Pro Cys Pro Tyr He Trp Ser Leu Asp Thr Gin Tyr Ser Lys Val 

50 55 60 

Leu Ala Leu Tyr Asn Gin His Asn Pro Gly Ala Ser Ala Ala Pro Cys 
65 70 75 80 

Cys Val Pro Gin Ala Leu Glu Pro Leu Pro He Val Tyr Tyr Val Gly 
10 85 90 95 

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

Lys Cys Ser 
115 

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

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 115 amino acids 

(B) TYPE: amino acid 

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

(ii) MOLECULE TYPE: protein 



(vii) IMMEDIATE SOURCE: 

(B) CLONE: TGF-beta-2 

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

(B) LOCATION: 1. .115 



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



Lys 
1 



Lys Arg Ala Leu Asp Ala Ala Tyr Cys Phe Arg Asn Val Gin Asp 
5 10 15 



WO 95/01801 



PCT/US94/07762 



-47- 



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

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

5 Gly Ala Cys Pro Tyr Leu Trp Ser Ser Asp Thr Gin His Ser Arg Val 

50 55 60 

Leu Ser Leu Tyr Asn Thr lie Asn Pro Glu Ala Ser Ala Ser Pro Cys 
65 70 75 80 

Cys Val Ser Gin Asp Leu Glu Pro Leu Thr lie Leu Tyr Tyr lie Gly 
10 85 90 95 

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

Lys Cys Ser 
115 

15 (2) INFORMATION FOR SEQ ID NO: 21: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 115 amino acids 

(B) TYPE: amino acid 

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

(ii) MOLECULE TYPE: protein 



(vii) IMMEDIATE SOURCE: 

(B) CLONE: TGF-beta-3 

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

(B) .LOCATION: 1. .115 



(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21: 



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



WO 95/01801 



PCT/US94/07762 



-48- 



Asn Cys Cys Val Arg Pro Leu Tyr He Asp Phe Arg Gin Asp Leu Gly 
20 25 30 

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

5 Gly Pro Cys Pro Tyr Leu Arg Ser Ala Asp Thr Thr His Ser Thr Val 

50 55 60 

Leu Gly Leu Tyr Asn Thr Leu Asn Pro Glu Ala Ser Ala Ser Pro Cys 
65 70 75 80 

Cys Val Pro Gin Asp Leu Glu Pro Leu Thr He Leu Tyr Tyr Val Gly 
10 85 90 95 

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



Lys Cys Ser 
115 



WO 95/01801 PCT/US94/07762 

-49- 

CLAIMS 

1 . Substantially pure growth differentiation factor-6 (GDF-6) and functional 
fragments thereof. 

2. An isolated polynucleotide sequence encoding the GDF-6 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 95/01801 



PCT/US94/07762 



-50- 

1 3. The antibodies of claim 1 1 , 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-6 associated disorder and detecting binding 
of the antibody. 

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

16. The method of claim 14, wherein the detecting is in vivo. 

17. The method of claim 16, wherein the antibody is detectably labeled. 

18. The method of claim 17, wherein the detectable label is selected from 
the group consisting of a radioisotope, a fluorescent compound, a 
bioluminescent compound and a chemiluminescent compound. 

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

20. The method of claim 19, wherein the antibody is detectably labeled. 

21 . The method of claim 20, wherein the label is selected from the group 
consisting of a radioisotope, a fluorescent compound, a bioluminescent 
compound, a chemoluminescent compound and an enzyme. 

22. A method of treating a cell proliferative disorder associated with 
expression of GDF-6, comprising contacting the cells with a reagent 
which suppresses the GDF-6 activity. 

23. The method of claim 22, wherein the reagent is an anti-GDF-6 antibody. 



WO 95/01801 PCT/US94/07762 

-51- 

24. The method of claim 22, wherein the reagent is a GDF-6 antisense 
sequence. 

25. The method of claim 22, wherein the cell is a placental cell. 

26. The method of claim 22, wherein the reagent which suppresses GDF-6 
activity is introduced to a cell using a vector. 

27. The method of claim 26, wherein the vector is a colloidal dispersion 
system. 

28. The method of claim 27, wherein the colloidal dispersion system is a 
liposome. 

29. The method of claim 28, wherein the liposome is essentially target 
specific. 

30. The method of claim 29, wherein the liposome is anatomically targeted. 

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

32. The method of claim 31, wherein the mechanistic targeting is passive. 

33. The method of claim 31, wherein the mechanistic targeting is active. 

34. The method of claim 33, wherein the liposome is actively targeted by 
coupling with a moiety selected from the group consisting of a sugar, 
a glycolipid, and a protein. 



WO 95/01801 PCT/US94/07762 

-52- 

35. The method of claim 34, wherein the protein moiety is an antibody. 

36. The method of claim 35, wherein the vector is a virus. 

37. The method of claim 36, wherein the virus is an RNA virus. 

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

39. The method of claim 38, wherein the retrovirus is essentially target 
specific. 

40. The method of claim 39, wherein the moiety for target specificity is 
encoded by a polynucleotide inserted into the retroviral genome. 

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

42. The method of claim 41 , wherein the protein is an antibody. 



WO 95/01801 



1/4 



PCT/l!S94/07762 




FIG. I 



SUBSTITUTE SHEET (RULE 26) 



WO 95/01801 



2/4 



PCT/US94/07762 





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UNTEEINATHONAL SEARCH RETORT 



International application No. 
PCT/US94/07762 



A. CLASSIFICATION OF SUBJECT MATTER 

IPC(5) tPlease See Extra Sheet. 

US CL :Please See Extra Sheet 
According to International Patent Classification (IPC) or to both national classification and IPC 



1. FIELDS SEARCHED) 



Minimum documentation searched (classification system followed by classification symbols) 
U.S. : 530/399, 397, 350; 536723.5, 23.51; 435/320.1, 252.3, 240.1, 7.1, 7.2; 424/85.8 



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) 
GEMEMBL SEQUENCE DATABASES, APS, DIALOG 



C. DOCUMENTS CONSDDERED TO BE RELEVANT 
Category 0 



A,P 
A,P 



A 



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



WO, A, 93/16099 (NEIDHARDT ET AL) 19 August 1993, 
see entire document. 

Nature, Volume 368, issued 14 April 1994, Storm et al, 
"Limb alterations in brachypodism mice due to mutations in 
a new member of the TGF B-superfamily", pages 639-643, 
see entire document. 

WO, A, 92/00382 (LEE) 09 January 1992, see entire 
document. 

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



Relevant to claim No. 



1-21 
1-21 



1-21 
1-21 



fx] Further documents are listed in the continuation of Box C. Q See patent family annex. 



T 

o- 
•r 



Spacol crtegomo of cited documenti: 

d ocumr nl defining the {jencml ctotc of the ort tvbich b cot considered 
Id bz of particular relevance 

cancer decuman published oa or after the mternnucaai filing date 

document crhicb may throw doubto on priority donn^o) or which b 
csied Co cniofaltoh the pubbcotioa dote of another citatum or other 
cpechl rec s og (co epecifted) 

docBsmesa referroQ to on oral disclosure, use, exhibition or other 



"X* 



d omnnrM p ufal bhe d prior to the mlerantionaJ filing dale but later man 
the pnomy dote claimed 



bier decuman publnbed after the micranxtonal filing dote or prionsy 
dote and not m conflict ty fan the f^irnt^ hui eiurf to imtiMntnt^ ^ 
prasiptt or theory underlying the invention 

dccumenl of parttcukir relgvonce; the claimed mvcatkm be 
conaidefed novel or cannot be coaaidered to involve on 
when the document b t^hcti alone 

documeot of porttcuibr releva&ce; the cbhned Eavearaoa entyrfft be 
cococdered to involve on inveative otep when (he document b 
combined with one or more other cucb documents, ouch combmottoa 
being obvious to o percoa chilled m the on 

documeot mrimbci of the came patent family 



Date of the actual completion of the international search 
05 OCTOBER 1994 



Name and mailing address of the ISA/US 
Commissioner of Potento and Trodemarfco 
Boa PCT 

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



Form PCT/1SA/210 (second sheet)(July 1992)* 



Date of mailing of the international search report 



Authorized officer 



ELIZABETH C KEM MERER 
Telephone No. (703) 308-0196 



ERER / 




INTERNATIONAL SEARCH REPORT 



International application No. 
PCT/US94/07762 



C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT 


Category* 


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


Relevant to claim No. 


A 


Journal of Biological Chemistry, Volume 268, Number 5, issued 
15 February 1993, McPherron et al, "GDF-3 and GDF-9: Two 
New Members of the Transforming Growth Factor-B Superfamily 
Containing a Novel Pattern of Cysteines' 1 , pages 3444-3449, see 
entire document. 


1-21 


A 


Molecular Endocrinology, Volume 4, Number 7, issued 1990, 
Lee, "Identification of a Novel Member (GDF-1) of the 
Transforming Growth Factor-B Superfamily-, pages 1034-1039, 
see entire document 


1-21 



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



INTERNATIONAL SEARCH REPORT 



International application No. 
PCT/US94/07762 



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



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

1. I I Claims Nos.: 

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



□ 



Claims Nos.: 

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



3. Q Claims Nos.: 

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



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



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



. pi As all required additional search fees were timely paid by the applicant, this international search report covers all searchable 
claims. 

- O M aU 5carchablc ckin* «>uld be searched without effort justifying an additional fee, this Authority did not invite payment 
of any additional fee. 

As only some of the required additional search fees were timely paid by the applicant, this international search report covers 
only those claims for which fees were paid, specifically claims Nos.: 
1*21 



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



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

No protest accompanied the payment of additional search fees. 



Form PCT/ISA/210 (continuation of first shcel(i))(July 1992)* 



INTERNATIONAL SEARCH REPORT 



Internationa! application No. 
PCT/US94/07762 



A. CLASSIFICATION OF SUBJECT MATTER: 
IPC (5): 

A61K 37/24, 37/36, 39/00; C07K 3/00, 13/00. 15/00. 17/00. C07H 17/00; C12Q 1/00; G01N 33/53; C12N 5/00, 
1/20, 15/00 

A. CLASSIFICATION OF SUBJECT MATTER: 
USCL : 

530/399, 397, 350; 536/23.5, 23.51; 435/320.1, 252.3, 240.1, 7.1, 7^2; 424/S5.8 

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

I. Claims 1-10, drawn to GDF-6 polypeptides, polynucleotides encoding same, vectors comprising the 

polynucleotides, and host cells. 

D. Claims 11-21, drawn to antibodies and a diagnostic method utilizing said antibodies. 

ID. Claims 22, 23, and 25, drawn to a method of treating disease with an antibody. 

IV. Claims 22, 24, and 25, drawn to a method of treating a disease with antasense polynucleotides. 

V. Claims 22 and 25-28, drawn to a method of treating disease utilizing gene therapy techniques. 

VI. Claims 29-42 drawn to targeted gene therapy techniques. 

The six groups of claims listed above are not so linked by a special technical feature within the meaning of PCT Rule 
13.2 so as to form a single inventive concept. Specifically, group I requires polypeptides which is not required by any 
of groups n-VI. Similarly, group II requires antibodies which are not required by groups IV or V. Although some 
claims of groups III and VI require antibodies, the methods of groups III and VI require consideration of disease states 
and therapies which are not required by group II. Groups III through VI are drawn to separate methods, in that each 
method requires elements not required by the others. For instance, group III requires consideration of antibody 
administration, which is not required by any of the other groups. Group IV requires consideration of antisense 
technology, which is not required by any of the other groups. Group V requires consideration of basic gene therapy 
techniques which is not required by the methods of groups ID or IV. Groups IV and V are separate in that group V 
requires consideration of liposome targeting, which is not required by any of the other groups. 



Form PCT/ISA/210 (extra shectXJuly 1992)* 



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