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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




PCT 

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 6 : 

C12N 15/12, C07K 14/475, 16/22, C12N 
15/62, A61K 38/18 



Al 



(31) International Publication Number: WO 98/49296 

(43) International Publication Date: 5 November 1998 (05.1 1.98) 



(21) International Application Number: PCT/US98/06324 

(22) International Filing Date: 31 March 1998 (31.03.98) 



(30) Priority Data: 

60/044,427 
60/045,157 



29 April 1997 (29.04.97) US 

30 April 1997 (30.04.97) US 



(71) Applicant (for all designated States except US): REGENERON 

PHARMACEUTICALS, INC. [US/US]; 777 Old Saw Mill 
River Road, Tarrytown, NY 10591-6707 (US). 

(72) Inventors; and 

(75) Inventors/Applicants (for US only): VALENZUELA, David, 
M. fCL/USJ; 216 Grange Street, Franklin Square, NY 
11010 (US). ROJAS, Eduardo, A. [CUUS]; Apartment 
304, 39 Longview Avenue, White Plains, NY 10605 (US). 
ECONOMIDES, Aris, N. [GR/US]; 12 Mt. Morris Park 
West, New York. NY 10027 (US). STAHL, Neil, E. 
[US/US]; R.D. #10, Kent Shore Drive, Carmel, NY 10512 
(US). 



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



Published 

With international search report. 



(74) Agents: COBERT, Robert, J. et al.; Regeneron Pharmaceuti- 
cals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY 
10591 (US). 



(54) TiUe: HUMAN CERBERUS PROTEIN 



(57) Abstract 

Human Cerberus proteins and related nucleic acids are provided. Included are proteins comprising a human cerberus domain having 
specific activity, particularly the ability to antagonize a bone morphogenic protein. The proteins may be produced recombinant^ from 
transformed host cells with the subject nucleic acids. Also provided are isolated hybridization probes and primers capable of specifically 
hybridizing with the disclosed genes, specific binding agents and methods of making and using the subject compositions. 



FOR THE PURPOSES OF INFORMATION ONLY 



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



AL 


Albania 


ES 


Spain 


LS 


Lesotho 


SI 


Slovenia 


AM 


Armenia 


Fl 


Finland 


LT 


Lithuania 


SK 


Slovakia 


AT 


Austria 


FR 


France 


LU 


Luxembourg 


SN 


Senegal 


AU 


Australia 


GA 


Gabon 


LV 


Latvia 


sz 


Swaziland 


AZ 


Azerbaijan 


GB 


United Kingdom 


MC 


Monaco 


TD 


Chad 


BA 


Bosnia and Herzegovina 


GE 


Georgia 


MD 


Republic of Moldova 


TG 


Togo 


BB 


Barbados 


GH 


Ghana 


MG 


Madagascar 


TJ 


Tajikistan 


BE 


Belgium 


GN 


Guinea 


MK 


The former Yugoslav 


TM 


Turkmenistan 


BF 


Burkina Faso 


GR 


Greece 




Republic of Macedonia 


TR 


Turkey 


BG 


Bulgaria 


HU 


Hungary 


ML 


Mali 


TT 


Trinidad and Tobago 


BJ 


Benin 


IE 


Ireland 


MN 


Mongolia 


UA 


Ukraine 


BR 


Brazil 


IL 


Israel 


MR 


Mauritania 


UG 


Uganda 


BY 


Belarus 


IS 


Iceland 


MW 


Malawi 


US 


United States of America 


CA 


Canada 


IT 


Italy 


MX 


Mexico 


uz 


Uzbekistan 


CF 


Central African Republic 


JP 


Japan 


NE 


Niger 


VN 


Viet Nam 


CG 


Congo 


KE 


Kenya 


NL 


Netherlands 


YU 


Yugoslavia 


CH 


Switzerland 


KG 


Kyrgyzstan 


NO 


Norway 


zw 


Zimbabwe 


CI 


Cote d'l voire 


KP 


Democratic People's 


NZ 


New Zealand 






CM 


Cameroon 




Republic of Korea 


PL 


Poland 






CN 


China 


KR 


Republic of Korea 


PT 


Portugal 






CU 


Cuba 


KZ 


Kazalcstan 


RO 


Romania 






CZ 


Czech Republic 


LC 


Saint Lucia 


RU 


Russian Federation 






DE 


Germany 


U 


Liechtenstein 


SD 


Sudan 






UK 


Denmark 


LK 


Sri Lanka 


SE 


Sweden 






EE 


Estonia 


LR 


Liberia 


SG 


Singapore 







WO 98/49296 



PCI7US98/06324 



HUMAN CERBERUS PROTEIN 

This International Application claims priority of U.S. Provisional Application 
60/045,157 filed April 30, 1997 and U.S. Provisional Application 60/044,427 filed 
April 29, 1997. All publications, patents and patent applications cited in this 
specification are hereby incorporated by reference as if each individual 
publication, patent or patent application was specifically and individually 
indicated to be incorporated by reference. 

INTRODUCTION 

Field of the Invention 

The field of this invention is proteins which regulate cell function, and in 
particular, induce neural development or antagonize bone morphogenic 
proteins. 

Background 

Natural regulators of cellular growth, differentiation and function have 
provided important pharmaceuticals, clinical and laboratory tools, and targets for 
therapeutic intervention. A variety of such regulators have been shown to have 
profound effects on basic cellular differentiation and developmental pathways. 
For example, the recently cloned Xenopus cerberus protein induces the 
formation of head structures in anterior endoderm of vertebrate embryos. 
Similarly, the noggin protein induces head structures in vertebrate embryos, and 
can redirect mesodermal fates from ventral fates, such as blood and 
mesenchyme, to dorsal fates such as muscle and notochord and can redirect 
epidermal fates to anterior neural fates. The activities of chordin are similar to 
those of noggin, reflecting a common mechanism of action - namely 
antagonizing bone morphogenic proteins (BMP) and thereby preventing their 
function. BMPs.have diverse biological activities in different biological contexts, 



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including the induction of cartilage, bone and connective tissue, and roles in 
kidney, tooth, gut, skin and hair development. 

Different members of the TGFP superfamily can instruct cells to follow different 
5 fates, for example TGFP induces neural crest to form smooth muscle, while 
BMP2 induces the same cells to become neurons. In Xenopus experiments, 
dissociated animal cap cells (prospective ectoderm) become epidermis in 
response to BMP4 but become mesoderm in response to activin. 

10 Since the sequence identity between activin and BMP4 is low, it is not surprising 
that they induce different fates. It is more surprising that members of the BMP 
subfamily, which are quite closely related in sequence, can induce distinct fates. 
A striking example results from implantation of a matrix impregnated with a 
BMP into muscle; when the effects are monitored histologically, BMP2, 4 and 7 

15 induce endochondral bone formation, whereas a related molecule BMP12/GDF7 
induces connective tissue similar to tendon. Similarly, BMP4 can induce cell 
death in the hindbrain neural crest, while the related protein dorsalin does not. 

Since different BMP family members can induce different fates, then BMP 
20 antagonists that have specificity in blocking subsets of BMPs could change the 
balance of BMPs that are presented to a cell, thus altering cell fate. In view of the 
importance of relative BMP expression in human health and disease, regulators 
of cellular function and BMP function in particular, such as noggin and cerberus, 
provide valuable reagents with a host of clinical and biotechnological 
25 applications. The present invention relates to a new family of regulators of 
cellular function. 



Relevant I Jtpraturp 

30 

Bouwmeester, T., et al. (1996) Nature 382: 595-601 describe cloning of the Xenopus 



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cerberus gene and provide a deduced amino acid sequence of Xenopus cerberus 
protein in addition to describing its biological activity. Lamb, T. M., et al. (1993) 
Science 262: 713-718; Smith, W. C, et al. (1992) Cell 70: 829-840; Smith, W. C, et 
al. (1993) Nature 361: 547-549; and Zimmerman, L. B v et al. (1996) Cell 86: 599-606 
5 describe the isolation and function of the noggin protein. Piccolo, S., et al. (1996) 
Cell 86: 589-598, Sasai, Y., et al. (1995) Nature 376: 333-336, and Sasai, Y., et al. 
(1994) Cell 79: 779-790 relate to the chordin protein. Enomoto et al. (1994) 
Oncogene 9: 2785-2791 and Ozaki, et al. (1996) Jpn. J. Cancer Res. 87: 58-61 describe 
human and murine homologs of the DAN gene. 

10 

SUMMARY OF THE INVENTION 

The invention provides methods and compositions relating to human cerberus 
protein and related nucleic acids. Included are proteins comprising a human 

15 cerberus domain and having human cerberus-specific activity. The proteins may 
be produced recombinantly from transformed host cells with the subject nucleic 
acids. The invention provides binding agents such as specific antibodies, and 
methods of making and using the subject compositions in diagnosis (e.g., genetic 
hybridization screens for human cerberus transcripts), therapy (e.g., gene therapy 

20 to modulate human cerberus gene expression) and in the biopharmaceutical 
industry (e.g., reagents for screening chemical libraries for lead pharmacological 
agents). 

Preferred uses for the subject human cerberus proteins include modifying the 
25 physiology of a cell comprising an extracellular surface by contacting the cell or 
medium surrounding the cell with an exogenous human cerberus protein under 
conditions whereby the added protein specifically interacts with a component of 
the medium and /or the extracellular surface to effect a change in the physiology 
of the cell. Also preferred are methods for screening for biologically active 
30 agents, which methods involve incubating a human cerberus protein in the 

presence of an extracellular human cerberus protein-specific binding target and a 
candidate agent, 



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under conditions whereby, but for the presence of the agent, the protein 
specifically binds the binding target at a reference affinity; detecting the binding 
affinity of the protein to the binding target to determine an agent-biased affinity, 
wherein a difference between the agent-biased affinity and the reference affinity 
5 indicates that the agent modulates the binding of the protein to the binding 
target. 

BRIEF DESCRIPTION OF THE FIGURE 

10 FIGURE 1 - Western Blot showing binding of human cerberus to human BMP2 
in lane 1. Addition of a human noggin deletion mutein blocks binding of 
human cerberus to human BMP2 as shown in lane 2. Control for non-specific 
binding shown in lane 3. 

15 DETAILED DESCRIPTION OF THE INVENTION 

The invention provides human cerberus protein which includes natural human 
cerberus protein and recombinant proteins comprising a human cerberus amino 
acid sequence, or a functional human cerberus protein domain thereof having an 

20 assay-discernable human cerberus-specific activity. Accordingly, the proteins 
may be deletion mutants of the disclosed natural human cerberus proteins and 
may be provided as fusion products, e.g., with non-human cerberus polypeptides. 
The subject human cerberus protein domains have human cerberus-specific 
activity or function and are functionally distinct from each other and from b57, 

25 DAN and noggin. 

Without being bound by theory, we have formulated hypotheses about the 
embryological effects of cerberus based on where it is expressed, and on the 
effects of RNA injection in embryos. Since cerberus is expressed in the Spemann 
30 organizer, we believe cerberus to be a mediator of some of the effects of the 
Spemann organizer, such as neural induction. Since cerberus is expressed in 
regions that will become heart primordium, we believe cerberus to influence 

4 



WO 98/49296 PCT/US98/06324 

either the dorsal-ventral pattern or anterior-posterior pattern of the neural plate. 
In the frog, cerberus occupies the anteriormost endomesoderm. This localized 
expression of cerberus in the frog may give rise to heart primordium. Although 
cerberus is unable to dorsalize ventral mesoderm and does not rescue axis 

5 formation in ventralized frog embryos, it has been known to promote formation 
of anterior neural structures such as cement gland, brain, and olfactory placodes. 
Cerberus exhibits homology to b57, a protein that has been shown to directly bind 
BMP-2 and BMP-4 and inhibit their biological actions. This effect has also been 
shown to be mediated by the unrelated protein noggin which is also expressed in 

10 the Spemann organizer and which has been shown to induce neural ectoderm. 
By inference, cerberus may also be a direct inhibitor of BMP activity, and this may 
account for its known biological effects. 

A number of applications for cerberus are suggested from its properties. 

15 Cerberus, like noggin and b57, may be useful in the study and treatment of heart 
disease and neurological disorders, as well as pathological conditions that arise 
from or involve heterotopic bone formation, cartilage or cartilagenous plaques. 
Furthermore, the cerberus cDNA may be useful as a diagnostic tool, such as 
through use of antibodies in assays for proteins in cell lines or use of 

20 oligonucleotides as primers in a PCR test to amplify those with sequence 
similarities to the oligonucleotide primer, and to see how much cerberus is 
present. The isolation of human cerberus, of course, also provides the key to 
isolate its putative receptor, other cerberus binding proteins, and /or study its 
antagonistic properties. 

25 

Human cerberus-specific activity or function may be determined by convenient 
jn vitro, cell-based, or in vivo assays - e.g., in_vitro binding assays, cell culture 
assays, in animals (e.g., immune response, gene therapy, transgenics, etc.), etc. 
Binding assays encompass any assay where the specific molecular interaction of a 
30 human cerberus protein with a binding target is evaluated. The binding target 
may be a natural binding target, or a non-natural binding target such as a specific 
immune protein such as an antibody, or a human cerberus specific agent such as 



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those identified in assays described below. Generally, binding specificity is 
assayed by bioassay (e.g., the ability to induce neuronal tissue from injected 
embryonic ectoderm), TGFp protein binding equilibrium constants (usually at 
least about 10 7 M' 1 , preferably at least about 10 8 M _l , more preferably at least about 
5 10 9 M* 1 ), by the ability of the subject protein to function as negative mutants in 
human cerberus-expressing cells, to elicit human cerberus specific antibody in a 
heterologous host (e.g., a rodent or rabbit), etc. 

The claimed proteins may be isolated or pure - an "isolated" protein is one that is 

10 no longer accompanied by some of the material with which it is associated in its 
natural state, and that preferably constitutes at least about 0.5%, and more 
preferably at least about 5% by weight of the total protein in a given sample; a 
"pure" protein constitutes at least about 90%, and preferably at least about 99% by 
weight of the total protein in a given sample. The subject proteins and protein 

15 domains may be synthesized, produced by recombinant technology, or purified 
from cells. A wide variety of molecular and biochemical methods are available 
for biochemical synthesis, molecular expression and purification of the subject 
compositions, see e.g., Molecular Cloning, A Laboratory Manual (Sambrook, et 
al., Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. 

20 Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, NY). An exemplary 
method for isolating natural human cerberus protein involves expressing a 
cDNA library (e.g., one derived from Xenopus ovarian cells) and assaying 
expression products for embryonic axis formation. This method and other 
suitable bioassays amenable to detecting human cerberus proteins have been 

25 described by Lemaire, P., et al., (1995) Cell 81:85-94; Smith, W. C, and Harland, R. 
M. (1992) Cell 70: 829-40; Smith, W. C, and Harland, R. M. (1991) Cell 67: 753-765; 
Piccolo, S., et al., (1996) Cell 86: 589-98; and Zimmerman, L. B., et aL, (1996) Cell 86: 
599-606. 

30 The subject proteins find a wide variety of uses including use as immunogens, 
targets in screening assays, bioactive reagents for modulating cell growth, 



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differentiation and /or function, etc. For example, the invention provides 
methods for modifying the physiology of a cell comprising an extracellular 
surface by contacting the cell or medium surrounding the cell with an exogenous 
human cerberus protein under conditions whereby the added protein specifically 

5 interacts with a component of the medium and /or the extracellular surface to 
effect a change in the physiology of the cell. According to these methods, the 
extracellular surface includes plasma membrane-associated receptors; the 
exogenous human cerberus refers to a protein not made by the cell or, if so, 
expressed at non-natural levels, times or physiologic locales; and suitable media 

10 include in vitro culture media and physiological fluids such as blood, synovial 
fluid, etc. Effective administrations of subject proteins may be used to reduce 
undesirable (e.g., ectopic) bone formation, inhibit the growth of cells that require 
a morphogenic protein (e.g., BMP-dependent neuroblastomas and gliomas), alter 
morphogen-dependent cell fate/differentiation in culture, such as with cells for 

15 transplantation or infusion, etc. The proteins may be may be introduced, 

expressed, or repressed in specific populations of cells by any convenient way 
such as microinjection, promoter-specific expression of recombinant enzyme, 
targeted delivery of lipid vesicles, etc. 

20 The invention provides natural and non-natural human cerberus-specific 

binding agents, methods of identifying and making such agents, and their use in 
diagnosis, therapy and pharmaceutical development. Human cerberus-specific 
binding agents include human cerberus-specific receptors, such as somatically 
recombined protein receptors like specific antibodies or T-cell antigen receptors 

25 (See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring 
Harbor Laboratory) and also includes other natural binding agents identified with 
assays such as one-, two- and three-hybrid screens, and non-natural binding 
agents identified in screens of chemical libraries such as described below. Agents 
of particular interest modulate human cerberus function. 

30 

The invention provides human cerberus nucleic acids, which find a wide variety 
of applications including use as translatable transcripts, hybridization probes, 



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PCR primers, diagnostic nucleic acids, etc., as well as use in detecting the presence 
of human cerberus genes and gene transcripts and in detecting or amplifying 
nucleic acids encoding additional human cerberus homolbgs and structural 
analogs. 

5 

The subject nucleic acids are of synthetic /non-natural sequences and/or are 
isolated, i.e., no longer accompanied by some of the material with which it is 
associated in its natural state, preferably constituting at least about 0.5%, more 
preferably at least about 5% by weight of total nucleic acid present in a given 

10 fraction, and usually recombinant, meaning they comprise a non-natural 

sequence or a natural sequence joined to nucleotide(s) other than that which it is 
joined to on a natural chromosome. Nucleic acids comprising the nucleotide 
sequence disclosed herein and fragments thereof, contain such sequence or 
fragment at a terminus, immediately flanked by a sequence other than that to 

15 which it is joined on a natural chromosome, or flanked by a native flanking 

region fewer than 10 kb, preferably fewer than 2 kb, which is immediately flanked 
by a sequence other than that to which it is joined on a natural chromosome. 
While the nucleic acids are usually RNA or DNA, it is often advantageous to use 
nucleic acids comprising other bases or nucleotide analogs to provide modified 

20 stability, etc. 

The amino acid sequences of the disclosed human cerberus protein is used to 
back translate human cerberus protein-encoding nucleic acids optimized for 
selected expression systems (Holler, et al. (1993) Gene 136: 323-328; Martin, et al. 
(1995) Gene 154: 150-166) or used to generate degenerate oligonucleotide primers 
and probes for use in the isolation of natural human cerberus encoding nucleic 
acid sequences ("GCG M software, Genetics Computer Group, Inc., Madison, WI). 
Human cerberus encoding nucleic acids may be part of expression vectors and 
may be incorporated into recombinant host cells, e.g., for expression and 
screening, for transgenic animals, for functional studies such as the efficacy of 
candidate drugs for disease associated with human cerberus mediated signal 
transduction, etc. Expression systems are selected and /or tailored to effect 



25 



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human cerberus protein structural and functional variants through alternative 
post-translational processing. 

The invention also provides for nucleic acid hybridization probes and 
5 replication /amplification primers having a human cerberus cDNA specific 
sequence and sufficient to effect specific hybridization with SEQ. NO. 1. 
Demonstrating specific hybridization generally requires stringent conditions, for 
example, hybridizing in a buffer comprising 30% formamide in 5 x SSPE (0.18 M 
NaCl, 0.01 M NaP0 4/ pH7.7, 0.001 M EDTA) buffer at a temperature of 42°C and 
10 remaining bound when subject to washing at 42°C with 0.2 x SSPE; preferably 
hybridizing in a buffer comprising 50% formamide in 5 x SSPE buffer at a 
temperature of 42°C and remaining bound when subject to washing at 42°C with 
0.2x SSPE buffer at 42°C. Human cerberus cDNA homologs can also be 
distinguished from other protein using alignment algorithms, such as BLASTX 
15 (Altschul, et al. (1990) Basic Local Alignment Search Tool, J. Mol. Biol. 215: 403- 
410). 

Human cerberus hybridization probes find use in identifying wild-type and 
mutant alleles in clinical and laboratory samples. Mutant alleles are used to 

20 generate allele-specific oligonucleotide (ASO) probes for high-throughput clinical 
diagnoses. Human cerberus nucleic acids are also used to modulate cellular 
expression or intracellular concentration or availability of active human 
cerberus. Human cerberus inhibitory nucleic acids are typically antisense - single 
stranded sequences comprising complements of the disclosed natural human 

25 cerberus coding sequences. Antisense modulation of the expression of a given 
human cerberus protein may employ antisense nucleic acids operably linked to 
gene regulatory sequences. Cells are transfected with a vector comprising a 
human cerberus sequence with a promoter sequence oriented such that 
transcription of the gene yields an antisense transcript capable of binding to 

30 endogenous human cerberus encoding mRNA. Transcription of the antisense 
nucleic acid may be constitutive or inducible and the vector may provide for 



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stable extrachromosomal maintenance or integration. Alternatively, single- 
stranded antisense nucleic acids that bind to genomic DNA or mRNA encoding a 
given human cerberus protein may be administered to the target cell, in or 
temporarily isolated from a host, at a concentration that results in a substantial 
5 reduction in expression of the targeted protein. An enhancement in human 
cerberus expression is effected by introducing into the targeted cell type human 
cerberus nucleic acids which increase the functional expression of the 
corresponding gene products. Such nucleic acids may be human cerberus 
expression vectors, vectors which upregulate the functional expression of an 
10 endogenous allele, or replacement vectors for targeted correction of mutant 
alleles. Techniques for introducing the nucleic acids into viable cells are known 
in the art and include retroviral-based transfection, viral coat protein-liposome 
mediated transfection, etc. 

15 The invention provides efficient methods of identifying agents, compounds or 
lead compounds for agents active at the level of human cerberus modulatable 
cellular function. Generally, these screening methods involve assaying for 
compounds which modulate human cerberus interaction with a natural human 
cerberus binding target. A wide variety of assays for binding agents are provided 

20 including protein-protein binding assays, immunoassays, cell based assays, etc. 
Preferred methods are amenable to automated, cost-effective high throughput 
screening of chemical libraries for lead compounds. 

In vitro binding assays employ a mixture of components including a human 
25 cerberus protein, which may be part of a fusion product with another peptide or 
polypeptide, e.g., a tag for detection or anchoring, etc. The assay mixtures 
comprise a natural human cerberus binding target. While native binding targets 
may be used, it is frequently preferred to use portions thereof as long as the 
portion provides binding affinity and avidity to the -subject human cerberus 
30 conveniently measurable in the assay. The assay mixture also comprises a 
candidate pharmacological agent. Candidate agents encompass numerous 
chemical classes, though typically they are organic compounds, preferably small 

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organic compounds, and are obtained from a wide variety of sources including 
libraries of synthetic or natural compounds. A variety of other reagents such as 
salts, buffers, neutral proteins, e.g., albumin, detergents, protease inhibitors, 
nuclease inhibitors, antimicrobial agents, etc., may also be included. The mixture 

5 components can be added in any order that provides for the requisite bindings 
and incubations may be performed at any temperature which facilitates optimal 
binding. The mixture is incubated under conditions whereby, but for the 
presence of the candidate pharmacological agent, the human cerberus specifically 
binds the cellular binding target, portion or analog with a reference binding 

10 affinity. Incubation periods are chosen for optimal binding but are also 
minimized to facilitate rapid, high throughput screening. 

After incubation, the agent-biased binding between the human cerberus and one 
or more binding targets is detected by any convenient way. For cell-free binding 

15 type assays, a separation step is often used to separate bound from unbound 
components. Separation may be effected by precipitation, immobilization, etc., 
followed by washing by, e.g., membrane filtration or gel chromatography. For 
cell-free binding assays, one of the components usually comprises or is coupled to 
a label. The label may provide for direct detection as radioactivity, luminescence, 

20 optical or electron density, etc., or indirect detection such as an epitope tag, an 
enzyme, etc. A variety of methods may be used to detect the label depending on 
the nature of the label and other assay components, e.g., through optical or 
electron density, radiative emissions, nonradiative energy transfers, or indirectly 
detected with antibody conjugates, etc. A difference in the binding affinity of the 

25 human cerberus protein to the target in the absence of the agent as compared 
with the binding affinity in the presence of the agent indicates that the agent 
modulates the binding of the human cerberus protein to the corresponding 
binding target. A difference, as used herein, is statistically significant and 
preferably represents at least a 50%, more preferably at least a 90% difference. 

30 

The invention provides for a method for modifying the physiology of a cell 
comprising an extracellular surface in contact with a medium, said method 

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comprising the step of contacting said medium with an exogenous human 
cerberus protein under conditions whereby said protein specifically interacts with 
at least one of a component of said medium and said extracellular surface to 
effect a change in the physiology of said cell. 

5 

The invention further provides for a method for screening for biologically active 
agents, said method comprising the steps of a) incubating a human cerberus 
protein in the presence of an extracellular human cerberus protein specific 
binding target and a candidate agent, under conditions whereby, but for the 
10 presence of said agent, said protein specifically binds said binding target at a 

reference affinity; b) detecting the binding affinity of said protein to said binding 
target to determine an agent-biased affinity, wherein a difference between the 
agent-biased affinity and the reference affinity indicates that said agent modulates 
the binding of said protein to said binding target. 

15 

One embodiment of the invention is an isolated human cerberus protein 
comprising the amino acid sequence as set forth herein or a fragment thereof 
having human cerberus-specific activity. 

20 Another embodiment of the invention is a recombinant nucleic acid encoding 
human cerberus protein comprising the amino acid sequence as set forth herein 
or a fragment thereof having human cerberus-specific activity. 

Still another embodiment is an isolated nucleic acid comprising a nucleotide 
25 sequence as set forth herein or a fragment thereof having at least 18 consecutive 
bases and sufficient to specifically hybridize with a nucleic acid having the 
sequence of set forth herein in the presence of natural human cerberus cDNA. 

Applicants have devised a method of screening for a molecule capable of 
30 competing with human cerberus for binding to a BMP comprising: 

a) contactings sample suspected of containing the molecule with the BMP in the 
presence of human cerberus under conditions in which the human cerberus is 

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capable of binding to the BMP; and b) detecting binding of the molecule to the 
BMP. 

In a preferred embodiment, the human cerberus is detectably labeled and 
5 includes, but is not limited to, human cerberus linked covalently or 

noncovalently to a radioactive substance, a fluorescent substance, a substance that 
has enzymatic activity, a substance that may serve as a substrate for an enzyme 
(enzymes and substrates associated with colorimetrically detectable reactions are 
preferred) or to a substance that can be recognized by an antibody molecule that is 
10 preferably a detectably labeled antibody molecule. 

By way of nonlimiting example, the method may be performed via an assay 
which is conceptually similar to an ELISA assay. For example, a BMP may be 
bound to a solid support, such as a plastic multiwell plate. As a control, a known 

15 amount of a molecule which contains the human cerberus BMP-binding domain 
and which has been Myc-tagged may then be introduced to the well and any 
tagged molecules which bind the BMP may then be identified by means of a 
reporter antibody directed against the Myc-tag. This assay system may then be 
used to screen test samples for molecules which are capable of i) binding to the 

20 tagged molecule or ii) binding to the BMP and thereby blocking binding to the 
BMP by the tagged molecule. For example, a test sample containing a putative 
molecule of interest together with a known amount of a tagged molecule which 
contains the human cerberus BMP-binding domain may be introduced to the 
well and the amount of tagged molecule which binds to the BMP may be 

25 measured. By comparing the amount of bound tagged molecule in the test well 
to the amount in the control well, samples containing molecules which are 
capable of blocking tagged molecule binding to the BMP may be identified. The 
molecules of interest thus identified may be isolated using methods well known 
to one of skill in the art. 

30 

Once a blocker of BMP binding is found, one of skill in the art would know to 
perform secondary assays to determine whether the blocker is binding to the 

13 



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tagged molecule or to the BMP, as well as assays to determine if the blocker 
molecule can neutralize the biological activity of the bound molecule. 

The present invention also provides for antibodies to the human cerberus 
5 protein described herein which are useful for detection of the protein in, for 
example, diagnostic applications. For preparation of monoclonal antibodies 
directed toward this human cerberus protein, any technique which provides for 
the production of antibody molecules by continuous cell lines in culture may be 
used. For example, the hybridoma technique originally developed by Kohler and 
10 Milstein (1975, Nature 256:495-497), as well as the trioma technique, the human 
B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and 
the EBV-hybridoma technique to produce human monoclonal antibodies (Cole 
et al., 1985, in "Monoclonal Antibodies and Cancer Therapy," Alan R. Liss, Inc. 
pp. 77-96) and the like are within the scope of the present invention. 

15 

The monoclonal antibodies for diagnostic or therapeutic use may be human 
monoclonal antibodies or chimeric human-mouse (or other species) monoclonal 
antibodies. Human monoclonal antibodies may be made by any of numerous 
techniques known in the art (^^ Teng et al, 1983, Proc. Natl. Acad. Sci. U.S.A. 
20 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79; Olsson et al., 1982, 
Meth. Enzymol. 92:3-16). Chimeric antibody molecules may be prepared 
containing a mouse antigen-binding domain with human constant regions 
(Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851, Takeda et al, 1985, 
Nature 314:452). 

25 

Various procedures known in the art may be used for the production of 
polyclonal antibodies to epitopes of the human cerberus protein described herein. 
For the production of antibody, various host animals can be immunized by 
injection with the human cerberus protein, or a fragment or derivative thereof, 
30 including but not limited to rabbits, mice and rats. Various adjuvants may be 
used to increase the immunological response, depending on the host species, and 
including but not limited to Freund's (complete and incomplete), mineral gels 



14 



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such as aluminum hydroxide, surface active substances such as lysolecithin, 
pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet 
hemocyanins, dinitrophenol, and potentially useful human adjuvants such as 
BCG (Bacille Calmette-Guerin) and Corynebacterium parvum . 

5 

A molecular clone of an antibody to a selected human cerberus protein epitope 
can be prepared by known techniques. Recombinant DNA methodology (see e.g., 
Maniatis et ah, 1982, Molecular Cloning, A Laboratory Manual, Cold Spring 
Harbor Laboratory, Cold Spring Harbor, New York) may be used to construct 
10 nucleic acid sequences which encode a monoclonal antibody molecule, or 
antigen binding region thereof. 

The present invention provides for antibody molecules as well as fragments of 
such antibody molecules. Antibody fragments which contain the idiotype of the 

15 molecule can be generated by known techniques. For example, such fragments 
include but are not limited to: the F(ab') 2 fragment which can be produced by 
pepsin digestion of the antibody molecule; the Fab' fragments which can be 
generated by reducing the disulfide bridges of the F(ab') 2 fragment, and the Fab 
fragments which can be generated by treating the antibody molecule with papain 

20 and a reducing agent. Antibody molecules may be purified by known techniques, 
e^ immunoabsorption or immunoaffinity chromatography, chromatographic 
methods such as HPLC (high performance liquid chromatography), or a 
combination thereof. 

25 The invention further provides for a method of using a human cerberus protein 
or fragment thereof as an antagonist of the activity of a bone morphogenic 
protein (BMP). Preferably, the invention provides for a method of antagonizing 
the function of a Bone Morphogenic Protein (BMP) which comprises contacting 
said BMP with human cerberus protein or a fragment thereof. The method of 

30 the invention is carried out under conditions whereby the human cerberus or 
fragment thereof binds to the BMP. In further preferred embodiments of the 

15 



WO 98/49296 PCT/US98/06324 

invention, the human cerberus or fragment thereof is used to antagonize the 
function of BMP2 or BMP4. 

Antagonists to BMPs may be useful for preventing and treating BMP-related 
disorders of animals, especially of humans. It was, therefore, an object of this 
invention to identify substances which effectively antagonize the function of 
BMPs in disease states in animals, preferably mammals, especially in humans. It 
was another object of this invention to prepare novel compounds which inhibit 
BMP. It was still another object of this invention to develop a method of 
antagonizing the functions of BMPs in disease states in mammals. It was also an 
object of this invention to develop a method of preventing or treating disorders 
relating to the function of BMPs. 

In addition to their roles in normal bone formation, the BMPs appear to be 
involved in diseases in which they promote abnormal bone growth. For 
example, BMPs have been reported to play a causative role in the disease known 
as Fibrodysplasia Ossificans Progressiva (FOP), in which patients grow an 
abnormal "second skeleton 7 ' that prevents any movement. 

Therefore, an object of the present invention is to provide a novel molecule for 
the treatment of diseases or disorders including, but not limited to, 
Fibrodysplasia Ossificans Progressiva (FOP). Since human cerberus binds BMPs, 
it offers hope as a therapeutic agent for this disease. Additionally, abnormal bone 
growth can occur after hip replacement surgery and thus ruin the surgical 
outcome. This is a more common example of pathological bone growth and a 
situation in which binders of BMPs such as human cerberus may be 
therapeutically useful. Human cerberus may also be useful for treating other 
forms of abnormal bone growth, such as the pathological growth of bone 
following trauma, burns or spinal cord injury. In addition, human cerberus may 
be useful for treating or preventing the undesirable actions of BMPs associated 
with the abnormal bone growth seen in connection with metastatic prostate 
cancer or osteosarcoma. 



16 



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In additional embodiments, the human cerberus nucleic acids, proteins, 
fragments and peptides of the invention may be used to antagonize BMP activity 
in mammals* 

The present invention also provides for compositions comprising a human 
cerberus molecule, as described herein and a suitable carrier. The active 
ingredient, which may comprise the human cerberus, should be formulated in a 
suitable carrier for systemic or local administration in vivo by any appropriate 
route including, but not limited to injection (e. g., intravenous, intraperitoneal, 
intramuscular, subcutaneous, endoneural, perineural, intraspinal, 
intraventricular, intravitreal, intrathecal etc.), by absorption through epithelial or 
mucocutaneous linings (e. g . , oral mucosa, rectal and intestinal mucosa, etc.); or 
by a sustained release implant, including a cellular or tissue implant. 

Depending upon the mode of administration, the active ingredient may be 
formulated in a liquid carrier such as saline, incorporated into liposomes, 
microcapsules, polymer or wax-based and controlled release preparations, or 
formulated into tablet, pill or capsule forms. 

The concentration of the active ingredient used in the formulation will depend 
upon the effective dose required and the mode of administration used. The dose 
used should be sufficient to achieve circulating plasma concentrations of active 
ingredient that are efficacious. Effective doses may be extrapolated from dose- 
response curves derived from in vitro or animal model test systems. 

The following examples are offered by way of illustration and not by way of 
limitation. 



17 



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Example 1 - Cloning and Sequencing of Human cerberus gene 



PCTAJS98/06324 



A human genomic library (Genome Systems, Inc. - Human release II BAC-4435) 
was hybridized to a mouse probe approximately 314 nucleotides in length that 
5 was obtained by PCR from a mouse EST clone (GenBank accession number 
AA120122; clone number 538769). DNA from human clones hybridizing to the 
mouse probe was digested with restriction enzymes, Southern blotted and 
hybridized. Those DNA fragments hybridizing to the mouse probe were 
purified, then subcloned using a commercial cloning kit (Zero Blunt PCR 

10 Cloning Kit, Invitrogen Cat# K2700-20) and sequenced using the ABI 373A DNA 
sequencer and Taq Dideoxy Terminator Cycle Sequencing Kit (Applied 
Biosystems, Inc., Foster City, CA). The sequence obtained corresponded to a 
segment of human cerberus encoding a polypeptide starting from the amino acid 
Threonine at position 170 (see SEQ ID No. 2) to a point beyond the stop codon. 

15 The RACE procedure was then used to obtain the full length human cerberus 
nucleotide sequence as follows. Oligonucleotides were designed based on the 
partial human sequence and used as primers for the reverse transcriptase 
reaction and for PCR. An approximately 1.2 Kb fragment was isolated and 
sequenced and found to contain the remainder of the human cerberus sequence. 

20 The sequence of human cerberus (SEQ ID NO. 1) was then further confirmed by 
PCR and by sequencing of a full length cerberus cDNA and also by partial direct 
sequencing of the human genomic clone described above. 

25 Example 2 - Construction of human Cerberus (hCer) expression plasmid pRG629 

A DNA fragment encoding the gene for hCerberus was PCR amplified from 
pMT21.hCer.Fc using the primers Nl-hCer (5'-AAACATGATGCAGGATGG 
CCGCCAG-3') and Cl-hCer (5'-GAGAGCGGCCGCTCATTAAGCTGAAACT 
30 CCTGGGATAAAGGAATCCTGGG-3'). The resulting 769 bp fragment was 

digested with Not 1 then ligated into the Pme 1-Not 1 sites of pRG461 a high copy 
vector encoding the gene for kanamycin resistance. This vector contains the 

18 



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phage 17 <t>l.l promoter which directs the transcription of genes inserted into the 
Pmel site. pRG461 was constructed at Regeneron. A clone was identified and 
named pRG629, the construct was confirmed by DNA sequence analysis, then 
transformed into E. coli strain RFJ143 by electroporation. RFJ143 is an E. Coli 
5 strain constructed at Regeneron and is essentially E. coli Kl 2 strain W3110 lacIQ 
ZpL8 Z+ Y+ fhuAA322-405 ara (DE3). 

Example 3 - Purification of human Cerberus protein 

10 E. coli strain RFJ143 containing pRG629 was grown in LB medium (Difco) and 
expression of hCerberus was induced by the addition of 1 mM IPTG. Induced 
cells were collected by centrifugation, resuspended in 10 volumes of 100 mM 
Tris-HCl, pH 8.5, 20 mM EDTA, and lysed by passage through a Niro-Soave Panda 
cell disrupter (Niro-Soave) to release inclusion bodies. The cell lysate was 

15 centrifuged and the pellet was resuspended in 10 volumes of 6 M guanidinium- 
HC1, 100 mM Tris-HCl, pH 8.5, 10 mM EDTA, 100 mM Na2S03, 10 mM Na2S406 
and stirred for 16 hr at room temperature. The solubilized inclusion bodies were 
fractionated on a Sephacryl S-300 column (Pharmacia) equilibrated in 8 M urea, 
50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA. Fractions containing 

20 hCerberus were pooled, diluted with 4 volumes of 6 M urea, 20 mM MES (2-(N- 
Morpholino)ethanesulfonic acid), pH 6.0 then loaded onto an SP-Sepharose 
(Pharmacia) column equilibrated with 6 M urea, 20 mM MES, pH 6.0 and eluted 
from the column with a linear gradient of NaCl in 6 M urea, 20 mM MES, pH 6.0. 
Purified cerberus was refolded by dilution with 10 volumes of buffer to 3.5 M 

25 urea, 50 mM Tris-HCl, pH 8.5, 0.1 mM EDTA, 0.5 mM cysteine followed by 

incubation at 4°C. After 2 days incubation, the refold mix was loaded onto a Q- 
Sepharose column (Pharmacia) equilibrated with 4 M urea, 50 mM Tris-HCl, pH 
9.5, 0.1 mM EDTA, 20% glycerol ad eluted with a linear NaCl gradient in the 
same buffer. Fractions containing hCerberus were pooled and dialyzed against 20 

30 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1 mM EDTA. The dialysate was acidified 
with 0.1 % TFA, loaded onto a Jupiter C5 column (Phenomenex) equilibrated in 

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0.1% TFA, 10% acetonitrile, and eluted with an increasing acetonitrile gradient 
from 30% to 50% at 1.3%/min. Fractions containing hCerberus were pooled, 
dried under vacuum, then resuspended in 20 mM Tris-HCl, pH 8.0, 150 mM 
NaCI, 0.1 mM EDTA. 

5 

Example 4 - Demonstration That Human Cerberus Binds to Human BMP2 

Human Cerberus (1 ^lg/ml) that was expressed in E. coli and refolded was co- 
incubated with hBMP2 (1 ng/ml) in the absence or in the presence of human 
10 noggin protein (hNGAB2, 2 ng/ml). Human noggin binds to BMP2 with high 
affinity. The hNGAB2 is a deletion mutein of human noggin that displays 
identical biological activity to human noggin but has reduced binding to heparin. 
Thus addition of noggin (hNGAB2) should inhibit binding of hCER to BMP2, if 
an excess of noggin is added over hCER and BMP2. 

15 

The formation of a stable complex between hCER and the BMPs was determined 
by immunoprecipitating hCER and associated proteins using an anti-hCER 
antisera bound to Protein G-Sepharose beads (Pharmacia). The binding reaction 
was carried out in binding buffer, which is comprised of 20 mM Tris pH 7.6, 150 

20 mM NaCI, 0.1% Tween 20 (TBST), 1 mg/ml bovine serum albumin (BSA). 

Binding was allowed to proceed for 1 hour, at 25°C, in a reaction volume of 1 ml, 
with continuous mixing to keep the Protein G-Sepharose in suspension, after 
which point the beads were spun down, washed once with TBST, moved to new 
eppendorf tubes, and washed 3 more times with TBST. Proteins bound to the 

25 beads were solubilized by addition of 25 \i\ of Laemmli SDS-PAGE sample buffer 
(see Sambrook, et al. - A Cloning Manual, Cold Spring Harbor Laboratory) and 
loaded onto 4 to 12% NuPAGE/MES gradient gels (Novex), which were run 
under reducing conditions. The proteins were subsequently transferred on 
Immobilon P (Millipore) and Western blotted for the presence of BMP2 or BMP4 

30 using polyclonal antisera raised against the respective proteins. 

As can be seen in Figure 1, hCER binds to hBMP2 (lane 1). Addition of hNGAB2 

20 



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PCTAJS98/06324 



blocks this interaction (lane 2), by binding to hBMP2 and blocking its ability to 
bind to hCER. This indicates that the epitope recognized by hCER on hBMP2 is 
the same or overlaps with the epitope recognized by noggin, or alternatively that 
binding of noggin to BMP2 and BMP4 sterically hinders the binding of hCER. 

5 There was no binding of hBMP2 to the beads if hCER was omitted from the 

reaction (lane 3), indicating that there is no non-specific binding of hBMP2 to the 
beads and that the observed binding is hCER-dependent. It should be noted that 
identical results have been obtained when examining the interaction of hCER 
with BMP4 and also when using different tagged forms of hCER, such as hCER- 

10 FLAG, hCER-myc and hCER-Fc. The tagged forms may be produced using 
standard genetic engineering techniques (see e.g., Molecular Cloning, A 
Laboratory Manual (Sambrook, et al., Cold Spring Harbor Laboratory), Current 
Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc., Wiley- 
Interscience, NY). 

15 

Example 5 - Tissue Expression of Human Cerberus 



We have examined the expression of human Cerberus by analysis of polyA+ 
mRNA prepared from different adult human tissues. TABLE 1 lists the tissues 
20 tested and the relative level of expression of hCerberus detected in these tissues. 



TABLE 1 



TISSUE 

25 heart 
brain 
placenta 
lung 
liver 

30 skeletal muscle 
kidney 
pancreas 



Relative Level of Expression of hCER 

undetectable 

undetectable 

undetectable 

undetectable 

undetectable 

undetectable 

undetectable 

undetectable 



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TISSUE 

spleen 
thymus 
5 prostate 
testis 
ovary 

small intestine 

colon (mucosa lining) 
10 peripheral blood leukocytes 

stomach 

thyroid 

spinal chord 

lymph node 
15 trachea 

adrenal gland 

bone marrow 

skeletal (muscle only) 

20 uterus (no endometrium) 
(muscle only) 

colon (no mucosa) 
(muscle only) 

25 

small intestine 

(muscle only) 

bladder (muscle only) 
30 heart (muscle only) 
stomach (muscle only) 
prostate (muscle only) 



TABLE 1 (cont'd) 

Relative Level of Expression of hCER 

undetectable 

undetectable 

undetectable 

undetectable 

undetectable 

medium 

low 

undetectable 

high 

very low 

very low 

medium 

undetectable 

undetectable 

undetectable 

undetectable 

low 

very low 

high (highest of all tissues screened) 

undetectable 
undetectable 
high 

undetectable 
22 



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References: 

Bouwmeester, T., Kim, S. H., Sasai, Y v Lu, B., and De, R. E. M. (1996). Cerberus is a 
head-inducing secreted factor expressed in the anterior endoderm of Spemann's 
organizer. Nature 382, 595*601. 

5 

Furuta, Y v Piston, D. W., and Hogan, B. L. (1997). Bone morphogenetic proteins 
(BMPs) as regulators of dorsal forebrain development. Development 224, 2203-12. 

Hemmati-Brivanlou, A., Kelly, O. G., and Melton, D. A. (1994). Follistatin, an 
10 antagonist of activin, is expressed in the Spemann organizer and displays direct 
neuralizing activity. Cell 77, 283-95. 

Piccolo, S., Sasai, Y., Lu, B„ and De, R. E. M. (1996). Dorsoventral patterning in 
Xenopus: Inhibition of ventral signals by direct binding of chordin to BMP-4. Cell 
15 86, 589-598. 

Smith, W. C, and Harland, R. M. (1992). Expression cloning of noggin a new 
dorsalizing factor localized to the spemann organizer in xenopus embryos. Cell 
70, 829-840. 

20 

Zimmerman, L. B., Jesus, E. J. M. D., and Harland, R. M. (1996). The Spemann 
organizer signal noggin binds and inactivates bone morphogenetic protein 4. Cell 
86, 599-606. 

25 

Although the foregoing invention has been described in some detail by way of 
illustration and example for purposes of clarity of understanding, it will be 
readily apparent to those of ordinary skill in the art in light of the teachings of 
this invention that certain changes and modifications may be made thereto 
30 without departing from the spirit or scope of the appended claims. 



23 



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Human cerberus nucleotide sequence - SEQ ID No. 1 



10 20 30 40 50 60 

ATG CAT CTC CTC TTA TTT CAG CTG CTG GTA CTC CTG CCT CTA GGA AAG ACC ACA CGG CAC CAG GAT 

70 80 90 100 110 120 130 

GGC CGC CAG AAT CAG AGT TCT CTT TCC CCC GTA CTC CTG CCA AGG AAT CAA AGA GAG CTT CCC ACA 

140 150 160 170 160 190 

GGC AAC CAT GAG GAA GCT GAG GAG AAG CCA GAT CTG TTT GTC GGA GTG CCA CAC CTT GTA GCC ACC 

200 210 220 230 240 250 260 

AGC CCT GCA GGG GAA GGC CAG AGG CAG AGA GAG AAG ATG CTG TCC AGA TTT GGC AGG TTC TGG AAG 

270 280 290 300 310 320 330 

AAG CCT GAG AGA GAA ATG CAT CCA TCC AGG GAC TCA GAT AGT GAG CCC TTC CCA OCT GGG ACC CAG 

340 350 360 370 380 390 

TCC CTC ATC CAG CCG ATA GAT GGA ATG AAA ATG GAG AAA TCT CCT CTT CGG GAA GAA GCC AAG AAA 

400 410 420 430 440 450 460 • 

TTC TGG CAC CAC TTC ATG TTC AGA AAA ACT CCG GCT TCT CAG GGG GTC ATC TTG CCC ATC AAA AGC 

470 480 490 500 510 520 

CAT GAA GTA CAT TGG GAG ACC TGC AGG ACA GTG CCC TTC AGC CAG ACT ATA ACC CAC GAA GGC TGT 

530 540 550 560 570 580 590 

GAA AAA GTA GTT GTT CAG AAC AAC CTT TGC TTT GGG AAA TGC GGG TCT GTT CAT TTT CCT GGA GCC 

600 610 620 630 640 650 660 

GCG CAG CAC TCC CAT ACC TCC TGC TCT CAC TGT TTG CCT GCC AAG TTC ACC ACG ATG CAC TTG CCA 



670 680 690 700 710 720 

CTG AAC TGC ACT GAA CTT TCC TCC GTG ATC AAG GTG GTG ATG CTG GTG GAG GAG TGC CAG TGC AAG 



730 740 750 760 770 780 790 

GTG AAG ACG GAG CAT GAA GAT GGA CAC ATC CTA CAT GCT GGC TCC CAG GAT TCC TTT ATC CCA GGA 



800 

GTT TCA GCT TGA 



24 



WO 98/49296 PCT/US98/06324 



HUMAN 



CERBERUS amino acid sequence - SEQilD No, 2. 



10 20 
Met His Leu Leu Leu Phe Gin Leu Leu Val Leu Leu Pro Leu Gly Lys Thr Thr Arg His Gin Asp 

30 40 
Gly Arg Gin Asn Gin Ser Ser Leu Ser Pro Val Leu Leu Pro Arg Asn Gin Arg Glu Leu Pro Thr 

50 60 
Gly Asn His Glu Glu Ala Glu Glu Lys Pro Asp Leu Phe Val Ala Val Pro His Leu Val Ala Thr 

70 80 
Ser Pro Ala Gly Glu Gly Gin Arg Gin Arg Glu Lys Met Leu Ser Arg Phe. Gly Arg Phe Trp Lys 
90 100 HO 

Lys Pro Glu Arg Glu Met His Pro Ser Arg Asp Ser Asp Ser Glu Pro Phe Pro Pro Gly Thr Gin 

120 130 
Ser Leu lie Gin Pro lie Asp Gly Met Lys Met Glu Lys Ser Pro Leu Arg Glu Glu Ala Lys Lys 

140 150 
Phe Trp His His Phe Met Phe Arg Lys Thr Pro Ala Ser Gin Gly Val lie Leu Pro lie Lys Ser 

160 170 
His Glu Val His Trp Glu Thr Cys Arg Thr Val Pro Phe Ser Gin Thr lie Thr His Glu Gly Cys 

180 190 
Glu Lys Val Val Val Gin Asn Asn Leu Cys Phe Gly Lys Cys Gly Ser Val His Phe Pro Gly Ala 
200 .210 220 

Ala Gin His Ser His Thr Ser Cys Ser His Cys Leu Pro Ala Lys Phe Thr Thr Met His Leu Pro 

230 240 
Leu Asn Cys Thr Glu Leu Ser Ser Val lie Lys Val Val Met Leu Val Glu Glu Cys Gin Cys Lys 

250 2 *° 
Val Lys Thr Glu His Glu Asp Gly His lie Leu His Ala Gly Ser Gin Asp Ser Phe He Pro Gly 
267 

Val Ser Ala g" 



25 



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PCTAJS98/06324 



WHAT IS CLAIMED IS: 

1. An isolated nucleic acid molecule encoding human cerberus. 

2. An isolated nucleic acid molecule according to claim 1, having a sequence 
selected from the group consisting of: 

(a) the nucleotide sequence comprising the coding region of the human 
cerberus as set forth in SEQ NO. 1; 

(b) a nucleotide sequence that hybridizes under stringent conditions to 
the nucleotide sequence of (a) and which encodes a molecule 
having the biological activity of the human cerberus; or 

(c) a nucleotide sequence which, but for the degeneracy of the genetic 
code would hybridize to a nucleotide sequence of (a) or (b), and 
which encodes a molecule having the biological activity of the 
human cerberus. 

3. A vector which comprises a nucleic acid molecule of claim 1 or 2. 

4. A vector according to claim 3, wherein the nucleic acid molecule is 
operatively linked to an expression control sequence capable of directing 
its expression in a host cell. 

5. A vector according to claim 3 or 4, which is a plasmid. 

6. Isolated human cerberus protein, or a fragment or derivative thereof 
having the biological activity of human cerberus. 

7. Isolated human cerberus protein, having the amino acid sequence as set 
forth in SEQ. NO. 2. 

8. A host-vector system for the production of human cerberus which 
comprises a vector of claim 3 or 4, in a host cell. 



26 



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PCTAJS98/06324 



9. A host-vector system according to claim 8, wherein the host cell is a 
bacterial, yeast, insect or mammalian cell. 

10. A method of producing human cerberus which comprises growing cells of 
a host-vector system of claim 8 or 9, under conditions permitting 
production of the human cerberus, and recovering the human cerberus so 
produced. 

11. An antibody which specifically binds the human cerberus of claim 6 or 7. 

12. An antibody according to claim 11, which is a monoclonal antibody. 

13. An antibody according to claim 11, which is a polyclonal antibody. 

14. A composition comprising human cerberus according to claim 6 or 7, and 
a carrier. 

15. A composition comprising an antibody according to claim 11, 12, or 13 and 
a carrier. 

16. Human cerberus according to claim 6 or 7, an antibody according to claim 
11, 12, or 13 or a composition according to claim 14 or 15, for use in a 
method of treatment of the human or animal body, or in a method of 
diagnosis. 

17. A polypeptide produced by the method of claim 10. 

18. A ligandbody which comprises human cerberus fused to an 
immunoglobulin constant region. 

19. The ligandbody of claim 18, wherein the immunoglobulin constant region 



27 



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PCT/US98/06324 



is the Fc portion of human IgGl. 

20. A ligandbody according to claim 18 or 19, for use in a method of treatment 
of the human or animal body, or in a method of diagnosis. 

21. A method of antagonizing the function of a Bone Morphogenic Protein 
(BMP) which comprises contacting said BMP with human Cerberus. 

22. The method of claim 21, wherein the BMP is BMP2 or BMP4. 

23. The method of claim 21 or 22, for use in treating BMP-related disorders of 
animals. 

24. The method of claim 23, for treatment of a human. 

25. The method of claim 24, wherein the BMP-related disorder is abnormal 
bone growth. 

26. The method of claim 24, wherein the BMP-related disorder is 
Fibrodysplasia Ossificans Progressiva (FOP). 

27. The method of claim 25, wherein the abnormal bone growth occurs 
following hip replacement surgery. 

28. The method of claim 25, wherein the abnormal bone growth occurs 
following trauma, a burn or a spinal cord injury or in connection with 
metastatic prostate cancer or osteosarcoma. 

29. A method of screening for a molecule capable of competing with human 
cerberus for binding to a BMP comprising: 

a) contacting a sample suspected of containing the molecule with the BMP 
in the presence of human cerberus under conditions in which the human 



28 



WO 98/49296 PCT/US98/06324 

cerberus is capable of binding to the BMP; and 
b) detecting binding of the molecule to the BMP. 

30. The assay of claim 29, wherein the BMP is BMP2 or BMP4. 



29 



WO 98/49296 PCTAJS98/06324 

1/1 



Figure 1 



inla-hCER + + + 

1 \iQ hCER + + - Standards 



hBMP2 



1jighBMP2 + + + 
2 \xQ hNGAB2 - + - nghBMP2 




CM CO 




SUBSTITUTE SHEET (RULE 26) 



INTERNATIONAL SEARCH REPORT 



Intei onal Application No 

PCT/US 98/06324 



A. CLASSIFICATION OF SUBJECT MATTER 

IPC 6 C12N15/12 C07K14/475 C07K16/22 C12N15/62 A61K38/18 



Acco/ding to International Patent ClassificatjonQPC) or to both national classification and IPC 



B. FIELDS SEARCHED 



Minimum documentation searched (classification system followed by classification symbols) 

IPC 6 C12N C07K A61K 



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 practical, search terms used) 



C. DOCUMENTS CONSIDERED TO BE RELEVANT 



Category 0 Citation ot document, with indication, where appropriate, of the relevant passages 



Relevant to claim No. 



DATABASE EMBL - EMEST13 

Entry MMAA20122, Acc.No. AA120122, 21 

November 1996 

MARRA, M. ET AL. : "mn32d09.rl Beddington 
mouse embryonic region Mus musculus cDNA 
clone 538769 5'." 
XP002072013 

cited in the application 
see the whole document 



-/- 



1-10,17 



m 



Further documents are listed in the continuation of box C. 



□ 



Patent family members are listed in annex. 



• Special categories of cited documents : 

"A" document defining the general state of the art which is not 

considered to be of particular relevance 
"E" earlier document but published on or after the international 

filing date 

V document which may throw doubts on priority claim(s) or 
which is cited to establish the publication date of another 
citation or other special reason (as specified) 

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

"P" document published prior to the international filing date but 
later than the priority date claimed 



T" later document published after the international tiling date 
or priority date and not in conflict with the application but 
cited to understand the principle or theory underlying the 
invention 

"X* document of particular relevance: the claimed invention 
cannot be considered novel or cannot be considered to 
involve an inventive step when the document is taken alone 

"Y" document of particular relevance; the claimed invention 

cannot be considered to involve an inventive step when the 
document is combined with one or more other such docu- 
ments, such combination being obvious to a person skilled 
in the art. 

document member of the same patent family 



Date of the actual completion ot the international search 

20 July 1998 


Date ot mailing ot the international search report 

3 0.07.98 


Name and mailing address ot the ISA 

European Patent Office, P.B. 5618 Patentlaan2 
NL - 2260 HV Rijswijk 
Tel. (+31-70) 340-2040. Tx. 31 6S1 epo nl, 
Fax: (+31*70) 340*3016 


Authorized officer 

Smalt, R 



Form PCT/tSA/210 (second sheet) (July 19&2) 



page 1 of 2 



INTERNATIONAL SEARCH REPORT 



Inte onal Application No 

PCT/US 98/06324 



C.(Continuatlon) DOCUMENTS CONSIDERED TO BE RELEVANT 



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



Relevant to claim No. 



p,x 



p,x 



BOUWMEESTER T ET AL: "CERBERUS IS A 
HEAD-INDUCING SECRETED FACTOR EXPRESSED IN 
THE ANTERIOR ENDODERM OF SPEMANN'S 
ORGANIZER" 
NATURE, 

vol. 382, 15 August 1996, 
pages 595-601, XP002066227 
cited in the application 
see the whole document 

BIBEN, C. ET AL. : "Murine cerberus 

homologue mCer-1: A candidate anterior 

patterning molecule." 

DEVELOPMENTAL BIOLOGY, 

vol. 194, 15 February 1998, 

pages 135-151, XP002072011 

See the whole document, particularly p. 

139, left-hand column. 

BELO, J. A. ET AL.: "Cerberus-like is a 
secreted factor with neutralizing activity 
expressed in the anterior primitive 
endoderm of the mouse gastrula." 
MECHANISMS OF DEVELOPMENT, 
vol. 68, November 1997, 
pages 45-57, XP002072012 
see the whole document 

LEMAIRE P ET AL: "The vertebrate 

organizer: structure and molecules" 

TRENDS IN GENETICS, 

vol. 12, no. 12, December 1996, 

page 525-531 XP004071057 

see the whole document 



1-10,17 



2-5,29, 
30 



2-5 



Form PCT/tSAtf to (continuation ot second sheet) (July 1992) 



page 2 of 2 



INTERNATIONAL SEARCH REPORT 



international application No. 

PCT/US 98/06324 



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



This International Search Report has not been established in respect of certain claims under Article I7(2)(a) for the following reasons: 



Claims Nos.: 

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

Please see Further Information sheet enclosed. 



| | 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: 



□ 



Claims Nos.: 

because ihey are dependent claims and are not drafted in accordance with the second and third sentences of Rule6.4(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: 



1 . I | As all required additional search fees were timely paid by the applicant, this International Search Report covers ail 
1 — 1 searchable claims. 

2. \^\ As all searchable claims could be searched without effort justifying an additional fee. this Authority did not invite payment 

of any additional fee. 



3. I I As only some of the required additional search fees were timely paid by the applicant, this International Search Report 
' — ' covers only those claims for which fees were paid, specifically claims Nos.: 



□ 



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



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

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



Form PCT/ISA/210 {continuation of first sheet (1))<July 1992) 



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