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WORLD INHELLECTUAL PROPERTY ORGAIsTlZATlON 
International Bureau 




PCX 

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification ^ : 

C12N 15/12, C07K 14/71, 14/56, 16/28, 
A61K 38/18, C12N 5/10 



Al 



(11) International Publication Number: WO 98/16641 

(43) International Publication Date: 23 April 1998 (23.04.98) 



(21) International Application Number: PCT/US97/ 18362 

(22) International Filing Date: 8 October 1997 (08.10.97) 



(30) Priority Data: 

08/729,452 
08/822,333 



II October 1996 (1K10.96) US 
20 March 1997 (20.03.97) US 



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

ERNMENT OF THE UNITED STATES OF AMERICA 
as represented by THE SECRETARY, DEPARTMENT OF 
HEALTH AND HUMAN SERVICES [US/USl; Suite 325, 
6011 Executive Boulevard. Rockville, MD 20852-3804 
(US). 

(72) Inventors; and 

(75) Inventors/Applicants (for US only): LUYTEN, Frank, P. 
[BE/BE]; Tervurenlaan 43, B-3080 Tervuren (BE). MOOS, 
Malcolm, Jr. [US/US]; 8507 Hazelwood Drive, Bethesda. 
MD 20814 (US). HOANG, Bang [US/US]; 4473 Groveland 
Road, University Heights, OH (US). WANG, Shouwen 
[CN/US]; 12630 Veirs Mill Road #315, Rockville. MD 
20853 (US). 



(74) Agent: SIMPSON, Andrew. H.; Knobbe. Martens, Olson & 
Bear, 16th floor, 620 Newport Center Drive, Newport Beach, 
CA 92660 (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, HU. IL, IS, JP, KE, KG. KP. KR, K2. 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. 
KE, LS. MW. SD, SZ. UG. ZW), Eurasian patent (AM. AZ, 
BY. KG, K2. 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. 

Before the expiration of the time limit for amending the 
claims and to he republished in the event of the receipt of 
amendments. 



(54) Title: ISOLATION AND METHOD OF USING TISSUE GROWTH-INDUCING FRZB PROTEIN 

(57) Abstract 

An isolated cDNA encoding a 
growth-inducing protein. Fizb, capable of 
stimulating bone, cartilage, muscle and nerve 
tissue formation. Frzb binds to and modulates 
the activity of Wnt growth factors which 
play a role in various developmental and 
neoplastic processes. The cDNA and protein 
sequences of human, bovine arid Xenopus 
Frzb are provided. Production and purification 
of recombinant Frzb are also described. 



CI) 


60 






§ 


50 




40 


ca 






c 


30 


o 


o 




a> 


20 


(0 








10 




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 


n 


Finland 


LT 


Lithuania 


SK 


Slovakia 


AT 


Austria 


FR 


France 


LU 


Luxembourg 


SN 


Senegal 


AU 


Australia 


GA 


Gabon 


LV 


Latvia 


5Z 


Swaziland 


AZ 


Azerbaijan 


GB 


United Kbigdom 


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 


Buitina 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 


COie d 'I 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 


Kazakstan 


RO 


Romania 






CZ 


Czech Republic 


LC 


Saint Lucia 


RU 


Russian Federation 






DE 


Gennany 


LI 


Liechtenstein 


SD 


Sudan 






DK 


Denmark 


LK 


Srt Lanka 


SE 


Sweden 






EE 


Estonia 


LR 


Liberia 


SG 


Sing^xne 







wo 98/16641 ^ PCT/US97/18362 

ISOLATION AND METHOD OF USING TISSUE 
GROWTH-INDUCING FRZB PROTEIN 
Field of the Invention 

The present invention relates to a protein isolated from cartilage capable of inducing skeletal morphogenesis, 

5 embryonic pattern formation and tissue specification. More particularly, the invention relates to the Frzb protein 
which induces in vivo cartilage, bone, neural and muscle tissue growth. Frzb also binds to the Wnt family of growth 
factors and modulates their biological activities 

Background of the Invention 
The discovery and IdentKlcation of diffusible factors that regulate skeletal morphogenesis have dramaticaiiy 

10 improved our understanding of the molecular events governing skeletal pattern formation. Genetic studies have 
confirmed the importance of these differentiation factors in the formation, growth and maintenance of the skeleton 
(Erlebacher et a!., Cell, 80:371 -37B, 1995). Likewise, non-diffusible molecules, including components of the 
extracellular matrix and ceil surface, are essential to patterning processes. One theory proposed for insect systems 
is that morphogenesis results from the |re)positioning of cells because of inherent characteristics such as differential 

15 adhesiveness (Nardi et al., J. EmbryoL Exp. MorphoL, 38:489-512, 1976). It is presently unknown whether 
analogous events occur in mammalian skeletal pattern formation. 

In Drosophila mefanogaster, the cuticle contains hairs and bristles arranged in a defined polarity, of which 
the pattern and orderly alignment reflect the polarity of the wing epidermis (Adler et al.. Genetics, 126:401-416, 
1990). Typically, these structures are aligned in parallel and point in the same direction as the body surface. 

20 Several genetic loci associated with epidermal cell polarity have been studied. One of the most thoroughly 
investigated is the frizzled [fz) locus, frizzled encodes an integral membrane protein having seven potential 
transmembrane domains. The fz locus is required for cellular response to a tissue polarity signal as well as 
intercellular transmission of that signal along the proximal-distal wing axis (Vinson et al., Ulature, 329:549-551, 1987; 
Vinson et al., Jyature, 338:263-264, 1989). Mutations of the // locus result in disruption of both cell-autonomous 

25 and noncell-autonomous functions of the // gene. Strong // mutations are associated with random orientation of 
wing hairs, while weaker mutations lead to hair and bristles randomly oriented parallel to neighboring cells with 
respect to the body axis (Vinson et al., lilatare, 329:549-551, 1987). Frizzled also regulates mirror-symmetric pattern 
formation in the Drosopfiila eye (Zheng et al., Development, 121:3045-3055, 1995). 

The rat and human homologs frinled-l and frizzled-2 (fz-1, fz-2) have been cloned and are expressed in a 

30 wide variety of tissues including kidney, liver, heart, uterus and ovary (Chan et al., 1 BioL Chem,, 267:25202-25297, 
1992; Zhao et aL, Genomics, 27:373-373, 1995). Six novel mammalian frizzled homologs have now been identified 
(Wang et al., J. Biol, Chem,, 271:4468-4476, 1996), each of which appears to be expressed in a distinct set of 
tissues during development or postnatally. 

The basic form and pattern of the skeleton derived from lateral plate mesoderm are first recognizable when 

35 mesenchymal cells aggregate into regions of high cell density called condensations which subsequently differentiate 
into cartilage and bone, and continue to grow by cell proliferation, cell enlargement and matrix deposition. Published 



wo 98/16641 ^ PCT/US97/18362 

PCT Application No. WO 96/14335 discloses the isolation, cloning and in vivo chondrogenic activity of cartilage^ 
derived morphogenetic proteins (COMPs) which are members of the TGF-^ superfamily. Genetic studies have 
demonstrated that disruption of condensations results in disturbed skeletal phenotypes (Eriebacher et la., Cell, 80:371- 
378, 1995). In humans, limb development takes place over a four week period from the fifth to the eighth week. 
5 The upper limbs develop slightly in advance of the lower limbs, although by the end of the period of limb development 
the two limbs are nearly synchronized. The most proximal parts of the limbs develop somewhat in advance of the 
more distal parts. 

Recently, the number of secreted factors implicated in both limb and axial patterning has increased steadily 
(Sive, Genes Dev., 7:1-12, 1993; Dawid, J. BioL Chem.. 269:6259-8262, 1994; Hogan, Genes Dev„ 10:1580-1594, 

10 1996). Some of these factors are expressed in the Spemann organizer, the region of the Xenopas embryo implicated 
in specification of the dorsal axis and critical to dorso-ventrat patterning of the vertebrate embryo. In contrast, the 
bone morphogenetic protein BMP-4 and Xwnt-8, a member of the Wnt family of growth factors, are expressed in 
presumptive ventral mesoderm and endoderm early in gastrutation, and are thought to act as positive ventral inducers 
(Hogan et aL, supra; DeRobertis et aL, IHature, 380:37-40, 1996; Christian et a!.. Genes Dev., 7:13-28, 1993). 

15 Several of these secreted factors are thought to produce their dorsalizing effects by binding to BMP4 or a related 
TGF-)9 class sigrial and inactivating it. No secreted factor with Wnt binding activity has been identified. 

Wnt proteins are implicated in a variety of developmental and neoplastic processes (Nusse et aL, Ceff, 
89:1073-1087, 1992; Parr et aL, Curr. Biol, 4:523-528, 1994; Moon, Bioessays, 15:91-97, 1993). The receptors 
for these proteins have not been identified. The Wnt family of proteins has been divided into two classes, I and II, 

20 based on their ability to induce axis duplication in Xenopus oocytes and their transforming activity in mammalian 
cells. Recently, Frizzled-class proteins were proposed as receptors for the Wnt growth factors (Wang et aL, J. Bioi. 
Chem., 271:4468-4476, 1996). This is supported by observations that Wingless protein (Wg), the Drosophila 
prototype of the Wnt family, binds to ceils transfected with the fmiledl gene \Dfi2\. Moreover, addition of Wg to 
cells transfected with Dfz2 causes increased accumulation of Armadillo, a Drosophila homologue of ^-catenin, an 

25 expected consequence of Wg signaling (Bhanot et aL, Nature, 382:225-230, 1996). In Xenopus embryos, 
overexpression of rat frizzled-1 (Rfz-1) resulted in recruitment of Xwnt-8 and Xenopus dishevelled, a component of 
the Wnt signaling pathway, to the plasma membrane (Yang-Snyder et at.. Current BioL, 6:1302-1306, 1996). 

There are few known proteins which induce skeletal morphogenesis, as well as induction of nerve and 
muscle tissue growth. There are no known secreted proteins which will bind to and modulate the function of the 

30 Wnt proteins. Such proteins have tremendous therapeutic applications. The present invention provides such a 
multifaceted protein. 

Summary of the Invention 

One embodiment of the present invention is an isolated polynucleotide having the nucleotide sequence shown 
in SEQ ID NO: 1, 3 or 23. 

35 Another embodiment of the invention is isolated Frzb protein having the amino acid sequence shown in SEQ 

ID NO: 2, 4 or 7. According to one aspect of this preferred embodiment, at least one acidic, basic, uncharged polar. 



wo 98/16641 ^ PCT/US97/18362 

•3- 

nonpolar or aromatic amino acid in the sequence shown in SEQ ID NO: 2, 4 or 7 is replaced with a different acidic, 
basic, uncharged polar, nonpolar or aromatic amino acid. Preferably, the protein having the amino acid sequence 
shown in SEQ ID NO: 2 is obtained by expression of a polynucleotide having the sequence shown in SEQ ID NO: 1. 
According to another aspect of this preferred embodiment, the protein having the amino acid sequence shown in SEQ 
5 ID NO: 4 is obtained by expression of a polynucleotide having the sequence shown in SEQ ID NO: 3. According to 
yet another aspect of this preferred embodiment, the protein having the amino acid sequence shown in SEQ ID NO: 
7 is obtained by expression of a polynucleotide having the sequence shown in SEQ ID NO: 23. 

Another embodiment of the invention is an isolated polynucleotide encoding a native Frzb protein, the 
polynucleotide capable of hybridizing to a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1 at 
10 55°C in 3 x SSC, 0.1% SOS. 

The present invention also provides an isolated Frzb protein encoded by the polynucleotide described in the 
preceding paragraph. 

Still another embodiment of the invention is an isolated recombinant Frzb protein having the amino acid 
sequence shown in SEQ ID NO: 2, 4 or 7. 
15 The present invention also provides isolated mammalian Frzb protein having a molecular weight of about 

36 kilodaltons. 

Another embodiment of the invention is a pharmaceutical composition for inducing cartilage, bone, nerve 
or muscle growth comprising the isolated Frzb protein encoded by a polynucleotide capable of hybridizing to a 
polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1 at 55°C in 3 x SSC, 0.1% SOS, or a Frzb 

20 protein having the amino acid sequence shown in SEQ ID NO: 2, 4 or 7, in a pharmaceutically acceptable carrier. 

Still another embodiment of the invention is a pharmaceutical composition comprising an isolated 
recombinant Frzb protein having the amino acid sequence shown in SEQ ID NO: 2 obtained by expression of a 
polynucleotide having the sequence shown in SEQ ID NO: 1, the amino acid sequence shown in ^EQ ID NO: 4 
obtained by expression of a polynucleotide having the sequence shown in SEQ ID NO: 3, or encoded by a 

25 polynucleotide capable of hybridizing to a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1 at 
55*^C in 3 x SSC, 0.1% SOS, in a pharmaceutically acceptable carrier. In one aspect of this preferred embodiment, 
the carrier comprises fibrin glue, freeze-dried cartilage grafts or collagen. The composition may further comprise 
cartilage progenitor cells, chondroblasts or chondrocytes. Alternatively, Frzb protein may be coated onto or mned 
with a resorbable or nonresorbable matrix. In another aspect of this preferred embodiment, Frzb Is mixed with a 

30 biodegradable polymer. 

A further embodiment of the invention is a method of treating a cartilage, bone, nerve or muscle disorder 
in a mammal in need thereof, comprising the step of administering to the mammal an effective cartilage, bone, nerve 
or muscle-inducing amount of any of the pharmaceutical compositions described hereinabove at the site of the 
disorder. Preferably, the administering step is intravenous, intrathecal, intracranial or intramuscular at the site of 

35 the disorder. Advantageously, the mammal is a human. 



wo 98/16641 ^ PCT/US97/18362 

Another embodiment of the invention is a method of stimulating cartilage formation in a mammal comprising 
the steps of combining a protein having the amino acid sequence shown in S£Q ID NO: 2, 4 or 1, or a protein 
encoded by a polynucleotide capable of hybridizing to a polynucleotide having the nucleotide sequence shown in SEQ 
ID NO: 1 at 55°C in 3 x SSC, 0.1 % SOS, with a matrix to produce a product that facilitates administration of the 

5 protein; and implanting the product into the body of a mammal to stimulate cartilage formation at the site of 
implantation. Preferably, the matrix comprises a cellular material. Advantageously, the mixing step additionally 
comprises mixing of viable chondroblasts or chondrocytes. In another aspect of this preferred embodiment, the 
implanting is subcutaneous or intramuscular. Preferably, the mammal is a human. 

Another embodiment of the present invention are isolated antibodies to the proteins having the amino acid 

10 sequences shown in SEQ ID NO: 2 or 4. These antibodies may be either polyclonal or monoclonal. 

The present invention also provides a method of modulating Wnt-mediated signaling in a cell, comprising 
contacting the cell with an effective Wnt-modulating amount of the isolated Frzb protein of Claim 3, a Frzb protein 
having the amino acid sequence shown in SEO ID NO: 2, 4 or 7 or an active Wnt modulating fragment thereof. 
Preferably, the cell is contacted w mo. Advantageously, the Wnt is Wnt-8, Wnt-1, Wnt-2, Wnt-3, Wnt-4, Wnt-5A, 

15 Wnt-5B, Wnt-6, Wnt7A or Wnt-7B. 

Another embodiment of the invention is a method of modulating Wnt-mediated signaling in a cell, comprising 
contacting the cell with a recombinant construct comprising the coding region of SEQ ID NO: 1, 3 or 23, or encoding 
an active Wnt-modutating fragment thereof, operabiy linked to a heterologous promoter in an expression vector. 
Preferably, the Wnt is Wnt-8, Wnt-l, Wnt'2, Wnt-3, Wnt4, Wnt-5A, Wnt-5B, Wnt-6, Wnt-7A or Wnt-7B. 

20 Still another embodiment of the invention is a method of inhibiting the growth of a Wnt growth factor- 

expressing tumor in a mammal, comprising the step of contacting the tumor with an effective tumor growth-inhibiting 
amount of the isolated Frzb proteins described above. In one aspect of this preferred embodiment, the tumor is a 
manunary or intestinal tumor. Preferably, the mammal is a human. 

The present invention also provides a method of inhibiting the growth of a Wnt growth factor-expressing 

25 tumor in a mammal, comprising the step of contacting said tumor with a recombinant construct comprising the coding 
region of SEO ID NO: 1, 2 or 23 operabty linked to a heterologous promoter in an expression vector. Preferably, 
the construct is injected into the tumor. Alternatively, the construct is systemically administered to the mammal. 
Advantageously, the expression vector is a plasmid vector, retroviral vector or adenoviral vector. 

Yet another embodiment of the invention are isolated antibodies to Frzb protein having the amino acid 

30 sequence shown in SEO ID NO: 2, 4 or 7. 

The present invention also provides a method of facilitating tissue growth or repair, comprising the steps 
of isolating cells from the tissue; introducing a recombinant construct expressing Frzb into the cells; and returning 
the cells to the tissue. Preferably, the recombinant construct comprises a retroviral vector, adenoviral vector, 
herpesvirus vector or adeno-associated viral vector. Advantageously, the tissue is cartilage, muscle, bone or neural 

35 tissue. 



wo 98/16641 PCT/US97/18362 

•5- 

Another embodiment of the invention is a method of identifying a compound which affects Frzb activity, 
comprising contacting isolated Frzb with the compound; and 

determining Frzb activity, wherein an increase in activity compared to Frzb atone indicates that said compound is a 
Frzb activator and a decrease in activity indicates that said compound is a Frzb inhibitor. In one aspect of this 
5 preferred embodiment, the determining step comprises an in vivo chondrogenesis assay. 

Brief Description of the Drawings 
Figure 1 shows the nucleotide (SEQ ID NO: 1] and deduced amino acid sequence (SEQ ID NO: 2) of bovine 
Frzb. The predicted gene product contains 325 amino acids with a putative signal peptide (boxed). The dashed 
underline indicates the tryptic peptide sequence used to isolate a cDNA fragment by RT-PCR. Two separate 
10 consensus polyadenylation sites are underlined. A ''TGA** termination codon is shown in the S'-untranslated region. 
The putative signal peptide cleavage site is indicated by the scissors. 

Figure 2A shows a comparison between the deduced amino acid sequences of bovine (SEO ID N0:2) and 
human (SEQ ID NO: 4) Frzb. The predicted 23 amino acid signal peptide is boxed. The asterisk indicates a potential 
N-linked glycosylation site. The putathre transmembrane region is underlined and bolded. 
15 Figure 2B shows a hydropathy plot of human Frzb from the deduced amino acid sequence. The plot was 

generated by the GeneWorks^^ program using the paradigm of Kyte and Doolittle. Hydrophobic residues are in the 
upper part of the graph. The arrowhead at the amino terminus indicates the potential signal peptide. The putative 
transmembrane domain is indicated by a downward arrow. N, C, and P are N-glycosylation, casein kinase 2 
phosphorylation, and protein kinase C phosphorylation sites, respectively. The stippled bar underneath the plot 
20 represents the frazied'\\kB domain. 

Figure 3 shows an amino acid sequence comparison of the N-terminal domain of bovine (amino acids 35-147 
of SEQ ID NO: 2) and human (amino acids 35-147 of SEQ ID NO: 4) Frzb, and their homology with amino acids 111- 
221 of rat fz-1 (SEQ ID NO: 5) and amino acids 53-163 of Drosophila frizzled (SEO ID NO: 6). Identical residues 
are denoted by shaded boxes. Gaps indicated by hyphens were introduced to optimize sequence alignment. Asterisks 
25 indicate conserved cysteine residues. The numbers to the right indicate amino acid residues for each protein. 

Figure 4 shows an amino acid sequence comparison between Xenopus Frzb (SEQ ID NO: 7), bovine Frzb and 
human Frzb. Amino acids identical among the three sequences are boxed. A consensus sequence (SEQ ID NO: 8) 
is shown. The putative signal peptide cleavage site is shown by the pair of scissors. 

Figure 5 shows that Frzb can block Wnt-8 signaling across cell boundaries. Ventral Xenopus blastomeres 
30 were injected with either prolactin (P) or Xfrzb (F) mRNA (50-100 pg per biastomere) as shown at the early 16 cell 
stage. At the late 16 celt stage, single blastomeres surrounded by those injected previously were injected with Xwnt- 
8 (W) mRNA (10 pg), and scored for secondary axes. The experiment was performed three times with similar results. 
Data were pooled for the graph shown in the figure. 

Figure 6 is a schematic diagram of the BFrzb and Frzb deletion constructs used for transfection of C0S7 

35 cells. 



wo 98/16641 - PCTAJS97/18362 

•b- 

Figure 7 is a graph illustrating the inhibition of Wnt-1 -mediated secondary axis formation by Frzb and the 
effect of various Frzb deletions on its ability to inhibit Wnt>1 mediated signaling. The constructs are shown in Figure 
6. 

Detailed Description of the Preferred Embodiments 

The present invention includes polynucleotides encoding Frzb protein isolated from various mammalian 
tissues, as well as the corresponding protein sequences and variations thereof. Bovine and human Frzb proteins 
exhibit 94% amino acid identity. An orthologue of Frzb protein, Xfrzb, is also present in Xenopus laem embryos 
and exhibits about 92% amino acid identity to the corresponding mammalian Frzb proteins in the conserved frizzled- 
related domain. Bovine articular cartilage extracts were prepared to characterize protein fractions capable of inducing 
cartilage formation when implanted subcutaneously into rats \in vivo chondrogenic acthfity). Trypsin digestion of 
highly purified chondrogenic protein fractions followed by polymerase chain reaction (PGR) using degenerate 
oligonucleotide primers derived from a 30 residue tryptic peptide of the purified protein led to identification of a 
cDNA encoding a 36 kDa protein. The amino-terminat domain of the deduced amino acid sequence exhibited about 
50% amino acid identity to the corresponding region of the Drosophila gene frizzled which is implicated in the 
specification of hair polarity during development. Because of its homology to frizzled, the protein was named Frzb. 

The nucleotide and protein sequences of bovine Frzb are set forth in SEQ ID NOS: 1 and % respectively. 
The nucleotide and protein sequences of human Frzb are set forth in SEQ ID NOS: 3 and 4, respectively. The Frzb 
protein sequences of the invention have the sequences shown in SEQ ID NOS: 2 and 4, or sequence variations 
thereof which do not substantially compromise the ability of these proteins to induce cartilage, bone, muscle and 
nerve tissue formation. It will be appreciated that Frzb proteins containing one or more amino acid replacements 
in various positions of the sequences shown in SEQ ID NOS: 2 and 4 are also within the scope of the invention. 
Many amino acid substitutions can be made to the native sequence without compromising its functional activity. 
This assertion is supported by the sequence data shown in Figure 4. Both the manunalian and Xenopus proteins have 
biological activity. The primary sequence divergence, particularly in the carboxyl terminal region of the molecule that 
contains the exon intron boundaries, is wider between the amphibian and mammalian forms of Frzb. These sequence 
differences do not materially alter the biological activity of the protein. 

Variations of these protein sequences contemplated for use in the present invention include minor insertions, 
deletions and substitutions. For example, conservative amino acid replacements are contemplated. Such replacements 
are, for example, those that take place within a family of amino acids that are related in the chemical nature of their 
side chains. The families of amino acids include the basic amino acids (lysine, arginine, histidine); the acidic amino 
acids (aspartic acid, glutamic acid); the non-polar amino acids (alanine, valine, leucine, isoleucine, proline, 
phenylalanine, methionine, tryptophan); the uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, 
serine, threonine, tyrosine) and the aromatic amino acids (phenylalanine, tryptophan and tyrosine). In particular, it 
is generally accepted that conservative amino acid replacements consisting of an isolated replacement of a leucine 
with an isoleucine or valine, or an aspartic acid with a glutamic acid, or a threonine with a serine, or a similar 



wo 98/16641 ^ PCT/US97/18362 

conservative replacement of an amino acid with a structurally related amino acid, in an area outside of the 
polypeptide's acthre site, will not have a major effect on the properties of the polypeptide. 

In fact, any protein derivative of SEQ ID NOS: 2 and 4, including conservative substitutions, non- 
conservative substitutions, mixtures thereof, as well as truncated peptides or sequence variations thereof may be 

5 tested as described in the following examples to determine their ability to induce cartilage, bone muscle and nerve 
tissue. Such routine experimentation will enable the skilled artisan to screen any desired Frzb protein. 

A portion of the isolated bovine fub cDNA sequence (SEQ ID NO: 1) was used to screen a human placental 
cDNA library under high stringency conditions (3 x SSC, 0.1% SDS. 55°C; see Example 3), resulting in isolation of 
a cDNA (SEQ ID NO: 3) encoding a protein having 94% identity to the bovine protein. The Xenopus cDNA sequence 

10 corresponding to the conserved frizzled-related region exhibits greater than 80% nucleotide sequence identity to both 
mammalian Frzb genes. Thus, any nucleotide sequence capable of hybridizing to the DMA sequence shown in SEQ 
to NO: 1 under these high stringency conditions is whhin the scope of the invention. 

Frzb is recovered in 105,000 x g supernatants of lysates prepared from Xenopus embryos or Frzb- 
transfected mammalian cells, indicating that Frzb is a soluble protein. Both mammalian and Xenopus Frzb are 

15 secreted from Xenopus oocytes injected with the respective mRNAs. In addition, secretion of Xenopus Frzb in soluble 
form was shown by incubation of oocytes with ^^S-methionine followed by analysis of culture supernatants by SOS- 
PAGE. Moreover, mammalian cells transf ected with a Frzb expression plasmid secrete Frzb into the culture medium. 

Both mammalian and Xenopus Frzb were subcloned into the pcDNA3 mammalian expression vector and 
expressed in Xenopus oocytes. This vector contains a CMV promoter which drives expression of the inserted gene. 

20 However, other heterologous promoters well known in the art are also contemplated including SV40 and RSV. Bovine 
and human Frzb were expressed in ATDC5, COSl and COS 7 cells and partially purified using heparin-Sepharose and 
Concanavalin A-Sepharose chromatography. The production of Frzb in insect expression systems, particularly 
baculovirus, is also within the scope of the invention. This protein preparation was used in the functional assays 
described in the examples presented below. Bovine Frzb was expressed in £ coG and purified from inclusion -bodies 

25 using Ni-NTA affinity chromatography. Many expression vectors suitable for use in eukaryotic expression systems 
are also within the scope of the present invention, including the LacSwitch^'^ inducible mammalian expression system 
(Stratagene, La Jolla, CA) and pcDNAS (Invitrogen, San Diego, CA). 

In situ hybridization and immunostaining of human embryonic sections demonstrate predominant expression 
surrounding the chondrifying bone primordia and subsequently in the chondrocytes of the epiphyses in a graded 

30 distribution that decreases toward the primary ossification center. Transcripts are present in the craniofacial 
structures but not in the vertebral bodies. Because it is expressed primarily in the cartilaginous cores of developing 
long bones during human embryonic and fetal development (6*13 weeks), has in vivo chondrogenic activity and is 
homologous to Drosophila frizzled, Frzb is intimately involved in skeletal morphogenesis via induction of cartilage and 
bone formation. 

35 As described in the Xenopus embryo experiments set forth below (Example 9), both bovine and Xenopus 

Frzb induce formation of secondary body axes which contain neural and muscle tissue, indicating that Frzb is an 



wo 98/16641 PCT/US97/18362 

•0- 

important protein component in the molecular pathway leading to initial specification of muscle and nerve in 
vertebrates. Further, both bovine and Xenopus Frzb induces molecular markers for muscle Imyo D, actin) and nerve 
(NCAM) tissue. This was determined by explanting ventral marginal zones during gastrulation (stage 10), followed 
by grafting onto oocytes expressing Frzb and culturing for an appropriate period of time. Explants were removed 

5 and assayed for expression of the particular marker. Untreated ventral marginal zones did not express these markers. 
These results have been obtained with both injection of ml^NA into developing vertebrate embryos and with Frzb 
protein secreted from Xenopus oocytes. Thus, overexpression of the gene encoding Frzb will induce the formation 
of nerve and muscle tissue in vertebrates. 

Wnt proteins are a large class of secreted proteins implicated in a wide variety of differentiation and 

10 developmental processes (Cuietal.,Z?mA?/;mCT/, 121:2177-2186, 1995; Bhanot et a!., to/tfA^, 382:225-230, 1996). 
When myc-tagged XWnt-8 and Frzb were cotransfected in mammalian cells, Frzb was co-immunoprecipitated with an 
antibody directed against myc (Example 15). When Frzb mRNA was coinjected with X-Wnt8 mRNA into Xenopus 
oocytes, Wnt-mediated induction of dorsal markers was blocked (Example 13). Thus, Frzb binds and inhibits Wnt-8 
during Xenopus gastrulation, thus preventing inappropriate ventral signaling in developing dorsal tissues. 

15 Because Wnts play critical roles in developmental processes and oncogenesis, Frzb is useful as a modulator of tissue 
formation and as a tumor suppressor agent. 

The cysteine-rich frizzled domain is required for binding of Frzb to Wnt*1 and Wnt-5. While several 
truncated versions of the frizzled domain co*immunoprecipitate with Wnt proteins, the inhibition of Wnt-1-driven axis 
duplication in Xenopus embryos was abolished upon modification of this domain. The C-terminal domain of Frzb 

20 appears to support its inhibitory activity, but is not required. The Frzb*Wnt protein interaction was demonstrated 
for both Wnt-8 and Wnt-1. Co-injection of Frzb and Wnt-5A did not inhibit the formation of the phenotype 
characteristic of Wnt-5A-injected Xenopus embryos. This suggests that the action of Frzb and other related secreted 
Wnt binding proteins is not always inhibitory, but may also be stimulatory. Thus, Frzb is capable of modulating Wnt 
activity. The determination of whether Frzb stimulates or inhibit signaling mediated by a particular Wnt protein can 

25 be made using an appropriate assay system for the Wnt protein of interest. 

To investigate the specificity of FrzbfWnt interactions, C0S7 cells were co-transf ected with Frzb and several 
HA-tagged Wnt family members (Example 17). In contrast to Frzb which was secreted into the medium in these 
transfected cells, no Wnt protein was detected in the supernatants. Thus, co-immunoprecipitation experiments were 
performed with C0S7 cell lysates and demonstrated a direct protein-protein interaction between Wnt-1, Wnt-8 and 

30 Frzb. As described in Example 18, Frzb co-immunoprecipitated with all of the Wnt proteins tested. Likewise, Wnts 
co-immunoprecipitated with Frzb. These findings demonstrate that Frzb has sufficient affinity for each of these to 
allow co-immunoprecipitation. Due to this direct interaction with many members of the Wnt protein family, it is likely 
that Frzb plays a pivotal role In modulating the activity of all these proteins, as well as Wnt proteins from other 
mammalian cell types. 

35 Lack of soluble Wnt proteins precluded classical binding studies. Frzb- Wnt interactions were investigated 

using different washing conditions after immunoprecipitation including a variety of salt concentrations and detergents. 



wo 98/16641 PCT/US97/18362 

•9- 

Increasing salt concentrations in the washing did not affect these interactions. Increasing SDS concentrations 
resulted in the loss of the interaction, although no differences were observed when Wnt-1 (class I) was compared 
to Wnt-5A (class II). Taken together, no washing conditions could be identified that suggested any differences in 
the nature of the interactions between Frzb and Wnt-1 or Wnt-5. 

5 As discussed in Example 19, the frizzled domain of Frzb is required and sufficient for Wnt binding as shown 

by immunoprecipitation experiments of C0S7 cell lysates co-transfected with Wnt-1 and several deletion constructs 
and Frzb protein was detected in the supernatant in all instances. The removal of the entire extracellular cysteine- 
rich N-terminal domain (CRD) resulted in loss of co-immunoprecipitation with the Wnts. Several modifications of the 
frizzled domain could be made without affecting the outcome of the co-immunoprecipitation experiments. 

10 Overexpression of Wnt protein leads to development of mammary tumors in mice (van Leeuwen et al., 

Seminars Cancer BioL, 6:127-133, 1995; Tsukamoto et al.. Cell, 56:619-525, 1988). Frzb is particularly useful for 
systemic or local administration directly into a tumor (e.g. in situ tumors), especially for "wnt-driven" tumors such 
as mammary and intestinal cancers. Determination of whether a tumor is "wnt-drhren** can be made by isolating DNA 
from the tumor and incubating the DNA with a labeled Wnt probe. Frzb can be combined with a pharmaceutically 

15 acceptable excipient and injected directly into a tumor, systemicalty administered, or the nucleotide sequence encoding 
bovine, human or Xenopus Frzb (SEQ ID NO: 1, 3 and 23, respectively) can be incorporated into an expression vector 
such as a plasmid, adenoviral vector or retroviral vector by methods well known In the art. These Frzb-containing 
constructs can be directly injected Into a tumor or administered systemicalty to a mammal. 

Frzb or constructs encoding Frzb also can be advantageously enclosed in micelles or liposomes. Liposome 

20 encapsulation technology is sell known. Liposomes can be targeted to a specific tissue, such as tumor tissue, 
through the use of receptors, ligands or antibodies capable of binding the targeted tissue. The preparation of these 
formulations is well known in the art (see, for example, Radin et al.. Math. EmymoL, 98:613-618, 1983). 

Frzb can be used for tissue regeneration, either alone or in conjunction with other morphogenetic proteins, 
Including Wnts, which are Implicated in many tissue specification processes. For example, Frzb in conjunction with 

25 endogenous Wnt may promote muscle formation and repair. Frzb can also be used to generate tissues or organs ex 
vivo from autologous, immortalized or xenogeneic cell sources. 

Frzb is contemplated for use in the therapeutic induction and maintenance of cartilage, bone, muscle and 
nerve tissue. For example, local injection of Frzb as a soluble agent Is contemplated for the treatment of subglottic 
stenosis, tracheomalacia, chondromalacia patellae and osteoarthrhic disease. Other contemplated utilities include 

30 healing of joint surface lesions (i.e. temporomandibular joint lesions or lesions induced post-traumatically or by 
osteochondritis) using biological delivery systems such as fibrin glue, f reeze dried cartilage grafts and collagens mixed 
with Frzb and locally applied to fill the lesion. Such mixtures can also be enriched with viable cartilage progenitor 
cells, chondroblasts or chondrocytes. Repair or reconstruction of cartilaginous tissues using resorbable or non- 
resorbable matrices (tetracalcium phosphate, hydroxyapatite) or biodegradable polymers (PLG, polylactic 

35 acid/polyglycolic acid) coated or mixed with Frzb is also within the scope of the invention. Such compositions may 
be used in maxillofacial and orthopedic reconstructive surgery. Frzb can also be used as a growth factor for cells 



wo 98/16641 PCT/US97/18362 

of the chondrocyte lineage in vitro. Cells expanded ex vivo can be implanted into an individual at a site where 
increased chondrogenesis is desired. 

The pharmaceutical composition comprising Frzb may also be used to treat or slow neurodegenerative (i.e. 
Huntington's disease, Alzheimer's disease, spinal cord injuries), myodegenerative (i.e. muscular dystrophy, myasthenia 

5 gravis, myotonic myopathies) and osteodegenerative disorders |i.e. osteoporosis, osteitis deformans). A Frzb-containing 
pharmaceutical composition is administered to an individual in need of facilitated neural, muscle, or bone cell growth 
in a growth-facilitating amount thereof. The Frzb protein will promote the growth of these tissues. Thus, Frzb is 
a grovnh factor or cytokine capable of inducing growth of a variety of tissues. It is also contemplated that Frzb 
will positively impact the growth of other tissues, including skin and blood vessels. Thus, Frzb-containing 

10 compositions may be used for stimulation of wound healing (i.e. lacerations, burns, surgical incisions), promotion of 
angiogenesis, to prevent rejection in tissue transplantation and as adjuvants to chemotherapy and immunotherapy. 

One embodiment of the invention is a pharmaceutical composition comprising the protein shown in SEQ ID 
NOS: 2 or 4, or sequence variations thereof, in a pharmaceutically acceptable carrier which may be supplied in unit 
dosage form. Frzb can be administered to an individual in need of facilitated neural, muscle cartilage and bone 

15 growth by numerous routes, including intravenous, subcutaneous, intramuscular, intrathecal, intracranial and topical. 
The compound is combined with a pharmaceutically acceptable carrier prior to administration. Such pharmaceutical 
carriers are known to one of ordinary skill in the art. 

The Frzb compositions for intravenous administration may be in the form of a sterile injectable preparation, 
such as a sterile injectable aqueous or oleaginous suspension. Frzb may be provided as either a bolus or continuous 

20 intravenous, intrathecal or intracranial drip infusion. Because the composKion will not cross the blood brain barrier, 
intrathecal (in the cerebrospinal fluid) or intracranial administration is required for treatment of neurodegenerative 
disorders. The suspension may be formulated according to methods welt known in the art using suitable dispersing 
or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution 
or suspension in a parenteraliy acceptable diluent or solvent, such as a solution in 1,3 butanediol. Suitable diluents 

25 include, for example, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils may 
be employed conventionally as a solvent or suspending medium, for this purpose, any bland fixed oil may be 
employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may likewise be used 
in the preparation of injectable preparations. 

The Frzb composition may be in soluble or microparticular form, or may be incorporated into microspheres 

30 or microvesicles, including micelles and liposomes. 

Contemplated daily dosages of Frzb for parenteral administration to patients with neurodegenerative, 
myodegenerative, and osteodegenerative disorders are between about 1 /ig and about 100 //g. Particularly preferred 
daily dosages are between about 10 jug and about 50 /yg. This dosage can be administered once per day, or split 
over 2, 3, 4 or more administrations. Contemplated daily dosages for systemic administration to patients with Wnt- 

35 driven tumors or for direct injection into a tumor are between about 100 //g and 1 mg. The exact dosage can be 



wo 98/16641 ^ ^ PCT/US97/18362 

determined by routine dose/response protocols known to one of ordinary skill in the art. In a preferred embodimenti 
administration of Frzb is continued until no further improvement In the particular disorder is observed. 

It is also anticipated that the fnb polynucleotides of the invention will have utility as diagnostic reagents 
for detecting genetic abnormalities associated with genes encoding Frzb. Such genetic abnormalities include point 
5 mutations, deletions or insertions of nucleotides. Diagnostic testing is performed prenatally using material obtained 
during amniocentesis or chorionic villus sampling. Any of several genetic screening procedures may be adapted for 
use with probes enabled by the present invention, including restriction fragment length polymorphism (RFLP) analysis, 
ligase chain reaction or PGR. Mutations in this gene indicate an increased risk of developmental abnormalities. 

Drug screening assays can be used to identify activators or inhibitors of the Frzb protein. For example, 
10 Frzb is incubated with a particular drug prior to the in vivo chondrogenesis assay described in Example 1 and 
compared to a control containing Frzb alone. An increase in cartilage growth in the presence of a drug compared 
to Frzb alone indicates activation of Frzb, while a decrease indicates inhibition of Frzb acth/ity. 

The isolation and partial sequencing of a chondrogenic activity present in bovine cartilage is described below. 

Exaniple 1 

15 Preparation and activity of articular cartilaoe extracts 

To characterize factors responsible for cartilage inductive activity in articular cartilage, a protein fraction 
containing potent cartilage inductive activity was isolated as described in PCT Publication No. WO 96/14335. 
Articular (metatarsophalangeal joints) cartilage extracts were prepared from newborn calves as described (Chang et 
al., J, BioL Chem., 269:28227-28234, 1994) to characterize protein fractions with in mo chondrogenic activity. 

20 Briefly, tissues were finely minced and homogenized with a Polytron (top speed, 2 x 30 seconds) in 20 volumes 1.2 
M guanidine hydrochloride, 0.5% CHAPS, 50 mM Tris-HCI, pH 7.2, containing protease inhibitors and extracted 
overnight at 4<'C as described by Luyten et al. U BioL Ciiem., 264:13377, 1989). Extracts were concentrated and 
exchanged with 6 M urea by diafiltration using an Uhrasette^'^ (Filtron Technology, Inc., MA) and applied to a 0.5 
i heparin-Sepharose (Pharmacia/LKB, Piscataway, NJ) column, the column was washed with 5 bed volumes of 6 M 

25 urea, 50 mM Tris HCI, pH 7.4, 0.15 M NaCI, then eluted with 2 volumes 1 M NaCI in the same buffer. 

In vivo chondrogenic activity was assayed in a subcutaneous implantation model in rats using a collagenous 
carrier (Luyten et al., 1 Biol, Chem., 264:13377-13380, 1989; Luyten et al., ibid,]. Briefly, a portion of each 
fraction was assayed by reconstitution with 25 mg guanidine insoluble collagenous residue of demineratized rat bone 
matrix according to procedures described by Luyten et aL (ibidX Implants yvere recovered after 10 days and alkaline 

30 phosphatase activity was measured as a biochemical indicator or cartilage and/or bone formation. Implants were 
also examined histologically for evidence or cartilage formation using standard procedures known to those of ordinary 
skill in the art. 

The 1 M NaCi eluate of articular cartilage, which contained biological activity, was concentrated by 
diafiltration and applied to a Sephacryl S-200 HR gel filtration column (XK 50/100, PharmaciafLKB). After molecular 
35 sieve chromatography, bioactive fractions were pooled and exchanged into "50 mM HEPES, pH 7.4, containing 0.15 
M NaCI, 10 mM MgS04, 1 mM CaCl2 and 0.1% (w/v) CHAPS using Macrosep^^ concentrators (Filtron). The 



wo 98/16641 ^2 PCT/US97/18362 

equilibrated sample was mixed with 1 ml ConA Sepharose (Pharmacia-LKB) previously washed with 20 volumes of 
the same buffer according to the procedure described by Paralkar et al. (Biochem. Biophys. Res, Commun,, 131:37, 
1989). After overnight incubation on an orbital shaker at 4''C, the slurry vvas packed into disposable 0.7 cm ID Bio- 
Rad columns (Bio Rad, Hercules, CA) and washed with 20 volumes of the HEPES buffer to remove unbound proteins. 
5 Bound proteins were eluted with 20 volumes of the same buffer containing 0.5 M methyl-D-mannopyranoside. The 
eluate was concentrated to 200 //t using Macrosep^'^ concentrators. Macromolecules were precipitated with 9 
volumes of absolute ethanol at 4°C overnight. The precipitate was redissolved in 1 ml 6 M urea, 50 mM Tris-HCI, 
pH 7.4. Bioactive bound protein was mixed with 2 x Laemmti SOS sample buffer (without reducing agents) and 
analyzed by 12% preparative SDS-PAGE. Gel elution of the separated protein fractions and testing for biological 
10 activity was performed as described by Luyten et al. (/M). Protein fractions from the 38 • 40 kOa region were 
obtained for bioassay by gel elution following SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and were found 
to be chondrogenic. 

Primary sequencing data from the bioactive fractions were determined by transfer to PVDF membranes for 
amino terminal sequencing (Moos et al., J. bioL Chem,, 263:6005-6008, 1988) or to nitrocellulose membranes for 

15 trypsin digestion as previously described (Aebersold et al., Proc, Natl. Acad. ScL VSA, 84:6970-6974, 1987; Tempst 
et al, Electrophoresis, 1 1:537-553, 1990). Tryptic peptides were separated by reverse-phase high performance liquid 
chromatography (HPLC) (Epifano et a!., Development, 121:1947-1956, 1995), and the sequence of individual peptides 
was determined using an Applied Biosystems Model 477A sequencer (Applied Biosystems, Foster City, CA) with 
modifications (Tempst et al., ibid:, Tempst et al., AnaL Biochem., 183:290-300, 1989), 

20 Example Z 

Reverse transcriptase-oolvmerase chain reaction (RT/PCR) 
Two degenerate oligonucleotide primers corresponding to the amino- and carboxyl-terminus of the 30 amino 
acid tryptic peptide 323 {ETVNLnSAGCLCPPLNVNEEYLIMGYEFP; SEQ ID NO: 9) were used in RT/PCR to clone 
cDNAs corresponding to peptide 323: 

25 323S: 5'- GA(A/G)AC(A/C/T)GT(C/G)AA(C/T)CT(C/G/T)TA. |C/T)AC(A/C/6m'3' (SEQ ID NO: 10); and 

323AS: 5'-(A/G)AA(Crr)TC(A/G)TA(A/C/G/T)CCCAT(A<C/G/T)AT-3' (SEQ ID NO: 11) 
For RTJPCR, first strand cDNA synthesis was performed with 1 /yg poly(A)'^ or 5 pg total RNA prepared from bovine 
articular chondrocytes using random hexanucleotide primers from the cDNA Cycle^^ kit (Invrtrogen corp., San Diego, 
CA) or 323AS. 323I323AS primer pairs were used in 30 cycles at 94''C for 1 min, 50°C for 1 min and 72''C for 

30 30 sec. PCR products were purified through a Probind™ membrane (Millipore), followed by subcloning with the TA 
Cloning^^ System (Invhrogen). This yielded a 90 base pair (bp) DNA fragment encoding the proper peptide sequence 
(dashed underline, fig. 1). The amino acid sequence deduced from the PCR product was the same as the tryptic 
peptide sequence. 

Other tryptic fragments were also sequenced by Edman degradation and had the following sequences: 
35 GVCISPEAIVTAID or H)GADFPM (SEQ ID NO: 12); QGCEPILIK (SEQ ID NO: 13); QGCEPILICAWPPLY (SEQ ID ND: 14) 
and ETVNLYTSAGCLCPPLNVNEEYLIMGYE (SEQ ID NO: 15). SEQ ID NO: 12 containing the D residue corresponds 



wo 98/16641 PCTAJS97/18362 

•13- 

to amino acids 145-163 of SEQ ID NO: 2. SEQ ID NO: 13 corresponds to amino acids 117-125 of SEQ ID NO: 2. 
SED ID l\ID: 14 is not found within SEQ ID NO: 2. SEQ ID NO: 15 corresponds to the sequence found within SEQ 
ID NO: 2 (ETVNLYTSSGCLCPPLNVNEEYLIMGYE; SEQ ID NO: 16) except for position 9 at which there is an alanine 
in SEQ ID NO: 13 and a serine in SEQ ID NO: 16. The proteins containing these amino acid sequences are most 
5 likely structurally and functionally related to the isolated cDNA. These peptides are useful in the design of 
oligonucleotide probes or in the generation of antisera for nucleic acid hybridization and expression cloning, 
respectively, of other members of the Frzb protein family. This will allow isolation of other Frzb-related proteins from 
any vertebrate species. 

cDNA clones were isolated and sequenced as described in the following example. 
10 Example 3 

Isolation and sequencing of cDNA clones 
Bovine articular cartilage total RNA was isolated as described (Luyten et al., Exp. Cell Res,, 210:224-229, 
1994). PolylA)*^ RNA was isolated using the PolyATract^*^ magnetic bead system (Promega, Madison, Wl). A cDNA 
library was constructed in a UNIZAP^^XR (Stratagene, La Jolla, CA) starting from bovine articular cartilage poly(A)^ 
15 RNA. The non-degenerate oligonucleotides designed from the 90 base pair fragment amplified by RT/PCR in Example 
2 used to screen the articular cartilage cDNA library were: 
323.23: 5'-GCTCTGGCT6CCTGTGTCCTCCACnAAC6-3' (SEQ ID NO: 17) 
323.40: 5'-CCTCCACTTAACGTTAATGAGGAGTATCTC-3' ISEQ ID NO: 18) 
Plaques hybridizing to both oligonucleotides were further purified using standard plaque hybridization procedures 
20 (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor laboratory Press, Cold Spring 
Harbor, NY). A 2.4 kb clone contained a single open reading frame (ORF) with two separate consensus 
polyadenylation sites and a poly A tail (Fig. 1). A 1.3 kb clone contained a single potyadenylation signal, a short 
poly A tail and a short 5'-noncoding region. Three other clones lacked the poly A tail but contained longer 5' ends. 
Because Northern analysis using a bovine cDNA probe revealed corresponding mRNA expression in placenta, a human 
25 placental cONA library was screened to isolate the human orthologue. 

Four clones ranging from 1.3 to 1.6 kb were analyzed and all contained the same open reading frame. All 
clones contained a consensus translation initiation site (Kozak, J. BioL Chem., 266:1 9867-1 9870, 1991) and an in- 
frame termination codon situated 144 base pairs upstream of the methionine start codon (Fig. 1). The size difference 
between the bovine and human cDNA inserts (2.4 kb vs. 1.3 kb) is due to a longer 3' untranslated region in the ~ 
30 bovine clone (Fig. 1). Based on sequences from these overlapping cDNA clones, the predicted size of both the human 
and bovine protein is 325 amino acids (Fig. 2A) (36.2 kDa). 

The bovine and human amino acid sequences are 94% identical. The deduced protein sequence of both the 
human and bovine cDNA revealed at least four structural domains (Figs. 1, 2A, 28). An amino-terminal hydrophobic 
stretch of 25 amino acids immediately downstream of the initiation methionine likely represents a signal peptide (von 
35 Heijne, NucL Acids Res., 14:46834690, 1986). A second hydrophobic region of 24 amino acids (residues 75-98), 
which represents a putative transmembrane domain, is followed be a region containing several potential 



wo 1>8/16641 PCT/US97/18362 

04. 

serine/threonine phosphorylation sites and a serine-rich carboxyl-termlnal domain (residues 301-325). Both homologs 
contain an N-linked glycosylation site at Asn 49, which is amino terminal of the putative transmembrane domain. 
A potential C-terminal glycosylation site in the bovine protein was not present in the human homolog. 

A search of the Gen Bank^*^ data base using the BLAST network service at the national Center for 

5 Biotechnology Information (NCBI) (Altschul et al, J. MoL BioL, 215:403410, 1990) indicated that Frzb has significant 
identity (about 50%) in the amino-terminal region (from amino acid 35-147) to Drosophila friziled and rat // proteins 
(Fig. 3). The homologous region begins shortly after the cleavage site of the predicted signal sequence. The 10 
cysteine residues in this region are conserved. 

Following isolation of the bovine cDNA, PGR was used to generate a 1 kb fragment containing Xhol sites 

10 at both ends. This fragment, representing the bovine open reading frame (bORF), was used to screen a human 
placenta ytgtll cDNA library (Clontech, Palo Alto, CA). Approximately 7x10^ plaques from the bovine library and 
3x10^ plaques from the human library were screened. Hybridizations were performed for 24 hours at 42°C in 6 
X SSC, 1 X Denhardt's solution, 0.01% yeast tRNA and 0.05% sodium pyrophosphate. The membranes were washed 
to a final stringency of 3 x SSC, 0.1% SDS at 55°C for 15 minutes (3 x SSC - 50 mM sodium citrate, pH 7.0, 

15 0.45 M NaCI). 

Sequencing was performed using the dideoxy chain termination method (Sanger et aL, Proc, NatL Acad. ScL 
USA, 74:5463-5467, 1977) and Sequenase™ Version 2.0 DMA polymerase according to the manufacturer's 
instructions (United States Biochemical Corp., Cleveland, OH). The sequencing data were obtained by primer walking 
and from subclones of restriction fragments into pBluescript SKI! (Stratagene). Compressions were resolved by 
20 performing the sequencing reactions in the presence of 7-deaza-6TP (U.S. Biochemical). 

Bovine Frzb was expressed in £ coB and purified therefrom as described 

below. 

Example 4 

Frzb Protein expression and antibody production 

25 The full-length bovine frzb gene was subcloned into the pcDNA3 mammalian expression vector (Invhrogen, 

San Diego, CA) under control of the CMV promoter and used to transfect ATDC5, COS-1 (ATCC CRL 1850) andCOS- 
7 (ATCC CRL 1651) cells using the LipofectAMINE™ reagent (6IBC0/BRL, Gaithersburg, MD) according to the 
manufacturer's instructions. A soluble, secreted Frzb protein was obtained from culture supernatants and partially 
purified by heparin-Sepharose and Concanavalin A-Sepharose chromatography. 

30 The bovine fnb open reading frame was subcloned in the proper orientation into the Xhol site of pET-2Ba(-i-) 

(Novagen, Madison, Wl) which contains an amino terminal stretch of six histidine residues to facilitate purification 
of the expressed protein as well as a T7 tag for immunodetection. The pET-bORF construct was used in the £ c^/a 
based pET System^*^ to obtain bovine Frzb fusion protein. Purification of protein product from inclusion bodies with 
Ni'NTA affinity chromatography (QIAGEN) was performed using decreasing pH steps according to the manufacturer's 

35 instructions. The affinity purified protein was visualized as a major band following Coomassie blue staining after 
SOS-PAGE. The identity of the fusion product was verified by immunoblotting using a T7 monoclonal antibody. 



wo 98/16641 _ PCT/US97/18362 

•lb- 
Rabbits were immunized with Birzb fusion protein for 6 months, 250 protein per boost, total of 10 injections. 
Following immunization, several rabbits were subsequently immunized with a synthetic peptide of 12 amino acids 
(residues 51-61 of Fig. 1) coupled to keyhole limpet hemocyanin (KLH) through a carboxyl-terminal cysteine. The 
resulting antisera were screened and titered in immunoblots using the Western-Light Ptus^*^ kit (TROPIX, MA) 
5 according to the manufacturer's protocol. Briefly, membranes were incubated overnight in blocking buffer (BF) 
containing 0.6% l-BLOCK^*^ (TROPIX) In phosphate buffered saline (PBS) and 0.1% Tween-20. The antiserum was 
diluted from 1:250 to 1:10,000 in BF. The membranes were washed three times for 5 min in BF after each 
incubation step. The membranes were incubated with secondary antibody at a dilution of 1:20,000 for 30 min, 
followed by AVOIX^^ (enzyme conjugate) incubation for 20 min. Blots were developed using the CSPD™ 

10 chemiluminescent substrate (TROPIX) and exposed to Kodak XAR-5 film for 1 to 10 min. Antiserum N374-PEP 
generated against residues 51-61 of Fig. 1 afforded the optimal signal to noise ratio in Western blots and was thus 
selected for further studies and immunohistochemical staining. This antibody detected a band migrating at the same 
apparent molecular weight as the Ni-NTA affinity purified protein as determined by Western blot analysis. This 
method can be used to generate antiserum to human Frzb, as well as any desired immunogenic fragment of bovine 

15 or human Frzb. 

Monoclonal antibodies to Frzb can also be generated using conventional hybridoma technology known to 
one of ordinary skill in the art. Briefly, three mice are immunized with 25 //g recombinant Frzb produced as 
described in above. Mice are inoculated at 3 week intervals with 20 //g Frzb per mouse (1/2 subcutaneously and 
1/2 intraperitoneally). Serum collected from each animal after the first inoculation reacts with Frzb as determined 
20 by immunoprecipitatlon. Three days after the final Inoculation, mice are sacrificed and the spleens harvested and 
prepared for eel! fusion. Splenocytes are fused with Sp2/0 Ag14 myeloma cells (ATCC CRL 1581) with polyethylene 
glycol (PEG). Following PEG fusion, cell preparations are distributed in 96-well plates at a density of 10^ cells per 
well and selected in hypoxanthlne/aminopterin/thymidine(HAT) medium containing 10% fetal calf serum and 100 U/ml 
interleukin-6. The medium is replaced with fresh HAT medium 10 days after plating. To identify hybridomas 
25 producing Mkbs which recognized Frzb epitopes, hybridoma supernatants are tested for the ability to 
immunopreclpitate purified Frzb or to detect Frzb by immunoblotting. 

As previously discussed, Frzb is a secreted soluble protein; however, to determine whether It also exists 
in a membrane-associated form, the following cell fractionation study was performed. 

Example 5 

30 Cell fractionation 

A full length 2.4 kilobase (kb) BamHIXbol fragment of bovine Frzb (Fig. 1) was cloned into the pcDNA3 

c 

expression vector (Invitrogen) to generate the construct pFrzb. C0S1 celts (1.6 x 10^ initial seeding density) were 
transf acted with 10 ;/g of either pFrzb or the control pcDNA3 vector per 100 mm dish using 120 //I 
LipofectAMINE^'^ reagent (GIBCO/BRL, Gaithersburg, MD). Transfection was carried out for 6 hours in serum-free 
35 OPTl-IVIEM® (GIBCO/BRL). Cells were Incubated at 37°C for 72 hours in serum-free OPTI-MEM^ with daily media 
changes. Conditioned media were then collected and concentrated 20-fold using a Centricon^'^ 10 microconcentrator 



wo 98/16641 PCT/US97/18362 

(Amicon, MA). Cells were scraped from the dishes and resuspended in lysis buffer (10 mM Tris HCI, 5 mM EDTA, 
1 mM phenylmethylsulfonyl fluoride (PMSF)). Cells were lysed using a syringe and a 25 gauge needle and the 
resulting lysate was collected. The lysate was centrifuged at 3,000 x g for 10 min to pellet debris, nuclei and non- 
tysed cells. Tlie resulting supernatant was centrifuged at 100,000 x g for 30 min. 

5 The resulting pellet, containing primarily membrane vesicles, microsomes and other particulates, was 

extracted successively with: 1) 10 mM Tris-HCI, pH 8.0, 6 M urea; 21 10 mM Tris-HCI, pH 8,0, 1% Triton X-100, 
6M urea; 3) 10 mM Tris-HCI, pH 8.0; and 4) 1% SOS in 1% Triton/6 M urea/10 mM Tris-HCI, pH 8.0. After each 
extraction, samples were centrifuged at 100,000 x g for 30 min. The extracts were then precipitated with an equal 
volume of 30% trichloroacetic acid (TCA) and re-dissoh/ed in SOS sample buffer. Equal amounts of cytosol, the 

1 0 membrane/particulate fraction and concentrated conditioned media were loaded and separated on 4-20% gradient Tris- 
glycine gels (Novex, San Diego, CA), blotted to Tropifluor^'^ PVDF membrane (TROPIX) using a GENIE^*^ 
electrophoretic blotter (Idea Scientific, Minneapolis, MN) and analyzed by immunoblotting as described in Example 4. 
The primary antiserum (N374-PEP) dilution was 1:1,000. The urea/SDS/Triton extract of the membrane pellet 
contained most of the Frzb protein. No protein was detected in the supernatants of the transfected cells or in 

15 untransf acted cells. 

Because the protein sequencing data were obtained from partially purified protein preparations of bovine 
articular cartilage extracts, similar cell fractionation studies were performed on supernatants and celt extracts of 
primary bovine articular chondrocyte cultures. Cells were grown to confluence in 100 mm dishes in Dulbecco's 
Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (PBS), then incubated for 48 hours in serum 

20 free OPTI-MEM*^ in the presence or absence of dextran sulfate (250 //g/ml) to improve recovery of soluble protein. 
Conditioned media and cell layers were processed as described above. Again, most of the protein was detected in 
the membrane associated fractions. The addition of dextran sulfate did not change this distribution. 

Thus, Frzb exists in both membrane-associated and soluble forms. Recent evidence suggests that the results 
of cell fractionation studies depend upon the cell or tissue type and are likely related to tell type specific differences 

25 in posttranslational proteolytic processing. Frzb Is secreted In soluble form in some, but not all, mammalian 
expression systems. Importantly, Frzb is soluble in frog embryos as described in Example 14. It is possible that Frzb 
may occur, and act. In both soluble and particulate forms. Nonetheless, the observation that Frzb can be secreted 
is highly significant in that soluble protein factors are more amenable to production and formulation. In secreted 
proteins, the signal peptide is cleaved from the preprotein to form the biologically active secreted molecule. In the 

30 mammalian cell expression systems used herein, cell lysates contained two Frzb bands as visualized by Western blots, 
one corresponding to the unprocessed protein containing the signal peptide, and one corresponding to the processed 
protein lacking the signal peptide. When Western blots were performed on a clarified lysate of Xenopus embryos, 
a single protein band was observed. 

Localization of mRNA encoding Frzb in human embryos was determined by in situ hybridization as described 

35 below. 



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Example 6 

In situ hybridization 

Serial sections of human embryos representing various stages of development were used for in vitro 
5 hybridization to explore the pattern of Frzb expression during embryonic development. Tissues from human embryos 
ranging from 6 to 13 weeks of gestation, estmiated on tha basis of crown-rump length and pregnancy records, were 
fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2L embedded in paraffin, cut serially at 5-7 /mi and 
mounted on satinated slides. These tissues were obtained from legally sanctioned procedures performed at the 
University of Zagreb Medical School, Zagreb, Croatia. The procedure for obtaining autopsy materials was approved 

10 by the Internal Review Board of the Ethical Committee at the University of Zagreb School of Medicine and the Office 
of Human Subjects Research of the National Institutes of Health, Bethesda, MD. In situ hybridization was performed 
as described previously {Pehon et a!., Development, 106:759-767, 1989; Vukicevic et al., 1 Histochem, Cytochem., 
42:869-875, 1994). Briefly, after a short prehybridization, sections were incubated overnight at 50^C in 50% 
formamide, 10% dextran sulfate, 4 x SSC, 10 mM dithiothreitol (DTT), 1 x Denhardt's solution, 500 //g/ml freshly 

15 denatured salmon sperm DNA and yeast tRNA with 0.2 • 0.4 ng/ml ^^S-labeled riboprobe (1 x 10^ cpm/jug) in a 
humidified chamber. Since the bovine Frzb open reading frame contained Xhol sites at both ends, this fragment was 
subcloned in both sense and antisense directions into the Xhol site of pBluescript SKII- vector and riboprobes were 
made using T7 RNA polymerase according to the manufacturer's instructions (Novagen). After hybridization, the 
sections were washed to a final stringency of 0.1 x SSC, 65°C for 2 x 15 min. After dehydration in a graded 

20 ethanol series containing 0.3 M ammonium acetate, slides were covered with NTB-2 emulsion (Kodak) and exposed 
for 1 • 3 weeks. The slides were then stained with 0.1% toluidina blue, dehydrated, cleared with xylene and 
mounted with Permount. 

Between 6 and 13 weeks, no hybridization was detected in most organs, including kidney, heart, muscle, 
intestine, liver, brain and lung. In contrast, strong hybridization was seen in the developing appendicular skeleton. 

25 At six weeks, Frzb transcripts were clearly visible surrounding the early cartilaginous rudiments of the developing 
limbs, as shown in the distal parts of the upper limb. Hybridization was apparent between neighboring areas of 
cartilaginous condensation in developing long bones. Subsequently, expression appeared within the cartilaginous cores 
of developing long bones. This was apparent in the proximal parts of the upper limb, which are more advanced in 
developmental state than the distal parts. Frzb was also present in the putathre limb primordia, thereby bridging the 

30 expression data obtained in early development to the localization in developing limbs. Additional experiments in 
developing limbs have revealed expression in the precartilaginous condensations and subsequently in the future joint 
interzones. 

In addition, Frzb was detected in the cartilage aniagen of several craniofacial bones and the epiphysial ends 
of the rib cage, while no signal was detected in the vertebral bodies at 6 weeks. At 13 weeks of gestation, Frzb 
35 transcripts were present in early chondroblasts of the tarsal bones of the foot, the carpal bones of the hand and 
the epiphysis of long bones. A striking feature of the expression pattern at this developmental stage was the 



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presence of a graded distribution, most prominent in the phalanges. The highest level of expression was observed 
at the epiphyses of long bones and at the periphery of cuboidal bones. The expression level then decreased with 
the appearance of chondrocyte hypertrophy and vascular invasion and appeared to be absent in the primary centers 
of ossification. Interestingly, at this stage of development, several layers of chondroblasts adjacent to the joint space 
5 did not show detectable transcripts, in sharp contrast to the prominent expression observed in other skeletal 
structures, no expression was apparent in the vertebral bodies at the stages examined. 
A Xenopus laevis orthologue of Frzb (Xfrzb) was isolated as described below. 

Example 7 
Isolation of XFrzb cDNA 

10 The primers 5'-TGGAACATGACTAAGATGCCC.3' {SEQ ID NO: 19) and 5'.CATATACTGGCAGCTCCTCG-3' 

(SEQ ID NO: 20) were used to label a region of the bovine Frzb cDNA sequence having a high degree of sequence 
identity to related genes from human and avian sources. 106 plaques from a Stage 20 Xenopus cDNA library 
prepared in ISH-lox (Novagen, Madison, Wl) were screened at low stringency (final stringency 35-BOC in 20 mM 
Na2HP04, pH. 7.2, 1 mM EDTA, 1% SDS) and purified plaques were characterized by direct sequencing (Wang et 

15 al., BioTechniques, 130-135, 1995). One 498 bp clone was 92% identical to a region of the bovine sequence. Two 
oligonucleotides, 5'-GTCnTTGGGAAGCCnCATGG.3'(SE0 ID NO: 21) and 5'.GCATCGT€GCAnTCACnTCA.3'(SE0 
ID NO: 22), corresponding to the 5' and 3' regions of this partial length clone, were used to screen duplicate lifts 
from a stage 13 library (Richter et a!., Proc, NatL Acad, ScL USA, 8086-8090, 1988). Plaques that hybridized to 
both oligonucleotides were further analyzed. Several clones containing a complete open reading frame were identified 

20 and sequenced. Two closely similar clones were isolated and one of these was chosen for further study. The 
nucleotide and deduced amino acid sequences of this Xfrzb clone is shown in SEQ ID NOS: 23 and 7, respectively. 

Xf rzb shares several features common to the mammalian proteins, including a consensus site for asparagine- 
iinked glycosylation, a conserved cysteine rich domain characteristic of Frizzled proteins, and a carboxyl terminal motif 
(amino acids 244-293) that appears homologous to the netrin-specific carboxyMerminal domain of C, elegans unc-6 

25 (Wadsworth et al., Bioessays, 16:355-362, 1996). 

Expression of Xfrzb was analyzed by in situ hybridization as described in Example 6. Expression begins 
early in gastrulation and continues as the embryo matures. Thus, it is present when many of the most important 
events in the establishment of the overall body plan of the developing embryo occur. It is expressed initially in the 
organizer region, extending beyond it during gastrulation. At the end of gastrulation, expression in this region 

30 abruptly ceases and then appears in primordial head mesoderm. Expression then becomes more localized, ultimately 
to a region corresponding to the developing pituitary gland. These observations are consistent with an important 
role in the induction of the nervous system and axial musculature, from which the majority of skeletal muscle is 
derived. Its expression in the pituitary suggests a prominent role in defining anterior mesodermal structures, including 
the pituitary itself. 



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Example 8 

Immunohistochemical staining 

Tissue sections were stained using the Vectastain elite ABC kit (Vector Laboratories, Buriingame, CA) 
according to the manufacturer's protocol. All embryos were embedded in JB4 resin (Polysciences, Warrenton, PA). 
For conventional histological analysis, 1-3-B5m sections were cut and stained with hematoxylin and eosin. Before 
staining, tissue sections were pretreated with chondroitinase ABC for 1 hour. The sections were blocked with PBS 
and 10% goat serum for 30 min, then incubated for 1 hour with primary antiserum (N374-PEP) at a dilution of 15 
/yg/ml in PBS containing 0.5% goat serum. In the controls, the primary antibody was replaced with normal pre* 
immune rabbit serum or secondary antibody alone. 

Immunohistochemical staining confirmed the presence of protein in developing skeletal structures, appearing 
within the cartilaginous cores of the developing long bones. The graded mRNA expression pattern detected by in situ 
hybridization, most prominent in the phalanges, was paralleled by the protein distribution. 

Example 9 

Ectopic expression of Frzb in Xenopus embryos 
Ectopic expression in developing Xenopus zvcbi'it^t induced formation of secondary body axes which 
contained neural and muscle tissue, but no notochord. This assay is an extremely stringent and specific test for the 
ability of a gene product to initiate a complex program of developmental events and indicates that Frzb can initiate 
the synthesis of nerve and muscle tissue. Further, overexpression of Xf rzb in explants fated to develop into ventral 
tissue induced molecular markers of muscle and nerve tissue. 



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Ultraviolet irradiation interrupts the normal mechanism for establishment of the dorso-anterior body axis, 
so that treated embryos did not develop dorsal structures (i.e. head, somites, neural tube, notochord) or the tissues 
comprising them. When irradiated enzymatically defoliiculated embryos were Injected m\\\ 50 mRNA encoding 
Xfrzb, a body axis was restored. The reconstituted axis contained a neural tube and dysmorphic somites, but no 
5 notochord. This experiment is an even more demanding test of the ability of a protein to initiate a complex 
developmental program. If a truncated construct, containing only the putath^e extracellular and transmembrane 
regions of the molecule, was used for injection with mRNA at the two cell stage of one btastomere, one half of the 
embryo appeared to develop normally, while the other was devoid of both muscle and neural tube; the notochord was 
normal bilaterally. This study evaluated the effects of ablating the function of Xfrzb, based on the premise that the 

10 defective molecule could act as a competitive inhibitor of endogenous Frzb. The effect produced by the defective 
Frzb was in essence the converse of what is observed if the unmodified gene is overexpressed. 

As an initial test of the ability of Frzb to play a role in patterning of the vertebrate embryo, the effects 
of overexpression and ectopic expression of Frzb in developing Xenopus embryos were evaluated. 

All embryos were embedded in JB4 resin (Potysciences, Warrenton, PA), for conventional histological 

15 analysis, 1-3 //m sections were cut and stained with hematoxylin and eostn; 10-20 sections were taken from 
embryos stained by in situ hybridization. Oarkfietd images of embryos were photographed with low angle oblique 
illumination and a Zeiss Stemi-6 dissecting microscope. Embryos cleared with benzyl alcohol/benzyl benzoate and the 
histological sections in Example 10 was photographed under diascopic illumination with a Nikon FX A microscope, 
the sections in Example 1 1 were photographed under multiple oblique illumination (Edge Scientific, Santa Monica, CA). 

20 Example 10 

Dorsalization of embryos by Frzb 
Enzymatically defoliiculated single ventral blastomeres at the 4 cell stage were injected with 50 ng bovine 
Frzb (Bfrzb) mRNA and cultured with oocyte Ringer's solution as previously described (Kay, Methods Cell Biol, 
36:657-669, 1991). Frogs and their embryos were maintained and manipulated using standard methods (Gordon, 

25 Methods Cell Biol, 16:125-139, 1977). mRNA injection was performed as described previously (Moos et al., 
Development, 121:4293-4301, 1995). Dorsal and ventral blastomeres were identified by size and pigment variations. 
Lithium treatment was for 1 hour at 0.1 M (Kao et al., Dev. BioL 127:64-77, 1988). UV irradiation was performed 
with a Stratalinker^^ (Stratagene). Animal cap explants were cultured in 0.7-1 x Marc's Modified Ringer's solution 
(Kay, supra.), Acthrin was a gift from the National Cancer Institute and bFGF was from GIBCO/Life Technologies 

30 (Gaithersburg, MD). 

Injection of Bfrzb mRNA into single ventral blastomeres produced duplicated posterior dorsal axes 
reproducibly. Muscle and neural tissues were apparent in frontal sections taken from these embryos, but notochord 
was absent. The frequency of axis duplication was approximately 15% (24/159; four independent experiments) with 
Bfrzb and somewhat less with the Xenopus gene. The difference may be due to the presence of a consensus 

35 translation initiation site (Kozak, J. BioL Chem., 266:19867-19870, 1991) in the bovine, but not the amphibian 
sequence. The phenotypes were identical in either case. When Frzb was injected into UV-irradiated enf>bryos which 



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are incapable of axis formation, dorsal axes were partially rescued in approximately 56% 137/66; three independent 
experiments). The rescued axes contained muscle and neural tube, but no notochord. Nevertheless, overexpression 
of Frzb in animal cap exptants did not induce markers for mesoderm (Brachyury IXbra)), neural tube (NCAM), or 
somites (muscle actin) (not shown). 
5 Example 11 

Expression of Xfrzb in developing Xenopus embrvo 
The Xfrzb open reading frame was subcloned into pCR-Script (Stratagene) to generate probes for in situ 
hybridization. Both Bfrzb and Xfrzb were subcloned into pSP64R1 (Dr. S. Sokol, Harvard University) for mRNA 
injection experiments. The pSPBAT Xwnt-S'"^^ plasmid used for mRNA injections and in vitro translation and the 

10 CSKA-X8 expression plasmid are described by Christian et al. (Genes Dev., 7:13-28, 1993). A pGEM SR-Xwnt-8 
plasmid (Smith et aU Ceil, 67:753-765, 1991) was used to generate probes for in situ hybridization, in vitro 
transcription was performed using mMessage mMachine or MEGAscript kits from Ambion (Austin, TX). The plasmid 
pLNCWntlHA, containing the open reading frame of mouse Wnti and a hemagglutinin (HA) tag near the C-terminus, 
was provided by Dr. J. Kitajewski (Columbia University). The Xlmf25 plasmid used for in situ hybridization analysis 

15 of MyoD is described by Scales et al. \Moi, Ceii. Biol., 1515-1524, 1990). The pfrzb expression plasmid is described 
by Hoang et al. U BioL Ciiem., 271:26131-26137, 1996). In situ hybridization was performed as outlined by 
Harland (Metiiods Cell BioL, 36:685-695, 1991), with modifications as described by Moos et al. Wevelopment, 
121:42934301, 1995). 

Xfrzb expression first became apparent in the late blastula (stage 9) by in situ hybridization. In early 
20 gastrulas (stage 10), mRNA expression was most apparent in the Spemann organizer. In later gastrulas (stage 10.5- 
11), there was expression in the blastopore lip that extended beyond the organizer as the blastopore lip progressed 
ventrally. At about stage 11, Xfrzb expression appeared in the dorsal midline. Examination of cleared embryos and 
corresponding histological sections revealed that this expression was in the involuted mesoderm which is thought to 
convey signals to the overlying neuroectoderm that participates in specification of the nervous system. Near the 
25 onset of neurulation, posterior expression was markedly reduced, and expression in the prechordal plate became 
apparent. The field of expression was then restricted progressively, stabiftzing in the putative pituitary and 
posteriorly in the vicinity of the proctodeum. Thus, Xfrzb is expressed at the appropriate time and place to 
participate in specification of the body axis. These results are consistent with RT-PCR analysis. 

Example 12 

30 Immunoblotting. immunoprecipitation and in vitro translation 

Embryos and oocytes were lysed by sonication on ice in 40 mM Tris base, 10 mM EDTA, 1 mM 
Phenylmethylsulfonyl fluoride (PMSF) in a volume of 10 //I/embryo or oocyte. In some experiments, 20,000 x g 
supernatants were extracted with an equal volume of 1,1,2-trichlorofluoroethane (Evans et al., Metitods<!ell BioL, 
36:117-132, 1991). in vitro translations were performed in the presence of ^^S-methionine with nuclease-tr«8ted 

35 rabbit reticulocyte lysate and canine pancreatic microsomal membranes (Promega, Madison. Wl) according to the 
manufacturer's instructions. )9-tactamase mRNA supplied with the kit was used as a positive control for translation 



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.22. 

and processing and as a negative control for nonspecific protein-protein interaction. SOS-PAGE was performed using 
Novex 10% Nu-PAGE gets (Novex, San Diego, CA). Samples from embryos were precipitated with 
methanollchloroform (Wessel et al., AnaL B/ochem., 138:141-143, 1984) prior to analysis. For metabolic labeling 
studies, gels were dried onto a single sheet of cellophane and imaged with BioMax MR2 film (Kodak) or a phosphor 
5 screen (Molecular Dynamics, Sunnyvale, CA). 

Immunoprecipitation was performed according to standard procedures (Harlow et al., Antibodies: a 
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988). Antiserum N374-PEP against 
Xfrzb was prepared as described (Hoang et al., supraX The clone 9E10 monoclonal antibody (Boehringer, 
Indianapolis, IN) was used for precipitation or detection of the c-myc epitope and hybridoma supernatant containing 

10 the 12CA5 monoclonal antibody was used for precipitation of the HA epitope. Immunoblot analyses of separated 
proteins were performed following transfer to nitrocellulose membranes using 1:20,000 dilutions of primary antisera 
and 1:100,000 dilutions of peroxidase-conjugated secondary antibody. Bands were detected with the Super Signal 
Ultra peroxidase substrate (Pierce, Rockford, IL). 

Endogenous Xfrzb could be detected in early gastrulas (stage 10) and all subsequent stages analyzed by 

15 immunoblot analysis. Xfrzb expression was unaffected by bFGF, enhanced by activin or lithium, and suppressed by 
UV irradiation as described for other genes expressed in the Spemann organizer (Slack, Curr, BioL, 4:118-126, 1994; 
Kao et aU Dev, BioL, 127:64-77, 1988). 

Example 13 

Blocking of Wnt-8 sionaling in vivo by Frzb 

20 When Xwnt*8 mRNA is injected during early embryogenesis, secondary dorsal axes with complete head 

structures are induced reliably (Smith et a!., supra.; Sokol et al.. Cell, 67:741-752, 1991). This phenomenon was 
used as an in vivo assay for Xwnt-8 activity. When prolactin mRNA was coinjected with Xwnt-8 message, 71% 
of the embryos (27/38) developed secondary axes. In contrast, when the prolactin mRNA was replaced by an 
identical amount of Frzb mRNA, axis duplications were suppressed fO/32 for Xfrzb; 1/36 for Bfrzb). Uninjected 

25 embryos did not display axial abnormalities. 

A Xwnt-8 expression plasmid under the control of the cytoskeletal actio (CSKA) promoter induces the 
ventrolateral marker Xpo (Sato et al., Development, 112:747-753, 1991) and suppresses induction of the dorsal 
marker goosecoid in activin-treated animal cap explants (Hoppler et al.. Genes Dev., 10:2805-2817, 1996). This 
effect was blocked completely in caps overexpressing a dominant-negative Xwnt-8. Our results confirmed that Xpo 

30 expression could be increased by Xwnt plasmid in activin-treated animal cap explants. Importantly, this effect was 
blocked by XFrzb. 

ADMP is a Spemann organizer specific marker that is induced by activin in animal cap explants (Moos et 
al., supraX Induction of ADMP by activin was suppressed in explants injected with Xwnt-8 plasmid; this suppression 
was rescued by Xfrzb. Frzb overexpression did not affect the expression level of Xwnt-8. Thus, Frzb appears to 
35 exert its dorsalizing effects by inhibiting the action of Xwnt-8. In a related experiment, the CSKA-Xwnt-8 plasmid 
was injected into dorsal blastomeres with or without Xfrzb mRNA. In this assay, CSKA-XWnt-6 plasmid produced 



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head defects (64/80 embryos, three independent experiments), as described previously (Christian et al.. Genes Dev., 
7:13-28, 1993). However, if Xfrzb mRNA was coinjected with the CSKA-Xwnt-8 plasmid, these defects were not 
observed (0/81 embryos). 

Induction of Siamois and Xnr3 proteins (Lemaire et ai., Ceff, 81:85-94, 1995; Smith et at., Ceff, 82:3746, 
5 1995) in animal cap explants Injected with Xwnt-8 mRNA has been used to assay Xwnt-8 signaling (Carnac et a!.. 
Development, 122:3055-3065, 1996; Yang-Snyder et ai., supraX Both Xfrzb and Bfrzb blocked the induction of 
these genes by Xwnt-8. 

Example 14 

Cell fractionation of Xenopus embryos and action across cell boundaries 

10 The subcellular distribution of Xfrzb expressed in vivo was analyzed. Endogenous Xfrzb protein was found 

in 105,000 X g supernatants isolated from Xenopus embryos, but could not be detected in cell pellets, in contrast 
to Bfrzb (Example 5). Further, Frzb was secreted by oocytes injected with Frzb mRNA. The apparent molecular 
weight of 33 kOa is consistent with removal of the putative signal sequence. Proteolytic processing likely accounts 
for the difference in molecular weight between secreted Frzb and Frzb contained in oocyte lysates. 

15 To determine whether Frzb could act across cell boundaries, an experimental design used to study the 

dominant-negative Xwnt-8 (Hoppler et al., supra,) was used. Xfrzb reduced the percentage of secondary axes induced 
by Xwnt-8 from 52% (46/88) to 10% (5/49) when the two mRNAs were injected into different cells. This indicates 
that the effects of Frzb on Xwnt-8 occur following secretion. 

Example 15 

20 Direct interaction of Frzb and X-Wnt8 proteins 

Direct interaction between Frzb and X-WntS proteins was demonstrated in two systems: rabbit reticulocyte 
lysate containing canine microsomal membranes, and transfected COS cells. COS 7 cells (1.6 X 10^ initial seeding 
density) were transfected with 5 //g of pfrzb (see Example 5) or pLNCWntIHA (see Example 11), or co-transfected 

with 4 /yg pfrzb and pLNCWntIHA in 100 mm dishes using 30 //I LipofectAMINE^'^ (Life Technologies, Inc., 

® 

25 Gaithersburg, MD). Transfections were performed for 6 hours in serum-free Opti-MEM I (Life Technologies). 
Thereafter, cells were incubated for 18 hours in media containing 10% fetal bovine serum. Subsequently, cells were 
cultured at 37°C for 24 hours in serum-free Opti-MEM I . Cells were extracted for 30 minutes on ice with 50 mM 
Tris-HCl, 150 mM NaCI, 1.0% NP40, 0.5% Deoxychotic acid and 0.1% SOS and centrifuged at 12,000 x g for 5 
minutes. Supernatants were saved for immunoprecipitation. 

30 Xwnt-8"^^^ Bfrzb, Xfrzb, and the )9-lactamase control mRNA were all translated and processed in vitro, 

either alone or in the following combinations: Wnt-8 + Frzb, Wnt-8 + )9-lac; Frzb )9-lac. As expected, the anti- 
myc antibody precipitated Xwnt-B'"^*^ but not ^-lactamase, Xfrzb or Bfrzb. Conversely, the 374-PEP antiserum, 
which recognized both mammalian and amphibian Frzb in immunoblots, precipitated both Xfrzb and Bfrzb, but neither 
Xwnt-S'"^^ nor ^ff-lactamase. However, when Xwnt-8^^^ and Frzb were cotranslated, both proteins were precipitated 

35 by either the myc-specific 9E10 monoclonal antibody or the 374-PEP antiserum. Identical results were obtained with 
Bfrzb. Neither reagent precipitated ^ lactamase cotranslated with Frzb or Xwnt-B'^V^ 



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These results were further supported by experiments in which C0S7 cells were co-transfected with 
expression plasnnids encoding Bfrzb and an HA-tagged murine Wnt-1, which belongs to the same functional class as 
Xwnt-8 (Nusse et zU Cell, 69:1073-1087, 1992). Cell lysates were immunoprecipitated with an antl-HA antibody, 
immunoblotted and probed with the Frzb specific 374-PEP serum. Frzb protein was detected only in lysates from 
5 cells transfected with both Frzb and Wnt-1. 

Example 18 

Frzb blocks MvoD expression 
Xwnt-8 was implicated in somite development using a carboxy-terminal deletion construct which acted in 
a dominant negative fashion (Hoppler et a1.. supra,). Because our data suggested that Frzb could also act as a Wnt 
10 inhibitor, we evaluated its effects on somite formation and MyoD expression, both of which are suppressed by the 
dominant negative Xwnt-8. When Xfrzb mRNA was injected radially into all blastomeres at the four cell stage, trunk 
development was grossly abnormal, resembling that seen in embryos overexpressing the dominant negative Xwnt-8. 
Furthermore, Xfrzb blocked MyoD expression both in gastrulating embryos and in activin-treated animal cap explants. 
At first glance, the ability of Frzb to induce partial dorsal axes or suppress MyoD expression appear to be 
15 incompatible. However, these observations can be reconciled by consideration of the cellular context in which 
overexpression of Frzb occurs. Ectopic gene expression may generate a secondary axis by a direct inductive effect, 
or indirectly by inhibition of a ventralizing signal. Frzb blocks the actions of Xwnt-8 as described in Example 13, 
but does not induce mesoderm, muscle or neural tissue when overexpressed in animal cap explants which do not 
express Xwnt-8 (Example 10). The dorsalizing actions of Frzb are thus likely to be indirect, resulting from inhibition 
20 of the ventralizing effects of Xwnt-8. 

Local overexpression of a molecule acting in such an indirect manner produces different effects than 
generalized overexpression. Injection of Frzb into a single blastomere within the expression domain of Xwnt-8 is 
expected to block its ventralizing acth/ity locally. Generation of a partial dorsal axis by Frzb (Example 10) is 
consistent with this prediction. On the other hand, generalized overexpression will block all actions of Xwnt-8 
25 throughout the embryo, including both its ventralizing acthrity and its effects on somite formation. 

Recently, a dominant-negative Xwnt-8 was shown to suppress development of the trunk and somites 
(Hoppler et a!.. Genes Dev., 10:2805-2817, 1996). When Frzb was overexpressed using an identical protocol (all 
blastomeres at the four cell stage), the same phenomenon was produced (Example 16). Thus, the induction of muscle 
tissue by local overexpression of Frzb in one type of experiment and suppression of somite development by 
30 generalized overexpression in another are compatible findings consistent with the conclusion that Frzb acts through 
inhibition of Xwnt-8 signaling. 

To investigate the specificity of Frzb/Wnt interactions, C0S7 cells were co-transfected with Frzb and several 
HA-tagged Wnt family members as described below. 



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Example 17 

Transient transfection of C0S7 cells 
Plasmids used for transfection of COS? cells were as follows. A c-term was made by deletion of most 
5 of the C-terminal region of Frzb from amino acids 160 to 316. A CRD was made by deletion of most of the frizzled 
domain of Frzb from amino acids 39 to 145 including nine conserved cysteines (C43-147). A 7C was made by 
deletion in the frizzled domain of amino acid 79 to 149 including seven conserved cysteines (C88-147). A 2C was 
made by a deletion in the frizzled domain from amino acid 124 to 149, including two conserved cysteines (C136- 
147). A 57-95 was made by deletion in the frizzled domain from amino acids 57 to 95 which interrupted the 
10 hydrophobic structure in the frizzled domain. These constructs are shown in Fig. 6. pFrzb-FLAG was made by 
replacement of the last seven residues of Frzb by a FLAG-tag. All constructs were subcloned into pCDNAS 
(Invitrogen). The following plasmids carried ten mouse Wnt gene family members: pLNCW1-HA, pLNCW2-HA, 
PLNCW3A.HA, pLNCWSB-HA, pLNCW4.HA, pLNCW5A.HA, pLNCWSB-HA, pLNCW6 HA, pLNCW7A.HA and pLNCW7B- 
HA. 

15 C0S7 cells (1.6 X 10^ initial seeding density) were transfected either with 5 //g ptasmid DNA, or co- 

transf acted with 4 //g for each plasmid per 100 mm dish using 30 LipofectAMlNE^^ reagent (GIBCO/BRL). 
Transfections were carried our for 6 hours in serum-free Opti MEM I (GIBCO/BRL). Equal amounts of 10% FBS in 
Opti-MEM 1*^ were added to the transfections and the cultures were continued for 18 hours, the ceils were then 
incubated at 37°C for 24 hours in serum-free Opti-MEM I . The cells were extracted for 30 minutes on ice with 

20 50 mM Tris-HCI, 150 mM NaCl 1.0% NP40, 0.5% deoxycholic acid and 0.1% SOS. 

Example 18 

Immunopreciprtations with HA and FLAG antiserum 
Fifty /j\ of protein A-agarose (Behringer Mannheim GmbH, Germany) was incubated with 100 jA hybridoma 
supernatant of anti-HA antibody 12CA5 in 450 /j\ of 50 mM Tris-HCI, pH 7.4, 150 mM NaCI by rotating overnight. 

25 100400 /yl of the cell tysates from transfected C0S7 cells and 0*300 /yl of RlPA buffer were added to the mixture 
to a final volume of 1 ml and incubation was continued for another hour. The agarose beads were washed by 
centrifugation at 12,000 x g for 20 seconds, followed by rotation for 20 minutes twice in 1 ml of "50 mM Tris-HCI, 
pH 7.4, 150 mM NaCI, pH 7.5; twice in 1 ml 50 mM Tris-HCI, pH 7.4, 500 mM NaCl and once in 50 mM Tris-HCI, 
pH 7.4, respectively, and centrifuged at 12,000 x g. After the last wash and centrifugation, pellets were suspended 

30 in 30-50 //I of 2 x Laemmli sample buffer with 4% )9-mercaptoethanol, boiled, separated on 4-20% gradient 
Tris/Glycine gels (Novex), blotted onto lmmobilon™-P membranes (Millipore) and analyzed by immunoblotting. 

Membranes were blocked for 30 min in blocking buffer (BF) consisting of 1 M Tris-HCI, pH 7.5, 0.9% NaCI, 
0.05% Tween-20 and 4% BSA. The primary antiserum (N374-PEP) was incubated with the membranes in 1/10 BF 
and 9/10 TBST buffer (10 mM Tris-HCI. pH 7.5, 0.1% Tween, 150 mM NaCI) at a dilution of 1:2,500. The 

35 membranes were washed four times for 5 min in TBST after each incubation step. The membranes were then 
incubated with the secondary antibody at a dilution of 1:10,000 for 60 minutes. Blots were developed using the 



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SuperSignal^^ CL substrate system (Pierce) for chemiluminescent detection and exposed to Kodak XAR-5 film for 
1 to 10 minutes. 

Fifty //I protein G-agarose (Behringer Mannheim) and 5 //g Anti FLAG M2 antibody (Eastman Kodak) were 
used for immunoprecipitation; a 1:1,000 dilution of Anti-HA-peroxidase antibody (HRP-HA) (Behringer Mannheim) was 
used for immunoblotting and SuperSignaF^ ULTRA substrate (Pierce) was used for the chemiluminescent detection. 

Frzb co immunoprecipitated with all the Wnts tested (Wnt-1, Wnt-2, Wnt-3A, Wnt<3B. Wnt4, Wnt-5A, Wnt- 
5B, Wnt-6, Wnt-7A and Wnt*7B). Likewise, using a flag tagged BFrzb cDNA, Wnts co-immunoprecipitated with Frzb. 
Thus, Frzb has sufficient affinity for each of these Wnt proteins to allow co-immunoprecipitation. 

Example 19 
Modulation of Wnt actwitv bv Frzb in vivo 

Injection of Wnt-T mRNA into Xenopus embryos resuhs in duplication of the dorsal axis and can easily be 
scored by direct inspection. Co-injection of Wnt-1 and Frzb resuhed in the complete inhibition of secondary axis 
formation due to blockade of Wnt signaling (Fig. 7). Removal of the entire frizzled domain abolished the inhibitory 
activity of Frzb. Substantial inhibition of Wnt-1 mediated axis duplication was also observed when the CRD only was 
co-injected with Wnt-1. The C-terminal domain plays a role in this effect, as inhibition was more efficient in the 
presence of this domain. This suggests a possible role of the C-terminal domain in the stabilization of its tertiary 
structure affecting the binding affinity to Wnts, a possible involvement in Frzb turnover and increased solubility of 
the protein, in contrast to the co-immunoprecipitation data, no inhibition was observed in this in vivo assay with 
any of the deletion constructs affecting the CRD domain. 

Wnt-5A was also co-injected with Frzb in Xenopus oocytes. Surprisingly, although Frzb binds Wnt-5A, no 
inhibition of Wnt-5A activity was observed. Co-injection of Wnt-5A and Frzb actually resulted in more pronounced 
changes in embryo phenotype. Thus, Frzb and related proteins are not ahvays inhibitory and can be considered 
modulators or Wnt acthrity. Frzb may restrict Wnt activities and strictly regulate boundaries in certain systems by 
immobilizing Wnts in the cell, to the cell membrane or the pericellular matrix, while in other systems Frzb may 
function as a soluble factor enhancing Wnt secretion and even providing transportation to neighboring cells. 



wo 98/16641 PCTAJS97/18362 

Example 20 

Treatment of Deep Knee Defects with Frzb 
A young patient having a large defect in the articular surface of the knee joint is identified. A periosteal 
flap is obtained from the bone beneath the joint surface of rib cartilage according to standard surgical procedures. 
5 The tissue flap is pre-incubated in a solution containing recombinant human, bovine or Xenopus Frzb protein. The 
Frzb treated periosteal flap is then attached over the lesion in the articular surface of the knee joint by a sewing 
procedure using, for example, resolvable material The joint is then closed and injected with a solution containing 
bovine, human or Xenopus Frzb protein in a pharmaceutically acceptable carrier. Injections are administered until 
cartilage repair is complete. The patient notices markedly less joint pain as the cartilage repair process progresses. 
10 Examination by arthroscopy indicates repair of the lesion within several weeks following the initial procedure. 

It is also contemplated that gene therapy protocols based on expression of Frzb cDNAs or genomic 
constructs can be used to facilitate in mo cartilage, bone, muscle and nerve repair. Therapy may be achieved by 
genetically altering synoviocytes, periosteal cells, chondrocytes, myoblasts, osteoblasts or neural cells by transf ection 
or infection with recombinant constructs directing expression of Frzb. Such altered celts can then be returned to 
15 the appropriate in vivo location. Gene transfer can be performed using numerous vectors well known in the art, 
including retroviruses, adenoviruses, herpesviruses and adeno-associated viruses. 

Both in vivo and ex vivo approaches are anticipated for continuous delivery of Frzb for treating neuro-, myo-, 
osteo- and chondrodegenerative disorders. In addition, inducible promoter constructs may be employed in gene 
therapy applications of the present invention. 



20 



VfO 98/16641 



-28- 

SEQUENCE LISTING 



PCT/US97/18362 



(1) GENERAL INFORMATION 

(i| APPLICANT: THE GOVERNMENT OF THE UNITED STATES OF AMERICA AS REPRESENTED BY THE 
SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES 

(ii) TITLE OF THE INVENTION: ISOLATION AND USE OF TISSUE GROWTH-INDUCING FRZB PROTEIN 

(iii) NUMBER OF SEQUENCES: 23 

(iv) CORRESPONDENCE ADDRESS: 

(A) ADDRESSEE: Knobbe, Martens, Olson & Bear 

(B) STREET: 620 Newport Center Drive, 18th Floor 

(C) CFTY: Newport Beach 
(0) STATE: CA 

(E) COUNTRY: U.SA 

(F) ZIP: 92660 

(V) COMPUTER READABLE FORM: 

(A) MEDIUM TYPE: Diskette 

(B) COMPUTER: IBM Compatible 

(C) OPERATING SYSTEM: DOS 

(0) SOFTWARE: FastSEQ Version 1.5 

(vO CURRENT APPLICATION DATA: 

(A) APPLICATION NUMBER: 

(B) FILING DATE: 

(C) CLASSIFICATION: 

(vii) PRIOR APPLICATION DATA: 

(A) APPLICATION NUMBER: 08/822,333 

(B) FILING DATE: 20-MAR-1997 

(vii) PRIOR APPLICATION DATA: 
(A) APPLICATION NUMBER: 08/729,452 



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PCTAJS97/18362 



(B) FILING DATE: 11-0CT-1996 

(vin) AHORNEY/AGENT INFORMATION: 

(A) NAME: Bartfeld, Neil S 

(B) REGISTRATION NUMBER: 39,901 

(C) REFERENCE/DOCKET NUMBER: NIH133.001QPC 

(ixl TELECOMMUNICATION INFORMATION: 

(A) TELEPHONE: 619-235-8550 

(B) TELEFAX: 619-235 0176 

(C) TELEX: 



(2) INFORMATION FOR SEQ ID N0:1: 

(0 SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 2374 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: Onear 

(ix) FEATURE: 

|A) NAME/KEY: Coding Sequence 
(B) LOCATION: 256...1230 
(D) OTHER INFORMATION: 



(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1: 



AATAGATGCC 6CG6CCCCAG AAGTCTTAGA CGTCGGGAAA GAGCACCCGG AGAGGCAGGG €0 

GCGGCGGCGG CTGGCGCTCG GCGCAGCTH TGGGACCCCA HGAGGGAAT TT6ATCCAAG 120 

GAAGCTGT6A GAHGCCGGG GGAGGAGAAG CTCCCATATC ATTGTGTCCA CHCCAGGGC 180 

GGGGAGGAGG AAACGGCGGA GCGGGCCTCT CGGCGTTCTC CGCACTIjCTG CACCCTGCCC 240 
CATCCTGCCG AGATC ATG GTC TGC G6G AGC CGA GGC GGG ATG CTG CTG CTG 291 
Met Val Cys Gly Ser Arg Gly Gly Met Leu Leu Leu 



wo 98/16641 



-30- 



PCT/US97/18362 



1 5 10 

CCG GCC GGG CTA CTC GCC CTG GCT GCG CTC TGC CTG CTC CGC GTG CCC 339 
Pro Ala Gly Leu Leu Ala Leu Ala Ala Leu Cys Leu Leu Arg Val Pro 
15 20 25 

GGA GCG CG6 GCG GCC GCC TGT GAG CCC GH CGC AH CCC CTG TGC AAG 387 
Gly Ala Arg Ala Ala Ala Cys Glu Pro Val Arg He Pro Leu Cys Lys 
30 35 40 

TCC CTG CCC TGG AAC ATG ACT AAG ATG CCC AAC CAC CTG CAC CAC AGC 435 
Ser Leu Pro Trp Asn Met Thr Lys Met Pro Asn His Leu His His Ser 
45 50 55 60 

ACC CAG GCC AAC GCC ATC CTG GCC ATC GAG CAG TTC GAA GGT CTG CTG 483 
Thr Gin Ala Asn Ala lie Leu Ala lie Glu Gin Phe Ghi Gly Leu Leu 
65 70 75 

GGC ACC CAC TGC AGC CCG GAT CTG CTC TTC HC CTC TGT GCT ATG TAC 531 
Gly Thr His Cys Ser Pro Asp Leu Leu Phe Phe Leu Cys Ala Met Tyr 
80 85 90 

GCG CCC ATC TGC ACC AH GAC HC CAG CAC GAG CCC ATC AAG CCC TGC S79 
Ala Pro lie Cys Thr lie Asp Phe Gin His Glu Pro lie Lys Pro Cys 
95 100 105 

AAG TCT GTG TGC GAG CGG GCC CGG CAG GGC TGT GAG CCC ATC CTC ATC 627 
Lys Ser Val Cys Ghi Arg Ala Arg Gin Gly Cys Glu Pro He Leu He 
110 115 120 

AAG TAC CGC CAC TCG TGG CCG GAA AGC CTG GCC TGC GAG GAG CTG CCA 675 
Lys Tyr Arg His Ser Trp Pro Glu Ser Leu Ala Cys Glu Glu Leu Pro 
125 130 135 140 



GTA TAT GAC CGC GGC GTG TGC ATC TCT CCG GAG GCC ATC GTC ACT GCC 
Val Tyr Asp Arg Gly Val Cys He Ser Pro €lu Ala He Val Thr Ala 



723 



wo 98/16641 

145 

GAC GGA GCC GAT TU CCT ATG GAT TCC AGT AAT GGA AAC TGT AGA GGA 771 
Asp Gly Ala Asp Phe Pro Met Asp Ser Ser Asri Gly Asn Cys Arg Gty 
160 165 170 

GCA AGC AGT GAA CGC T6C AAA TGT AAA CCA GTC AGA GCT ACA CAG AAG 819 
Ala Ser Ser Gtu Arg Cys Lys Cys Lys Pro Val Arg Ala Thr Gin Lys 
175 180 185 

ACC TAT TTC CGA AAC AAT TAC AAC TAT GTC ATT CGG GCT AAA GTT AAA 867 
Thr Tyr Phe Arg Asn Asn Tyr Asn Tyr Val lie Arg Ala Lys Val Lys 
190 195 200 

GAA ATA AAG ACC AAG TGT CAT GAT GTG ACT GCA GTA GTG GAG GTG AAG 915 
Glu He Lys Thr Lys Cys His Asp Val Thr Ala Val Val Glu Val Lys 
205 210 215 220 

GAG AH HA AAG GCT TCT CTG GTA AAC AH CCA AGG GAA ACT GTG AAC 963 
Glu lie Leu Lys Ala Ser Leu Val Asn He Pro Arg Glu Thr Val Asn 
225 230 235 

CTT TAT ACC AGC TCT GGC TGC CTG TGT CCT CCA CH AAC GH AAT GAG 1011 
Leu Tyr Thr Ser Ser Gly Cys Leu Cys Pro Pro Leu Asn Val Asn Glu 
240 245 250 

GAG TAT CTC ATC ATG GGC TAC GAA GAT GAA GAG CGC TCC AGA TTA CTG 1059 
Glu Tyr Leu He Met Gly Tyr Glu Asp Glu Glu Arg Ser Arg Leu Leu 
255 260 265 

HG GTA GAA GGT TCT AH GCT GAG AAA TGG AAG GAT CGA CH GGT AAA 1107 
Leu Vat Glu Giy Ser lie Ala Glu Lys Trp Lys Asp Arg Leu Gly Lys 
270 275 280 

AAA GTT AAG CGG TGG GAT ATG AAG CTC CGT CAT CTT GGA CTG AAT ACA 1155 
Lys Val Lys Arg Trp Asp Met Lys Leu Arg His Leu Gly Leu Asn Thr 



PCT/US97/18362 



150 155 



wo 98/16641 



•32- 



PCT/US97/I8362 



285 290 295 300 

AGT GAT TCT AGC CAT AGT GAT TCC ACT CAG AGT CAG AAG CCT GGC AGG 1203 
Ser Asp Ser Ser His Ser Asp Ser Thr Gin Ser Gin Lys Pro Gly Arg 
305 310 315 

AAT TCT AAC TCC CGG CAA GCA CGC AAC TAAATCCTGA AATGCAGAAA ATCCTCA 1257 
Asn Ser Asn Ser Arg Gin Ala Arg Asn 
320 325 

GTGGACnCC TAnAAGACT TGCAHGCTG GACTAGCAAA GGCAAATTGC ACTAHGCAC 1317 
GTCATAGTCT ATTTTnAGC CACAAAAATC AGGTGGTAAC TGATAHACT TCTATTTTn 1377 
CTTTTGTTTT CTGCTTTTCT CCTTCCCCCA nCCCTTTH TGTGGTCTGA GTACAGATCC 1437 
HAAATATAT TATATGTATT CTATHCACT AATCATGGGA AAACTGHCT HGCAATAAT 1497 
AATAAAHAA ACATGHGAT ACCAGGGCCT CTHGCTGGA GTAAATGHA ATTTGCTGn 1557 
CTGCACCCAG AHGGGAATG CAATAHGGA TGCAAAGAGA GATHCTGGT ATACAGAGAA 1 61 7 
AGCTAGATAG GCTGTAAAGC ATACTTTGCT GATCTAAHA CAGCCTCAH CHGCATGCC 1677 
THTGGCAH CTCCTCACGC TTAGAAAGH CTAAATGTTT ATAAAGGTAA AATGACAGH 1 737 
TGAAATCAAA TGCCAACAGG CAGAGCAATC AAGCACCAGG AAGCATTTAT GAAGAAATGA 1 797 
CACATGAGAT GAATTATHG CAAGATTGGC AGGAAGCAAA ATAAATAGCA HAGGAGCTG 1 857 
GGGATAGAGC ATHTGCCTG ACTGAGAAGC ACAACTGAAG CTAGTAGCTG nGGGGTGTT 1917 
AACAGCAGCA TnTTCTTTT GACGATACAT nGTHGTCT GTGAATATAT TGATCAGCAT 1 977 
TAGAGCAGTG GAHGTGACC AGACATCAGG TGHATCAGC ATAGCTCTGT TTAATTTGCT 2037 
TCCTTTTAGA TGAACGCAn GGTGTCTTTT TTTTCncn TTAAAATAAA TCTCCCHGC 2097 
TGCAnTGAC CAGGAAAAGA AAGCATATAT GCATGT6CAC CGGGCTGTTA TTTTTAAGAT 2157 
ATGTAGCTCT ATAAAACGCT ATAGTCAAAA GATGGTAAAA TGTGCAAGAT TCTGGGTGTG 2217 
TGTAHAATG TGTGTGTGTC CGCATACACT CACACTCAAG CTGAAGTGAA CGACAGGCCT 2277 
GTGCACTGGC CTGCACTTTA TCATTrGGAT HGTGCTGH TAATGCTCAG TAAAATATGC 2337 
TTAATAAAAG GAAAAAAAAA AAAAAAAAAA AAAAAAA 2374 

(2) INFORMATION FOR SEQ ID N0:2: 

(i| SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 325 amino acids 

(B) TYPE: amino acid 

10 STRANDEDNESS: single 



wo 98/16641 

(D) TOPOLOGY: linear 



-33- 



(ii) MOLECULE TYPE: protein 
(v) FRAGMENT TYPE: internal 

(xi) SEQUENCE DESCRIPTION: SEQ 10 N0:2: 

Met Val Cys Gly Ser Arg Gly Gly Met Leu Leu Leu Pro Ala Gly Leu 
15 10 15 

Leu Ala Leu Ala Ala Leu Cys Leu Leu Arg Val Pro Gly Ala Arg Ala 

20 25 30 

Ala Ala Cys Glu Pro Va! Arg He Pro Leu Cys Lys Ser Leu Pro Trp 

35 40 45 

Asn Met Thr Lys Met Pro Asn His Leu His His Ser Thr Gin Ala Asn 

50 55 60 

Ala lie Leu Ala lie Glu Gin Phe Glu Gly Leu Leu Gty Thr His Cys 
65 70 75 80 

Ser Pro Asp Leu Leu Phe Phe Leu Cys Ala Met Tyr Ala Pro He Cys 

85 90 95 

Thr lie Asp Phe Gin His Glu Pro lie Lys Pro Cys Lys Ser Val Cys 

100 105 110 

Glu Arg Ala Arg Gin Gly Cys Glu Pro lie Leu He Lys Tyr Arg His 

115 120 125 

Ser Trp Pro Glu Ser Leu Ala Cys Glu Glu Leu Pro Val Tyr Asp Arg 

130 135 140 

Gly Val Cys He Ser Pro Glu Ala lie Val Thr Ala Asp Gty Ala Asp 
145 150 155 160 

Phe Pro Met Asp Ser Ser Asn Gly Asn Cys Arg Gly Ala Ser Ser Glu 

165 170 175 

Arg Cys Lys Cys Lys Pro Val Arg Ala Thr Gin Lys Thr Tyr Phe Arg 

180 185 190 

Asn Asn Tyr Asn Tyr Val lie Arg Ala Lys Val Lys Glu lie Lys Thr 

195 200 205 

Lys Cys His Asp Val Thr Ala Val Val Glu Val Lys Glu lie Leu Lys 

210 215 220 

Ala Ser Leu Val Asn He Pro Arg Glu Thr Val Asn Leu Tyr Thr Ser 



wo 98/16641 PCT/US97/18362 

-On* 

225 230 235 240 

Ser Gly Cys Leu Cys Pro Pro Leu Asn Val Asn Glu Glu Tyr Leu He 

245 250 255 

Met Gly Tyr Glu Asp Gtu Glu Arg Ser Arg Leu Leu Leu Val Glu Gly 

260 265 270 

Ser He Ala Gtu Lys Trp Lys Asp Arg Leu Gly Lys Lys Val Lys Arg 

275 280 285 

Trp Asp Met Lys Leu Arg His Leu Gly Leu Asn Thr Ser Asp Ser Ser 

290 295 300 

His Ser Asp Ser Thr Gin Ser Gin Lys Pro Gly Arg Asn Ser Asn Ser 
305 310 315 320 

Arg Gin Ala Arg Asn 
325 

(2) INFORMATION FOR SEQ ID N0:3: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1484 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ix) FEATURE: 

(A) NAME/KEY: Coding Sequence 

(B) LOCATION: 208...1182 
(0) OTHER INFORMATION: 



(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3: 

CGG6GCCTGG GCGGSAGGGG CGGTGGCTGG AGCTCGGTAA AGCTCGTGGG ACCCCATTGG «0 
GGGAAHTGA TCCAAGGAAG CGGTGAHGC CGGGGGAGGA GAAGCTCCCA GATCCTTGTG 120 
TCCACTTGCA GCGGGGGAGG CGGAGACGCG GAGCGGGCCT TTTGGCGTCC ACT43CGCGGC 180 
TGCACCCTGC CCCATCCTGC CGGGATC ATG GTC TGC GGC AGC CCG €GA 6GG ATG 234 
Met Val Cys Gly Ser Pro Gly Gly Met 



wo 98/16641 

1 

CTG CTG CTG CGG GCC GGG CTG CTT GCC CTG GCT GCT CTC TGC CTG CTC 282 
Leu Leu Leu Arg Ala Gly Leu Leu Ala Leu Ala Ala Leu Cys Leu Leu 
10 15 20 25 

CGG GTG CCC GGG GCT CGG GCT GCA GCC TGT GAG CCC GTC CGC ATC CCC 330 
Arg Val Pro Gly Ala Arg Ala Ala Ala Cys Glu Pro Val Arg lie Pro 
30 35 40 

CTG TGC AAG TCC CTG CCC TGG AAC ATG ACT AAG ATG CCC AAC CAC CTG 378 
Leu Cys Lys Ser Leu Pro Trp Asn Met Thr Lys Met Pro Asn His Leu 
45 50 55 

CAC CAC AGC ACT CAG GCC AAC GCC ATC CTG GCC ATC GAG CAG TTC GAA 426 
His His Ser Thr Gin Ala Asn Ala lie Leu Ala He Glu Gin Phe Glu 
60 65 70 

GGT CTG CTG GGC ACC CAC TGC AGC CCC GAT CTG CTC TTC HC CTC TGT 474 
Gly Leu Leu Gly Thr His Cys Ser Pro Asp Leu Leu Phe Phe Leu Cys 
75 80 85 

GCC ATG TAC GCG CCC ATC TGC ACC AH GAC HC CAG CAC GAG CCC ATC 522 
Ala Met Tyr Ala Pro He Cys Thr He Asp Phe Gtn His Glu Pro lie 
90 95 100 105 

AAG CCC TGT AAG TCT GTG TGC GAG CGG GCC CGG CAG GGC TGT GAG CCC 570 
Lys Pro Cys Lys Ser Val Cys Glu Arg Ala Arg "Gin Gly Cys Glu Pro 
110 115 120 

ATA CTC ATC AAG TAC CGC CAC TCG TGG CCG GAG AAC CTG fiCC TGC GAG 618 
lie Leu He Lys Tyr Arg His Ser Trp Pro Glu Asn Leu Ala Cys Glu 
125 130 135 



PCT/U$97/18362 

-Jo- 



GAG CTG CCA GTG TAC GAC AGG GGC GTG TGC ATC TCT CCC GAG GCC ATC 
Glu Leu Pro Val Tyr Asp Arg Gly Val Cys lie Ser Pro Glu Ala lie 



666 



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-36- 



PCTAJS97/18362 



140 145 150 

GH ACT GCG GAC GGA GCT GAT HT CCT ATG GAT TCT AGT AAC GGA AAC 714 
Val Thr Ala Asp Gly Ala Asp Phe Pro Met Asp Ser Ser Asn Gly Asn 
155 160 165 

TGT AGA GGG GCA AGO AGT GAA CGC TGT AAA TGT AAG CCT AH AGA GCT 762 
Cys Arg Gly Ala Ser Ser Glu Arg Cys Lys Cys Lys Pro lie Arg Ala 
170 175 180 185 

ACA CAG AAG ACC TAT TTC CGG AAC AAT TAC AAC TAT GTC AH CGG GCT 810 
Thr Gin Lys Thr Tyr Phe Arg Asn Asn Tyr Asn Tyr Val lie Arg Ala 
190 195 200 

AAA on AAA GAG ATA AAG ACT AAG TGC CAT GAT GTG ACT GCA GTA GTG 858 
Lys Val Lys Glu lie Lys Thr Lys Cys His Asp Val Thr Ala Val Val 
205 210 215 

GAG GTG AAG GAG AH CTA AAG TCC TCT CTG GTA AAC ATT CCA CGG GAC 906 
Glu Val Lys Glu He Leu Lys Ser Ser Leu Val Asn lie Pro Arg Asp 
220 225 230 

ACT GTC AAC CTC TAT ACC AGC TCT GGC TGC CTC TGC CCT CCA CTT AAT 954 
Thr Val Asn Leu Tyr Thr Ser Ser Gly Cys Leu Cys Pro Pro Leu Asn 
235 240 245 

GTT AAT GAG GAA TAT ATC ATC ATG GGC TAT GAA GAT GAG GAA CGT TCC 1002 
Val Asn Ghi Ghi Tyr lie lie Met Gly Tyr Glu Asp Glu Glu Arg Ser 
250 255 260 265 

AGA TTA CTC TTG GTG GAA GGC TCT ATA «CT GAG AAG TGG AAG GAT CGA 1050 
Arg Leu Leu Leu Val Glu Gly Ser lie Ala Glu Lys Trp Lys Asp Arg 
270 275 280 



CTC GGT AAA AAA GTT AAG CGC TGG GAT ATG AAG CH CGT CAT CH GGA 
Leu Gly Lys Lys Val Lys Arg Trp Asp Met Lys Leu Arg His Leu Gly 



1098 



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-37- 



PCTAJS97/18362 



285 290 295 

CTC AGT AAA AGT GAT TCT AGC AAT AGT GAT TCC ACT CAG AGT CAG AAG 1 146 
Leu Ser Lys Ser Asp Ser Ser Asn Ser Asp Ser Thr Gin Ser Gin Lys 
300 305 310 

TCT GGC AGG AAC TCG AAC CCC CGG CAA GCA CGC AAC TAAATCCCGA AATACA 1198 
Ser Gly Arg Asn Ser Asn Pro Arg Gin Ala Arg Asn 
315 320 325 

AAAAGTAACA CAGTGGACTT CCTAHAAGA CnACHGCA HGCTGGACT AGCAAAGGAA 1258 
AATTGCACTA nGCACATCA TAHCTAHG TTTACTATAA AAATCATGTG ATAACTGAH 1 318 
ATTACnCTG TTTCTCTTn GGHTCTGCT TCTCTCTTCT CTCAACCCCT HGTAATGGT 1 378 
TTGGGGGCAG ACTCHAAGT ATAHGTGAG TTTTCTATTT CACTAATCAT GAGAAAAACT 1438 
GTTCTTTTGC AATAATAATA AAHAAACAT GCTGHAAAA AAAAAA 1484 

(2) INFORMATION FOR SEQ ID N0:4: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 325 amino acids 

(B) TYPE: aniino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: Gnear 

(ii) MOLECULE TYPE: protein 
(v) FRAGMENT TYPE: internal 

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4: 

Met Val Cys Gly Ser Pro Gly Gly Met Leu Leu Leu Arg Ala Gly Leu 
1 5 10 15 

Leu Ala Leu Ala Ala Leu Cys Leu Leu Arg Val Pro Gly Ala Arg Ala 

20 25 30 

Ala Ala Cys Glu Pro Val Arg lie Pro Leu Cys Lys Ser Leu Pro Trp 

35 40 45 

Asn Met Thr Lys Met Pro Asn His Leu His His Ser Thr Gin Ala Asn 



wo 98/16641 PCTAJS97/18362 

•38- 

50 55 60 

Ala lie Leu Ala lie Glu Gin Phe Glu Gly Leu Leu Gly Thr His Cys 
65 70 75 80 

Ser Pro Asp Leu Leu Phe Phe Leu Cys Ala Met Tyr Ala Pro lie Cys 

85 90 95 

Thr He Asp Phe Gin His Glu Pro He Lys Pro Cys Lys Ser Val Cys 

100 105 110 

Glu Arg Ala Arg Gin Gly Cys Glu Pro lie Leu He Lys Tyr Arg His 

115 120 125 

Ser Trp Pro Gtu Asn Leu Ala Cys Glu Glu Leu Pro Val Tyr Asp Arg 

130 135 140 

Gly Val Cys He Ser Pro Glu Ala He Val Thr Ala Asp Gly Ala Asp 
145 150 155 160 

Phe Pro Met Asp Ser Ser Asn Gly Asn Cys Arg Gly Ala Ser Ser Glu 

165 170 175 

Arg Cys Lys Cys Lys Pro He Arg Ala Thr Gin Lys Thr Tyr Phe Arg 

180 185 190 

Asn Asn Tyr Asn Tyr Val He Arg Ala Lys Vat Lys Glu He Lys Thr 

195 200 205 

Lys Cys His Asp Val Thr Ala Val Val Glu Val Lys Gtu He Leu Lys 

210 215 220 

Ser Ser Leu Val Asn He Pro Arg Asp Thr Val Asn Leu Tyr Thr Ser 
225 230 235 240 

Ser Gly Cys Leu Cys Pro Pro Leu Asn Val Asn Gtu Gtu Tyr He He 

245 250 255 

Met Gly Tyr Glu Asp Glu Glu Arg Ser Arg Leu Leu Leu Val Glu Gly 

260 265 270 

Ser He Ala Gtu Lys Trp Lys Asp Arg Leu Gly Lys Lys Val Lys Arg 

275 280 285 

Trp Asp Met Lys Leu Arg His Leu Gly Leu Ser Lys Ser Asp Ser Ser 

290 295 300 

Asn Ser Asp Ser Thr Gin Ser Gin Lys Ser Gly Arg Asn Ser Asn Pro 
305 310 315 320 

Arg Gin Ala Arg Asn 
325 



wo 98/16641 

(2) INFORMATION FOR SEQ ID N0:5: 



.39- 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 111 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5: 

Cys Gin Pro lie Ser lie Pro Leu Cys Thr Asp lie Ala Tyr Asn Gin 
1 5 TO 15 

Thr He Met Pro Asn Leu Leu Gly His Thr Asn Gin Glu Asp Ala G!y 

20 25 3D 

Leu Glu Val His Gin Phe Tyr Pro Leu Val Lys Val Gin Cys Ser Ala 

35 40 45 

Glu Leu Lys Phe Phe Leu Cys Ser Met Tyr Ala Pro Val Cys Thr Val 

50 55 60 

Leu Glu Gin Ala Leu Pro Pro Cys Arg Ser Leu Cys Glu Arg Ala Gin 
65 70 75 80 

Gly Cys Glu Ala Leu Met Asn Lys Phe Gly Phe Gin Trp Pro Asp Thr 

85 90 95 

Leu Lys Cys Glu Lys Phe Pro Val His Gly Arg Gly Glu Leu Cys 
100 105 110 

(2) INFORMATION FOR SEQ ID N0:8: 

Ii) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 111 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 



wo 98/16641 PCTAJS97/18362 

40- 

fxi) SEQUENCE DESCRIPTION: SEQ ID N0:6: 

Cys Glu Pro lie Thr lie Ser lie Cys Lys Asn lie Pro Tyr Asn Met 
1 5 10 15 

Thr lie Met Pro Asn Leu He Gly His Thr Lys Gin Glu Glu Ala Gly 

20 25 30 

Leu Glu Val His Gin Phe Ala Pro Leu Val Lys lie Gly Cys Ser Asp 

35 40 45 

Asp Leu Gin Leu Phe Leu Cys Ser Leu Tyr Val Pro Val Cys Thr lie 

50 55 60 

Leu Glu Arg Pro lie Pro Pro Cys Arg Ser Leu Cys Glu Ser Ala Arg 
65 70 75 80 

Val Cys Glu Lys Leu Met Lys Thr Tyr Asn Phe Asn Trp Pro Glu Asn 

85 90 95 

Leu Glu Cys Ser Lys Phe Pro Val His Gly Gly Glu Asp Leu Cys 
100 105 110 

(2) INFORMATION FOR SEQ ID N0:7: 

(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 319 amino acids 
IB) TYPE: amino acid 
(C) STRANDEDNESS: single 
(0) TOPOLOGY: linear 



(xO SEQUENCE DESCRIPTION: SEQ ID N0:7: 

Met Ser Pro Thr Arg Lys Leu Asp Ser Phe Leu Leu Leu Val lie Pro 
1 5 10 15 

Gly Leu Val Leu Leu Leu Leu Pro Asn Ala Tyr Cys Ala Ser Cys Glu 

20 25 30 

Pro Va! Arg lie Pro Met Cys Lys Ser Met Pro Trp Asn Met Thr Lys 

35 40 45 

Met Pro Asn His Leu His His Ser Thr Gin Ala Asn Ala lie Leu Ala 
50 55 60 



wo 98/16641 , , PCT/US97/18362 

41- 

lie Glu Gin Phe 6lu Gly Leu Leu Thr Thr Gtu Cys Ser Gin Asp Leu 
65 70 75 80 

Leu Phe Phe Leu Cys Ala Met Tyr Ala Pro He Cys Thr lie Asp Phe 

85 90 95 

Gin His Glu Pro He Lys Pro Cys Lys Ser Val Cys Glu Arg Ala Arg 

100 105 110 

Ala Gly Cys Glu Pro lie Leu lie Lys Tyr Arg His He Trp Pro Glu 

115 120 125 

Ser Leu Ala Cys Glu Glu Leu Pro Val Tyr Asp Arg Gly Val Cys lie 

130 135 140 

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

Asp Phe Pro Met Asp Ser Asn Asn Gty Asn Cys Gly Ser Thr Ala Gly 

165 170 175 

Glu His Cys Lys Cys Lys Pro Met Lys Ala Ser Gin Lys Thr Tyr Leu 

180 185 190 

Lys Asn Asn Tyr Asn Tyr Val lie Arg Ala Lys Val Lys Glu Val Lys 

195 200 205 

Val Lys Cys His Asp Ala Thr Ala He Val Glu Val Lys Glu lie Leu 

210 215 220 

Lys Ser Ser Leu Val Asn lie Pro Lys Asp Thr Val He Leu Tyr Thr 
225 230 235 240 

Asn Ser Gly Cys Leu Cys Pro Gin Leu Val Ala Asn Glu Glu Tyr He 

245 250 255 

lie Met Gly Tyr Glu Asp Lys Glu Arg Thr Arg Leu Leu Leu Val Glu 

260 265 270 

Gly Ser Leu Ala Glu Lys Trp Arg Asp Arg Leu Ala Lys Lys Val Lys 

275 280 285 

Arg Trp Asp Gin Lys Leu Arg Arg Pro Arg Lys Ser Lys Asp Pro Val 

290 295 300 

Ala Pro lie Pro Asn Lys Asn Ser Asn Ser Arg Gin Ala Arg Ser 
305 310 315 

(2) INFORMATION FOR SEQ ID N0:8: 



(1) SEQUENCE CHARACTERISTICS: 



wo 98/16641 



42- 



(A) LENGTH: 318 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



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

Met Val Cys Gly Ser Gly Gly Met Leu Leu Leu Ala Gty Leu Leu Ala 
1 5 10 15 

Leu Ala Ala Leu Leu Leu Arg Val Pro Gly Ala Arg Ala Ala Ala Cys 

20 25 30 

Glu Pro Val Arg lie Pro Leu Cys Lys Ser Leu Pro Trp Asn Met Thr 

35 40 45 

Lys Met Pro Asn His Leu His His Ser Thr Gin Ala Asn Ala lie Leu 

50 55 60 

Ala lie Glu Gtn Phe Glu Gly Leu Leu Gly Thr His Cys Ser Pro Asp 
65 70 75 80 

Leu Leu Phe Phe Leu Cys Ala Met Tyr Ala Pro lie Cys Thr lie Asp 

85 90 95 

Phe Gin His Glu Pro lie Lys Pro Cys Lys Ser Val Cys Glu Arg Ala 

100 105 110 

Arg Gin Gly Cys Glu Pro lie Leu lie Lys Tyr Arg His Ser Trp Pro 

115 120 125 

Giu Ser Leu Ala Cys Glu Glu Leu Pro Val Tyr Asp Arg Gly Val Cys 

130 135 140 

lie Ser Pro Glu Ala lie Val Thr Ala Asp Gly Ala Asp Phe Pro Met 
145 150 155 160 

Asp Ser Ser Asn Gly Asn Cys Arg Gly Ala Ser Ser Glu Arg Cys Lys 

165 170 175 

Cys Lys Pro Arg Ala lie Gin Lys Thr Tyr Phe Arg Asn Asn Tyr Asn 

180 185 190 

Tyr Val lie Arg Ala Lys Val Lys Glu He Lys lie Lys Cys His Asp 

195 200 205 

Val Thr Ala Val Val Gtu Val Lys Glu lie Leu Lys Ser Ser Leu Val 
210 215 220 



wo 98/16641 

-43- 

Asn He Pro Arg Asp Thr Val Asn Leu Tyr Thr Ser Ser Gly Cys Leu 
225 230 235 240 

Cys Pro Pro Leu Asn Val Asn Glu Glu Tyr lie lie Met Gly Tyr Glu 

245 250 255 

Asp Glu Glu Arg Ser Arg Leu Leu Leu Val Glu Gly Ser lie Ala Glu 

260 265 270 

Lys Trp Lys Asp Arg Leu Gly Lys Lys Val Lys Arg Trp Asp Met Lys 

275 280 285 

Leu Arg His Leu Gly Leu Ser Asp Ser Ser Ser Asp Ser Thr Gin Ser 

290 295 300 

Gin Lys Pro Gly Arg Asn Ser Asn Ser Arg Gin Ala Arg Asn 
305 310 315 

(2) INFORMATION FOR SEQ ID N0:9: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 30 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ 10 NOrO: 

Ghi Thr Val Asn Leu Tyr Thr Ser Ala Gly Cys Leu Cys Pro Pro Leu 
15 10 15 

Asn Val Asn Glu Glu Tyr Leu lie Met Gly Tyr Glu Phe Pro 
20 25 30 

121 INFORMATION FOR SEQ ID N0:10: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 21 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: singfe 



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PCT/US97/18362 



(D) TOPOLOGY: linear 
in) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:10: 
6ARACHGTSA AYCTBTAYAC N 21 
(2) INFORMATION FOR SEQ ID N0:11: 

(1) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 18 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: Knear 

(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:11: 
RAAYTCRTAN CCCATNAT 18 
12) INFORMATION FOR SEQ ID N0:12: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 19 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ix) FEATURE: 

(A) NAME/KEY: Other 

(B) LOCATION: 13...13 

ID) OTHER INFORMATION: Aspartic Acid or Histidin 



wo 98/16641 



45- 



PCT/US97/18362 



(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12: 

Gly Val Cys lie Ser Pro Glu Ala lie Val Thr Ala Xaa Gly Ala Asp 
1 5 10 15 

Phe Pro Met 



(2) INFORMATION FOR SED ID N0:13: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 9 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 
ID) TOPOLOGY: Knear 



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

Gbi Gly Cys Glu Pro lie Leu He Lys 
1 5 

(2) INFORMATION FOR SEQ ID N0:14: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 15 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14: 

Gin Gly Cys Glu Pro He Leu He Cys Ala Trp Pro Pro Leu Tyr 
15 10 15 



(2) INFORMATION FOR SEQ ID N0:15: 



wo 98/16641 



46- 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 28 amino acids 

(B) TYPE: amino acid 

(C) STRAI\IDEDNESS: single 
ID) TOPOLOGY: linear 

(xi) SEQUENCE DESCRIPTION: SEQ 10 N0:15: 

Glu Thr Val Asn Leu Tyr Thr Ser Ala Gly Cys Leu Cys Pro Pro Leu 
1 5 10 15 

Asn Val Asn Glu Glu Tyr Leu He Met Gly Tyr Glu 
20 25 

(2) INFORMATION FOR SEQ ID N0:16: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 28 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16: 

Glu Thr Val Asn Leu Tyr Thr Ser Ser Gly Cys Leu Cys Pro Pro Leu 
1 5 10 15 

Asn Val Asn Glu Glu Tyr Leu lie Met Gly Tyr Glu 
20 25 

(2) INFORMATION FOR SEQ 10 N0:17: 

|i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 30 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 



wo 98/16641 



•47- 



PCTAJS97/18362 



(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: cDNA 

(xi) SEOUENCE DESCRIPTION: SEQ ID N0:17: 

BCTCTGGCTG CCTGTGTCCT CCACTTAACG 30 

(2) INFORMATION FOR SEQ ID N0:18: 

(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
IB) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
ID) TOPOLOGY: Bnear 



(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18: 
CCTCCACHA ACGHAATGA GGAGTATCTC 30 
(2) INFORMATION FOR SEQ ID N0:19: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 21 base pairs 

(B) TYPE: nucleic acid 

(C) STRANOEDNESS: single 

(D) TOPOLOGY: linear 

(xi) SEOUENCE DESCRIPTION: SEQ ID N0:19: 
T6GAACAT6A CTAAGATGCC C 21 



(2) INFORMATION FOR SEQ ID N0:20: 



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PCT/US97/18362 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 20 base pairs 

(B) TYPE: nucleic acid 

10 STRANDEDNESS: single 
(D) TOPOLOGY: Enear 

Ixi) SEQUENCE DESCRIPTION: SEQ ID N0:20: 
CATATACTGG CAGCTCCTCG 20 
(2) INFORIMATION FOR SEQ ID N0:21: 

(il SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 22 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(xQ SEQUENCE DESCRIPTION: SEQ ID N0:21: 
GTCTTTTGGG AAGCCTTCAT GG 22 
(2) INFORMATION FOR SEQ ID N0:22: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 22 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(xi) SEQUENCE DESCRIPTION: SEQ ID NQ:22: 



GCATCGTGGC ATHCACTTT CA 



22 



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49- 



PCT/US97/18362 



(2) INFORMATION FOR SEG ID N0:23: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1291 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



(xi) SEQUENCE OESCRIPTION: SEQ ID N0:23: 



TTTACTGTGC CAGTCHCCC TGTAACCAGC GACCTGTAH CCCCCAAGTA AGCCTACACA 60 

TACAGGTTGG GCAGAATAAC AATGTCTCCA ACAAGGAAAT TGGACTCAn CCTGCTACTG 1 20 

GTCATACCTG GACTGGTGCT TCTCTTAHA CCCAATGCH ACTGT€CnC GTGTGAGCCT 180 

GTGCGGATTC CCATGTGCAA ATCTATGCCA TGGAACATGA CCAAGATGCC CAACCATCTC 240 

CACCACAGCA CTCAAGCCAA TGCTATCCTG GCAAnGAAC AGrfTGAAGG TTTGCTGACC 300 

ACTGAATGTA GCCAGGACCT rFTGnCTTT CTGTGTGCCA TGTATGCCCC CATTTGTACC 360 

ATCGATnCC AGCATGAACC AAHAAGCCT TGCAAGTCCG TGTGCGAAAG GGCCAGGGCC 420 

GGCTGTGAGC CCATTCTCAT AAAGTACCGG CACACTTGGC CAGAGAGCCT GGCATGTGAA 480 

GAGCTGCCCG TATATGACAG AGGAGTCTGC ATCTCCCCAG ACGCTATCGT CACAGTGGAA 540 

CAA6GAACAG AHCAATGCC AGACHCCCC ATGGAHCAA ACAATG6AAA HGCGGAAGC 600 

ACGGCAGGTG AGCACTGTAA ATGCAAGCCC ATGAAGGCH CCCAAAAGAC 6TATCTCAAG 660 

AATAAnACA AHATGTAAT CAGAGCAAAA GTGAAAGAGG TGAAAGTGAA ATflCCACGAC 720 

GCAACAGCAA HGTGGAAGT AAA6GAGATT CTCAAGTCH CCCTAGTGAA CATTCCTAAA 780 

GACACAGTGA CACTGTACAC CAACTCAGGC TGCTTGTGCC CCCAGCTTGT TGCCAATGAG 840 

6AATACATAA HATGGGCTA TGAAGACAAA 6AGCGTACCA GGCHCTACT A6T6GAA6GA 900 

TCCHGGCCG AAAAATG6AG AGATCGTCH 6CTAAGAAAG TCAAGC^TG 6GATCAAAAG 960 

CnCGACGTC CCAG6AAAAG CAAAGACCCC GTG6CTCCAA HCCCAACAA AAACAGCAAT 1020 

TCCAGACAAG CGCGTAGTTA 6ACTAACGGA AAGGTGTATG 6AAACTCTAT GGACHTGAA 1080 

ACTAAGATH GCATTGHGG AAGAGCAAAA AAGAAATTGC ACTACAGCAC fiHATAHCT 1 140 

ATTGTrTACT ACAAGAAGCT GGTHAGHG AHGTAGHC TCCTnCCTT CnTTTTrTA 1200 

TAACTATATT GCACGTGHC CAGGCAGIFT ATCAACTTCC AGTCACAGAG CAGTGACTGA 1260 
ATGTAGCTAA GAGCCTATCA TCTGATCACT A 1291 



wo 98/16641 PCT/US97/18362 

•50- 

WHAT IS CLAIMED IS: 

1. An isolated polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1, 3 or 23. 

2. An Isolated polynucleotide encoding a native Frzb protein, said polynucleotide capable of hybridizing 
to a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1 at SS^'C in 3 x SSC, 0.1% SOS. 

5 3. An isolated Frzb protein encoded by the polynucleotide of Claim 2. 

4. An isolated recombinant Frzb protein having the amino acid sequence shown in SEQ ID NO: 2, 4 

or 7. 

5. The isolated Frzb protein of Claim 4, wherein at least one acidic amino acid contained therein is 
replaced with a different acidic amino acid. 

10 6. The isolated Frzb protein of Claim 4, wherein at least one basic amino acid contained therein is 

replaced with a different basic amino acid. 

7. The isolated Frzb protein of Claim 4, wherein at least one nonpolar amino acid contained therein 
is replaced with a different nonpolar amino acid. 

8. The isolated Frzb protein of Claim 4, wherein at least one uncharged polar amino acid contained 
15 therein is replaced with a different uncharged polar amino acid. 

9. The isolated Frzb protein of Claim 4, wherein at least one aromatic amino acid contained therein 
is replaced with a different aromatic amino acid. 

10. The protein having the amino acid sequence shown in the SEQ ID NO: 2 of Claim 4, wherein said 
protein is obtained by expression of a polynucleotide having the sequence shown in SEQ ID NO: 1. 

20 11. The protein having the amino acid sequence shown in the SEQ ID NO: 4 of Claim 4, wherein said 

protein is obtained by expression of a polynucleotide having the sequence shown in SEQ ID NO: 3. 

12. The protein having the amino acid sequence shown in the SEQ ID NO: 7 of Claim 3, wherein said 
protein is obtained by expression of a polynucleotide having the sequence shown in SEQ ID NO: 23. 

13. Isolated mammalian Frzb protein having a molecular weight of about 38 kilodaltons. 

25 14. A pharmaceutical composition for inducing cartilage, bone, nerve or muscle growth comprising the 

isolated Frzb protein of Claim 3, or a Frzb protein having the amino acid sequence shown in SEQ ID NO: 2, 4 or 7, 
in a pharmaceuticaily acceptable carrier. 

15. The composition of Claim 6, wherein said carrier comprises fibrin glue, freeze-dried cartilage grafts 
or collagen. 

30 16. The composition of Claim 7, wherein said composition further comprises cartilage progenitor cells, 

chondroblasts or chondrocytes. 

17. The composition of Claim G, wherein said Frzb protein is coated onto or mixed with a resorbable 
or nonresorbable matrix. 

18. The composition of Claim 6, wherein said Frzb protein is mixed with a biodegradable polymer. 



wo 98/16641 PCT/US97/18362 

•5 1- 

19. A method of treating a cartilage, bone, nerve or muscle growth disorder in a mammal in need 
thereof, comprising the step of administering to said mammal an effective cartilage, bone, nerve or muscle-inducing 
amount of the pharmaceutical composition of Claim 6 at the site of said disorder. 

20. The method of Claim 19, wherein said disorder is selected from the group consisting of subglottic 
stenosis, tracheomalacia, chondromalacia patellae, osteoarthritis, joint surface lesions, neurodegenerative disorders, 
myodegenerative disorders and osteodegenerathre disorders. 

21. The method of Claim 19, wherein said administering step is intravenous, intrathecal, intracranial, 
intramuscular or subcutaneous. 

22. The method of Claim 19, wherein said mammal is a human. 

23. A method of stimulating cartilage formation in a mammal, comprising the steps of: 
combining the isolated Frzb protein of Claim 3, or a Frzb protein having the amino acid sequence 

shown in SEQ ID NO: 2, 4 or 7, with a matrix to produce a product that facilitates administration of said 
protein; and 

implanting said product into the body of a mammal to stimulate cartilage formation at the site of 
implantation. 

24. The method of Claim 23, wherein said matrix comprises a cellular material. 

25. The method of Claim 23, wherein said combining step additionally comprises mixing of viable 
chondroblasts or chondrocytes. 

26. The method of Claim 23, wherein said implanting is subcutaneous or intramuscular. 

27. The method of Claim 23, wherein said mammal is a human. 

28. A method of modulating Wnt-mediated signaling in a cell, comprising contacting said cell with an 
effectwe Wnt modulating amount of the isolated Frzb protein of Claim 3, a Frzb protein having the amino acid 
sequence shown in SEQ ID NO: 2, 4 or 7, or a Wnt-modulating fragment thereof. 

29. The method of Claim 28, wherein said cell is contacted in v'm, 

30. The method of Claim 28, wherein said Wnt is selected from the group consisting of Wnt-8, Wnt-1, 
Wnt-2, Wnt-3, Wnt-4, Wnt-5A, Wnt-5B, Wnt-6, Wnt-7A and Wnt-7B. 

31. A method of modulating Wnt-mediated signaling in a cell, comprising contacting said cell with a 
recombinant construct comprising the coding region of SEQ ID NO: 1, 3 or 23, or a region encoding an active Wnt- 
modulating fragment thereof, operably linked to a heterologous promoter in an expression vector. 

32. The method of Claim 31, wherein said Wnt is selected from the group consisting of Wnt-8, Wnt-1, 
Wnt-2, Wnt.3, Wnt-4, Wnt-5A, Wnt-5B, Wnt-6, Wnt-7A and Wnt-7B. 



wo 98/16641 PCT/US97/18362 

•52- 

33. A method of inhibiting the growth of a Wnt growth factor-expressing tumor in a mammal, 
comprising the step of contacting said tumor with an effective tumor growth-inhibiting amount of the isolated Frzb 
protein of Claim 3, or a Frzb protein having the amino acid sequence shown in SEQ ID NO: 2, 4 or 7. 

34. The method of Claim 33, wherein said tumor is a mammary or intestinal tumor. 
5 35. The method of Claim 33, wherein said mammal is a human. 

36. A method of inhibiting the growth of a Wnt growth factor-expressing tumor in a mammal, 
comprising the step of contacting said tumor with a recombinant construct comprising the coding region of SEQ ID 
NO: 1, 3 or 23 operably linked to a heterologous promoter in an expression vector. 

37. The method of Claim 36, wherein said construct is injected into said tumor. 

10 38. The method of Claim 38, wherein said recombinant construct is systemically administered to said 

mammal. 

* 

39. The method of Claim 36, wherein said expression vector is a plasmid vector, retroviral vector or 
adenoviral vector. 

40. Isolated antibodies to Fab protein having the amino acid sequence shown in SEQ ID NO: 2, 4 or 

15 7. 

41. The antibodies of Claim 40, wherein said antibodies are polyclonal. 

42. The antibodies of Claim 40, wherein said antibodies are polyclonal. 

43. A method of facilitating tissue growth or repair, comprising the steps of: 
isolating cells from said tissue; 

20 introducing a recombinant construct expressing Frzb into said celts; and 

returning said cells to said tissue. 

44. The method of Claim 43, wherein said recombinant construct comprises a retroviral vector, 
adenoviral vector, herpesvirus vector or adeno-associated viral vector. 

45. The method of Claim 43, wherein said tissue is selected from the group consisting of cartilage, 
25 muscle, bone and neural tissue. 

46. A method of identifying a compound which affects Frzb activity, comprising: 
contacting isolated Frzb with said compound; and 

determining Frzb acthrity, wherein an increase in activity compared to Frzb atone indicates that 
said compound is a Frzb activator and a decrease in activity indicates that said compound is a Frzb 
30 inhibitor. 

47. The method of Claim 46, wherein said determining step comprises an in vivo chondrogenesis assay. 

48. An isolated Frzb-derived peptide having an amino acid sequence shown in a SEQ ID NO: selected 
from the group consisting of SEQ ID NO: 12, 13, 14, 15 and 16. 

49. A recombinant construct comprising the coding region of SEQ ID NO: 1, 3 or 23 operabty linked 
35 to a heterologous promoter in an expression vector. 

50. The recombinant construct of Claim 49, wherein said expression vector is eukaryotic. 



wo 98/16641 PCT/US97/18362 

51. The recombinant construct of Claim 49, wherein said expression vector is prokaryotic. 

52. The recombinant construct of Claim 49, wherein said expression vector is pcDNA3. 

53. A cultured mammalian cell line containing the recombinant construct of Claim 49. 

54. An isolated recombinant Frzb protein containing amino acids 33-319 of SEQ ID NO: 7, 33-325 of 
5 SEQ ID NO: 2 or 33-325 of SEQ ID NO: 4. 



wo 98/16641 PCT/US97/18362 

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INTERNATIONAL SEARCH REPORT 



International i calton No 

PCT/US 97/18362 



A. CLASSIFICATION OF SUBJECT MATTER 

IPC 6 C12N15/1Z C07K14/71 C07K14/51 C07K16/28 A61K38/18 
C12N5/10 



Acco rding to Intemationai Patent dassiftoation (IPC) or to both national otostifioation and IPC 

B. FIELDS SEARCHED 

Minimum documentation saarohed (claasificaiion system followed by classifoallon symbols) 

IPC 6 C12N C07K A61K 



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



Electronic date base consulted during the tntemational search (name of data base and, where practical, search terms used) 



C. DOCUMENTS CONSIDERED TO BE RELEVANT 



Category 


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


Relevant to No. 


A 


WO 96 14335 A (US HEALTH ;LUYTEN FRANK P 






(US); MOOS MALCOLM JR (US); CHANG STEVEN) 






17 May 1996 






cited in the application 






see the whole document 




A 


WANG Y ET AL.: 'A large family of 






putative transmembrane receptors 






homologous to the product of the 






Drosophila tissue polarity gene frizzled" 






JOURNAL OF BIOLOGICAL CHEMISTRY, 






vol. 271. no. 8, 23 February 1996, 






pages 4468-4476. XP002O56247 






cited in the application 






see the whole document 











0 



Further documents am Bated in the continuation of trax 0. 



Patent family members are ftsted in annex. 



Special categories of cited documents : 

*A' document defining the general state of the art which is not 
considered to be of parttoular relevanoe 

'E' earlier documerrt but published on or after the international 
filing date 

'L' document which may throw doubts on priorAy claim(8) or 
which is cited to establish the publication date of another 
citation or other special reason (as specified) 

'O* document referring to an oral dtsclosure, use, exhbition or 
other means . . 

*P* document published prior to the international fifing date but 
later than the priority date claimed 



T* later document published after the intomatlonal fillrtg date 
or priority date and not in conflict with the applioatton but 
cited to understand the principle or theory underlying the 



'X* document of particular relevance; the claimed invention 
cannot bB considered novel or cannot be considered to 
invohre 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- 
mente, such combination being obvious to a person skilled 
In the art 

*&' document member of the same patent family 



Date of the aotuol oomplebon of the international search 

20 February 1998 


Date of mailing of the intemational search report 

11.03.98 


Name and mailing address of the ISA 

European Patent Offioe, P.B. 5816 Patentlaan 2 
NL • 2280 HV Rijswi^ 
Tel. (+31-70) 340-2040, Tx. 31 651 epo nl. 
Fax: (+31-70) 340-3016 


Authorized offioer 

Oderwald. H 



Forni PCT/ISA/210 (second sheet) (July 1992) 



page 1 of 2 



INTERNATIONAL SEARCH REPORT 



International , 



'cation No 



PCT/US 97/18362 



C.(Conllnuatjon) DOCUMENTS CONSIDERED TO BE RELEVANT 


Category ^ 


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


Relevant to claim No. 


A 


LUYTEN F P ET AL: "PURIFICATION AND 
PARTIAL AMINO ACID SEQUENCE OF OSTEOGENIN, 
A PROTEIN INITIATING BONE DIFFERENTIATION" 
JOURNAL OF BIOLOGICAL CHEMISTRY, 
vol. 264, no. 23, 15 August 1989, 
pages 13377-13380, XPG0OO35793 
cited in the application 
see the whole document 




P.X 


HOANG B ET AL: "Primary structure and 
tissue distribution of FRZB, a novel 
protein related to Drosophila frizzled, 
suggest a role in skeletal morphogenesis." 
JOURNAL OF BIOLOGICAL CHEMISTRY, (1996 OCT 
18) 271 (42) 26131-7. JOURNAL CODE: HIV. 
ISSN: 0021-9258., XP0e2056248 
see the whole document 


1-54 


P.X 


MAYR T ET AL.: "Human Fritz mRNA, 
complete cds." 
EMBL SEQUENCE DATABASE, 
3 April 1997, HEIDELBERG, GERMANY, 
XP0O2056439 
see the whole document 


2 


P.X 


MAYR T ET AL.: "Mus musculus Fritz (mfiz) 
mRNA, complete cds." 
EMBL SEQUENCE DATABASE, 
3 April 1997, HEIDELBERG, GERMANY, 
XP002O5644O 
see the whole document 


2 


P.X 


RATTNER A ET AL.: "A family of secreted 
proteins contains homology to the 
cysteine-rich ligand-binding domain of 
frizzled receptors" 

PROCEEDINGS OF THE NATIONAL ACADEMY OF 

SCIENCES OF THE UNITED STATES OF AMERICA, 

vol. 94, April 1997, 

pages 2859-2863, XP002056441 

see abstract; figure 1 

see page 2859, paragraph 6 - page 2860, 

paragraph 1 

see page 2860, paragraph 5 - paragraph 7 


2 


E 


WO 97 48275 A (UNIV CALIFORNIA) 24 

December 1997 

see the whole document 


1-9,11, 
13-54 


T 


WANG S ET AL: "Frzb, a secreted protein 

CApVCbaCU III LIIC OpCillOIIII Uiyaill^Ci , UlTiUS 

and inhibits Wnt-8." 

CELL, (1997 MAR 21) 88 <6) 757-66. JOURNAL 
CODE: CQ4. ISSN: 0092-8674.. XPOO2056442 
see the whole document 


28-39 



Form PCT/ISA/210 (continuation of second Bheet) (Juty 1992) 



page 2 of 2 



INTERNATIONAL SEARCH REPORT 



tntematio. ipplicatton No. 

PCT/US 97/18362 



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 ol certain claims under Article 17(2)(a) for the following reasons: 
1. [x] Claims Nos.; 

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

see FURTHER INFORMATION sheet PCT/ISA/21G 



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. Claims f^.: 

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

Box II Observations where unity of Invention Is lacking (Continuation of item 2 of first sheet) 

This International Searching Authority found multiple inventions in this international application, as follows: 



1 . I I As all required additional search fees were timely paid by the applicant, the International Search Report covers all 

J searchatie claims. 

2. I I As all search at)le 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 adcfitional search fees were timely paid t>y the applicant, this Intemattonat Search Report 
' ' covers only those claims for which fees were paid, specifically claims Nos.: 



4. I I Nn required additional search fees were timely paid by the applicant. Consequently, this International Search Report is 
restricted to the 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 j No protest accompanied the paymerrt of additional search fees. 

Form PCT/lSA/21 0 (continuation of first sheet (1 )} (July 1 992) 



INTERNATIONAL SEARCH REPORT 



International Application No. PCT/US 97/18352 



FURTHER INFORMATION CONTINUED FROM PCT/ISA/ 210 



Remark : Although claims 19-39 are directed to a method of treatment of 
the human/animal body , the search has been carried out and based on the 
alleged effects of the compound/composition. 



INTERNATIONAL SEARCH REPORT 

tnformatiun on patent family members 



International . 



Icatlon No 



PCT/US 97/18362 



Patent docuirent 
cited in search report 



Publication 
date 



Patent family 
nnember(8) 



Putilication 
date 



WO 9614335 A 
WO 9748275 A 



17-05-96 
24-12-97 



AU 1120295 A 
NONE 



31-05-96 



Fofm PCT/lSA/210 (palent lanity annex) tJuty 1992) 



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