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PCT 



WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCI) 



(51) International Patent Classification 5 : 

C07K 15/06, 3/02, 3/20, 13/00, A61K 35/12 
A61K 37/12, A01N 63/02, A23J 1/10 



Al 



(11) International Publication Number: WO 91/02744 

(43) Internationa] Publication Date: 7 March 1991 (07.03.91) 



(21) International Application Number : PCT/US90/04745 

(22) International Filing Date: 21 August 1990 (21.08.90) 



(30) Priority data: 
397,779 



21 August 1989 (21.08.89) US 



(60) Parent Application or Grant 

(63) Related by Continuation 
US 

Filed on 



397,779 (CIP) 
21 August 1989(21.08.89) 



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

LABORATORIES, INC. [US/US]; 2500 Faber Place, 
Palo Alto, CA 94303 (US). 

(72) Inventors ; and 

(75) Inventors/ Applicants (for US only) : BENTZ, Hanne [DE/ 
USJ; 36125 Toulouse Street, Newark, CA 94560 (US). 
NATHAN, Ranga [CA/US]; 6104 Robertson Avenue, 
Newark, CA 94560 (US). ROSEN, David, M. [US/USJ; 
3655 Shadyhollow Court, San Jose, CA 95148 (US). 
DASCH, James, R. [US/US]; 3181 Morris Drive, Palo 
Alto, CA 94303 (US). SEYEDIN, Saeid, M. IIR/US]; 
20761 Lowena Court, Saratoga, CA 95070 (US). PIEZ, 
Karl, A. [US/US]; 1120 Bay Laurel Drive, Menlo Park, 
CA 94025 (US). OGAWA, Yasushi [US/US]; 310 Faral- 
Ion Avenue, Pacifica, CA 94044 (US). ANDREWS, Wil- 
liam, H. [US/US]; 780 Fathom Drive, San Mateo, CA 
94404 (US). STEPHENSON, Frank, H. [US/US]; 710 



Moraga, Apartment C, San Francisco, CA 94122 (US). 
HEDGPETH, Joel [US/US]; 200 Cresta Vista, San 
Francisco, CA 94127 (US). 

(74) Agents: CIOTTI, Thomas, E. et al.; Irell & Manella, 545 
Middlefield Road, Suite 200, Menlo Park, CA 94025 
(US). 

(81) Designated States: AT (European patent), AU, BE (Euro- 
pean patent), CA, CH (European patent), DE (Euro- 
pean patent)*, DK (European patent), ES (European pa- 
tent), FR (European patent), GB (European patent), IT 
(European patent), JP, LU (European patent), NL (Eu- 
ropean patent), SE (European patent), US. 

Published 

With international search report. 

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



(54) Title: BONE-SPECIFIC PROTEIN 



(H,N)-Ala-Lys-Tyr-Asn-Lys-Ile-Lys-Ser-Arg-Gly- 
12 
Ile-Lys-Ala-Asn-R -Phe-Lys-Lys-Leu-R - 

Asn-Leu-R^-Phe-Leu-Tyr-Leu-Asp-His-Asn- 

Ala-Leu-Glu-Ser-Val-Pro-Leu-Asn-Leu-Pro- 

Glu-Ser-teu-Arg-val-Ile-His-Leu-Gln-Phe- 
4 

Asn-Asn- I le-R -Ser- Ile-Thr-Asp-Asp-Thr- 
Phe-Cys-Lys-Ala-Asn-Asp-Thr-Ser-Tyr-Ile- 
Arg-Asp-Arg- I le-Glu-Glu-I le-Arg-Leu-Glu- 
Gly-Asn-Pro-R 5 -R 6 -Leu-Gly-Lys-His-Pro- 
Asn-Ser-Phe-Ile-Cys-Leu-Lys-Arg-Leu-Pro- 
Ue-Gly-Ser-Tyr-R - ( COOH ) , 



(57) Abstract 



ti) 



DEMORALIZED BONE POWDER (0.5 N HCL) 



4 M GUANIDINE HYDROCHLORIDE EXTRACT 



SEPHACRYLS - 200 (20 - 36KD MW) 



CM - CELLULOSE (10- I 50 mM NaCL) 



C0NCANAVALIN - A (0.5 M METHYL OC - D - MANNOPYRANOSIDE) 



HEPARIN - SEPHAROSE (0. 1 - 0.5 M NaCL) 



CIS- REVERSED PHASE - HPLC (42 - 45% ACETONOTRILE) 



Proteins, having sequence (I) where R 1 is Ala ou Thr, R2 is Asn or His, R 3 is Thr or Ser, R 4 is Ala or Thr, R 5 is He or 
Val, R6 is Val or He and R 7 is Phe or He, is disclosed. These proteins are present in osteoblasts and osteocytcs and may be 
used as markers to follow bone and/or cartilage metabolism. 



* See back of page 



DESIGNATIONS OF "DE" 



Until further notice, any designation of "DE" in any international application 
whose international filing date is prior to October 3, 1990, shall have effect in the 
territory of the Federal Republic of Germany with the exception of the territory of the 
former German Democratic Republic. 



FOR THE PURPOSES OF INFORMATION ONLY 

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



AT 


Austria 


ES 


Spain 


MC 


Monaco 


AU 


Australia 


Fl 


Finland 


MC 


Madagascar 


BB 


Barbados 


FR 


France 


ML 


Mali 


BE 


Belgium 


CA 


Gabon 


MR 


Mauritania 


BF 


Burkina Fasso 


GB 


United Kingdom 


MW 


Malawi 


BG 


Bulgaria 


CR 


Greece 


NL 


Netherlands 


BJ 


Benin 


HU 


Hungary 


NO 


Norway 


BR 


Brazil 


rr 


Italy 


PL 


Poland 


CA 


Canada 


jp 


Japan 


RO 


Romania 


CF 


Central African Kc public 


KP 


Democratic People's Republic 


SO 


Sudan 


CC 


Congo 




or Korea 


SE 


Sweden 


CH 


Switzerland 


KR 


Republic or Korea 


SN 


Senegal 


CM 


Cameroon 


LI 


Liechtenstein 


su 


Soviet Union 


DE 


Germany 


LK 


Sri Lanka 


TD 


Chad 


DK 


Denmark 


U) 


Luxembourg 


TC 


Togo 










US 


United States of America 



WO 91/02744 



PCT/US90/04745 



5 BONE-SPECIFIC PROTEIN 

Description 

Technical Field 
1° The present invention relates to protein 

chemistry. More particularly, . it relates a protein that 
occurs specifically in bone, antibodies to such proteins, 
and immunoassays for detecting such proteins. 

15 Background Art 

Others have fractionated bone in an attempt to 
identify proteins which can stimulate the formation of new 
bone when placed in contact with living systems. (Urist, 
M. R., Clin Orthop (1968) 56:37; Science (1965) j.50:893; 

20 Reddi, A. H. , et al., Proc Natl Acad Sci (USA) (1972) 
£9:1601.) A "bone morphogenic protein" (BMP) was 
extracted from demineralized bone using urea or guanidine 
hydrochloride and reprecipitated according to the 
disclosures in U.S. Patents Nos . 4 , 294 ,753 and 4,455,256 

25 to Urist. Urist subsequently reported (Urist, M. R., Clin 
Orthop Rel Res (1982) 162:219) that ion exchange 
purification of this crude protein mixture yielded an 
activity which was unadsorbed to carboxymethyl cellulose 
resin (CMC) at pH 4.8. Urist 's reports in Science (1983) 

30 220 :680-685, Proc Natl Acad Science (USA) (1984) 81:371- 
375, and U.S. Pat. No. 4,789,732 describe BMPs having 
molecular weights of 17,500 and 18,500 daltons . Urist 's 
patent publication, EPA Publication No. 0212474, describes 
BMP fragments of 4,000 to 7,000 daltons obtained by 

35 limited proteolysis of BMP. 



WO 91/02744 



PCT/US90/04745 



-2- 

U.S. Patent No. 4 , 608 , 199 describes a bone- 
derived protein of 30,000-32,000 daltons. The protein is 
described as being water soluble and having no affinity 
for concanavalin A (ConA). 
5 WO 88/00205 reports four proteins, designated 

BMP-1, BMP-2 Class I, BMP-2 Class II and BMP-3, that are 
alleged to have osteogenic activity by themselves or in 
combination with other factors. Sequences are provided 
for each of these proteins which show no homology to the 
10 sequence (see below) of the protein of the present 
invention . 

Commonly owned U.S. 4,434,094 reported the 
partial purification of a bone -generation-stimulating, 
bone-derived protein by extraction with chaotropic agents, 

15 fractionation on anion and cation exchange columns, and 
recovery of the activity from a fraction adsorbed to CMC 
at pH 4.8. This new protein fraction was termed 
"osteogenic factor" (OF) and was characterized as having a 
molecular weight below about 30,000 daltons. 

20 Commonly owned U.S. Patent No. 4,774,332 

describes two proteins that were purified to homogeneity 
using a purification procedure that is similar in part to 
that disclosed in U.S. 4,434,094. Those two proteins 
eluted from CMC at about a 150-200 mM NaCl gradient. 

25 These two proteins were originally called cartilage- 
inducing factor (CIF) A and CIF B. CIF A was subsequently 
found to be identical to a previously identified protein 
now called transforming growth factor betal (TGF-betal). 
CIF B has been found to be a novel form of TGF-beta and is 

30 now known as TGF-beta2 . These proteins and homologous 
proteins exhibiting similar activity are collectively 
referred to as TGF-beta. 

Commonly owned U.S. Patent No. 4,627,982 
concerns a partially purified bone-inducing factor present 

35 in the CMC-bound fraction of U.S. 4,434,094 that elutes in 
the portion of the NaCl gradient below that in which the 



WO 91/02744 



t 



PCT/US90/0474S 



-3- 

major portions of TGF-betal and TGF-beta2 elute (i.e., 
below about 150 mM NaCl). The present invention relates 
to the identification of an ingredient of that fraction, 

5 Disclosure of the Invention 

One aspect of the invention is a substantially 
pure polypeptide that is found in bone and has the 
following sequence: 

10 

( H~N ) -Ala-Ly s -Tyr-As n-Lys - 1 le-Lys -Ser-Arg-Gly- 

12 
Ile-Lys-Ala-Asn-R -Phe-Lys-Lys-Leu-R - 

Asn-Leu-R 3 -Phe-Leu-Tyr-Leu-Asp-His-Asn- 

Ala-Leu-Glu-Ser-Val-Pro-Leu-Asn-Leu-Pro- 

Glu-Ser-Leu-Ar g-Val - I le-His -Leu-Gln-Phe- 
4 

Asn-Asn- I le-R -Ser- I le-Thr-Asp-Asp-Thr- 
Phe-Cys-Lys-Ala-Asn-Asp-Thr-Ser-Tyr-Ile- 
Arg-Asp-Arg-Ile-Glu-Glu-Ile-Arg-Leu-Glu- 
Gly-Asn-Pro-R 5 -R 6 -Leu-Gly-Lys-His-Pro- 
Asn-Ser-Phe-Ile-Cys-Leu-Lys-Arg-Leu-Pro- 
Ile-Gly-Ser-Tyr-R 7 -(COOH) , 

12 3 
where R is Ala or Thr, R is Asn or His, R is Thr or 

Ser, R 4 is Ala or Thr, R^ is lie or Val, R^ is Val or lie 

n 

25 and R is Phe or lie, and substantially pure polypeptides 
that are and substantially homologous thereto. 

Deglycosylated analogs of the above-described 
polypeptides are another aspect of the invention. 

Further aspects of the invention are recombinant 

30 materials (i.e., recombinant DNA, recombinant vectors, and 
recombinant cells or microorganisms) and processes for 
producing the polypeptides of the invention, antibodies 
specific to the polypeptides , and immunoassays for the 
polypeptides . 

35 



15 



20 



WO 91/02744 



i 



PCT/US90/04745 



-4- 

Brief Description of the Drawings . 
In the drawings: 

Figure 1 is a flow chart of the process that was 
used to isolate the bovine species of the bone-specific 
5 protein of the invention from demineralized bovine bone. 

Figure 2 is a graph of the optical densities 
(absorbances at 280 run) of the gel filtration fractions of 
the gel filtration fractions of the example (1fC). 

Figure 3 is a graph of the optical densities 
10 (absorbances at 280 nm) of eluate fractions from the 
preparative ion exchange chromatography of the example 
(ID). 

Figure 4 is a graph of the optical densities 
(absorbances at 280 nm) of eluate fractions from the 
15 cross-linked ConA chromatography step of the example (VE); 

Figure 5 is a graph of the optical densities 
(absorbances at 280 nm) of eluate fractions from the 
heparin-sepharose chromatography step of the example (UF); 
Figure 6 is a graph of the optical densities 
20 (absorbances at 230 nm) of the gradient fractions from the 
C18-RP-HPLC chromatography step of the example '( If G) ; 

Figure 7 is a table showing results of amino 
acid sequencing of the bovine isolate of the invention and 
locations of the sequenced fragments in the overall 
25 sequence. 

Figure 8 is a photograph of an autoradiograph of 
SDS-PAGE analyses of the purified bovine protein that are 
described in the example (?H) (lanes A and C show 
glycosylated protein; lanes B and D show enzymatically 
30 deglycosylated protein) . 

Figure 9 is a schematic diagram illustrating the 
structure of the bovine gene that encodes the mature 
bovine species of the protein of the invention. Various 
restriction sites are indicated. 



WO 91/02744 



PCT/US90/0474S 



-5- 

Figure 10 is a restriction map of the region of 
the human gene that encodes the human species of the 
protein. 

Figure 11, in parts A-E, shows the DMA sequence 
5 and deduced amino acid sequence of the mature human spe- 
cies of the protein of the invention in comparison with 
the sequences of the bovine gene and protein. Part A 
shows the preliminary unconfirmed sequence of a putative 
"amino terminal" precursor exon of the human gene for the 

10 prepropolypeptide. The extent of this exon has not been 
determined. Bases of uncertain identity in the DNA 
sequence are represented by numbers. The signal sequence 
indicated in the figure is putative and was identified by 
the von Heijne algorithm. Part B shows the sequence of 

15 another precursor exon, designated -1, that is downstream 
of the amino terminal exon and upstream of the exon that 
encodes the amino terminal of the mature protein. Parts 
C-E show the sequences of exons 1-3 which encode the 
mature protein. Asterisks designate points of identity 

20 between the bovine and human DNA sequences. Amino acid 
numbering is relative to the mature sequence with the 
first amino acid thereof designated n l". Consensus 3' and 
5' exon splice sites are indicated by YYYYYYYYYYYNYAG and 
JAGGTRAGT/ respectively. 

25 Figure 12 is a schematic diagram of the 

mammalian expression vector phOIFIS which contains the 
human gene that encodes the human species of the invention 
protein . 

30 Modes of Carrying Out the Invention 

Isolation of Protein from Bone 

It is believed that the protein of the present 
invention has been highly conserved among mammalian 
35 species — i.e., corresponding proteins from different 

mammalian species (herein called "species analogs") will 



WO 91/02744 



PCT/US90/04745 



-6- 

have substantially homologous amino acid sequences that 
vary from the bovine or human proteins described herein, 
if at all, in one or more amino acid residue additions, 
deletions or substitutions and/or substantially similar 
5 glycosylation patterns. The amino acid sequences of 
"substantially homologous" proteins will usually be at 
least 50% identical, more usually at least 80% identical, 
and preferably at least 90% identical to the bovine/human 
amino acid sequence described herein. Such proteins may 

10 be derived from bone or other tissues of diverse mammalian 
origin or synthesized using recombinant DNA procedures. 
The term is intended to include muteins or analogs of the 
native protein that are altered- in manners known in the 
art, such as by substitution of cysteines with neutral 

15 (uncharged) amino acids to avoid improper disulfide 

bonding, by substitution or elimination of residues in the 
asparagine-linked glycosylation sites of the proteins to 
alter glycosylation patterns, by substitution of 
methionines to make the molecules less susceptible to 

20 oxidation, by conservative substitution of other residues, 
by chemical modification of one or more residues, by 
substitution with nonnatural amino acids or by elimination 
or alteration of side-chain sugars. The source of protein 
prepared by purification from native sources is 

25 advantageously porcine or bovine long bone because of its 
ready availability. 

The process for isolating the protein from bone 
is as follows. The bone is first cleaned using mechanical 
or abrasive techniques, fragmented, and further washed 

30 with, for example, dilute aqueous acid preferably at low 
temperature. The bone is then demineralized by removal of 
the calcium phosphates in their various forms, usually by 
extraction with stronger acid. These techniques are 
understood in the art, and are disclosed, for example, in 

35 U.S. 4,434,094. The resulting preparation, a 



WO 91/02744 



PCI7US90/04745 



-7- 

demineralized bone, is the starting material for the 
preparation of the protein from native sources. 

The initial extraction is designed to remove the 
nonfibrous (e.g. , noncollagenous ) proteins from the 
5 deminefalized bone. This can be done with the use of 
chaotropic agents such as guanidine hydrochloride (at 
least about 4 molar), urea (8 molar) plus salt, or sodium 
dodecylsulf ate (at least about 1% by volume) or such other 
chaotropic agents as are known in the art (Termine et al., 

10 J Biol Chem (1980) 255:9760-0772; and Sajera and Hascall, 
J Biol Chem (1969) 244:77-87 and 2384-2396). The extrac- 
tion is preferably carried out at reduced temperatures to 
reduce the likelihood of digestion or denaturation of the 
extracted protein. A protease inhibitor may be added to 

15 the extract ant, if desired. The pH of the medium depends 
upon the extractant selected. The process of extraction 
generally takes on the order of about 4 hr to 1 day. 

After extraction, the extractant may be removed 
by suitable means such as dialysis against water, preceded 

20 by concentration by ultrafiltration if desired. Salts can 
also be removed by controlled electrophoresis, or by 
molecular sieving, or by any other means known in the art. 
It is also preferred to maintain a low temperature during 
this process so as to minimize denaturation of the 

25 proteins. Alternatively, the extractant chaotropic agent 
need not be removed, but rather the solution need only be 
concentrated, for example, by ultrafiltration. 

The extract, dissolved or redissolved in 
chaotropic agent, is subjected to gel filtration to obtain 

30 fractions of molecular weight in the range of about 20 , 000 
to 36,000 daltons . Gel sizing is done using standard 
techniques, preferably on a Sephacryl S-200 column at room 
( 10°C-25°C ) temperature . 

The sized fraction is then subjected to ion 

35 exchange chromatography using CMC at approximately pH 4.5- 
5.2 preferably about 4.8, in the presence of a nonionic 



WO 91/02744 



PCT/US90/04745 



chaotropic agent such as 6M urea. . Other cation exchangers 
may be used, including those derived from polyacrylamide 
and cross-linked dextran; however cellulosic cation 
exchangers are preferred. Of course, as in any ion 
5 exchange procedure, the solution must be freed of 
competing ions before application to the column. The 
protein is adsorbed on the column and is eluted in an 
increasing salt concentration gradient in the range of 
about 10 mM to about 150 mM. This fraction is designated 

10 M CMB-1 M for convenience. 

CMB-1 is lyophilized and the dry CMB-1 is dis- 
solved in aqueous sodium deoxycholate (DOC), pH 8.0. This 
solution is affinity chromatographed on an equilibrated 
column of ConA cross-linked to resin. The ConA-bound 

15 material is eluted from the resin with aqueous DOC 

containing a displacement carbohydrate. This fraction is 
designated "CAB-1" for convenience. 

CAB-1 is reequilibrated for heparin-sepharose 
chromatography by desalting on a GH-25 column equilibrated 

20 on heparin-sepharose buffer, 6M urea, 0.1M NaCl, 50 mM 
Tris-HCl pH 7.2. The desalted fraction is loaded onto a 
heparin-sepharose column. After washing, bound material 
is eluted from the column using the same buffer at a 0.5M 
NaCl salt concentration. The resulting eluate is 

25 designated "HSB-l " for convenience. 

HSB-1 is diluted and adjusted to pH 2 and loaded 
onto a C18-RP-HPLC column. Bound proteins were gradient 
eluted from the column using a solvent -consisting of 90% 
acetonitrile in 0.1% aqueous TFA (Solvent B) . The protein 

30 of the invention elutes at approximately 47-50% of solvent 
B (42-45% acetonitrile) by volume. 

Proteins eluted by the C18 chromatography were 
iodinated by the chloramine-T method. Analysis of the 
fraction by SDS-PAGE and autoradiography shows a major 

35 broad band at 20,000 to 28,000 daltons comprising the 



WO 91/02744 



PCT/US90/04745 



-9- 

protein. The "smearing" of the protein is believed to 
mainly be the result of heterogeneity in the glycosylation 
of the molecule or perhaps variable post-translational 
modification or proteolytic degradation. After enzymatic 
5 or chemical deglycosylation, SDS-PAGE analysis of the 
pre ;.ein gives a single band of approximately 10 f 000 
daltons . Reduction of the deglycosylated protein with 
dithiothreitol does not affect its migration. 

Initial amino acid sequence analysis of the 
10 glycosylated protein so isolated from bovine bone yielded 
the following internal sequence in the N-terminal portion 
of the protein: 

-Lys-Tyr-Asn-Lys-Ile-Lys-Ser-Arg-Gly-Ile-Lys- 
15 Ala-Asn-Thr-Phe-Lys-Lys-Leu-His-Asn-Leu-Ser-Phe-X-Tyr-Thr- 
Asp-His-Asn-Ala-Leu-Glu- 

The initial amino acid (Lys) in the above sequence is 
nearest to the N-terminal. Initially, the nature of the 

20 signal obtained for the residue designated X did not 

permit this residue to be identified. Repeated sequencing 
of the entire peptide and sequencing of oligopeptides 
generated from endoproteinase Lys-C (an enzyme that 
cleaves proteins at Lys residues) and endoproteinase Glu-C 

25 (an enzyme that cleaves proteins at Glu residues) digests 
have revealed that the above sequence is preceded by an 
Ala residue which is the N-terminus, that the residue 
designated X is Leu, that the second Thr residue (the 26th 
residue in the above sequence) was incorrect and that this 

30 residue is actually a Leu residue, and that the isolate 
consists of a protein of approximately 106 amino acids. 
Figure 7 provides a summary of these sequence analyses. 
The symbol "CHO" designates a carbohydrate substitueat. 
The symbol "COOH" represents a carboxyl group and 

35 designates the carboxy terminus. The first column (on the 



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PCIYUS90/04745 



10 



15 



20 



25 



-10- 

left) provides the sequence analysis of the N-terminal 
fragment described above. The second, fourth, and sixth 
columns give the sequences of three major Lys-C fragments 
of the isolate. The third and fifth columns give the 
sequences of two Glu-C fragments. 

Subsequent isolation of the gene for this 
protein confirmed the sequence shown in Figure 7 with the 
sole exception that the deduced sequence lacked the Asp 
residue at the carboxy terminal. Accordingly, the 
sequence for the native bovine protein is as follows: 

(H 2 N)-Ala-Lys-Tyr-Asn-Lys-Ile-Lys-Ser-Arg-Gly- 
Ile-Lys-Ala-Asn-Thr»Phe-Lys-Lys-Leu»His- 
Asn-Leu-Ser-Phe-Leu-Tyr-Leu-Asp-His-Asn- 
Ala-Leu-Glu-Ser-Val-Pro-Leu-Asn-Leu-Pro- 
Glu-Ser-Leu-Arg-Val-Ile-His-Leu-Gln-Phe- 
Asn-Asn-Ile-Thr-Ser-Ile-Thr-Asp-Asp-Thr- 
Phe-Cys-Lys-Ala-Asn-Asp-Thr-Ser-Tyr-Ile- 
Arg-Asp-Arg-Ile-Glu-Glu-Ile-Arg-Leu-Glu- 
Gly-Asn-Pro-Val-Ile-Leu-Gly-Lys-His-Pro- 
Asn-Ser-Phe-Ile-Cys-Leu-Lys-Arg-Leu-Pro- 
Ile-Gly-Ser-Tyr-Ile-(COOH) , 

The sequence of the corresponding human protein 
was determined by obtaining the human gene using DNA 
probes based on the bovine DNA sequence, sequencing the 
human gene and deducing the amino acid sequence of the 
protein encoded thereby. The sequence of the human 
protein was found to be as follows. 



30 



(H 2 N)-Ala-Lys-Tyr»Asn-Lys-Ile-Lys-Ser-Arg-Gly» 
Ile-Lys-Ala-Asn-Ala-Phe-Lys-»Lys-Leu-Asn- 
Asn-Leu-Thr-Phe-Leu-Tyr-Leu-Asp-His-Asn- 
Ala-Leu-Glu-Ser-Val-Pro-Leu-Asn-Leu-Pro- 
35 Glu-Ser-Leu-Arg-Val-Ile-His-Leu-Gln-Phe- 



WO 91/02744 



PCT/US90/04745 



-11- 

Asn-Asn-Ile-Ala-Ser-Ile-Thr-Asp-Asp-Thr- 
Phe-Cys-Lys-Ala-Asn-Asp-Thr-Ser-Tyr-Ile- 
Arg-Asp-Arg-Ile-Glu-Glu-Ile-Arg-Leu-Glu- 
Gly-Asn-Pro-Ile-Val-Leu-Gly-Lys-His-Pro- 
5 Asn-Ser-Phe-Ile-Cys-Leu-Lys-Arg-Leu-Pro- 
Ile-Gly-Ser-Tyr-Phe-(COOH) , 

A comparison of the human sequence with the 
bovine sequence shows that there are seven differences at 

10 positions 15, 20 , 23, 54, 84, 85 and 105 of the sequence. 
Accordingly, at least the residues at those positions may 
be interchanged. It is possible, of course, that 
sequences of other mammalian or. avian species may exhibit 
other differences. 

15 In the course of obtaining the genes for the 

mature bovine and human proteins it was discovered that 
the genes each encode a precursor segment. Portions of 
the precursor segments for the bovine and human proteins 
are shown in Figure 11, parts A and B. Accordingly, it is 

20 believed that the protein occurs as a prepropolypeptide 
and is processed into the mature protein defined by the 
sequences indicated above. Polypeptides comprising the 
mature protein sequence and including a portion or all of 
the precursor segments are intended to be within the scope 

25 of the invention. 

The invention provides the protein in 
substantially pure form in which it is essentially free of 
other molecules with which it is associated in nature. In 
this regard, the term "substantially pure" intends a 

30 composition containing less than about 30% by weight 
contaminating protein, preferably less than about 10% 
contaminating protein, and most preferably less than about 
5% by weight contaminating protein. The term 
"substantially pure" is used relative to proteins with 

35 which the protein is associated in nature and is not 

intended to exclude compositions in which the protein is 



WO 91/02744 



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-12- 

admixed with nonproteinaceous carriers or vehicles, or 
proteinaceous carriers or vehicles, provided other 
protein(s) with which it is associated naturally are 
absent. The invention also provides the protein in novel 
5 partially glycosylated or totally deglycosylated forms 
(both of which are referred to herein as 
"deglycosylated" ) . 

Based on the above amino acid sequences, 
oligonucleotide probes which contain the codons for a por- 

10 tion or all of the determined amino acid sequences are 
prepared and used to screen DNA libraries for 
substantially homologous genes that encode related 
proteins. The homologous genes -may be from other species 
of mammals or animals (e.g., avians) or may represent 

15 other members of a family of related genes. The basic 
strategies for preparing oligonucleotide probes and DNA 
libraries, as well as their screening by nucleic acid 
hybridization, are well known to those of ordinary skill 
in the art. See, e^., DNA CLONING: VOLUME I (D.M. Glover 

20 ed. 1985); NUCLEIC ACID HYBRIDIZATION (B.D. Hames and S.J. 
Higgins eds. 1985) ; OLIGONUCLEOTIDE SYNTHESIS (M.J. Gate 
ed. 1984); T. Maniatis, E.F. Frisch & J. Sambrook, 
MOLECULAR CLONING: A LABORATORY MANUAL (1982). 

First, a DNA library is prepared. Since the 

25 initially identified protein was bovine, it was logical to 
probe a bovine library first, find full length clones and 
use the full length bovine clones to probe libraries of 
other mammalian species to identify the protein gene (and 
thus the amino acid sequences) of other species. The 

30 library can consist of a genomic DNA library. Bovine and 
human genomic libraries are known in the art. See , e.g . , 
Lawn et al-, Cell (1978) 15:1157-1174. DNA libraries can 
also be constructed of cDNA prepared from a poly-A RNA 
(mRNA) fraction by reverse transcription. See , e.g. , U.S. 

35 Patent Nos. 4,446,235; 4,440,859; 4,433,140; 



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4,431,740; 4,370,417; 4,363,877. The mRNA is isolated 
from an appropriate cell line or tissue that expresses the 
factor. Libraries from cells involved in bone formation 
(e.g., osteoblasts) or from osteotumors (e.g., 
s osteosarcoma lines) are likely sources to probe for the 
nucleic acids that encode the protein. cDNA (or genomic 
DNA) is cloned into a vector suitable for construction of 
a library. A preferred vector is a bacteriophage vector, 
such as phage lambda. The construction of an appropriate 
1Q library is within the skill of the art. 

Once the library is constructed, oligonucleo- 
tides to probe the library are prepared and used to 
isolate the desired genes. The oligonucleotides are 
synthesized by any appropriate method. The particular 
is nucleotide sequences selected are chosen so as to 

correspond to the codons encoding the known amino acid 
sequences of the protein. Since the genetic code is 
redundant, it will often be necessary to synthesize 
several oligonucleotides to cover all, or a reasonable 
2Q number, of the possible nucleotide sequences which encode 
a particular region of a protein. Thus, it is generally 
preferred in selecting a region upon which to base the 
probes, that the region not contain amino acids whose 
codons are highly degenerate. it may not be necessary, 
25 however, to prepare probes containing codons that are rare 
in the mammal from which the library was prepared. In 
certain circumstances, one of skill in the art may find it 
desirable to prepare probes that are fairly long, and/or 
encompass regions of the amino acid sequence which would 
3Q have a high degree of degeneracy in corresponding nucleic 
acid sequences, particularly if this lengthy and/or 
degenerate region is highly characteristic of the protein. 
Probes covering the complete gene, or a substantial part 
of the genome, may also be appropriate, depending upon the 
35 expected degree of homology. Such would be the case, for 
example, if a cDNA of a bovine protein was used to screen 



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a human gene library for the corresponding human protein 
gene. It may also be desirable to use two probes (or sets 
of probes), each to different regions of the gene, in a 
single hybridization experiment. Automated oligonucleo- 
5 tide synthesis has made the preparation of large families 
of probes relatively straightforward. While the exact 
length of the probe employed is not critical, generally it 
is recognized in the art that probes from about 14 to 
about 20 base pairs are usually effective. Longer probes 

10 of about 25 to about 60 base pairs are also used. 

The selected oligonucleotide probes are labeled 
with a marker, such as a radionucleotide or biotin using 
standard procedures. The labeled set of probes is then 
used in the screening step, which consists of allowing the 

15 single-stranded probe to hybridize to isolated ssDNA from 
the library, according to standard techniques. Either 
stringent or permissive hybridization conditions could be 
appropriate, depending upon several factors, such as the 
length of the probe and whether the probe is derived from 

20 the same species as the library, or an evolutionarily 

close or distant species . The selection of the appropri- 
ate conditions is within the skill of the art. See gener- 
ally , NUCLEIC ACID HYBRIDIZATION, supra . The basic 
requirement is that hybridization conditions be of suf- 

25 ficient stringency so that selective hybridization occurs; 
i.e., hybridization is due to a sufficient degree of 
nucleic acid homology (e.g., at least about 75%), as op- 
posed to nonspecific binding. Once a clone from the 
screened library has been identified by positive 

30 hybridization, it can be confirmed by restriction enzyme 
analysis and DNA sequencing that the particular library 
insert contains a gene for the protein. 

Alternatively, a DNA coding sequence for a 
protein can be prepared synthetically from overlapping 

35 oligonucleotides whose sequence contains codons for the 
amino acid sequence of the protein. Such oligonucleotides 



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are prepared by standard methods and assembled into a 
complete coding sequence. See, e.g. , Edge, Nature (1981) 
292:756; Nambair et al., Science (1984) 223:1299; Jay et 
al w J Biol Chem (1984) 259:6311. 
5 Accordingly recombinant polynucleotides that 

encode the polypeptides may be prepared and isolated by 
one or more of the above described techniques. The term 
"recombinant polynucleotide" as used herein denotes a 
polynucleotide of genomic, cDNA, semisynthetic or 

10 synthetic origin which, by virtue of its origin or 

manipulation (1) is not associated with all or a portion 
of the nucleic acid with which it is associated in nature 
or in the form of a library, (2} is linked to a 
polynucleotide to which it is not linked in nature or in a 

15 library, or (3) is not found in nature or in a library. 

The DNA sequence coding for the protein can be 
cloned in any suitable vector, identified, isolated, and 
thereby maintained in a composition substantially free of 
vectors that do not contain the coding sequence of the 

20 protein (e.g., other library clones). Numerous cloning 
vectors are known to those of skill in the art,* and the 
selection of an appropriate cloning vector is a matter of 
choice. Examples of recombinant DNA vectors for cloning 
and the host cells which they transform include 

25 bacteriophage lambda ( E. coli ), pBR322 ( E. coli ), 
PACYC177 ( E. coli ), pKT230 (gram-negative bacteria), 
pGV1106 (gram-negative bacteria) , pLAFRl (gram-negative 
bacteria), pME290 (non -E. coli gram-negative bacteria), 
pHV14 ( E. coli and Bacillus subtilis ), pBD9 (Bacillus), 

30 plJ61 ( Streptomyces ) , pUC6 ( Streptomyces ) , actinophage C31 
( Streptomyces ) , YIp5 (yeast), YCpl9 (yeast), and bovine 
papilloma virus (mammalian cells). See generally , DNA 
CLONING: VOLUMES I.& II, supra ; MOLECULAR CLONING: A 
LABORATORY MANUAL, supra . 

35 in one embodiment of the present invention, the 

coding sequence for gene encoding the protein is placed 



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under the control of a promoter, ribosome binding site 
(for bacterial and eucaryotic expression)/ and optionally 
an operator (collectively referred to herein as "control" 
sequences), so that the DNA sequence encoding the protein 
5 (referred to herein as the "coding" sequence) is 

transcribed into RNA and the RNA translated into protein 
in the host cell transformed by the vector. The coding 
sequence may or may not contain a signal peptide or leader 
sequence. The determination of the point at which the 

10 precursor protein begins and the signal peptide ends is 
easily determined from the N-terminal amino acid sequence 
of the precursor protein. The protein can also be 
expressed in the form of a fusion protein, wherein a 
heterologous amino acid sequence is expressed at the 

15 N-terminal. See , e.g. , U.S.. Patents Nos . 4,431,739; 
4,425,437. 

The recombinant vector is constructed so that 
the protein coding sequence is located in the vector with 
the appropriate control sequences , the positioning and 

20 orientation of the protein coding sequence with respect to 
the control sequences being such that the coding sequence 
is transcribed under the control of the control sequences 
(i.e., by RNA polymerase which attaches to the DNA 
molecule at the control sequences ) . The control sequences 

25 may be ligated to the coding sequence prior to insertion 
into a vector, such as the cloning vectors described 
above. Alternatively, the coding sequence can be cloned 
directly into an expression vector which already contains 
the control sequence and an appropriate restriction site 

30 downstream from control sequences. For expression of the 
protein coding sequence in procaryotes and yeast, the 
control sequences will be heterologous to the coding 
sequence. If the selected host cell is a mammalian cell, 
the control sequences can be heterologous or homologous to 

35 the protein coding sequence, and the coding sequence can 
be genomic DNA, cDNA or synthetic DNA. Either genomic or 



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cDNA coding sequence may be expressed in yeast. If 
secretory expression in eukaryotic cells is necessary or 
desirable, sequences such as the yeast alpha factor signal 
sequence or other sequences that direct secretion are 
5 included in the control sequence. If glycosylation 

similar to the native molecule is desired, the gene may be 
expressed in yeast or mammalian cells (COS, CHO, or CV-1 
cells) using vectors and procedures known in the art. 
A number of procaryotic expression vectors are known in 

10 the art. See , e.g. , U.S. Patent Nos. 4,440,859; 

4,436,815; 4,431,740; 4,431,739; 4,428,941; 4,425,437; 
4,418,149; 4,411,994; 4,366,246; 4,342,832. See also 
British Patent Specifications GB 2,121,054; GB 2,008,123; 
GB 2,007,675; and European Patent Specification 103,395. 

15 Yeast expression vectors are known in the art. See , e.g. , 
U.S. Patent Nos. 4,446,235; 4,443,539; 4,430,428. See 
also European Patent Specifications 103,409; 100,561; and 
96,491. 

Recombinant protein can be produced by growing 

20 host cells transformed by the expression plasmid described 
above under conditions whereby the protein is produced. 
The protein is then isolated from the host cells and 
purified. If the expression system secretes protein into 
growth media, the protein can be purified directly from 

25 cell -free media. If the recombinant protein is not 

secreted, it is isolated from cell lysates. The selection 
of the appropriate growth conditions and recovery methods 
are within the skill of the art or are apparent from the 
recovery methods used to isolate the native proteins. The 

30 recombinant protein may be recovered by affinity 

chromatography using the antibodies produced in accordance 
with the invention. Recombinant protein may be 
unglycosylated or have a different glycosylation pattern 
than the native molecule depending upon the host that is 

35 used to produce it. The proteins are useful for making 



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antibodies that recognize sequential epitopes of the 
protein, and are useful as bone markers. 

Either native, deglycosylated, or synthetic 
(recombinant) protein can be used to produce antibodies, 
5 both polyclonal and monoclonal. The term "antibody" is 
intended to include whole Ig of any isotype or species as 
well as antigen binding fragments, chimeric constructs and 
single chain antibodies. If polyclonal antibodies are 
desired, purified protein is used to immunize a selected 

1Q mammal (e.g., mouse, rabbit, goat, horse, etc.) and serum 
from the immunized animal later collected and treated 
according to known procedures. Compositions containing 
polyclonal antibodies to a variety of antigens in addition 
to the protein can be made substantially free of anti- 

15 bodies which do not bind specifically to the protein 
bodies by passing the composition through a column to 
which protein has been bound. After washing, polyclonal 
antibodies to the protein are eluted from the column. 
Monoclonal anti-protein antibodies can also be readily 

2Q produced by one skilled in the art. The general methodol- 
ogy for making monoclonal antibodies by hybridomas is well 
known. Immortal, antibody-producing cell lines can also 
be created by techniques other than fusion, such as direct 
transformation of B lymphocytes with oncogenic DNA, or 

25 transfection with Epstein-Barr virus. See , e . q . , 
Schreier, M. , et al., HYBRIDOMA TECHNIQUES (1980); 
Hammerling et al., MONOCLONAL ANTIBODIES AND T-CELL 
HYBRIDOMAS (1981); Kennett et al., MONOCLONAL ANTIBODIES 
(1980). 

30 B Y employing the bone-specific protein (native, 

deglycosylated or synthetic) as an antigen in the im- 
munization of the source of the B-cells immortalized for 
the production of monoclonal antibodies, a panel of 
monoclonal antibodies recognizing epitopes at different 

35 sites on the protein molecule can be obtained. Antibodies 
which recognize an epitope in the binding region of the 



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protein can be readily identified .in competition assays 
between antibodies and protein. Antibodies which 
recognize a site on the protein are useful, for example, 
in the purification of the protein from cell lysates or 
5 fermentation media, in characterization of the protein and 
in identifying immunologically related proteins. Such 
immunologically related proteins (i.e., that exhibit 
common epitopes with the protein) are another aspect of 
the invention. In general, as is known in the art, the 

10 anti-protein antibody is fixed (immobilized) to a solid 

support, such as a column or latex beads, contacted with a 
solution containing the protein, and separated from the 
solution. The protein, bound to the immobilized 
antibodies, is then eluted. Antibodies to the protein may 

15 be used to identify osteoblasts and osteocytes by 

conventional immunoassay procedures. Such identification 
may be used to follow bone and/or cartilege turnover. 

20 Examples 

The following is intended to further illustrate 
processes for preparing the proteins of the invention and 
their use in preparing antibodies. These -examples are not 
intended to limit the invention in any manner. 

25 

A. Preparation of Demineralized Bone 

Bovine metatarsal bone was obtained fresh from 
the slaughterhouse and transported on ice. Bones were 
cleaned of all periosteum and marrow with high pressure 

30 water, crushed into fragments using a liquid-nitrogen- 
cooled grinder and pulverized into powder using a liquid- 
nitrogen-cooled mill . The pulverized bone was washed four 
times for 20 minutes in 4°C deionized water (8 liters/kg). 
The bone was then washed overnight with the same volume of 

35 deionized water at 4°C. The bone powder was demineralized 
for 5 hr in 0.5 N HC1 (21 liter/kg) at 4°C. The acid was 



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decanted, and the demineralized bone powder was washed 
several times with 4°C deionized water until the wash 
reached a pH>3. The excess water was removed on a suction 
filter. 

5 

B. Extraction of Noncollagenous Proteins 

Demineralized bone powder was extracted with 4M 
guanidine-HCl, 10 mM EDTA pH 6.8 (2 liters/kg bone powder) 
for 16 hr at 4°C The suspension was suction-filtered to 
10 recover the guanidine-HCl-soluble fraction and 

concentrated at least 5-fold by ultrafiltration using a 
10,000 dalton cut-off membrane (S10Y10 Amicon spiral 
cartridge) . 

15 C. Gel Filtration 

The extract from IfB, redissolved in 4M 
guanidine-HCl f was fractionated on a Sephacryl S-200 
column equilibrated in 4M guanidine-HCl, 0.02% sodium 
azide, 10 mM EDTA, pH 6.8. Fractions were assayed by 

20 their absorbance at 280 nm and the fractions were combined 

as shown in Figure 2. The fraction indicated by < > in 

Figure 2 constitutes a low molecular weight (LMW, 10,000- 
30,000 daltons) protein fraction possessing the greatest 
activity. This fraction was pooled and dialyzed against 6 

25 changes of 180 volumes of deionized water and lyophilized. 
All operations except lyophilization and dialysis (4°C) 
were conducted at room temperature. 

D. Ion Exchange Chromatography 

30 The pooled fraction from If C was dissolved in 6M 

urea, 10 mM NaCl, 1 mM NEM, 50 mM sodium acetate, pH 4.8 
and centrifuged at 10,000 rpm for 5 min. The supernatant 
was fractionated on a CM52 (a commercially available CMC) 
column equilibrated in the same buffer. Bound proteins 

35 were eluted from the column using a 10 mM to 400 mM NaCl 
gradient in the same buffer, and a total volume of 350 ml 



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at a flow rate of 27 ml/hr. Proteins eluted with 10-150 

mM NaCl (the < > of Figure 3) were collected and 

dialyzed against 6 changes of 110 volumes of deionized 
water for 4 days and lyophilized. All of the foregoing 
5 operations were conducted at room temperature except 
dialysis (4°C) . 

E . ConA Chromatography 

The fraction obtained in step D above was 

10 enriched by affinity chromatography using concanavalin A 
(ConA)-Sepharose 4B (Pharmacia). In order to minimize 
leaching of ConA from the column during chromatography, 
the~resin was cross-linked with. glutaraldehyde essentially 
as described by K.P. Campbell, D.H. MacLennan, J Biol Chem 

15 (1981) 256:4626, Briefly, resin was pelleted (500 x g, 
5 min) and washed twice with 4 volumes of 250 mM NaHC0 3 , 
pH 8.8. The resin was then equilibrated in the same 
buffer for 6-8 hrs at 4°C. After pelleting, the resin was 
cross-linked by the addition of 4 volumes of 250 mM 

20 NaHCO^, pH 8.8, 250 mM methyl-alpha-D-mannopyranoside 
(alpha-MM), 0.03% glutaraldehyde with gentle mixing for 
1 hr at room temperature. The reaction was quenched by 
washing the resin twice in 1M Tris-HCl, pH 7.8. The resin 
was stored in the same buffer containing 0.01% Thimersol 

25 at 4 C until use. 

Samples for ConA chromatography were solubilized 
in 1% deoxycholate at pH 8.0. Any small amount of 
precipitate was removed by centrif ugation 12,000 x g, 5 
minutes . 

30 Prior to chromatography, cross-linked resin was 

first equilibrated with >5 column volumes of 50 mM Tris, 
pH 8.0 followed by >5 column volumes of 1% sodium 
deoxycholate. Samples were loaded and nonbound fractions 
collected by washing with 1% DOC. Elution was monitored 

35 by OD 280" Bound material was eluted with 0.5M alpha-MM in 
1% DOC as shown in Figure 4. 



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F. Chromatography on Heparin-Sepharose 

The bound fraction eluted from the ConA column 
was reequilibrated by chromatography on a GH-25 column 
5 (Pharmacia) equilibrated in 6M urea, 0.1M NaCl, 50 mM 
Tris-HCl pH 7-2 heparin-sepharose buffer. Approximately 
80 mg (1 mg/ml) were loaded on a 25 ml large heparin 
sepharose column (Pharmacia). The column was washed of 
all unbound material. Then bound proteins were eluted 
10 with the same equilibrating buffer but containing 0.5M 
NaCl as shown in Figure 5. About 5-8 mg of heparin- 
sepharose bound proteins were recovered. 

G. Chromatography on C18-RP-HFLC 

15 The pH of the heparin-bound fraction was lowered 

below 5 by adding TFA. Final purification of the hepar in- 
bound fraction was achieved using reversed phase HPLC. 
The columns used were a Vydac TP-RP18 4.6 mm x 25 cm and 
1.0 x 25 cm. Solvent A was 0.1% aqueous trif luoroacetic 

20 acid (TFA) and B 90% acetonitrile in A. Bound proteins 
were eluted from the column with a 32-62% B solvent gradi- 
ent at a rate of 1%/min. The protein composition eluted 
between 47-50% solvent B as shown in Figure 6. 140-200 ug 
protein were recovered. Amino acid composition and amino 

25 acid sequences of the protein were determined using 

standard procedures and are described above and shown in 
Figure 7 . 

H. Deglycosylation 

30 Glycopeptidase F cleaves N-linked oligo- 

saccharides at the innermost N-acetylglucosamine residue. 
High mannose, hybrid and complex oligosaccharides are 
susceptible to the enzyme. Protein was iodinated by the 
chloramine-T method. Labeled protein was digested for 12- 

35 15 hours with 6.7 units/ml glycopeptidase F (Boehringer 
Mannheim) in 0.1M Tris-HCl , pH 7.4, 10 mM EDTA, at 37°C. 



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Both the glycosylated and deglycosylated forms 
were analyzed by sodium dodecyl sulfate/ 15% polyacrylaraide 
slab gels prepared according to standard methods. Figure 
8 is a photograph of the autoradiograph . 

5 

I . Isolation of Bovine Protein Gene 

The protein is designated "OIF" in the drawings. 

The following four 20-mer oligonucleotide probes 
were synthesized using a Biosearch 8600 DNA synthesizer, 
10 The sequences of these probes were derived from the amino 
acid sequence of the bovine protein that was isolated from 
bone. 

GCNAARTAYAAYAARATQAA 
1 5 TTYCGNTTRTGNAARTTYTT 
CTRCTRTGNAARACRTTYCG 
CTYCCNTTRGGNCANTAWGA 

where A is adenine, C is cytosine, G is guanine, T is 

20 thymine, N is A, C, G or T, Q is A, C, or T, R is A or G, 
W is A, G or T and Y is C or T. 

These probes were used to analyze a lambda 
bacteriophage "library" containing DNA fragments from 
bovine liver. The lambda phage vector, EMBL3 (Frischauf, 

25 A.M., et al., J Mol Biol (1983) 170:827) was purchased 
(Stratagene, 1190 North Torrey Pines Road, La Jolla, CA 
92037) and used as described. Bovine liver was collected 
at a slaughterhouse and quickly frozen in liquid nitrogen. 
The frozen tissue was pulverized and lysed with sarkosyl 

30 NL-97A and proteinase K. Cellular DNA was purified by 
CsCl density gradient centrif ugation, treated with Sau3A, 
and fractionated by sucrose gradient centrif ugation after 
phenol-chloroform extraction. 

The DNA was concentrated to 1 mg/ml and 1 ug was 

35 mixed with 1 ug EMBL3 DNA. The mixture was treated with 
DNA ligase as described by the supplier and packaged via 



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the "gigapack kit" (Stratagene) to make a library stock. 
Approximately 10,000 viable phage were plated on each of 
60 plates (150 mm) (see Molecular Cloning: A Laboratory 
Manual, Maniatis, Fritsch and Sambrook, Cold Spring Harbor 
5 Laboratory, Cold Spring Harbor, New York (1983)).* 

Phage plaques were transferred to nitrocellulose 
filters (4 replicates per plate). Absorbed DNA was de- 
natured by treatment with 0.5M NaOH, 1.5M NaCl. Filters 
were neutralized in 0.5M Tris, pH 8.0, 1.5M NaCl, washed 
10 with 2XSSC, air dried, and baked for 2 hr at 80°C under 
vacuum. 

Phage containing the gene were identified by 
32 

hybridizing P-labeled oligonucleotides with filters 
containing DNA transferred from phage plaques. Plaques 

15 hybridizing in duplicate to at least two of the four 

oligonucleotides were purified and further characterized. 
From three experiments representing a total of 6 x 10 5 
plaques (2-4 bovine genome equivalents) only two plaques 
were shown on final analysis to contain sequences compat- 

20 ible with the protein sequence. These two phage (bOIF21 
and bOIF39) were shown by restriction site mapping and 
Southern analysis to contain identical sequences in the 
bOIF region although the extent of bovine DNA in the two 
was different. 

25 Sequencing of these clones revealed the gene 

structure shown in Figure 9 and the DNA sequence of Figure 
11. As shown, the mature bovine sequence is encoded by 
three exons, designated 1, 2, and 3 in Figure 9. Exon 1 
encodes the first 17 amino acids, Exon 2 amino acids 18 

30 through 49, and Exon 3 the remaining residues. The pre- 
cursor segment of the prepropolypeptide is encoded by a 
portion of Exon 1 and separate exons upstream from Exon 1. 

To confirm the splicing of the exons, a bovine 
cancellous bone mRNA library was prepared using the 

35 polymerase chain reaction (PCR) with primers selected from 
within Exons 1 and 3. A clone containing the bOIF cDNA 



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sequence was isolated and the nucleotide sequence 
determined* The resulting sequence confirmed the exon 
splicing contemplated by Figures 9 and 11. 

5 J- Isolation of Human Protein Gene 

Human fetal liver DNA was isolated, treated with 
Sau3A as was described for bovine DNA (above). Phage 
plaques (2.5 x 10 5 ) form the EMBL3 human liver DNA library 
(generated as described above) and a similar number of 

10 plaques from a human liver DNA library produced in lambda 
phage Charon 4a (Lawn, R.M., et al.. Cell (1978) 15:1157) 
were probed with a radioactively-labeled (EcoRI) DNA frag- 
ment (Fragment 1, Figure 9) containing the first exon of 
the bovine gene. All positive appearing plaques (13 from 

15 the EMBL3 library and 6 from the Charon 4a) were isolated 
and reprobed with the radioactive exon 1 DNA fragment as 
well as a second DNA fragment containing exon 3 (Fragment 
2, Figure 9) of the bovine gene. Only one phage from the 
EMBL3 library (phage 41) hybridized to both DNA fragments 

20 and a second phage (phage 28) from the gene library 

hybridized with only the exon 1 fragment upon rescreening. 
All other phage did not hybridize with the radioactive 
probes upon rescreening, indicating that their original 
identification was either an artifact of hybridization or 

25 that the human protein cognate DNA was lost upon 
replication during isolation of the phage. 

Phage 41 and 28 were subjected to restriction 
site mapping and Southern blot analysis to locate the 
sequences for the human probes and the DNA sequence of 

30 these regions was determined. 

Sequencing of these clones showed that the human 
gene structure paralleled that shown in Figure 9, that is 
the human gene for the mature protein comprises three 
exons of identical length to the bovine exons. A restric- 

35 tion map of the human gene region is shown in Figure 10. 
It was further determined that the human protein, as the 



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bovine, occurs as a prepropolypeptide with the precursor 
segment being encoded by a portion of Exon 1 and upstream 
exons. The human genomic DNA sequence is shown in Figure 
11. 

5 

K. Construction of Mammalian Expression Vector Containing 
Human Protein Gene 

A Hindlll fragment of the human protein gene 
(site 15422 to site 25814 in Figure 10) was cloned into a 
10 Hindlll site of plasmid pSC614 (see Figure 12) in 

transcriptional alignment with the SV40 promoter or in the 
opposite orientation to yield plasmids phOIF17 and phOIF18 
respectively. Figure 12 is a schematic diagram of plasmid 
phOIF18. 

15 

L. Transfection of COS-7 Cells with phOIF18 

COS-7 cells were transfected with phOIF18 (see 
Feigner, P.L., et al., Proc Natl Acad Sci (USA) (1987) 
84:7413). After transfection the cells were allowed to 
20 grow in medium with or without serum (5%). In these 

experiments only the cells grown in serum-containing media 
synthesized the protein. 

M. Production and Testing of Antibodies to the Protein 
25 M.l. Production of Polyclonal Antibodies 

Polyclonal antibodies to (1) a synthetic 30-mer 
polypeptide having a sequence corresponding to the amino 

acids 1-30 of Figure 7 except for a Leu > Asn 

substitution at position 25 and (2) the native protein 
30 purified from bone as described above were prepared and 
characterized as follows. 

Antiserum to the 1-30-mer was raised in a rabbit 
by injecting the rabbit with 500 ug of the polypeptide in 
complete Freund's adjuvant (CFA) , followed by boosts of 
35 500 ug of the polypeptide in incomplete Freund's adjuvant 
(ICFA) at approximately three week intervals. The 



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antiserum was obtained after the fourth boost and had a 
titer as measured by ELISA of >1: 10,000. Rabbit antiserum 
to the native protein was raised similarly using an 
initial injection of 50 ug protein in CFA followed by 
5 boosts of 50 ug protein in ICFA. This antiserum had a 
titer of >1:10 / 000 by ELISA. 

The antiserum to the 1-30-mer was tested in 
Western blots on the purified native protein, 
deglycosylated native protein, and on crude native protein 

10 (Con-A bound material), all fixed post-blotting with 0.2% 
glutaraldehyde. The antiserum detected the purified 
native protein at >1 ug and also recognized the 
deglycosylated protein and the crude protein. The 
antiserum to the native protein recognized the native 

15 protein at >100 ng in Western blots. 

M.2. Production of Monoclonal Antibodies 
Murine monoclonal antibodies to the purified 
native protein were prepared as follows. From two fusions 

20 25 positive wells were identified by immunoprecipitation. 
A group of female Balb/c mice was injected 
intraperitoneally (IP) with 10-20 ug of purified native 
protein in CFA. The animals were boosted with 10-20 ug of 
protein in ICFA. Following the third boost, the mice were 

25 bled and serum antibody titers against the protein checked 
by ELISA. Two animals were found to have titers of 
>1:40,000. They were given a final intravenous (IV) 
injection of 20 ug protein four days prior to the fusion. 

Fusion to the SP2/0 myeloma (<5M3659 B f NIGMS 

30 Human Genetic Mutant Cell Repository, Camden, NJ) was 

performed essentially according to the protocol of Oi and 
Herzenberg, "Immunoglobul in-producing Hybrid Cell Lines" 
in Selected Methods in Cellular Immunology , Mishell and 
Shiigi, eds., W.H. Freeman and Co., San Francisco, pp. 

35 357-362, (1980). Spleen cells from the animals were mixed 
with SP2/0 at a ratio of 5:1. 50% polyethylene glycol 



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-28- 

1500 (Boehringer-Mannheim Biochemicals , Indianapolis, IN) 

was used as the fusagen. Cells were plated at 10^ cells/ 

well along with resident peritoneal cells at 4 x 10^ 

cells /well in DMEM with high glucose (4.5 g/1) sup- 

5 plemented with 20% FCS (Hyclone Laboratories, Logan, UT) , 

2 mM L-glutamine, 2 mM sodium pyruvate, nonessential amino 

acids, penicillin and streptomycin. In this procedure, 

aminopterin was replaced by azaserine (Sigma) according to 

the procedure by Larrick et al., Proc Natl Acad Sci (USA) 

10 (1983) 80:6376, and added along with thymidine and 

hypoxanthine on day 1 after the fusion. 

From two fusions 25 positive wells were identi- 

125 

fied by immunoprecipitation of . I-labeled protein. 

All 25 were also positive in an ELISA against 

15 the protein. In addition, several other wells were 

positive by ELISA but negative by immunoprecipitation. 
The supernatant from one uncloned well (.3B2.17, previously 
designated F013-3B2) was particularly positive and was 
used in a Western blot. In this testing synthetic 

20 peptides corresponding to amino acid segments 1-30, 62-95 
and 76-105 of the protein sequence were made arid 1-2 ug of 
each was applied to separate lanes in the gel. Blots were 
probed with 50-100 ug/ml of purified antibody. This anti- 
body recognized >300 ng protein as well as deglycosylated 

25 protein. The antibody also picked up the protein in a 
crude fraction (total Con-A bound) and was found to 
recognize the C-terminal peptide (76-105) but not the N- 
terminal peptide (1-30). Another clone, designated 
2C11.6, was found to recognize the internal 62-95 segment. 

30 Clones 3B2.17 and 2C11.6 were subcloned by limiting dilu- 
tion and were found to be stable and to be IgG isotype. 
These clones have been deposited in the American Type 
Culture Collection (ATCC) on 5 April 1989 under the provi- 
sions of the Budapest Treaty. Their ATCC designations 
'35 are, respectively, HB10099 (3B2.17) and HB10098 (2C11.6). 



WO 91/02744 



t 



PCT/US90/04745 



-29- 

M. 3 . Immunostaininq With Antibodies 
Rat fetuses (19 days old) and 3 day old rats 
were used for tissue sections. Tissue was fixed in 10% 
formalin and 5u sections were prepared in paraffin. The 
5 sections were treated with xylene (deparaf f inized) .and 
washed with Tris-buf fered saline (TBS), pH 7.6, three 
times . The washed sections were then contacted with a 
mixture of TBS, 0.05% Tween, 0.5% bovine serum albumin 
(BSA), 10% normal mouse serum (NMS), for 1 hr at 25°C or 

10 overnight at 4°C. Monoclonal antibody 3B2 (see M.2) at 5 
ug/ml in TBS / Tween/ BSA/ NMS was added and the sections were 
incubated for 1 hr at room temperature. The sections were 
then washed three times in TBS /-Tween and contacted with 
biotinylated goat anti-mouse antibody for 10 min at room 

15 temperature. The sections were then washed again three 
times with TBS/Tween and contacted with streptavidin- 
horseradish peroxidase for 10 min at room temperature. 
Thereafter, the sections were washed a final three times 
with TBS/Tween and contacted with substrate. After 

20 substrat treatment, the sections were washed in water, 
counterstained with Mayer's hematoxylin, washed again in 
water, dehydrated in 100% ethanol, and mounted. 

The most intenst staining occurred in 
hypertrophic calcifying cartilage in the growth plate. 

25 There was also a clear staining pattern in osteoblasts and 
osteocytes with the darkest in more mature cells. No 
detectable staining was observed in soft tissue. A faint 
pattern was seen in bone itself. 

Modifications of the above-described modes of 

30 carrying out the invention that are obvious to those of 
skill in the arts relevant to the invention are intended 
to be within the scope of the following claims. 



35 



WO 91/02744 



PCT/US90/04745 



-30- 

Claims 

1. A substantially pure polypeptide having the 
following amino acid sequence: 

5 . 

(H 0 N) -Ala-Lys-Tyr-Asn-Lys-Ile-Lys-Ser-Arg-Gly- 

12 
Ile-Lys-Ala-Asn-R -Phe-Lys-Lys-Leu-R - 

Asn-Leu-R 3 -Phe-Leu-Tyr-Leu-Asp-His-Asn- 

Ala-Leu-Glu-Ser-Val-Pro-Leu-Asn-Leu-Pro- 

10 Glu-Ser-Leu-Arg-Val-Ile-His-Leu-Gln-Phe- 

4 

Asn-Asn-Ile-R -Ser-Ile-Thr-Asp-Asp-Thr- 
Phe-Cys-Lys-Ala-Asn-Asp-Thr-Ser-Tyr-Ile- 
Ar g-Asp-Arg- I le-Glu-Glu- I le-Arg-Leu-Glu- 
Gly-Asn-Pro-R 5 -R 6 -Leu-Gly-Lys-His-Pro- 
15 Asn-Ser-Phe-Ile-Cys-Leu-Lys-Arg-Leu-Pro- 
I le-Gly-Ser-Tyr-R 7 - ( GOOH ) , 

1 2 3 

where R is Ala or Thr, R is Asn or His, R is Thr or 

4 5 6 

Ser, R is Ala or Thr, R is lie or Val, R is Val or lie 
7 

20 and R is Phe or lie, and substantially pure polypeptides 
that are and substantially homologous thereto or are 
immunologically related thereto. 

2. The polypeptide of claim 1 wherein R 1 is 

25 Ala, R 2 is Asn, R 3 is Thr, R 4 is Ala, R 5 is lie, R 6 is 
7 

Val, and R is Phe. 

3. The polypeptide of claim 1 wherein R 1 is 
Thr, R 2 is His, R 3 is Ser, R 4 is Thr, R 5 is Val, R 6 is 

30 He, and R 7 is He. 

4 . A substantially pure prepropolypeptide 

comprising 

(a) the polypeptide of claim 1 and 
35 (b) at least a portion of a precursor segment 

having the sequence shown in Figure HA or Figure 11B, 

or a sequence substantially homologous thereto. 



WO 91/02744 



PCT/US90/04745 



-31- 



5 . A substantially pure prepropolypeptide 

comprising 

(a) the polypeptide of claim 2 and 
5 (b) at least a portion of a precursor segment 

having the human sequence shown in Figure 11, Parts A and 
B ' 

or a sequence substantially homologous thereto. 

10 6 . A substantially pure prepropolypeptide 

comprising 

(a) the polypeptide of claim 3 and 

(b) at least a portion of a precursor segment 
having the bovine sequence shown in Figure 11, Part B, 

15 or a sequence substantially homologous thereto. 

7. A polypeptide having t e following amino 
acid sequence: 

20 (H 2 N)-Ala-Lys-Tyr-Asn-Lys-Ile-Lys-Ser-Arg-Gly- 

l y 
Ile-Lys-Ala-Asn-R -Phe-Lys-Lys-Leu-R - 

Asn-Leu-R 3 -Phe-Leu-Tyr-Leu-Asp-His-Asn- 

Ala-Leu-Glu-Ser-Val-Pro-Leu-Asn-Leu-Pro- 

Glu-Ser-Leu-Arg-Val-Ile-His-Leu-Gln-Phe- 

25 Asn-Asn-Ile-R -Ser-Ile-Thr-Asp-Asp-Thr- 

Phe-Cys -Lys -Ala-Asn-Asp-Thr-Ser-Tyr- I le- 
Arg-Asp-Arg-Ile-Glu-Glu-Ile-Arg-Leu-Glu- 
Gly-Asn-Pro-R 5 -R 6 -Leu-Gly-Lys-His-Pro- 
Asn-Ser-Phe-Ile-Cys-Leu-Lys-Arg-Leu-Pro- 

30 Ile-Gly-Ser-Tyr-R 7 -{COOH) , 

1 2 ? 

where R is Ala or Thr, R is Asn or His, R is Thr or 

4 5 A 

Ser, R is Ala or Thr, R is lie or Val, R D is Val or He 
7 

and R is Phe or He, wherein said polypeptide is 
35 deglycosylated relative to a native polypeptide having 
said sequence and deglycosylated polypeptides that are 
substantially homologous thereto or are immunologically 
related thereto. 



WO 91/02744 



PCT/US90/04745 



-32- 



8 . Antibody that binds to a polypeptide of 
claim 1, 2, 3, or 7 . 

5 9 . A recombinant polynucleotide encoding a 

polypeptide of claim 1, 2 or 3. 

10. A recombinant polynucleotide encoding a 
prepropolypeptide of claim 4, 5, or 6 . 

10 

11. A recombinant vector containing a 
recombinant polynucleotide of claim 9 and capable of 
directing the expression of the- polypeptide encoded 
thereby. 

15 

12 . A recombinant vector containing a 
recombinant polynucleotide of claim 10 and capable of 
directing the expression of the prepropolypeptide encoded 
thereby . 

20 

13. A recombinant host cell or microorganism 
containing the recombinant vector of claim 11 and capable 
of permitting expression of said polypeptide. 

25 14. A recombinant host cell or microorganism 

containing the recombinant vector of claim 12 and capable 
of permitting expression of said polypeptide. 



30 



35 



WO 91/02744 



1 / 17 



PCT/US90/04745 



DEMORALIZED BONE POWDER (0.5 N HCL) 

I 

4 M GUANIDINE HYDROCHLORIDE EXTRACT 

I 

SEPHACRYLS - 200 (20 - 36KD MW) 



CM - CELLULOSE (10- 1 50 mM NaCL) 



CONCANAVALIN - A (0.5 M METHYL ot - D - MANNOPYRANOSIDE) 



HEPARIN - SEPHAROSE (0. 1 - 0.5 M NaCL) 



C I 8 - REVERSED PHASE - HPLC (42 - 45% ACETONOTRILE) 

FIG. I 



SUBSTITUTE SHEET 



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PCT/US90/04745 



2 / 17 



E 1.0 




—i ■ 1 ■ 1 ■ 1 — 

25 35 45 55 
FRACTION NUMBER 



65 



FIG. 2 




UJ 

o 
< 

00 

o 

CO 
CD 
< 



300 



200 



100 



h — • — r 
20 40 60 80 100 
FRACTION NUMBER 



O 
id 
Z 

E 



FIG. 3 




20 40 
FRACTION NUMBER 



FIG. 4 



SUBSTITUTE SHEET 



WO 91/02744 



3 / 17 



PCT/US90/04745 



1.5 

E 




< 0.04—- 1 r— 1 ' 1 ■ 1— — -H 

0 4 8 12 16 20 24 
FRACTION NUMBER 



FIG. 5 




0.0 4- — • 1 • 1 » 1 ' I 

0 10 20 30 40 

Minutes 



SUBSTITUTE SHEET 



WO 91/02744 



A / 17 



PCT/US90/04745 



FIG. 7-1 



AA * 


N- 


LYS-C 


GLU-C . 


LYS-C 


GLU-C 


LYS-C 


1 


ALA 












2 


LYS 












3 


TYR 












4 ' 


ASN 












5 


LYS 












e 


ILE 












7 


LYS 












e 


SB* 












9 


/FG 












10 


GLY 












1 1 


ILE 












12 


LYS 












13 


ALA 












14 


ASN 












15 


THR 












ie 


PHE 












17 


LYS 












18 


LYS 


(LYS) 










10 


LEU 


LEU 










.2 0 


HIS 


HIS 










21 


ASN 


ASN 










22 


LEU 


LEU 










23 


sm 


sm 










24 


PHE 


PHE 










25 


LEU 


LEU 










26 


TYR . 


TYR 










27 


LEU 


LEU 










28 


ASP 


ASP 










29 


HIS 


HIS 










30 


ASN 


ASN 










31 


ALA 


ALA 










32 


LfU 


LEU 










33 


GUJ 


GLU 










34 




sra 










35 




VAL 










38 




PRO 










37 




LEU 










38 




ASN 










39 




LEU 










40 




PRO 










41 




GLU 










42 




sm 


sm 








43 




LEU 


LEU 








44 






Are 








45 






VAL 








46 






ILE 








47 






HIS 








46 






LEU 








49 






GLN 








50 






PHE 








51 






ASN 








52 






ASN 









SUBSTITUTE SHEET 



WO 91/02744 



5 / 17 



PCT/US90/04745 



FIG. 7-2 



5 3 






II c 

ILc 








5 4 






Inn 








D O 














5 e 






II c 
ILc 








a 7 






TUB 
Inn 








C D 






nor 








59 






A5r 








60 






Inn 








6 1 






PHE 








62 






(XS 








63 






LYS 








64 






ALA 


ALA 






65 














66 








ASP 






67 








THR 






66 








SER 






69 














70 








ILE 






71 














72 








ASP 






73 








ARG 






74 








ILE 






75 








GUI 






76 








GLU 






77 








ILE 






78 








ARS 






79 








LEU 






60 








GW 






61 








GLY 


GLY 




82 








ASN 


ASN 




83 








PFO 


PRO 




84 








VAL 


VAL 




85 








ILE 


ILE 




86 








LEU 


t CI 1 

LeU 




87 








GLY 


GLY 




68 








LYS 


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89 










nib 




90 










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9 1 










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ASM 




©2 










Sen 




93 










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O A 
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9 5 










OS 




96 










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97 










LYS 




98 










ARG 


ARG 


99 










LEU 


LEU 


100 










PRO 


PRO 


101 










ILE 


ILE 


102 










aY 


GLY 


103 










SER 


SER 


I'M 










TYR 


TYR 


105 












vnsasami 



SUBSTITUTE SHEET 



WO 91/02744 



6/ 17 



PCT/US90/04745 



FIG. 8 



ENZ- + - + 
I I 5B I 1 5D 
I 1 l 1 



45K - 
30K - 




SOBSlJIUJi SHEET 



WO 91/02744 PCIYUS90/0474S 

7/17 



Bovine OIF 
Gene Region 
13.7 Kbp 



AL1 5567 
:COR1 5635 
XBA1 5860 

HIND1 11 6896 
ECOR1 7677 
SAL1 8226 
NSI1 8301 
ECOR1 8783 
XBA1 8846 



ECOR1 1015 
GU1 1365 



.HIND1 11 9192 
HIND1 11 9213 
JECOR1 9693 
ECOR1 9718 
BGLII 9904 
3AMH1 10462 
3GLI1 10479 
BAMH1 10970 
ISI1 11111 
ECOR1 11886 
| BAMH1 12781 
L, JH1ND11 1 12790 



r 



OIF 



Exon1-> Exon3-> 



L 



Fragment 



Exon2-> 



Fragment 2 



Phage 39 



Phage 21 



FIG. 9 



SUBSTITUTE SHEET 



WO 91/02744 



8/ 17 



PCT/US90/04745 



FIG. 10 



NHE1 1900 

NHE1 4554 
HIND3 4956 
5PE1 5043 
COR5 5815 
IND3 7515 



ECOF 



HIND3 15422 
5PE1 17072 
ECOR5 17403 
XBA1 18572 
NHE1 19379 
3SSH2 20179 
XBA1 20879 

HIND3 25814 



XBA1 25870 
XBA1 28343 

IND3 30512 
IND3 32459 



J! 



N-terminal Exon-> 



Exon-1 -> Exon2-> 
Exon1-> 

Exon3-> 




FIG. 1 2 



SOESTITOTE SHEET 



WO 91/02744 PCT/US90/04745 

9/ 17 

AMINO TERMINAL EXON OF preproOIF 



A AGC TTT AAA TAT TGC TTC GAT GGT CTG AAT TTT TAT 
T TCG AAA TTT ATA ACG AAG CTA CCA GAC TTA AAA ATA 

TTC CAG GGA AAA AGA GAG TTT TGT CCC ACA GTC AGC 
AAG GTC CCT TTT TCT CTC AAA ACA GGG TGT CAG TCG 

AGG CCA CTA GTT TAT TAA CTT CCA GTC ACC TTG ATT 
TCC GGT GAT CAA ATA ATT GAA GGT CAG TGG AAC TAA 
▲ 

SPE1 

Signal Sequence 
*** *** *** *** *** *** *** *** *** 

Met Lys Thr Leu Gin Ser Thr Leu Leu 
TTT GCT AAA ATG AAG ACT CTG CAG TCT ACA CTT CTC 
AAA CGA TTT TAG TTC TGA GAC <3TC AGA TGT GAA GAG 

▲ 

PST1 

*** *** *** *** *** *** *** *** *** *** 
Leu Leu Leu Leu Val Pro Leu lie Lys Pro Ala Pro 
CTG TTA CTG CTT GTG CCT CTG ATA AAG CCA GCA CCA 
GAC AAT GAC GAA CAC GGA GAC TAT TTC GGT CGT GGT 

Pro Thr Gin Gin Asp Ser Arg lie lie Tyr Asp Tyr 
CCA ACC CAG CAG GAC TCA CG7 ATT ATC TAT GAT TAT 
GGT TGG GTC GTC CTG AGT GCO TAA TAG ATA CTA ATA 

Gly Thr Asp Asn Phe Glu Glu Ser lie Phe Ser Gin 
GGA ACA GAT AAT TTT GAA GAA TCC ATA TTT AGC CAA 
CCT TGT CTA TTA AAA CTT CTT AGG TAT AAA TCG GTT 



Asp Tyr Glu Asp Lys Tyr 
GAT TAT GAG GAT AAA TAC 
CTA ATA CTC CTA TTT ATG 



Leu Asp Gly Lys lie Leu 
CTG GAT GGA AAA ATA TTA 
GAC CTA CCT TTT TAT AAT 



Arg Tyr Phe lie Phe Tyr Ser Lys Phe Ser Phe Leu 
AGG TAC TTT ATT T8C TAT TCT AAA TTT AGC TT8 CTA 
TCC ATG AAA TAA A9G ATA AGA TTT AAA TCG AA9 GAT 

Asn Thr Ala 
AAT ACT GCC C 
TTA TGA CGG G 



FIG.11-1 

PART A 

SUBSTITUTE- SHEET 



WO 91/02744 



PCT/US90/04745 



10/17 















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11/17 



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PCT/US90/04745 



12/ 17 



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WO 91/02744 



PCT/US90/04745 



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SUBSTITUTE SHEET 



INTERNATIONAL SEARCH REPORT 



Interna tional Application No. PCT/US90/04745 
CLASSIFICATION OF SUBJECT MATTER (It several classification symbols apply, indicate all) » 



According to International Patent Classification (IPC) or to both National Classification and IPC 

IPC (5): C07K 15/06,3/02,3/20,13/00; A61K 35/12,37/12; 
A01N 63/02; A23J 1/10 U.S. CL. 42 4/95 

II. FIELDS SEARCHED ~ ~ 



Minimum Documentation Searched 7 

Classification System 



Classification Symbols 



U.S. 



424/95; 514/2, 12, 21; 530/350, 353, 355, 356 
530/ 414, 416, 417, 540 



Documentation Searched other than Minimum Documentation 
to the Extent that such Documents are Included in the Fields Searched • 



III. DOCUMENTS CONSIDERED TO BE RELEVANT » 



Category * 



Citation of Document, " with indication, where appropriate, of the relevant passages * 



Relevant to Claim No. ia 



A 
A 



A 
A 



US, A, 4,294,753 (URIST) 13 OCTOBER 1981 
See entire document. 

US, A, 4,434,094 (SEYEDIN ET AL) 

28 FEBRUARY 1984; See entire document. 

US, A, 4,455,256 (URIST) 19 JUNE 1984 
See entire document. 

US, A, 4,608,199 (CAPLAN ET AL) 

26 AUGUST 1986; See entire document. 

US, A, 4,627,982 (SEYEDIN ET AL) 

09 DECEMBER 1986; See entire document. 

US, A, 4,774,322 (SEYEDIN ET AL) 

27 SEPTEMBER 1988; See entire document. 

US, A, 4,789,732 (URIST) 06 DECEMBER 1988 
See entire document. 

US, A, 4,795,804 (URIST) 03 JANUARY 1989 
See entire document. 



1-7 
1-7 
1-7 
1-7 
1-7 
1-7 
1-7 
1-7 



* Special categories of cited documents: 10 
M A" document defining the general state of the art which is not 
considered to be of particular retevance 

"E" earlier document but published on or after the international 
filing date 

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

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

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



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

"X" document of particular relevance; the claimed invention 
cannot be considered novel or cannot be considered to 
involve an inventive step 

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

document member of the same patent family 



IV. CERTIFICATION 



Date of the Actual Completion of the International Search 



19 DECEMBER 1990 



Date of Mailing of this International Search Report 



3 17J AM 1991 




FormPCT/lSA/210 (second sheer) (Rov.11-87) 



International Application No. PCT/US90/04745 



III. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM THE SECOND SHEET) 



Category * | Citation of Document, with indication, where appropriate, of the relevant passages j Relevant to Claim No 



US, A, 4,810,691 (SEYEDIN ET AL) 
07 MARCH 1989; See entire document. 

US, A, 4,816,442 (McPHERSON ET AL) 
28 MARCH 1989; See entire document . 

WO, Al, WO88/00205 (WANG ET AL) 

14 JANUARY 1988; See entire document. 

EP, A2, 0212474 (URIST ET AL) 04 MARCH 1987 
See entire document. 

Reddi et al., Proc. Nat. Acad. Sci. USA 
(1972) Vol. 69: 1601-1605. See entire 
document . 

Urist et al., Scienu (1965) Vol. 150 : 
893-899. See entire document. 

Urist et al., Clin. Orthop. (1968) Vol. 56: 
37-50. See entire document. 

Urist et al., Clin. Orthop. Rel. Res. (1982) 
Vol. 162 : 219-232. See entire document. 

Urist et al., Science (1983) Vol. 220 : 
680-685. See entire document. 

Urist et al., Proc. Nat. Acad. Sci. USA 
(1984) Vol. 81.: 371-375. See entire document 

EP, A3, 0128041 (BAYLINK) 12 DECEMBER 1984 
See entire document. 

Jennings et al., Meth. Enzymol. (1987) 
Vol. 146: 281-283. See entire document. 



1-7 
1-7 
1-7 
1-7 
1-7 

1-7 
1-7 
1-7 
1-7 
1-7 
1-7 
1-7 



Form PCT/1SW210 (txtra sheet) (Rev.11-87) 



" rte "" > """' N °- PCT/DS90/04745 



FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET 



V.Q OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABLE 1 

This international search report has not been established in respect of certain claims under Article 17(2) (a) for the following reasons: 
1.n Claim numbers . because they relate to subject matter i- not required to be searched by this Authority, namely: 



2.Q Claim numbers . . because they relate to parts of the international application that do not comply with the prescribed require- 
ments to such an extent that no meaningful international search can be carried out specifically: 



3-0 Claim numbers , because they are dependent claims not drafted in accordance with the second and third sentences of 

PCT Rule 6.4(a). 

VLgJ OBSERVATIONS WHERE UNITY OF INVENTION IS LACKING 2 

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

Group I: Claims 1-7, drawn to a polypeptide. 
Group II: Claim 8, drawn to an antibody. 

Group III: Claims 9 & 10, drawn to a recombinant polynucleotide 
Group IV: Claims 11-14, drawn to a recombinant vector and host 
cell • 

1. n As all required adStionafseerch fees were timely paid by the applicant, this international search report covers all searchable claims 

of the international application. 

2. (^1 As only some of the required additional search fees were timel> paid by the applicant, this international search report covers only 

those claims of the international application for which fees were paid, specifically claims: 



3. ffi No 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 claim numbers: 

1-7 

4. n As all searchable claims could be searched without effort justifying an additional fee, the International Searching Authority did not 

invite payment of any additional fee. 

Remark on Protest 
n The additional search fees were accompanied by applicant's protest. 
f~l No protest accompanied the payment of additional search fees. 



Form PCT/lSA/210 (supplemental sheet (2 (Rev. 11-67) 



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