Skip to main content

Full text of "USPTO Patents Application 09804625"

See other formats


J 



Europalsches Patentamt 
European Patent Office 
Office europeen des brevets 



0 Publication number: 



0 401 055 

A2 



EUROPEAN PATENT APPLICATION 



© Application number: 90306058.0 
© Date of filing: 04.06.90 



© int.ciAC12N 15/12, A61K 37/02, 
C07K 13/00, C12P 21/02 



© Priority: 02.06.89 US 360826 

@ Date of publication of application: 
05.12.90 Bulletin 90/49 

© Designated Contracting States: 

AT BE CH DE DK ES FR GB GR IT LI LU NL SE 



© Applicant: CHIRON CORPORATION 
4560 Horton Street 
Emeryville California 94608(US) 

© Inventor: Kiefer, Michael C. 
401 Wright Court 
Clayton, California 94517(US) 
Inventor: Masiarz, Frank R. 
148 Marview Way 

San Francisco, California 94131 (US) 
Inventor: Barr, Philip J. 
67 Bay Forest Drive 
Oakland, California 94611 (US) 



© Representative: Bannerman, David Gardner et 
al 

Withers & Rogers 4 Dyer's Buildings Holborn 
London, EC1N2JT(GB) 



© Bone Calcification factor. 



© The isolation, identification and production by 
recombinant methods of bone calcification factor, a 
22KD polypeptide, are disclosed. The peptide has 



FIG. I 



CM 
< 

in 
in 



ttmrn LEU MLimfe 



ly'qji ttk c 



c m at err ere etc 
t tc& cts en ere a* 

h-otis-r? 

— im LCD MD 
U TV VM. LED LED PRO 



T TAT KA TAC OA Tit 
tTIT St/m TAC TO TAT 

r ra blt Tntsalm 



calcification-inducing activity when implanted with 
matrix Gla protein into mammals. 

FIG. I (CONT.) 



Hum* AAX 
tomm AAL 


AAT 
AAT 


GGG 
EEA 


06 
CTE 


6T6 
GTE 


CCA CGA TTC CAE 
CCA CJEA TTt CAE 


ACC 
ACC 


CCC TAC TTC CAE 
CEC TAC TTC CAE 


TCA ETC 
TCA CTE 


Hibmm ASK 
Bonne ASH 


ASH 
ASM 


aT 

CLY 


LEU 
LEU 


YAL 
YAL 


alagly pue cm 

ALA GLY ME GUI 


SER 
SEX 


ARE TYR PIE CLU 
ARE TYR PIE CLU 


SER YAL 
SER VAL 


Kimwi CTG 
Bonn CTE 


CAT 
GAT 


C6C 
CEC 


GAS 
CAE 


TGG 

TBS 


CAE TTT TAC TCT 
CAA TTT TAC TCC 


TCT 

TET 


CEC TAC AfiC ARE 
CEC TAC ACC AAE 


RCA TCC 
RCA TCC 


ButM LEU 
Room LEU 


ASP 
AST 


ARE 
ARC 


GUI 

CJU 


YRP 
TRP 


GUI HE TYR CYS 
GUt rUE TYR CYS 


CYS 
CYS 


ARC TYR .SCR LYS 
ARC TYR SER LYS 


ARE CYS 
ARE CYS 


Himmc CCA 
Bow* CCA 


TAT 
TAT 


TCC 
TCC 


T6C 
TSC 


TEC 

Tse 


CTA ACA ACA CAA 
CTC RCA AGA CAA 


TAT 
TAT 


CCA CET CAC TAT 
CCA EEC CAC TAT 


SET CAC 
EST CAC 


fkMAN PRO 

Bonn fro 


TTR 
TYK 


SCR 
SEX 


CYS 
CYS 


TRP 
TRf 


UEll TUt THR CLU 
LEU THt THR CLD 


TYR 

TYR 


PRO ELY HIS TTR 
PRO ELY HIS TYI 


CLY CU> 
CLY GUI 



o 
o. 

LU 



C AGktASTiC* 



Human CU) tCT ASP MET IUE SER YYR ASM TYR ASP YYR TYR fUEl ARE CLY ALA 
Bovua CUi MET ASP TET XLE SER TYR ASM TYR ASP TYR TYR WET! ARC CLY ALA 



Ruuh ACA ACC ACT TTC TCT CCA CTE CAA ACC EAT CEC CAE TEC RIB TTC ATA 
Rome ACA ACC ACT TTC TCT CCA CTE CAA AGE CAT CEC CAC TEC AAA TTC ATA 



Human ATE TEC C6G ATE ACT CM TAC CAC TET EAR TTT CCA AAT CTT TAfi 
Rome AT6 TSC CES ATE ACT CAC TAT CAC TCT CAA TTT CCA ART CTT TAC 

I TYR ASP CYS CUI PBE ALA ASH VAL - 
3 TYR AST CYS CUI PHE ALA ASM KM. . 



Xerox Copy -Centre 



1 



EP 0 401 055 A2 



2 



BONE CALCIFICATION FACTOR 



This invention relates to a class of mature 
native mammalian proteins which initiates calcifica- 
tion and which is named herein as bone calcifica- 
tion factor (BCF). Representative of this class are 
human and bovine BCF, for which the full length 
coding sequences are provided herein. The BCF is 
provided by isolation from bone sources and by 
synthesis using recombinant DNA techniques. 



BACKGROUND OF THE INVENTION 

It is known that demineralized bone matrix in- 
duces new bone formation when implanted in the 
soft tissue by a process generally designated as 
matrix induced bone formation (see Urist, M.R., 
Science , 150 : 893-899 (1965)). There have been 
numerous efforts to extract and identify the active 
material (or materials) which induces this process, 
and it has been generally referred to in the litera- 
ture as bone morphogenetic protein(s) (BMP). It is 
uncertain whether BMP is a single material or a 
mixture of materials, and there does not appear to 
be agreement among the investigators as to which 
material, if any, is the bone morphogenetic protein. 

The therapeutic use of BMP offers consider- 
able advantages over use of traditional bone graft 
materials. While not intended to be limited by any 
theory, one hypothesis assumes that BMP trans- 
forms tissue cells into osteoblasts (cells that manu- 
facture bone). During a process that replicates nor- 
mal human fetal development, BMP-induced 
osteoblasts form cartilage which, over a period of 
several months, evolve into solid bone. Thus BMP 
may be useful for replacing bone that has been 
destroyed by disease or accident, for use in treat- 
ment of scoliosis victims, for treatment of mal- or 
mis-formed bone, for use in healing of a fracture, 
etc. 

It is thus an object of the present invention to 
produce a functional bone calcification factor or a 
component thereof, which is a 22 KD protein iden- 
tified by its entire amino acid sequence, which 
initiates calcification. 

It is another object of the present invention to 
produce this biologically active 22 KD protein by 
recombinant DNA technology. 

It is yet another object of the present invention 
to construct nucleic acid screening probes for iso- 
lation of the gene comprising the 22 KD BCF, 

It is yet another object of the present invention 
to provide an amino acid sequence of mature 22 
KD BCF which can be thus prepared by direct 
biochemical synthesis or from constituent amino 
acids by peptide synthesis, for example as by the 



Merrifield method, and particularly by use of auto- 
mated peptide synthesis technology. 

These and other objects of the invention will be 
apparent from the following description of the pre- 
5 ferred embodiments and from practice of the inven- 
tion. 



SUMMARY OF THE INVENTION 

10 

The present invention provides a class of ma- 
ture native mammalian proteins (the class is 
termed herein as BCF), represented by native hu- 
man and bovine BCF described herein, which ini- 

75 tiate calcification in vivo which is important for 
formation of bone. The human and/or bovine BCF 
can be used to identify and isolate other mamma- 
lian BCF proteins which may or may not be ho- 
mologous (in their nucleotide and amino acid se- 

20 quences) to human or bovine BCF and which ex- 
hibit the BCF biological activity. It is recognized 
that there may be allelic variations in BCF within a 
species, and such allelic variants are also within 
the scope of the class of proteins provided by the 

25 present invention. 

The present invention further provides polypep- 
tides which are analogs of BCF, such as BCF 
muteins, fusion proteins, comprising BCF or BCF 
domains, and BCF fragments. The term fusion pro- 

30 tein includes a protein comprising a complete BCF 
sequence or a BCF domain, and a heterologous N- 
or C-terminal sequence <such as a signal sequence 
or sequence which protects the protein from deg- 
radation). A BCF mutein is a protein substantially 

35 homologous to a native BCF sequence (e.g„ a 
minimum of about 75%, 85%, 90% or 95%" ho- 
mologous) wherein at least one amino acid is dif- 
ferent. A BCF fragment or domain is an amino acid 
sequence of sufficient length from a BCF protein 

40 such that it is identifiable as having been derived 
from such BCF protein. The origin of a particular 
peptide can be determined, for example, by com- 
paring its sequence to those in public databases. 
The present invention further provides a 22 KD 

45 bone calcification factor having the human and 
bovine amino acid sequences shown in FIG. 1. The 
present invention also provides methods of prepar- 
ing the 22 KD bone calcification factor (BCF) by 
recombinant DNA techniques. 

so The present invention provides the DNA se- 
quence encoding BCF, which may be used to 
construct vectors for expression in host systems by 
recombinant DNA techniques. 

The present invention also provides therapeutic 
compositions comprising BCF and matrix Gla pro- 



2 



3 



EP 0 401 055 A2 



4 



tein (MGP) for initiating calcification and methods 
for inducing calcification in vertebrates by introduc- 
ing in vivo at the desired site an effective calcifica- 
tion initiating amount of BCF and MGP. The iden- 
tity of MGP was first reported by Price, Urist and 
Otawara in Biochem. Biophys. Res. Comm . 
117:765-771 (1983). 

BRIEF DESCRIPTION OF THE DRAWINGS 

FIG. 1 depicts the DNA sequences and en- 
coded amino acid sequences of human and bovine 
BCF including their signal sequences; 

FIG. 1A depicts the amino acid sequence of 
human BCF (hBCF) without its signal peptide; 

FIG. 1 B depicts the amino acid sequence of 
bovine BCF (bBCF) without its signal peptide; 

FIG. 1C depicts the DNA sequence encoding 
hBCF without its signal sequence; 

FIG. 1D depicts the DNA sequence encoding 
bBCF without its signal sequence; 

FIG. 2 illustrates the sequence of human 
BCF tryptic fragment no. 41, a two-fold degenerate 
45-mer oligonucleotide probe (probe A), and a sec- 
ond probe B, designed therefrom, consisting of 64 
18-mers which are complementary to all possible 
codons shown. 

FIG. 3 illustrates probe C (derived from 
clone Ost 3-7) and four BCF cDNA clones isolated 
from a bovine cDNA library (bb1.1-7) and two hu- 
man osteosarcoma cDNA libraries (Ost 1-7, Ost 3-7 
and Ost 3-17). The length, coding region (boxed) 
and partial restriction map of the clones is in- 
cluded. The Ncol and Spel sites 0 are only 
present in the human BCF sequences. 

FIG. 4 illustrates oligonucleotide adapters 
(boxed) used to prepare BCF expression vectors 
which are secreted from yeast using the alpha- 
factor signal peptide. 

FIG. 4A illustrates the junction between the 
BCF-encoding DNA and promoter in an expression 
vector used to express unsecreted BCF in yeast. 

FIGS. 5, 6 and 7 are photomicrographs of 
the quadriceps pouches of mice 21 days after 
implantation of a composite of recombinant hBCF 
and MGP, showing initiation of calcification. 

DETAILED DESCRIPTION OF THE INVENTION 

The BCF according to the present invention 
may be obtained, free of other osteoinductive asso- 
ciated factors, directly from bone sources, by pre- 
parative peptide synthesis using chemical methods 
(such as the Merrifield synthesis method) or by 
recombinant DNA technology. 

As more particularly described in Example 1, 
BCF may be obtained by purification from human, 



bovine, or other vertebrate bone from partially puri- 
fied extracts (e.g., U.S. Patent 4,795,804 and re- 
ferences cited therein) by preparative gel elec- 
trophoresis and electroelution of the 22 K protein. 

s BCF may also be obtained by recombinant 

DNA methods, such as by screening reverse tran- 
scripts of mRNA, or by screening genomic libraries 
from any cell. The DNA may also be obtained by 
simply synthesizing the DNA using commonly 

70 available techniques and DNA synthesizing appara- 
tus. Synthesis may be advantageous because 
unique restriction sites may be introduced at the 
time of preparing the DNA, thereby facilitating the 
use of the gene in vectors containing restriction 

75 sites not otherwise present in the native source. 
Furthermore, any desired site modification in the 
DNA may be introduced by synthesis, without the 
need to further modify the DNA by mutagenesis. 
In general, DNA encoding BCF may be ob- 

20 tained from human, bovine or other sources by 
construction a cDNA library from mRNA isolated 
from bones of the vertebrate; and screening with 
labeled DNA probes encoding portions of the hu- 
man or chains in order to detect clones in the 

25 cDNA library that contain homologous sequences; 
or by polymerase chain reaction (PCR) amplifica- 
tion of the cDNA (from mRNA) and subcloning and 
screening with labeled DNA probes; and then ana- 
lyzing the clones by restriction enzyme analysis 

30 and nucleic acid sequencing so as to identify full- 
length clones and, if full-length clones are not 
present in the library, recovering appropriate frag- 
ments from the various clones and ligating them at 
restriction sites common to the clones to assemble 

35 a clone encoding a full-length molecule. Particularly 
preferred DNA probes are set forth in the accom- 
panying examples. Any sequences missing from 
the library may be obtained by the 3 extension on 
the complementary mRNA of synthetic oligodeox- 

40 y nucleotides identified by screening cDNA in the 
library (so-called primer extension), or homologous 
sequences may be supplied from known cDNAs 
derived from human or bovine sequences as 
shown in FIG. 1 . 

45 The practice of the present invention will em- 
ploy, unless otherwise indicated, conventional mo- 
lecular biology, microbiology, and recombinant 
DNA techniques within the skill of the art. Such 
techniques are explained fully in the literature. See 

so e.g., Maniatis, Fritsch & Sambrook, "Molecular 
Cloning: A Laboratory Manual" (1982); "DNA Clon- 
ing: A Practical Approach," Volumes I and II (D.N. 
Glover ed. 1985); "Oligonucleotide Synthesis" 
(M.J. Gait ed. 1984); "Nucleic Acid Hybridization" 

55 (B.D. Hames & S.J. Higgins eds. 1985); 
"Transcription And Translation" (B.D. Hames & S.J. 
Higgins eds. 1964); "Animal Cell Culture" (R.I. 
Freshney ed. 1986); "Immobilized Cells And En- 



3 



5 



EP 0 401 055 A2 



6 



zymes" (IRL Press, 1986); B. Perbal, "A Practical 
Guide To Moleculr Cloning" (1984). 

In describing the present invention, the follow- 
ing terminology will be used in accordance with the 
definitions set out below. 

The term "osteoinductive associated factors" 
includes factors known in the art which are present 
in mammalian bone or other mammalian tissue and 
tend to co-purify with BMP or BMP activity. Such 
factors include proteins which have been isolated 
from bone having reported molecular weights of 34 
KD, 24 KD, 18.5 KD, 17.5 KD, 17 KD, 16.5 KD, 14 
KD (as cited in U.S. Patent No. 4,761,471), and 6 
KD (reported by Price, P.S., et al., from PNAS , 73, 
pp. 1447-1451, 1976). 

A "replicon" is any genetic element (e.g., plas- 
mid, chromosome, virus) that functions "as an 
autonomous unit of DNA replication in vivo ; i.e.. 
capable of replication under its own control. 

A "vector" is a replicon, such as plasmid, 
phage or cosmid. to which another DNA segment 
may be attached so as to bring about the replica- 
tion of the attached segment. 

A "double-stranded DNA molecuie" refers to 
the polymeric form of deoxyribonucleotides 
(adenine, guanine, thymine, or cytosine) in its nor- 
mal, double-stranded helix. This term refers only to 
the primary and secondary structure of the mol- 
ecule, and does not limit it to any particular tertiary 
forms. Thus, this term includes double-stranded 
DNA found, inter alia, in linear DNA molecules 
(e.g., restriction fragments), viruses, plasmids, and 
chromosomes. In discussing the structure of par- 
ticular double-stranded DNA molecules, sequences 
may be described herein according to the normal 
convention of giving only the sequence in the 5' to 
3' direction along the nontranscribed strand of DNA 
(i.e., the strand having a sequence homologous to 
the mRNA). 

A DNA "coding sequence" is that portion of a 
DNA sequence, the transcript of which is translated 
into a polypeptide in vivo when placed under the 
control of appropriate regulatory sequences. The 
complementary DNA strand will be understood to 
be that strand which is transcribed. The boundaries 
of the coding sequence are determined by a start 
codon at the 5 (amino) terminus and a translation 
stop codon at the 3' (carboxy) terminus. A coding 
sequence can include, but is not limited to, pro- 
caryotic sequences, cDNA from eucaryotic mRNA, 
genomic DNA sequences from eucaryotic (e.g., 
mammalian) DNA, and even synthetic DNA "se- 
quences. A polyadenylation signal and transcription 
termination sequence will usually be located 3 to 
the coding sequence. 

A "promoter sequence" is a DNA regulatory 
region capable of binding RNA polymerase in a 
cell and initiating transcription of a downstream (3' 



direction) coding sequence. For purposes of defin- 
ing the present invention, the promoter sequence is 
bounded at its 3' terminus by the translation start 
codon of a coding sequence and extends upstream 

5 (5 direction) to include the minimum number of 
bases or elements necessary to initiate transcrip- 
tion at levels detectable above background. Within 
the promoter sequence will be found a transcription 
initiation site (conveniently defined by mapping 

70 with nuclease S1), as well as protein binding do- 
mains (consensus sequences) responsible for the 
binding of RNA polymerase. Eucaryotic promoters 
will often, but not always, contain "TATA" boxes 
and "CAT" boxes. Procaryotic promoters contain 

75 Shine-Dalgarno sequences in addition to the -10 
and -35 consensus sequences. 

A coding sequence is "under the control" of 
the promoter sequence in a cell when RNA poly- 
merase which binds the promoter sequence tran- 

20 scribes the coding sequence into mRNA which is 
then in turn translated into the protein encoded by 
the coding sequence. 

A cell has been "transformed" by exogenous 
DNA when such exogenous DNA has been intro- 

25 duced into the cell membrane. Exogenous DNA 
may or may not be integrated (covalently linked) to 
chromosomal DNA making up the genome of the 
cell. In procaryotes and yeast, for example, the 
exogenous DNA may be maintained on an epi- 

30 somal element such as a plasmid. With respect to 
eucaryotic cells, a stably transformed cell is one in 
which the exogenous DNA has become integrated 
into a chromosome so that it is inherited by daugh- 
ter cells through chromosome replication. This sta- 

35 bility is demonstrated by the ability of the 
eucaryotic cell to establish cell lines or clones 
comprised of a population of daughter cells con- 
taining the exogenous DNA. A "clone" is a popula- 
tion of cells derived from a single cell or common 

40 ancestor by mitosis. A "cell line" is a clone of a 
primary cell that is capable of stable growth in vitro 
for many generations. 

Two DNA sequences are "substantially ho- 
mologous" when at least about 85% (preferably at 

45 least about 90%, and most preferably at least 
about 95%) of the nucleotides match over the 
defined length of the DNA sequences. Sequences 
that are substantially homologous can be identified 
in a Southern hybridization experiment under, for 

so example, stringent conditions as defined for that 
particular system. Defining appropriate hybridiza- 
tion conditions is within the skill of the art. See, 
e.g., Maniatis et al., supra ; DNA Cloning, Vols. I & 
II, supra ; Nucleic Acid Hybridization, supra . 

55 A "heterologous" region of the DNA construct 
is an identifiable segment of DNA within a larger 
DNA molecule that is not found in association with 
the larger molecule in nature. Thus, when the het- 



4 



7 



EP 0 401 055 A2 



8 



erologous region encodes a mammalian gene, the 
gene will usually be flanked by DNA that does not 
flank the mammalian genomic DNA in the genome 
of the source organism. Another example of a 
heterologous coding sequence is a construct where 
the coding sequence itself is not found in nature 
(e.g., a cDNA where the genomic coding sequence 
contains introns, or synthetic sequences having 
codons different than the native gene). Allelic vari- 
ations or naturally-occurring mutational events do 
not give rise to a heterologous region of DNA as 
defined herein. 

A composition comprising "A" (where "A" is a 
single protein, DNA molecule, vector, etc.) is sub- 
stantially free of "B" (where "B" comprises one or 
more contaminating proteins, DNA molecules, vec- 
tors, etc.) when at least about 75% by weight of 
the proteins, DNA, vectors (depending on the cate- 
gory of species to which A and B belong) in the 
composition is M A\ Preferably, "A" comprises at 
least about 90% by weight of the A + B species in 
the composition, most preferably at least about 
99% by weight. It is also preferred that a composi- 
tion, which is substantially free of contamination, 
contain only a single molecular weight species 
having the activity or characteristic of the species 
of interest. 

As more particularly described in the following 
examples, human and bovine cDNA libraries were 
initially probed for sequences encoding BCF se- 
quences using labeled oiigodeoxynucleotides 
whose sequences were based on a partial amino 
acid sequence determined from analysis of purified 
protein samples derived from bone described here- 
in. However, it is realized that once being provided 
with non-chromosomal DNA encoding human and 
bovine BCF and their leader sequences as de- 
scribed herein, one of ordinary skill in the art would 
recognize that other precisely hybridizing probes 
may be prepared from the described sequences in 
order to readily obtain the remainder of the desired 
human or bovine gene. 

The non-chromosomal DNA provided by the 
present invention is novel, since it is believed that 
the naturally-occurring human and bovine genes 
(chromosomal) contain introns (transcribed se- 
quences, the corresponding amino acids of which 
do not appear in the mature protein). Hence, the 
term "non-chromosomal" excludes the DNA se- 
quences which naturally occur in the chromosomes 
of human or bovine cells. The present invention 
also encompasses the non-chromosomal cDNA se- 
quences derivable from the DNA sequences dis- 
closed herein. 

Vectors are used to amplify the DNA which 
encodes the chains, either in order to prepare 
quantities of DNA for further processing (cloning 
vectors) or for expression of the chains (expression 



vectors). Vectors comprise plasmids, viruses 
(including phage), and integratable DNA fragments, 
i.e., fragments that are integratable into the host 
genome by recombination. Cloning vectors need 

5 not contain expression control sequences. How- 
ever, control sequences in an expression vector 
include a transcriptional promoter, an optional oper- 
ator sequence to control transcription, a sequence 
encoding suitable rRNA ribosomal binding sites (for 

70 prokaryotic expression), and sequences which con- 
trol termination of transcription and translation. The 
expression vector should preferably include a se- 
lection gene to facilitate the stable expression of 
BCF and/or to identify transformants. However, the 

75 selection gene for maintaining expression can be 
supplied by a separate vector in cotransformation 
systems using eukaryotic host cells. 

Suitable vectors generally will contain replicon 
(origins of replication, for use in non-integrative 

20 vectors) and control sequences which are derived 
from species compatible with the intended expres- 
sion host. By the term "replicable" vector as used 
herein, it is intended to encompass vectors contain- 
ing such replicons as well as vectors which are 

25 replicated by integration into the host genome. 
Transformed host cells are cells which have been 
transformed or trahsfected with vectors containing 
BCF encoding DNA. The expressed BCF will be 
deposited intracellular^ or secreted into either the 

30 periplasmic space or the culture supernatant, de- 
pending upon the host cell selected and the pres- 
ence of suitable processing signals in the ex- 
pressed peptide, e.g. homologous or heterologous 
signal sequences."" 

35 Suitable host cells are prokaryotes or 
eukaryotic cells. Prokaryotes include Gram nega- 
tive or Gram positive organisms, for example E. 
coli or bacilli. Eukaryotic cells include yeast, higher 
eukaryotic cells such as established cell lines of 

40 mammalian origin, or insect cells. Expression in 
insect cells may be accomplished using host cells 
and insect expression vectors as disclosed by Luc- 
kow, V.A., and Summers, M.B., Biotechnology 
6:47-55 (1976). 

45 " Expression vectors for host cells ordinarily in- 
clude an origin of replication, a promoter located 
upstream from the BCF coding sequence, together 
with a ribosome binding site, a polyadenylation 
site, and a transcriptional termination sequence. 

so Those of ordinary skill will appreciate that certain of 
these sequences are not required for expression in 
certain hosts. An expression vector for use with 
microbes need only contain an origin of replication 
recognized by the host, a promoter which will func- 

55 tion in the host and a selection gene. 

An expression vector is constructed according 
to the present invention so that the BCF coding 
sequence is located in the vector with the appro- 



5 



9 



EP0 401 055 A2 



10 



priate regulatory sequences, the positioning and 
orientation of the 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., RNA polymerase which 
binds to the DNA molecule at the control se- 
quences transcribes the coding sequence). The 
control sequences 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 con- 
trol sequences and an appropriate restriction site. 
For expression of BCF in procaryotes and yeast, 
the control sequences will necessarily be heterolo- 
gous to the coding sequence, if the host cell is a 
procaryote, it is also necessary that the coding 
sequence be free of introns (e.g., cDNA). If the 
selected host cell is a mammalian cell, the control 
sequences can be heterologous or homologous to 
the BMP coding sequence, and the coding se- 
quence can either be genomic DNA containing 
introns or cDNA. Either genomic or cDNA coding 
sequences can be expressed in yeast. 

Expression vectors must contain a promoter 
which is recognized by the host organism. Promot- 
ers commonly known and available which are used 
in recombinant DNA construction include the 0- 
lactamase (penicillinase) and lactose promoter sys- 
tems, a tryptophan (trp) promoter system and the 
tac promoter. While these are commonly used, 
other known microbial promoters are suitable. 

In addition to prokaryotes, eukaryotic cells 
such as yeast are transformed with BCF encoding 
vectors. Saccharomyces cerevisiae , or common 
baker's yeast, is the most commonly used among 
lower eukaryotic host microorganisms. However, a 
number of other strains are commonly available 
and useful herein. Yeast vectors generally will con- 
tain an origin of replication from the 2 micron yeast 
plasmid or an autonomously replicating sequence 
(ARS), a promoter, DNA encoding BCF, sequences 
tor polyadenylation and transcription termination, 
and a selection gene. 

Suitable promoting sequences in yeast vectors 
include the promoters for the glycolytic enzymes 
such as enolase, 3-phosphoglycerate kinase, 
glyceraldehyde-3-phosphate dehydrogenase, hex- 
okinase, pyruvate decarboxylase, phosphofruc- 
tokinase, glucose-6-phosphate isomerase, 3- 
phosphoglycerate mutase, pyruvate kinase, 
triosephosphate isomerase, phosphoglucose 
isomerase, and glucokinase. 

Other yeast promoters, which have the addi- 
tional advantage of transcription controlled by 
growth conditions are the promoter regions for al- 
cohol dehydrogenase 1 or 2, isocytochrome C, 
acid phosphatase, as well as enzymes responsible 



for maltose and galactose utilization. 

Higher eukaryotic cell cultures may be used, 
whether from vertebrate or invertebrate cells, in- 
cluding insects, and the procedures of the propa- 
s gation thereof are known. See, for example. Tissue 
Culture , Academic Press, Kruse and Patterson, edi- 
tors (1973). 

Suitable host cells for expressing BCF in high- 
er eukaryotes include: monkey kidney CVI line 

10 - transformed by SV40 (COS-7, ATCC CRL 1651); 
baby hamster kidney cells (BHK, ATCC CRL 10); 
Chinese hamster ovary-cells-DHFR (described by 
Urlaub and Chasin. PNAS (USA) 77: 4216 (1980)); 
mouse Sertoli cells (TM4, Mather, J.P., Biol. Re- 

75 prod. 23:' 243-251 (1980)); monkey kidney ceils 
(CVI ATCC CCL 70); african green monkey kidney 
cells (VERO-76, ATCC CRL-1587); human cervical 
carcinoma cells (HELA, ATCC CCL 2); canine kid- 
ney cells (MDCK, ATCC CCL 34); buffalo rat liver 

20 cells (BRL 3A, ATCC CRL 1442); human lung cells 
(W138, ATCC CCL 75); human liver cells (Hep G2, 
HB 8065); mouse mammary tumor (MMT 060652, 
ATCC CCL 51); rat hepatoma cells (HTC, Ml , 54, 
Baumann, M., et al.. J. Cell Biol. 85: 1-8 (1980)) 

25 and TRI cells (Mather, J.P., et aT7 Annals N.Y. 
Acad. Sci. 383 : 44-68 (1982)). Commonly used 
promoters are derived from polyoma, Adenovirus 2, 
and Simian Virus 40 (SV40). It will be appreciated 
that when expressed in mammalian tissue, the re- 

30 combinant BCF may have higher molecular weight 
due to glycosylation. It is therefore intended that 
partially or completely glycosylated forms of BCF 
having molecular weights greater than that pro- 
vided by the amino acid backbone are within the 

35 scope of this invention. 

A number of procaryotic expression vectors are 
known in the art. See, e.g., U.S. Patent Nos. 
4,440,859; 4,436,815; 4,431740; 4,431,739; 
4,428,941; 4,425,437; 4,418,149; 4,411,994; 

40 4,366,246; 4,342,832; see also U.K. Pub. Nos GB 
2,121,054; GB 2.008,123; GB 2,007,675; and Eu- 
ropean Pub. No. 103,395. Preferred procaryotic 
expression systems are in E. coli. Other preferred 
expression vectors are those for use in eucaryotic 

45 systems. An exemplary eucaryotic expression sys- 
tem is that employing vaccinia virus, which is well- 
known in the art. See. e.g., Macket et al. (1984) J. 
Virol . 49:857; "DNA Cloning," Vol. II, pp. 191-211, 
supra ; PCT Pub. No. WO 86/07593. Yeast expres- 

eo sion vectors are known in the art. See, e.g.. U.S. 
Patent Nos. 4,446,235; 4,443,539; 4,430^28; see 
also European Pub. Nos. 103,409; 100,561; 96,491. 
Another expression system is vector pHS1 , which 
transforms Chinese hamster ovary cells. The use of 

55 the vector is described in PCT Pub. No. WO 
87/02062, the disclosure of which is incorporated 
herein by reference. 

Mammalian tissue can be cotrartsformed with 



6 



11 



EP 0 401 055 A2 



12 



DNA encoding a. selectable marker such as 
dihydrofolate reductase (DHFR) or thymidine 
kinase and DNA encoding BCF. If wild type DHFR 
protein is employed, it is preferable to select a host 
cell which is deficient in DHFR, thus permitting the 
use of the DHFR coding sequence as marker for 
successful transfection in hgt" medium, which 
lacks hypoxanthine, glycine, and thymidine. An ap- 
propriate host cell in this case is the Chinese 
hamster ovary (CHO) cell line deficient in DHFR 
activity, prepared and propagated as described by 
Urlaub and Chasin, 1980, Froc. Nat. Acad. Sci . 
(USA) 77: 4216. 

Recently, expression vectors derived from Bac- 
clovirus for use in insect cells have become known 
in the art. 

Depending on the expression system and host 
selected, BCF is produced by growing host cells 
transformed by an expression vector described 
above under conditions whereby the BCF protein is 
expressed. The enzyme protein is then isolated 
from the host cells and purified. If the expression 
system secretes the enzyme into -growth media, 
the protein can be purified directly from cell-free 
media. If the BCF protein is not secreted, it is 
isolated from cell lysates. the selection of the ap- 
propriate growth conditions and recovery methods 
are within the skill of the art. 

The recombinantiy made BCF is recovered 
from transformed cells in accordance with per se 
known procedures. Preferably, an expression vec- 
tor will be used which provides for secretion of 
BCF from the transformed cells, thus the cells may 
be separated by centrifugation. The BCF generally 
is purified by general protein purification tech- 
niques, including, but not limited to, size exclusion, 
ion-exchange chromatography, HPLC, and the like. 

Once a coding sequence for BCF has been 
prepared or isolated, it can be cloned into any 
suitable vector or replicon and thereby maintained 
in a composition which is substantially free of vec- 
tors that do not contain a BCF coding sequence 
(e.g., free of other library clones). Numerous clon- 
ing" vectors are known to those of skill in the art. 
Examples of recombinant DNA vectors for cloning 
and host cells which they can transform include the 
various bacteriophage lambda vectors (E. coli), 
pBR322 (E. coli), pACYC177 (E. coli), pKT230 
(gram-negative bacteria), pGVH06 (gram-negative 
bacteria), pLAFRI (gram-negative bacteria). 
pME290 (non-E. coli gram-negative bacteria), 
pHV14. (E. coli and Bacillus subtilis), pBD9 
(Bacillus), plJ61 (Streptomyces), pUC6 
(Streptomyces), actinophage, +C31 

(Streptomyces), Ylp5 (Saccharomyces), YCp19 
(Saccharomyces), and bovine papilloma virus 
(mammalian cells). See generally , DNA Cloning: 
Vols. I & II, supra; T. Maniatis et al., supra; B. 



Perbal, supra . 

It is further intended that calcification-initiating 
fragments of BCF are within the scope of the 
present invention. Such active fragments may be 
5 produced, for example, by pepsin digestion of 
BCF. The active fragments may be identified by 
the in vivo and/or in vitro assays described herein- 
below. 

Alternatively the BCF may be made by con- 

io ventional peptide synthesis using the principles of 
the Merrifield synthesis and preferably using com- 
mercial automatic apparatus designed to employ 
the methods of the Merrifield synthesis. Peptides 
prepared using the Merrifield synthesis may be 

rs purified using conventional affinity chromatography, 
gel filtration and/or RP-HPLC. 

FIG. 1 shows the aligned nucleotide and de- 
duced amino acid sequences for both bovine and 
human BCF for maximum amino acid sequence 

20 identity. Amino acids not conserved in both species 
are boxed. The putative initiation codon is located 
at position -17 followed by a stretch of amino acids 
showing strong hydrophobicity characteristics of 
signal peptides. A putative signal peptide cleavage 

25 site is indicated by the arrow. The putative mature 
proteins begin at position 1(Gln), and contain 183 
amino acids, of which 96.2% are identical. The 
derived molecular weight of human BCF is 21 ,967 
and for bovine BCF is 21,984. The underlined 

30 sequences are those from which the 
oligonucleotide probes are derived. 

Substantially pure BCF, higher molecular 
glycosylated forms thereof, or active fragments 
thereof, or the nontoxic salts thereof, combined 

35 with a pharmaceutically acceptable carrier to form 
a pharmaceutical composition, may be adminis- 
tered to mammals, including humans, either intra- 
venously, subcutaneously, percutaneously, in- 
tramuscularly or orally. 

40 Such proteins are often administered in the 
form of pharmaceutically acceptable nontoxic salts, 
such as acid addition salts or metal complexes, 
e.g., with zinc, iron or the like (which, are consid- 
ered as salts for purposes of this application). Illus- 

45 trative of such acid addition salts are hydrochloride, 
hydrobromide, sulphate, phosphate, maleate, ace- 
tate, citrate, benzoate, succinate, malate. ascor- 
bate, tartrate and the like. If the active ingredient is 
to be administered in tablet form, the tablet may 

50 contain a binder, such as tragacanth, corn starch or 
gelatin; a disintegrating agent, such as alginic acid; 
and a lubricant, such as magnesium stearate. If 
administration in liquid form is desired, sweetening 
and/or flavoring may be used, and intravenous ad- 

55 ministration in isotonic saline, phosphate buffer so- 
lutions or the like may be effected. 

Pharmaceutical compositions will usually con- 
tain an effective amount of BCF in conjunction with 



7 



13 



EP 0 401 055 A2 



14 



a conventional, pharmaceutical^ acceptable carrier. 
The dosage will vary depending upon the specific 
purpose for which th*e protein is being adminis- 
tered, and dosage levels in the range of about 0.1 
ug to about 100 milligrams per Kg. of body weight 
may be used. 

Implants of recombinant BCF, when mixed with 
matrix Gla protein (MGP) (see Example 7), will 
initiate calcification. The BCF may be either the 
human or bovine form or mixtures thereof. Simi- 
larly, the MGP may be any mammalian form there- 
of, preferably human, bovine or mixtures thereof. 
Matrix Gla protein (MGP) may be isolated in the 
course of preparation of bone morphogenetic pro- 
tein (BMP) from demineralized gelatinized bovine 
cortical bone by the methods of Urist et al. [See: 
Price et aL, Proc. Natl. Acad. Sci. USA 73:1447, 
1976; Urist, M.R.. Huo. Y.K. Brownell, A.G., Hohl, 
W.M. Buyske, J., Lietze, A., Tempst, P., Humkapil- 
lar, M., and DeLange, R.J.: Purification of bovine 
bone morphogenetic protein by hydroxyapatite 
chromatography. Proc. Natl. Acad. Sci . 81 :371-375, 
1984 and Urist, M.R., Chang, J.J.. Lietze, A., Huo, 
Y.K., Brownell, A.G., and DeLange, R.J.: Methods 
of preparation and bioassay of bone morphogenetic 
protein and polypeptide fragments. In: Barnes, D., 
and Sirbaska, DA (eds.): Methods in Enzymology , 
vol. 146. New York, Academic Press, 1987, pp. 
294-312]. A preparation containing MGP is first 
separated from other bone matrix protein by hollow 
fiber ultrafiltration through a 10 K pore-size filter. 
Under dissociated conditions in 6M urea and 0.02 
M edetic acid (EDTA), the MGP assumes an elon- 
gated structure in which proteins with 14 to 15 K 
molecular weight (M r ) pass through a 10 K filter. 
The MGP is further purified by ion exchange 
chromatography (Berg. R.A. In: Methods in En- 
zymology, 1982, vol. 82:372-398). 

Furthermore, to initiate calcification BCF and 
MGP may be mixed with any combination of one 
or more other proteins, particularly, with one or 
more other proteins, derived from bone. Such mix- 
tures may not only initiate calcification, but may 
also induce cartilage formation and bone growth. 

Implants of mixtures of BCF and MGP induce 
calcification in the quadriceps compartment. The 
BCF cDNA may also be utilized in a diagnostic test 
for identifying subjects having defective BCF- 
genes, defective BCF or autoantibodies directed 
against BCF; or to detect levels of BCF, which may 
be an indication of osteoporosis. 

Preparations of BCF may be assayed in vivo 
according to the method described by Urisfet al., 
Methods in Enzymology (D. Barnes and D.ATsfr- 
baska, Eds.), vol. 146, pp. 294-312, Academic 
Press, N.Y. (1987), and in vitro by the method of 
Sato and Urist, Clin. Orthop ., 183:180-187 (1984) 
as modified by Kawamura and Urist, Dev. Biol., 



130:435-442 (1988). all of which are incorporated 
by reference herein. 

It is preferred that the BCF be admixed with 
matrix Gla protein (MGP) to form a delivery system 

5 comprising these two proteins. The amount of MGP 
in the composition is not believed to be critical and, 
for convenience, equal portions of BCF and MGP 
may be used in dosages in the range of about 0.1 
ug (combined weight of BCF and MGP) to 100 

w mg/Kg. body weight. 

The BCF and MGP may be implanted as a 
time-release composition encapsulated, for in- 
stance, in liposomes or other time-release mem- 
branes, natural or synthetic, which are absorbable 

75 by the host subject. The purification protocols, de- 
scribed in detail below, allow for the first time the 
purification of native BCF in sufficient quantity and 
at a high enough purity to permit accurate amino 
acid sequencing. The amino acid sequences de- 

20 rived from the purified BCF allow for the design of 
probes to aid in the isolation of native BCF nucleic 
acid sequence, or the design of synthetic nucleic 
acid sequences encoding the amino acid sequence 
of BCF. 

25 Specific anti-sera or monoclonal antibodies 
(described below) can be made to a synthetic or 
recombinant BCF peptide having the sequence or 
fragments of the sequence of amino acid residues, 
such as those shown in Figures 1A or 1B. An 

30 example is the tryptic fragment shown in FIG. 2, 
and antibodies thereto can be used to im- 
munoprecipitate any BCF present in a selected 
tissue, cell extract or body fluid. Purified BCF from 
this source can then be sequenced and used as a 

36 basis for designing specific probes as described 
above. Antibodies to other regions that diverge 
from known BCF can also be used. Also useful as 
antigens are purified native or recombinant BCF. 
As mentioned above, a ON A sequence encod- 

40 ing BCF can be prepared synthetically rather than 
cloned. The DNA sequence can be designed with 
the appropriate codons for the BCF amino acid 
sequence. In general, one will select preferred 
codons for the intended host if the sequence will 

45 be used for expression. The complete sequence is 
assembled from overlapping oligonucleotides pre- 
pared by standard methods and assembled into a 
complete coding sequence. See , e.g., Edge (1981) 
Nature 292:756; Nambair, et al.~(1984) Science 

so 223:1299; Jay et al. (1984) J. BioL Chem . 
259:6311. 

Synthetic DNA sequences allow convenient 
construction of genes which will express BCF an- 
alogs or "muteins". Alternatively, DNA encoding 
55 muteins can be made by site-directed mutagenesis 
of native BCF genes or cDNAs, and muteins can 
be made directly using conventional polypeptide 
synthesis. Muteins altered, for example, by the 



8 



15 



EP 0 401 055 A2 



16 



substitution of acidic residues (e.g., Glu or Asp) 
could have reduced activity toward membrane- 
bound or complex substrates or have anti-sense 
therapeutic uses for overproduction of BCF. 

Site-directed mutagenesis is conducted using a 
primer snthetic oligonucleotide complementary to a 
single stranded phage DNA to be mutagenized 
except for limited mismatching, representing the 
desired mutation. Briefly, the synthetic 
oligonucleotide is used as a primer to direct syn- 
thesis of a strand complementary to the phage, 
and the resulting double-stranded DNA is trans- 
formed into a phage-supporting host bacterium. 
Cultures of the transformed bacteria are plated in 
top agar, permitting plaque formation from single 
cells which harbor the phage. 

Theoretically, 50% of the new plaques will con- 
tain the phase having, as a single strand, the 
muted form; 50% will have the original sequence. 
The resulting plaques are hybridized with kinased 
synthetic primer at a temperature which permits 
hybridization of an exact match, but at which the 
mismatches with the original strand are sufficient to 
prevent hybridization. Plaques which hybridize with 
the probe are then picked, cultured, and the DNA 
recovered. . 

Native, recombinant or synthetic BCF peptides 
(full length or subunits) can be used to produce 
both polyclonal and monoclonal antibodies. If poly- 
clonal antibodies are desired, purified BCF peptide 
is used to immunize a selected mammal (e.g., 
mouse, rabbit, goat, horse, etc.) and serum from 
the immunized animal later collected and treated 
according to known procedures. Compositions con- 
taining polyclonal antibodies to a variety of anti- 
gens in addition to BCF can be made substantially 
free of antibodies which are not anti-BCF by im- 
munoaffinity chromatography. 

Monoclonal anti-BCF antibodies can also be 
readily produced by one skilled in the art form the 
disclosure herein. The general methodology for 
making monoclonal antibodies by hybridomas is 
well known. Immortal, antibody-producing cell lines 
can also be created by techniques other than fu- 
sion, such as direct transformation of B lym- 
phocytes with oncogenic DNA, or transfection with 
Epstein-Barr virus. See, e.g., M. Schreier et al., 
"Hybridoma Techniques ""(1980); Hammerling et 
al., "Monoclonal Antibodies And T-cell 
Hybridomas'' (1981); Kennett et al., "Monoclonal 
Antibodies" (1980); see also U.S. Patent Nos. 
4,341 ,761 ; 4,399,1 21 ; 4,427,783; 4,444,887; 
4,451,570; 4,466,917; 4,472,500; 4,491,632; 
4,493,890. 

Panels of monoclonal antibodies produced 
against BCF peptides can be screened for various 
properties; i.e., isotype, epitope, affinity, etc. Of 
particular interest are monoclonal antibodies that 



neutralize the activity of BCF. Such monoclonals 
can be readily identified in BCF activity assays. 
High affinity antibodies are also useful in im- 
munoaffinity purification of native or recombinant 
5 BCF. 

Antibodies to BCF forms described herein 
(both polyclonal and monoclonal) may be used to 
inhibit or to reverse arterial calcification. An appro- 
priate therapeutic method would be to treat the 

10 patient with an effective dose of anti-BCF anti- 
bodies through a conventional intravenous route. In 
the treatment of local, acute inflammation, treat- 
ment with anti-BCF antibody would be indicated, 
perhaps by intramuscular injection. These com- 

75 positions may also be useful in targeting various 
forms of tumors, since tumors are known to some- 
times calcify, suggesting the presence of BCF. 
BCF antagonists, such as BCF muteins, could also 
be used in place of antibodies. 

20 The determination of the appropriate treatment 
regimen (i.e., dosage, frequency of administration, 
systemic vs. local, etc.) is within the skill of the art. 
For administration, the antibodies will be formulated 
in a unit dosage injectable form (solution, suspen- 

25 sion, emulsion, etc.) in association with a phar- 
maceutical^ acceptable parenteral vehicle. Such 
vehicles are usually nontoxic and nontherapeutic. 
Examples of such vehicles are water, saline, Ring- 
er's solution, dextrose solution, and Hank's solu- 

30 tion. Nonaqueous vehicles such as fixed oils and 
ethyl oleate may also be used. A preferred vehicle 
is 5% (w/w) human albumin in saline. The vehicle 
may contain minor amounts of additives, such as 
substances that enhance isotonicity and chemical 

as stability, e.g., buffers and preservatives. The anti- 
body is Typically formulated in such vehicles at 
concentrations of about 1 ug/ml to 10 mg/ml. 

Anti-BCF antibodies will also be useful in di- 
agnostic applications. The present invention con- 

40 templates a method, particularly a diagnostic meth- 
od, in which a sample from a human (or other 
mammal) is provided, and the amount of BCF is 
quantitatively measured in an assay. For example, 
employing anti-BCF antibodies in a quantitative im- 

45 munoassay could be used to detect genetic defi- 
ciency in BCF. Antibody specific for BCF could be 
formulated into any conventional immunoassay for- 
mat; e.g., homogeneous or heterogeneous, 
radioimmunoassay or ELISA. The various formats 

so are well known to those skilled in the art. See, e.g., 
"Immunoassay" A Practical Guide" (D.W. Chan 
and M.T. Perlstein, eds. 1987) the disclosure of 
which is incorporated herein by reference. 

In general, recombinant production of BCF can 

55 provide compositions of that BCF substantially free 
of other proteins having osteoinductive associated 
functions. The ability to obtain high levels of purity 
is a result of recombinant expression systems 



9 



17 



EP 0 401 055 A2 



18 



which can produce BCF in substantial quantities 
vis-a-vis in vivo sources. Thus, by applying con- 
ventional techniques to recombinant cultures, BCF 
compositions can be produced that are substan- 
tially more pure than the compositions available 
from bone sources. 

Purified BCF will be particularly useful as a tool 
in the design and screening of calcification inhibi- 
tors. First, milligram amounts of the material are 
obtainable according to the present invention. Mil- 
ligram amounts are capable of crystallization to 
permit three dimensional studies using X-ray dif- 
fraction and computer analysis. This may permit 
deduction concerning the shape of the molecule, 
thus defining proper shapes for substances usable 
as inhibitors of the activity normally exhibited by 
BCF. Generally, antagonists have been "peptides" 
whose interactions with a factor which is inhibited 
are stabilized by modification of the "residues" 
participating in the peptide bond so as to enhance 
the ability of the "peptide" to interact specifically 
with converting factor. Thus the peptide bond joins 
specifically chosen carboxyiic acids and amines 
(not necessarily amino acids). 

These "peptides" are configured in a three 
dimensional array so as to complement the con- 
tours of the intended target, converting enzyme. A 
similar lock and key spatial arrangement may result 
from molecules designed complementary to the 
surface contours of the BCF of the invention. It is 
understood that "surface" includes convolutions 
which may race inward, and specifically includes 
the active site. Furthermore, "complementary" is 
understood to mean that, in addition to spatial 
conformations which "fit", interactions between the 
protein and the molecule which matches its surface 
contours are attractive and positive. These inter- 
actions may be hydrogen bonding, ionic, or hy- 
drophobic affinity. 

Accordingly, the invention contemplates pep- 
tide antagonists or agonists (2-15 amino acids) to 
BCF which are characterized by three dimensional 
contours complementary to the three dimensional 
contours on the surface of recombinant BCF. By 
peptide in this context is meant that the antagonist 
or agonist contains carboxyiic acid amide bonds 
corresponding to one less than the number of 
residues. The carboxyiic acid and amine partici- 
pants need not be a-amino acids. 

Second, even without the assistance of a three 
dimensional structure determination, purified BCF 
of the invention is of significance as a reagent in 
screening BCF inhibitors in vitro as an ad hoc 
approach to evaluation. Impure BCF preparations 
currently available yield confusing data due to the 
impact of the impurities on the test results. For 
example, contaminants which turn out to be them- 
selves inhibitors, activators, or substrates for BCF 



may interfere with the evaluation. Thus, a substan- 
tial improvement in current screening techniques 
for BCF inhibitors would be effected by the avail- 
ability of the purified BCF protein. 

5 It will be understood that this description and 

disclosure of the invention is intended to cover all 
changes and modifications of the invention which 
are within the spirit and scope of the invention. It is 
within the knowledge of the art to insert, delete or 

70 substitute amino acids within the amino acid se- 
quence of a BCF without substantially affecting the 
calcification and bone growth inducing activity of 
the molecule. The invention is expressly stated to 
be broad enough to include intentional deletions, 

75 additions or substitutions. Furthermore, it is recog- 
nized that one skilled in the art could recombinan- 
tly produce such modified proteins. 

Native, recombinant or synthetic BCF peptides 
(full length or subunits) can be further used to 

20 produce both polyclonal and monoclonal anti- 
bodies. If polyclonal antibodies are desired, purified 
BCF is used to immunize a selected mammal (e.g., 
mouse, rabbit, goat, horse, etc.) and serum fToTn 
the immunized animal later collected and treated 

25 according to known procedures. Compositions con- 
taining polyclonal antibodies to a variety of anti- 
gens in addition to BCF can be made substantially 
free of antibodies which are not anti-BCF by im- 
munoaffinity chromatography. 

30 Monoclonal anti-BCF antibodies can also be 
readily produced by one skilled in the art from the 
disclosure herein. The general methodology for 
making monoclonal antibodies by hybridomas is 
well known. Immortal, antibody-producing cell lines 

35 can also be created by techniques other than fu- 
sion, such as direct transformation of B lym- 
phocytes with oncogenic DNA, or transfection with 
Epstein-Barr virus. See, e.g., M. Schreier et al M 
"Hybridoma Techniques"~(1980); Hammerling "et 

40 al„ "Monoclonal Antibodies And T-cefi 
Hybridomas" (1981); Kennett et al., "Monoclonal 
Antibodies" (1980); see also U.S. Patent Nos. 
4,341 ,761 ; 4,399,1 21 ; 4,427,783; 4,444,887, 
4,451,570; 4,466.917; 4,472,500; 4,491,632; 

45 4,493,890. 

Panels of monoclonal antibodies produced 
against BCF peptides can be screened for various 
properties; i.e., isotype. epitope, affinity, etc. Of 
particular interest are monoclonal antibodies that 

so neutralize the activity of BCF. Such monoclonals 
can be readily identified in BCF activity assays. 
High affinity antibodies are also useful in im- 
munoaffinity purification of native or recombinant 
BCF. 

55 Anti-BCF antibodies will also be useful in di- 
agnostic applications. For example, bone isolated 
from osteoporosis patients may show that it is 
deficient in BCF. Thus, the present invention con- 



10 



19 



EP 0 401 055 A2 



20 



templates a method, particularly a diagnostic meth- 
od, in which a bone sample from a human (or other 
mammal) is provided, and 'the amount of BCF is 
quantitatively measured in an assay. Antibody spe- 
cific for BCF could be formulated into any conven- 
tional immunoassay format; e.g., homogeneous or 
heterogeneous, radioimmunoassay or ELISA. The 
various formats are well known to those skilled in 
the art. See, e.g., "Immunoassay: A Practical 
Guide" (D.W. Chan and M.T. Perlstein, eds. 1987) 
the disclosure of which is incorporated herein by 
reference. Quantitative assays other than im- 
munoassays could also be used to measure the 
relative levels of BCF compared to a standard or 
prior observed BCF level in a patient. 

The following examples are provided by way of 
illustration but are not intended to limit the inven- 
tion in any way. 



EXAMPLE 1 



Sequence Analysis of BCF 



The 22K proteins of interest, partially purified 
from human and bovine sources as described by 
Urist, et al., Proc. Nat. Acad. Sci. USA , 81, 371-375 
(1984), were further purified to homogeneity by 
preparative gel electrophoresis and electroelution 
(M.W. Hunkapiller, E. Lujan, F. Ostrander and LE. 
Hood, Methods in Enzymology , 9V. 227-236 
(1983)). This purification showed that the initial 
partially purified samples contained, in addition to 
the 22K BCF, other mammalian proteins at 34K, 
19K, 14K and 6K. After precipitation with acetone 
(W. H. Konigsberg and L Henderson, Methods in 
Enzymology , 91: 254-259 (1983)) and quantitation 
by amino acid analysis (B.A. Bidlingmeyer, S.A. 
Cohen and T.L. Tarvin, Journal of Chromatography , 
336: 93-104 (1984)), the material was reduced un- 
deF denaturing conditions with 2-mercaptoethanol 
and cysteine residues were derivatized with 4-vinyl- 
pyridine (M. Friedman, LG. Krull and J.F. Cavins, 
Journal of Biological Chemistry , 245 : 3868-3871 
(1970)). After exhaustive dialysis to remove the 
denaturant, protein recovery was assessed by a 
repetition of amino acid analysis. The proteins were 
digested with TPCK-trypsin in the presence of 2M 
urea to generate unblocked peptide fragments suit- 
able for sequence analysis (G. Allen, Sequencing 
°* Proteins and Peptides , pages 51-62 (1981), 
Elsevier/North Holland Publishing Company, Am- 
sterdam, Holland). Products of the"digestion were 
resolved by reverse-phase high performance liquid 
chromatography using gradients of acetonitrile or 



acetonitrile/isopropanol in aqueous trifluoroacetic 
acid (J.E. Shively, Methods of Protein Microcharac- 
terization , pages 41-87 (1986), Humana Press, Clif- 
ton, New Jersey). Peptide fractions were subjected 

5 to automated Edman degradation using an Applied 
Biosystems 470A protein sequencer (M.W. Hun- 
kapiller, R.M. Hewick, W.J. Dreyer and LE. Hood, 
Methods in Enzymology , Sh: 399-413 (1983)). The 
phenylthiohydantoin amino acid derivatives were 

70 identified by chromatography on an Applied 
Biosystems 120A PTH analyzer (M.W. Hunkapiller. 
Applied Biosystems , User Bulletin Number 14 
(1985), Applied Biosystems, Foster City. Califor- 
nia). The hBCF sequence determined by this meth- 

75 od is confirmed by the sequence deduced from .the 
human cDNA in FIG. 1 . 



EXAMPLE 2 

20 



RNA Isolation 

25 . mRNA was isolated from fresh 7-month old calf 
bones (obtained from Rancho Veal Meat Packers, 
Petaluma, CA) or from human osteosarcoma cells. 

Calf femur midshafts were scraped free of con- 
nective tissue and marrow, broken into coarse frag- 

30 ments, and frozen at -80 'C. Human osteosarcoma 
cells were also frozen at -80* C. RNA was isolated 
from both the frozen tissues by the guanidinium 
thiocyanate/CsCl method (Maniatis^T., Fritsch, E.F. 
and Sambrook, J. Molecular Cloning: A Laboratory 

35 Manual (Cold Spring Harbor Lab., Cold~Spring Har- 
bor, NY (1982); Freeman, G.J., Clayberger, C, De- 
Kruyff, R., Rosenblum, D.S. and Cantor. H. Proc. 
Natl. Acad. Sci.: USA , 80: 4094-4098 (1983)). An 
osterizer was used to pulverize the bovine tissue 

40 directly in the guanidinium thiocyanate extraction 
solution. Poly(A)* RNA was purified by a single 
fractionation over oligo(dT)-Cellulose (Maniatis, et 
al., supra ). 

45 

Construction of the cDNA Libraries 

First strand cDNA was synthesized from bovine 
matrix or human osteosarcoma poly(A) RNA using 

so conditions similar to Okayama and Berg (Okayama, 
H. and Berg, P. Molec. and Cell Biol. 3: 4094-4094 
(1983)). About 5ug of poly(A) RNA in 20ul 5mM 
tris-hydrochloride (pH 7.5) was heated to 65' C for 
3 min, then quick cooled on wet ice and imme- 

55 diately adjusted (at room temperature) to contain 
50mM Tris-hydrochloride (pH 8.3 at 42* C), 8mM 
MgCI 2 , 30mM KCI. 10mM dithiothreitol, 2mM each 
of dATP, dGTP, dTTP and [a-^PJdCTP- 



11 



21 



EP 0 401 055 A2 



22 



(~300cpm/pmol), 60 units RNasin, and 2.5 ug oligo 
(dT)i2-i8 (total volume 40 ml). The reaction was 
initiated by the addition of 50-60 units of cloned 
moloney murine leukemia virus reverse transcrip- 
tase and continued for 60 min at 42 °C. Double- 
stranded (ds) cDNA synthesis and EcoRl linker 
addition were performed by two different methods. 
Initially, the second cDNA strand was synthesized 
by the method of Wickens et al. (Wickens, M.P., 
Buell, G.N. and Schimke, R.T. J. Biol. Chem. 253: 
2483-2495 (1978)). The hairpin loop was removed 
from the ds cDNA by SI nuclease, methylated with 
EcoRl methylase, made blunt-ended with T* DNA 
polymerase, ligated to phosphorylated EcoRl link- 
ers and finally digested with EcoRl (Maniatis, et al., 
supra) . Later, the second cDNA strand was syn- 
thesized by the method of Gubler and Hoffman 
(Gubler, U. and Hoffman, B.J. Gene 25: 263-269 
(1983)) as modified by Aruffo and Seed~(Aruffo, A. 
and Seed, B. Proc. Natl. Acad. ScL: USA 74: 8573- 
8577 (1987)). The ds cDNA was then ligated to 
asymmetrically (hemi) phosphorylated EcoRl 
adapters (see oligonucleotide synthesis) as de- 
scribed by Aruffo and Seed, supra , phosphorylated 
with Ta polynucleotide kinase (Maniatis, et al., 
supra) , adjusted to 0.5M NaCl, 25mM EDTA and 
heated at 75 °C for 15 min to inactivate the poly- 
nucleotide kinase. The ds cDNA prepared by both 
procedures was separated from uniigated 
linkers/adapters by chromatography on Biogel A- 
15m and recovered by ethanol precipitation. cDNA 
was ligated to XZAP arms (Stratagene) with T* 
DNA ligase (New England Biolabs) as described by 
supplier, but included 15% polyethylene glycol 
(PEG) 8000 (Sigma), a modification described by 
Pfeiffer and Zimmerman (Pheiffer, B.H. and Zim- 
merman, S.B. Nucl. Acids Res. 11: 7853-7871 
(1983)). The ligated DNA was recovered by cen- 
trifugation (12,000 xg), washed with chloroform, 
dried, resuspended in 4ul water and incubated with 
an in vitro packaging extract (Stratagene) according 
to supplier. Recombinant phage were propagated 
in E. coli BB4 (Stratagene). 



EXAMPLE 3 



Synthesis of Oligonucleotides 



Oligonucleotides were synthesized by the 
phosphoramidite method with an Applied 
Biosystems (Foster City, CA) model 380A syn- 
thesizer, purified by polyacrylamine gel elec- 
trophoresis and desalted on a Waters SEP-PAK 
fiB) cartridge. A 10-mer oligonucleotide 



(5 CCGAATTCGG3 ) was synthesized and used as 
the EcoRl linker for cDNA library construction. Prior 
to ligation, the linker was phosphorylated with Ta 
polynucleotide kinase (Maniatis, T., Fritsch, E.F. 

5 and Sambrook, J M Molecular Cloning: A Laboratory 
Manual (Cold Spring Harbour Lab., Cold Spring 
Harbor, NY, 1982)). A 14-mer oligonucleotide 
(5CCTGTAGATCTCCG3) and a 18-mer 
oligonucleotide (5'AATTCGGAGATCTACAGG3') 

to were synthesized and used as the EcoRl adapters. 
The 14-mer was phosphorylated (Maniatis, et aL, 
supra ) and subsequently heated to 95* C for 15 
min to inactivate polynucleotide kinase, prior to 
annealing with the 18-mer. These asymmetrically 

j 5 phosphorylated adapters also contained an internal 
Bglll restriction enzyme site; Based on the amino 
acid sequence of the human BCF tryptic fragment, 
a two-fold degenerated 45-mer oligonucleotide 
probe was designed (FIG. 2, probe A) following the 

20 rules of Lathe (Lathe, R., J. Mol. Biol. 183 : 1-12 
(1985). The two oligonucleotide probes (A and B) 
were synthesized based on the amino acid se- 
quence of a purified tryptic peptide of the human 
bone calcification factor (hBCF) (FIG. 2). 

25 

EXAMPLE 4 



30 

Screening of the cDNA Libraries 



35 a. Human osteosarcoma libraries. 

Approximately 300,000 recombinant phage 
were plated (50,000 phage/1 37mm dia. plate) in E. 
coli BB4, grown for 5-6 h at 37 *C, transferred to 

40 nitrocellulose filters (Millipore, HATF 137), pro- 
cessed according to Benton and Davis (Benton, 
W.D. and Davis, R.W., Science 196 : 180 (1977)) 
and screened with oligonucleotide probe A. Eigh- 
teen putative hBCF cDNA clones from 300,000 

45 recombinants were identified. Southern blot analy- 
ses (described below) of the cDNA inserts were 
performed using probe A and also probe B (FIG. 
2), a fully degenerate 18-mer oligonucleotide con- 
tained within probe A. Most of the clones hybridiz- 

50 ed to both probes. The probe was end-labeled with 
Ja polynucleotide kinase and [7 32 -P]ATP (Maniatis, 
et aL, supra ) to a specific activity of 1-2x10 8 
cpm/ug. The filters were prehybridized for 1-2 h at 
37° C in 20% (vol/vol) formamide, 5xSSC (1xSSC 

55 = 0.15 M sodium chloride/0.1 5M sodium citrate, 
pH 7), 5x Denhardt's solution (1x Denhardt's solu- 
tion = 0.02% polyvinylpyrrolidone/0/02% 
Filcoll/0/02% bovine serum albumin), 10% dextran 



12 



23 



EP 0 401 055 A2 



24 



sulfate, 50mM sodium phosphate pH 6.8, 1 mM 
sodium pyrophosphate, 0.1% NaDodSO* and 
50ug/ml denatured salmon sperm DNA. Labeied 
probe was added to a concentration of lO G cpm/ml 
and hybridization was continued overnight at 37* C 
with gentle shaking. The filters were washed twice 
(20 min/wash) in 2XSSC, 0.1% NaDodSO* at 55° C 
and exposed to Kodak XAR-2 film with a Dupont 
Lightning Plus intensifying screen overnight at - 
80* C. Areas of plaques giving signals on duplicate 
filters were picked, replated and rescreened as 
above until pure plaques were obtained. 

Two of the double positive clones (Ost 3-7 and 
Ost 3-17, FIG. 3) were sequenced and shown to 
contain identical overlapping sequences as well as 
a region encoding the tryptic fragment (FIG. 1 
underlined and FIG. 2). Ost 3-17 contains the com- 
plete mature protein coding sequence, but not the 
complete signal peptide, as is evident from the 
bBCF cDNA shown in FIG. 1. 

An additional 300,000 recombinant phages 
from two different osteosarcoma cDNA libraries 
were later plated and screened as above but with 
the following changes: (1) The hybridization mix 
contained 40% formamide, 5xSSC, 5X Denhardt's 
solution, 10% PEG 8000, 50mM sodium phosphate 
pH 6.8, 0.5% NaDodSOA and 50ug/ml denatured; 
(2) the filters washed at 65 *C in 2xSSC, 0.1% 
NaDodSO*; and (3) the probe was a 240bp. DNA 
fragment obtained by digesting cDNA clone Ost 3- 
7 with Bglll and Asp718 (probe C, FIG. 3). The 
probe was purified and labeled by the oligo-primer 
method (Feinberg, A.P. and Vogelstein, B„ Anal. 
Biochem. 137 : 266-267 (1984)) to a specific activity 
of >1 x 10 s cpm/ug. Approximately 20 clones from 
each library gave strong hybridization signals and 
restriction enzyme analysis of these clones iden- 
tified several that were longer than Ost 3-17. One 
of these, Ost 1-7 (FIG. 3), was sequenced and 
judged to be full length based on its homology to 
the bovine BCF cDNA clone, described below. 



b. Bovine bone matrix cDNA library. 

Approximately 300,000 recombinants from the 
bovine bone matrix cDNA library were screened 
with probe C (FIG. 3), a 240b.p. Bglll - Asp 718 
DNA fragment from Ost 3-7, under the conditions 
described for probe A except that formamide was 
omitted from the hybridization solution. The filters 
were washed at 55 °C in 2xSSC, 0.1% NaDodSO*. 
Twenty-four positive plaques were identified. Clone 
bb1 .1 -7 (FIG. 4), which contained the largest insert, 
was sequenced and shown to contain sequences 
homologous to hBCF. The deduced amino acid 
sequence of the bBCF cDNA indicates that amino 
acid -4 is not the initiation codon due to the pres- 



ence of a Val at this position. The most likely 
initiation codon is located at amino acid position 
-17 which is 28 nucleotides beyond the s' end of 
the hBCF Ost 3-17 clone. The Met at position -17 
5 is also preceded by an acceptable ribosome bind- 
ing site. 



EXAMPLE 5 

70 



Subcloning, Sequencing and Analysis 

75 

Recombinant plasmids were released in the 
Bluescript SK(-)vector from XZAP by the M13 
rescue/excision protocol described by the supplier 
(Stratagene). The plasmids were propagated in E. 

20 coli BB4, and plasmid DNA was isolated by the 
alkaline lysis method (Maniatis, et a!., supra ). cDNA 
inserts were excised with either EcoRI or Bglll 
restriction enzymes (Boehringer-Mannheim), puri- 
fied by polyacrylamide gel electrophoresis 

25 (Maniatis, et at., supra) and passage over an Elutip- 
d column "(Schleicher and Schuell) and subcloned 
into the M13 sequencing vectors (Yanisch-Perron, 
C. Viera, J. and Messing, J., Gene 33: 103-119 
(1985)). DNA sequencing was performed by the 

30 dideoxy chain termination method (Sanger, F. Nic- 
klen, S. and Coulson. A.R., Proc. Nati. Acad. Sci. 
USA 74: 5463-67 (1977)) using M13 primers as 
well as specific internal primers. Ambiguous re- 
gions were resolved 7-deaza-2-deoxyguanidine- 

35 triphosphate (Barr, P.J., Thayer, R.M., Layboum, 
P., Najarian, R.C., Seela, F., and Totan, D., Biotech- 
niques 4: 428-32 (1986)) and sequenase (U.S. 
BiochemTcals). 

40 

a. Northern Blot 

Poly (A)* RNA was fractionated on a 1.4% 
agarose gel in the presence of formaldehyde 

45 (Lehrach, H„ Diamond, D., Wozney, J.M. and 
Boedtker, H., Biochemistry 16: 4743-51 (1977)) and 
directly transferred to nitrocellulose according to 
Thomas (Thomas, P., Proc. Natl. Acad. Sci. USA 
77: 5201-5 (1980)). Filters were hybridized with 

so probe C as previously described (EXAMPLE 4, 
Screening of the cDNA Libraries) in the 40% for- 
mamide containing hybridization solution and were 
washed at 55° C in 2xSSC, 0.1% NaDodSO* and 
O.lxSSC, 0.1% NaDodS04 with autoradiography 

55 following each set of washings. 

RNA transfer blot analysis demonstrates the 
presence of two mRNA forms of «0.9 and 1.8kB in 
human osteosarcoma tissue. The two forms were 



13 



25 



EP 0 401 055 A2 



26 



also observed in human placenta but were absent 
in a human liver cell line, HEPG2. The two forms 
were also observed in bovine bone matrix cells 
with the larger form predominating. A trace of the 
large species could also be seen in bovine bone 
marrow. The two mRNAs are most likely generated 
by differential polyadenylation at the 2 sites 
(AATAAA) found in the 3 untranslated region. 



b. Southern Blot 



1. Genomic 

lOug of genomic DNA (Clontech) was digested 
with EcoRI, fractionated on 0.7% agarose gels and 
transferred to nitrocellulose according to Maniatis. 
et a!., supra . Hybridization and washing conditions 
were identical to those described in a. above. 

Genomic DNA transfer blot analysis suggests 
that hBCF and bBCF are single copy (or low copy) 
genes due to the few bands seen in the EcoRI 
digest. 



2. cDNA Clones 

DNA from cDNA clones were digested with 
EcoRI or BglH, fractionated on 1 .0% agarose gels, 
transferred to nitrocellulose (Maniatis, et al., supra) 
and hybridized with probe A as previously de- 
scribed or with probe B in a tetramethylammonium 
chloride containing hybridization solution under 
conditions described by Wood (Wood, W.I., Git- 
schier, J., Laskey, L. and Lawn, R., Proc. Natl. 
Acad, Sci. USA 82: 1585-88 (1985)). 



EXAMPLE 6 



Expression of hBCF in Yeast 



Yeast expression vectors were constructed for 
intracellular production or secretion of hBCF from 
the ADH2/GAPDH regulatable promoter. Since the 
first cDNA clone did not contain DNA encoding a 
signal peptide, methionine-4, was used as the N- 
terminal amino acid for these constructions. Subse- 
quent cDNA analyses, and identification of a clas- 
sical signal peptide, and a signal peptidase 
cleavage site, allowed construction of a yeast a- 
factor/hBCF fusion with glutarnine +1 as the N- 
terminal amino acid of recombinant hBCF. For 
cloning into expression vectors, natural Nco-1, Bgl- 



1 and Spe-1 sites were used together with the 
synthetic oligonucleotide adapters shown (FIG. 4). 
Since Spe-1 and Xba-1 give the same restriction 
enzyme overhang, the initial construction was sim- 

5 ply an insertion of the Nco-1 /SPE-1 hBCF gene 
into Nco-1 /Xba-1 digested pBSlOOhaFGF, a vector 
containing ADH2/GAPDH promoter and GAPDH 
terminator elements flanking a synthetic human 
acidic fibroblast growth factor (FGF) gene. The 

to haFGF gene contains an Nco-1 and unique Xba-1 
sites. The resulting plasmid, pBSlOOhBCF, was 
used for further constructions. Thus, the Nco-1/Sal- 
1 fragment containing the hBCF gene was cloned 
together with BAMH1 /Nco-1 fragments encoding 

75 the GAPDH and ADH2/GAPDH promoters into 
BamH1/Sal-1 digested pBS24.1. The resulting plas- 
mids, pBS24A/GhBCFKQ (-4 to 183) and pBS24 
GAPhBCF (-3 to 183), were used to direct intracel- 
lular expression of hBCF. For secretion, BgM/Sal-1 

20 fragments were excised and cloned into pBS24.1 
together with BamH1 /Xba-1 fragments encoding 
the ADH2/GAPDH promoter, the yeast o-factor se- 
cretory signal/leader sequence, and the synthetic 
linkers shown in FIG. 4 (boxed), for expression of 

25 hBCF(-4 to 183) and hBCF(1-183). yeast cells 
transformed with expression plasmids encoding 
ADH2/GAPDH promoter-hBCF (-4-183) and GAPDH 
promoter-hBCF(-4-183) fusions, as analyzed by 
SDS-PAGE and Coomassie blue staining of total 

30 proteins, showed no expression as compared with 
control yeast cells. Therefore, to study secretion 
systems, the pBS24 plasmids containing a-factor 
leader-hBCF (-3-183) and hBCF (1-183) fusions 
were -constructed. In each case, transcription was 

35 driven by the ADH2/GAPDH promoter. Yeast strain 
AB110 was transformed with the yeast expression 
plasmids, and yeast supernatants were analyzed 
by precipitation with 10% trichloroacetic acid and 
separation by SDS-PAGE. High levels of expres- 

40 sion of the approximately 22kD product was ob- 
served by Coomassie blue staining, when com- 
pared with control yeast cells transformed with the 
parent yeast vector pBS24. The transformant 
AB110 (pBS24.1 22kQ) is deposited under acces- 

45 sion number ATCC 20948. The active lot of yeast 
cells comprised a pool of two lots, KO-2 and KQ-3, 
from which the recombinant BCF was isolated and 
purified as follows. 

50 

Lot KQ-2 

The cells were removed by centrifugation from 
the fermentation medium, and the medium con- 
55 centrated using a YM10 Amicon spiral cartridge. 
The concentrate was diafiltered into water, then 20 
mM Tris-CI, 1 mM EDTA, 3 M NaCI, pH 7.5. This 
was passed over a column of prep grade Superose 



14 



27 



EP 0 401 055 A2 



28 



12 at 4* C, then at room temperature. The 22KBCF 
did not stick to either column. The flowthrough was 
purified by adsorption onto Superose 12 HR 10/30, 
and eluted with the same buffer but with 1 M NaCi. 



Lot KQ-3 

The cells were removed and the medium con- 
centrated as described above. The concentrate was 
diafiltered against water, then 20 mM Tris-Cl, 1 mM 
EDTA, pH 7.5. This was loaded onto a Mono-Q HR 
10/10 column, washed, and eluted with a gradient 
to 0.5 M NaCl. The 22KBCF-containing fractions 
were identified by gel electrophoresis, pooled, ad- 
justed to 3 M NaCl with solid NaCl, and loaded 
onto a Superose 12 HR 10/30 column. The 22 
KBCF was eluted with buffer containing 1 M NaCl. 



Lot KQ-2/3 

The Superose eluates (from Lots KQ-2 and 
KQ-3) were pooled, concentrated using YM 10 
membrane in Amicon stirred cell, dialyzed versus 
water, and lyophilized. 

Alternatively, the recombinant BCF may be iso- 
lated and purified as follows. 

The media is removed from the cells and con- 
centrated approximately ten fold. The pH is ad- 
justed to 7.5, the concentrate is diluted to a con- 
ductivity below 5 mS/cm, then applied to Fast Flow 
Q ion-exchange resin pre-equilibrated with 50mM 
Tris/Hcl, 1mM EDTA, 1mM PMSF, pH 7.5. The 
column is washed with 1 column volume of the 
above buffer and eluted using a 0-1 M NaCl salt 
gradient in the above buffer. The 22KBCF is eluted 
at a conductivity of 20-30 mS/cm, which is con- 
firmed using SDS-PAGE. The 22KBCF containing 
fractions are pooled, the pH maintained at 7.5 and 
made 4M with respect to Urea, concentrated, and 
run over a S-100 sizing column in 4M Urea, 
100mM Tris/HCI, 1mM EDTA, 1mM PMSF at pH 
7.5. The 22KBCF containing fractions are identified 
by SDS-PAGE, pooled, concentrated, and dialysed 
against 10mM ammonium bicarbonate pH 7.8. The 
22 KBCF is then lyophilized and may be stored dry 
at4"C. 



EXAMPLE 7 



A sample of purified recombinant human BCF- 
(rhBCF) expressed as in Example 6, 1 mg (lot KQ 
2/3), was dissolved in water containing 5 mg of 
human fibrin. The rBCF-fibrin composite was 
lyophilized and implanted in the mouse thigh mus- 



cle pouch. In another mouse, bovine matrix Gla 
protein was dissolved in 6M urea containing 1 mg 
of human rhBCF and dialyzed against water. The 
precipitate and supernatant were lyophilized to pre- 

5 pare a composite of MGP and hBCF proteins. The 
composite as implanted in the quadriceps pouch. 
For controls, the contralateral thighs were implant- 
ed with bovine matrix Gla protein plus albumin in 
one mouse; in the other mouse the control con- 

70 sisted of 5 mg of human fibrin and 1.0 mg of 
albumin. 

Microradiographs of the rBCF-matrix Gla pro- 
tein composite showed areas of calcified tissue. 
Histological sections showed small round cells, 

75 multinucleated cells, macrophages with hyperplasia 
and hypertrophy of mesenchymal type cells. There 
were plates of calcified ground substance but no 
cartilage or bone cells. FIG. 5 is a photomicrograph 
showing hyperplasia and hypertrophy of connective 

so tissue cells on the surface of a composite implant 
(indicated by P) of recombinant protein hBCF (Lot 
KQ 2/3, 1 mg) and 1 mg of human matrix Gla 
protein. Calcification of the implanted proteins is 
indicated by CP in the quadriceps* pouch of a 

25 mouse on day 21. The fibrous connective tissue 
envelope is indicated by F. 

FIG. 6 is a photomicrograph showing islands of 
calcified protein (CP) induced by a composite of 
recombinant hBCF and biological human matrix Gla 

30 protein. Note the large foam cell (arrow). The entire 
implant is enclosed in a fibrous capsule (F) by day 
21. 

FIG. 7 is a photomicrograph showing a com- 
posite of recombinant hBCF and matrix Gla protein 

35 on day 21. Note the highly vascular interior of the 
implant including small round celts, multinucleated 
cells, macrophages, calcifying intercellular sub- 
stance (CP) and large foam cells (arrow). All of the 
implanted protein and reactive tissue was enclosed 

40 in a fibrous envelope (F) on day 21 . 



Claims 

45 1. A composition comprising a polypeptide se- 

lected from mammalian bone calcification factor 
and analogs thereof substantially free of other 
osteoinductive associated factors. 

2. A composition according to Claim 1, wherein 
so said mammalian bone calcification factor is human 

BCF. 

3. A composition according to -Claim 1, wherein 
said mammalian bone calcification factor is bovine 
BCF. 

55 4. A composition according to Claim 2 wherein 
said polypeptide comprises an amino acid se- 
quence as shown in FIG. 1 A or a fragment thereof. 
5. A composition according to Claim 3 wherein 



15 



29 



EP 0 401 055 A2 



30 



said polypeptide comprises an amino acid se- 
quence as shown in FIG. 1 B or a fragment thereof. 

6. A composition according to Claim 4, wherein 
said polypeptide comprises an amino terminus se- 
lected from the signal peptide sequence met-asp- 
leu-ser-leu-leu-trp-val-leu-leu*pro-leu-val-thr-met- 
ala-trp-gly and sequences of said signal peptide 
sequence derived by deletion of one or more ami- 
no acids from the amino terminus of said signal 
peptide sequence. 

7. A composition according to Claim 5, wherein 
said polypeptide comprises an amino terminus se- 
lected from the signal peptide sequence met-asp- 
leu-thr-leu-leu-trp-val-leu-leu-pro-leu-val-thr-val-ala- 
trp-gly and sequences of said signal peptide se- 
quence derived by removal of one or more amino 
acids from the amino terminus of said signal pep- 
tide sequence. 

8. Non-chromosomal DNA as shown in FIG. 

1C. 

9. DNA complementary to the sequence shown 
in FIG. 1C. 

10. Non-chromosomal DNA as -shown in FIG. 

1D. 

11. DNA complementary to the sequence 
shown in FIG. 1 D. 

12. Non-chromosomal DNA according to Claim 
8 further comprising the 5 sequence selected from 
the leader sequence ATG GAC CTC AGT CTT 
CTC TGG GTA CTT CTG CCC CTA GTC ACC 
ATG GCC TGG GGC and sequences derived by 
removal of one or more deoxy nucleotides from the 
5 -terminus of said leader sequence. 

13. Non-chromosomal DNA according to Claim 
10 further comprising the 5 sequence selected 
from the leader sequence ATG GAC CTC ACT 
CTT CTG TGG GTG CTT CTG CCA CTG GTC 
ACC GTG GCT TGG GGA and sequences derived 
by removal of one or more deoxynucleotides from 
the 5-terminus of said leader sequence. 

14. A method comprising the steps of 

(a) constructing a vector which includes the 
DNA sequence shown in FIG. 1C or a fragment 
thereof, 

(b) transforming a host cell with said vector, 

and 

(c) culturing the resultant transformed cell 
under conditions to express the peptide encoded 
by said DNA sequence or fragment thereof. 

15. A method comprising the steps of 

(a) constructing a vector which includes the 
DNA sequence shown in FIG. 1 D or a calcification- 
initiating fragment thereof, 

(b) transforming a host cell with said vector, 

and 

(c) culturing the resultant transformed cell 
under conditions to express the peptide encoded 
by said DNA sequence or fragment thereof. 



16. A method according to Claim 14 wherein 
said DNA sequence further comprises a 5 se- 
quence selected from the leader sequence ATG 
GAC CTC AGT CTT CTC TGG GTA CTT CTG 

5 CCC CTA GTC ACC ATG GCC TGG GGC and any 
sequence derived by removal of one or more deox- 
ynucleotides from the 5 -terminus of said leader 
sequence. 

17. A method according to Claim 15 wherein 
10 said DNA nucleotide sequence further comprises a 

5 sequence selected from the leader sequence 
ATG GAC CTC ACT CTT CTG TGG GTG CTT 
CTG CCA CTG GTC ACC GTG GCT TGG GGA 
and any sequence derived by removal of one or 
75 more deoxynucleotides from the 5'-terminus of 
said leader sequence. 

18. A method according to any of Claims 14 to 
17 wherein said host cell is eukaryotic. 

19. A method according to Claim 18 wherein 
20 said host comprises yeast. 

20. A method according to Claim 18 wherein 
said vector comprises a GAPDH promoter which 
controls expression of said peptide. 

21. A method according to Ciaim 18 wherein 
25 said promoter comprises an ADH2/GAPDH pro- 
moter which controls expression of said peptide. 

22. A replicable vector comprising DNA ac- 
cording to any of Claims 8, 10, 12 or 13. 

23. A host cell transformed with a replicable 
30 vector according to Claim 22. 

24. A composition for including calcification, 
comprising an effective calcification-inducing 
amount of a mixture of mammalian bone calcifica- 
tion factor or an analog thereof, and matrix Gla 

35 protein. 

25. A composition according to claim 24 
wherein said factor comprises human bone cal- 
cification factor. 

26. A composition according to Claim 24 
40 wherein said factor comprises bovine bone cal- 
cification factor. 

27. A composition according to Claim 25 
wherein said factor has an amino acid sequence as 
shown in Fig. 1 A, or a fragment thereof. 

.45 28. A composition according to Claim 26 
wherein said factor has an amino acid sequence as 
shown in Fig. 1B, or a fragment thereof. 

29. A method for inducing calcification for the 
formation of bone in a vertebrate, comprising the 

so step of administering to said vertebrate in a phar- 
maceutical ly effective manner an effective 
calcification-inducing amount of a composition ac- 
cording to any one of Claims 24 to 28. 

30. A diagnostic method comprising the steps 
55 of providing a bone sample from a mammal and 

measuring the amount of mammalian bone cal- 
cification factor in said sample in a quantitative 
assay. 



16 



31 EP 0 401 055 A2 32 



31. A composition comprising antibodies rec- 
ognizing an epitope unique to mammalian bone 
calcification factor. 



w 



15 



20 



25 



30 



35 



40 



45 



50 



55 



17 



EP 0 401 055 A2 



FIG. I 



Human GCCAAAATCCCAGGCAGC AT6 GAC CTC AGT CTT CTC TGG GTA CTT CT6 CCC 
Bovine AGAAGCCCAGACGGC ATG GAC CTC ACT CTT CTG TGG 6TG CTT CTG CCA 

h-Ostl-7 -17 -10 K0st3-I7 

Human HET ASP LEU SER LEU LEU TRP VAL LEU LEU PRO 

Bovine HET ASP LEU THR LEU LEU TRP VAL LEU LEU PRO 



Human CTA GTC AGC ATG GCC TGG GGC CAG TAT GGC GAT TAT GGA TAC CCA TAC 
Bovine CTG GTC ACC GTG GOT TGG GGA, CAG TAT GGT GAC TAT GGG TAC TCC TAT 

{ I K— 0st3-7 



Human LEU VAL THR 
Bovine LEU VAL THR 



HET 
VAL 



ALA TRP GLY'GLN TYR GLY ASP TYR GLY TYR 
ALA TRP GLY GLN TYR GLY ASP TYR GLY TYR 



PRO 
SER 



TYR 
TYR 



Human CAG CAG TAT CAT GAC TAC AGC GAT GAT GGG TGG GTG AAT TTG AAC CGG 
Bovine CAT CAG TAC CAT GAC TAC AGT GAC GAT GGG TGG GTG AAT CTG AAC CGG 



Human 
Bovine 



GLN 
HIS 



GLN TYR HIS ASP TYR SER ASP ASP GLY TRP VAL ASN LEU ASH ARG 
GLN TYR HIS ASP TYR SER ASP ASP GLY TRP VAL ASN LEU ASN ARG 



Human CAA GGC TTC AGC TAC CAG TGT CCC CAG GGG CAG GTG ATA GTG GCC GTG 
Bovine CAG GGC TTC AGC TAC CAG TGT CCC CAC GGG CAG GTG GTG GTG GCC GTG 



Human GLN GLY LEU SER TYR GLN CYS PRO 
Bovine GLN GLY LEU SER TYR GLN CYS PRO 



GLN 
HIS 



GLY GLN VAL 
GLY GLN VAL 



ILE 
VAL 



VAL ALA VAL 
VAL ALA VAL 



Human AGG AGC ATC TTC AGC AAG AAG GAA GGT TCT GAC AGA CAA TGG AAC TAC 
Bovine AGG AGC ATC TTC AAC AAG AAG GAA GGT TCC GAC AGA CAG TGG AAC TAC 



Human ARG SER ILE PIE 
Bovine ARG SER ILE PHE 



SER 
ASN 



LYS LYS GLU GLY SER ASP ARG GLN TRP ASN TYR 
LYS LYS GLU GLY SER ASP ARG GLN TRP ASN TYR 



Human GCC TGC ATG CCC ACG CCA CAG AGC CTC GGG GAA CCC ACG GAG TGC TGG 
Bovine GCC TGC ATG CCC ACA CCC CAG AGC CTG GGG GAG CCT ACG GAG TGC TGG 

Human ALA CYS HET PRO THR PRO GLN SER LEU GLY GLU PRO THR GUI CYS TRP 
Bovine ALA CYS HET PRO THR PRO GLN SER LEU GLY GLU PRO THR GLU CYS TRP 



Human TGG GAG GAG ATC AAC AGG GCT GGC ATG GAA TGG TAC CAG ACG TGC TCC 
Bovine TGG GAG GAG ATC AAC AGG GCT GGA ATG GAA TGG TAC CAG ACA TGC TCC 

Human TRP GLU GLU ILE ASN ARG ALA GLY HET GLU TRP TYR GLN THR CYS SER 
Bovine TRP GLU GLU ILE ASN ARG ALA GLY HET GLU TRP TYR GLN THR CYS SER 



EP 0 401 055 A2 



FIG. I (CONT.) 



Human AAC AAT GGG CTG GTG GCA GGA TTC CAG AGC CGC TAC TTC GAG TCA GTG 
Bovine AAC AAT GGA CTG GTG GCA GGA TTC CAG AGC CGC TAC TTC GAG TCA GTG 

Human ASN ASN GLY LEU VAL ALA GLY PHE GLN SER ARG TYR PHE GUI SER VAL 
Bovine ASN ASN GLY LEU VAL ALA GLY PHE GLN SER ARG TYR PHE GLU SER VAL 

Human CTG GAT CGG GAG TGG CAG TTT TAC TGT TGT CGC TAC AGC AAG AGA TGC 
Bovine CTG GAT CGC GAG TGG CAA TTT TAC TGC TGT CGC TAC AGC AAG AGA TGC 

Human LEU ASP ARG GLU TRP GLN PHE TYR CYS CYS ARG TYR SER LYS ARG CYS 
Bovine LEU ASP ARG 6LU TRP GLN PHE TYR CYS CYS ARG TYR SER LYS ARG CYS 

Human CCA TAT TCC TGC TGG CTA ACA ACA GAA TAT CCA GGT CAC TAT GGT GAG 
Bovine CCA TAT TCC TGC TGG. CTG ACA ACA GAA TAT CCA GGC CAC TAT GGT GAG 

Human PRO TYR SER CYS TRP LEU THR THR GLU TYR PRO GLY HIS TYR GLY GLU 
Bovine PRO TYR SER CYS TRP LEU THR THR GLU TYR PRO GLY HIS TYR GLY GUI 



Human GAG ATG GAC ATG ATT TCC TAC AAT TAT GAT TAC TAT ATC C6A GGA GCA 
Bovine GAG ATG GAC ATG ATT TCC TAC AAT TAT GAT TAC TAT ATG C6A GGG GCA 



Human GLU MET ASP MET ILE SER TYR ASN TYR ASP TYR TYR 
Bovine GLU MET ASP NET ILE SER TYR ASN TYR ASP TYR TYR 



ILE 
NET 



ARG GLY 
ARG GLY 



Human ACA ACC ACT TTC TCT GCA GTG GAA AGG GAT CGC CAG TGG AAG TTC ATA 
Bovine ACA ACC ACT TTC TCT GCA GTG GAA AGG GAT CGC CAG TGG AAA TTC ATA 

Human THR THR THR PHE SER ALA VAL GLU ARG ASP ARG GLN TRP LYS PHE ILE 
Bovine THR THR THR PHE SER ALA VAL GLU ARG ASP ARG GLN TRP LYS PHE ILE 



Human ATG TGC CGG ATG ACT GAA TAC GAC TGT GAA TTT GCA AAT GTT TAG 
Bovine ATG TGC CGG ATG ACT GAC TAT GAC TGT GAA TTT GCA AAT GTT TAG 



Human HET CYS ARG HET THR 
Bovine HET CYS ARG HET THR 



GLU 
ASP 



TYR ASP CYS GUI PHE ALA ASN VAL 
TYR ASP CYS GLU PHE ALA ASN VAL 





cc 








>< 


W 


| 














ft 








to 


h3 




o 




M 




a: 












8 












ft. 

Ml 








to 








< 


h 


3 












*a 


§ 










3 






Oi 






>« 


to 






H 


< 






ft 


































W 


B 


< 




to 


















3 


e> 












Oi 


OS 






AS 


TY 






to 


ft 






H 


&a 






X 


CO 






ct 


D 








W 






















1 
















52 


















3 






ft 








>< 




c 




H 




id 








E 




O 


Z 


. 3 




DC 


to 


X 




P< 


< 



EP 0 401 055 A2 





EP 0 401 055 A2 



CD 

e> 



c 

> 

o 
(0 



PC 

PC 
►« 

to 

ft 

>< 

a 
cp 







04 


w 


g 


PC 






H 


z 




z 


CO 


g 




< 








•* 

a 






g 










ft 






H 








>; 


PC 






to 


CD 


CP 


to 




to 






M 






X 


CP 


Oi 


Q 


D 


to 








Cm 


CP 


pc 


to 


to 


W 






to 


(J 




PC 




to 






>< 


r~ . 
C-* 








P$ 


z 


10 


^ 


to 


< 


H 




to 


It 


W 


M 


W 


S3 


S3 


to 


0< 


Oi 


p- 


w 


^ 


to 




H 




M 


Z 




PC 






W 


CP 




to 


to 






M 




1 


w 


cp 




PC 


cp 




JM 




J 


H 






pc 






w 


to 




to 


< 


3 





Pk 




to 






„ -1 












PC 


Z 


DO 


PC 






EC 


w 


H 


cp 


04 


to 


g 


PC 


Pu 


PK 


Pk 








0* 










04 








PC 


K 






H 


< 




>* 


D 


PC 


to 






w 




$ 




to 


u 


D 


Eh 


s 5' 


to 


W 






>« 


^ 


§ 


CP 


CJ 


OC 




w 


PC 


W 








to 


C2> 


o« 


E-4 






>* 


w 


HP 








cp 






04 


9 


O 






PC 


PC 






fi. 
**4 


rid! 




0 




Z 




04 


X 


to 


1 


PC 


H 


<; 




H 




w 




D 






w 




04 


M 










>* 


CD 


w 




g 












to 


D 


z 


04 










u 


O 














'3 


S> 








H 




s 


DC 


O4 


0: 




>• 


OC 




1 


H 




to 






to 


to 


PC 


to 






w 




u 


u 


to 



PC 

to 



04 

PC 

» 

i 

PC 
H 
PC 

O 
CO 
»4 

O* 
PC 
H 

to 
o 

PC 
14 
CO 

o 

PC 

o< 

to 
u 
o 

PC 

< 



e> to ^ 

5 ^ s 

< > 

H 04 Z 

e g 5? 



PC 



to 



i 

CP 



1 



OC o w 
^ PC w 
H 3 O4 



04 04 



H > 
PC < 

to 



3 



PC CP to 

|H |M 

bi < U 



04 

PC 
H 
04 

to 
< 



W PC PC 

h3 W « 

M tO H 

H W H 

S S g 

O4 PC CP 

a e § 



to 



g i 

p PC H 

3 e s 



3 



w 



to 

Oi 



EP 0 401 OSS A2 



o 
o 













H 












< 


1 














O 




1 




& 




P 




Eh 












3 


g 




























1 










u 






u 


8 










u 



c 

ID 



i 
s 

u 

I 

s 

u 

s 

u 

< 

u 
u 



I 

3 

u 

s 

u 

H 
H 

&5 
% 

U 
O 

u> 
u 

si 



o 




O 


o 


< 








< 






eg 


3 


u 




a 


< 


u 


•< 


u 






u 




< 


u 








U> 




ft 


O 






.&* 


H 


o 




a 




< 

o 


H 






a 


CD 


O 




H 


O 


U 


«: 


<< 


u 


y 


a 


e> 




o 


*t; 




CD* 
<< 


o 




< 


u 






u> 


i 












u 


s 








a- 












O 


U 




H 


U 






o 


% 


U 


o 








3 


& 


3 


3 




u 






o 








o 


eg 


U 


tr> 




a 


o 


8 


o 


H 


O 




O 


H 


'SI 


CD 


CD 




H 


U 




a 




i 


o 


u 




Eh 









< 




H 


< 








fcH 


cd 












u 


u 




u 






H 












Eh 




u 




H 


< 


U 


y 


&H 








£ 










U 


Eh 


H 






L> 


O 


U 


U 


< 








CD 


CP 


u 






Eh 


U 


u 


o 


H 




r i 


U 


H 






g 




CD 


CD 


< 








<< 


H 


Eh 






o 














a 


O 










u 


H 




E< 




Eh 


o 




< 






y 


Eh 












H 


H 






P. 


f$ 


Eh 










O 








ft 




o 


H 


< 


Eh 




Eh 








U 


6 




§ 














U 


u 




O 






CD 


o 




Eh 


Eh 




CD 


o 








g 


H 


H 


s. 






o 


C!> 


U 


o 


U 




Eh 








H 




U 




Eh 


s 


Eh 




CD 




O 




Eh 


o 




o 


U 






u> 




u 


Eh 






u 








o 






i 


1 


s 


















H 




o 


< 






O 


H 


a 










a 


a 






% 


u 


c? 


u 








u 




o 




a 




Eh 




Eh 




o 




U 


< 




Eh 




u 


e> 


U 




u> 




H 




Eh 


<* 






Eh 



8 

Eh 



EP 0 401 055 A2 





u 


cd 


cd 














U 






o 




















cd 




H 


cd 


CD 




IS 










CD 


s 




u 


CD 






< 


cd 






CD 




CJ 




H 

8 


CD 


? 

cd 








u 






S 


< 






CD 


u 








u 








cj 




u 




u 


o 




GT 


GT 






< 


H 


£2 




















































S 


Si 
















U 






g 













8 



a 



o 

H 

CJ 

a 

cd 

cd 
cd 



cd 

CJ 
CJ 

§ 

u 

u 
u 
cj 

< 



u 
u 



0) 

c 

> 
o 

03 






<2 


52 
















3 




y 


u 




CJ 




H 




< 










y 


y 


cj 


u 


g 






CD 












a 


O 


< 




CD 


cd 


u 


r . 


CJ 




r > 




U 




H 




3 


CD 


gs 


3 








cd 




U 


< 




< 


CD 










3 




CJ 


y 




CD 


H 


g 


CJ 








id! 


H 


< 


H 




U 


cd 




CJ 


CD 


cd 


H 


<« 


cd 


* 




C5> 


cd 


u 




H 




cd 


U 


a 




cd 


CD 


CD 






CD 


CD 






H 


H 




CD 




U 








CD 


3 


a 


CD 


H 






(J 


(J 




i 


CD 


a 






O 
















§ 




cd 








cd 




CD 








H 


U 


8 




U 


U 






CD 


u 






H 


CD 






CD 


H 


U 




< 


3 






U 




8 




H 


< 







t! 


CD 




CD 






CD 


o 






CD 


















< 


u 




rj 


CD 


y 


U 


H 




CD 


H 




r % 






H 


< 






3 






H 


CD 


ft 


IS 




CD 






H 












cS 


% 


3 




y 


CD 


H 


< 


H 




H 


CD 




CJ 






U 






H 


CD 




H 


H 






a 






H 




CD 


CJ 


CD 




H 






H 






H 


CD 


9 




CD 






U 






8 




















1 


















1 




s 








H 






CD 


CD 




CD 


CD 





EP 0 401 055 A2 




EP 0 401 055 A2 



FIG. 4A 



A/G PROMOTER 



•4 -1 +1 

MEllALA.TRRGLY.GLN.TYR. GLY. . . 



OR GAPDH 

PROMOTER 



CC.ATG.GbC.TGG.GGC.CAG.TAT.GGC. . . 
GG.TAC.CQG.ACC.C CG.GTC.ATA.CCa , 

BgM 

INTRACELLULAR 



1 

NCO-1 



FIG. 4 

a-FACTOR L- ■ * 22K GENE 
^3. 




SER.LEU.ASP.LYS. ARG.lALA.TRP.GLY.GLN.TYR. GLY. . 



TCT CTA.GAT.AAA.AGA.GCC.TGG.GGC.CAG.TAT.GGC. . 



A6A.GAT.C tTA.TTT.TCT.CGG. ACC.CCG.GTp J\TA.CCG. 



Xba-1 BgM 

a-FACTOR L « 1 ' 22KQ GENE 



Jgln 



SER.LEU.ASP.LYS. ARGjGLN.TYR.GLY. . 



TCT jCTA.GA T.AAA.AGA.C AG.TAr r.GGC. 



AGA.GAT.CTA.TTT.TCT.G1CJVTA. CCG. 



T 



Xba-1 BgM 

SECRETION 
(A/G PROMOTER) 



EP 0 401 055 A2 



FIG. 5 




EP 0 401 055 A2 



FIG.6 



EP 0 401 055 A2 



FIG. 7 





Europalsches Patentamt 
European Patent Office 
Office europ€en des brevets 



l 



© Publication number: 



0 401 055 A3 



® 



EUROPEAN PATENT APPLICATION 



© Application number: 90306058.0 
© Date of filing: 04.06.90 



© int. Cl. 5 : C12N 15/12, A61K 37/02, 
C07K 13/00, C12P 21/02 



© Priority: 02.06.89 US 360826 

© Date of publication of application: 

05.12.90 Bulletin 90/49 

® Designated Contracting States: 

AT BE CH DE DK ES FR GB GR IT LI LU NL SE 

® Date of deferred publication of the search report: 

07.08.91 Bulletin 91/32 



© Applicant: CHIRON CORPORATION 
4560 Horton Street 
Emeryville California 94608(US) 



© Inventor: Kiefer, Michael C. 
401 Wright Court 
Clayton, California 94517(US) 
Inventor: Masiarz, Frank R. 
148 Marview Way 

San Francisco, California 94131(US> 
Inventor: Barr, Philip J. 
67 Bay Forest Drive 
Oakland, California 94611 (US) 



Representative: Hartley, David et al 
4 Dyer's Buildings Holborn 
London EC1N 2JT(GB) 



© Bone Calcification factor. 



© The isolation, identification and production by 
recombinant methods of bone calcification factor, a 
22KD polypeptide, are disclosed. The peptide has 



calcification-inducing activity when implanted 
matrix Gla protein into mammals. 



with 



FIG. I 



FIG. I (CONT.) 



00 

< 

in 
in 



CL 
LU 



gccaaaatcccaggcagc ate cac ctc act ctt ctc teg gta ctt ctc etc 

AGAAGCCCASACGGC ATG SAC CTC ACT CTT CT5 TSS ST6 CTT CTC CCA 
I — 0«1 1 "7 -IT -K> K0it3-I7 

fCrASPLEDSERLEOLEDTftPVALUaiLDPRO 
NET ASP LEU THR LEU LEO TRP VAL LEO LED PRO 



Hunan CTA CTC ACC ATS GCC TGG CGC CAG TAT GGC GAT TAT GGA TAC CCA TAC 
Bonn CTG CTC ACC GT6 6CT TGG GGA CAG TAT GET SAC TAT Ctt TAC TCC TAT 

, , H 1 I h— Oil 3-7 _ 

Human LEU VAL TWt pCt] ALA TRP GLY'gUI TTR GLY ASP TYR GLV TYRW8S]TtR 
to vim LEU VAL THRlVAUALA TRP GLT GLN TYB GLT ASP TYR GLY TYRgfflTTR 

Human CAC<«AC TAT CAT 6AC TAC AGC GAT GAT 666 TGG GTS AAT TTG AAC CCS 
Bovxm CAT CAS TAC CAT GAC TAC ACT GAC GAT GG& TGG GTG AAT CTG AAC CBS 

Hunan {GUfjGLN TYR HIS ASP TYR SER ASP ASP GLY TRP VAL ASH LEU ASM ARC 
U GLN TYR HIS ASP TYR SER ASP ASP GLY TV VAL ASM LEU ASM AM 



Kukam CAA GGC TTC AGC TAC CAG TGT CCC CAG GGG CAG GTG ATA GTG CCC GTG 
BovxNi CAG GGC TTC AGC TAC CAG TGT CCC CAC GGG CAG GTG CTS CTG CCC CTG 



KuNAH GLN GLY LED SER TYR 6LN CYS PROK 
Bovnm GLN GLY LEU SER TYR GLN CYS PRO I 



UGLY ELM VALULE] VAL AU VAL 
0 GLY GLN VALffAUVAL ALA VAL 



Bumah AG6 AGC ATC TTC AGC AA6 AAG GAA GET TCT GAC AGA CAA TGG AAC TAC 
Bovnn AGS AGC ATC TTC AAC AAG AAG GAA GST TCC GAC AGA CAG TCC AAC TAC 



Kukam ARC SER ZU PIE E 
Bovxnk ARG SER XL£ PKEU 



9 LYS LYS 6LU GLY SER ASP ARG GLN TRP ASM TYR 
LYS LYS GUI GLY SER ASP ARG GLN TIP ASM TYR 



Human GCC TEC ATG CCC AC6 CCA CAG AGC CTC GGG GAA CCC ACC GAG TGC TCfi 
BovM CCC TGC ATG CCC ACA CCC CAG AGC CTG GGG GAG CCT ACS GAG TGC TGG 

Hunan ALA CYS NET PRO THR PRO GLN SER LEU GLY GiO PRO THR GUI CYS TIP 
Bovxm AUCYSfCrP1ttTWPR0GLN$ai£l)aYGUIPinTACUICnTltP 

Hunan TGG GAG GAG ATC AAC AGS GCT GGC ATG GAA TGG TAC CAG ACS TGC TCC 
Bovua TGG GAG GAG ATC AAC AGG GCT GGA ATG GAA TGG TAC CAG ACA TGC TCC 

Hunan TRP GUI GLU XLE ASM ARG ALA GLY H£T GLI) TRP TYR GLN THR CYS SER 
Bovsmr TRP GLD GLU XLE ASH ARG ALA GLY PCT GUI TRP TYR GUI TUR CYS SER 



Bovxm 


AAC 
AAC 


AAT GGG CTG 
AAT GGA CTG 


GTG 
GTG 


GCA 
GCA 


GGA 
GGA 


TTC 
TTC 


CAG 

CAG 


AGC CGC 
AGC CGC 


TAC 
TAC 


TTC 
TTC 


CAG 
GAG 


TCA GTG 
TCA GTG 


KlMAM 

Bovxm 


ASH 
ASM 


ASM GLY LEU 
ASM GLY LED 


VAL 
VAL 


ALA 
ALA 


GLY 
GLY 


PHE 
PHE 


GLN 
GLN 


SER ARG 
SER ARG 


TYR 
TYR 


PKE 
PHE 


GUI 
GLU 


SER VAL 
SER VAL 


Human 

BOVXME 


CTG 
CTG 


GAT CGG GAG 
GAT CGC GAG 


TGG 
TGG 


CAG 
CAA 


TTT 
TTT 


TAC 
TAC 


TGT 
TGC 


TGT CGC 
TGT CGC 


TAX 
TAC 


AGC 
AGC 


AAG 
AAG 


AGA TGC 
AGA TGC 


HUMAN 
BOVXME 


LEU 
LED 


ASP ARG GLU 
ASP ARG GLU 


TRP 
TRP 


GUI 

em 


PHE 
PHE 


TYR 
TYR 


CTS 
CTS 


CYS ARG 
CYS ARG 


TYR 
TYR 


SER 

sa 


LYS 
LYS 


ARG CYS 
ARG CTS 


Kukam 
Bovxnr 


CCA 
CCA 


TAT TCC TGC 
TAT TCC TGC 


TGG 
TGG 


CTA 
CTG 


ACA 
ACA 


ACA 
ACA 


GAA 
6AA 


TAT -CCA 
TAT CCA 


GET 
GGC 


CAC 

ate 


TAT 
TAT 


66? GAG 
GGT GAG 


Human 
Bovxmx 


PRO 
PRO 


TYR SER CYS 
TYR SER CYS 


TRP 

TRP 


LED 
LEU 


thr 

THR 


THR 
THR 


GLU 
GLU 


TYR PRO 
TYR PRO 


GLY 
GLY 


BIS 
HIS 


TYR 

TYR 


GLY GLU 
GLY GLU 



Kukam GAG ATG GAC ATG ATT TCC TAC AAT TAT GAT TAC TAT ATC C£A GGA GCA 
Bovxm GAG ATG GAC ATG ATT TCC TAC AAT TAT GAT TAC TAT ATG CGA GGG GCA 



Kukam GLU BET ASP HET ILE SER TYR ASM TYR ASP TYR TYR& 
BovxKe GUI PET ASP HET ILE SER TYR ASH TYR ASP TYR TYr{| 



Pars gly ala 
1ar6 gly ala 



Human ACA ACC ACT TTC TCT GCA GTG GAA AGG GAT CGC CAS TGG AAG TTC ATA 
Bovznc ACA ACC ACT TTC TCT GCA GTG GAA AGG GAT CGC CAG TGG AAA TTC ATA 

Hunan THR THR THR PHE SER ALA VAL GLU ARG ASP ARG GUI TRP LYS PHE XLE 
Bovxm THR THR THR PHE SER ALA VAL GLU ARG ASP ARG GLN TRP LYS PHE XLE 



ATG TGC CGG ATG ACT GAA TAC GAC TGT GAA TTT GCA AAT GTT TAG 
BovxNt ATG TGC CGG ATG ACT GAC TAT GAC TGT GAA TTT GCA ART GTT TAG 



Human MET CYS ARG MET THRU 
Bovxmb MET CYS ARG HET THR {J 



i] TYR ASP CYS GLU PHE ALA ASM VAL • 
J TYR ASP CYS GUI PHE ALA ASM VAL - 



Xerox Copy Centre 



J 



European Patent 
Office 



PARTIAL EUROPEAN SEARCH REPORT 

which under Rule 45 of the European Patent Convention 
shall be considered, for the purposes of subsequent 
proceedings, as the European search report 



Application number 

EP 90306058 
- page 1 - . 



DOCUMENTS CONSIDERED TO BE RELEVANT 



Category 



Citation of document with Indication, where appropriate, 
of relevant passages 



Relevant 
to claim 



CLASStRCATIONOFTH 
APPLICATION 0nt. Cl.f 



X 
Y 

D,X 



EP-A-0148155 (THE DOW CHEMICAL COMPANY) 
* whole document; in particular page 7, 
line 5 - page 8, line 14, table 4 * 



WO-A- 8800205 (GENETICS INSTITUTE , INC.) 
* whole document * 



PROCEEDINGS OF THE NATIONAL ACADEMY 
OF SCIENCES OF THE UNITED STATES OF 
AMERICA 

vol. 81, no. 2,, January 1984, pages 371- 
375, Washington DC, OS ; M . R . URIST et 
al.: "Purification of bovine bone morpho- 
genetic protein by hydroxyapatite chroma- 
tography" 

* abstract; page 373, column 2; page 
374, column 2 - page 375, column 1 * 



./2 



1-5, 
24-28, 
30,31 
6-22 



1-3, 

24-26 

6-22 



1,3 



C12N15/12 
A61K37/02 
C07K13/00 
C12P21/02 



TECHNICAL FIELDS 
SEARCHED {InL Qffi 



INCOMPLETE SEARCH 



The Search Division considers that the present European patent application does not comply with 

the provisions ot the European Patent Convention to such an extent that it is not possible to carry 

out a meaningful search into the state of the art on the basis of someot the claims. 

Claims searched completely: 1-28,30,31 

Claims searched incompletely: - 

Clai ms not searched: 2 9 

Reason for the limitation ot the search: 



C12N15/12 
C12N15/16 
C07K13/00 
C07K15/00 
A61K37/22 
A61K37/36 
A61K35/32 



Article 52(4) EPC 



Place of search 


Date of completion of the search 


Examiner 


Berlin 


28.03.1991 


P. JULIA Y BALLBE 



CATEGORY OF CITED DOCUMENTS 

X : particularly relevant if taken alone 

Y : particularly relevant If combined with another 

document of the same category 
A : technological background 
O : non-written disclosure 
P : intermediate document 



T : theory or principle underlying the invention 
E : earlier patent document, but published on, or 

after the filing date 
D : document cited in the application 
L : document cited for other reasons 



& : member of the same patent family, corresponding 
document 




European Patent 
Office 



PARTIAL EUROPEAN SEARCH REPORT 



Application number 
EP 90306058 
- page 2 - 



DOCUMENTS CONSIDERED TO BE RELEVANT 



Category 



Citation of document wttt Indication, wnere appropriate, of relevant 
passage* 



D,X 
D,A 



P,X 



P,X 



EP-A-0212474 (UNIVERSITY OF CALIFORNIA) 
* whole document * 



EP-A-0196056 (CHIRON CORPORATION) 

* page 9, line 27 - page 10, line 21; 
page 15, line 11 - page 16, line 21; 
table 18 * 



WO-A-9003733 (INTERNATIONAL GENETIC 
ENGINEERING, INC.) 
* whole document * 



EP-A-0336760 (COLLAGEN CORPORATION) 
* whole document * 



Relevant 

to claim 



1-3 

14,15, 
24-26 



18-21 



1-3, 

24-26, 

30,31 



1-3, 

24-26, 

31 



CLASSIFICATION OF THE 
^ 



APPLICATION (Int. CI J 



TECHNICAL FIELOSL 
SEARCHED (Int. Cv9 



EPO Pom 1S0U MJt 



This Page is Inserted by IFW Indexing and Scanning 
Operations and is not part of the Official Record 

BEST AVAILABLE IMAGES 

Defective images within this document are accurate representations of the original 
documents submitted by the applicant. 

Defects in the images include but are not limited to the items checked: 

□ BLACK BORDERS 

□ IMAGE CUT OFF AT TOP, BOTTOM OR SIDES 
□f FADED TEXT OR DRAWING 
[^BLURRED OR ILLEGIBLE TEXT OR DRAWING 

□ SKEWED/SLANTED IMAGES 

□ COLOR OR BLACK AND WHITE PHOTOGRAPHS 

□ GRAY SCALE DOCUMENTS 

□ LINES OR MARKS ON ORIGINAL DOCUMENT 

□ REFERENCE(S) OR EXHIBIT(S) SUBMITTED ARE POOR QUALITY 

□ OTHER: ; 

IMAGES ARE BEST AVAILABLE COPY. 
As rescanning these documents will not correct the image 
problems checked, please do not report these problems to 
the IFW Image Problem Mailbox.