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J) 



Europaisches Patentamt 
European Patent Office 
Office europeen des brevets 



© Publication number: 



0 212 474 

A2 



© EUROPEAN PATENT APPLICATION 

© Application number: 86110935.3 
© Date of filing: 07.08.86 



© int. ci/ : C07K 15/06 , C12N 15/00 , 
A61K 35/12 



@ Priority: 07.08.85 US 763479 

® Date of publication of application: 
04.03.87 Bulletin 87/10 

© Designated Contracting States: 

AT BE CH DE FR GB IT LI LU NL SE 



© Applicant: University of California 
2490 Channing Way 
Berkeley California 97420(US) 

© Inventor: Urist, Marshall R. 
796 Amalfl Drive 
Pacific Palisades, CA 90272(US) 

© Representative: Kinzebach, Werner, Dr. 
Patentanwalte Reitstotter, Kinzebach & 
Partner Sternwartstrasse 4 Postfach 86 06 49 
D-8000 Munchen 86(D£) 



© Bone morphogenetic agents, 

© The production and isolation of bone mor- 
phogenetic peptide agents displaying the osteoin- 
ductive and immunoreactive activity of bone mor- 
phogenetic protein (BMP) and deriving from natural, 
synthetic or recombinant DNA sources, and said 
agents. 



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BONE MORPHOGENETIC AGENTS 



This application is a continuation-in-part ap- 
plication of copending application Serial No. 
523.606. fiied August 16. 1983, which in turn is a 
continuation-in-part application of application Serial 
No. 260.726. filed May 5. 1981. now Patent No. 
4.455.256. which in turn is a continuation applica- 
tion of application Serial No. 174.906, filed August 
4, 1980. now Patent No. 4.294,753. 

This invention was made with Government sup- 
port under Grant No. DE 02103-20 with the Na- 
tional Institutes of Health and the University of 
California. The Government* has certain rights in 
this invention. 



FIELD OF INVENTION 

This invention relates to bone morphogenetic 
agents which are peptides that cause bone tissue 
to form and/or grow. More particularly, the present 
invention relates to bone morphogenetic peptides 
comprising at least an active portion of the osteoin- 
ductive and immunoreactive domain of the bone 
morphogenetic protein molecule, said peptide 
agents obtained from whatever source, including 
natural, synthetic or recombinant DNA sources, and 
their production, isolation, amino acid sequence 
and use. 



BACKGROUND OF THE INVENTION 

It has been demonstrated by the applicant that 
demineralized bone matrix induces new bone for- 
mation when implanted in soft tissue. Freshly ex- 
cised bone, demineralized in cold dilute HCI for 4 
hours and implanted in brain, muscle subcutis, and 
bone defects, induces differentiation of postfetal 
mesenchymal-type perivascular connective tissue 
cells into cartilage and bone (Urist. M.R., Science. 
150: 893-899. 1965). The process is generally des- 
ignated as matrix induced bone formation. Further, 
the inductive principle has been discovered and 
extracted; it is a material designated bone mor- 
phogenetic protein (BMP). Reference is made to 
Patent No. 4.294,753: Patent No. 4.455.256; and 
pending Application Serial No. 523,606. fiied Au- 
gust 16. 1983. See also: Urist. M.R., et ah, Proc. 
Soc. Exp. Biol. Med .. 173: 194-199. 1983; and. 
Urist. M.R., et al., Proc. Natl. Acad. Sci . (USA) 81: 
371-375, 1984. These patents, application and re- 
ferences are incorporated herein by reference. 



It is believed that BMP transforms tissue cells 
into osteoblasts, cells that manufacture bone. Hy- 
pothetically. the process is classified as phenotypic 
transformation and the BMP is a paracrine mol- 
5 ecule. As distinguished from malignant transforma- 
tion by a carcinogen, phenotypic transformation is 
a self-limited host-regulated development process. 
During a process that replicates normal human 
fetal development, BMP-induced osteoblasts to 

io form cartilage, which, over a period of several 
months, evolved into solid bone. The use of BMP 
implants by physicians offers considerable advan- 
tages over traditional bone graft operations. The list 
of present and potential uses for BMP includes 

75 almost every site in the human body: for use in 
replacing bone that has been destroyed by disease 
or accident; for use in treatment of scoliosis vic- 
tims; for use in treatment of mal or misformed 
bone; for use in healing two edges of a fracture, 

20 and the like. 

Currently, the process for isolation of BMP is 
relatively lengthy and expensive. Whereas milli- 
gram doses of BMP are required to induce bone 
formation in vivo , only microgram and picogram 

25 doses of hormones, vitamins and metabolites are 
needed to produce measureable biological reac- 
tions. One objective of the present invention was to 
identify and produce functional peptide segments 
of BMP that yield enhanced biological activity per 

30 unit of implanted bone morphogenetic material. An- 
other objective was to create and produce biologi- 
cally active peptides that are sequenced and then 
produced in quantity by direct biochemical synthe- 
sis from the constituent amino acids, for example, 

as by the Merrifield method. Another objective of the 
present invention was to provide bone mor- 
phogenetic peptides that are sequenced and pro- 
duced in quantity by expression of "genes" by 
recombinant DNA technology, or are used to con- 

40 struct nucleic acid screening probes for the isola- 
tion of the gene comprising the osteoinductive re- 
gion of BMP for expression of bone morphogenetic 
peptides by recombinant DNA technology. Obtain- 
ing and characterizing peptide segments with bone 

45 morphogenetic activity is a key step in several 
commercially feasible processes to manufacture in 
quantity an efficient bone morphogenetic agent. 

50 SUMMARY OF THE INVENTION 

The present invention concerns bone mor- 
phogenetic peptide agents comprising at least an 
active portion of the osteoinductive and im- 
munoreactive domain of the BMP molecule. 



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Bone morphogenetic peptides having a range 
of relative molecular weights (M r ) of about 4K to 
about 7K were produced by limited proteolysis of 
BMP from bovine bone and limited proteolysis of 
human bone matrix non-coliagenous proteins. 
These bone morphogenetic peptide products were 
designated BMP-p and comprised osteoinductive 
and immunoreactive segments of the BMP mol- 
ecule. Tables I and 111 contain the amino acid 
composition of two species of BMP-p, one - 
(bovine) having a M r of about 4.1 K and the other - 
(human) having a M r of about 4.7K t 0.3K. The N- 
terminal sequence of the first 36 amino acids of 
purified human BMP-p having a M r of about 4.7K ± 
0.3K is given in Table IV. ♦ 

Bone morphogenetic agents that comprise 
osteoinductive and immunoreactive domains of nat- 
ural BMP molecules are prepared using the amino 
acid sequence of BMP-p, such as is given in Table 
IV, by conventional peptide synthesis procedures, 
e.g., the Merrifield method. 

The full or partial amino acid sequence of 
BMP-p or of Table IV is employed by one skilled in 
the art of genetic engineering to synthesize ON A 
sequences which code on expression for bone 
morphogenetic peptides, or to synthesize DNA se- 
quences which can be used as screening probes to 
isolate genes coding for the osteoinductive and/or 
immunoreactive regions of BMP. The synthetic 
DNA or the natural gene is inserted into a recom- 
binant vehicle which is introduced into an appro- 
priate host and expresses a bone morphogenetic 
peptide comprising an amino acid sequence sub- 
stantially homologous - with the amino acid se- 
quence of Table IV. Bone morphogenetic peptides 
produced by these methods comprise osteoinduc- 
tive and immunoreactive domains of BMP discov- 
ered and isolated by the applicant as described in 
Patent No. 4,294,753; Patent No. 4,455*256; and 
pending Application Serial No. 523,606, filed Au- 
gust 16, 1983. 

This invention also provides for the use of bone 
morphogenetic agents comprising osteoinductive 
and immunoreactive domain of BMP to induce car- 
tilage and/or bone formation in animals and hu- 
mans capable of benefiting from such induction. 

This invention further provides a process for 
the production and purification of bone mor- 
phogenetic peptides from limited pepsin or trypsin 
proteolysis of bone morphogenetic protein (BMP). 
The bovine BMP-p and human BMP-p generated 
by limited proteolysis were isolated by any of one 
or more of: sequential differential precipitation, gel 
filtration, hydroxyapatite (HA) chromatography, and 
high pressure liquid chromatography (HPLC). The 
smallest functional unit of the bovine BMP-p seg- 
ments isolated by high pressure liquid chromatog- 



raphy (HPLC) had a M r of about 4.1 K. The M r of 
the smallest active human BMP-p isolated at this 
time was about 4.7K i 0.3K. 

5 

DETAILED DESCRIPTION OF INVENTION 

There are numerous examples of the role of 
proteases in biological systems, generation of 

70 hormones, activation of enzymes, assembly of 
fibrils, blood coagulation, etc. See, Neurath. H. - 
(1980) in Protein Folding . Edited by K. Jaenick, 
Elservier Biomedical Press, New York, N.Y. pp. 501 
-524. Neither the inactive precursor nor the cell 

75 membrane receptors of either BMP or BMP-p are 
known: 

Without intending to be limited by theory, it is 
thought that invivo limited proteolysis of BMP oc- 
curs by the action of intracellular proteinases on 

20 BMP enroutet o formation of a nucleosome, and 
BMP induced DNA differentiation. According to this 
theory the BMP molecule is constructed of do- 
mains, hinges and fringes, with the active domain 
of the BMP molecule surrounding a hydrophobic 

25 core that is inaccessible to limited proteolysis, and 
the structure of BMP is reduced and rearranged to 
generate smaller core peptides with the same or 
enhanced biological activity. Based on this hypoth- 
esis, experiments were undertaken to generate 

30 osteoinductive peptide fragments in vitro by means 
of limited proteolysis. 

As more particularly described in Examples I, II 
and III below, bovine and human BMP were par- 
tially purified and subjected to limited pepsin or 

35 trypsin proteolysis. A group of osteoinductive and 
immunoreactive peptides were generated having a 
M r of about 4K to about 7K. These products are 
called bone morphogenetic peptides, and desig- 
nated BMP-p. Sources other than bovine or human 

40 BMP are employed equally as well. 

The observation is that the kinetics of pepsin 
proteolysis includes a rate limiting step during 
which hydrolysis is Diphasic. In phase I, pepsin 
cleaves the fringes and hinges of the BMP mol- 

45 ecule, sparing the active -domain. Hydrolysis accel- 
erates in phase II to rapidly degrade BMP-p. In 
individual batches, the densest population of 8MP- 
p molecules reflects cleavage at various bonds 
among the hinges and fringes of the BMP mol- 

so ecule, sparing the active domain, with statistically 
most frequent M r of 5.3K ± 1.0K, and ranging from 
4K to 7K. 

Example I -details the method of prod ction and 
. isolation of novel bone morphogenetic peptides - 
55 (BMP-p) from bovine BMP. Example II details the 
method of production and isolation of BMP-p from 
human BMP. 



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In Example I, a 0.5M CaCl, -6M urea inorganic 
solvent mixture was used for the initial extraction of 
BMP from bovine bone. Urea was initially selected 
to avoid the relatively high expense of using 
guamdine hydrochloride (GuHCI) on batches of 
bones large enough to prepare significant quan- 
tities of BMP. Whenever urea was used, the N- 
termmal amino adds of BMP and BMP-p proved to 
be blocked, possibly by a firm isocyanate bond, 
and attempts to determine the N-terminal amino 
acid sequence of BMP and BMP-p by automated 
Edman degradation were unsuccessful. Since N- 
termmal blocking groups in proteins are usually not 
removable without degrading the protein, it was 
desirable to obtain BMP and BMP-p that could be 
sequenced. This became pcJssible once if was dis- 
covered that BMP extracted with GuHCI contained 
an unblocked N-terminal amino acid. 

Example II details the use of a high purity 
grade GuHCI in place of the urea employed in 
Example I; and the production of BMP and BMP-p 
with an unblocked N-terminal amino acid. 

The BMP-p generated by limited pepsin prot- 
eolys,s or trypsin proteolysis of human or bovine 
BMP was isolated by means of a combination of 
sequential differential precipitation, gel filtration, 
hydroxyapatite chromatography, and high pressure 
liquid chromatography. Various biologically-func- 
tional BMP-p species were characterized by M p 
immunoreactivity with BMP antibody, isoelectric fc^ 
cusing. amino acid composition analysis, and/or 
amino acid sequencing. 

The sequence of the first thirty-six amino acids 
of the N-terminal segment of human BMP-p has 
been determined. 



Example I 

Bovine bone morphogenetic protein (bBMP) 
was purified or partially purified from bovine bone 
by methods described in detail in the previously 
tioT Pate " tS ' pub,ications - and copending applica- 

The purification process steps comprised: de- 
m.neraiizing bone tissue: treating the demoralized 
bone tissue under aqueous conditions with a water 
soluble neutral salt and a solubilizing agent for the 
BMP, the agent being selected from the group 
consisting of urea and guanidine. and thereby 
transforming the bone collagen to gelatine and 
extracting BMP into the. solution of solubilizing 
agent: and separating the solubilizing agent and 
neutral salt from the solution, thereby precipitating 
BMP m the aqueous medium. In the present Exam- 
ple I, urea was used. 



Punfied BMP and partially purified BMP yield 
the same bone morphogenetic peptide products 
upon limited pepsin proteolysis as described below 
in detail for partially purified BMP. 

s Referring to the drawing. Figure 1 depicts the 

steps of pepsin limited proteolysis of bovine BMP 
One gram of the partially purified BMP (See Table 
1. step 8. Urist fi aj. 1984) was digested for 2 
hours at 37«C in 2 liters of 0.01 N HCI containing 

«> 10 ug pepsin (Sigma Co.. St Louis. MO) per mg 
protein. a ' 

The partially purified BMP starting material 
compnsed an assortment of about 15 proteins with 
Mr ranging from 2.5K to 90K. After incubation in 

' S ™T SL 2 h ° UrS * Pr0t8inS Mr ranQ e to", 

30K to 90K were hydrolyzed and the peptide pro- 
ducts of the limited pepsin treatment were sepa- 
rated into 3 groups: 



20 



ss 



Group I -HCI insoluble/water insoluble 
Group II -HCI soluble/water insoluble 
Group III -HCI soluble/water soluble 



See Figure 1. 

When partially purified bovine BMP was in- 
cubated in COIN HCI for 2 hours in 2. 5. and 10 ug 

,„ * P ?™ P6r m9 0f protein (en2 y me to Protein ratio 
M 1:100). the HCI soluble/water insoluble components 
of the supernatant consistently induced bone for- 
mation. Other ratios (1:200 and 1:500) failed to 
produce this separation. 

Initially, the pepsin cleaved peptide products 
as were separated into HCI soluble (Group II and III) 
and HCI insoluble (Group I) parts. The Group I (HCI 
insoluble) peptide products were separated from 
the HCI soluble products (Groups I and III) by 
centrifugation at 50,000 g. washed 3X in 1 liter of 
40 Solf dei ° ni2ed Water " 3,1(1 col| ected by lyophiliza- 

The HCI soluble (II and III) peptides were sepa- 
rated by dialysis against deionized water (3X) until 
formation of a water insoluble precipitate was corn- 
's plete. This precipitate {Group II) was -separated 
from the supernatant (Group III) by centrifugation at 
50.000 g for 1 hour, and by washing 3X in cold 
water. Group II and III were lyophilized separately 
As described below these three groups of pep- 
so bdes produced by limited pepsin proteolysis were 
further fractionated, characterized by M r , and 
bioassayed in vivo or invitro. 

Molecular weight (M,) determinations were 
SS Zlf * V , SDS - PAGE ^rylamide gel elec- 
ss frophoresis. Lyophilized fractions were solubilized 
by incubation for 24 hours in 0.06 M f ris-HCI (pH 
6.8) containing 2 M urea and 0.2% SOS Five 
nucroliters (2.5% mg/ml) of each sample were ap- 



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plied to a 12.6% gel with a 3% stacking gel and 
were electrophoresed at 25mA. The gels were 
stained with 0.25% Coomassie brilliant blue R-250 
in methanol/acetic acid/H,0 (5:1:5). The molecular 
weights were determined by using protein stan- 
dards (Pharmacia), with a range of M r 94,000- 
14,000 and by peptide standards (Pharmacia) with 
a range of M r 17,200-1,600. The relative molecular 
mass (M r ) was calculated by plotting the logarithm 
of molecular weight versus distance of migration of 
6 standard peptides relative to the distance of the 
unknown. (Swank and Munkres. 1971, Anna!. 
Biochem. 39: 462.) 



Bone morphogenetic activity was also assayed 
in tissue cultures. BMP was added to a CMRL - 
(G1BCO) culture medium. The inductive activity per 
0.1 to 1 .0 mg of partially purified polypeptides was 
measured by counting the number of cartilage cells 
per low power field. Purified BMP was assayed in 
tissue cultures of 10 HPLC fractions, 10 mg/ml in 
triplicate by the method of Sato and Urist (1984), 
Clin. Orthop. 184: 180-187. 

Under the conditions specified, nearly all of the 
BMP activity was recovered and accounted for by 
the sum of the activities of Group I (78%) and 
Group II (22%). Group II! peptides had no BMP 
activity and consequently were not isolated or fur- 
ther characterized. 

As shown in Figure 2, the products of prot- 
eolysis formed through about the first 2 hours of 
incubation maintained high BMP activity. Between 
2 and 3 hours the sum of the HCI soluble and HC! 
insoluble products returned BMP activity almost as- 
high as the partially purified BMP starting material. 
It was not until about 4 hours or more of incubation 
that the total BMP activity fell below about 50%. 

The partially purified BMP starting material 
consisted of an assortment of about 15 proteins 
with an M r range from 2.5K to 90K. After incubation 
in pepsin for 2 hours, proteins in the M r range of 
30K to 90K were hydroiyzed. The remaining poly- 
peptides were partially digested peptides or spared 
peptides in the M r range of 2.5K to 30K. Although 
the quantity of induced bone formation was about 4 
times greater in implants of HCI insoluble than 



Bone morphogenetic activity was determined 
by implantation of lyophilized partially purified pep- 
tides and/or chromatographic fractions of peptides 
in the hindquarter muscles of Swiss-Webster strain 

5 mice. For controls, samples of the M r 38.000, 
24,000, 15,000, 12.000, 4,000, 8,500, 3.000 and 
2,500 products of pepsin proteolysis were implant- 
ed in the contralateral hindquarter muscle pouches. 
The quantity of new bone was measured by 

70 correlated observations on roentgenograms, excis- 
ed wet ossicle weights, histological sections, 
histomorphometric data, and **Ca uptake by cal- 
cified new bone. The histomorphimetry was^ per- 
formed on roentgenograms of histologically valid 

is deposits of bone using the random point analysis 
method with the following formula: 

= % BMP-induced 
bone 

formation 

25 soluble groups, the SOS Page patterns were equal- 
ly cleared of proteins with M r greater than 30K, and 
the individual components were about the same in 
number. 

Group I and II peptides were further fractionat- 
30 ed as described below, and the bone inducing 
activity of both groups was always associated with 
peptides with M r less than 7K and greater than 3K. . 

Lyophiii2ed Group II products were weighed 
and 0.7g were redissolved in .05M phosphate buff- 
as er, pH 6.8 in 6M urea. The buffered solution was - 
applied to a Sephacryl S-200 column (5 cm 1 95 
cm) with downward flow regulated by a peristaltic 
pump and collected in 5 fractions. Two fractions 
with'M, greater than 30,000 were pooled, dialyzed 
40 against water, and relyophilized for bioassay of 
new bone formation induced per mg of implanted 
protein. Three fractions with M , proteins less than 
30.000 were pooled and similarly prepared. The 
proteins with M r less than 30,000 were further 
45 fractionated by hydroxyapatite (HA) chromatog- 
raphy, as follows. An 80 mg sample was applied to 
HA columns (2.5 * 40 cm) and eluted along a 
stepwise gradient of concentrations of 0.01 to 0.05, 
.0.05 to O.2., 0.2 to 0.5M phosphate buffer in *6M 
so urea. The proteins were collected in 9 f ractions, 
examined by SDS gel electrophoresis/ desalted in 
sacs with a pore si2e of 2,000 molecular weight by 
dialysis against water, lyophilized and implanted for 
BMP activity. 

ss Peptides having M f less than 10K were sub- 
fractionated using Sephadex G-S0 (ultrafine) molec- 
ular sieve chromatography. " Samples of freeze- 
dried peptides were dissolved in 0.01 M sodium 



— Points on radio-opaque bone X 100 
Points on quadracepts muscle compartment 



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phosphate (pH 7.0) containing 6M urea and 
charged to a column (1.5 * 100 cm) equilibrated in 
the same buffer. Fractions of approximately 20 ml 
each were collected, combined within each peak, 
dialyzed exhaustively against cold, deionized water 5 
and lyophilized. The resulting fractions were ana- 
lyzed by SDS-PAGE peptide gels by the methods 
of Swank & Munkres, (1971). 

Group I lyophilized products were dissolved in 
100 ml of 6M urea and purified by sequential HA u 
chromatography and G-50 gel filtration by the 
above described procedure. 

Further purification of the peptides isolated by 
HA chromatography was accomplished by HPLC 
by methods previously described (Urist etaJL, is 
1984) and detailed below in Example II. Both HCI 
insoluble and HCI soluble groups of pepsin cleaved 
peptides yielded the M f 4K to 7K bBMP-p, includ- 
ing a large population of M r 5.6K ± 1.5K molecules, 
the smallest of which had an M r of about 4.1 K. 20 

The group II products of pepsin digestion that 
were fractionated first by Sephacryl S-200 gel filtra- 
tion and then by HA chromatography yielded' a 
broad spectrum of proteins and peptides. Approxi- 
mately 95% of the products which had a M r less 25 
than 3K had no BMP activity. Nearly all of the bone 
inductive activity consisted of proteins and pep- 
tides in the M f range of 2.5K to 30K. Slab gel 
preparative electrophoresis preparations were 
made of HA chromatographic fractions and cuts 30 
were made from the areas of peptides in the M r 
range 4K to 7K, and in the M r range 2.K to 3K. The 
peptides in the gel slices were eluted with 6M urea, 
and reelectrophoresed. The combination of protein 
and peptide standards validated the assigned M r of 35 
the peptides in the range of about 4K to about 7K. 
Only the components in this range induced car- 
tilage formation invitro and bone formation invivo . 
The peptides in the M r range 2K to 3K or lower- - 
(isolated by HA Chromatography) did not induce 40 
bone formation. 

HA chromatography of the Group I products of 
pepsin digestion yielded the same constituents (in 
somewhat different proportions) as obtained by HA 
chromatography of the Group II products discussed 46 



above. Peptide fractions with M r range 4K to 7K 
were eluted with 0.15 to 0.25M concentrations of 
phosphate ion, and found to induce bone formation. 
Peptides eluted at higher phosphate in concentra- 
tions had a M r of 1K to 2.5K and did not induce 
bone formation. 

By means of Sephadex G-50 gel filtration 
chromatagraphy, the fractions with high BMP activ- 
ity were resolved into 9 subfractions. Only one 
subtraction, further purified by HPLC and consist- 
ing of a peptide with a M f of about 4.1 K, induced 
bone formation. 

Biologically active fractions obtained by either 
G-50 or HA chromatography were separated into 
10 subfractions with the aid of a reverse phase 
HPLC column (Urist et aL, 1984). All 10 were bic- 
assayed in tissue cultures of connective tissue out- 
growths of muscle. The subtraction containing pep- 
tide with an M r of 4.1 K induced differentiation of 
cartilage. The quantity of cartilage varied from 2 to 
9% of the total area of tissue in 6 serial sections of 
the culture. The nine other subfractions with com- 
ponents of higher and lower M r failed to induce 
cartilage cell differentiation in 27 other cultures 
prepared under identical conditions. 

The pi .of M r 4K to 7K peptides isolated by HA 
chromatography and HPLC was determined by 
isoelectric focusing by the method of Righetti and 
Drysdale in Isoelectric Focusing . Amsterdam, North 
Holland, Publ. Co., 1976, using a micropH probe - 
(Microelectrodes, Inc.) for pH measurements on the 
cold gels (5% polyacrylamide and 2% ampholyte). 
Focusing was at 10 W at 2°C for 3.5 hours. The pi 
is about 3.9. 

Amino acid analysis was performed on an ami- 
no acid analyzer (Modified Beckman 121). Cystine 
and half cystine analysis was performed following 
performic acid oxidation. Table 1 presents the ami- 
no, acid composition of the bovine BMP-p 4.1 K 
peptide. The composition is characteristic of a 
composite of acidic polypeptides. 



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Table I 

AMINO ACID ANALYSIS OP BOVINE BMF-p OF M 4.1K + 1.5K 

Putative 

"Moles Mole * Res/41Ses Residing 



Amino Acid 



Lysine 

Histidine 

Arginine 

Aspartic Acid 

Threonin 

Serine (corr. 
by 7.5%) 

Glutamic Acid 

Proline 

Glycine 
Alanine 

1/2 Cystine 

(Cysteic Acid) 
Valine 
Methionine 
Isoleucine 
Leucine 
Tyrosine 
Phenylalanine 



5.28 


5 . 26 


2 1 fi 

£ . JL D 


(2) 


2.96 




1 • <i 


/ 1 \ 

(i) 


5.66 


5. 64 




i *j 


13.01 


12.97 


5.32 


(5) 


3.93 


3.92 


1.61 


(2) 


4.68 


4.67 


1.91 


(2) 


17.98 


17.93 


7.35 


(7) 


6.57 


6.55 


2.69 


(3) 


5.82 


5.80 


2.34 


(2) 


6.08 


6.06 


2.48 


(2-: 



5.12 
4.92 
(0.94) 
3.39 
6.88 
4.78 
-3.2,1 
100.30 



The M r 5.6K t 1 .5K group of peptides, and the 
M f 4.1 K peptide were further purified by hydropho- 
bic column chromatography, and analysed in the 
gas/liquid phase protein sequenator (Hewick, et aL, 
1981, J. BioL Chem. 256 1990.) The M f 4.1 K BMP- 
p product of pepsin limited proteolysis had a bloc- 
ked N-terminus, presumably the same as that of 
the M r 18.5K BMP from which it was derived. 

Bovine BMP-p was tested for immunologic ac- 
tivity by means of the dot EL1SA (Hawkes, et aL, 
Anr Biochem. 119: 142-147, 1982) and found to 
cross react with mouse anti-bovine BMP (M r 
18,500). Thus the immunoreactive and bioactive 
segments of bBMP-p (M r 4.1 K) and bBMP (18.5K) 
appeared to be identical. 



5.10 
4.91 

3.38 
6.86 
4.77 
3.23 



2.09 (2) 
2.01 (2) 
(0.38) 

1.39 (1-2) 
2.81 (3) 
1.96 (2) 
1.32 (1-2) 
100.00 39-42 

+ 
Trp 



40 



45 



SO 



55 



Ultrafiltration using M r 10K pore size has also 
been used to separate digested high from low M r 
peptides of Group I and I! components. 

From 120 implants of (1 to 10 mg samples) 
and ten 15 mg samples of partially purified BMP-p 
(assayed in triplicate) the percentages of the 
quadraceps compartment occupied by induced 
bone formation were calculated. The partially puri- 
fied BMP displaced 50 to 90% of the quadracepts. 
The 0.01 N HCI soluble/water insoluble products of 
limited pepsin proteolysis, Group II, displaced 20 to 
75%; 0.01 N HCI insoluble/water insoluble, "Group I. 
50 to 84%; 0.01 N HCI soluble/water soluble! 
Group-Ill, 0%. The volume of the deposit was 
directly proportion to the doses in the range of 1 to 



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14 



10 mg. Doses larger than 10 mg were found to 
produce more bone than the quadracepts compart- 
ment could contain and caused the deposits to 
grow across the pelvis into the contralateral limb. 

Implants of 5 mg of partially purified BMP-p - 
(M r 5.6K + 1.5K) derived from either Group I or II, 
isolated by sequential HA chromatography and S- 
200 or G-50 gel filtration induced formation of bone 
deposits occupying 32% of the volume of the 
quadraceps compartment The wet weight of a 
deposit of the volume of histologically valid bone 
dissected free of muscle tissue was about 200 mg. 

Purified. M r 4.1 K BMP-p, isolated by HPLC, 
induced differentiation of cartilage in tissue culture 
and represented the smallest unit of polypeptide 
structure with biologic activity. 



EXAMPLE II 

By the mechanism of pepsin limited proteolysis 
of human BMP (hBMP), a group of peptides were 
generated having M,of 4K to 7K and the osteoin- 
ductive and immunoreactive properties of BMP. 

Peptides having an M r of 4K to 7k were sepa- 
rated from higher and- lower M r polypeptides by S- 
200 gel filtration of a 4M GuHCI solution of the 
products of pepsin proteolysis. All of the osteoin- 
ductive activity was concentrated in the peptides 
with M r of 4K to 7k. None was found in fractions 
with M r greater than 7K or lower than 3K. By 
means of G-50 filtration of these fractions, a group 
of peptides with a M r of 5.5K ± 0.8K was isolated. 
This group also induced differentiation of cartilage 
and bone in a muscle pouch in the mouse thigh. 

Further purification of the G-50 isolated hBMP- 
p with HPLC produced peptides with M r of 4.7K + 
0.3K. Different biochemists working on three sepa- 
rate batches of about 1.5 kilos each of fresh wet 
bone, produced hBMP-p having M r of 4.7K, 5.0K, 
and 4.4K. The 4.7K human BMP-p induced car- 
tilage in tissue cultures. 

The materials and methods were as follows: 
Rve kg of human cortical bone were excised at 
autopsies of 22 to 51 year old men within 12 hours 
after accidental death following the guidelines of 
the Anatomical Gifts Act of California. The dia- 
physeal cortical bone was extensively washed, me- 
chanically demarrowed. defatted in 1:1 chloroform- 
methanol, fro2en in liquid N 3 , and ground into 0.5 
mm 3 particles in a Wiley Mill. The ground bone 
particles were dimrneralized in 0.6N HCI for 48 
nours, washed, and the hBMP extracted with 4M 
guanidine HCI (GuHCI) containing sulfhydryl group 
enzyme inhibitors and differential precipitation in 
1.5M GuHCI containing 5 mmoles/1 of N-ethyl mal- 
eiamide. Ostenonectin was removed with 0.2% Tri- 
ton X-100, and matrix gla protein (MGP) by ultrafil- 



tration through a hollow fiber cartidge HIP10-8. 
approximate cut-off M r 10,000. One gram of the 
1.5M GuHCI insoluble proteins was digested for 2 
hours in a solution of pepsin (Sigma Co. St Louis) 
s 10 ug/mg (enzyme to protein ratio 1:100) in 0.01 N 
HCI at 37°C. The pepsin-cleaved HCI 
insoluble/water insoluble and HCI soluble/water in- 
soluble fractions were separated from the HCI 
soluble/water soluble proteins by centrifugation at 
10 50,000 g. for one hour. Hydrolysis was terminated 
by dialysis by raising the the pH to 7.0 with 0.01 M 
NaOH and by dialysis against cold water in Spec- 
trophor tubing, (pore size cutoff, mol. wt 2.000 
Spectrum Co. Los Angeles, CA). 
75 The COIN HCI soluble products were lyophiliz- 

ed, weighed, and 0.7 g were redissolved in .05M 
phosphate buffer, ph 6.8 in 4M GuHCI. The buf- 
fered solution was applied to a Sephacryl S-200 
column (5cm 1 95cm) with downward flow regu- 
20 lated by a peristaltic pump and collected in 5 
fractions. Two fractions with M r greater than 30,000 
were pooled, dialyzed against water, and re- 
lyophilized for bioassay of new bone formation 
induced per mg of implanted protein. Three frac- 
25 tions with M r less than 30,000 were pooled and 
similarly prepared. Eighty mg samples of the pro- 
teins with M r less than 30,000 were applied to a 
hydroxyapatite column (HA) (2.5 * 4.0 cm) and 
eluted along a stepwise gradient of concentrations 
30 of 0.01 to 0.05, 0.05 to 0.2, 02 to 0.5M phosphate 
buffer. The proteins were collected in 9 fractions, 
examined by SDS gel electrophoresis, desalted in 
sacs (pore size, mol. wt., 2,000) by dialysis against 
cold water, lyophilzied and implanted for BMP ac- 
35 tivity. 

Peptides (M P less than 10K) were subfrac- 
tionatcd usinq Sephadex G-50 Ultrafine molecular 
sieve chromatography. Samples of freeze-dried 
peptides were dissolved in 0.01 M sodium phos- 

40 phate (pH 7.0) containing 4M GuHCI and charged 
to a column (1.5 1 100 cm) equilibrated in the 
same buffer. Fractions of approximately 20 ml each 
were collected, combined within each peak, dia- 
lyzed exhaustively against cold, deionized water 

45 and lyophilized. The resulting fractions were ana- 
lyzed by SDS-PAGE peptide gels by the methods 
of Swank & Munkres (1971). 

The pepsin cleaved 0.01 N HCI insoluble, water 
insoluble products remaining from the two hour 

so limited pepsin proteolysis were separated from the 
0.01 N HCI soluble products by centrifugation at 
50,000 g. washed 3 X in 1 liter of cold deionized 
water, and collected by lyoohiiization. The lyophiliz- 
ed proteins and peptides were dissolved in 4M 

55 GuHCI and similarly fractionated by the above de- 



8 



15 



0 212 474 



16 



scribed procedures. Matrix gla protein (MGP) per- 
sisting insoluble aggregates containing high BMP 
activity, was removed by solubilization in 4 M 
GuHCI and ultrafiltration. 

Reverse phase high performance liquid s 
chromatography (HPCL) was performed on M f 
5,500 ± 1,000 protein fractions isolated by 
Sephadex G-50 chromatography. These fractions 
were dissolved in 4M guandinium hydrochloride 
buffered with 0.01 M phosphate (pH 7.2). After io 
centrifugation through a nylon 66 membrane {to 
eliminate any vestige of undissolved protein), the 
sample was charged to a Vydac (C«) column (4.6 
mm ■ 25 cm) (to. City) and eluted with a gradient 
of 0,1% trifluoracetic acid (TFA) in, water and 0.1% 75 
TFA in acetonitrile (AN). The gradient was started 
at 20% AN and continued to 35% AN in 15 min- 
utes (1%. per minute). From 35% AN to 50% AN 
was accomplished in 10 minutes after which the 
column was reequilibrated. The higher concentra- 20 
tion of TFA served to effect better resolution of 
closely eluting materials and also allowed better 
correlation between analytical and preparative 
chromatographies. After appropriate fractions were 
collected, the TFA and AN were eliminated by 25 
vacuum centrifugation. ihe samples were recovered 
in a dry state by lyophilization. 

For determination of M„ protein fractions were 
examined by sodium dodecyl sulfate 
polyacrylamide slab gel electrophoresis (SDS 30 
PAGE). The lyophilized proteins were solubiiized 
by incubation for 24 hours in 0.06M Tris-HCI (pH 
6.8) containing 2M urea and 02% SDS. Five 
microliters (2.5 mg/ml) of each sample were ap- 
plied to a 12.6% gel with a 3% stacking gel and 35 
were electrophoresed at 25 mA. The gels were 
stained with 0.25% Coomaissie brilliant blue R-250 
in methanol/acetic acid/H,0 (5:1:5). The molecular 
weights were determined by using protein stan- 
dards (Pharmacia) with a range of M f 94,000- 40 
14,400 and by peptide standards (Pharmacia) with 



a range of M r 17,200-1,600. The relative molecular 
mass (M r ) was calculated by plotting the logarithm 
of molecular weight versus distance of migration of 
6 standard peptides relative to the distance of the 
unknown. (Swank & Munkres). 

The population density of human BMP-p mol- 
ecules was concentrated in the region of M r of 5.5K 
i 0.8K. Bovine BMP-p of Example I included mol- 
ecules with M r as smail as 4.1 K + 0.3K. 

Bioassay of BMP-p for osteoinductive activity 
was determined by implantation of 128 lyophilized 
preparations in the hindquarter muscles of Swiss- 
Webster strain mice. For controls, 10 samples each 
of osteonectin (M , 38,000), M r 24,000, MGP (M r 
14,000 to 15,000), and M r 12,000, 8.500, 4,000, 
3,000, and 2,500 products of pepsin proteolysis 
were implanted in the contralateral hindquarter 
muscle pouches. 

The quantity of new bone was measured by 
correlated observations on roentgenograms, excis- 
ed wet ossicle weights, histological sections, 
histomorphometric data, and "Ca uptake by cal- 
cified new bone as described above in Example I. 
Histomorphometric data were obtained by random 
point analysis. 

Human BMP-p was soluble in CMRL (GIBCO) 
culture medium containing 15% fetal calf serum, 
and assayed also in tissue cultures of 10 HPLC 
fractions, 10 ug/ml, in triplicate by the method of 
Sato and Urist. 

Table II summarizes results of bioassay of 
osteoinductive proteins and peptides, including by- 
products of various stages of purification. Bone 
matrix non-collagenous proteins (separated by dif- 
ferential precipitation from solutions of GuHCI) had 
a low level of osteoinductive activity. Partial pu- 
rification by HA chromatography increased the spe- 
cific activity 25X as measured by histomorphome- 
try and 950X by incorporation of tt Ca into calcifying 
new bone. Further purification increased the quan- 
tity of induced bone formation in both parameters. 



50 



9 



17 0 212 474 18 

Table IT 

BIOASSAY OP PROTEINS COMPARED WITH PEPTIDES 
GENERATED BY LIMITED PEPSIN PROTEOLYSIS 



uuinpoijencs 


Bone Histo- 


4 5 

Ca uptake 




morphometric 


by calfifying 




2 2 
mm /nun 


new bone 




of muscles 


cpm/rag 






implanted 






protein 


1.5M GuHCl Insoluble/water 






soluble non-collagenous 






proteins, 90 to 30K 


0.5 


302 ± 104 


38K Triton X-100 soluble 






proteins 


0 


20 i 09 



34K CL-6B Isolated 
osteonectin 



25 - 11 



30 + 24 + 22K 1.5M GuHCl 
Soluble proteins 



31 ± 



11_ 



24K Hydroxy apatite unbound 
protein 



26 ± 



08 



17. 5K BMP + 15 + 

14K ola Protein 



12.4 2660 - 500 



14K Bone ola Protein 



45 - 10 



Pepsin generated 

7 to 14 K polypeptides 



5.5 + 0.8K G-50 Isolated 
polypeptides 



28.5 3696 * 9flO 



13.0 



2964 - 608 



4.7K HPLC Purified 
polypeptides 



Cartilage in 
tissue cultures 



N.D. 



The pi of peptides isolated by. HA chromatog- 
raphy and HPLC was determined by isoelectric 
focusing by the method of Righetti and Drysdaie - 
{1976) using a micropH- probe (Microelectrodes. 
Inc.) for pH measurements on the cold gels {5% 
polyacrylamide and 2% ampholyte). Focusing was 
at 10 W at 2°C for 3.5 hours. The pi of the M f 
4.7K peptide was about 7.1 t 0.1. 



45 



50 



Amino acid analyses were performed on an 
amino acid analyzer (modified -Beckman 121). 
Cystine and half cystine analysis was performed 
following performic acid oxidation. The -composition 
was characteristic of a neutral peptide. The amino 
acid composition of the M f 4.7K peptide is pre- 
sented in Table III, 



55 



10 



19 



0 212 474 

Table III 
31S OF M r 4 
ISOLATED BY REVERSAL PHASE HPLC 



20 



AMINO ACiD ANALYSIS OF M f 4.7K HUMAN BMP-p 



Putative 



nMoles; 


nGrams 


Mole %r 


Residues 


Residue 


Lys 


4.31 


552.4 


4.64 


2.36 


(21 


His 


2.30 


315.4 


2.48 


1.26 


(1) 


Arg 


16.69 


2606.6 


17.97 


9.12 


(9) 


Asx 


7.63 


878.1 


8.22 


4.17 


(4) 


Thr 


0.85 


85.9 


0.92 


0.47 


(0-1) 


Ser 


5.08 


442.3 


5.47 


2.78 


(3) 


(corr . 












by 10%) 












Glu 


18.79 


2426.0 


20.23 


10.27 


(10) 


Pro 


7.54 


732.2 


8.12 


4.12 


(4) 


Gly 


2.16 


123.2 


2.33 


1.18 




Ala 


6.24 


443.5 


6.72 


3.41 


(3) 


1/2 Cys 


6.30 


3.3 


1.35 


3.1 


(2} 


(Cysteic 










Acid) 












Val 


4.18 


414.4 


4.50 


2.28 


(2) 


Met 


1.26 


165.3 


1.36 


0.69 


(1) 


lie 


4.00 


452.6 


4.31 


2.19 


(2) 


Leu 


4.68 


529.5 


5.04 


2.56 


(2-3) 


Tyr 


5.32 


8158 . 1 


5.73 


2.81 


(3) 


Phe 


1.83 


269.3 


1.97 


1.00 


(1) 




92.86 


11,304.8* 


100.01 


50-52 












+ Trp 



* 11,304. 8nGrams = 11. 39; or 113g in 1.0ml (32% 
protein) 



Amino acid sequences of the HPLC purified 
peptides were analyzed in the gas/liquid phase 
protein sequenator. The partial amino acid se- 
quence of the N-terminal segment of HPLC purified 
human BMP-p is shown in Table IV. 



50 



The amino acid sequence of the first 15 resi- 
dues of the N-terminal segment of hBMP-p was 
reproducible on 3 separate batches of bone. The 
sequence of residues 17 to 27 were determined 
with less certainty, and 28 to 36 with interesting 
inconsistencies *hat require special investigation. 



55 



11 



21 



0 212 474 



22 



Table IV 



PARTIAL AMINO ACID SEQUENCE OP hBMP-p 

1 2 3 4 5 

NH. - ILE - PRO - GLN - GLN - ARG 



6 7 8 9 10 

- ARG - TRP - ARG - ALA - LYS 

11 12 13 14 IS 

- VAL - GLN - ASN - ARG - ILE 



(Less certain 
than 1-15) 

21 22 23 24 25 
- LYS - PRO - VAL - HIS - GLU 



26 27 | 28 29 30 
— - LEU - ASM - lAPn - CYS - ALA 

(Less certain 31 32 33 34 35 

than 17-27) - ASP - GLY - TYR - ARG - LEU 

36 
- CYS - 



16 17 18 19 20 

(ALA?)- ARG - ASP - SER - TYR 



EXAMPLE III 

Trypsin proteolysis of purified or partially puri- 
fied BMP generated BMP-p. The trypsin derived 
BMP-p were purified, bio-assayed and character- 
ized by the same methods that were reported for 
isolation of pepsin-derived BMP-p. 

One g of crude partially purified BMP was 
digested for intervals of 15 min., 30 mia. 1 hr. and 
4 hrs. in solutions of trypsin (TPCK-treated. bovine 
pancrease, Sigma, St. Louis), 5 mg per g of total 



protein in O.IM Tris pH 7.20 at 37°C. At the des- 
ignated interval, hydrolysis was terminated by the 
addition of IN HCI to produce a pH of 1.0. The 
solution was centrifuged at 20.000 rpm for 10 min. 

so to separate the acid soluble from the acid insoluble 
precipitates. The precipitate was washed in cold 
water 3*. The acid soluble supernatant was trans- 
ferred to Spectrapor tubing (pore size 2.000 MW. 
Spectrum, Co., Los Angeles), and dialyzed against 

55 ionized water (3 s ). A water insoluble precipitate 
that formed inside the sac was separated from the 
water soluble components by centrifugatton at 



12 



23 



0 212 474 



24 



50,000 g. for 1 hr. f and by washing the precipitate 
3* in deionized cold water. The water soluble 
supernatant and the washed precipitates were sep- 
arately collected, iyophilized, and weighed. 

The Iyophilized products were weighed and 5 
redissolved in .05M phosphate buffer, pH 6.8 in 4M 
GuHCI for $-200 gel filtration. The solutions were 
applied to a Sephacryl S-200 column (5 cm x 95 
cm) with downward flow regulated by a peristaltic 
pump and collected in 5 fractions. Two fractions io 
with M f greater than 30,000 were pooled, dialyzed 
against water, and relyophilized for bioassay of 
new bone formation induced per mg of implanted 
protein. Three fractions with M r proteins less than 
30,000, were pooled and similarly prepared. is 

The proteins with M r less than 30,000 were 
subfractionated using Sephadex G-50 (Ultrafine) 
molecular sieve chromatography. Samples of 
freeze-dried polypeptides were dissolved in 0.01 M 
sodium phosphate (pH 7.0) containing 6M urea and so 
charged to a column (1.5 * 100 cm) equilibrated in 
the same buffer. Fractions of approximately 20 ml 
each were collected, combined within each peak, 
dialyzed extensively against cold deionized water, 
and Iyophilized. The resulting fractions were ana- 25 
lyzed by SDS-PAGE peptide gels by the methods 
of Swank & Munkres. 

Further purification of the polypeptides, iso- 
lated by G-50 gel filtration was accomplished by 
HPLC using a hydrophobic column (Ultrapore, 30 
reverse-phase Spherogel, Beckman). 

Implants of crude partially purified BMP in- ■ 
duced formation of cartilage and -bone in mice. The 
deposits replaced approximately 4% of the volume 
of the quadriceps muscle compartment Implants of 35 
the products of trypsin-iimited proteolysis, both 
acid-insoluble and acid-soluble fractions, induced 
cartilage and bone tissue formations that filled the 
entire quadriceps compartment and sometimes 
even extended across the midline. The reaction to 40 
the acid insoluble/water soluble proteins produced 
formation of fibrous connective tissue only. 

The deposits of bone induced by implants of 
products of limited trypsin proteolysis had the 
same structure as deposits produced by implants 45 
of BMP. The bone consisted of a shell of lamellar 
bone filled with bone marrow, and a core of woven 
bone containing inclusions of cartilage and chondro 
osteoid. 



EXAMPLE IV 

Bone morphogenetic agents are prepared by 
conventional chemical synthesis of peptides from 55 
constituent amino acids comprising the amino acid 
sequence (or substantially homologous equiv- 
alents) of BMP-p such as is given in Table IV. A 



method of chemical synthesis of peptides is the 
Merrifield method. Merrifield, R.B., J. Am. Chem. 
Soc,, 85:2194, 1963; Merrifield. R.B., Science. 
150:178, 1965. 

Such synthetic bone morphogenetic peptide 
agents comprise the osteoinductive and im- 
munoreactive domain of natural BMP. 



EXAMPLE V 

By methods known to one skilled in the art of 
genetic engineering, synthetic DNA "genes" cod- 
ing on expression for bone morphogenetic agents 
are constructed comprising the codon sequence for 
the amino acid sequence for BMP-p such as is 
given in Table IV. The required DNA sequence is 
determined from the amino acid sequence and the 
genetic code, and a "gene" is chemically syn- 
thesized and inserted into a recombinant DNA vec- 
tor. The recombinant vector is introduced into a 
host cell, such as E. ccji • in which it is replicated 
and expressed when the cell is cultured. During 
growth of the host cell in culture, the host cell 
machinery transcribes mRNA from the synthetic 
gene and translates it into bone morphogenetic 
peptide agents. Variables such as vector choice, 
host choice, position of the synthetic gene in the 
recombinant vector, etc., are manipulated to en- 
gineer in Vivo cultures that are efficient factories 
for the production of bone morphogenetic peptide 
agents displaying the osteoinductive and im- 
munoreactive activity of natural BMP or BMP-p. 

For these methods available to one skilled in 
the art of genetic engineering, see for example, 
Maniatis, T„ Fritsch, E.F., and Sambrook, J.. 
(1982) Molecular Cloning, A Laboratory Manual. 
Manual , Cold Spring Harbor Laboratory, Cold 
Spring Harbor. New York. 



EXAMPLE VI 

By methods available to one skilled in the art 
of genetic engineering (see Maniatis et ah above), 
bone morphogenetic agents are produced by ex- 
pressing cloned cDNA or cloned fragments of ver- 
tebrate DNA which comprise the codon sequence 
for part or all of the amino acid sequence for BMP- 
p given in Table IV. 

Using known recombinant DNA techniques, 
fragments of a vertebrate genome or cDNA are 
inserted into vectors, and the recombinant vectors 
are introduced into host organisms. Novel 
radiolabelled nucleic acid probes of single stranded 
DNA or RNA comprising codons for some of all of 
the amino acid sequence for BMP-p "given in Table 
IV are synthesized chemically. These probes (or 



13 



25 



0 212 474 



26 



the complementary strands of these specific nu- 
cleic acids) are used to screen host colonies for ' 
complimentary sequences and thereby to select 
recombinant clones with a DNA sequence compris- 
ing the osteoinductive and immunoreactive domain 5 
of BMP. Selected colonies are cultured and tested 
for the production of peptides that are osteoinduc- 
tive or immunoreactive with anti-BMP antibody. 

Alternatively, novel radiolabeled nucleic acid 
probes comprising codons for some or all of the w 
amino acid sequence for BMP-p given in Table IV 
are used to identify and select by hybridization 
RNA. cDNA or fragments of vertebrate ON A that 
comprise codons for the osteoinductive region of 
BMP. Those selected are used to construct recom- 75 
binant vectors for the production of bone mor- 
phogenetic agents in a host 

Although the instant disclosure sets forth ail 
essential information in connection with the inven- 
tion, the numerous publications cited herein may 20 
be of assistance in understanding the background 
of the invention and the state of the art Accord- 
ingly, ail of the publications cited are hereby incor- 
porated by reference into the present disclosure. 

\\ w\\ be understood Wis description and 25 
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 
substitute amino acids within the amino acid se- 00 
quence of a given BMP-p without substantially af- 
fecting the bone morphogenetic activity of the mol- 
ecule. The invention is expressly stated to be 
broad enough to include intentional deletions, addi- 
tions or substitutions. Furthermore, it is recognized 35 
that one skilled in the art could recombinants 
produce such modified proteins. 



Claims 



40 



1- A bone morphogenetic agent comprising a 
peptide displaying osteoinductive activity similar to 
bone morphogenetic protein (BMP), said peptides 
obtained from whatever source, including natural, 4$ 
synthetic or recombinant DNA sources. 

2. The bone morphogenetic peptide agent of 
claim 1, comprising amino acid sequences of N- 
terminal segments of bone morphogenetic protein - 
(BMP), said N-terminal segments of BMP having a 50 
range of relative molecular weights (M f ) from about 

4K to about 7K, said peptide agent obtained from 
whatever source, including single or multiple amino 
acid additions, substitutions, deletions or insertions. 

3. The bone morphogenetic peptide agent of 55 
claim 1 which are characterized by containing the 
following amino acid sequence: -ILE -PRO -GLN - 
GLN -ARG -ARG -TRP -ARG -ALA -LYS -VAL - 



GLN -ASN -ARG -ILE-. and including single or 
multiple amino acid additions, substitutions, dele- 
tions or insertions which provide a peptide similar 
in osteoinductive activity of BMP. 

4. The bone morphogenetic peptide agent of 
claim 3. including the following amino acid se- 
quence: -ARG -ASP -SER -TYR -LYS -PRO -VAL - 
HIS -GLU -LEV -ASN-. 

5. The bone morphogenetic peptide agent of 
claim 4, including the following amino acid se- 
quence: -ARG-CYS - ALA -ASP -GLY -TYR -ARG - 
LEU -CYS-. 

6. The bone morphogenetic. peptide agents of 
claim 1 derived from proteolysis of BMP, and hav- 
ing, a range of relative molecular weights (M f ) from 
about 4K to about 7K. 

7. The bone morphogenetic peptide agent of 
claim 6 wherein said proteolysis of BMP is by 
pepsin, 

8. The bone morphogenetic peptide agent of 
claim 6 wherein said proteolysis of BMP is by 
trypsin. 

9. The bone morphogenetic peptide agent of 
claim 6, 7 % or 8 wherein said proteotv*ed BMP \s 
human BMP. 

1 0. The bone morphogenetic peptide agent of • 
claim 9 wherein said human BMP-p has a relative 
molecular weight (M r ) of about 4.7K t 0.3K. 

11. The bone morphogenetic peptide agent of 
claim 6, 7 or 8 wherein said proteolyzed BMP is 
bovine BMP. 

12. The bone morphogenetic peptide agent of 
claim 11; wherein said bovine BMP-p has a relative 
molecular weight (M f ) in the range of about 4.1 K. 

13. The bone morphogenetic peptide agent of 
claim 1 derived from direct chemical* synthesis 
from constituent amino acids and wherein said 
peptide comprises amino acid sequences of BMP- 
p, including single or multiple amino acid additions, 
substitutions, deletions or insertions. 

14. The bone morphogenetic peptide agent of 
claim 1 derived from expression of DNA sequences 
comprising sequences of codons which on expres- 
sion code for a peptide similar in osteoinduction 
activity of BMP or BMP-p, said DNA sequences 
from whatever source obtained, including natural, 
synthetic or semi-synthetic sources. 

15. The bone morphogenetic peptide agent of 
claim 14, wherein said DNA expressed is char- 
acterized by a. sequence of codons which on ex- 
pression code for a peptide containing the amino 
acid sequence: -ILE -PRO -GLN -GLN -ARG -ARG 
-TRP -ARG -ALA -LYS -VAL -GLN -ASN -ARG - 
ILE-. including single or multiple base substitutions, 
additions, deletions or insertions which express a 
peptide similar in immunoreactive or osteoinductive 
activity of BMP-p. 



14 



27 



0 212 474 



28 



16. A process for selecting DNA sequences 
coding on expression for a peptide displaying the 
immunoreactive or osteoinductive activity of BMP 
or BMP-p from a group of DNA sequences from 
whatever source, comprising selecting the DNA s 
sequences that hybridize to a nucleic acid probe 
containing the DNA or the RNA sequences or the 
complements thereof that code for the amino acid 
sequences of BMP-p or the amino acid sequence: - 

ILE -PRO -GLN -GLN -ARG. -ARG -TRP -ARG -ALA 70 
-LYS -VAL -GLN -ASN -ARG -1LE-, and said probe 
including single or multiple base substititions, addi- 
tions, deletions or insertions. 

17. A nucleic acid probe specific for selecting 
nucleic acid sequences coding on expression for a 75 
peptide displaying the immunoreactive or osteoin- 
ductive activity of bone morphogenetic protein - 
(BMP) comprising the codon sequence or the com- 
plement thereof for part or all of the amino acid 
sequences of BMP-p, or comprising part or all of 20 
the amino acid sequence of Table IV said probe 
including single or multiple base substitutions, ad- 
ditions, deletions or insertions. 

18. A DNA sequence which expresses a bone 
morphogenetic peptide agent and which hybridizes 25 
to the probe of claim 17. 

19. A process for producing a DNA sequence 
coding on expression for a peptide displaying the 
immunoreactive or osteoinductive activity of BMP 

or BMP-p, comprising direct chemical synthesis of 30 
DNA containing a sequence of codons for the ami- 
no acid sequence of BMP-p f including single or 
multiple base additions, substitutions, deletions or 
insertions, or comprising enzymatic synthesis of 
cDNA from an RNA template containing -codons for 35 
the amino acid sequence of BMP-p f including sin- 
gle or multiple base additions, substitutions, dele- 
tions or insertions. 

20. A method of producing a recombinant DNA 
molecule capable of expressing a bone mor- 40 
phogenetic agent comprising the step of introduc- 
ing into a cloning vehicle a DNA sequence selected 
according to claim 16 or produced according to 
claim 19. 



21. A recombinant DNA molecule comprising a 
cloning vehicle having inserted therein a DNA se- 
quence selected according to the process of claim 
16 or produced according to the process of claim 
19. 

22. A host transformed with a recombinant 
DNA molecule according to claim 21. 

23. A method of producing a bone mor- 
phogenetic peptide agent displaying the im- 
munoreactive or osteoinductive activity of BMP or 
BMP-p comprising the steps of transforming an 
appropriate host with a recombinant molecule ac- 
cording to claim 21, culturing said host, and col- 
lecting said peptide. 

24. A method of producing a bone mor- 
phogenetic peptide agent displaying the im- 
munoreactive or osteoinductive activity of BMP 
comprising proteolyzing BMP for a period of time 
to produce BMP-p having relative molecular 
weights in the range from about 4K to about 7K. 

25. The method of claim 24, wherein said prot- 
eolysis of BMP is by pepsin. 

26. The method of claim 24, wherein said prot- 
eolysis of BMP is by trypsin. 

27. A method for producing a bone mor- 
phogenetic peptide agent displaying the im- 
munoreactive or osteoinductive activity of BMP 
comprising direct chemical synthesis of said pep- 
tide from constituent amino acids, said peptide 
containing the amino acid sequence of BMP-p, 
including single or multiple amino acid additions, 
substitutions, deletions or insertions. 

28. A composition for inducing bone formation 
which comprises a bone morphogenetic peptide 
agent selected from the group consisting of pep- 
tides according to any one of claims 1-8, 13-15. 

29. A method for inducing bone formation in 
vertebrates which comprises administering to said 
vertebrate in a pharmaceutical^ acceptable man- 
ner an effective amount of a composition according 
to claim 28. 



55 



15 



0 212 474 



FIGURE 1 

PEPSIN LIMITED PROTEOLYSIS OF BOVINE BMP 

Partially Purified 
BMP ' 

(Step 8, PNAS 81:371-375, 1984) 
+ 

Pepsin (10 ug/mg Protein) 
in 0.01N HC1, 2h 

Group I 



HC1 soluble peptides. 
Dialyze (2K MW pore 

size vs. HjO.) 



HC1 insoluble/^O 
insoluble peptides, 
(80% of the total 
BMP activity.) 



Group II 



Group III 



HC1 soluble/water 
insoluble peptides. 
(10 to 20% of the 
total BMP activity.)* 



HC1 soluble/water 
soluble peptides 
(No BMP activity. ) 



0 212 474 



v 

FIGURE 2 



DECLINE IN BMP 
ACTIVITY BY PEPSIN PROTEOLYSIS 



ICC 



0 « HC1 CONTROL 

□ = HC1-SOLUBLE 

WATER INSOLUBLE 

A = HC1-INSOLUBLE 

WATER-INSOLUBLE 



75 



so 



25 




JL 



-L 



A 



L5 2 2.3 3 3.5 W 

HOURS, PEPSIN PROTEOLYSIS 



*5 



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