EP0853667A2 - Bone stimulating factor - Google Patents

Abstract

Polypeptides which increase or promote mammalian bone growth, related nucleotide sequences, antibodies, diagnostic kits and treatments. Subsequences of the polypeptide Asp Ser Asp Leu Tyr Ala Glu Leu Arg Cys Met Cys Ile Lys Thr Thr Ser Gly Ile His Pro Lys Asn Ile Gln Ser Leu Glu Val Ile Gly Lys Gly Thr His Cys Asn Gln Val Glu Val Ile Ala Thr Leu Lys Asp Gly Arg Lys Ile Cys Leu Asp Pro Asp Ala Pro Arg Ile Lys Lys Ile Val Gln Lys Lys Leu Ala Gly Asp Glu Ser Ala Asp have been shown to promote growth. Subsequences include Asp Ser Asp Leu Tyr Ala Glu Leu Arg Cys Met Cys Ile Lys Thr Thr Ser Gly Ile His Pro Lys Asn Ile Gln Ser; Ile Lys Thr Thr Ser Gly Ile His Pro Lys Asn Ile Glu Ser; and Cys Met Cys Ile Lys Thr Thr Ser Gly Ile His Pro Lys Asn Ile Gln.

Description

BONE STIMULATING FACTOR

The present invention relates to polypeptides which stimulate bone growth Understanding of issues related to bone growth and strength has progressed over the years, a summary being provided in, for example, international patent application No PCT/CA 94/00144, published on September 15, 1994 under WO 94/20615, United States Patent No 5,320,970 and European patent application No 92 302 446, published under 505 210 on September 23, 1992 the contents of which applications are incorporated herein by reference

By way of background to the present invention, described below, neutrophil- activating peptide (NAP-2, SEQ ID NO 1 ) and a variant of NAP-2, here termed "NAP-2V (SEQ ID NO 2) have been known for some time (Walz, A , and Baggio ni, (1989) Biochem Biophys Res Commun 159 969) British Patent No 2 231 872 (British Patent No 2 231 872 Inventors M Baggiohni. K J Clemetson, and A Walz Published June 14, 1990 ), describes the ammo acid sequence of NAP-2 and three apparently naturally occurring variants including NAP-2V The other two variants have an additional four (SEQ ID NO 3) and three (SEQ ID NO 4) ammo acids at the N-terminal of the NAP-2 sequence NAP-2 is a subsequence of β-thromboglobulm (β-TG, SEQ ID NO 5) which has an additional eleven ammo acids at the N-terminal end β-TG is itself a subsequence of connective tissue- activating peptide (CTAP-III, SEQ ID NO 6) which has an additional four ammo acids at the N- terminal CTAP-III is a subsequence of platelet basic protein (PBP, SEQ ID NO 7) which has an additional nine am o acids at the N-termmal

NAP-2 along with ιnterleukιn-8 (human IL-8, SEQ ID NO 8, porcine IL-8 SEQ ID NO 9) and melanoma growth-stimulating activity (MGSA) have been assigned to a subfamily known as the α-chemokines The α-chemokιnes have in common with each other four cysteine residues at highly conserved positions, which enclose the core region of the molecules as described by Brandt et a/ (Ehlert, J E , F Peterson, M H G Kubbuta, J Gerdes, H -D Flad, and E Brandt, (1995) J Biol Chem 2706338) Brandt et al found an apparently naturally occurring C-termmus truncated variant of NAP-2, lacking the last four ammo acids of NAP-2, that displays enhanced increase in potency to stimulate neutrophil degranulation Brandt et al also synthesized variants lacking the final one, two, three five and six ammo acids of the C-termmus of NAP-2 All of these C-truncated polypeptides exhibited a moderate increase in potency over NAP-2 with the exception of the sequence having only the first sixty-four ammo acids of NAP-2 Brandt et al discussed the possible significance of the sequence modifications with respect to the structure of NAP-2 and its function

Platelet factor 4 (PF4, SEQ ID NO 10) is a seventy ammo acid polypeptide (Hermodson M G Schmer and K Kurachi, (1977) J Biol Chem 252 6276 Morgan, F J G S Begg C N Chesterman, (1979) Thromb Haemost 42 1652) PF4 has been shown to inhibit proliferation of two osteoblastic osteosarcoma cell lines Saos-2 and G-292 (United States Patent No 5,304,542 Inventor D M Tatakis Issued April 19, 1994) Indomethacin apparently did not affect PF4-ιnduced inhibition of the cell proliferation Particular fragments, PF4(58-70), PF4(47-70) and monomeric low-affinity PF4 (LAPF4), which is 50% homologous to PF4 and contains an α- helical C-termmus were also suggested as being useful PF4 and such related polypeptides were thus described as being useful in a method for inhibiting proliferation of osteoblasts, in among other things, humans suffering from osteoporosis

The first 70 ammo acids of NAP-2V and the sequence of PF4 are about 51% homologous and the positions of the four cysteine residues are conserved between the two polypeptides It has now been shown that NAP-2, NAP-2V, as well as subsequences of

NAP-2V also show bone stimulatory effects, while certain subsequences do not display bone stimulatory activity NAP-2V-(1-26) (SEQ ID NO 11) and NAP-2V-( 13-26, gln²⁵-glu²⁵) (SEQ ID NO 12) were found to increase the observed bone apposition rate, the latter of the two being more potent than the former NAP-2V-( 10-26) (SEQ ID NO 13) appeared to cause a small increase in the observed bone apposition rate although the statistical significance of the observed increase was questionable NAP-2V-(11-26) (SEQ ID NO 14) and NAP-2V-( 12-26) (SEQ ID NO 15) were found to have no effect on observed bone mineral apposition rate

The invention thus includes a polypeptide which promotes bone growth in mammals, where the polypeptide includes an ammo acid sequence corresponding to SEQ ID NO 2 with (a) from 6 to about 12 ammo acids deleted from the N-termmus of SEQ ID NO 2, (b) 7 to about 49 ammo acids deleted from the C-terminus of SEQ ID NO 2, or both (a) and (b), wherein the sequence includes no cysteine residues or at least two cysteine residues or a functionally equivalent homologue

In another aspect, a polypeptide of the present invention is an ammo acid sequence corresponding to SEQ ID NO 11 up to 69 am o acids in length, or corresponding to SEQ ID NO 11 with from 6 to about 12 ammo acids deleted from the N-terminus of SEQ ID NO 11 , or corresponding to SEQ ID NO 11 with from 6 to about 9 ammo acids deleted from the N-termmus of SEQ ID NO 11 , wherein the sequence includes no cysteine residues or at least two cysteine residues, or a functionally equivalent homologue Alternatively, the invention is a polypeptide which promotes bone growth in mammals where the polypeptide has an ammo acid sequence corresponding to SEQ ID NO 12 up to 69 ammo acids in length or a functionally equivalent homologue, or having an ammo acid sequence consisting essentially of the ammo acid sequence corresponding to SEQ ID NO 11 , or a conservatively substituted variant thereof or a polypeptide having an ammo acid sequence consisting essentially of the ammo acid sequence corresponding to SEQ ID NO 12 or a conservatively substituted variant thereof, or a polypeptide having an am o acid sequence consisting essentially of the ammo acid sequence corresponding to SEQ ID NO 13, or a conservatively substituted variant thereof

A polypeptide of the present invention can have at least two cysteine residues which are located at positions corresponding to the tenth and twelfth positions of SEQ ID NO 2

A polypeptide of the present invention can have, if desired or necessary, one or the other or both of the N-termmal ammo acid and the C-termmal ammo acid protected by a protecting group

The polypeptide can be synthetic and the am o acid sequence can have a molecular weight in the range of from about 1000 to 4000, or from about 1500 to about 3000 or more preferably from about 1500 to about 1800

In another aspect, the invention is a first polypeptide comprising a sequence of ammo acids sufficiently dup cative of a second polypeptide having an am o acid sequence corresponding to SEQ ID NO 2 with (a) from 6 to about 12 am o acids deleted from the N- terminus of SEQ ID NO 2 (b) 7 to about 49 am o acids deleted from the C-terminus of SEQ ID NO 2, or both (a) and (b), wherein the sequence includes no cysteine residues or at least two cysteine residues, or a functionally equivalent homologue, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide The DNA sequence of NAP-2V disclosed by Walz ef al (British Patent No 2 231 872 Inventors M Baggiohni, K J Clemetson, and A Walz Published June 14, 1990

Neutrophil-activating peptιde-2 and processes for the production of NAP-2, B-TG, CTAP-III and PBP) is identified here as SEQ ID NO 16

It will, of course, be understood by those skilled in the art that portions of the nucleic acid sequence identified as SEQ ID NO 16 correspond to sequences coding for the polypeptides identified as SEQ ID NO 1 , NO 11 , NO 12 (Glu-GIn) or NO 13 For example nucleic acids 37 through 78 of SEQ ID NO 16 encode the am o acid subsequence identified as SEQ ID NO 12 in which the penultimate ammo acid of the subsequence is glutamine

"Stringent hybridization conditions" takes on its common meaning to a person skilled in the art here Appropriate stringency conditions which promote nucleic acid hybridization, for example, 6x sodium chloride/sodium citrate (SSC) at about 45°C are known to those skilled in the art The following examples are found in Current Protocols in Molecular Biology John Wiley & Sons NY (1989), 6 3 1-6 3 6 For 50 ml of a first suitable hybridization solution mix together 24 ml formamide 12 ml 20x SSC 0 5 ml 2 M Tris-HCl pH 7 6 0 5 ml 100x Denhardt's solution 2 5 ml deionized H₂0, 10 ml 50% dextran sulfate and 0 5 ml 10% SDS A second suitable hybridization solution can be 1% crystalline BSA (fraction V) 1 mM EDTA 0 5 M Na₂HPO_< pH 7 2, 7% SDS The salt concentration in the wash step can be selected from a low stringency of about 2x SSC at 50°C to a high stringency of about 0 2x SSC at 50°C Both of these wash solutions may contain 0 1% SDS In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22 °C to high stringency conditions, at about 65 °C The cited reference gives more detail, but appropriate wash stringency depends on degree of homology and length of probe If homology is 100%, a high temperature (65°C to 75°C) may be used If homology is low, lower wash temperatures must be used However, if the probe is very short (<100bp), lower temperatures must be used even with 100% homology In general one starts washing at low temperatures (37°C to 40°C), and raises the temperature by 3-5°C intervals until background is low enough not to be a major factor in autoradiography Alternatively, such a first polypeptide is a sequence of am o acids sufficiently dup cative of a second polypeptide having an ammo acid sequence corresponding to SEQ ID NO 11 up to 69 ammo acids in length, or corresponding to SEQ ID NO 11 with from 6 to about 12 ammo acids deleted from the N-termmus of SEQ ID NO 11 , or corresponding to SEQ ID NO 11 with from 6 to about 9 ammo acids deleted from the N-termmus of SEQ ID NO 11 , wherein the sequence includes no cysteine residues or at least two cysteine residues or a functionally equivalent homologue, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide

The first polypeptide can have a sequence of am o acids sufficiently duplicative of a second polypeptide having an ammo acid sequence corresponding to SEQ ID NO 12 up to 69 ammo acids in length, or a functionally equivalent homologue, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide

The first polypeptide can have a sequence of ammo acids sufficiently duplicative of a second polypeptide having an am o acid sequence corresponding to to SEQ ID NO 11 , or a conservatively substituted variant thereof, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide, or it can have a sequence of ammo acids sufficiently duplicative of a second polypeptide comprising an ammo acid sequence corresponding to SEQ ID NO 12, or a conservatively substituted variant thereof, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide, or it can have a sequence of ammo acids sufficiently duplicative of a second polypeptide having an ammo acid sequence corresponding to SEQ ID NO 13, or a conservatively substituted variant thereof, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide A bone growth polypeptide of the invention can be of any suitable length, and particularly can be up 14amιno acids in length or up to 20 am o acids in length or up to 30 ammo acids in length, or up to 40 ammo acids in length, or up to 50 ammo acids in length, or up to 60 ammo acids or more in length

The invention includes any number of chimeric bone stimulating factors made having an ammo acid sequence of polypeptides of the present invention In another aspect, the invention is an agent for use in prevention and treatment of a bone reduction related disease which includes any polypeptide or polypeptides of the present invention as an active ingredient

The invention is, alternatively, a pharmaceutical composition for promoting bone growth, having a therapeutically effective amount of a polypeptide or polypeptides of the present invention

The invention includes a method of increasing bone growth in a mammal by administering a therapeutically effective amount of a polypeptide having an am o acid sequence of a polypeptide or polypeptides of the present invention

The invention includes use of a polypeptide or polypeptides of the present invention for the treatment of osteoporosis Alternatively, the use of a polypeptide or polypeptides can be to promote bone growth in a mammal

The invention includes use of the polypeptide or polypeptides in the preparation of a medicament for use in promoting bone growth or the treatment of osteoporosis The invention includes a diagnostic kit for determining the presence of a polypeptide or polypeptides of the present invention The kit can includes an antibody to a said polypeptιde(s) linked to a reporter system wherein the reporter system produces a detectable response when a predetermined amount of the polypeptιde(s) and the antibody are bound together The invention includes an antibody synthesized using a polypeptide consisting of an ammo acid sequence identified as SEQ ID NO 11 , SEQ ID NO 12. or SEQ ID NO 13 or a conservatively substituted variant thereof

More generally, the invention includes an antibody which binds to a polypeptide or polypeptides of the present invention, synthesized using the polypeptιde(s) The invention includes an isolated DNA fragment which encodes the expression of any of the polypeptides of the present invention, and DNA which differs from the fragment due to the degeneracy of the genetic code

The invention includes a vector comprising a DNA sequence which encodes the expression of any of any of the polypeptides of the present invention The invention includes a process for producing a polypeptide of the invention which includes the steps of a) preparing a DNA fragment containing a nucleotide sequence which encodes the polypeptide, b) incorporating said DNA fragment into an expression vector to obtain a recombinant

DNA fragment which contains the DNA fragment and is capable of undergoing replication, c) transforming a host cell with the recombinant DNA fragment to isolate a transformant which can express the polypeptide, and d) culturmg the transformant to allow the transformant to produce the polypeptide and recovering the polypeptide from resulting cultured mixture In yet another aspect, the invention is a synthetic polypeptide up to 65 ammo acids in length, having in vivo bone stimulatory activity in mammals, having an ammo acid sequence which is at least about 19% conserved in relation to the ammo acid sequence identified as SEQ ID NO 2, a conservatively substituted variant thereof, or a functionally equivalent homologue The synthetic polypeptide can also be at least about 22%, 25%, 28%, 31%. or

35% conserved in relation to the ammo acid sequence identified as SEQ ID NO 2, a conservatively substituted variant thereof, or a functionally equivalent homologue

Such a synthetic polypeptide can have at least 49 ammo acids deleted from the sequence The polypeptide can have no cysteine residues or at least two cysteine residues

The polypeptide can have a molecular weight in the range of from about 1000 to 4000

In another aspect, the present invention is a first polypeptide having a sequence of ammo acids sufficiently duplicative of a second polypeptide which includes an ammo acid sequence of any of the synthetic polypeptides, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide

The invention includes a chimeric bone stimulating factor comprising an ammo acid sequence of any of the synthetic polypeptides

An agent of the present invention for use in prevention and treatment of a bone reduction related disease can include one or more of the synthetic polypeptides

As well a pharmaceutical composition of the present invention for promoting bone growth can include a therapeutically effective amount of a one or more of the synthetic polypeptides

The invention includes a method of increasing bone growth in a mammal by administering a therapeutically effective amount of one or more of the synthetic polypeptides Such a synthetic polypeptide can be used for the treatment of osteoporosis or to promote bone growth in a mammal

Such a synthetic polypeptide can be used in the preparation of a medicament for use in promoting bone growth or the treatment of osteoporosis The invention includes a diagnostic kit for determining the presence of one or more the synthetic polypeptides, the kit including antιbody(ιes) to a the polypeptιde(s) linked to a reporter system wherein the reporter system produces a detectable response when a predetermined amount of the polypeptιde(s) and the antιbody(ιes) are bound together

The invention includes an antibody which binds to one or more of the synthetic polypeptides, synthesized using the polypeptide

Further still, the invention includes an isolated DNA fragment which encodes the expression of any of the synthetic polypeptides, and DNA which differs from the fragment due to the degeneracy of the genetic code The invention includes a vector which includes such a DNA sequence The invention includes a process for producing any of the synthetic polypeptides, which includes a) preparing a DNA fragment containing a nucleotide sequence which encodes a polypeptide, b) incorporating said DNA fragment into an expression vector to obtain a recombinant DNA fragment which contains the DNA fragment and is capable of undergoing replication, c) transforming a host cell with the recombinant DNA fragment to isolate a transformant which can express the polypeptide, and d) culturmg the transformant to allow the transformant to produce the polypeptide and recovering the polypeptide from resulting cultured mixture

The invention includes a polypeptide exhibiting bone stimulatory activity in mammals, the polypeptide being up to 65 ammo acids in length and havmg the sequence identified as SEQ ID NO 11 , SEQ ID NO 12, or SEQ ID NO 13, analogues thereof wherein the am o acids in the sequence may be substituted, deleted or added, so long as the bone stimulatory activity in mammals derived the three dimensional structure of the sequence is preserved and conjugates of each of the polypeptides or analogues thereof, wherein if the polypeptide sequence contains a cysteine residue, there are at least two cysteine residues Such a polypeptide can be substantially pure and the ammo acid sequence can have a molecular weight in the range of from about 1000 to about 4000, or from about 1500 to about 3000 or from about 1500 to about 1800 The invention is also a first polypeptide that includes a sequence of am o acids sufficiently duplicative of a second polypeptide having an ammo acid sequence corresponding such a polypeptide or a functionally equivalent homologue thereof, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide, or a chimeric bone stimulating factor including such an ammo acid sequence, or an agent for use in prevention and treatment of a bone reduction related disease which includes such a polypeptide as an active ingredient, or a pharmaceutical composition for promoting bone growth, having a therapeutically effective amount of such a polypeptide, or a method of increasing bone growth in a mammal by administering a therapeutically effective amount of such a polypeptide, having an ammo acid sequence of a polypeptide defmed in claim 56 or 57, or the use of such a polypeptide for the treatment of osteoporosis or to promote bone growth in a mammal, or the use of such a polypeptide in the preparation of a medicament for use in promoting bone growth or the treatment of osteoporosis, or a diagnostic kit for determining the presence of such a polypeptide, the kit including an antibody to a the polypeptide linked to a reporter system wherein the reporter system produces a detectable response when a predetermined amount of the polypeptide and the antibody are bound together, or an antibody which binds to such a polypeptide, synthesized using the polypeptide, or an isolated DNA fragment which encodes the expression of any such polypeptide, or which differs from the fragment due to the degeneracy of the genetic code, or a vector including a DNA sequence which encodes the expression of any such polypeptide, or a process for producing such a polypeptide, which process includes a) preparing a DNA fragment containing a nucleotide sequence which encodes the polypeptide, b) incorporating the DNA fragment into an expression vector to obtain a recombinant DNA fragment which contains the DNA fragment and is capable of undergoing replication, c) transforming a host cell with the recombinant DNA fragment to isolate a transformant which can express the polypeptide, and d) culturing the transformant to allow the transformant to produce the polypeptide and recovering the polypeptide from resulting cultured mixture

Finally, the invention includes an isolated DNA sequence encoding the ammo acid sequence of any of the polypeptides of the invention, or an analogue thereof, wherein the ammo acids in the sequence may be substituted, deleted or added, so long as bone stimulatory activity in mammals derived from the three dimensional conformation of the sequence is preserved in a polypeptide comprising the ammo acid sequence, sequences which hybridize to the DNA and encode an ammo acid sequence of a polypeptide which displays bone stimulatory activity in mammals and DNA which differs from the sequence due to the degeneracy of the genetic code DESCRIPTION OF THE DRAWINGS

Figure 1 shows the bone apposition rate (μm per day) for rats injected with 25 nmol (N=5 in both cases) of chemically synthesized polypeptides having the sequences of NAP-2 (SEQ ID NO 1) and NAP-2V (SEQ ID NO 2), respectively, compared to that of a group of control (N=5) rats The error bars are ± 1 S D

Figure 2 graphically illustrates the observed bone mineral apposition rate (μm per day) for rats injected with chemically synthesized polypeptides of A, NAP-2V-(1-26) (SEQ ID NO 11 ), B, NAP-2V-( 13-26, gln²⁵-glu²⁵) (SEQ ID NO 12, ), C, NAP-2V-( 10-26) (SEQ ID NO 13), D, NAP-2V-(11-26) (SEQ ID NO 14), and E, NAP-2V-( 12-26) (SEQ ID NO 15) The first bar in the graph represents the control The number of rats used for the determinations were 4, 4, 3, 4, 4, and 4, respectively The error bars are ± 1 S D

Figure 3 illustrates the ammo acid sequences of polypeptides tested, corresponding ammo acids aligned with each other, the active peptides being shown above the line and sequences which were not found to stimulate bone growth being below the line Approximate molecular weights are shown below the sequence identification numbers

MATERIALS, METHODS AND RESULTS

Polypeptides having the sequences of NAP-2 (SEQ ID NO 1 ) and NAP-2V (SEQ ID NO 2) were chemically synthesized directly according to standard methods and experiments were conducted to determine whether the chemically synthesized polypeptides displayed activity

Experiments were conducted simultaneously on three groups of male Sprague-Dawley rats, there being five rats in each group Each rate weighed between 250 and 350 g Each rat of the first group was given, by subcutaneous injection into the left gluteus maximus region, 200 μl of a 1% aqueous acetic acid solution containing 25 nmol (about 191 μg) of NAP-2 (SEQ ID NO 1 ) Each rat of the second group was similarly given 200 μl of a 1% aqueous acetic acid solution containing 25 nmol (about 207 μg) of NAP-2V (SEQ ID NO 2) Each rat of the third group, the control group, was similarly given 200 μl of 1% acetic acid solution

Immediately following administration of the test solution, 300 μl of an aqueous solution of tetracycline hydrochloride was administered intramuscularly into the right gluteus maximus the concentration of tetracycline being sufficient to obtain a dosage of about 24mg/kg of rat body weight A second dose of tetracycline hydrochloride solution was administered about 48 hours after the first dose The rats were sacrificed about 24 hours after administration of the second dose of tetracycline Sections of the lower metaphysis of the right femur were used for bone measuring the bone mineral apposition rate Processing of the bone material for measurement has been described previously See, for example, international patent application No. PCT/CA 94/00144 published under No WO 94/20615 on September 15, 1994 The results obtained are summarized in Table One and Figure 1

TABLE ONE: Comparison of the Group Arithmetic Means of Bone Apposition Rates (μm/day) Among Groups Administered with NAP-2, NAP-2V and control solutions shown in Figure 1

Control SEQ ID NO 1 SEQ ID NO:2

Mean 0.99 μm/d 1.23 μm/d 1.28 μm/d

S.D. 0.04 0.05 0 08

N 5 5 5 t d f P

Control Group vs 7.91 8 <0.001 SEQ ID NO:1

Control Group vs 7.03 8 O.001 SEQ ID NO:2

SEQ ID NO: 1 vs 1.15 8 >0.20 SEQ ID NO.2

Polypeptides having the sequence corresponding to either SEQ ID NO 1 or SEQ ID NO:2 have thus been found to stimulate bone growth.

In a second set of experiments, twenty-four male Sprague-Dawley rats (Charles River Laboratory) were divided into six groups of four Each of the first group, the control group, was injected in the right thigh with 200 μl of 0 1 % acetic acid solution The rats of the other groups were each injected in the right thigh with about 200 μl of a 0 1% acetic acid solution containing about 25 nmol of a chemically synthesized polypeptide as follows

oup Polypeptide SEQ ID NO

A NAP-2V-(1-26) 11

B NAP-2V-(13-26; gln²⁵-glu²⁵) 12

C NAP-2V-( 10-26) 13

D NAP-2V-(11-26) 14

E NAP-2V-( 12-26) 15

Immediately after injection of the polypeptide (control) solution, each rat was injected in the right gluteus maximus with 200 μl of a 1M tetracycline hydrochloride (Sigma Chemical) solution This dosage of tetracycline is about 16 mg per kg of animal body weight About 48 hours later, each rat was injected in the left gluteus maximus with the same dosage of tetracycline Twenty-four hours later the rats were sacrificed by C0₂ narcosis and the right femur taken for bone mineral apposition rate determination One rat died during the course of the experiments

Immediately after dissection a bone sample was fixed in 10% formaldehyde solution at pH 7 4 Later the same day, a 1 1 H₂0-acetone solution was exchanged for the formaldehyde solution This was exchanged twice the following day with acetone This was exchanged the following day by a 1 1 acetone-Spurr's medium solution which was exchanged later the same day with Spurr s medium The following day each sample was embedded in a fresh change of Spurr's medium and cured at 60°C for 24 hours followed by curing at 80°C for 24 hours

Each cured block was cut into 400 μm thick sections using a Leitz saw microtome equipped with a diamond charged blade The relatively thick sections were ground down between two ground glass plates pre-roughened with carborundum powder to a final thickness of about 10 μm, water being used as the grinding lubricant The thin sections were dried and mounted unstained in Permount (Fisher)

Measurements were made using a Leitz scanning light microscope photometer MPV-CD magnifying the sections 16X, as described in international patent application No PCT/CA94/00144

The results obtained are shown in Table Two and Figure 2

Table TWO: Comparison of Group Arithmetic Means of Bone Apposition Rates (μm per day) shown in Figure 2.

Control Group A Group B Group C Group D Group E

Mean 1 08 1 47 1 70 1 18 1 07 1 04

S D 0 08 0 05 0 09 0 08 0 07 0 08

N 4 4 3 4 4 4 t d f P

Control vs Group A 7 90 6 <0 001

Control vs Group B 9 69 5 <0 001

Group A vs Group B 4 87 5

As graphically illustrated in Figure 2 NAP-2V-(1-26) (SEQ ID NO 11 ) and NAP-2V-( 13-26, gln²⁵-glu²⁵) (SEQ ID NO 12) act to stimulate bone growth the latter of these two polypeptides displaying greater activity

NAP-2V-( 10-26) (SEQ ID NO 13) appeared to cause a small increase in the observed bone apposition rate although the significance of the observed increase was questionable NAP-2V-(11-26) (SEQ ID NO 14) and NAP-2V-( 12-26) (SEQ ID NO 15) were found to have no effect on observed bone mineral apposition rate The sequence of NAP-2V- (10-26) retains both the cys¹⁰ and cys¹² residues The sequences of NAP-2V-(11-26) and NAP-2V-( 12-26) each retain the cys¹² residue The sequence of NAP-2V-(13-26, gln²⁵-glu²⁵) retains neither of the cys¹⁰ and cys¹² residues All of these NAP-2V subsequences lack the cys³⁶ and cys⁵² residues present in the parent NAP-2V It may be that the reduced activity of NAP-2V-( 10-26), NAP-2V-(11-26) and NAP-2V-( 12-26) is due to spontaneous intermolecular disulfide bonding that prevents a polypeptide-receptor interaction required for the bone stimulatory effect, but this is not known for certain The sequence of NAP-2V-( 13-26, gln²⁵-glu²⁵) is different from the corresponding subsequence of NAP-2V at the 25 position a glutamic acid residue being present in place of the glutamine residue It would of course be expected that the subsequence having the glutamine residue as occurs in NAP-2V would also act to stimulate bone growth in mammals It has been postulated that NAP-2 contains two internal disulfide bonds between Cys-5 and Cys-31 , and Cys-7 and Cys-47, respectively (Baggiolmi, M , Clemetson, K J , Walz, A Internationai Patent Appication No PCT/EP89/01389, published June 14 1990 under WO90/06321 ) By extension, sequences and subsequences disclosed herein that contain the corresponding cysteine residues would likely contain similar linkages therebetween

It will of course be understood, without the intention of being limited thereby, that a variety of other substitutions of ammo acids is possible while preserving the structure responsible for the bone stimulatory effect of the subsequences of NAP-2V disclosed herein Conservative substitutions are described in the patent literature, as for example in United States Patent No 5 2264,558 It is thus expected, for example, that interchange among non¬ polar aliphatic neutral ammo acids, glycine, alanine, proline, valine and isoleucine, would be possible Likewise substitutions among the polar aliphatic neutral am o acids, serine threonine, methionine asparagine and glutamine could possibly be made Substitutions among the charged acidic ammo acids, aspartic acid and glutamic acid, could probably be made as could substitutions among the charged basic ammo acids, lysine and arginine

Substitutions among the aromatic ammo acids, including phenylalanine histidine tryptophan and tyrosine would also likely be possible These sorts of substitutions and interchanges are well known to those skilled in the art Other substitutions might well be possible A peptide containing an ammo acid sequence that can be aligned with that of SEQ ID NO 11 or SEQ ID NO 12 and having 50% or homology therewith may retain at least part of the bone stimulating effect thereof Of course it would also be expected that the greater percentage of homology say 60%, 70%, 80%, 90%, or more, could increase the degree of retained bone stimulating activity

Insofar as deletion of one or more ammo acids is concerned, it is likely that deletions of a small number of ammo acids from each end of either sequence might be possible, bearing in mind the observation that the deletions to obtain SEQ ID NOs 14 and 15 yield polypeptides which do not appear to enhance bone growth Further, symmetrical, or nearly symmetrical deletions would likely be the most possible to be made while retaining the three-dimensional configuration Internal deletions, although likely to be possible to some limited extent, should be few Additions of ammo acids could very likely be made at the ends of the sequence, and as with deletions, symmetrical or nearly symmetrical additions to the carboxy and ammo terminals are likely to be possible Internal additions, although likely to be possible to some limited extent, should be few

Of the above-listed modifications to the sequence, terminal additions are most likely to be most useful, as such a modification can serve a variety of functions an identifying group as for use in a radioimmunoassay, or a linking group, as examples

A polypeptide of the present invention can be improved with respect to possible degradation, as might occur in the body in the presence of protease, for instance, by protection of the C-terminus, the N-termmus, or both the C-termmus and N-termmus of the polypeptide

As used herein, "protected" terminal am o group refers to a terminal ammo group (N-termmus) coupled with any of various amino-terminal protecting groups that can be employed in peptide synthesis Examples of suitable groups include acyl protecting groups, for example, formyl, acetyl, benzoyi, tπfluoroacetyl, succinyl, and methoxysuccinyl, aromatic urethane protecting groups, for example benzyloxycarbonyl, and aliphatic urethane protecting groups, for example f-butoxycarbonyl or adamantyloxycarbonyl (Gross and Mienhofer eds ,

The Peptides vol 3, pp 3 to 88 (Academic Press, New York, 1981))

As used herein, "protected" terminal carboxyl group refers to a terminal carboxyl group (C-terminus) coupled with any of various carboxy-terminal protecting groups As will be readily apparent to a person skilled in the art, suitable groups include f-butyl, benzyl or other acceptable groups linked to the terminal carboxyl group through an ester or ether bond

Compounds within the scope of this invention can be synthesized chemically be means well known in the art such, for example solid phase peptide synthesis The synthesis is commenced from the carboxy-terminal end of the peptide using an α-amino protected ammo acid f-Butyloxycarbonyl (Boc) protective groups, or other suitable protective groups can be used (Stewart ef al "Solid-Phase Peptide Synthesis ' W H Freeman Co San Francisco (1969), Merrifield, J Am Chem Soc 85 2149-2154 (1963), Vale ef a/ , Sc/e/7ce 213, 1394-1397 (1981), and Marke ef al J Am Chem Sci 103, 3178 (1981)) Synthetic methods are also described in "Principles of Peptide Synthesis" M Bodansky Ed (Spnng-Verlag 1984) These and other methods of peptide synthesis are also exemplified by U S Patent Nos 3.862,925, 3,842,067, 3,972,859, 4,105 602, 4,683,291 4,244 946 and 4,305,872

Compounds may also be synthesized using manual or automatic techniques, for example, an Applied BioSystems 430A Peptide Synthesizer (Foster City, California) or a Biosearch SAM 11 automatic peptide synthesizer (Biosearch, Inc , San Rafael, California) Compounds of the present invention and compositions containing them find use in numerous therapeutic and prophylactic applications in the prevention and treatment of bone reduction related to a disease Compounds can thus be used as treatments to promote bone growth, in the treatment of osteoporosis, for example, by any suitable route The preferred routes are suitable for delivery of polypeptide-type compounds to the bloodstream of a subject, bearing in mind proper storage and handling conditions required for polypeptides such as those described herein

Thus the present invention also provides compositions containing an effective amount of compounds of the present invention, including the nontoxic addition salts, amides and esters thereof, which may, alone, serve to provide the treatment benefits described above Such compositions can also be provided together with physiologically tolerable liquid, gel or solid diluents adjuvants and excipients

In the above examples involving subsequences of NAP-2V, about 75 nmol of polypeptide per kg of bodyweight of animal was used per administration In practice, particularly as human subjects are concerned, the daily dosage may well be between 0 01 and 300 mg or more per kg of bodyweight More preferably, the dosage would be in the neighbourhood of from about 0 1 to about 30 mg per kg of bodyweight It may be that the preferred frequency of administration would be greater or less than once per day, depending upon the route of administration, convenience, and the variation of effectiveness of treatment with frequency of and amount used per administration The dosage administered also depends on the subject and to which effect such administration is to give The dosage of any one or more of the compounds will depend on many factors including the specific compound or combination of compounds being utilized, the mode of administration, and the mammal being treated Dosages of a particular compound or combination of compounds can be determined using conventional considerations, for example, by customary comparison of the differential activities of the subject compounds and that of a known agent that is by means of an appropriate pharmacological protocol in which for example, bone density of subjects is measured over time Pharmaceutical preparations include any of the compounds prepared as an injectable solution, including an injectable solution prepared just prior to use, for promoting bone growth and/or treatment of osteoporosis An injectable can be either a liquid solution or suspension, solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared The preparation may also be emulsified The active polypeptide is often mixed with diluents and excipients which are physiologically tolerable and compatible with the polypeptide Suitable diluents and excipients are, for example, water, saline, dextrose glycerol or the like, and combinations thereof In addition if desired, the compositions can contain minor amounts of auxiliary substances such as wetting or emulsifying agents stabilizing or pH buffering agents, and the like

Pharmaceutical preparations include the employment of the compounds in admixture with conventional excipients, that is, pharmaceutically acceptable organic or inorganic carrier substances which do not deleteriously react with the compounds, and which possibly enhance the storage and handling stability of the compounds The preparative procedure may include the sterilization of the pharmaceutical preparations The compounds may be mixed with auxiliary agents such as lubricants, preservatives, stabilizers, salts for influencing osmotic pressure, etc , which do not react deleteriously with the compounds

The compositions are conventionally administered parenterally, by injection, for example either subcutaneously or intravenously Additional formulations which are suitable for other modes of administration include suppositories, intranasal aerosols, and, in some cases, oral formulations For suppositories, traditional binders and excipients may include, for example, polyalkylene glycols or triglycerides, such suppositories may be formed from mixtures containing the active ingredient in the range of 0 5% to 10%, preferably 1%-2% Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like These compositions take the form of solutions, suspensions, tablets, pills capsules, sustained release formulations, or powders, and contain 10% - 95% of active ingredient, preferably 25% - 70% These oral formulations include formulations designed to protect the peptide until it can be absorbed The peptide compounds may be formulated into the compositions as neutral or salt forms Pharmaceutically acceptable non-toxic salts include the acid addition salts (formed with the free ammo groups) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like Salts formed with the free carboxyl groups may be derived from inorganic bases such as, for example, sodium, potassium ammonium, calcium, or ferric hydroxides and such organic bases as isopropylamine trimethylamine, 2-ethylamιno ethanol histidme procaine and the like The compounds of the invention can be homopolymeπzed to themselves (i e , (peptide)-) or, heteropolymeπzed to one another The compounds can also be conjugated to biocompatible polymeric compounds, such as BIOPOL™ (WR Grace & Co -Conn )

If prepared using recombinant techniques, a DNA sequence encoding a desired polypeptide of the present invention is synthesized using standard automated techniques, or the coding sequences or portions thereof are retrieved from cDNA or genomic libraries This DNA is ligated into suitable expression vectors and these vectors are transformed into appropriate hosts A variety of expression vector/host cell systems can be used, including both procaryotic and eukaryotic culture systems Procaryotes most frequently are represented by various strains of E coli

However, other microbial strains may also be used, such as bacilli, for example bacillus subtilis, various species of Pseudomonas, ot other bacterial strains In such procaryotic systems, plasmid vectors which contain replication origins, and control sequences derived from a species compatible with the host are used For example, E coli is typically transformed using derivatives of pBR322, a plasmid derived from an E coli species (Bolivar et al , (1977) Gene 2 95 Commonly used procaryotic control sequences, which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta- lactamase (penicillmase), lactose (1nc) promoter systems (Chang et al , (1977) Nature 198 1056), the tryptophan (trp) promoters system (Goeddel et al , (1990) Nucleic Acids Res 8 4057), and the lambda-derived P_L promoter and N-gene ribosome binding site (Shimatake et al , (1981 ) Nature 292 128) However, any available promoter system compatible with procaryotes can be used

The expression systems useful in the eukaryotic systems of the invention comprise promoters derived from appropriate eukaryotic genes A class of promoters useful in yeast, for example, include promoters for synthesis of glycolytic enzymes, including alcohol dehydrogenase promoters, glyceraldehyde-3-phosphate dehydrogenase promoter (Holland & Holland, (1980) J Biol Chem 25 2596), alpha-factor promoter (Bitter et al , (1984) Proc Natl Acad Sci 81 5330), the gal promoter (Johnston & David, (1984) Mol Cell Biol 4 1440) those for 3-phosphoglycerate kinase (Hitzeman et al , (1980) J Biol Chem 256 1385) or the Leu2 gene obtained from YEp13 (Broach J , et al , (1978) Gene 8 121 )

Suitable mammalian promoters include the early and late promoters from SV40 (Fiers et al , (1978) Nature 273 113) or other viral promoters such as those derived from polyoma, adenovirus II, bovine papilloma virus or avian sarcoma viruses Suitable viral and mammalian enhancers are cited above In the event plant cells are used as an expression system the nopaline synthesis promoter is appropriate (Depicker, A et al (1982) J Mol Appl Gen 1 56) The expression systems are included on replication vectors or are caused to integrate into the chromosome of a recombinant host For systems wherein the vectors include a replication system, these may be low or high copy number, usually having copy numbers of fewer than about 1000, although in certain situations, runaway vectors may be employed. Whether provided on a vector intended for integration or in a replication system, the sequence encoding a polypeptide of the invention may be ligated in tandem with an amplifying gene such as dihydrofolate reductase, metallothioneins, thymidine kinase, or the like In procaryotic systems, both the amplifying gene and the target gene can be under the regulation of the same transcnptional and translational regulatory regions Usually, the vector will include a marker which allows for selection of host cells containing the expression system; the nature of these markers depends on the host and is understood in the art. In addition to required regulators such as promoters, additional sequences such as enhancers can also be employed to enhance the level of transcription. If the polypeptide is to be secreted, an upstream sequence encoding signal peptides such as those described in U.S Pat. Nos 4,336,336, 4,338,397; and 4,546,082 may be employed The signal sequence is enzymatically cleaved as the polypeptide product is secreted

Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described by Cohen, S.N., (1972) Proc Natl Acad Sci USA 69:2110; or the RbCI method described in Maniatis et al., Molecular Cloning: A Laboratory Manual (1982) Cold Spring

Harbor Press, p. 254 is used for procaryotes or other cells which contain substantial cell wall barriers Infection with Agrobacteπum tumefaciens (Shaw, C.H., (1938) et al., Gene 23:315) is used for certain plant cells. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, (1978) Virology 52:2:546 is preferred Transformations into yeast are carried out, for example, according to the method of Van Solmgen, P., et al., (1977) J Bacter 130:946; and Hsiao, CL, et al., (1979) Proc Natl

In general, after construction of a suitable expression system, the system is transfected into the appropriate host and successful transformants are selected by markers contained on the expression vectors. Successfully transformed colonies are then cultured in order to produce the desired polypeptide. It is sometimes preferred that a promoter which can be controlled by regulating conditions in the environment be used so that the cells can be grown under conditions where the gene encoding the desired polypeptide of the invention is not expressed, and then production of the polypeptide induced by appropriate manipulation of conditions For example, if the trp promoter is used in E coli, the cells are grown in the presence of tryptophan and expression is then induced by diminution of tryptophan concentration or by addition of a tryptophan analogue such as indolylacetic acid. If the gene is under control of the PL promoter, the cells are grown at relatively low temperature such as at about 35°C , to a suitable cell density, and the temperature is then elevated to activate this promoter If produced in bacterial hosts as a mature intracellular polypeptide, the N-terminal methionine may or may not be cleaved In mammalian systems, for example, the use of the metallothionein promoter permits induction by addition of heavy metals or glucocorticoids This protocol is preferred to prevent premature accumulation of the polypeptide which might be harmful to the growth of the cell

The polypeptide can be produced mtracellularly, or in secreted form by construction of vectors in which the peptide is preceded by a signal peptide workable in the appropriate host

The polypeptide is recovered from the medium or from the cells using suitable techniques generally known in the art, and purified by, for example, ion exchange chromatography, ammonium sulfate precipitation, gel permeation chromatography, and so forth The polypeptide made available by the invention disclosed herein can be used to obtain antisera thereto (Stites, D P and A I Terr 1991 In Basic & Clinical Immunology, 7th Ed Appleton and Lange, Norwalk, Connecticut and San Matea California) Methodology and products can be developed using an antibody to a polypeptide for use in detecting the polypeptide with which the antibody binds This apparently having been accomplished at least for the polypeptide having the sequence of CTAP-III (SEQ ID NO 3) (Baggiohni, M , Clemetson, K J , Walz, A Internationai Patent Appication No PCT/EP89/01389, published June 14, 1990 under WO90/06321 ) Methodology and products can be developed using an antibody to a polypeptide for use in detecting the polypeptide with which the antibody binds For example, an antibody can be linked to or conjugated with a reporter system which is set up to indicate positively binding of the polypeptide to the antibody Well known reporter systems include radioimmuno assays (RIAs) or immunoradiometπc assays (IRMAs) Alternatively, an enzyme-linked immunosorbent assay (ELISA) would have in common with RIAs and IRMAs a relatively high degree of sensitivity, but would generally not rely upon the use of radioisotopes A visually detectable substance may be produced or at least one detectable in a spectrophotometer An assay relying upon fluroescence of a substance bound by the enzyme being assayed could be used It will be appreciated that there are a number of reporter systems which may be used, according to the present invention to detect the presence of a particular polypeptide With standardized sample collection and treatment polypeptide presence above a threshold amount in blood serum could well be determined Such an antibody-linked reporter system could be used in a method for determining whether blood serum of a subject contains a deficient amount of the polypeptide Given a normal threshold concentration of such a polypeptide in blood serum of a given type of subject, test kits could thus be developed A further advantage may be obtained through chimeric forms of the protein, as known in the art A DNA sequence encoding the entire protein, or a portion of the protein, could thus be linked with a sequence coding for the C-termmal portion of £ coli β- galactosidase to produce a fusion protein, for example An expression system for human respiratory syncytial virus giycoproteins F and G is descnbed in United States Patent No 5,288,630 issued February 22, 1994 and references cited therein, for example

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(l) APPLICANT:

(A) NAME- OSTEOPHARM LIMITED

(B) STREET: 2395 Speakman Drive

(C) CITY: Mississauga

(D) PROVINCE: Ontario

(E) COUNTRY: CA

(F) POSTAL CODE (ZIP) : L5K 1B3

(A) NAME: TAM, Cherk Shmg

(B) STREET: 1072 Rectory Lane

(C) CITY: Oakville

(D) PROVINCE: Ontario {E) COUNTRY: CA

(F) POSTAL CODE (ZIP) L6M 2B7

(ll) TITLE OF INVENTION: BONE STIMULATING FACTOR (ill) NUMBER OF SEQUENCES: 16

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette, 3 1/2 inch, 1 4 Mb storage

(B) COMPUTER: COMPAQ, IBM PC compatible

(C) OPERATING SYSTEM: MS-DOS 5.1

(D) SOFTWARE: WORD PERFECT

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NO:

(B) FILING DATE: 26-SEPTEMBER-1996 (Vl) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 60/004,314

(B) FILING DATE: 26-SEPTEMBER-1995

(2) INFORMATION FOR SEQ ID NO:1

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH. 70 ammo acids (D) TOPOLOGY: linear

(XI) SEQUENCE DESCRIPTION: SEQ ID NO:1

Ala Glu Leu Arg Cys Met Cys Ile Lys Thr Thr Ser Gly Ile His Pro 1 5 10 15

Lys Asn Ile Gin Ser Leu Glu Val Ile Gly Lys Gly Thr His Cys Asn 20 25 30

Gin Val Glu Val Ile Ala Thr Leu Lys Asp Gly Arg Lys Ile Cys Leu 35 40 45 Asp Pro Asp Ala Pro Arg Ile Lys Lys Ile Val Gin Lys Lys Leu Ala 50 55 60

Gly Asp Glu Ser Ala Asp 65 70

(2) INFORMATION FOR SEQ ID NO:2

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 75 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2

Asp Ser Asp Leu Tyr Ala Glu Leu Arg Cys Met Cys Ile Lys Thr Thr 1 5 10 15

Ser Gly lie His Pro Lys Asn Ile Gin Ser Leu Glu Val Ile Gly Lys 20 25 30

Gly Thr His Cys Asn Gin Val Glu Val Ile Ala Thr Leu Lys Asp Gly 35 40 45

Arg Lys Ile Cys Leu Asp Pro Asp Ala Pro Arg Ile Lys Lys Ile Val 50 55 60

Gin Lys Lys Leu Ala Gly Asp Glu Ser Ala Asp 65 70 75

(2) INFORMATION FOR SEQ ID NO:3

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 74 ammo acids (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3

Ser Asp Leu Tyr Ala Glu Leu Arg Cys Met Cys Ile Lys Thr Thr Ser 1 5 10 15

Gly Ile His Pro Lys Asn Ile Gin Ser Leu Glu Val Ile Gly Lys Gly 20 25 30

Thr His Cys Asn Gin Val Glu Val Ile Ala Thr Leu Lys Asp Gly Arg 35 40 45

Lys Ile Cys Leu Asp Pro Asp Ala Pro Arg Ile Lys Lys Ile Val Gin 50 55 60

Lys Lys Leu Ala Gly Asp Glu Ser Ala Asp 65 70

(2) INFORMATION FOR SEQ ID NO:4

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 73 amino acids

(B) TYPE: ammo acid (D) TOPOLOGY, linear

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:4

Asp Leu Tyr Ala Glu Leu Arg Cys Met Cys Ile Lys Thr Thr Ser Gly 1 5 10 15

Ile His Pro Lys Asn Ile Gin Ser Leu Glu Val Ile Gly Lys Gly Thr 20 25 30

His Cys Asn Gin Val Glu Val Ile Ala Thr Leu Lys Asp Gly Arg Lys 35 40 45

Ile Cys Leu Asp Pro Asp Ala Pro Arg Ile Lys Lys Ile Val Gin Lys 50 55 60

Lys Leu Ala Gly Asp Glu Ser Ala Asp 65 70 (2) INFORMATION FOR SEQ ID NO:5

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 81 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5

Gly Lys Glu Glu Ser Leu Asp Ser Asp Leu Tyr Ala Glu Leu Arg Cys 1 5 10 15

Met Cys Ile Lys Thr Thr Ser Gly Ile His Pro Lys Asn Ile Gin Ser 20 25 30

Leu Glu Val Ile Gly Lys Gly Thr His Cys Asn Gin Val Glu Val Ile 35 40 45

Ala Thr Leu Lys Asp Gly Arg Lys Ile Cys Leu Asp Pro Asp Ala Pro 50 55 60

Arg Ile Lys Lys Ile Val Gin Lys Lys Leu Ala Gly Asp Glu Ser Ala 65 70 75 80

Asp

(2) INFORMATION FOR SEQ ID NO: 6

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 85 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6

Asn Leu Ala Lys Gly Lys Glu Glu Ser Leu Asp Ser Asp Leu Tyr Ala 1 5 10 15

Glu Leu Arg Cys Met Cys Ile Lys Thr Thr Ser Gly Ile His Pro Lys 20 25 30

Asn Ile Gin Ser Leu Glu Val Ile Gly Lys Gly Thr His Cys Asn Gin 35 40 45

Val Glu Val Ile Ala Thr Leu Lys Asp Gly Arg Lys Ile Cys Leu Asp 50 55 60

Pro Asp Ala Pro Arg Ile Lys Lys Ile Val Gin Lys Lys Leu Ala Gly 65 70 75 80

Asp Glu Ser Ala Asp 85 (2) INFORMATION FOR SEQ ID NO:7

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 94 ammo acids

(B) TYPE: ammo acid (D) TOPOLOGY: linear

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 7

Ser Ser Thr Lys Gly Gin Thr Lys Art Asn Leu Ala Lys Gly Lys Glu

1 5 10 15

Glu Ser Leu Asp Ser Asp Leu Tyr Ala Glu Leu Arg Cys Met Cys Ile 20 25 30

Lys Thr Thr Ser Gly Ile His Pro Lys Asn lie Gin Ser Leu Glu Val 35 40 45

Ile Gly Lys Gly Thr His Cys Asn Gin Val Glu Val Ile Ala Thr Leu 50 55 60

Lys Asp Gly Arg Lys Ile Cys Leu Asp Pro Asp Ala Pro Arg Ile Lys 65 70 75 80

Lys Ile Val Gin Lys Lys Leu Ala Gly Asp Glu Ser Ala Asp

85 90

(2) INFORMATION FOR SEQ ID NO: 8

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 79 amino acids

(B) TYPE: am o acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8

Glu Gly Ala Val Leu Pro Arg Ser Ala Lys Glu Leu Arg Cys Gin Cys 1 5 10 15

Ile Lys Thr Tyr Ser Lys Pro Phe His Pro Lys Phe Ile Lys Glu Leu 20 25 30

Arg Val Ile Glu Ser Gly Pro His Cys Ala Asn Thr Glu Ile Ile Val 35 40 45

Lys Leu Ser Asp Gly Arg Glu Leu Cys Leu Asp Pro Lys Glu Asn Trp 50 55 60

Val Gin Arg Val Val Glu Lys Phe Leu Lys Arg Ala Glu Asn Ser 65 70 75 (2) INFORMATION FOR SEQ ID NO: 9

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 103 base pairs (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9

Met Thr Ser Lys Leu Ala Val Ala Phe Leu Ala Val Phe Leu Leu Ser 1 5 10 15

Ala Ala Leu Cys Glu Ala Asp Val Leu Ala Arg Val Ser Ala Glu Leu 20 25 30

Arg Cys Gin Cys Ile Asn Thr His Ser Thr Pro Phe His Pro Lys Phe 35 40 45 lie Lys Gle Leu Arg Val Ile Gle Ser Gly Phe His Cys Glu Asn Ser 50 55 60

Glu Ile Ile Val Lys Leu Val Asn Gly Lys Glu Val Cys Leu Asp Pro 65 70 75 80

Lys Glu Lys Trp Val Gin Lys Val Val Gin Ile Phe Leu Lys Arg Thr 85 90 95

Glu Lys Gin Gin Gin Gin Gin 100

(2) INFORMATION FOR SEQ ID NO: 10

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 70 ammo acids

(B) TYPE: amino acid (D) TOPOLOGY- linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10

Glu Ala Glu Glu Asp Gly Asp Leu Gin Cys Leu Cys Val Lys Thr Thr 1 5 10 15

Ser Gin Val Arg Pro Arg His Ile Thr Ser Leu Glu Val Ile Lys Ala 20 25 30

Gly Pro His Cys Pro Thr Ala Gin Leu Ile Ala Thr Leu Lys Asn Gly 35 40 45

Arg Lys Ile Cys Leu Asp Leu Glu Ala Pro Leu Tyr Ly- Lys Ile Ile 50 55 60

Lys Lys Leu Leu Glu Ser 65 70 (2) INFORMATION FOR SEQ ID NO:11

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 amino acids (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11

Asp Ser Asp Leu Tyr Ala Glu Leu Arg Cys Met Cys Ile Lys Thr Thr 1 5 10 15

Ser Gly Ile His Pro Lys Asn Ile Gin Ser 20 25

(2) INFORMATION FOR SEQ ID NO:12

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid (D) TOPOLOGY, linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12

Ile Lys Thr Thr Ser Gly Ile His Pro Lys Asn Ile Glu Ser 1 5 10

(2) INFORMATION FOR SEQ ID NO: 13

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 ammo acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(XI) SEQUENCE DESCRIPTION: SEQ ID NO:13

Cys Met Cys Ile Lys Thr Thr Ser Gly Ile His Pro Lys Asn Ile Gin 1 5 10 15

Ser

(2) INFORMATION FOR SEQ ID NO:14

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14

Met Cys Ile Lys Thr Thr Ser Gly Ile His Pro Lys Asn Ile Gin Ser 1 5 10 15 (2) INFORMATION FOR SEQ ID NO:15

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15

Cys Ile Lys Thr Thr Ser Gly Ile His Pro Lys Asn Ile Gin Ser 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:16

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 228 base pairs

(B) TYPE: nucleic acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16

GAC AGT GAC TTG TAT GCT GAA CTC CGC TGC ATG TGT ATA AAG ACA ACC 48 Asp Ser Asp Leu Tyr Ala Glu Leu Arg Cys Met Cys Ile Lys Thr Thr 1 5 10 15

TCT GGA ATT CAT CCC AAA AAC ATC CAA AGT TTG GAA GTG ATC GGG AAA 96 Ser Gly Ile His Pro Lys Asn Ile Gin Ser Leu Glu Val Ile Gly Lys 20 25 30

GGA ACC CAT TGC AAC CAA GTC GAA GTC ATA GCC ACA CTG AAG GAT GGG 146 Gly Thr His Cys Asn Gin Val Glu Val Ile Ala Thr Leu Lys Asp Gly 35 40 45

AGG AAA ATC TGC CTG GAC CCA GAT GCT CCC AGA ATC AAG AAA ATT GTA 192 Arg Lys Ile Cys Leu Asp Pro Asp Ala Pro Arg Ile Lys Lys Ile Val 50 55 60

CAG AAA AAA TTG GCA GGT GAT GAA TCT GCT GAT TAA 228

Gin Lys Lys Leu Ala Gly Asp Glu Ser Ala Asp TER 65 70 75

Claims

1 A polypeptide which promotes bone growth in mammals, comprising an ammo acid sequence corresponding to SEQ ID NO 2 with (a) from 6 to about 12 ammo acids deleted from the N- terminus of SEQ ID NO 2, (b) 7 to about 49 ammo acids deleted from the C-termmus of SEQ ID NO 2, or both (a) and (b), wherein the sequence includes no cysteine residues or at least two cysteine residues, or a functionally equivalent homologue

2 A polypeptide which promotes bone growth in mammals comprising an ammo acid sequence corresponding to SEQ ID NO 11 up to 69 ammo acids in length, or corresponding to SEQ ID NO 11 with from 6 to about 12 ammo acids deleted from the N-terminus of SEQ ID NO 11 , or corresponding to SEQ ID NO 11 with from 6 to about 9 ammo acids deleted from the N-termmus of SEQ ID NO 11 , wherein the sequence includes no cysteine residues or at least two cysteine residues, or a functionally equivalent homologue

3 A polypeptide which promotes bone growth in mammals comprising an ammo acid sequence corresponding to SEQ ID NO 12 up to 69 ammo acids in length, or a functionally equivalent homologue

4 A polypeptide comprising an ammo acid sequence consisting essentially of the ammo acid sequence corresponding to SEQ ID NO 11, or a conservatively substituted variant thereof

5 A polypeptide comprising an ammo acid sequence consisting essentially of the ammo acid sequence corresponding to SEQ ID NO 12, or a conservatively substituted variant thereof 6 A polypeptide comprising an ammo acid sequence consisting essentially of the ammo acid sequence corresponding to SEQ ID NO 13, or a conservatively substituted variant thereof

7 The polypeptide of claim 1 wherein the at least two cysteine residues are located at positions corresponding to the tenth and twelfth positions of SEQ ID NO 2

8 The polypeptide of claim 1 wherein any substitution of an ammo acid is a conservative substitution

9 The polypeptide of any of claims 1 to 8 wherein one or the other or both of the N-termmal am o acid and the C-termmal ammo acid includes a protecting group

10 A polypeptide of any of claims 1 to 8 wherein the polypeptide is synthetic and the ammo acid sequence has a molecular weight in the range of from about 1000 to 4000 11 A polypeptide of any of claims 1 to 8 wherein the ammo acid sequence has a molecular weight in the range of from about 1500 to about 3000

12 A polypeptide of any of claims 1 to 8 wherein the am o acid sequence has a molecular weight in the range of from about 1500 to about 1800

13 A first polypeptide comprising a sequence of ammo acids up to 69 ammo acids in length and sufficiently duplicative of a second polypeptide comprising an ammo acid sequence corresponding to SEQ ID NO 2 with (a) from 6 to about 12 ammo acids deleted from the N- termmus of SEQ ID NO 2, (b) 7 to about 49 ammo acids deleted from the C-termmus of SEQ ID NO 2, or both (a) and (b), wherein the sequence includes no cysteine residues or at least two cysteine residues, or a functionally equivalent homologue, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide

14 A first polypeptide comprising a sequence of ammo acids up to 69 ammo acids in length and sufficiently duplicative of a second polypeptide comprising an ammo acid sequence corresponding to SEQ ID NO 11 up to 69 ammo acids in length, or corresponding to SEQ ID

NO 11 with from 6 to about 12 ammo acids deleted from the N-termmus of SEQ ID NO 11 , or corresponding to SEQ ID NO 11 with from 6 to about 9 ammo acids deleted from the N-termmus of SEQ ID NO 11 , wherein the sequence includes no cysteine residues or at least two cysteine residues or a functionally equivalent homologue, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide

15 A first polypeptide comprising a sequence of ammo acids up to 69 ammo acids in length and sufficiently duplicative of a second polypeptide comprising an ammo acid sequence corresponding to SEQ ID NO 12 up to 69 am o acids in length, or a functionally equivalent homologue, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encodmg the second polypeptide

16 A first polypeptide comprising a sequence of am o acids up to 69 ammo acids in length and sufficiently duplicative of a second polypeptide comprising an am o acid sequence corresponding to to SEQ ID NO 11 , or a conservatively substituted variant thereof, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide

17 A first polypeptide comprising a sequence of ammo acids up to 69 ammo acids in length and sufficiently duplicative of a second polypeptide comprising an ammo acid sequence corresponding to SEQ ID NO 12, or a conservatively substituted variant thereof such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide

18 A first polypeptide comprising a sequence of am o acids up to 69 ammo acids in length and sufficiently duplicative of a second polypeptide comprising an ammo acid sequence corresponding to SEQ ID NO 13 or a conservatively substituted variant thereof, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide

19 A first polypeptide of any of claims 13 to 18 up to 60 ammo acids in length 20 A first polypeptide of any of claims 13 to 18 up to 50 ammo acids in length

21 A first polypeptide of any of claims 13 to 18 up to 40 ammo acids in length

22 A first polypeptide of any of claims 13 to 18 up to 30 am o acids in length 23 A first polypeptide of any of claims 13 to 18, up to 20 ammo acids in length wherein the ammo acid sequence includes no cysteine residues or at least two cysteine residues

24 A first polypeptide of any of claims 13 to 18, up to 14 ammo acids in length

25 A chimeric bone stimulating factor comprising an ammo acid sequence of any of claims 1 to 8 or claims 13 to 18

26 An agent for use in prevention and treatment of a bone reduction related disease which comprises a polypeptide of any of claims 1 to 8 or claims 13 to 18 as an active ingredient

27 A pharmaceutical composition for promoting bone growth, comprising a therapeutically effective amount of a polypeptide of any of claims 1 to 8 or claims 13 to 18 28 A method of increasing bone growth in a mammal by administering a therapeutically effective amount of a polypeptide having an ammo acid sequence of a polypeptide defined in any of claims

1 to 8 or claims 13 to 18

29 The use of a polypeptide of any of claims 1 to 8 or claims 13 to 18 for the treatment of osteoporosis 30 The use of a polypeptide of any of claims 1 to 8 or claims 13 to 18 to promote bone growth in a mammal

31 The use of a polypeptide having a sequence according to any of claims 1 to 8 or claims 13 to

18 in the preparation of a medicament for use in promoting bone growth or the treatment of osteoporosis 32 A diagnostic kit for determining the presence of a polypeptide of any of claims 1 to 8 or claims

13 to 18 comprising an antibody to a said polypeptide linked to a reporter system wherein the reporter system produces a detectable response when a predetermined amount of the polypeptide and the antibody are bound together

33 An antibody synthesized using a polypeptide consisting of an ammo acid sequence identified as SEQ ID NO 11 SEQ ID NO 12, or SEQ ID NO 13 or a conservatively substituted variant thereof

34 An antibody which binds to a polypeptide defined in any of claims 1 to 8 or claims 13 to 18, synthesized using the polypeptide

35 An isolated DNA fragment which encodes the expression of any of the polypeptides of claims 1 to 8 or claims 13 to 18, and DNA which differs from the fragment due to the degeneracy of the genetic code

36 A vector comprising a DNA sequence which encodes the expression of any of the polypeptides of claims 1 to 7 or 9

37 A process for producing a polypeptide of any of claims 1 to 8 or claims 13 to 18 which comprises a) preparing a DNA fragment containing a nucleotide sequence which encodes said polypeptide, b) incorporating said DNA fragment into an expression vector to obtain a recombinant DNA fragment which contains said DNA fragment and is capable of undergoing replication, c) transforming a host cell with said recombinant DNA fragment to isolate a transformant which can express said polypeptide, and d) culturmg said transformant to allow the transformant to produce said polypeptide and recovering said polypeptide from resulting cultured mixture

38 A synthetic polypeptide up to 65 ammo acids in length, having in vivo bone stimulatory activity in mammals, having an ammo acid sequence which is at least about 19% conserved in relation to the ammo acid sequence identified as SEQ ID NO 2, a conservatively substituted variant thereof, or a functionally equivalent homologue

39 A synthetic polypeptide up to 65 ammo acids in length, having in vivo bone stimulatory activity in mammals, having an am o acid sequence which is at least about 22% conserved in relation to the am o acid sequence identified as SEQ ID NO 2, a conservatively substituted variant thereof; or a functionally equivalent homologue 40 A synthetic polypeptide up to 65 ammo acids in length, having in vivo bone stimulatory activity in mammals, having an ammo acid sequence which is at least about 25% conserved in relation to the ammo acid sequence identified as SEQ ID NO.2, a conservatively substituted variant thereof, or a functionally equivalent homologue

41 A synthetic polypeptide up to 65 ammo acids in length, having in vivo bone stimulatory activity in mammals, having an am o acid sequence which is at least about 28% conserved in relation to the am o acid sequence identified as SEQ ID NO 2, a conservatively substituted variant thereof, or a functionally equivalent homologue

42 A synthetic polypeptide up to 65 am o acids in length, having in vivo bone stimulatory activity in mammals, having an ammo acid sequence which is at least about 31 % conserved in relation to the ammo acid sequence identified as SEQ ID NO 2, a conservatively substituted variant thereof, or a functionally equivalent homologue

43 A synthetic polypeptide up to 65 am o acids in length, having in vivo bone stimulatory activity in mammals, having an ammo acid sequence which is at least about 35% conserved in relation to the ammo acid sequence identified as SEQ ID NO.2, a conservatively substituted variant thereof, or a functionally equivalent homologue

44 A polypeptide of claim 38, 39, 40, 41 , 42 or 43 having at least 49 ammo acids deleted from said sequence

45 A polypeptide of claim 38 or 39, having at least 58 am o acids deleted from said sequence

46 A polypeptide of claim 38 having at least 61 ammo acids deleted from said sequence 47 A polypeptide of claim 38, 39, 40 41 , 42 or 43 wherein the sequence includes no cysteine residues or at least two cysteine residues 48 A polypeptide of claim 29, 30, 31 or 32 wherein the polypeptide has a molecular weight in the range of from about 1000 to 4000

49 A first polypeptide comprising a sequence of am o acids up to 69 ammo acids in length and sufficiently duplicative of a second polypeptide comprising an ammo cid sequence of claim 38, 39, 40, 41 , 42 or 43 such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide

50 A first polypeptide of claim 49, up to 60 ammo acids in length

51 A first polypeptide of claim 49, up to 50 am o acids in length

52 A first polypeptide of claim 49, up to 40 ammo acids in length 53 A first polypeptide of claim 49, up to 30 ammo acids in length

54 A first polypeptide of claim 49, up to 20 ammo acids in length wherein the ammo acid sequence includes no cysteine residues or at least two cysteine residues

55 A first polypeptide of claim 49, up to 14 ammo acids in length

56 A chimeric bone stimulating factor comprising an ammo acid sequence of claim 38, 39, 40, 41 , 42 or 43

57 An agent for use in prevention and treatment of a bone reduction related disease which comprises a polypeptide of claim 38, 39, 40, 41 , 42 or 43

58 A pharmaceutical composition for promoting bone growth, comprising a therapeutically effective amount of a polypeptide of claim 38, 39, 40, 41 , 42 or 43 59 A method of increasing bone growth in a mammal by administering a therapeutically effective amount of a polypeptide having an ammo acid sequence of a polypeptide defined in claim 38, 39, 40, 41 42 or 43

60 The use of a polypeptide of claim 38, 39, 40, 41 , 42 or 43 for the treatment of osteoporosis

61 The use of a polypeptide of claim 38, 39, 40, 41 , 42 or 43 to promote bone growth in a mammal

62 The use of a polypeptide having a sequence according to claim 38, 39, 40, 41 , 42 or 43 in the preparation of a medicament for use in promoting bone growth or the treatment of osteoporosis

63 A diagnostic kit for determining the presence of a polypeptide of claim 38, 39, 40, 41 , 42 or 43 comprising an antibody to a said polypeptide linked to a reporter system wherein the reporter system produces a detectable response when a predetermined amount of the polypeptide and the antibody are bound together

64 An antibody which binds to a polypeptide defined in claim 38, 39 40 41 42 or 43 synthesized using the polypeptide

65 An isolated DNA fragment which encodes the expression of any cf the polypeptides of claim 38, 39, 40, 41 , 42 or 43, and DNA which differs from the fragment due to the degeneracy of the genetic code 66 A vector comprising a DNA sequence which encodes the expression of any of the polypeptides of claim 38, 39, 40 41 42 or 43

67 A process for producing a polypeptide of any of claim 38, 39, 40 41 , 42 or 43, which comprises a) preparing a DNA fragment containing a nucleotide sequence which encodes said polypeptide, b) incorporating said DNA fragment into an expression vector to obtain a recombinant DNA fragment which contains said DNA fragment and is capable of undergoing replication, c) transforming a host cell with said recombinant DNA fragment to isolate a transformant which can express said polypeptide, and d) culturing said transformant to allow the transformant to produce said polypeptide and recovering said polypeptide from resulting cultured mixture

68 A polypeptide exhibiting bone stimulatory activity in mammals, the polypeptide being up to 65 ammo acids in length and having the sequence identified as SEQ ID NO 11 , SEQ ID NO 12, or SEQ ID NO 13, analogues thereof wherein the am o acids in the sequence may be substituted, deleted or added, so long as the bone stimulatory activity in mammals derived the three dimensional structure of the sequence is preserved, and conjugates of each of the polypeptides or analogues thereof, wherein if the polypeptide sequence contains a cysteine residue, there are at least two cysteine residues 69 A polypeptide of any of claim 68 wherein the polypeptide is substantially pure and the ammo acid sequence has a molecular weight in the range of from about 1000 to about 4000, or from about 1500 to about 3000, or from about 1500 to about 1800

70 A first polypeptide comprising a sequence of ammo acids up to 69 ammo acids in length and sufficiently duplicative of a second polypeptide comprising an ammo acid sequence corresponding an ammo acid of claim 68 or 69, or a functionally equivalent homologue thereof, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide

71 A first polypeptide of claim 70, up to 60 ammo acids in length

72 A first polypeptide of claim 70, up to 50 ammo acids in length 73 A first polypeptide of claim 70, up to 40 ammo acids in length

74 A first polypeptide of claim 70, up to 30 ammo acids in length

75 A first polypeptide of claim 70, up to 20 ammo acids in length wherein the ammo acid sequence includes no cysteine residues or at least two cysteine residues

76 A first polypeptide of claim 70 up to 14 ammo acids in length 77 A chimeric bone stimulating factor comprising an ammo acid sequence of claim 68 or 69 78 An agent for use in prevention and treatment of a bone reduction related disease which comprises a polypeptide of claim 68 or 69 as an active ingredient 79 A pharmaceutical composition for promoting bone growth comprising a therapeutically effective amount of a polypeptide of claim 68 or 69

80 A method of increasing bone growth in a mammal by administering a therapeutically effective amount of a polypeptide having an am o acid sequence of a polypeptide defined in claim 68 or 69

81 The use of a polypeptide of claim 68 or 69 for the treatment of osteoporosis

82 The use of a polypeptide of claim 68 or 69 to promote bone growth in a mammal

83 The use of a polypeptide having a sequence according to claim 68 or 69 in the preparation of a medicament for use in promoting bone growth or the treatment of osteoporosis 84 A diagnostic kit for determining the presence of a polypeptide of claim 68 or 69 comprising an antibody to a said polypeptide linked to a reporter system wherein the reporter system produces a detectable response when a predetermined amount of the polypeptide and the antibody are bound together

85 An antibody which binds to a polypeptide defined in claim 68 or 69, synthesized using the polypeptide

86 An isolated DNA fragment which encodes the expression of any of the polypeptides of claim 68 or 69, and DNA which differs from the fragment due to the degeneracy of the genetic code

87 A vector comprising a DNA sequence which encodes the expression of any of the polypeptides of claim 68 or 69 88 A process for producing a polypeptide of claim 68 or 69, which comprises a) preparing a DNA fragment containing a nucleotide sequence which encodes said polypeptide, b) incorporating said DNA fragment into an expression vector to obtain a recombinant DNA fragment which contains said DNA fragment and is capable of undergoing replication c) transforming a host cell with said recombinant DNA fragment to isolate a transformant which can express said polypeptide, and d) culturmg said transformant to allow the transformant to produce said polypeptide and recovering said polypeptide from resulting cultured mixture 89 An isolated DNA sequence encoding the ammo acid sequence set forth in any of claims 1 to 8, 32 to 37 or 56 or 57, or an analogue thereof, wherein the ammo acids in the sequence may be substituted deleted or added so long as bone stimulatory activity in mammals derived from the three dimensional conformation of the sequence is preserved in a polypeptide comprising the ammo acid sequence sequences which hybridize to the DNA and encode an ammo acid sequence of a polypeptide which displays bone stimulatory activity in mammals, and DNA which differs from the sequence due to the degeneracy of the genetic code