BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates generally to the modulation of bone cell growth and resorption, and particularly to a factor derived from pancreatic extracts that inhibits the resorption of bone, lowers serum calcium, stimulates bone cells to proliferate, and increases the formation of bone.

Brief Description of the Background Art Bone is a tissue which must be broken down and rebuilt by bone cells for its optimal function. The cells which have the capacity to degrade (or resorb) bone are unique bone cells called osteoclasts. The osteoclast is a multinucleated cell which has the capacity to form pits (resorption lacunae) in bone tissue. This property is the mechanism by which osteoclasts cause bone resorption. The cells which are responsible for making bone are osteoblasts. These are mononuclear cells which synthesize the structural proteins of bone and then mineralize these structural proteins to form the hard tissue. Both osteoclasts and osteoblasts are controlled by separate factors which can either stimulate or inhibit their activity. Only some of these factors have been identified. Factors which inhibit the activity of osteoclasts decrease the process of bone resorption. Factors which stimulate the activity of osteoblasts may lead to the formation of new bone.

The pancreas produces a myriad of factors which influence body functions and distant tissue. For example, the pancreas is the source of insulin and glucagon, which affect carbohydrate metabolism, and a number of other more recently described peptides derived from the specialized islet cell tissue

of the pancreas. It also produces a number of digestive enzymes which are important in the degradation of protein ingested in the food and present in the gastrointestinal tract. Recently, a newly discovered peptide has been described in the pancreatic tissue of patients with diabetes. This peptide, which is called amylin, is a 37 amino acid single chain polypeptide with an estimated molecular weight of 3.8 kilodaltons and has approximately 50% homology with calcitonin gene-related peptide. Amylin has been shown to have hypocalcemic activity in vivo and inhibit osteoclastic bone resorption in vitro (Westermark et al, (1989) Proc. Natl. Acad. Sci. USA. 84'- 3881-3885; Datta et al, (1989) Biochem. Biophys. Res. Commun.. 162: 876-881. However, amylin clearly has properties which differ from the subject of this application.

The existence of active factors in pancreatic tissue was first described by Takaoka et al. (Takaoka el al. (1969) Acta Med. Nagasaki 13: 28-35). Takaoka described the hypocalcemic action of the pancreatic extract and its effect in alleviating symptoms of myasthenia gravis. He named the composition containing these activities PX, for pancreatic extract. Partially purified pancreatic factor (PX) has been observed to decrease the level of blood- ionized calcium in mice and inhibit osteoclast formation in vitro (Yoneda et al. (1991) FEBS Letters 278(2) 171-174), and prevents the progression of hypocalcemia in tumor-bearing mice (Yoneda et al. (194) Cancer Res. 54:2509-2514). Purified porcine pancreatic factor demonstrated a high sequence homology with human elastase IIIB (Yoneda et al. (1993) J. Bone and Mineral Research 8 (Suppl. 1): 230).

Hypocalcemic properties of PX (Yoneda et al. 1993) were mimicked in all in vitro assays by human recombinant elastase IIIB, a protein first identified by Tani and co-workers by genomic gene cloning (Tani et al. (1988) J. Biol. Chem. 263: 1231-1239). Tani et al., however, did not demonstrate hypocalcemic activity by elastase IIIB, but suggested that in addition to digestive function, elastase IIIB might function in cholesterol metabolism.

Another report of a hypocalcemic factor from porcine pancreas (Tomomura et al. (1992) FEBS LETS 301: 277-281) described a chymotrypsin- like protease, caldecrin, that shared several features with PX (family of serine proteases, similar elution position from RP HPLC, molecular mass). On the other hand, the hypocalcemic action of caldecrin was observed only in PTH-

stimulated ⁴⁵ Ca release from fetal rat long bones and not in 1 ,25-dihydroxy vitamin D3, interleukin-1a, or prostaglandin E1 -stimulated bone resorption. PX and human recombinant elastase IIIB in the same assay inhibit bone resorption induced by either PTH or 1 ,25-dihydroxy vitamin D3. While treatment with PMSF, a potent serine protease inhibitor, apparently increased the activity of caldecrin, it blocked the activity of PX in vitro (Yoneda et al. (1993) J.Bone and Mineral Research 8 (Suppl.1) 230) and in vivo. These features clearly differentiate the biological activities observed in PX from those described for caldecrin.

Newbould et al. recently described osteoid production in an adenocarcinoma of pancreatic acinar cells (Newbould et al. (1992) Pancreas Z: 611 -615).

SUMMARY OF THE INVENTION

The present applicants have shown that PX has osteoinductive properties in vitro and in vivo. One of the aspects of the present invention is the preparation and provision in substantially pure form of a modulator of bone cell function. The present modulator comprises a factor possessing dual activities: osteoblast grovyth stimulation and inhibition of bone resorption . Such a factor would be useful in both diagnostic and therapeutic applications. For example, antagonists of this peptide factor, such as antibodies, fragments thereof, and small molecules exhibiting like function, could be employed in diagnostic assays and related kits, for the detection of the factor itself and thereby the detection of the conditions responsible for the aberrations from normal levels of the factor in question. In this fashion, both the overproduction and the underproduction of the modulator or factor could be monitored. Thus, such assays will be useful in the clinical diagnosis and assessment of those diseases in which excess or decreased production of this factor occurs, and for monitoring treatment efficacy. Further, either the agonists or the antagonists to the factor could be used as part of a drug screen to identify new agents that could themselves, serve as modulators of bone cell presence and growth.

A further aspect of the present invention relates to the therapeutic applications to which the present modulator or factor, its agonists and antagonists may be directed. For example, the present modulator may be used

in the treatment of bone diseases characterized by abnormal osteoblast and osteoclast function, such as osteoporosis. Monoclonal or polyclonal antibodies or antagonists to this polypeptide would be useful to treat diseases where there is either loss or overgrowth of bone. Such antibodies could also be used in assays for detecting this factor.

As stated above, the pancreatic-derived bone-active factor or modulator of the invention is a protein with separate functions, which include the stimulation of bone cell proliferation and the inhibition of bone resorption.

This factor may play a critical role in normal osteoclast formation, in the formation of the normal bone marrow cavity and disease states related to osteoclast function and development. It may also play a critical role in bone formation. Antagonists to the bone resorbing activity may be responsible for stimulating bone resorption in a number of disease states such as osteopetrosis. In one embodiment, this factor may be useful in the treatment of diseases where there is increased bone resorption such as osteoporosis, malignant diseases involving the skeleton or Paget's disease of bone. Antagonists to the factor may be responsible for slowing bone formation in disease states where there is excessive bone formation such as osteopetrosis or osteoblastic metastasis. In one embodiment, this factor may be useful in the treatment of diseases where there is decreased bone formation such as osteoporosis. This factor may be also useful in bone injuries, in repairing bone defects, or hastening fracture repair.

Accordingly, it is a principal object of the present invention to provide one or more modulators or factors, their agonists and antagonists, all hereinafter alternately referred to as modulators, that exhibit the ability to control bone resorption and bone cell growth.

It is a further object of the present invention to provide modulators as aforesaid that are capable of use in the diagnosis of maladies involving aberrations or other variations in bone cell stasis, and bone size, density and strength.

It is a yet further object of the present invention to provide assays and other test kits directed to the diagnosis of maladies and other conditions

involving bone cell growth, and to the discovery of drugs and other agents that may be effective as modulators as defined herein.

It is a still further object of the present invention to provide therapeutic methods and corresponding pharmaceutical compositions capable of treating the maladies and conditions relating to bone cell stasis as aforesaid, such as osteopetrosis and osteoporosis, that utilize as an active ingredient thereof one or more of the modulators as defined herein.

It is a still further object of the present invention to provide the pancreas- derived bone active factor comprising one of the modulators as defined herein, in substantially pure form so that it may be used in treatment of bone diseases and injuries.

Other and further objects, features and advantages will be apparent from the following description of the presently preferred embodiments of the invention, given for the purposes of disclosure when taken into conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

The invention will be more readily understood from a reading of the following specification and by reference to the accompanying drawings forming a part thereof:

FIGURE 1 shows the effects of pancreatic extract on bone resorption in organ cultures. In each case, PX inhibited bone resorption. The following legends should be considered as part of the Figure.

⁴⁵ Ca release was measured after 120h culture. ^* Significantly different from control (p<0.005).

"Significantly different from bones treated with no pancreatic extract (p<0.01).

FIGURE 2 shows the effects of PX on the capacity of mononuclear cells and mouse bone marrow cells to form multinucleated cells with osteoclast characteristics (capacity to multinuclearity, increase in tartrate-resistant acid

phosphatase). The following legends should be considered as part of the Figure.

^* Significantly different from control (p<0.01).

"Significantly different from cultures treated with 1 ,25(OH)2D3 alone (p<0.01).

FIGURE 3 shows the effect of partially-purified pancreatic extract on Ca ²⁺ in mice.

FIGURE 4 shows the effects of pancreatic extracts on blood ionized calcium in nude mice bearing MH-85 human squamous cell tumors. The following legend should be considered as part of the Figure.

'significantly lower than PBS-treated group (p<0.01, Student's t test).

FIGURE 5 shows the effect of partially-purified pancreatic extract on body weight of MH-85-bearing nude mice, compared with control non-tumor- bearing nude mice and MH-85 tumor-bearing mice not injected with pancreatic extract. The following legends should be considered as part of the Figure. 'Significantly different from control animals at day 0 (p<0.05). "Significantly different from control animals at day 10

(p<0.01).

"•"Significantly different from MH-85 tumor-bearing animals untreated with pancreatic extract at day 10 (p<0.05).

FIGURE 6 comprises three graphs shows the effects of PX on food intake. Panel a demonstrates the data from non tumor bearing mice; Panel b presents the data respecting nude mice bearing MH-85 tumors; and Panel c. demonstrates the data respecting mice with MH-85 tumors receiving PX . The following legends should be considered as part of the Figure. 'Significantly different from control (p<0.01). "Significantly different from MH-85-bearing animals untreated with pancreatic extract (p<0.01).

FIGURE 7 shows the effects of PX on formation of osteoclasts from murine marrow cultures stimulated by MH-85 conditioned media. PX inhibited the formation of these osteoclasts.

FIGURE 8 shows the effects of PX on bone resorption in organ cultures of fetal rat long bones which have been stimulated with the conditioned media harvested from MH-85 squamous cell tumors. Bone resorption was inhibited by PX.

FIGURE 9 shows the effects of PX on the survival of nude mice bearing MH-85 tumors. PX prolongs survival of tumor-bearing mice.

FIGURE 10 presents the results of chromatographic separation with a

DEAE-cellulose column where biological activity stimulating 3H thymidine incorporation in MG-63 cells was eluted with 0.2 M NaCI.

FIGURE 11 represents the results of the repetition of the procedure described with respect to FIGURE 10 with the active fractions recovered from the FIGURE 10 procedure.

FIGURE 12 shows the results of measurements of proliferative activity that were made with the fractions obtained from the procedure of FIGURE 11 that were subjected to isoelectrofocusing.

FIGURE 13 shows the effect of PX on D3-stimulated ⁴⁵ Ca release in fetal long bone. The particular fractions tested were derived from prepared by HPLC from fractions subjected to isoelectrofocusing.

FIGURE 14 presents a comparison of the amino acid sequences of PX (SEQ ID No: 1 ) and human elastase IIIB (SEQ ID No.: 2), and reveals the homology between them.

FIGURE 15 shows the effects of anion exchange chromatography of crude pancreatic extracts on DE-52 anion exchange columns. Activity was monitored as a percent increase in DNA synthesis in MG-63 human bone cells, or inhibition of bone resorption in organ cultures of fetal rat long bones.

FIGURE 16 shows size exclusion chromatography of PX obtained from

DE-52 anion exchange columns. Activity was measured as proliferation of MG- 63 cells and percent inhibition of osteoclastic bone resorption. These data

show the separation of MG-63 cell proliferation and bone resorbing activity from the capacity to stimulate alkaline phosphatase activity in MG-63 cells.

FIGURE 17 shows the effect of human elastase IIIB on ⁴⁵ Ca release in a fetal long bone assay. The effect was neutralized with rabbit polyclonal antibodies against hEIIIB.

FIGURE 18 shows the effect of PX on ⁴⁵ Ca release in a fetal long bone assay. The effect was similar to that of hEIIIB even to the extent that it was neutralized with rabbit polyclonal antibodies against hEIIIB.

FIGURE 19 shows the effect of PMSF on human recombinant elastase III B activity. Treatment with PMSF followed by dialysis abolished hrEIIIB activity.

FIGURE 20 shows the effect of 1 ,25 D3 and human recombinant elastase III B on mouse calvarial assay. In the absence of 1,25 D3 hrEIIIB had no effect.

FIGURE 21 shows the effect of human recombinant elastase III B on rat osteoclast pit formation.

FIGURE 22 shows the effect of PX and human recombinant elastase III B on serum ionized calcium levels in swiss mice. Both elastase III B and PX reduced hypercalcemia.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

It will be readily apparent to one skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

In its broadest aspect, the present invention is directed to the discovery of certain factors, defined herein as modulators, that are capable of functioning to control the growth and resorption of bone. A particular factor, isolated from pancreatic extracts (and alternately referred to herein as PX) demonstrates the stated abilities. The present invention is also predicated on the discovery that this factor (PX) demonstrates a significant homology with a protein known as

human elastase III B, and that neither material has previously demonstrated the activities of a modulator as disclosed and defined herein.

Accordingly, the invention extends to the discovery of the specific materials named herein and to their agonists, antagonists, cognate molecules and other materials that may either mimic or inhibit the particular activities of interest herein, all of these materials considered herein as modulators, and to the diagnostic and therapeutic utilities to which these materials may be directed.

Several biological activities for osteoblasts and osteoclasts have been isolated and substantially purified from porcine pancreatic extracts. Pancreatic tissue is the source of many hormones and factors which influence distant tissues, and we have found that it also contains endogenous bone cell stimulatory activities. Among these activities is a novel factor which stimulates the proliferation of human MG-63 osteosarcoma cells which have the characteristics of osteoblasts (bone forming cells). This factor is not any of the known bone growth factors which have been shown to promote proliferation of human bone cells. In one embodiment, this factor has a molecular weight of about 28 kilodaltons. The same factor also exhibits another activity - an inhibition of osteoclastic bone resorption.

The present invention arose out of the discovery of a biochemical purification scheme which allows, for the first time, the preparation of these bone cell stimulatory factors in substantially homogeneous form. The invention encompasses this pancreas-derived bone activator factor per se, as well as synthetically produced pancreas-derived bone activator factor, naturally and pharmaceutically acceptable salts and pharmaceutically acceptable derivatives. The term "PX", which as used hereinafter, also refers to the for pancreas-derived bone activator factor, also encompasses biologically active fragments thereof.

The invention also concerns compositions, such as diagnostic compositions, containing modulators such as the particular pancreatic-derived bone activator factor, and methods of using these in treatment and diagnosis. As stated earlier, the modulators of the present invention may be used as part of diagnostic methods and corresponding kits, for the detection and monitoring

of conditions involving aberrant or unwanted bone depletion or accretion, such as the conditions of osteoporosis and osteopetrosis. Similarly, a drug screen may be devised involving one or more of the modulators, for the express purpose of identifying yet further such modulators for either diagnostic or therapeutic use.

In other aspects, the invention concerns monoclonal and polyclonal antibodies to these factors as well as uses of these antibodies therapeutically and diagnostically. The invention also contains an assay for this peptide. The invention also concerns synthetic antagonists which neutralize the biological activity of these factors and can be used therapeutically. These antagonists were synthesized based on our knowledge of amino acid sequence of these factors.

A polypeptide of approximately 28,000 daltons has been isolated in an electrophoretically homogeneous form. This polypeptide stimulates the activity of osteoblasts and inhibits the activity of osteoclasts.

Briefly, purification of PX was achieved by the sequential use of extraction in acetone, ammonium sulphate precipitation, lyophilization, DE52 anion exchange chromatography, isoelectric focussing and C18 reverse phase high performance liquid chromatography.

As used herein, the term "PX" refers to naturally occurring as well as synthetically produced pancreatic-derived bone activator factor having a molecular weight of approximately 28,000 daltons, as well as biologically active fragments thereof.

As used herein the term "salts" refers to both salts of carboxy groups of the polypeptide or protein chain and to acid addition salts of amino groups of the polypeptide chain. Salts of the carboxy group may be formed with either in¬ organic or organic bases by means known in the art per se. Inorganic salts include, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like. Salts with organic bases include those formed, for example, with amines such as triethanolamine, arginine, lysine, piperidine, caffeine, procaine and the like. Acid addition salts include, for example, salts with mineral acids such as,

for example, hydrochloric acid or sulfuric acid, and salts with organic acids such as, for example, acetic acid or oxalic acid.

Derivatives may also be prepared from the functional groups which occur at side chains on the residues of the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain diagnostically or therapeutically acceptable. These derivatives may, for example, include: any fragments formed from proteolytic cleavage; synthetic or natural proteins with 75% or greater homology to the molecule, either to the whole molecule or to a portion or portions thereof.

Both the salts and the derivatives encompassed by the invention are those which are therapeutically or diagnostically acceptable, i.e., those which do not destroy the biologic or immunogenic activity of PX.

The term "PAGE" is electrophoresis as performed on a polyacrylamide gel and separates proteins or peptides on the basis of charge. If sodium dodecylsulfate (SDS) is incorporated into the gel (SDS-PAGE), the surface active nature of the SDS results in a uniform negative charge on the peptide or protein which is a function of size. The result is that separation is based on molecular size. Native PAGE denotes the employment of this technique without the presence of SDS, and thus proteins are separated on the basis of charge.

Under suitable circumstances, chromatographic procedures may be carried out, preferably in a narrow bore column containing a fine particle resin under increased pressure to enhance the effectiveness of separation, i.e., by high pressure liquid chromatography. Concentration and salt removal are commonly used precursors to certain chromatographic or separation techni- ques employed in the invention. Salt removal may be performed by, for example, dialysis or gel filtration or by a relatively recently developed technique called control pore glass (CPG) chromatography. A number of gel filtration and concentration techniques are also used. Certain commercial available materials are especially useful. Salt removal is generally necessary if ion exchange or other techniques which depend on total ionic strength are employed. These preparation methods and the extent to which they are required for particular separation procedures are well known in the art.

The term "specific activity" refers to the activity of the factor in assays described in this application and known in the art related to the amount of protein by weight in the sample. As specified in the current disclosure, the activity of the factor is measured according to the assay procedure set forth hereinbelow in Example 10.

The techniques for detectably labelling the homogeneous factors and the monoclonal antibodies thereto of the present invention with a radiolabel, an enzyme label, or a fluorescent label are well known to those of skill in the art. Reference can be made to Chard, An Introduction To Radioimmunoassay And Related Techniques. North-Holland Publishing Co., Amsterdam-NY-Oxford (1978), The Enzyme-Linked Immunoadsorbent Assay (ELISA) by Voller, A., et aL, Dynatech Europe Borough House, Rue du Pre, Guernsey, Great Britain, and Radioiodination Techniques. Review 18. Amersham Corporation, by A. E. Bolton, all herein incorporated by reference. Preferably, the purified PX is labelled with ¹²⁵ l using the Bolton/Hunter reagent which involves succinylation of the free N-terminals and lysine.

"Homogeneity" is defined as the substantial absence of other proteins or peptides normally associated with PX in nature.

Monoclonal and polyclonal antibodies may be prepared using the method of Mishell, B. B., et §1, Selected Methods In Cellular Immunology. (W.H. Freeman, ed.) San Francisco (1980). The anti-PX antibodies will be useful in the diagnosis and monitoring of diseases where osteoblast function is altered, such as osteoporosis. Likewise, therapeutic protocols may be devised that utilize the modulators of the invention, either individually, or in combinations with each other where effective, and in pharmaceutical compositions suitable for individual therapeutic circumstances and corresponding strategies. Administration of the modulators or compounds useful in the method of present invention may be by parenteral, intravenous, intramuscular, subcutaneous, rectal or any other suitable means. The dosage administered may be dependent upon the age, weight, kind of concurrent treatment, if any, and nature of the immune dysfunction being treated. The effective compound useful in the method of the present invention may be employed in such forms as capsules, liquid solutions, suspensions or elixirs for

oral administration, or sterile liquid forms such as solutions or suspensions. Any inert carrier is preferably used, such as saline, or phosphate-buffered saline, or any such carrier in which the compounds used in the method of the present invention have suitable solubility properties for use in the method of the present invention.

The term "animal" is meant to include mammals such as humans, dogs, cats, horses, cows, mice etc.

Having now generally described the invention, a more complete understanding can be obtained by reference to the following specific examples. These examples are provided for the purpose of illustration only and are not intended to be limiting unless otherwise specified.

EXAMPLE 1: ASSAYS FOR BONE RESORPTION. OSTEOCLAST FORMATION AND OSTEOBLAST PROLIFERATION

Bone resorption was assessed by determining ⁴⁵ Ca release from fetal rat long bones in organ culture as described (Raisz (1965) J. Clin. Invest. 44: 103-116). Bones were labeled with 50 μCi ⁵ CaCl2 (ICN Radiochemicals, Costa Mesa, CA) by injecting the mothers 1 day before removal of the fetuses. Long bones (radius and ulna) were dissected from 19-day-old fetal rats with removal of cartilage and connective tissue. Bones were cultured in 0.5 ml serum-free BGJ medium (Sigma Chemical Co., St. Louis, MO) supplemented with 1 mg/ml bovine serum albumin. Bones were incubated on steel grids at the interphase between medium and a 95% air, 5% CO2 atmosphere at 37°C.

All bones were precultured in medium for 24 h to remove exchangeable ⁴⁵ Ca. Bones were then cultured for 120 h in BGJ medium supplemented with 1 mg/ml bovine serum albumin and factors to be tested. Medium was collected at days 2 and 5. At the end of culture, bones were placed in 50% trichloroacetic acid for 1 h and ⁴⁵ Ca radioactivity in medium and trichloroacetic acid extract of bones was measured in a liquid scintillation counter. Bone resorption is stimulated by a variety of known bone resorption stimulants. ⁴⁵ Ca release, measured after 120 h culture, demonstrated that bone resorption stimulated by different factors was inhibited or reduced by administration of PX. Bone resorption was assessed as the percent of total ⁴⁵ Ca that was released into the medium. Results from the performance of this assay are presented in Figure 1 , where fetal rat long bones were stimulated by PTH, 11-1 a, PGE2, TNF,

TGF-a, and 1,25 (OH)2D3 as well as control. In all instances but the control, PX significantly reduced bone resorption.

Osteoclast formation was assessed by counting multinucleated cells which stained for the characteristic enzyme tartrate-resistant acid phosphatase (TRAP(+)MNC) number in mouse bone marrow cultures according to the method previously described (Takahashi et al, (1988) Endocrinology 122: 1373-1382). Bone marrow cells harvested from 4 to 6 week old male C57BL mice (Harlan Industries, Houston, TX) were inoculated onto 24-well plates (2 x 10 ⁶ cells/0.5 ml/well) and cultured in alpha minimal essential medium (a-MEM) supplemented with 10% fetal calf serum (FCS) (Hyclone) in the presence of 10" ⁸ M 1.25D3 with or without partially-purified PX for 6 days. The cultures were washed in PBS, fixed in 60% acetone in citrate buffer pH 5.4, air dried and stained for TRAP using a commercial kit (Sigma). All TRAP(+)MNC (cells stained in red with more than 3 nuclei) were counted manually under light microscopy. The results from this assay, presented in Figure 2, demonstrate that the addition of PX causes a significant reduction in osteoclast formation when compared to cultures treated with 1 ,25 (OH)2U3 alone.

Osteoblast proliferation was assessed using human osteoblastic osteosarcoma MG-63 cells. These cells (5 x 1θ3/well) were cultured in 96-well plates for 44 h in a-MEM supplemented with 0.2% bovine serum albumin in the presence of serially-diluted (1:2) PX which was fractionated on anion exchange or gel filtration column chromatography. The cells were then incubated with 0.2 μCi/well ^H-thymidine (ICN Radiochemicals) for a further 4 h. At the end of incubation, the cells were detached by treatment with trypsin- EDTA solution (GIBCO) and harvested onto glass fiber strips using a multiple automated cell harvester (Cambridge Technology Inc., Watertown, MA). The radioactivity was measured by liquid scintillation counting.

EXAMPLE 2: ASSAY FOR HYPOCALCEMIC ACTIVITY

Eight to 10 week-old male Balb/c mice (Harlan Sprague-Dawley) were given crude or partially-purified PX intraperitoneally every day for 5 days. Thirty μl of blood was drawn by puncturing the orbital plexus with calcium- titrated sodium heparinate-coated tubes (Corning) under ether anesthesia. Blood ionized calcium (Ca ²⁺ ) was determined using a Ciba-Coming calcium- pH analyzer (Model 634). Twelve mice were divided into 2 groups (6

animals/group) after their Ca ⁺ was measured. Pancreatic extract was administered intraperitoneally (1 mg) in 0.1 ml phosphate-buffered saline (PBS) once a day for 5 days. As shown in Figure 3 and Table 1 , the Ca ²⁺ of the PX-treated group was significantly lower than PBS-treated group (p<0.01 ).

TABLE 1

EXAMPLE 3: EFFECTS OF PX ON CAPACITY OF HUMAN TUMORS TO CAUSE INCREASES IN BLOOD CALCIUM AND INCREASE BONE RESORPTION For these studies, a well characterized human tumor called MH-85 was used. The MH-85 tumor was isolated from a human squamous cell carcinoma of the maxilla (Yoneda et al (1989. Oral Surg. 68: 604-611 ) and has been serially transplanted in nude mice (Balb/c/nu/nu, male, 4 to 6 week-old, Harlan Sprague-Dawley) for more than 5 years in our laboratory. Similar to the observation in the human patient with this tumor, marked hypercalcemia with increased osteoclastic bone resorption and cachexia with anorexia and decrease in body weight was induced in nude mice following subcutaneous inoculation of MH-85 under the skin of the right dorsal portion and subsequent tumor growth (Yoneda et al, .1991 . J. Clin. Oncol. 9: 468-477). Determination of Ca ²⁺ , body weight, tumor size and food consumption was performed as described.

MH-85 cells were established in culture from the tumor formed in nude mice as described previously (Yoneda et al, ⁽ 1991 . J. Clin. Oncol. 9: 468-477) and grown in a-MEM (Hazleton Biologies Inc., Lenexa, KA) supplemented with

10% FCS (Hyclone Laboratories Inc., Logan, UT) and 1% penicillin- streptomycin solution (GIBCO Laboratories, Grand Island, NY). Serum-free culture supematants of MH-85 (MH-85 CM) were harvested from 48h-cultures of confluent MH-85 cells.

Eighteen MH-85-bearing nude mice at 6 weeks after tumor implantation were divided into 2 groups (9 animals/group) and their Ca ²⁺ was determined using a Ciba-Coming calcium pH analyzer (Model 634). The pancreatic extract (1 mg) in 0.1 ml phosphate-buffered saline was administered intraperitoneally to the first group once a day for 9 days. The second group was treated with vehicle alone. Blood (30 μl) was drawn from the orbital plexus. Normal values for Ca ²⁺ in nude mice are between 0.95 and 1.25 mmol/L. The data demonstrate that the calcium values for the PX-treated group after 2 weeks following the commencement of PX treatment, were significantly lower than those of the PBS-treated group (p<0.01, Student's t test). The pancreatic extracts inhibited the increase in blood ionized calcium seen in the tumor- bearing nude mice. Figure 4 shows that crude pancreatic extracts cause a decrease in blood ionized calcium in MH-85 tumor-bearing mice. Similar data is shown in Table 2, below. In animals to which pancreatic extract was administered 6 weeks after tumor implantation (day 0) for 9 days, PX increased the body weight of these cachectic animals.

TABLE 2

Effects of partially-purified PX on Ca ²⁺ in MH-85 tumor-bearing nude mice

at 4 weeks. + Three animals died 13, 17 and 23 days after initiation of PX injection.

Figure 5 demonstrates that pancreatic extracts increase the body weight of nude mice carrying MH-85 tumors, while Figure 6 demonstrates that PX exhibits a positive effect on food intake as it increases the body weight in nude mice bearing MH-85 tumors. PX effected an increase in the body weight compared with untreated nude mice bearing MH-85 tumors where body weight progressively decreased. Food intake was measured every day and calculated as: amount given amount left/animal number. Animals were given pancreatic extract at 6 weeks after tumor implantation (day 0) for 9 days. Non tumor bearing mice are shown in Panel a for comparison.

Moreover, MH-85 conditioned media increased the formation of osteoclasts in murine marrow cultures (Figure 7), and increased bone resorption in organ cultures of fetal rat long bones (Figure 8). In each case, these effects of MH-85 conditioned media were inhibited by pancreatic extracts. In addition, the necrotic effect of MH-85 tumors was delayed by the administration of PX (Figure 9).

EXAMPLE 4: PURIFICATION OF BIOLOGICAL ACTIVITY Methods of Extraction of Pancreatic Tissue

Acetone powder of porcine pancreas (Sigma) (600 g) was suspended in 0.1M Tris-HCI (pH 8.0) containing 2% NaCI for 1h with gentle shaking and centrifuged at 10,000 g for 45 min. The supernatants were fractionated with 30 to 60% acetone, and centrifuged at 5,000 g for 30 min. The pellets were then dissolved in distilled water, dialyzed against 500-fold distilled water and precipitated with 80% ammonium sulfate at pH 7.0. The precipitates were dissolved in distilled water, dialyzed against 500-fold distilled water and lyophilized (crude PX).

Supernatants of porcine pancreas homogenates in 0.1 M Tris-HCI (pH

8.0) containing 2% NaCI were fractionated with 30 to 60% acetone. The resultant pellets were dissolved in water, dialysed and precipitated with 80% ammonium sulfate. The precipitates were dissolved in distilled water, dialysed and lyophilized (crude PX). Crude PX was run through DE-52 (Whatman Labsales, Hillsboro, OR) anion exchange column (4.8 x 60 cm, Kontes, Vineland, NJ) which was equilibrated with 0.1M Tris-HCI buffer (pH 7.5). Protein was eluted by step-wise washes with 0.1, 0.2, and 1 M NaCI in 0.1 M

Tris-HCI buffer (pH 7.5) at a flow rate of 120 ml/h. Fractions of 20 ml were collected. Biological activity stimulating ³ H thymidine incorporation in human osteosarcoma cells MG-63 was eluted with 0.2 M NaCI (Figure 10). Active fractions were pooled, dialyzed against 0.1 M Tris-HCI pH 7.5, and rechromatographed in DEAE-Sephacel column (4.8 x 10 cm, Kontes) equilibrated in the same buffer. The column was eluted with stepwise NaCI washes as above. Active material stimulating proliferation in MG-63 cells was eluted in the early region (fr. 61-80) of 0.2 M NaCI wash (Figure 11).

These fractions were pooled, acidified to pH 5, dialyzed against 20 mM ammonium acetate, made 10% in glycerol and subjected in five separate batches to isoelectrofocusing in ampholytes of pH range 3-10 (Miniphor, Rainin). Protein with an isoelectric point of 4.3-4.5 (Miniphor fr. 12-15) showed an inhibition of ⁴⁵ Ca release in a fetal rat long bone assay. This material also stimulated proliferation in MG-63 and C433 cell lines and was inhibitory in MC- 3T3 cells (Figure 12).

Miniphor fractions 12-15 were pooled and chromatographed in C18 RP HPLC (2.1 mm x 15 cm, Vydac) in a gradient of 0-70% acetonitrile in 0.1% TFA at a flow rate of 0.15 ml/min. Biologically active material was eluted with 48% acetonitrile (Figure 13). Protein purified by C18 chromatography was over 95% homogeneous in SDS-PAGE in 10-15% acrylamide (PHAST System, Pharmacia LKB) and migrated as a single band of 28 kD under reducing and non-reducing conditions. The amino acid sequence of the first 32 N-terminal residues showed 88% homology with human elastase IIIB (Figure 14). A summary of the protein purification scheme is given in Table 3.

TABLE 3 PX PURIFICATION

Step Protein Activity Specific Activity

Porcine pancreas 500 g N/D N/D acetone powder

Ammonium sulfate 10 g N/D N/D

DEAE 1 9 9 500 μg/ml 0.0002

DEAE 2 400 mg 120 μg/ml 0.0008

Miniphor 30 mg 40 μg/ml 0.025

stimulated ⁴⁵ Ca release.

The results of further examples of purifications conducted in accordance with the invention are presented in Figures 15 and 16. Figure 15 presents the results of anion exchange chromatography performed on a DE-52 column. The resulting fractions were then subjected to testing for Absorbance (A2βθ)-

MG-63 proliferation-stimulating activity, and bone resorption-inhibiting activity. Figure 16 contains the results of size exclusion chromatography of DE-52- purified material such as that prepared and tested in Figure 15. A Sephacryl S-200 column was employed and the resulting fractions were tested for the same properties as those of Figure 15.

EXAMPLE 5: COMPARISON OF BIOLOGICAL EFFECTS OF PX AND HUMAN ELASTASE IIIB

Human recombinant elastase IIIB (750 ng/ml) fully inhibited 1.25D3- stimulated ⁴⁵ Ca release in a fetal rat long bone assay. The inhibition was seen at elastase IIIB concentrations as low as 75 ng/ml. Neutralizing rabbit polyclonal antibodies against human recombinant elastase IIIB reversed the inhibition seen with elastase. When ⁴⁵ Ca release was stimulated with calcitonin (100 mU/ml), the antibody did not have effect on bone resorption (Figure 17). Similarly to elastase, homogeneous PX (120 ng/ml) inhibited ⁴⁵ Ca release in a fetal rat long bone assay. The effect of PX was neutralized with anti-elastase IIIB rabbit antibody (Figure 18).

Elastase IIIB inhibition of ⁴⁵ Ca release was abolished by a treatment of human recombinant elastase IIIB (75 ng/ml) with 1 mM PMSF for 1 h at 4 C, followed by an extensive dialysis (Figure 19). Thus, biological effect of PX is due to proteolytic activity and the serine residue at the active site is necessary for inhibition of bone resorption in rat long bone assay.

Human recombinant elastase IIIB also inhibited in a dose-dependent manner l ,25D3-stimulated ⁴⁵ Ca release in fetal rat calvarial assay. In the absence of L25D3, elastase IIIB had no effect (Figure 20).

In an isolated rat osteoclast assay, human recombinant elastase IIIB (75 750 ng/ml) reduced 1 ,25D3-stimulated bone resorption and decreased numbe of pits per slice as well as overall pit area (Figure 21).

Effects of human recombinant elastase IIIB and PX on serum ionized calcium levels were determined in ICR Swiss mice. Hypercalcemia was induced by injections of IL-1 (0.1 μg per injection, 4 times a day). The control group received IL-1 alone; elastase IIIB (0.5 μg per injection, twice daily) and PX (5 μg per injection, twice daily) were injected over calvariae of IL-1 -treated mice. Both elastase IIIB and PX reduced hypercalcemia (Figure 22).

EXAMPLE 6: PREPARATION OF MONOCLONAL ANTIBODIES TO PX Monoclonal antibodies (MAb) to PX were made using an in vitro immunization technique (Van Ness et al. (1983) Nature 301: 425-427). Spleen cells of 8 to 12 week-old female Balb/C mice were immunized with purified PX in the presence of 100 μg N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP, Sigma), 125 μg lipopolysaccharide (LPS, Difco) and 500 ml culture supernatants of concanavalin A (50 μg/ml)-stimulated spleen cells (5 x 10 ⁶ /ml) in 5 ml IMDM supplemented with 20% FBS in 6-well plates for 4 days. The immunized spleen cells were then hybridized with mouse myeloma FO cells (kindly provided by Dr. Eguchi, Kaneka, Japan) at a ratio of 2:1 in the presence of 50% polyethylene glycol (1500, Boehringer-Manheim). After the hybridization, the cells were suspended in 120 ml IMDM supplemented with 10% FBS and 0.5 ml cell suspension were inoculated onto each well in 48-well plates. After 24 hours, 5 x 10 ⁵ /well thymocytes in 0.5 ml HAT medium were plated onto each well as a feeder layer. The plates were cultured for 14 days in HAT medium (Flow) and then for 7 to 10 days in HT medium (Flow). The cells were fed fresh medium every 2 days. The culture supernatants harvested from the wells in which growing hybridoma cells cover 50% of the surface area were screened for their crossreactivity with purified PX by enzyme-linked immunosorbent assay (ELISA).

EXAMPLE 7: POLYCLONAL ANTIBODIES TO PX

Rabbits (male, 6 to 8 week-old) were subcutaneously injected (10 sites/animal, 100 μl/site) with 1.3 mg keyhole limpet hemocyanin (KLH)- conjugated purified PX and 2 ml complete Freund's adjuvant. Four weeks after the first immunization, animals were boosted intramuscularly with 1.3 mg KLH-

conjugated purified PX and 2 ml incomplete Freund's adjuvant 3 times. At each booster, blood was drawn from an ear vein and tested for its titer against purified PX by ELISA.

EXAMPLE 8: PREPARATION OF ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR PX

The ELISA plates (Nunc) were coated with 100 ng/50 μl/well purified PX overnight at 4°C, washed with 200 μl/well phosphate-buffered saline (PBS) twice, incubated with 200 μl/well blocking buffer (1% bovine serum albumin in PBS) for 1 hour at 37°C and rinsed with 250 μl washing buffer (0.05% Tween 20 in PBS). The plates were then incubated with 200 μl/well hybridoma culture supernatants to be tested for overnight at 4°C and rinsed with 250 μl/well washing buffer 3 times. After rinsing, the plates were incubated with 200 μl/well peroxidase-conjugated goat anti-mouse immunoglobulin G antibodies for 1 hour at 37°C, rinsed with 250 μl/well washing buffer 5 times and incubated with 200 μl/well O-phenylenediamine dihydrochloride (OPD) solution (4 mg OPD and 10 μl 30% hydrogen peroxide in 10 ml water) for 10 to 20 minutes. The reaction was stopped by adding 80 μl/well 2.5M sulfuric acid. The absorbance was read at 492 nm.

EXAMPLE 9: IDENTIFICATION OF RECEPTORS FOR PX

To understand the mechanism by which PX affects bone cell function, it will be necessary to characterize the receptor-mediated signal transduction mechanism responsible for cell activation. These mechanisms may be identified by any one or a combination of the studies such as those described below.

For these studies, one may use PX labeled with iodine at tyrosine residues using the Bolton-Hunter reaction. A suitable target cell is then selected. To date, the most suitable of the target cells appears to be MG-63 cells. The cells are then characterized by Scatchard analysis to determine the receptor number, receptor binding affinity and number of receptor sites, and to determine saturability and specificity of the ligand-receptor interaction.

Thereafter, the receptor is isolated and purified, and traditional techniques for isolation and purification may be employed. Two approaches to receptor identification may be employed. Accordingly, the receptor may be

identified by affinity labeling. A chemical cross-linking agent which covalently crosslinks labeled factor to the receptor will be used. The factor covalently bound to an inert matrix will be utilized to construct an affinity column. The receptor will be solubilized by non-ionic detergents. The receptor will then be eluted from the affinity column in an acidic urea solution, dialyzed and then pH increased to lead to renaturation of the receptor.

The alternative approach to the identification of the receptor is to use the technique of expression cloning. Advances in expression cloning of receptors have been made recently with the use of the slide autoradiography approach which is used to detect the cDNA. This approach is an order of magnitude more sensitive than previous detection methods and has greatly eased the screening of plasmid-based cDNA expression libraries for receptors with low abundance mRNAs. Receptors identified in this manner include the receptor for GM-CSF, PTH and calcitonin. The technique for the cloning of the low affinity receptor for human GM-CSF was described by Gearing et al (1989) Embo J. 8: 3667-3676.

The cDNA library from MG-63 cells may be used, which cells express the receptor, and for which the receptor has been characterized. The cDNA library is subfractionated into 500 pools of 2x10 ⁴ clones each and then DNA from each pool is transfected into 1.5 x 10 ⁶ cells by electroporation. This technique detects single clones from pools of 2x10 ⁴ clones (compared with 1x10 ³ clones using previous methods). Following transfection, the cells are cultured for 48 hours on glass slides and incubated with a labeled peptide. Following the 48 hour incubation period, the slides are fixed and dipped in photographic emulsion and the positive pools identified. Individual clones expressing the PX receptor are identified. The recombinant receptor and ligand are cross-linked and this complex is compared with the cross-linked natural receptor from MH-85 cells covalently linked to the ligand. Provided the characteristics of the cross-linked receptors from COS cells and MH-85 cells are similar, we will then proceed to sequence the cloned cDNA for the receptor.

The cloning of the PX receptor should open up a number of different avenues for further research. It should help elucidate the signal transduction mechanisms for PX on cells in the osteoclast and osteoblast lineages. It should determine which cell types in the osteoclast and osteoblast lineages

acquire PX responsiveness during differentiation. In addition, it is possible that soluble forms of the receptor may be useful as agonists or antagonists to PX and may be useful in blocking bone resorption in those circumstances where PX is overexpressed, or stimulating bone formation. Similar findings have been found previously with cytokine receptors.

EXAMPLE 10: CLONING AND EXPRESSION OF RECOMBINANT HUMAN ELASTASE IIIB.

The pMAL-2 vector (New England Biolabs, Beverly, Mass.) was used for expression of human elastase IIIB (EIIIB) gene. The gene fragment coding for proelastase IIIB was inserted downstream from the malE gene of E. coli which encodes maltose binding protein (MBP) (Guan et al. (1987) Gene 67: 21-23; Maina et al. (1988) Gene 74: 365-373), resulting in the expression of an MBP fusion protein. The proteolytic cleavage site for the factor Xa was modified by oligonucleotide-directed mutagenesis to a TEV protease site, and this construct was subsequently used to obtain the recombinant product. However, the original construct can also be used as a more costly alternative for generating recombinant EIIIB. Recombinant fusion protein was expressed as a soluble protein in E. coli upon induction with IPTG. The fusion protein was partially purified from E. coli lysate by affinity purification in amylose resin (Nagai et al. (1984) Nature 309:810-812: Nagai et al. (1987) Methods in Enzymol. 153: 461- 481). The fusion protein was activated either by trypsin alone or by a sequential digestion with TEV protease and trypsin to an active form of elastase IIIB. The final product showed a 28kD band in reducing SDS-PAGE, cross-reacted with rabbit polyclonal antibodies against native elastase IIIB, and exhibited elastase enzymatic activity using a chromogenic elastase substrate, N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide (SAPL).

This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all respects illustrative and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

NAME: OSTEOSA, INC.

STREET: 2040 Babcock Road, Suite 201 CITY: San Antonio STATE: Texas

COUNTRY: USA POSTAL CODE: 78229

(i) APPLICANT: NAME: YONEDA, Toshiyuki

STREET: 3530 Hunter's Sound

CITY: San Antonio

STATE: Texas

COUNTRY: USA POSTAL CODE: 78230

(i) APPLICANT:

NAME: IZBICKA, Elzbieta STREET: 7738 Apple Green CITY: San Antonio

STATE: Texas COUNTRY: USA POSTAL CODE: 78240 (i) APPLICANT:

NAME: MUNDY, Gregory R.

STREET: 3719 Morgan's Creek

CITY: San Antonio

STATE: Texas COUNTRY: USA

POSTAL CODE: 78230

(ii) TITLE OF INVENTION: Modulators of Bone Cell Function and Uses Thereof

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Rhone-Poulenc Rorer Inc. (B) STREET: 500 Arcola Rd. 3C43

(C) CITY: Collegeville

(D) STATE: PA

(E) COUNTRY: USA

(F) ZIP: 19002

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(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: PCT

(B) FILING DATE: (C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/363,092

(B) FILING DATE: 20-DEC-1994 (viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Savitzky, Martin

(B) REGISTRATION NUMBER: 29,699

(C) REFERENCE/DOCKET NUMBER: A1172-WO <ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (610)454-3816

(B) TELEFAX: (610)454-3808

(2) INFORMATION FOR SEQ ID NO: 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 amino acids

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

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: N-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Sus scrofa

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

Val Val Asn Gly Glu Asp Ala Val Pro Tyr Ser Trp Pro Trp Gin Val 1 5 10 15

Ser Leu Gin Tyr Glu Lys Asn Gly Val Phe Gin His Thr Gly Gly 20 25 30

(2) INFORMATION FOR SEQ ID NO:2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 amino acids

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

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 :

Val Val Asn Gly Glu Asp Ala Val Pro Tyr Ser Trp Pro Trp Gin Val

1 5 10 15

Ser Leu Gin Tyr Glu Lys Ser Ser Ser Phe Tyr His Thr Cys Gly Gly 20 25 30