WO2001032197A2 - Methods of using lp8, a pdgf-related protein, to treat musculoskeletal disorders - Google Patents

Abstract

The invention provides compositions and methods for treating osteopenia, osteoporosis, arthritis, sarcopenia, peridontal disease or promoting bone growth comprising the administration of a therapeutically effective amount of LP8, a fragment thereof, an analog thereof, and/or an antagonist or agonist thereof.

Description

METHODS OF USING P8, A PDGF-RELATED PROTEIN, TO TREAT MUSCULOSKELETAL DISORDERS

BACKGROUND OF THE INVENTION This invention relates to recombinant DNA technology. In particular the invention relates to therapeutic uses of LP8, a platelet-derived growth factor (PDGF) homolog.

Recently, much attention has been paid to the use of growth factors to accelerate wound healing, particularly of skin. Growth factors are agents that cause cells to migrate, differentiate, transform, or mature and divide. These factors are polypeptides that can be isolated from a variety of normal and malignant mammalian cell types. Some growth factors can be produced by genetically- engineered microorganisms such as bacteria (e.g. E. coli ) and yeasts (See, e.g., Chapters 10 and 11 of Molecular and Cellular Biology of Wound Repair (1986) , incorporated herein by reference) . Included among the growth factors are epidermal growth factor (EGF) , transforming growth factors alpha and beta, fibroblast growth factor (FGF) , insulin-like growth factor (IGF), nerve growth factor (NGF) , and PDGF. These molecules are described in United States Patent No. 4,939,135, incorporated herein by reference. The use of PDGF to accelerate wound healing in skin and connective tissue has been investigated (Antoniades et al., Proc. Natl. Acad. Sci. USA 88:565-569 (1991); Cromack et al., J. Trauma 30:S129-133 (1990); Ross et al . , Philos. Trans. R. Soc. Lond. (Biol.) 327:155-169 (1990)). Human PDGF is believed to be the major mitogenic growth factor in serum for connective tissue. PDGF has been shown to induce mitogenesis in arterial smooth muscle cells, fibroblast cell lines, and glial cells (See e . g. Deuel et al, J. Biol. Chem., 256(17), 8896-8899 (1981); Heldin et al, J. Cell Physiol., 105, 235 (1980) (brain glial cells); Raines and Ross, J. Biol. Chem., 257, 5154 (1982) (monkey arterial smooth muscle cells) ) . PDGF is also believed to be a che o-attractant for fibroblasts, smooth muscle cells, monocytes, and granulocytes . Because of its apparent abilities to induce mitogenesis and to attract fibroblasts to the site of wounds, PDGF could have therapeutic utility m the repair of injured or traumatized connective tissues. PDGF was initially described by Ross et al . as a factor found in whole blood serum that is capable of supporting the growth of fibroblasts m culture (Proc. Natl. Acad. Sci. USA, 71:1207-1210 (1974)). PDGF was subsequently isolated from platelets and from serum, with the native unreduced PDGF being identified as a 27-35 kd dimeπc protein. Reduction of PDGF yields two or more subunits in a molecular weight range of approximately 18 kd and 16 kd, respectively referred to as the "A" and "B" subunits. The A chain is approximately 35% homologous to the B chain. The PDGF B chain from human platelets comprises a 109 ammo acid cleavage product of a 241 ammo acid precursor polypeptide (Johnsson et al, EMBO Journal, 3(5), 921-928 (1984)). PDGF is believed to be biologically active only dimeric form. Biologically active PDGF dimers can take the form of a PDGF A-B heterodimer, a PDGF B-B homodi er, or a PDGF A-A homodimer (see, e.g. Hannmk et al, Mol. Cell. Biol., 6, 1304-1314 (1986)). Each monomeπc subunit of the biologically active dimer, irrespective of whether it is an A chain or a B chain, contains eight cysteine residues, some of which form interchain disulfide bonds to hold the dimer together. The 109 amino acid sequence (PDGF B. sub.109) identified as the mature form of PDGF B, has been produced in yeast and other eucaryotic host cells.

Sarcopenia is a major determinant of age-related disabilities that is characterized by a decline in muscle mass, muscle weakness, and increased fatigability (See, e.g. P. Balagopal et.al., Endocrine, 7, 57-60, (1997); K. Short and K. Nair, J. Endocrinol . Invest . , 22, 95-105, (1999)). These changes produce substantial physical disability in the elderly. The quality and quantity of muscle depends on the integrity of a continuous remodeling process that includes breakdown of old proteins and synthesis of new ones. The maintenance of muscle is determined by a delicate balance between these two processes, implying that a decline in muscle mass occurs when protein breakdown exceeds synthesis. The quality of life of individuals suffering from sarcopenia and other related musculoskeletal disorders would be improved by a compound that impeded or reversed the degenerative process of these conditions.

Similarly, as knowledge about bone growth and strength has progressed over the years, various approaches to the treatment and prevention of diseases involving reduction in bone mass have been contemplated (for review, see WO 94/20615 published on Sep. 15, 1994, the contents of which are incorporated herein by reference) . More specifically, international patent application published Sep. 17, 1992 under No. 92/15615 describes a protein, derived from porcine pancreas, which depresses serum calcium levels and is used in the treatment of bone disorders that cause elevation of serum calcium levels . European Patent Application No. 504 938 published Sep. 23, 1992 describes the use of di- or tri-peptides, which inhibit cysteine proteases, in the treatment of bone diseases. An international patent application published Sep. 3, 1992 under No. 92/14481 discloses a composition for inducing bone growth, the composition containing activin and bone morphogenic protein. European Patent Application No. 499 242 published Aug. 19, 1992 describes the use of cell growth factor compositions thought to be useful for treating bone diseases involving bone mass reduction. Such growth factors are thought to induce osteoblast proliferation. International patent application published June 25, 1992 under No. 92/10515 describes a drug containing the human N-terminal fragment 1-37 of parathyroid hormone (PTH) . Finally, European Patent Application No. 451 867 published Sep. 16, 1991 describes parathyroid hormone antagonists for treating osteoporosis.

A newly identified PDGF homolog, referred to herein as LP8, is described in co-pending United States Patent Application, Serial No. 09/237,705, herein incorporated by reference. The P8 protein (SEQ ID NO: 2) is a member of the cysteine knot family. As contemplated by the present invention, LP8 and related molecules are useful in treating a variety of diseases and conditions. LP8 and related molecules, e.g. functional fragments thereof, are useful in treating a variety of diseases and conditions including osteoporosis, various forms of arthritis, sarcopenia, and in promoting connective tissue grafts and the success of organ transplants. Antagonists of LP8 are also useful in treating cancers including sarcomas. In another embodiment, the present invention relates to a pharmaceutical compound comprising an LP8 fragment together with one or more pharmaceutically acceptable diluents, carriers, or exipients

The methods of the present invention will be particularly useful for treatment of osteopenia, osteoporosis, bone or muscle loss due to malignancy, or endocrine disorders or arthritis, or immobility and disuse, and in the treatment of fractures by enabling the patient to reverse previous bone loss . The method can be used for treatment of cancer or bone or muscle disorders in all animals, e.g. mammals particularly humans, cattle, horses, dogs and cats.

The invention also provides a method of prophylactically increasing or maintaining bone density and/or bone quality in a subject having a substantially normal bone density comprising the step of administering an effective amount of LP8 , a fragment thereof, an analog thereof, or an LP8 agonist.

In another embodiment the present invention relates to a method for treating arthritis by the administration of a therapeutically effective amount of LP8 , a fragment thereof, or an analog thereof.

In one embodiment the present invention relates to a method for treating osteoporosis by the administration of a therapeutically-effective amount of LP8, a fragment thereof, or an analog thereof.

In one embodiment the present invention relates to a method for treating osteopenia by the administration of a therapeutically-effective amount of LP8 , a fragment thereof, or an analog thereof. In yet another embodiment, the present invention relates to the use of LP8, a fragment thereof, or an analog thereof in the manufacture a medicament for treating osteoporosis and/or osteopenia. In yet another embodiment, the present invention relates to the use of LP8, a fragment thereof, or an analog thereof in the manufacture a medicament for treating arthritis .

In yet another embodiment, the present invention relates to the use of LP8, a fragment thereof, or an analog thereof in the manufacture a medicament for treating cancer.

In one embodiment the present invention relates to use of an LP8 C-terminal fragment having biological activity to treat diseases, conditions, or disorders described herein.

In a preferred embodiment of the present invention an LP8 fragment comprising residues from about 233 through 345 of SEQ ID NO: 2 is used to treat the diseases, conditions, or disorders described herein. Definitions

The term "analog" or "functional analog" refers to a modified form of LP8 in which at least one amino acid substitution and/or deletion has been made in SEQ ID NO: 2 or a fragment thereof such that said analog retains substantially the same biological activity in vivo and/or in vitro as the unmodified LP8 or fragment thereof.

The terms "complementary" or "complementarity" as used herein refer to the capacity of purine and pyrimidine nucleotides to associate through hydrogen bonding to form double stranded nucleic acid molecules. The following base pairs are related by complementarity: guanine and cytosine; adenine and thy ine; and adenine and uracil. As used herein, "complementary" means that the aforementioned relationship applies to substantially all base pairs comprising two single-stranded nucleic acid molecules over the entire length of said molecules. "Partially complementary" refers to the aforementioned relationship in which one of two single-stranded nucleic acid molecules is shorter in length than the other such that a portion of one of the molecules remains single-stranded.

The term "conservative substitution" or "conservative amino acid substitution" as used herein refers to a replacement of one or more amino acid residue (s) in a P8 protein according to the substitutions designated in Table 1.

"Fragment thereof" refers to a fragment, or sub-region of an P8 nucleic acid or protein molecule, such that said fragment comprises 5 or more amino acids, or 10 or more nucleotides that are contiguous in the parent protein or nucleic acid molecule.

"Functional fragment, " as used herein, refers to an isolated sub-region, or fragment of a protein, or sequence of amino acids that, for example, comprises a functionally distinct region such as an active site on an enzyme, or a binding site for a ligand or other substrate, or a binding site for a receptor. Functional fragments may be produced by subcloning methods, or as natural products of alternative splicing processes.

"Functionally-related" as used herein is applied to proteins or peptides that are predicted to be functionally similar or identical to P8 or fragment thereof, for example, the ability to activate PDGF receptor.

Functionally related molecules are expected in cases of proteins or peptides that have chemical similarities in their amino acid composition and/or sequence compared with LP8. For example, one or more conservative amino acid substitutions or deletions in the LP8 sequence would not be expected to alter the function of LP8 protein. "Host cell" refers to any eucaryotic or procaryotic cell that is suitable for propagating and/or expressing a cloned gene contained on a vector that is introduced into said host cell by, for example, transformation or transfection, or the like. LP8 refers to a nucleic acid, or gene, or cDNA (e.g. SEQ ID N0:1 and/or coding region therein) and/or to a protein (SEQ ID NO: 2 and/or analogs or functional fragments and equivalents thereof) . LP8 is a member of the PDGF family of proteins. "LP8 short" refers to a C-terminal fragment of LP8 comprising residues from about 233 through 345 of SEQ ID NO: 2 and having biological activity, e.g. the ability to activate Map Kinase and/or to stimulate growth of BalbC cells . The term "homolog" or "homologous" designates a relationship of partial identity or similarity of sequence between nucleic acid molecules or protein molecules at one or more regions within said molecules.

The term "hybridization" as used herein refers to a process in which a single-stranded nucleic acid molecule joins with a complementary strand through nucleotide base pairing. "Selective hybridization" refers to hybridization under conditions of high stringency. The degree of hybridization depends upon, for example, the degree of homology or relatedness, the stringency of hybridization, and the length of hybridizing strands. " Isolated nucleic acid compound" refers to any specific RNA or DNA molecule, however constructed or synthesized or isolated, which is locationally distinct from its natural location, and which is substantially free of other larger or smaller nucleic acid compounds.

A "nucleic acid probe" or "probe" as used herein is a labeled nucleic acid compound which hybridizes with another nucleic acid compound. "Nucleic acid probe" means a single stranded nucleic acid sequence that will combine with a complementary or partially complementary single stranded target nucleic acid sequence to form a double-stranded molecule. A nucleic acid probe may be an oligonucleotide or a nucleotide polymer. A probe will usually contain a detectable moiety which may be attached to the end(s) of the probe or be internal to the sequence of the probe.

The term "orthologue" or "orthologous" refers to two or more genes or proteins from different organisms that exhibit sequence homology.

The term "paralogue" or "paralogous" refers to two or more genes or proteins within a single organism that exhibit sequence homology.

The term "plasmid" refers to an extrachromosomal genetic element. The plasmids disclosed herein are commercially available, publicly available on an unrestricted basis, or can be constructed from readily available plasmids in accordance with published procedures. A "primer" is a nucleic acid fragment which functions as an initiating substrate for enzymatic or synthetic elongation of, for example, a nucleic acid molecule. The term "promoter" refers to a nucleic acid sequence that directs transcription, for example, of DNA to RNA. An inducible promoter is one that is regulatable by environmental signals, such as carbon source, heat, or metal ions, for example. A constitutive promoter generally operates at a constant level and is not regulatable.

"Recombinant DNA cloning vector" as used herein refers to any autonomously replicating agent, including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DNA segments can or have been incorporated.

The term "recombinant DNA expression vector" or "expression vector" as used herein refers to any recombinant DNA cloning vector, for example a plasmid or phage, in which a promoter and other regulatory elements are present thereby enabling transcription of an inserted DNA, which may encode a protein. The term "stringency" refers to hybridization conditions. High stringency conditions disfavor non- homologous base pairing. Low stringency conditions have the opposite effect. Stringency may be altered, for example, by temperature and salt concentration. "Low stringency" conditions comprise, for example, a temperature of about 37° C or less, a formamide concentration of less than about 50%, and a moderate to low salt (SSC) concentration; or, alternatively, a temperature of about 50° C or less, and a moderate to high salt (SSPE) concentration, for example 1M NaCl.

"High stringency" conditions comprise, for example, a temperature of about 42° C or less, a formamide concentration of less than about 20%, and a low salt (SSC) concentration; or, alternatively, a temperature of about 65° C, or less, and a low salt (SSPE) concentration. For example, high stringency conditions comprise hybridization in 0.5 M NaHP0 , 7% sodium dodecyl sulfate (SDS), 1 M EDTA at 65°C (Ausubel, F.M. et al . Current Protocols in Molecular Biology, Vol. I, 1989; Green Inc. New York, at 2.10.3) .

"SSC" comprises a hybridization and wash solution. A stock 20X SSC solution contains 3M sodium chloride, 0.3M sodium citrate, pH 7.0.

"SSPE" comprises a hybridization and wash solution. A IX SSPE solution contains 180 mM NaCl, 9mM Na₂HP0 , 0.9 M NaH₂P0 and 1 mM EDTA, pH 7.4. "Substantially pure, " used in reference to a peptide or protein, means separation from other cellular and non- cellular molecules, including other protein molecules. A substantially pure preparation would be about at least 85% pure; preferably about at least 95% pure. A "substantially pure" protein can be prepared by a variety of techniques, well known to the skilled artisan, including, for example, the IMAC protein purification method.

"Treating" as used herein describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of a protein of the present invention to prevent the onset of the symptoms or complications, alleviating the symptoms or complications, or eliminating the disease, condition, or disorder. Treating as used herein includes the administration of the protein for cosmetic purposes.

The term "vector" as used herein refers to a nucleic acid compound used for introducing exogenous or endogenous DNA into host cells. A vector comprises a nucleotide sequence which may encode one or more protein molecules. Plasmids, cosmids, viruses, and bacteriophages, in the natural state or which have undergone recombinant engineering, are examples of commonly used vectors. The various restriction enzymes disclosed and described herein are commercially available and the manner of use of said enzymes including reaction conditions, cofactors, and other requirements for activity are well known to one of ordinary skill in the art. Reaction conditions for particular enzymes were carried out according to the manufacturer's recommendation.

The LP8 gene encodes a protein that is related to the PDGF family of proteins. The LP8 cDNA comprises a DNA sequence specified herein by SEQ ID NO:l, the coding region being defined by residues 276 through 1310 of SEQ ID NO:l. Those skilled in the art will recognize that owing to the degeneracy of the genetic code, numerous "silent" substitutions of nucleotide base pairs could be introduced into the coding sequence identified as SEQ ID NO : 1 without altering the identity of the encoded amino acid(s) or protein product. All such substitutions are intended to be within the scope of the invention.

The LP8 gene is expressed prominently in epithelial cells and in smooth muscle cells. The LP8 protein stimulates the proliferation of multiple cell types and therefore appears to be a mitogenic factor for these cell types .

The LP8 protein is membrane-bound in vivo. A secreted form of LP8 can be produced by removing a portion of the sequence at the amino terminus and fusing therefore, any suitable signal peptide to facilitate secretion from an expression host cell. For example, residues from about 1 through about 22 of SEQ ID NO: 2, or residues from about 1 through about 15 of SEQ ID NO: 2 can be removed and replaced with the kappa light chain signal sequence, or any other suitable signal peptide, for example the protrypsin signal peptide or other signal peptide.

Exemplary functional fragments of LP8 include the regions from about residues 1 through about 29 of SEQ ID N0:2, and from about residues 30 through about 80 of SEQ ID

NO:2. These regions define a kinase domain receptor (KDR) binding site.

Amino acid substitutions can be made in the LP8 molecule in accordance with the following Table. Modifications of LP8 peptides made in accordance with the

Table are expected to retain the biological activity of LP8 based on art recognized substitutability of certain amino acids (See e.g. M. Dayhoff, In Atlas of Protein Sequence and Structure, Vol. 5, Supp . 3, pgs 345-352, 1978). Functionality of analogs is easily tested in an assay that measures endothelial cell mitogenic activity, for example.

ORIGINAL RESIDUE EXEMPLARY SUBSTITUTIONS

ALA SER, THR

ARG LYS

ASN HIS, SER

ASP GLU, ASN

CYS SER

GLN ASN, HIS

GLU ASP, GLU

GLY ALA, SER

HIS ASN, GLN

ILE LEU, VAL, THR

LEU ILE, VAL

LYS ARG, GLN, GLU, THR

MET LEU, ILE, VAL

PHE LEU, TYR

SER THR, ALA, ASN

THR SER, ALA

TRP ARG , SER

TYR PHE

VAL ILE, LEU, ALA

PRO ALA

Functionally related proteins and peptides

Structurally-related analogs having biological activities that are similar or identical to LP8, for example, the ability to induce mitogenesis, in vivo or in vitro, are also contemplated by the present invention. Said analogs, while being functionally related, comprise amino acid sequences that differ in one or more positions from SEQ ID NO: 2. Functional analogs of LP8 can be generated by deletion, insertion, inversion, and/or substitution of one or more amino acid residues in said LP8. Substitution analogs can generally be made by solid phase or recombinant techniques in which, for example, single or multiple conservative amino acid substitutions are made, for example, according to Table 1. Generally, in the case of multiple substitutions, it is preferred that less than ten residues be changed in any given molecule, most preferably between one to five residues are changed in any given molecule, such that about between 90% to 99% of residues are identical with the sequence of SEQ ID NO: 2; alternatively, such that about between 95% to 99% of residues are identical with SEQ ID NO : 2.

One embodiment of the instant invention provides fragments of the LP8 proteins and analogs that may or may not be biologically active.

The instant invention provides fragments of LP8 that retain biological activity. Such fragments are useful, among other things, as therapeutic agents.

In one embodiment, the invention relates to C-terminal fragments of LP8 comprising at least 50 contiguous residues from about region 80 through 345 of SEQ ID NO: 2, said fragments retaining biological activity in vivo and/or in vitro. A preferred embodiment relates to fragment LP8 short which is defined herein by residues from about 233 through 345 of SEQ ID NO: 2.

Fragments of the LP8 proteins and analogs may be generated by any number of suitable techniques, including chemical synthesis of any portion of SEQ ID NO: 2, proteolytic digestion of SEQ ID NO : 2 , or most preferably, by recombinant DNA mutagenesis techniques, well known to the skilled artisan. See. e . g. K. Struhl, "Reverse biochemistry: Methods and applications for synthesizing yeast proteins in vi tro, " Meth . Enzymol . 194, 520-535. For example, in a preferred method, a nested set of deletion mutations are introduced into a nucleic acid sequence encoding LP8 (e.g. residues 276 through 1310 of SEQ ID NO:l) such that varying amounts of the protein coding region are deleted, either from the amino terminal end, or from the carboxyl end of the protein molecule. This method can also be used to create internal fragments of the intact protein in which both the carboxyl and amino terminal ends are removed. Several appropriate nucleases can be used to create such deletions, for example _3al31, or in the case of a single stranded nucleic acid molecule, mung bean nuclease. For simplicity, it is preferred that the LP8 gene be cloned into a single-stranded cloning vector, such as bacteriophage M13, or equivalent. If desired, the resulting gene deletion fragments can be subcloned into any suitable vector for propagation and expression of said fragments in any suitable host cell.

Functional fragments of the proteins disclosed herein may be produced as described above, preferably using cloning techniques to engineer smaller versions of the intact gene, lacking sequence from the 5' end, the 3' end, from both ends, or from an internal site. Fragments may be tested for biological activity using any suitable assay, for example, the ability of a protein fragment to induce mitogenesis, in vivo or in vi tro .

Those skilled in the art will recognize that the LP8 gene could be obtained by a plurality of recombinant DNA techniques including, for example, hybridization, polymerase chain reaction (PCR) amplification, or de novo DNA synthesis . (See e.g., T. Maniatis et al . Molecular Cloning: A Laboratory Manual, 2d Ed. Chap. 14 (1989)). Methods for constructing cDNA libraries in a suitable vector such as a plasmid or phage for propagation in procaryotic or eucaryotic cells are well known to those skilled in the art. [See e . g. Maniatis et al . Supra] . Suitable cloning vectors are well known and are widely available .

The LP8 gene, or fragment thereof, can be isolated from a tissue in which said gene is expressed, for example, placenta. In one method, mRNA is isolated, and first strand cDNA synthesis is carried out. A second round of DNA synthesis can be carried out for the production of the second strand. If desired, the double-stranded cDNA can be cloned into any suitable vector, for example, a plasmid, thereby forming a cDNA library. Oligonucleotide primers targeted to any suitable region of SEQ ID NO : 1 can be used for PCR amplification of LP8. See e.g. PCR Protocols: A Guide to Method and Application, Ed. M. Innis et al . , Academic Press (1990) . The PCR amplification comprises template DNA, suitable enzymes, primers, and buffers, and is conveniently carried out in a DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk, CT) . A positive result is determined by detecting an appropriately-sized DNA fragment following agarose gel electrophoresis.

One embodiment of the present invention relates to the substantially purified protein encoded by the LP8 gene.

Skilled artisans will recognize that the proteins of the present invention can be synthesized by a number of different methods, such as chemical methods well known in the art, including solid phase peptide synthesis or recombinant methods. Both methods are described in U.S. Patent 4,617,149, incorporated herein by reference. The principles of solid phase chemical synthesis of polypeptides are well known in the art and may be found in general texts in the area. See, e . g. , H. Dugas and C. Penney, Bioorganic Chemistry (1981) Springer-Verlag, New York, 54-92. For example, peptides may be synthesized by solid-phase methodology utilizing an Applied Biosystems 43OA peptide synthesizer (Applied Biosystems, Foster City, CA) and synthesis cycles supplied by Applied Biosystems. The proteins of the present invention can also be produced by recombinant DNA methods using the cloned LP8 gene. Recombinant methods are preferred if a high yield is desired. Expression of the cloned gene can be carried out in a variety of suitable host cells, well known to those skilled in the art. For this purpose, the LP8 gene is introduced into a host cell by any suitable means, well known to those skilled in the art. While chromosomal integration of the cloned gene is within the scope of the present invention, it is preferred that the gene be cloned into a suitable extra-chromosomally maintained expression vector so that the coding region of the LP8 gene is operably-linked to a constitutive or inducible promoter.

The basic steps in the recombinant production of the LP8 protein are: a) constructing a natural, synthetic or semi- synthetic DNA encoding LP8 protein;

b) integrating said DNA into an expression vector in a manner suitable for expressing the LP8 protein, either alone or as a fusion protein;

c) transforming or otherwise introducing said vector into an appropriate eucaryotic or prokaryotic host cell forming a recombinant host cell,

d) culturing said recombinant host cell in a manner to express the LP8 protein; and

e) recovering and substantially purifying the LP8 protein by any suitable means, well known to those skilled in the art. Procaryotes may be employed in the production of recombinant LP8 proteins (including, e.g., LP8 short), fragments thereof, or analogs thereof. For example, the Escherichia coli K12 strain 294 (ATCC No. 31446) is particularly useful for the prokaryotic expression of foreign proteins. Other strains of E. coli , bacilli such as Bacillus subtilis , enterobacteriaceae such as Salmonella typhimurium or Serratia marcescans, various Pseudomonas species and other bacteria, such as Streptomyces, may also be employed as host cells in the cloning and expression of the recombinant proteins of this invention.

Promoter sequences suitable for driving the expression of genes in procaryotes include β-lactamase [ e . g. vector PGX2907, ATCC 39344, contains a replicon and β-lactamase gene], lactose systems [Chang et al . , Nature_ (London) , 275:615 (1978); Goeddel et al . , Nature (London), 281:544 (1979)], alkaline phosphatase, and the tryptophan (trp) promoter system [vector pATHl (ATCC 37695)], which is designed to facilitate expression of an open reading frame as a trpE fusion protein under the control of the trp promoter. Hybrid promoters such as the tac promoter (isolatable from plasmid pDR540, ATCC-37282) are also suitable. Still other bacterial promoters, whose nucleotide sequences are generally known, may be ligated to DNA encoding the protein of the instant invention, using linkers or adapters to supply any required restriction sites. Promoters for use in bacterial systems also will contain a Shine-Dalgarno sequence operably-linked to the DNA encoding the desired polypeptides . These examples are illustrative rather than limiting. Vectors

Another aspect of the present invention relates to recombinant DNA cloning vectors and expression vectors comprising the nucleic acids of the present invention. The preferred nucleic acid vectors are those which comprise DNA. A preferred recombinant DNA vectors comprise an isolated DNA sequence that encodes residues 233 through 345 of SEQ ID NO: 2.

The skilled artisan understands that choosing the most appropriate cloning vector or expression vector depends upon a number of factors including the availability of restriction enzyme sites, the type of host cell into which the vector is to be transfected or transformed, the purpose of the transfection or transformation (e.g., stable transformation as an extrachromosomal element, or integration into the host chromosome) , the presence or absence of readily assayable or selectable markers (e.g., antibiotic resistance and metabolic markers of one type and another) , and the number of copies of the gene desired in the host cell.

Vectors suitable to carry the nucleic acids of the present invention comprise RNA viruses, DNA viruses, lytic bacteriophages, lysogenic bacteriophages, stable bacteriophages, plasmids, viroids, and the like. The most preferred vectors are plasmids. When preparing an expression vector the skilled artisan understands that there are many variables to be considered, for example, whether to use a constitutive or inducible promoter. The practitioner also understands that the amount of nucleic acid or protein to be produced dictates, in part, the selection of the expression system. Regarding promoter sequences, inducible promoters are preferred because they enable high level, regulatable expression of an operably-linked gene. The skilled artisan will recognize a number of suitable promoters that respond to a variety of inducers, for example, carbon source, metal ions, and heat. Other relevant considerations regarding an expression vector include whether to include sequences for directing the localization of a recombinant protein. For example, a sequence encoding a signal peptide preceding the coding region of a gene is useful for directing the extracellular export of a resulting polypeptide.

The present invention also provides a method for constructing a recombinant host cell capable of expressing LP8 proteins and fragments thereof (e.g. LP8 short), said method comprising transforming or otherwise introducing into a host cell a recombinant DNA vector that comprises an isolated DNA sequence that encodes SEQ ID NO : 2 or fragment thereof. A suitable host cell is any eucaryotic cell that can accomodate high level expression of an exogenously introduced gene or protein, and that will incorporate said protein into its membrane structure. Vectors for expression are those which comprise SEQ ID NO:l or fragment thereof. Transformed host cells may be cultured under conditions well known to skilled artisans such that SEQ ID NO: 2 is expressed, thereby producing a recombinant LP8 protein in the recombinant host cell. The proteins of this invention may be synthesized either by direct expression or as a fusion protein comprising the protein of interest as a translational fusion with another protein or peptide which may be removable by enzymatic or chemical cleavage. It is often observed in the production of certain peptides in recombinant systems that expression as a fusion protein prolongs the life span, increases the yield of the desired peptide, or provides a convenient means of purifying the protein. This is particularly relevant when expressing mammalian proteins in procaryotic hosts. A variety of peptidases (e.g. enterokinase and thrombin) which cleave a polypeptide at specific sites or digest the peptides from the amino- or carboxy-termini ( e . g. diaminopeptidase) of the peptide chain are known. Furthermore, particular chemicals { e . g. cyanogen bromide) will cleave a polypeptide chain at specific sites. The skilled artisan will appreciate the modifications necessary to the amino acid sequence (and synthetic or semi-synthetic coding sequence if recombinant means are employed) to incorporate site- specific internal cleavage sites ( See e . g. , P. Carter, "Site Specific Proteolysis of Fusion Proteins", Chapter 13, in Protein Purification: From Molecular Mechanisms to Large Scale Processes, American Chemical Society, Washington, D.C. (1990) ) .

In addition to procaryotes, a variety of amphibian expression systems such as frog oocytes, and mammalian cell systems can be used. The choice of a particular host cell depends to some extent on the particular expression vector used. Exemplary mammalian host cells suitable for use in the present invention include HepG-2 (ATCC HB 8065), CV-1 (ATCC CCL 70), LC-MK2 (ATCC CCL 7.1), 3T3 (ATCC CCL 92), CHO-K1 (ATCC CCL 61), HeLa (ATCC CCL 2), RPMI8226 (ATCC CCL 155), H4IIEC3 (ATCC CCL 1600), C127I (ATCC CCL 1616), HS- Sultan (ATCC CCL 1484), and BHK-21 (ATCC CCL 10), for example . A wide variety of vectors are suitable for transforming mammalian host cells. For example, the pSV2- type vectors comprise segments of the simian virus 40 (SV40) genome required for transcription and polyadenylation. A large number of plasmid pSV2-type vectors have been constructed, such as pSV2-gpt, pSV2-neo, pSV2-dhfr, pSV2-hyg, and pSV2-b-globin, in which the SV40 promoter drives transcription of an inserted gene. These vectors are widely available from sources such as the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland, 20852, or the Northern Regional Research Laboratory (NRRL) , 1815 N. University Street, Peoria, Illinois, 61604.

Promoters suitable for expression in mammalian cells include the SV40 late promoter, promoters from eukaryotic genes, such as, for example, the estrogen-mducible chicken ovalbumin gene, the interferon genes, the glucocorticoid- inducible tyrosine aminotransferase gene, the thymidine kinase gene promoter, and the promoters of the major early and late adenovirus genes and the cytomegalovirus promoter. Plasmid pRSVcat (ATCC 37152) comprises portions of a long terminal repeat of the Rous Sarcoma virus, a virus known to infect chickens and other host cells. This long terminal repeat contains a promoter which is suitable for use in the vectors of this invention. H. Gorman et al . , Proc . Nat . Acad. Sci . (USA) , 79, 6777 (1982). The plasmid pMSVi (NRRL B-15929) comprises the long terminal repeats of the Murine Sarcoma virus, a virus known to infect mouse and other host cells. The mouse metallothionein promoter has also been well characterized for use in eukaryotic host cells and is suitable for use in the present invention. This promoter is present in the plasmid pdBPV-MMTneo (ATCC 37224) which can serve as the starting material for the construction of other plasmids of the present invention. Transfection of mammalian cells with vectors can be performed by a plurality of well known processes including, but not limited to, protoplast fusion, calcium phosphate co-precipitation, electroporation and the like. See, e.g., Maniatis et al . , supra .

Some viruses also make appropriate vectors . Examples include the adenoviruses, the adeno-associated viruses, the vaccinia virus, the herpes viruses, the baculoviruses, and the rous sarcoma virus, as described in U.S. Patent 4,775,624, incorporated herein by reference.

Eucaryotic microorganisms such as yeast and other fungi are also suitable host cells. The yeast Saccharomyces cerevisiae is the preferred eucaryotic microorganism. Other yeasts such as Kluyveromyces lactis and Pichia pas toris are also suitable. For expression in Saccharomyces , the plasmid YRp7 (ATCC-40053 ) , for example, may be used. See, e . g. , L. Stinchcomb et al . , Nature, 282, 39 (1979); J. Kingsman et al . , Gene, 7, 141 (1979); S. Tschemper et al . , Gene, 10, 157 (1980). Plasmid YRp7 contains the TRPl gene which provides a selectable marker for use in a trpl auxotrophic mutant.

An expression vector carrying the cloned LP8 gene or fragment thereof is transformed or transfected into a suitable host cell using standard methods. Cells that contain the vector are propagated under conditions suitable for expression of the recombinant LP8 protein. For example, if the recombinant gene has been placed under the control of an inducible promoter, suitable growth conditions would incorporate the appropriate inducer. The recombinantly-produced protein may be purified from cellular extracts of transformed cells by any suitable means .

In a preferred process for protein purification, the LP8 gene is modified at the 5 ' end to incorporate several histidine residues at the amino terminus of the LP8 protein. This "histidine tag" enables a single-step protein purification method referred to as "immobilized metal ion affinity chromatography" (IMAC), essentially as described in U.S. Patent 4,569,794, which hereby is incorporated by reference. The IMAC method enables rapid isolation of substantially pure recombinant LP8 protein starting from a crude extract of cells that express a modified recombinant protein, as described above.

Other embodiments of the present invention comprise isolated nucleic acid sequences that encode SEQ ID NO: 2, or related nucleic acids that are at least about 75% identical to the coding region of SEQ ID NO : 1 , or to their complementary sequence, or nucleic acids that hybridize to the coding region of SEQ ID NO : 1 under high stringency conditions and encode a protein that has mitogenic activity, or nucleic acid that hybridize to the coding region of SEQ ID NO : 1 under high stringency conditions and encode a protein that is at least about 75% identical with SEQ ID NO: 2; alternatively, a protein that is at least about 85% identical with SEQ ID NO : 2 ; preferably, a protein that is at least about 90 to 95% identical with SEQ ID

NO: 2; and most preferably, a protein that is at least 95% identical with SEQ ID NO:2. The percent identity between nucleic acids or proteins is determined by any suitable comparison algorithm, well known to the skilled artisan. By percent identity is meant the number of residues that are identical between optimally aligned nucleic acids (or proteins) , divided by the total length (including gaps) of the shortest sequence of the pair or group being compared. Nucleic acid or protein sequences are optimally aligned to achieve the greatest degree of similarity, allowing for gaps, using any suitable algorithm, for example, a dynamic programming algorithm (See e.g. Smith and Waterman, J. Mol. Biol. 147, 195 (1985), BLASTA (Altschul et al . J. Mol. Biol. 215, 403 (1990); or FASTA (Lipman & Pearson, Science, 227, 1435 (1985), herein incorporated by reference. Such alignments are carried out with the paramters set to maximize the alignment score obtained for a pair of sequences being compared.

The LP8 cDNA (viz. SEQ ID NO : 1 ) and related nucleic acid molecules that encode SEQ ID NO: 2, or functional fragments thereof, or analogs, may be produced by chemical synthetic methods. The synthesis of nucleic acids is well known in the art. See, e . g. , E.L. Brown, R. Belagaje, M.J. Ryan, and H.G. Khorana, Methods in Enzymology, 68:109-151 (1979). Fragments of the DNA sequence corresponding to the LP8 gene may be generated using a conventional DNA synthesizing apparatus, such as the Applied Biosystems Model 380A or 380B DNA synthesizers (Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404) using phosphoramidite chemistry, thereafter ligating the fragments so as to reconstitute the entire gene. Alternatively, phosphotriester chemistry may be employed to synthesize the nucleic acids of this invention. ( See, e.g., M.J. Gait, ed. , Oligonucleotide Synthesis, A Practical Approach, (1984)).

In an alternative methodology, namely PCR, the DNA sequences disclosed and described herein, comprising, for example, a portion or all of SEQ ID NO:l can be produced from a plurality of starting materials. For example, starting with a cDNA preparation (e.g. cDNA library) derived from a tissue that expresses the LP8 gene, suitable oligonucleotide primers complementary to SEQ ID NO:l or to any sub-region therein, are prepared as described in U.S. Patent No. 4,889,818, hereby incorporated by reference. Other suitable protocols for the PCR are disclosed in PCR Protocols: A Guide to Method and Applications, Ed. Michael A. Innis et al . , Academic Press, Inc. (1990). Using PCR, any region of the LP8 gene can be targeted for amplification such that full or partial length gene sequences may be produced.

The ribonucleic acids of the present invention may be prepared using polynucleotide synthetic methods discussed supra, or they may be prepared enzymatically, for example, using RNA polymerase to transcribe a LP8 DNA template.

The most preferred systems for preparing the ribonucleic acids of the present invention employ the RNA polymerase from the bacteriophage T7 or the bacteriophage SP6. These RNA polymerases are highly specific, requiring the insertion of bacteriophage-specific sequences at the 5 ' end of the template to be transcribed. See, Maniatis et al . , supra .

This invention also provides nucleic acids, RNA or DNA, that are complementary to the coding region of SEQ ID NO:l, or fragment thereof. For the purpose of identifying compounds having utility as agonists or antagonists of LP8, it would be desirable to identify compounds that bind LP8 proteins. A method for determining agents that bind LP8 proteins comprises contacting the LP8 protein with a test compound and monitoring binding by any suitable means.

The instant invention provides a screening system for discovering compounds that bind LP8 proteins, said screening system comprising the steps of :

a) preparing a LP8 protein;

b) exposing said LP8 protein to a test compound;

c) quantifying the binding of said compound to LP8 protein by any suitable means.

Utilization of the screening system described above provides a means to identify compounds that may alter the biological function of LP8. This screening method may be adapted to large-scale, automated procedures such as a

PANDEX® (Baxter-Dade Diagnostics) system, allowing for efficient high-volume screening of potential therapeutic agents .

In such a screening protocol LP8 is prepared as described herein, preferably using recombinant DNA technology. A test compound is introduced into a reaction vessel containing the LP8 protein or fragment thereof. Binding of LP8 by a test compound is determined by any suitable means. For example, in one method radioactively- labeled or chemically-labeled test compound may be used. Binding of the protein by the compound is assessed, for example, by quantifying bound label versus unbound label using any suitable method. Binding of a test compound may also be carried out by a method disclosed in U.S. Patent 5,585,277, which hereby is incorporated by reference. In this method, binding of a test compound to a protein is assessed by monitoring the ratio of folded protein to unfolded protein, for example by monitoring sensitivity of said protein to a protease, or amenability to binding of said protein by a specific antibody against the folded state of the protein. The foregoing screening methods are useful for identifying a ligand, for example, an antagonist of a LP8 protein, as a lead to a pharmaceutical compound for the treatment of cancer, or for inhibiting tumor growth or for inhibiting tissue growth, for example smooth muscle tissue growth. A ligand that binds LP8, or related fragment thereof, is identified, for example, by combining a test ligand with LP8 under conditions that cause the protein to exist in a ratio of folded to unfolded states. If the test ligand binds the folded state of the protein, the relative amount of folded protein will be higher than in the case of a test ligand that does not bind the protein. The ratio of protein in the folded versus unfolded state is easily determinable by, for example, susceptibility to digestion by a protease, or binding to a specific antibody, or binding to chaperonin protein, or binding to any suitable surface.

In another embodiment, the present invention relates to therapeutic applications for LP8 proteins comprising the administration of a therapeutically effective amount of a LP8 protein or fragment thereof, or analog thereof to treat a variety of diseases and/or conditions related to abnormal musculoskeletal structure, function, or metabolism including, but not limited to, the treatment and/or prevention of osteopenia, osteoporosis, sarcopenia, or arthritis .

In another embodiment, the present invention relates to a method for treating and/or preventing musculoskeletal conditions such as osteopenia, osteoarthritis, sarcopenia, or osteoporosis, comprising the administration of a therapeutically effective amount of LP8 , or analog thereof, or functional fragment thereof. In another embodiment, the present invention relates to a method for treating wounds or bone fractures comprising the administration of a therapeutically effective amount of LP8 , or analog thereof, or functional fragment thereof. In another embodiment, the present invention relates to a method for treating periodontal diseases comprising the administration of a therapeutically effective amount of LP8, or analog thereof, or functional fragment thereof. LP8 functions like PDGF to stimulate smooth muscle cells and fibroblast cell proliferation. Moreover, biochemical studies have shown that LP8 can activate the PDGF receptor (PDGF-R) as well as downstream signaling events such as MAP Kinase activation. These results suggest that LP8 may exert its biological influence through the PDGF-R. Thus, LP8 can mimic PDGF function and play a role in cell proliferation, including muscle cell proliferation, wound healing, and bone growth.

PDGF has been shown to increase bone mass and close cartilage growth plates in the long bones of rats, and to stimulate fibrosis in connective tissues, in vivo (B.

Mitlak et al . J. Bone & Miner. Res., 11:238-247, (1996)). Like PTH, a known anabolic agent to increase skeletal mass, PDGF also stimulates interstitial collagenase, a step thought to be necessary to stimulate bone turnover, thereby increasing the proportion of new matrix. In vitro, PDGF stimulates proliferation of osteoprogenitor cells in fetal rat calvaria, and the rate of bone collagen synthesis (See e.g. Hock, J. and Canalis, E., Endrocrin., 134, (1994)). PDGF also stimulates proliferation of muscle cells and chondrocytes , suggesting significant effects on the homeostasis of these tissues. For therapeutic utility, an effective amount of LP8, a fragment or analog thereof is administered to an organism in need thereof in a dose between about 0.1 and 1000 ug/kg body weigh . In practicing the methods contemplated by this invention, LP8, a fragment thereof, or an analog thereof can be administered in a single daily dose or in multiple doses per day. The amount per administration will be determined by the physician and depend on such factors as the nature and severity of the disease, and the age and general health of the patient. Accordingly, patients at risk of bone deterioration may be given a regular dose of LP8, a fragment thereof, an analog thereof, or an LP8 agonist to prevent bone deterioration. Patients at greatest risk for bone deterioration are post-menopausal women and men above the age of 60. By "normal bone density" is meant within two standard deviations of the mean value for race, age and sex.

The present invention also provides a pharmaceutical composition comprising as the active agent a LP8 polypeptide, or a fragment or analog thereof, or a pharmaceutically acceptable non-toxic salt thereof, and a pharmaceutically acceptable solid or liquid carrier. For example, compounds comprising LP8, a fragment thereof, or an analog thereof can be admixed with conventional pharmaceutical carriers and excipients, and used in the form of tablets, capsules, elixirs, suspensions, syrups, wafers, and the like. The compositions comprising LP8, a fragment thereof, or an analog thereof will contain from about 0.1% to 90% by weight of the active compound, and more generally from about 10% to 30%. The compositions may contain common carriers and excipients such as corn starch or gelatin, lactose, sucrose, microcrystallme cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, and alginic acid. The compounds can be formulated for oral or parenteral administration.

For intravenous (IV) use, the LP8 protein is administered in commonly used intravenous fluid (s) and administered by infusion. Such fluids, for example, physiological saline, Ringer's solution or 5% dextrose solution can be used.

For intramuscular preparations, a sterile formulation, preferably a suitable soluble salt form of the LP8 protein, for example SEQ ID NO : 2 , such as the hydrochloride salt, can be dissolved and administered in a pharmaceutical diluent such as pyrogen-free water (distilled) , physiological saline or 5% glucose solution. A suitable insoluble form of the compound may be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, e.g. an ester of a long chain fatty acid such as ethyl oleate.

The present invention also provides a pharmaceutical composition comprising as the active agent an antagonist of LP8, or a pharmaceutically acceptable non-toxic salt thereof, and a pharmaceutically acceptable solid or liquid carrier. For example, compounds comprising LP8 antagonist can be admixed with conventional pharmaceutical carriers and excipients, and used in the form of tablets, capsules, elixirs, suspensions, syrups, wafers, and the like. The compositions comprising LP8 will contain from about 0.1% to 90% by weight of the active compound, and more generally from about 10% to 30%. The compositions may contain common carriers and excipients such as corn starch or gelatin, lactose, sucrose, microcrystallme cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, and alginic acid. The compounds can be formulated for oral or parenteral administration.

Skilled artisans will recognize that IC50 values are dependent on the selectivity of the compound tested. For example, a compound with an IC50 which is less than 10 nM is generally considered an excellent candidate for drug therapy. However, a compound which has a lower affinity, but is selective for a particular target, may be an even better candidate. The skilled artisan will recognize that any information regarding the bindmg potential, inhibitory activity, or selectivity of a particular compound is useful toward the development of pharmaceutical products.

The following examples more fully describe the present invention. Those skilled m the art will recognize that the particular reagents, equipment, and procedures described are merely illustrative and are not intended to limit the present mvention in any manner.

EXAMPLE 1 RT-PCR Amplification of LP8 Gene from mRNA

A LP8 gene is isolated by reverse transcriptase PCR (RT-PCR) usmg conventional methods. Total RNA from a tissue that expresses the LP8 gene, for example placenta, is prepared using standard methods. First strand cDNA synthesis is achieved using a commercially available kit (Superscript™ System; Life Technologies) in conjunction with specific primers directed at any suitable region of SEQ ID NO:l, for example between residues 276 and 1310.

Amplification is carried out by adding to the first strand cDNA (dried under vacuum) : 8 μl of 10X synthesis buffer (200 mM Tris-HCl, pH 8.4; 500 mM KC1, 25 mM MgCl , 1 ug/ul BSA) ; 68 μl distilled water; 1 μl each of a 10 uM solution of each primer; and 1 μl Taq DNA polymerase (2 to 5 U/μl) . The reaction is heated at 94° C for 5 min. to denature the RNA/cDNA hybrid. Then, 15 to 30 cycles of PCR amplification are performed using any suitable thermal cycle apparatus. The amplified sample may be analyzed by agarose gel electrophoresis to check for an appropriately- sized fragment.

EXAMPLE 2 Production of a Vector for Expressing LP8 in a Host Cell An expression vector suitable for expressing LP8 or fragment thereof in a variety of procaryotic host cells, such as E. coli is easily made. The vector contains an origin of replication (Ori), an ampicillin resistance gene (Amp) useful for selecting cells which have incorporated the vector following a tranformation procedure, and further comprises the T7 promoter and T7 terminator sequences in operable linkage to a LP8 coding region. Plasmid pET28A (obtained from Novogen, Madison WI) is a suitable parent plasmid. PET28A is linearized by restriction with endonucleases Ndel and BamHI and ligated to a DNA fragment bearing Ndel and BamHI sticky ends comprising the coding region of the LP8 gene as disclosed by SEQ ID NO:l viz. residues 276 through 1310, or fragment thereof.

The LP8 gene used in this construction may be slightly modified at the 5' end (amino terminus of encoded protein) in order to simplify purification of the encoded protein product. For this purpose, an oligonucleotide encoding 8 histidine residues is inserted after the ATG start codon. Placement of the histidine residues at the amino terminus of the encoded protein serves to enable the IMAC one-step protein purification procedure.

An expression vector suitable for expressing LP8 short in a variety of procaryotic host cells, such as E. coli is also easily made. The vector contains an origin of replication (Ori), an ampicillin resistance gene (Amp) useful for selecting cells which have incorporated the vector following a tranformation procedure, and further comprises the T7 promoter and T7 terminator sequences in operable linkage to a coding region for LP8 short, e.g. residues 972-1310 of SEQ ID N0:1. Plasmid pET28A (obtained from Novogen, Madison WI) is a suitable parent plasmid.

PET28A is linearized by restriction with endonucleases Ndel and BamHI and ligated to a DNA fragment bearing Ndel and BamHI sticky ends comprising the coding region of LP8 short . A DNA encoding LP8 short was constructed as follows: Forward primer: 5 ' -actcccaagctttccagagtggtggatctgaacc-3 ' ; Reverse primer: 5 ' -agtcgcggatccctatcctcctgtgctccctctgc-3 ' . The short form cDNA was PCR amplified suing the following conditions: 94 C for 2 min.; 94 C for 30 sec, 60 C for 1 min, 72 C for 1 min; 25 cycles, then 72 C for 5 min. The PCR product was purified and digested with Hindlll and BamHI and the fragment ligated into HindiII/BamHI digested pCMVFlag-1. The sequence was confirmed by DNA sequence analysis. For recombinant expression, the plasmid was transfected into 292 T cells and the LP8 short purified using an anti-Flag antibody column. The LP8 short nucleic acid used in this construction may (if desired) be modified at the 5' end (amino terminus of encoded protein) in order to simplify purification of the encoded protein product. For this purpose, an oligonucleotide encoding 8 histidine residues is inserted after an appropriately placed ATG start codon. Placement of the histidine residues at the amino terminus of the encoded protein serves to enable the IMAC one-step protein purification procedure.

EXAMPLE 3 Recombinant Expression of a Secreted LP8 Protein An expression vector that carries an ORF encoding residues 16 though 345 of SEQ ID NO: 2 and further comprising a kappa chain signal peptide fused at the amino terminus of said residue 16 was operably-linked to an CMV promoter in plasmid pcDNA3 and transfected into 293 T cells using standard methods. Transfectants were analyzed for transient expression of LP8 using Western blot analysis. The results showed that the truncated LP8 protein was secreted into the culture medium. In another expression system, an expression vector that comprises the preprotrysin leader sequence followed by Flag peptide fused to the amino terminus of an ORF encoding residues 23 through 345 of SEQ ID NO: 2 was operably-linked to a CMV promoter in plasmid pFLAG and transfected into 293 T cells using standard methods. Transfectants were analyzed for transient expression of LP8 using Western blot analysis. The results showed that the LP8 protein was secreted into the culture medium.

EXAMPLE 4 Detecting Ligands that Bind LP8 Using a Chaperonin Protein Assay

The wells of an ELISA plate are coated with chaperonin by incubation for several hours with a 4 μg/ml solution of the protein in Tris-buffered Saline (TBS: 10 mM Tris-HCl, pH 7.5 , 0.2 M NaCl) . The plates are then washed 3 times with TBS containing 0.1% Tween-20 (TBST). Then, a mixture of LP8 protein (sufficient amount to saturate about 50% of the binding sites on chaperonin) and test compound (10^~9 to 10^~5 M) in about 50 μl volume is added to each well of the plate for an incubation of about 60 minutes. Aliquots of the well solutions are then transferred to the wells of fresh plates and incubated for 60 minutes at room temperature, followed by 3 washes with TBST. Next, about 50 μl of an antibody specific for LP8 plus 5% nonfat dry milk are added to each well for a 30 minute incubation at room temperature. After washing, about 50 μl of goat anti- rabbit IgG alkaline phosphatase conjugate at an appropriate dilution in TBST plus 5% nonfat dry milk are added to each will and incubated 30 minutes at room temperature. The plates are washed again with TBST and 0.1 ml of 1 mg/ml p- nitrophenylphosphate in 0.1% diethanolamine is added. Color development (proportional to bound alkaline phosphatase antibody conjugate) is monitored with an ELISA plate reader. When test ligand binding has occurred, ELISA analysis reveals LP8 in solution at higher concentrations than in the absence of test ligand. EXAMPLE 5 Use of LP8 to Treat Osteoporosis Experimental models of postmenopausal osteoporosis are known in the art. Germane to this invention is the ovariectomized rat model which is provided in US 5,393,763 which is incorporated herein by reference.

An additional demonstration of the method of treating or preventing osteoporosis due to estrogen deprivation would be as follows: One hundred patients would be chosen, who are healthy postmenopausal women, aged 45-60 and who would normally be considered candidates for estrogen replacement therapy. This includes women with an intact uterus, who have had a last menstrual period more than six months, but less than six years. Patients excluded for the study would be those who have taken estrogens, progestins, or corticosteroids six months prior to the study or who have ever taken bis-phosphonates .

Fifty women (test group) would receive LP8 , a fragment or analog thereof. The other fifty women (control group) would receive a matched placebo per day. Both groups would receive calcium carbonate tablets (648 mg) per day.

A baseline examination of each patient includes quantitative measurement of urinary calcium, creatinine, hydroxyproline, and pyridinoline crosslinks. Blood samples are measured for serum levels of osteocalcin and bone- specific alkaline phosphatase. Baseline measurements would also include a uterine examination and bone mineral density determination by photon absorptiometry .

The study would continue for at least six months, and each patient would be examined for changes in the above parameters. During the course of treatment, the patients in the treatment group would show a decreased change in the biochemical markers of bone resorption as compared to the control group. Also, the treatment group would show little or no decrease in bone mineral density compared to the control group. Both groups would have similar uterine histology.

EXAMPLE 6 Endothelial Cell Growth Assay For conducting the cell growth assay, approximately 3000 human umbilical vein endothelial cells were grown in 96 well TC plates in 150 μl Media 199/10% fetal calf serum with and without LP8. In some experiments, E. coli produced LP8 was used, in other experiments a mammalian produced LP8 containing a FLAG peptide was used. LP8 proteins were added to a final concentration of 0.1 ng/ml - lμg/ml . After 24 hours post-plating, approximately 0.25 μCi ³H- thymidine was added to each well. Forty-eight hours later, plates were frozen at -70 °C, thawed, cells harvested onto filter paper and the samples counted in a scintillation counter . EXAMPLE 7

Assay for LP8 Antagonist Assay reactions are set up essentially as described in Example 9, except that a compound to be tested for LP8 antagonist activity is included at the step of adding conditioned medium to bovine capillary endothelial cells. Multiple assays can be set up in which a constant amount of conditioned medium is incubated with varying amounts of test compound, for example from about 10 ng/ml to about 100 μg/ml . For conducting the cell growth assay, bovine capillary endothelial cells are maintained in DMEM containing 20% calf serum according to the method of Ferrara, Biochem. Biophys. Res. Co m. , 161, 851-58, 1989. Cells are plated at about 8 x 10³ cells per well in 12 well plates in DMEM supplemented with 10% calf serum, 2 mM glutamine, and antibiotics. Conditioned medium from transiently transfected 293 cells 72 hours post-transfection is added and cell number determined after 5 days.

EXAMPLE 8 LP8 and LP8 short Exposure to Cell Proliferation Panel

LP8 proteins produced in mammalian cells showed mitogenic activity on BalbC/3T3 cells while E. coli expressed LP8 did not (see Figure 1) . All positive controls (i.e. human FGF-2) showed the activity expected. The following cell panels were exposed to LP8 , or fragments or analogs thereof and assayed for resulting cell proliferation:

CTLL.6 (murine) Cytotoxic T-cell line, from C57/B1/6 mice, lymphoblast morphology, IL-2 dependent

MTS proliferation assays

Growth Medium: RPMI 1640/L-Glu + 10%FBS + ImM Sodium

Pyruvate + lOmM HEPES + 5xl0^"5 M 2-Me + 2ng/ml IL-2 Assay Medium: RPMI 1640/L-Glu + 10%FBS + ImM Sodium

Pyruvate + lOmM HEPES + 5xl0^"5 M 2-Me

T1165.17 (murine)

Plasmacytoma cell line (originates from B-cell), from ascites tumors of Balb/CanPt mice IL-1 dependent MTS proliferation assay Growth Medium: RPMI 1640/L-Glu + 10%FBS + ImM Sodium Pyruvate + lOmM HEPES + 5xl0^"5 M 2-Me + 2ng/ml rhIL-1 Assay Medium: RPMI 1640/L-Glu + 10%FBS + ImM Sodium Pyruvate + lOmM HEPES + 5xl0^"5 M 2-Me

BalbC/3T3 Clone A31 (murine)

From 14 - 17 -day-old Balb/C mouse embryos, fibroblast morphology, non-tumorigenic , contact-inhibited

³H Thymidine uptake proliferation assays Growth Medium: DMEM + 10% Calf Serum

Assay medium: DMEM + 2% Plasma Dialyzed Calf Serum

TF .1 (human)

Erythroleukemia, lymphoblast morphology hGM-CSF dependent

MTS proliferation assays

Growth Medium: RPMI 1640/L-Glu + 10%FBS + ImM Sodium Pyruvate + lOmM HEPES + 5xl0^"5 M 2-Me + 5ng/ml rhGM-CSF Assay Medium: RPMI 1640/L-Glu + 10%FBS + ImM Sodium Pyruvate + lOmM HEPES + 5xl0^"5 M 2-Me

MCF-7

Human breast cancer cell line, epithelial morphology

³H Thymidine uptake proliferation assays Responds by growth to insulin

Growth Medium: Eagles MEM (w/o phenol red) + 10%FBS + ImM

Sodium Pyruvate + Nonessential Amino Acids + L-Glu + lμg/ml insulin

Assay Medium: Eagles MEM (w/o phenol red) + ImM Sodium Pyruvate + Nonessential Amino Acids + L-Glu + lOμg/ml human transferrin HUVEC

Human umbilical vein endothelial cells, primary ³H Thymidine uptake proliferation assays Growth Medium: Clonetics complete endothelial growth medium

Assay Medium: Medium 199 + 10%FBS

EXAMPLE 9 LP8 and LP8 short Stimulates BalbC/3T3 Cell Proliferation In these experiments approximately 5000 BalbC/3T3 cells were seeded per well in a 96 well plate in DMEM/10% calf serum. The cells were grown for two days in an incubator to achieve approximately 90-100% confluence. Then, cells were starved in DMEM/2% dialyzed calf serum for 24 hours in the incubator and growth factors were added diluted in DMEM/2% dialyzed calf serum. The final volume per well was 200 μl . Cells were incubated an additional 16- 18 hours.

To assess the effects of LP8, LP8 short, and other growth factors on cell proliferation, each well received

0.25uCi ³H-thymidine during a 2 hour pulse in the incubator. Cells were then harvested and counted in a scintillation counter. Human PDGF, isolated from human platelets, was purchased from R & D Systems (cat # 120-HD-001) . Figure 1 shows that LP8 and stimulated thymidine uptake when added at 10-1000 ng/ml protein. Figure 3 shows similar effect of LP8 on human dermal fibroblasts. EXAMPLE 10 LP8 and LP8 Short Stimulates Human Aortic Smooth Muscle

Cells Approximately 5000 human aortic smooth muscle cells were seeded per well in a 96 well plate containing smooth muscle growth media (SMGM) from Clonetics. Cells were grown overnight in an incubator. After overnight incubation, SMGM was replaced with 100 μl/well smooth muscle basal media (SMBM) . Cells were starved in SMGM for 48 hours in the incubator. Next, growth factors diluted in SMBM were added to each well. Human PDGF, isolated from human platelets, was purchased from R & D Systems (cat # 120-HD-001) . The final volume per well was 200 μl . Cells were incubated for approximately 20 hours. To assess the effect on cell proliferation approximately 0.25 μCi ³H-thymidine was added to each well for 4 hours in the incubator. Cells were harvested and the quantity of radioactivity taken up by the cells was determined using a scintillation counter. Figure 2 shows LP8 cell proliferative effects on human aortic smooth muscle cells as measured by ³H-thymidine .

EXAMPLE 11 Activation of MAP Kinase by LP8 and LP8 short BalbC/3T3 cells were plated in 24 well dishes in DMEM/ 10% calf serum. The following day the cells were about 80% confluent. At this time, the medium was replaced with DMEM-no serum and the cells were serum starved for about five hours at 37 °C . Cells were then stimulated with LP8 and/or LP8 short for 10 min. at room temperature in DMEM-no serum. After stimulation, the ligand containing medium was aspirated and about 100 μl of lysis buffer was added to the cells. (Lysis Buffer is 50 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, ImM EDTA, ImM pervanadate . ) After lysis, about 15 μl of the lysate was analyzed by SDS-PAGE gel. After electrophoresis the samples were transferred to a membrane and Western blot analysis was performed using phospho-MAP Kinase antibodies (New England Biolabs (NEB); see Figure 4). Alternatively, lysates were first immunoprecipitated using anti-PDGFRβ antibodies (NEB) and then subjected to Western blot analysis using phosphotyrosine antibodies (NEB) ; see Figure 5). Generally, PDGFRβ activation leads to tyrosine phosphorylation of several intracellular substrates including but not limited to PLC-γ and SHP-2 and eventual activation of MAP kinases (ERK1 and ERK2 ) . Despite the absence of any detectable physical interaction between LP8 and PDGFRβ, LP8 stimulation of cells mimics the effects of the PDGFRβ receptor-mediated signaling pathway whereby PDGFRβ becomes tyrosine phosphorylated and MapK is activated (Figure 5) . LP8 also activates Gsk3 and Akt (data not shown) which further suggests that LP8 may activate the PI3 kinase pathway. Furthermore, treatment of cells with a specific inhibitor of PDGFR (see Figure 5) or down- regulating PDGFRβ by chronic treatment of cells with PDGF-BB (data not shown) completely blocks PDGF-BB as well as LP8 mediated responses .

EXAMPLE 12 LP8 Stimulates Rat Metatarsal Bone Growth In Vitro Newborn Sprague Dawley rats (Harlan, Indianapolis, IN) were sacrificed at day 0 and the metatarsals surgically removed and placed in BGJ medium (Life Technologies, Rockville, MD) without serum and containing an antibiotic- antimycotic solution. The metatarsals were cultured for 7 days in the presence of vehicle, PDGF (100 ng/ml +/- Genistein 100 μM) , or LP8 (10 ng/ml and 100 ng/ml +/- Genistein 100 μM) , in a 96 well round bottom petri dish under 5% C0₂ at 37° C. Genistein is a boad based tyrosine kinase inhibitor. The medium was changed every 24 hours during the 7 day treatment period. Metatarsals were imaged under a light microscope and changes in mineralization quantified using Image Pro™ analysis software package.

Anabolic activity was quantified over the 7-day period as the increase in endochondral ossification measured as the longitudinal extension of the mineralized region (see Figures 6 and 7) . Results are expressed as percent change relative to the vehicle control.

EXAMPLE 13 Use of LP8 to Treat Sarcopenia The efficacy of LP8 in treating sarcopenia is evaluated in elderly males between the ages of 55 - 100.

Test subjects are monitored at regular intervals for muscle mass, muscle weakness and increased fatigability, as described, for example, in Fiatarone et al . , JAMA, 263:3029-3034, (1990), herein incorporated by reference. Medical histories including interviews with family members are taken. Based on the clinical tests, physical examinations, and medical histories subjects are identified as having sarcopenia for the test group. Once per day treatments with a LP8 protein are administered as an bolus of 2.5 mg/kg by any suitable route of administration.

Treatment with the LP8 protein is continued for from four days to at least four week. Test subjects are monitored throughout the test period for changes in muscle mass, muscle weakness and fatigability.

Claims

WE CLAIM :

1. A method for promoting bone growth in a mammal by administration of a therapeutically effective amount of LP8 protein, a fragment thereof, or an analog thereof.

2. A method of treatment for bone fractures comprising the administration of an effective amount of LP8, a fragment thereof, or an analog thereof to the fracture site.

3. A method of prophylactically increasing or maintaining bone density in a subject having a substantially normal bone density comprising the step of administering an amount of LP8, a fragment thereof, or an analog thereof to said subject effective to stimulate osteoblast proliferation.

4. A method for treating osteoporosis by administration of a therapeutically effective amount of LP8, or a fragment thereof, or analog thereof.

5. A method for treating arthritis by administration of a therapeutically effective amount of an LP8, or a fragment thereof, or analog thereof.

6. A method for treating sarcopenia by administration of an effective amount of LP8 , or a fragment thereof, or analog thereof.

7. A method for treating periodontal disease by administration of an effective amount of LP8, or a fragment thereof, or analog thereof.

8. A method for preventing cartilage degradation or promoting cartilage differentiation and function by administration of an effective amount of LP8, or a fragment thereof, or analog thereof.

9. A method for wound healing by administration of an effective amount of LP8, or a fragment thereof, or analog thereof .

10. An LP8 C-terminal fragment having biological activity.

11. An LP8 fragment as in Claim 7 comprising residues 233 through 345 of SEQ ID NO: 2.

12. A pharmaceutical compound comprising an LP8 fragment of Claims 10 and 11 and one or more pharmaceutically acceptable diluents, carriers, or excipients .