METHODS FOR MODULATING CELL FUSION INVENTOR: Agnes Vignery RELATED APPLICATIONS This application is related to U. S. provisional patent application Serial No.

60/105,387 filed October 23,1998 which is herein incorporated by reference in its entirety.

INVENTIONS MADE UNDER FEDERAL SPONSORED RESEARCH This invention was partially made with government support under National Institutes of Health Grants No. RO1-AR35004, DE12110 and AR08395.

FIELD OF THE INVENTION The invention relates generally to cell surface proteins whose expression is inducible, transient and specific for the induction of cell fusion. The invention relates specifically to methods that alter fusion by modulating the expression or function of these proteins.

BACKGROUND OF THE INVENTION Mononuclear phagocytes are omnipresent mononucleated cells which have the potential, in specific instances, to fuse and become osteoclasts or multinucleated giant cells. These cells are primarily associated with bone resorption and chronic inflammatory reactions, respectively. It is considered reasonable to assume that macrophage multinucleation results from a fusion event similar to that occurring in virus-cell, myoblast-myoblast and sperm-oocyte fusion. While the molecular events involved in macrophage fusion remain enigmatic, the understanding of the mechanisms by which viruses fuse with host cells in order to introduce their nucleic acids has made some recent progress (White, 1990, Annu. Rev. Physiol. 52: 675-697). It is now well established that the binding and fusion of viruses with host cells is mediated by viral proteins which use

cell surface molecules as viral receptors. Thus, virus and host cell plasma membrane binding is mediated by a"receptor-ligand"type of interaction. For instance, the human immunodeficiency virus (HIV), which causes AIDS, binds CD4 molecules on T lymphocytes and macrophages (Dalgleish et al., 1984, Nature 312: 763-767; Klatzman et al., 1984, Nature 312: 767-768). It is believed that viral fusion proteins act by virtue of a stretch of hydrophobic amino acids known as a fusion peptide.

Antibodies with anti-fusion activity directed against myoblasts (Wakelam, 1989, Curr. Top. Membr. Transp. 32: 87-112) and sperm cells (Yanagimachi, 1989, Curr. Top.

Membr. Transp. 32: 3-43) have been generated. These antibodies recognize surface proteins that may mediate the actual adhesion-fusion process, similar to viral fusion proteins. Indeed, the antigen recognized by the anti-sperm cell antibody has been shown to contain a putative fusion peptide and an integrin ligand domain (Blobel et al., 1992, Nature 356: 248-252; Wolfsberg et al., 1993, Proc. Natl. Acad. Sci. USA 90: 10783- 10787). This is the first indication that mammalian cell fusion may be mediated, like virus-cell fusion, by cell surface proteins which have the capacity to act in concert as a ligand and a fusion molecule.

Because of the numerous similarities between osteoclasts and giant cells (including their destructive capabilities) and because the molecular mechanisms controlling macrophage fusion remain elusive, there is a need to investigate the mechanism by which macrophages fuse with one another to become multinucleated.

SUMMARY OF THE INVENTION The invention comprises a method of modulating cell fusion, comprising the step of administering to the cell an agent that modulates the expression of Macrophage Fusion Receptor (MFR) or CD44. This method encompasses both up-regulation and down- regulation of these proteins.

The invention further comprises a method for inhibiting cell fusion, comprising the step of contacting a cell with an agent that blocks the function of MFR or CD44 on the cell by selectively binding to MFR or CD44. Such agents include soluble MFR or fragments thereof, soluble SHP-1 protein or fragments thereof, anti-MFR antibodies, soluble CD44 or fragments thereof, anti-CD44 antibodies, hyaluronic acid, chondroitin sulfate A,

chondroitin sulfate B, and osteopontin.

Another embodiment of the invention includes methods wherein the agent reduces the severity of a pathological state mediated by cell. Such pathological states may be characterized by alterations in at least one physiological process such as giant cell formation, macrophage multinucleation, osteoclast formation, myoblast fusion and sperm- oocyte fusion. These methods may be used to produce alterations in bone resorption, muscle development and fertilization and may be used to treat pathological states such as osteosclerosis, osteoporosis, osteopetrosis, pyknodysostosis, Paget's disease, myoblastoma, myosarcoma, sarcoidosis, viral infection, giant cell myocarditis, autoimmune responses associated with surgical transplants and implants, and infertility.

The invention further comprises methods of screening for agents which modulate MFR or CD44 comprising the steps of incubating cells under fusogenic conditions in the presence of an agent; and determining whether MFR or CD44 is up-regulated or down-regulated in the presence or absence of the agent, wherein up-regulation or down-regulation of MFR or CD44 identifies the agent as a modulator.

Further embodiments of the invention include methods of identifying an agent that modulates MFR or CD44 mediated cell fusion, comprising contacting a cell which expresses MFR or CD44 with the agent in the presence of a labeled MFR or CD44 ligand which modulates cell fusion; and determining whether the agent modulates the interactions of the labeled ligand with MFR or CD44, thereby determining whether the agent modulates cell fusion. MFR or CD44 ligands which may be labeled include anti-MFR antibodies, SHP-1, anti-CD44 antibodies, hyaluronic acid, chondroitin sulfate A, chondroitin sulfate B, and osteopontin. The invention also includes pharmaceutical compositions comprising the agents identified in the above mentioned methods.

BRIEF DESCRIPTION OF THE DRAWINGS Figure1 Recognition of recombinant MFR by monoclonal antibody 10C4 in COS-7 cells transiently transfected with MFR and endogenously expressed MFR in fusogenic peritoneal macrophages.

Figure 2 Northern blot analysis of MFR. Each lane contained approximately 2 ug of total RNA from non-fusing (NF) and fusing (F) peritoneal (P) or alveolar macrophages

(A) or mRNA from the indicated tissue.

Figure 3 Kinetics of MFR expression on macrophages induced to fuse in vitro.

MFR, CD4 and MHCII cell surface expression was determined by ELISA on alveolar macrophages cultured under fusogenic conditions.

Figure 4 (A) Soluble MFR (extracellular domain) blocks macrophage fusion in vitro by binding to macrophages. Cells were cultured for four days in the presence of soluble MFR (GST-MFRe), glycosylated MFR (D-myc-his-MFRe) or GST alone (GST) before being fixed and examined for multinucleation under 5x magnification. (B) Specific binding of soluble MFR to macrophages. (C) Specific binding of soluble CD4 (GST- CD4e) to macrophages. (D) Specific binding of soluble, glcosylated MFR to macrophages.

Figure 5 (A) Kinetics of CD44 expression on macrophages induced to fuse in vitro.

CD44, CD4 and MHCII cell surface expression was determined by ELISA on alveolar macrophages cultured under fusogenic conditions. (B) Northern blot analysis of CD44.

Each lane contained approximately 2 gg of total RNA from non-fusing (NF) and fusing (F) peritoneal (Po) or alveolar macrophages (As).

Figure 6 CD44 ligands inhibit macrophage fusion in vitro. Cells were cultured for four days in the presence of increasing concentrations of hyaluronic acid (HA), chondroitin sulfate A (CSA) or osteopontin (OP) before being fixed and examined for multinucleation under 5x magnification.

Figure 7 Hyaluronic acid decreases CD44 cell surface expression. Alveolar macrophages were cultured under fusogenic conditions in the absence or presence of bovine serum albumin, chondroitin sulfate A, chondroitin sulfate B, hyaluronic acid or osteopontin (1 mg/ml) for three days followed by determination of CD44 expression by ELISA.

Figure 8 Recombinant, Recombinant, soluble inhibits macrophage macrophage fusion. Alveolar macrophages were cultured under fusogenic conditions in the absence or presence of the extracellular domain of CD44 (GST-CD44e) or calreticulin (GST-Cal) for three days. (B) Specific binding of soluble CD44 or calreticulin to macrophages.

Figure 9 (A) High expression of CD44 on fusing macrophages in vivo. Frozen sections from bone implants were incubated with either monoclonal anti-CD44 antibody

or mouse IgGl (B) followed by goat anti-mouse IgG F (ab') 2 fragments conjugated to Cy3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT I. General Description The present invention is based in part on genes that are expressed in macrophages and transiently induced at the onset of fusion. The genes encode proteins known as Macrophage Fusion Receptor (MFR) and CD44 (CD44) predicted to consist of 509 and 364 amino acids, respectively. These proteins can serve as targets for agents that can be used to modulate the expression or activity of the proteins. For example, agents may be identified which modulate physiological processes associated with bone resorption, muscle development and fertilization. Pathological states associated with alterations in these physiological processes may be treated by altering cell fusion through modulation of MFR or CD44.

Additionally, these proteins provide a novel target for screening of synthetic small molecules and combinatorial or naturally occurring compound libraries to discover novel therapeutics to modulate fusion between cells.

II. Specific Embodiments A. Methods for Modulation of Protein Expression or Function The identification of the roles of MFR and CD44 in cell fusion has led to the discovery of methods to identify compounds that are capable of modulating the expression of these proteins. Molecules that up-or down-regulate MFR or CD44 are therefore part of the invention. Modulation is defined here as either an increase or decrease, in activation, function or synthesis of MFR or CD44, including an increase or decrease in the activity, function or synthesis, of their ligands or activators. Modulation is further defined as the inhibition, induction, agonism, antagonism, up-regulation or down-regulation of the expression or function of an MFR or CD44 gene or gene product.

Modulation further includes an increase or decrease in the degradation of the MFR or CD44 mRNA in a cell, their protein products, ligands or activators. Modulation may therefore be achieved in a number of ways. For example, by administration of molecules that can either stabilize or destabilize the binding of MFR and CD44 with their ligands.

SHP-1, a protein-tyrosine phosphatase with two Src homology 2 (SH2) domains, may interact and modulate MFR because MFR has several tyrosine phosphorylation sites.

Such molecules therefore encompass polypeptide products, including those encoded by the DNA sequences of the MFR or CD44 gene or DNA sequences containing various mutations. These mutations may be point mutations, insertions, deletions or spliced variants of the MFR or CD44 gene. This invention also includes truncated polypeptides encoded by the DNA molecules described above. These polypeptides being capable of interfering with interaction of MFR or CD44 with other proteins.

A further embodiment of this invention includes the modulation of MFR or CD44 function by altering expression of the MFR or CD44 gene, the use of antisense gene therapy being an example. Down-regulation of MFR or CD44 expression may be accomplished by administering an effective amount of at least one antisense oligonucleotide. These antisense molecules can be fashioned from the DNA sequence of the MFR or CD44 gene or sequences containing various mutations, deletions, insertions or spliced variants. Another embodiment of this invention relates to the use of isolated RNA or DNA sequences derived from the MFR or CD44 gene that encode functional portions of the gene, for instance, regions responsible for cell fusion. These sequences may contain various mutations such as point mutations, insertions, deletions or spliced variant mutations of MFR or CD44 gene and can be useful in gene therapy.

Molecules that increase or decrease the degradation of MFR or CD44 may also be used to modulate their functions, and are within the scope of the invention.

Phosphorylation of MFR or CD44 may alter protein stability, therefore kinase inhibitors may be used to up-regulate or down-regulate its function. For example, tyrosine kinase inhibitors which inhibit phosphorylation of tyrosine at the immunoreceptor tyrosine-based inhibition motif (ITIM) domains (amino acids 435-505 of SEQ ID NO: 2) may be used to modulate MFR.

Modulation of MFR or CD44 may also be accomplished by the use of polyclonal or monoclonal antibodies or fragments thereof directed against the MFR or CD44. Such molecules are within the claimed invention. For example, monoclonal antibody 10C4 which binds to MFR can be used to modulate the function of MFR.

This invention further includes small molecules with the three-dimensional

structure necessary to bind with sufficient affinity to block MFR or CD44 interactions with other proteins. MFR or CD44 blockade resulting in decreased cell fusion make these small molecules useful as therapeutic agents in pathological states associated with altered cell fusion. For example, blockade of CD44 by hyaluronic acid may be used to block macrophage fusion associated with osteoclast formation and thus be used to treat a pathological state such as osteoporosis.

The agents discussed above represent various effective therapeutic compounds in treating pathological states associated with altered cell fusion. Applicants have thus provided agonists and antagonists and methods of identifying agonists and antagonists that are capable of modulating MFR or CD44.

As described above, an embodiment of the invention relates to antisense or gene therapy. It is now known in the art that altered nucleic acid or DNA molecules can be tailored to provide a specific selected effect, when provided as antisense or gene therapy.

The native DNA segment coding for MFR and CD44 has, as do all other mammalian DNA strands, two strands; a sense strand and an antisense strand held together by hydrogen bonds. The mRNA coding for MFR and CD44 has a nucleotide sequence identical to the sense strand, with the expected substitution of thymidine by uridine. Thus, based upon the knowledge of the MFR and CD44 sequences, synthetic oligonucleotides can be synthesized. These oligonucleotides can bind to the DNA and RNA coding for MFR or CD44. The active fragments of the invention, which are complementary to mRNA and the coding strand of DNA, are usually at least about fifteen nucleotides, more usually at least twenty nucleotides, preferably thirty nucleotides and more preferably may be fifty nucleotides or more. There is no upper limit, other than a practical limit, on the maximal size of such a nucleic acid molecule in that the nucleic acid molecule can include a portion of a gene, an entire gene, or multiple genes, or portions thereof. The binding strength between the sense and antisense strands is dependent upon the total hydrogen bonds.

Therefore, based upon the total number of bases in the mRNA, the optimal length of the oligonucleotide sequence may be easily calculated by the skilled artisan. The sequence may be complementary to any portion of the sequence of the mRNA. For example, it may be proximal to the 5'-terminus or capping site or downstream from the capping site, between the capping site and the initiation codon and may cover all or only a portion of

the non-coding region or the coding region. The particular site (s) to which the antisense sequence binds will vary depending upon the degree of inhibition desired, the uniqueness of the sequence, the stability of the antisense sequence, etc.

In the practice of the invention, expression of MFR or CD44 may be down-regulated by administering an effective amount of the synthetic antisense oligonucleotide sequences described above. The oligonucleotide compounds of the invention bind to the mRNA coding for human MFR or CD44 thereby inhibiting expression (translation) of these proteins. The isolated DNA sequences containing various mutations such as point mutations, insertions, deletions or spliced mutations of MFR or CD44 are useful in gene therapy as well. In another embodiment, cell fusion may be up- regulated by selective expression of MFR or CD44 via conventional gene therapy methods.

Antisense oligonucleotides can also be used as tools in vitro to further determine the biological function of genes and proteins. Oligonucleotide phosphorothioates (PS-oligos) have also shown great therapeutic potential as antisense-mediated inhibitors of gene expression. Various methods have been developed for the synthesis of antisense oligonucleotides. See Agrawal et al., 1993 (Methods of Molecular Biology: Protocols for Oligonucleotides and Analogs, Humana Press) and Eckstein et al., 1991 (Oligonucleotides and Analogues: A Practical Approach, Oxford University Press).

B. Methods to Identify Agents that Modulate Expression of MFR or CD44.

Another embodiment of the present invention provides methods for identifying agents that modulate the expression of a nucleic acid encoding a protein of the invention such as MFR or CD44. Examples of MFR and CD44 are embodied in SEQ ID NO: 2 and SEQ ID NO: 4, respectively, although any MFR or CD44 molecules may be used. Such molecules may include naturally occurring variants of SEQ ID NO: 2 or SEQ ID NO: 4 as well as engineered variants that comprise, for instance, conservative substitutions, deletions or insertions. When appropriate, all variants, regardless of source, will retain the ability to facilitate cell fusion (see Saginario et al., 1998, Mol. Cell. Biol. 18: 6213-6223; Sterling et al., 1998, J. Cell Biol. 143: 1-11 for various fusion assays, herein incorporated by reference in their entirety). Such assays may utilize any available means of monitoring

for changes in the expression level of the nucleic acids of the invention. As used herein, an agent is said to modulate the expression of a nucleic acid of the invention, for instance a nucleic acid encoding the protein having the sequence of MFR or CD44, if it is capable of up-or down-regulating expression of the nucleic acid in a cell.

In one assay format, cell lines that contain reporter gene fusions between the open reading frame defined by nucleotides 234-1763 of SEQ ID NO: 1 (see Fujioka et al., 1996, Mol. Cell. Biol. 16: 6887-6899 herein incorporated by reference in its entirety) or nucleotides 176-1204 of SEQ ID NO: 3 and any assayable fusion partner may be prepared.

Numerous assayable fusion partners are known and readily available including the firefly luciferase gene and the gene encoding chloramphenicol acetyltransferase (Alam et al., 1990, Anal. Biochem. 188: 245-254). Cell lines containing the reporter gene fusions are then exposed to the agent to be tested under appropriate conditions and time. Differential expression of the reporter gene between samples exposed to the agent and control samples identifies agents which modulate the expression of a nucleic acid encoding the MFR or CD44.

Additional assay formats may be used to monitor the ability of the agent to modulate the expression of a nucleic acid encoding a protein of the invention such as MFR or CD44. For instance, mRNA expression may be monitored directly by hybridization to the nucleic acids of the invention. Cell lines are exposed to the agent to be tested under appropriate conditions and time and total RNA or mRNA is isolated by standard procedures such those disclosed in Sambrook et al., 1985 (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press).

Probes to detect differences in RNA expression levels between cells exposed to the agent and control cells may be prepared from the nucleic acids of the invention. It is preferable, but not necessary, to design probes which hybridize only with target nucleic acids under conditions of high stringency. Only highly complementary nucleic acid hybrids form under conditions of high stringency. Accordingly, the stringency of the assay conditions determines the amount of complementarily which should exist between two nucleic acid strands in order to form a hybrid. Stringency should be chosen to maximize the difference in stability between the probe-target hybrid and potential probe- non-target hybrids.

Probes may be designed from the nucleic acids of the invention through methods known in the art. For instance, the G+C content of the probe and the probe length can affect probe binding to its target sequence. Methods to optimize probe specificity are commonly available in Sambrook et al., 1985 (Molecular Cloning: A Laboratory Manual.

Cold Spring Harbor, NY. Cold Spring Harbor Laboratory Press); or Ausubel et al., 1995 (Current Protocols in Molecular Biology, Greene Publishing Company).

Hybridization conditions are modified using known methods, such as those described by Sambrook et al., 1985 and Ausubel et al., 1995 as required for each probe.

Hybridization of total cellular RNA or RNA enriched for polyA RNA can be accomplished in any available format. For instance, total cellular RNA or RNA enriched for polyA RNA can be affixed to a solid support and the solid support exposed to at least one probe comprising at least one, or part of one of the sequences of the invention under conditions in which the probe will specifically hybridize. Alternatively, nucleic acid fragments comprising at least one, or part of one of the sequences of the invention can be affixed to a solid support, such as a porous glass or solid surface silicon wafer. The wafer can then be exposed to total cellular RNA or polyA RNA from a sample under conditions in which the affixed sequences will specifically hybridize. Such glass wafers and hybridization methods are widely available, for example, those disclosed by Beattie, 1995 (W09511755). By examining for the ability of a given probe to specifically hybridize to an RNA sample from an untreated cell population and from a cell population exposed to the agent, agents which up or down regulate the expression of a nucleic acid encoding MFR or CD44 are identified.

Hybridization for qualitative and quantitative analysis ofmRNA may also be carried out by using a RNase Protection Assay (i. e., RPA, see Ma et al. 1996, Methods 10: 273-238). Briefly, an expression vehicle comprising cDNA encoding the gene product and a phage specific DNA dependent RNA polymerase promoter (e. g., T7, T3 or SP6 RNA polymerase) is linearized at the 3'end of the cDNA molecule, downstream from the phage promoter, wherein such a linearized molecule is subsequently used as a template for synthesis of a labeled antisense transcript of the cDNA by in vitro transcription. The labeled transcript is then hybridized to a mixture of isolated RNA (i. e., total or fractionated mRNA) by incubation at 45°C overnight in a buffer comprising 80% formamide, 40 mM

PIPES, pH 6.4,0.4 M NaCI and 1 mM EDTA. The resulting hybrids are then digested in a buffer comprising 40 Rg/ml ribonuclease A and 2 ug/ml ribonuclease. After deactivation and extraction of extraneous proteins, the samples are loaded onto urea/polyacrylamide gels for analysis.

In another assay format, agents which effect the expression of the instant gene products, cells or cell lines would first be identified which express said gene products physiologically (e. g., see for example, Figure 2 for tissue distribution via Northern blot, however, RPA may serve the identical purpose of expression selection). Cell and/or cell lines so identified would be expected to comprise the necessary cellular machinery such that the fidelity of modulation of the transcriptional apparatus is maintained with regard to exogenous contact of agent with appropriate surface transduction mechanisms and/or the cytosolic cascades. Further, such cells or cell lines would be transduced or transfected with an expression vehicle (e. g., a plasmid or viral vector) construct comprising an operable non-translated 5'-promoter containing end of the structural gene encoding the instant gene products fused to one or more antigenic fragments, which are peculiar to the instant gene products, wherein said fragments are under the transcriptional control of said promoter and are expressed as polypeptides whose molecular weight can be distinguished from the naturally occurring polypeptides or may further comprise an immunologically distinct tag. Such a process is well known in the art (see Maniatis et al., 1992, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press).

Cells or cell lines transduced or transfected as outlined above would then be contacted with agents under appropriate conditions; for example, the agent in a pharmaceutically acceptable excipient contacted with cells in an aqueous physiological buffer such as phosphate buffered saline (PBS) at physiological pH, Eagles balanced salt solution (BSS) at physiological pH, PBS or BSS comprising serum or conditioned media comprising PBS or BSS or serum incubated at 37°C. Said conditions may be modulated as deemed necessary by one of skill in the art. Subsequent to contacting the cells with the agent, said cells will be disrupted and the polypeptides from disrupted cells are fractionated such that a polypeptide fraction is pooled and contacted with an antibody to be further processed by immunological assay (e. g., ELISA, immunoprecipitation or Western blot). The pool of proteins isolated from the"agent contacted"sample will be

compared with a control sample where only the excipient is contacted with the cells and an increase or decrease in the immunologically generated signal from the"agent contacted" sample compared to the control will be used to distinguish the effectiveness of the agent.

In order to assay MFR or CD44 expression of the present invention in a physiologically relevant manner, cells may be assayed under conditions which model cell fusion. For example, some model systems include incubation of cells under fusogenic conditions (Vignery et al., 1990, J. Bone Min. Res. 5: 637-644).

The agents identified by the above methods can be, as examples, peptides, small molecules, vitamin derivatives, as well as carbohydrates. A skilled artisan readily recognizes that there is no limit as to the structural nature of the agents.

C. Methods to Identify Agents that Modulate at least one Activity of MFR or CD44.

Another embodiment of the present invention provides methods for identifying agents that modulate at least one activity of MFR or CD44. Such methods or assays may utilize any means of monitoring or detecting the desired activity.

In one format, the relative amounts of a protein of the invention between a cell population that has been exposed to the agent to be tested compared to an unexposed control cell population may be assayed. In this format, probes such as specific antibodies are used to monitor the differential expression of the protein in the different cell populations. Cell lines or populations are exposed to the agent to be tested under appropriate conditions and time. Cellular lysates may be prepared from the exposed cell line or population and a control, unexposed cell line or population. The cellular lysates are then analyzed with the probe.

In another assay format, the agent to be tested may be screened for the ability to bind or interact with MFR or CD44. For examples, see Kenakin, 1987 (Pharmacological analysis of drug receptor interaction, Raven Press). Such assays include direct binding assays or indirect assays such as competitive binding assays. For instance, a method of identifying an agent that modulates CD44 mediated cell fusion may be used which comprises contacting a cell which expresses CD44 with an agent in the presence of labeled hyaluronic acid and determining whether the agent modulates the interactions of the labeled hyaluronic acid with CD44, thereby determining whether the agent modulates cell

fusion.

Antibody probes used in the described methods may be prepared by immunizing suitable mammalian hosts in appropriate immunization protocols using the peptides, polypeptides or proteins of the invention if they are of sufficient length, or if desired, or if required to enhance immunogenicity, conjugated to suitable carriers. Methods for preparing immunogenic conjugates with carriers such as BSA, KLH, or other carrier proteins are well known in the art. In some circumstances, direct conjugation using, for example, carbodiimide reagents may be effective; in other instances linking reagents such as those supplied by Pierce Chemical Company, may be desirable to provide accessibility to the hapten. The hapten peptides can be extended at either the amino or carboxy terminus with a cysteine residue or interspersed with cysteine residues, for example, to facilitate linking to a carrier. Administration of the immunogens is conducted generally by injection over a suitable time period and with use of suitable adjuvants, as is generally understood in the art. During the immunization schedule, titers of antibodies are taken to determine adequacy of antibody formation.

While the polyclonal antisera produced in this way may be satisfactory for some applications, for pharmaceutical compositions, use of monoclonal preparations is preferred. Immortalized cell lines which secrete the desired monoclonal antibodies may be prepared using the standard method of Kohler and Milstein, 1975 (Nature 256: 495-497) or modifications which effect immortalization of lymphocytes or spleen cells, as is generally known. The immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigen is the peptide hapten, polypeptide or protein. When the appropriate immortalized cell culture secreting the desired antibody is identified, the cells can be cultured either in vitro or by production in ascites fluid.

The desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant. Fragments of the monoclonals or the polyclonal antisera which contain the immunologically significant portion can be used as antagonists, as well as the intact antibodies. Use of immunologically reactive fragments, such as the Fab, Fab' of F (ab') z fragments is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin.

The antibodies or fragments may also be produced, using current technology, by

recombinant means. Antibody regions that bind specifically to the desired regions of the protein can also be produced in the context of chimeras with multiple species origin, for instance, humanized antibodies.

Agents that are assayed in the above method can be randomly selected or rationally selected or designed. As used herein, an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences involved in the association of the a protein of the invention alone or with its associated substrates, binding partners, etc. An example of randomly selected agents is the use a chemical library or a peptide combinatorial library, or a growth broth of an organism.

As used herein, an agent is said to be rationally selected or designed when the agent is chosen on a non-random basis which takes into account the sequence of the target site and/or its conformation in connection with the agent's action. As described in the Examples, there are immunoglobulin-like domains, putative binding sites for SH2 and SH3 domains, immunoreceptor tyrosine-based inhibition motifs (ITIM), glycosylation and phosphorylation sites in MFR and CD44. Agents can be rationally selected or rationally designed by utilizing the peptide sequences that make up these sites. For example, a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to the extracellular domain of MFR starting at amino acid 28 and ending at amino acid 383 of SEQ ID NO: 2.

The agents of the present invention can be, as examples, peptides, small molecules, vitamin derivatives, as well as carbohydrates. A skilled artisan readily recognizes that there is no limit as to the structural nature of the agents of the present invention.

Dominant negative proteins, DNA encoding these proteins, antibodies to these proteins, peptide fragments of these proteins or mimics of these proteins may be introduced into cells to affect function."Mimic"used herein refers to the modification of a region or several regions of a peptide molecule to provide a structure chemically different from the parent peptide but topographically and functionally similar to the parent peptide. For examples, see Meyers, 1995 (Molecular Biology and Biotechnology, VCH Publishers).

The peptide agents of the invention can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art. In addition, the DNA encoding these peptides may be synthesized using commercially available oligonucleotide

synthesis instrumentation and produced recombinantly using standard recombinant production systems. The production using solid phase peptide synthesis is necessitated if non-gene-encoded amino acids are to be included.

Another class of agents of the present invention are antibodies immunoreactive with critical positions of proteins of the invention. As discussed above, antibody agents are obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of the protein intended to be targeted by the antibodies.

D. Uses for Agents that Modulate at Least One Activity of MFR or CD44.

As provided in the Examples, the proteins and nucleic acids of the invention, such as MFR or CD44, are expressed on macrophages during cell fusion. Agents that modulate, up-regulate or down-regulate the expression of the protein, or agents such as agonists or antagonists which modulate at least one activity of the protein may be used to modulate biological and pathologic processes associated with the protein's function and activity.

As used herein, a subject can be any mammal, so long as the mammal is in need of modulation of a pathological state or biological process mediated by MFR or CD44 cell fusion. The term"mammal"is meant an individual belonging to the class Mammalia.

The invention is particularly useful in the treatment of human subjects.

As used herein, the phrases"pathological state"or"pathological condition"in reference to the expression or function of either MFR or CD44 includes, but is not limited to osteosclerosis, osteoporosis, osteopetrosis, pyknodysostosis, Paget's disease, myoblastoma, myosarcoma, sarcoidosis, viral infection, giant cell myocarditis, autoimmune responses associated with surgical transplants and implants, and infertility.

The pathological state can be characterized by alterations in any of the following physiological processes: giant cell formation, macrophage multinucleation, osteoclast formation, myoblast fusion, and sperm-oocyte fusion.

Pathological state refers to a category of physiological processes which produce a deleterious effect. For example, expression of a protein of the invention may be associated with osteoclast formation from multinucleated macrophages and subsequent degradation of bone. As used herein, an agent is said to modulate a pathological process when the

agent reduces the degree or severity of the process. For instance, Paget's disease (Osteitis Deformans) may be prevented or disease progression modulated by the administration of agents which down-regulate or modulate in some way the expression or at least one activity of either MFR or CD44.

As used herein, an agent is said to modulate a pathological process when the agent reduces the degree or severity of the process. For example, an agent is said to modulate osteoporosis when the agent reduces the formation of osteoclasts.

E. Methods of Treating Pathological Conditions.

The agents of the present invention can be provided alone, or in combination with other agents that modulate a particular pathological process. For example, an agent of the present invention can be administered alone or in combination with other known drugs, to facilitate the treatment of myoblastoma, myosarcoma and granuloma. Furthermore, the present invention can be used to treat any pathological state associated with giant multinucleated cells such as viral infections, sarcoidosis, or autoimmune responses associated with surgical transplants or implants such as synthetic prosthesis. Examples of pathological states include, but are not limited to, autoimmune responses such as giant cell myocarditis associated with cardiac transplants or viral infections such as the measles virus (Morbillovirus). As used herein, two agents are said to be administered in combination when the two agents are administered simultaneously or are administered independently in a fashion such that the agents will act at the same time.

The agents of the present invention can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes.

Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

The present invention further provides compositions containing one or more agents which modulate expression or at least one activity of a protein of the invention. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. Typical dosages comprise 0.1 to 100 pg/kg body weight. The preferred dosages comprise 0.1 to 10, ug/kg body weight. The most preferred

dosages comprise 0.1 to 1 gg/kg body weight.

In addition to the pharmacologically active agent, the compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action. Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts. In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.

Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent for delivery into the cell.

The pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient.

Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.

In practicing the methods of this invention, the compounds of this invention may be used alone or in combination, or in combination with other therapeutic or diagnostic agents. In certain preferred embodiments, the compounds of this invention may be co- administered along with other compounds typically prescribed for these conditions according to generally accepted medical practice. The compounds of this invention can be utilized in vivo, ordinarily in mammals, such as humans, sheep, horses, cattle, pigs, dogs, cats, rats and mice, or in vitro.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following

working examples therefore, specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

EXAMPLES Example 1: Detection of MFR on Macrophages with Anti-MFR Antibodies.

Rat alveolar and peritoneal macrophages were obtained from twelve-week old Fisher rats from Charlers River (Kingston, NY) by tracheobronchial and peritoneal lavage.

Cells were cultured in fusogenic conditions in MEME supplemented with 5% human serum as previously described (Vignery, 1989, Am. J. Pathol. 135: 565-570). COS-7 cells were transiently transfected with either an expression vector encoding the MFR protein (pK-RSV-MFR) or not at all as previously described (Saginario et al., 1998, Mol. Cell.

Biol. 18: 6213-6223). The recombinant protein monoclonal antibody 10C4 has been previously described (Saginario et al., 1995, Proc. Natl. Acad. Sci. USA 92: 12210-12214).

Rat alveolar macrophages, peritoneal macrophages and COS-7 cells were collected, plated in six-well plastic dishes at 1 x 10'cells per milliliter, cultured for the indicated times, metabolically labeled with 35S-methionine and subjected to immunoprecipitation with protein A-sepharose-lOC4-conjugated beads as previously described (Saginario et al., 1995, Proc. Natl. Acad. Sci. USA 92: 12210-12214). Peritoneal macrophage lysates were incubated with protein A-sepharose beads as a control. The immunoprecipitates were analyzed by SDS-PAGE and autoradiography.

Monoclonal antibody 10C4 precipitated from radiolabeled COS-7 cells transiently transfected with pK-RSV-MFR a protein of 120 kDa (Figure 1), confirming that monoclonal 10C4 recognizes MFR. In addition, this antibody precipitated from radiolabeled, fusogenic peritoneal macrophages a protein of 120 kDa (Figure 1) which was also identified as MFR.

Example 2: Expression of MFR mRNA in Fusogenic and Non-fusogenic Macrophages.

A multiple-tissue Northern blot was purchased from Clontech (Palo Alto, CA).

Each lane contained approximately 2 ug of tissue-derived polyA mRNA. The blot was

hybridized with a 32P-labeled PCR-generated DNA probe according to the manufacturer's instructions. Alveolar and peritoneal macrophages were isolated, and part of the cells were cultured in a fusogenic milieu for seventy-two hours. Total RNA was isolated by the guanidinium isothiocyanate-cesium chloride method (Glover, 1985, DNA Cloning: A Practical Approach, IRL Press); 8 pg was electrophoretically separated in formaldehyde- agarose gels, blotted onto a nylon membrane and hybridized with a 32P-labeled PCR- generated DNA probe corresponding to the full-length MFR cDNA. Signals on the autoradiogram were determined by using a Linotype-Hell scanner with Multi-Analyst software (Bio-Rad Laboratories, Hercules, CA).

Northern blot analysis revealed a 4.2 kilobase MFR transcript in both freshly isolated and fusing alveolar and peritoneal macrophages (Figure 2). MFR transcripts were, however, more abundant in alveolar than peritoneal cells. MFR transcripts were detected in all tissues examined but at low abundance.

Example 3: Transient Induction of MFR Expression at the Onset of Fusion.

To determined the levels of MFR, CD4 and MHCII cell surface expression by ELISA, 5 x 104 alveolar macrophages were plated and fixed at room temperature in 4% paraformaldehyde for ten minutes and then incubated in 100 gl of PBS supplemented with 5% dry milk for two hours. The cells were subsequently reacted overnight with IgGl, anti-rat MFR, anti-rat CD4 or anti-rat MHCII (10-50 ug/ml). Following three PBS washes for ten minutes each, the cells were incubated at room temperature for two hours with goat anti-mouse IgG-HRP conjugate. The cells were again washed three times. Surface MFR expression was determined by incubating the cells for five minutes in 100 gl of HRP substrate (Moss Inc., Pasadena, MD). Optical density at 650 nm was measured with a ELISA plate reader (Corning).

To investigate the kinetics of MFR expression, levels of cell surface MFR and of two other macrophage surface proteins were determined by ELISA as a function of time in culture. MFR expression peaked within two days after plating the cells with decreasing and then leveling off thereafter (Figure 3). The peak level of MFR expression was approximately two and a half times greater than that of either CD4 or MHCII, as well as that of MFR sixty minutes after plating. Neither CD4 nor MHCII expression was altered

by fusion.

Example 4: Soluble MFR Blocks Macrophage Fusion.

Soluble MFR (MFRe, amino acids 28-383 of SEQ ID NO: 2) was expressed using two different systems. First, MFRe was expressed as a GST fusion protein using pGEX-4T (Pharmacia Biotech). PCR amplification of MFRe was performed with a sense primer (nucleotides 114-150) that lacked the leader sequence and an antisense primer (nucleotides 1145-1181) that did not include the transmembrane domain of MFR using full-length MFR cDNA as the template. Both primers were designed to contain EcoRI sites. The amplified PCR fragment was digested with EcoRI and inserted in frame into the EcoRI site of pGEX-4T-1. This construct was used to transform Escherichia coli strain BL-21.

GST-MFRe was isolated from two liters of bacterial culture using the bulk GST purification modules as recommended by the manufacturer. The eluted protein was extensively dialyzed against PBS. A pGEX-calreticulin construct (GST-Cal) was used as the control.

Second, MFRe was expressed as a fusion protein containing a C-terminal Myc epitope and polyhistidine tag (Invitrogen). PCR amplification was performed with the sense primer which included the leader sequence (nucleotides 5-41) and the antisense primer used above using full length MFR cDNA as a template. The PCR product was digested with EcoRI and inserted into the EcoRI site of pcDNA3.1B (GibcoBRL). The recombinant protein was purified from culture supernatant using the Xpress protein purification system (Invitrogen) according to the manufacturer's instructions and extensively dialyzed against PBS. Approximately 250 pg of recombinant Myc-His-MFRe was deglycosylated in its native form with incubation with 10 units if N-glycosidase F for forty-eight hours at 37°C in 0.1 M sodium phosphate. Deglycosylation was analyzed by comparative Western blot analysis of both native and N-glycosidase F treated Myc-His- MFRe using an anti-Myc antibody (Invitrogen).

For fusion assays using recombinant fusion proteins, freshly isolated rat alveolar macrophages were plated and cultured in fusogenic milieu supplemented with GST- MFRe, GST-CD4e, GST, Myc-His-MFRe or deglycosylated Myc-His-MFRe (D-Myc- His-MFRe) at various concentrations. The cells were examined for four days and fusion

was graded blindly by three individuals on a scale of one (absence of fusion) to five (fusion greater than 90%).

To investigate whether soluble MFRe altered fusion, alveolar macrophages were cultured in the in the absence or presence of either GST alone, GST-CD4e, GST-MFRe, D-Myc-His-MFRe or Myc-His-MFRe and fusion was monitored morphologically and determined (Figure 4). Alveolar macrophages cultured in fusogenic milieu reached 99% fusion within four days. The addition of 50 nM GST-MFRe completely blocked macrophage aggregation and therefore fusion. The addition of 100 nM GST or GST- CD4e had no effect on fusion. Myc-His-MFRe had no effect on fusion while the deglycosylation of Myc-His-MFRe (D-Myc-His-MFRe) appeared to restore its capability to block fusion.

Example 5: Soluble MFR Binds Directly to MFR on the Cell Surface.

For binding assays using recombinant fusion proteins, freshly isolated rat alveolar macrophages were plated in ninety-six well plates overnight in fusogenic milieu supplemented with GST-MFRe, GST-CD4e, GST, Myc-His-MFRe or deglycosylated Myc-His-MFRe (D-Myc-His-MFRe) at various concentrations and times. Binding proceeded overnight at 4°C. Medium was removed and replaced with fresh medium allowing dissociation which proceeded for three hours. The medium was then removed and all wells and the cells were fixed for thirty minutes in 4% paraformaldehyde. Cells were rinsed three times with PBS and blocked for one hour with 5% dry milk in PBS. following three washes in PBS, the cells were incubated with rabbit anti-goat IgG-HRP for thirty minutes. Following three final washes, 100 pl of HRP substrate was added to each well and the optical density at 650 nm was measured on an ELISA plate reader. Specific binding was determined as the difference in average OD values between the two sets of cells (i. e., between total and non-specific binding values for each ligand concentration).

To ensure that GST-MFRe inhibited fusion by virtue of interacting with specific binding sites on the surface of macrophages, cells were cultured for twenty-four hours in a fusogenic milieu and then incubated overnight at 4°C with increasing concentrations of GST-MFRe or GST-CD4e. Both GST-MFRe and GST-CD4e specifically bound to macrophages in a dose-dependent and saturable manner (Figure 4) while GST did not. D-

Myc-His-MFRe appeared to bind to macrophages in a specific and dose-dependent manner while Myc-His-MFRe did not. These results indicate that the core peptide of MFR plays an important role in cellular macrophage interactions.

Example 6: Detection of CD44 on Macrophages with Anti-CD44 Antibodies.

The levels of CD44, CD4 and MHCII cell surface expression were determined by ELISA. Alveolar macrophages per well plated in 96-well dishes were cultured for the indicated times. The minimum culture time after plating in each experiment was one hour in order to secure the adherence of the cells to the wells. Cells were fixed at room temperature in 4% paraformadehyde for ten minutes, then incubated in 100 « u1 of PBS supplemented with 5% dry milk for two hours. The cells were subsequently reacted overnight with IgGl, anti-rat CD44 (10-50 ug/ml), anti-rat CD4 (100 ug/ml) or anti-rat MHCII (10-50 Fg/ml). After three PBS washes, the cells were incubated at room temperature for two hours with goat anti-mouse IgG horseradish peroxidase conjugate.

The cells were again washed with PBS and surface CD44 expression determined by incubating the cells for five minutes in 100 « of HRP substrate (Moss, Pasadena, MD).

Optical density measurements were made using a Molecular Devices kinetic reader (Sunnyvale, CA).

To investigate the kinetics of CD44 expression during induction of fusion, rat alveolar macrophages were plated in fusogenic conditions and CD44 expression was determined by ELISA as a function of time after plating. CD44 expression increased to reach a peak at a time that varied between twenty-four and forty-eight hours and declined thereafter (Figure 5). In some experiments, a dramatic increase was detected as early as five to ten hours after plating. This increase confirmed the morphological observation that CD44 expression was less abundant in multinucleated than mononucleated fusing macrophages. In contrast to CD44, the expression of CD4 and MHCII remained low, and was not altered by fusogenic conditions in macrophages (Figure 5). When subjected to Northern blot analysis, CD44 transcripts were found to be abundant in freshly isolated macrophages and increased by 36% in fusing alveolar macrophages and 13% in fusing peritoneal macrophages (Figure 5). This indicates that the regulation of CD44 mRNA expression in macrophages may be both transcriptional and post-transcriptional.

Example 7: Inhibition of Cell Fusion by CD44 Ligands.

Rat alveolar macrophages were plated in flat bottom 96-well tissue culture dishes that had been treated overnight at 37°C with hyaluronic acid (1 mg/ml) or chondroitin sulfate A or chondroitin sulfate B (1 mg/ml). The cells were cultured for four days in MEM supplemented with different concentrations of hyaluronic acid, chondroitin sulfate A or chondroitin sulfate B and osteopontin. In experiments in which HA was obtained from Pharmacia, hyaluronic acid was added to the cells every day in fresh medium.

Competition binding studies were preformed by supplementing monoclonal antibody anti-CD44 with hyaluronic acid (1 mg/ml), chondroitin sulphate A (1 mg/ml,), chondroitin sulphate B (1 mg/ml) or osteopontin (1 uM). CD44 expression was determined by ELISA, as described above.

To analyze whether CD44 ligands, hyaluronic acid (HA), chondroitin sulfate A (CSA) and osteopontin (OP), all of which are extracellular matrix components, altered multinucleation, alveolar macrophages were cultured in fusogenic conditions in the presence of increasing concentrations of hyaluronic acid, chondroitin sulfate A or osteopontin. Although alveolar macrophages are all fused after four days of culture under fusogenic conditions, the addition of hyaluronic acid, Chondroitin sulfate A or osteopontin prevented multinucleation in a dose-dependent manner (Figure 6). In the presence of lmg/ml hyaluronic acid, and to a lesser degree lmg/ml chondroitin sulfate A and 1 p. M osteopontin, macrophages failed to form giant cells.

Example 8: Down-regulation of CD44 Expression with CD44 ligands.

To determine the mechanism by which hyaluronic acid and to a lesser extent chondroitin sulfate A and osteopontin inhibited macrophage multinucleation, fusing alveolar macrophages were cultured in the presence of chondroitin sulfate A, hyaluronic acid or osteopontin and CD44 expression was determined by ELISA as described above.

Hyaluronic acid significantly reduced the increase in CD44 expression associated with cell fusion in a dose-dependent manner (Figure 7). This reduction was not observed in the presence of either chondroitin sulfate A or osteopontin. BSA, which was used as a negative control, had no effect on CD44 expression.

Example 9: Soluble CD44 Blocks Macrophage Fusion.

The recombinant extracellular domain of CD44 (CD44e, aminon acids 16-267 of SEQ ID NO: 4) was expressed using two different systems. CD44e was expressed as a fusion protein using the GST fusion protein system (Pharmacia Biotech). PCR amplification of this region was performed with a sense primer (nucleotides 158-195) and an antisense primer (nucleotides 878-913), using rat macrophage cDNA as template (derived from total RNA). The primers were designed to allow digestion of the PCR product with EcoRI and ZhoI with subsequent ligation into frame of EcoRI-XhoI cut pGEX-4T-1 vector. The resulting construct encoded a fusion protein of-50 kDa and was used to transform Escherichia coli strain BL-21. Soluble GST-CD44e was isolated from one liter of bacterial culture using the bulk GST purification module as described by the manufacturer. The eluted protein was extensively dialyzed against PBS and stored at -70°C until ready for use.

CD44e was expressed in mammalian cells with Myc-His fusion tag using the mammalian expression vector pcDNA3.1-Myc-His (Invitrogen). PCR amplification of the extracellular region was performed with a sense primer (nucleotides 86-122) an antisense primer (nucleotides 878-913) and rat macrophage cDNA as a template. The PCR product was digested with EcoRI and ligated into the EcoRI site of pcDNA3.1-Myc-His-B. This construct was used to transfect COS-7 cells using lipofectamine (GibcoBRL) according to the manufacturer's instructions. Transfected ells were cultured for seventy-two hours in Opti-MEM reduced serum medium (GibcoBRL). The recombinant protein was purified from culture supernatant using the Xpress protein purification system (Invitrogen).

A recombinant soluble form of the extracellular domain of CD44 fused to GST (GST-CD44e) was generated to assess the effects of soluble CD44 on macrophage fusion.

The addition of soluble CD44 to fusing macrophages strongly prevent multinucleation in a dose-dependent manner (Figure 8). Neither GST-calreticulin (GST-Cal) nor GST alone, used as controls, prevented multinucleation. Both GST-CD44e and GST-Cal, however, bound to macrophages in a saturable and dose-dependent manner (Figure 8).

Example 10: Expression of CD44 Macrophages Fusing in. vivo.

Tissues were prepared from both control and experimental rats implanted with

bone particles as previously described (Vignery et al., 1989, Am. J. Pathol. 135: 565-570).

The implants and the rat tissues were quick frozen and cut to 6 pm frozen sections using a Reichert-Jung cryostat (Leica, Deerfield, IL). The sections were first incubated overnight in 5% dry milk in PBS, then for two hours with anti-CD44, anti-CD4, anti-MHCII or mouse IgGI. Sections were then incubated for one hour with goat anti-mouse Cy3- F (ab') 2. After three washes with PBS the sections were imaged at 550 nm using the Cy3 excitation filter block on an Olympus microscope.

To investigate whether fusing macrophages expressed detectable levels of CD44, rat osteoclast-like cells were induced in vivo by implanting syngeneic bone particles intramuscularly and sections were obtained from bone implants, as well as from long bones, brain, liver, spleen, lymph nodes, kidney, lung, striated muscle and pancreas.

Sections were reacted with monoclonal antibodies directed against CD44, CD4 or MHCII, followed by goat anti-mouse IgG conjgated to Cy3. Although each of these tissues hosts resident macrophages and many of them contain cells such as lymphocytes and epithelial cells known to express CD44, only osteoclast-like cells from the bone implants exhibited a strong fluorescent signal that was not detected in the presence of anti-CD4, anti-MHCII or IgGl (Figure 9). The signal was restricted to both mono-and multinucleated cells that were closely apposed to the bone implants. In larger cells attached to the implants, the signal appeared stronger distal to the bone (i. e., concentrated in the non-adherent domain of the plasma membrane. This is the domain that faces the incoming fusing macrophages.

None of the surrounding cells, such as muscle cells, fibroblasts or endothelial cells expressed a detectable level of fluorescence.

Although the present invention has been described in detail with reference to examples above, it is understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. All cited patents and publications referred to in this application are herein incorporated by reference in their entirety.