Abstract:
A pharmaceutical composition is described that can be used for treating or prevention of diseases association with bone resorption, particularly of a metastatic carcinoma. In certain aspects, the composition is based on a polypeptide which includes the leading 215 amino acids of the human osteoprotegerin followed by the Fc portion of the human IgG1 protein. Pharmaceutical formulations are provided that are suitable for administering the pharmaceutical composition into primates via subcutaneous and intravenous routes.

Description:
FIELD OF THE INVENTION 
       [0001]    Generally, the invention relates to the field of biological pharmaceuticals as well as their use in conditions associated with bone resorption, for example in oncology. More specifically, the invention relates to an osteoprotegerin-derived composition that binds to receptor activator of NF-kappaB ligand (RANKL). 
       BACKGROUND 
       [0002]    The approaches described in this section could be pursued, and are not necessarily approaches that have previously been conceived or pursued. Therefore, unless otherwise indicated, it should not be assumed that any of the approaches described in this section qualify as prior art, merely by virtue of their inclusion into this section. 
         [0003]    Bone metastases are a common complication of both solid tumors and hematologic cancers with an incidence of 15-75% in patients with solid tumors and nearly 100% in patients with multiple myeloma. Cancers that are most likely to metastasize to bone include breast, lung, prostate, thyroid and renal cancers. Rate of bone metastases in different types of cancer is as follow:
       Multiple myeloma—70-95%—Breast cancer—65-75%—Prostate cancer—65-75%—Lung cancer—30-40%—Renal cancer—40% —Bladder cancer—20-25%—Melanoma—14-45%.       
 
         [0005]    Skeletal complications of bone metastases account for significant morbidity due to pain, pathologic fractures, spinal cord compression, and other nerve-compression syndromes. 
         [0006]    Bone metastases can be osteolytic, osteoblastic, or mixed. Normal bone remodeling is controlled by osteoblasts and osteoclasts in a balanced sequence. Receptor activator of nuclear factor KB (RANK) ligand (RANKL), a member of the tumor necrosis factor family, is expressed on the surface of osteoblasts. RANKL binds the receptor RANK on osteoclast precursors, which leads to signaling via TNF receptor-associated factors (TRAFs) and ultimately activation of nuclear factor KB in the nucleus, inducing differentiation into mature osteoclasts which degrade or resorb bone. Other osteoclast-activating factors include parathyroid hormone-related protein, interleukins, and chemokines. A decoy receptor for RANKL, osteoprotegerin (OPG), is present in bone marrow and secreted by osteoblasts and acts as a balance between the osteoblasts and osteoclasts. 
         [0007]    In the setting of bone metastases in cancer, the cross talk between RANKL, RANK, and OPG is disrupted. Osteoclast activation is enhanced when metastases release interleukins, parathyroid hormone-related protein, and other factors that up regulate RANKL expression. These factors may also inhibit OPG. In addition, growth factors released from bone lesions stimulate the growth of tumor cells, setting up a vicious cycle (Roodman G D. Mechanisms of bone metastasis. N Engl J Med 2004; 350:1655-64; Vallet S, smith M R, Rage N. Novel bone-targeted strategies in oncology. Clin Cancer Res 2010;16:4084-93; Marathe A, Peterson M C, Mager D E. Integrated cellular bone homeostasis model for denosumab pharmacodynamics in multiple myeloma patients. J Pharmacol Exp Ther 2008; 326:555-562; George S, Brenner A, Sarantopoulos J, Bukowski R M. RANK ligand: effects of inhibition. Curr Oncol Rep 2010;12: 80-86). 
         [0008]    Human OPG (GenBank: U94332.1) is a 401 amino acid protein which contains a signal peptide of 21 amino acids, that is cleaved before glutamic acid 22 giving rise to a mature soluble protein of 380 amino acid. OPG is a member of the tumor necrosis factor receptor (TNFR) family, comprising four cysteine-rich TNFR like domains in its N-terminal portion. OPG has been shown to have a role in the development of bone, and mice lacking the OPG gene had an osteoporotic phenotype and gross skeletal abnormalities. 
         [0009]    OPG, which is produced by osteoblasts and bone marrow stromal cells, acts as a secreted decoy receptor with no apparent direct signaling function. OPG acts by binding to its natural ligand—osteoprotegerin ligand (OPGL), which is also known as RANKL. The binding between OPG and RANKL prevents RANKL from activating its cognate receptor RANK, which is an osteoclast receptor vital for osteoclast differentiation, activation and survival. 
         [0010]    Recombinant OPG exists in monomeric and dimeric forms of apparent molecular weights of about 55 kDa and about 110 kDa, respectively. Truncation of the N-terminal domain to residue cysteine 185 results in OPG inactivation, presumably by disrupting a disulfide bond of the TNFR-like domain, whereas truncation of the C-terminal portion of the protein to residue 194 does not alter biological activity. 
         [0011]    Overexpression of OPG in transgenic mice leads to profound osteopetrosis characterized by a near complete lack of osteoclasts in the mice. Conversely, ablation of the OPG gene causes severe osteoporosis in mice, indicating an important physiological role of OPG in regulating bone resorption. The secretion of OPG and RANKL from osteoblasts and stromal cells is regulated by numerous hormones and cytokines. The relative levels of OPG and RANKL production are thought to control the extent of bone resorption: expression of RANKL increases bone resorption, whereas excess OPG has the opposite effect. Recombinant OPG blocks the effects of the vast majority of the factors which stimulate osteoclasts, in vitro and in vivo. OPG also inhibits bone resorption in a variety of animal disease models, including ovariectomy, induced osteoporosis, humoral hypercalcemia of malignancy, and experimental bone metastasis. Therefore, OPG might represent an effective therapeutic option for diseases associated with excessive osteoclast activity (Kostenuik P J, Shalhoub V., Curr Pharm Des. 2001 May; 7(8):613-35). 
         [0012]    RANK/RANKL pathway is well-known target that has proved to be the effective treatment for bone metastasis. Denosumab is a high affinity monoclonal antibody that binds to human RANKL and inhibits its interactions with RANK, thus having a similar to OPG mode of action. Denosumab is a full-length human monoclonal anti-RANKL antibody of the IgG2 subclass, consisting of 2 heavy chains, and 2 light chains of the kappa subclass, produced in Chinese hamster ovary (CHO) cells. Denosumab under the trade name Prolia was approved by U.S. Food and Drug Administration (FDA) for prevention and treatment of osteoporosis in postmenopausal women. Denosumab under the trade name Xgeva was approved by U.S. Food and Drug Administration (FDA) for the prevention of skeletal-related events in patients with bone metastases from solid tumors. Further clinical trials of denosumab for other bone remodeling related conditions are currently under way, i.e. for bone metastases from other forms of cancer (Lipton A et al. Randomized Active-Controlled Phase II Study of Denosumab Efficacy and Safety in Patients With Breast Cancer-Related Bone MetastasesJ Clin Oncol 25:4431-4437 (2007); Neville-Webbe H L, Coleman R E. Bisphosphonates and RANK ligand inhibitors for the treatment and prevention of metastatic bone disease. Eur J Cancer 2010; 46:1211-1222; Santini D, Galluzzo S, Zoccoli A, Pantano F, Fratto M E, et al. New molecular targets in bone metastases. Canc Treat Rev 2010; 36S3:S6-10). 
         [0013]    It would therefore be desirable to have a therapeutic composition that is capable of binding to RANKL and is based on the naturally occurring OPG molecule, which, while having an acceptable pharmacological profile, has a broader therapeutic potential. 
       SUMMARY OF THE INVENTION 
       [0014]    This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. 
         [0015]    In certain aspects, the present invention provides for pharmaceutical composition containing a polypeptide that binds human RANKL with a Kd value of no more than about 5×10 −13 M. The polypeptide comprises a first amino acid sequence comprising amino acids 1 through 215 of human osteoprotegerin (GenBank: U94332.1). The polypeptide further comprises a second amino acid sequence comprising amino acids 103 through 329 of human immunoglobulin gamma-1 Fc (GenBank: J00228.1). The polypeptide may comprise amino acid sequence of SEQ ID NO. 1. 
         [0016]    In certain aspects, the present invention provides for a therapeutic composition. The composition comprises a polypeptide that binds to human RANKL. The polypeptide comprises a biologically active portion of human osteoprotegerin and a Fc portion of human immunoglobulin gamma-1. The polypeptide binds human RANKL with a Kd value of no more than about 5×10 −13 M. 
         [0017]    The polypeptide may exhibit a half-life in systemic circulation in Cynomolgus monkey of at least 48 hours after a subcutaneous administration of the therapeutic composition at a dose of 3 mg/kg. The polypeptide may exhibit a half-life in systemic circulation in Cynomolgus monkey of at least 38 hours after a subcutaneous administration of the therapeutic composition at a dose of 10 mg/kg.
   1. The therapeutic composition may also contain about 25 mM sodium phosphate, from about 50 mM to about 100 mM NaCl, from about 20 to about 25 mM L-Arginine hydrochloride, while having pH value from about 6.3 to about 6.8. The therapeutic composition may also contain about 10 mg/mL sucrose. The therapeutic composition may also contain from about 10 mg/mL to about 25 mg/mL mannitol.   
 
         [0019]    In certain aspects, the present invention provides for a use of a substance for manufacture of a medicament for the treatment or prevention of a disease associated with bone resorption or remodeling. The substance comprises a polypeptide that binds to human RANKL. The polypeptide comprises a first amino acid sequence comprising amino acids 1 through 215 of human osteoprotegerin. The polypeptide further comprises a second amino acid sequence comprising amino acids 103 through 329 of human immunoglobulin gamma-1 Fc. The first amino acid sequence in the polypeptide may precede the second amino acid sequence. The polypeptide may comprise amino acid sequence of SEQ ID NO. 1. The disease associated with bone resorption or remodeling may be a carcinoma, a breast cancer, a prostate cancer, multiple myeloma, a bone sarcoma, bone metastases due to solid tumors, osteoporosis, rheumatoid arthritis, or psoriatic arthritis. 
         [0020]    In certain aspects, the present invention provides for a method of treating or preventing a disease associated with bone resorption or remodeling. The method comprises administering to a patient in need for treating or preventing a disease associated with bone resorption or remodeling a therapeutically effective amount of a pharmaceutical composition comprising a polypeptide that binds to human RANKL. The polypeptide comprises a first amino acid sequence comprising amino acids 1 through 215 of human osteoprotegerin. The polypeptide further comprises a second amino acid sequence comprising amino acids 103 through 329 of human immunoglobulin gamma-1 Fc. The first amino acid sequence in the polypeptide may precede the second amino acid sequence. The polypeptide may comprise amino acid sequence of SEQ ID NO. 1. The disease associated with bone resorption or remodeling may be a carcinoma, a breast cancer, a prostate cancer, multiple myeloma, a bone sarcoma, bone metastases due to solid tumors, osteoporosis, rheumatoid arthritis, or psoriatic arthritis. 
         [0021]    These and other aspects and advantages of the invention described herein will become apparent upon consideration of the Figures and detailed description below. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0022]    The following drawings and descriptions are provided to aid in the understanding of the invention: 
           [0023]      FIG. 1  shows association and dissociation curves of Denosumab at various rshRANKL concentrations generated by BIAcore X100; 
           [0024]      FIG. 2  shows association and dissociation curves of polypeptide of SEQ ID NO. 1 at different RANKL concentrations generated by BIAcore X100; 
           [0025]      FIG. 3  shows representative size-exclusion (SEC) HPLC chromatograms of polypeptide of SEQ ID NO. 1 analyzed at 0 days time point (panel A), 67 days time point (panel B) and 176 days time point (panel C); 
           [0026]      FIG. 4  shown primate single polypeptide of SEQ ID NO. 1 dose linearity for C max  and AUC last  for administered doses of 0.3, 3, 10 and 30 mg/kg for the subcutaneous route of administration; and 
           [0027]      FIG. 5  shown primate single polypeptide of SEQ ID NO. 1 dose study results corrected by the dose for C max  and AUC last  for administered doses of 0.3, 3, 10 and 30 mg/kg for the subcutaneous route of administration. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0028]    The teachings disclosed herein are based, in part, upon engineering of a protein molecule comprising a biologically active N-terminal portion of OPG which is fused to the Fc portion of a human IgG. To enable recombinant production of such OPG-derived protein molecule, a DNA expression vector has been constructed for overproducing the protein molecule in a heterologous protein expression system, and mammalian cells have been prepared stably expressing the protein molecule to a high expression level. Design, preparation and preliminary characterization of composition of matter of the present teachings are disclosed, in part, in an International Patent Application Publication No. WO/2013/147899, published on Oct. 3, 2013, which is incorporated herein by reference in the entirety. 
         [0029]    The protein molecule from the recombinant source formed homo-dimmers and homo-tetramers in solution. A protein purification procedure has been devised allowing obtaining a physiologically relevant substantially pure homo-dimeric preparation of the protein molecule. Unexpectedly, purified protein molecule demonstrates an exceptionally high degree of binding affinity for RANKL in an in vitro binding assay. Pharmaceutical formulations were devised allowing subcutaneous and intravenous administration of the protein molecule into primates. Thus formulated protein molecule exhibits an acceptable pharmacokinetics profile upon subcutaneous and intravenous administration into primates. Even further, thus formulated protein molecule exhibits substantial systemic exposure upon subcutaneous administration into humans. 
         [0030]    The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and in the specific context where each term is used. Certain terms are discussed below or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the invention and how to make and use them. The scope or meaning of any use of a term will be apparent from the specific context in which the term is used. “About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typically, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated. 
         [0031]    The methods of the invention may include steps of comparing sequences to each other, including wild-type sequence to one or more mutants (sequence variants). Such comparisons typically comprise alignments of polymer sequences, e.g., using sequence alignment programs and/or algorithms that are well known in the art (for example, BLAST, FASTA and MEGALIGN, to name a few). The skilled artisan can readily appreciate that, in such alignments, where a mutation contains a residue insertion or deletion, the sequence alignment will introduce a “gap” (typically represented by a dash, or “A”) in the polymer sequence not containing the inserted or deleted residue. 
         [0032]    The methods of the invention may include statistical calculations, e.g. determination of IC50 or EC50 values, etc. The skilled artisan can readily appreciate that such can be performed using a variety of commercially available software, e.g. PRISM (GraphPad Software Inc, La Jolla, Calif., USA) or similar. 
         [0033]    “Homologous,” in all its grammatical forms and spelling variations, refers to the relationship between two proteins that possess a “common evolutionary origin,” including proteins from superfamilies in the same species of organism, as well as homologous proteins from different species of organism. Such proteins (and their encoding nucleic acids) have sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or by the presence of specific residues or motifs and conserved positions. However, in common usage and in the instant application, the term “homologous,” when modified with an adverb such as “highly,” may refer to sequence similarity and may or may not relate to a common evolutionary origin. 
         [0034]    The term “sequence similarity,” in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin. 
         [0035]    The terms “protein” and “polypeptide” are used interchangeably. In general, OPG-derived proteins of the present teachings for use in mammals are expressed in mammalian cells that allow for proper post-translational modifications, such as CHO or HEK293 cell lines, although other mammalian expression cell lines are expected to be useful as well. It is therefore anticipated that the OPG-derived proteins may be post-translationally modified without substantially effecting their biological function. 
         [0036]    In certain aspects, functional variants of OPG-derived protein molecules of the present teachings include fusion proteins having at least a biologically active portion of the human OPG and one or more fusion domains. Well known examples of such fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chain constant region (e.g., an Fc), maltose binding protein (MBP), or human serum albumin. A fusion domain may be selected so as to confer a desired property. For example, the OPG polypeptide portion may be fused with a domain that stabilizes the OPG polypeptide in vivo (a “stabilizer” domain), optionally via a suitable peptide linker. The term “stabilizing” means anything that increases the half life of a polypeptide in systemic circulation, regardless of whether this is because of decreased destruction, decreased clearance, or other pharmacokinetic effect. Fusions with the Fc portion of an immunoglobulin are known to confer desirable pharmacokinetic properties on certain proteins. Likewise, fusions to human serum albumin can confer desirable properties. Other types of fusion domains that may be selected include multimerizing (e.g., dimerizing, tetramerizing) domains and functional domains that confer an additional biological function, e.g. promoting accumulation at the targeted site of action in vivo. 
         [0037]    In certain aspects, the present invention provides for a polypeptide comprising the leading 215 amino acids of the human OPG (GenBank: U94332.1), followed by 227 amino acids of the Fc portion of the human Ig Gamma-1 (GenBank: J00228.1). In an example embodiment, the protein molecule of the present invention comprises amino acid sequence of SEQ ID NO. 1. 
         [0000]    
       
         
               
               
             
               
               
               
             
           
               
                 hOPG-hIgG1-Fc polypeptide 
                   
               
               
                 (SEQ ID NO. 1) 
                   
               
             
          
           
               
                 MNKLLCCALV FLDISIKWTT QETFPPKYLH YDEETSHQLL CDKCPPGTYL KQHCTAKWKT 
                 60 
                   
               
               
                   
               
               
                 VCAPCPDHYY TDSWHTSDEC LYCSPVCKEL QYVKQECNRT HNRVCECKEG RYLEIEFCLK 
                 120 
               
               
                   
               
               
                 HRSCPPGFGV VQAGTPERNT VCKRCPDGFF SNETSSKAPC RKHTNCSVFG LLLTQKGNAT 
                 180 
               
               
                   
               
               
                 HDNICSGNSE STQKCGIDVT LCEEAFFRFA VPTKFDKTHT CPPCPAPELL GGPSVFLFPP 
                 240 
               
               
                   
               
               
                 KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV 
                 300 
               
               
                   
               
               
                 LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL 
                 360 
               
               
                   
               
               
                 TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC 
                 420 
               
               
                   
               
               
                 SVMHEALHNH YTQKSLSLSP GK 
                 442 
               
             
          
         
       
     
         [0038]    In certain aspects, the present invention provides for a recombinant DNA molecule having an open reading frame coding for a polypeptide comprising the leading 215 amino acids of the human OPG followed by 227 amino acids of the Fc portion of the human Ig Gamma-1, optionally connected via a flexible linker. In an example embodiment, the recombinant DNA molecule of the present invention comprises nucleotide sequence of SEQ ID NO. 2. 
         [0000]    
       
         
               
               
             
               
               
               
             
           
               
                 hOPG-hIgG1-Fc DNA 
                   
               
               
                 (SEQ ID NO. 2) 
                   
               
             
          
           
               
                 ATGAATAAGC TGCTGTGCTG TGCCCTCGTG TTTCTCGATA TAAGCATTAA GTGGACTACC 
                 60 
                   
               
               
                   
               
               
                 CAGGAGACAT TCCCTCCTAA GTATCTGCAC TATGACGAGG AGACAAGCCA TCAGCTGCTG 
                 120 
               
               
                   
               
               
                 TGCGATAAGT GTCCTCCTGG GACCTATCTC AAACAACATT GTACAGCCAA ATGGAAGACA 
                 180 
               
               
                   
               
               
                 GTCTGCGCTC CATGTCCTGA CCACTACTAC ACCGACTCTT GGCATACTAG CGACGAATGT 
                 240 
               
               
                   
               
               
                 CTGTATTGTT CACCCGTGTG CAAGGAGCTG CAATACGTGA AACAGGAATG CAATAGGACA 
                 300 
               
               
                   
               
               
                 CATAACCGCG TGTGTGAATG CAAAGAGGGC AGGTATCTGG AGATCGAATT TTGTCTGAAG 
                 360 
               
               
                   
               
               
                 CACCGGAGCT GCCCACCCGG CTTTGGAGTG GTCCAGGCCG GGACTCCCGA GAGAAACACT 
                 420 
               
               
                   
               
               
                 GTGTGCAAAA GATGCCCAGA CGGATTCTTT TCAAACGAGA CATCTTCTAA GGCACCATGT 
                 480 
               
               
                   
               
               
                 CGGAAGCACA CTAACTGTTC CGTCTTTGGG CTGCTGCTCA CCCAGAAGGG CAATGCCACC 
                 540 
               
               
                   
               
               
                 CACGATAATA TTTGCTCCGG AAACTCCGAA TCCACCCAAA AGTGCGGGAT AGATGTTACC 
                 600 
               
               
                   
               
               
                 CTCTGCGAAG AGGCATTCTT CCGCTTCGCT GTTCCTACCA AGTTCGACAA AACTCACACA 
                 660 
               
               
                   
               
               
                 TGCCCACCGT GCCCAGCTCC GGAACTCCTG GGCGGACCGT CAGTCTTCCT CTTCCCCCCA 
                 720 
               
               
                   
               
               
                 AAACCCAAGG ACACCCTCAT GATCTCCCGG ACCCCTGAGG TCACATGCGT GGTGGTGGAC 
                 780 
               
               
                   
               
               
                 GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG TGGACGGCGT GGAGGTGCAT 
                 840 
               
               
                   
               
               
                 AATGCCAAGA CAAAGCCGCG GGAGGAGCAG TACAACAGCA CGTACCGTGT GGTCAGCGTC 
                 900 
               
               
                   
               
               
                 CTCACCGTCC TGCACCAGGA CTGGCTGAAT GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC 
                 960 
               
               
                   
               
               
                 AAAGCCCTCC CAGCCCCCAT CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA 
                 1020 
               
               
                   
               
               
                 CCACAGGTGT ACACCCTGCC CCCATCCCGG GATGAGCTGA CCAAGAACCA GGTCAGCCTG 
                 1080 
               
               
                   
               
               
                 ACCTGCCTGG TCAAAGGCTT CTATCCCAGC GACATCGCCG TGGAGTGGGA GAGCAATGGG 
                 1140 
               
               
                   
               
               
                 CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGTTGG ACTCCGACGG CTCCTTCTTC 
                 1200 
               
               
                   
               
               
                 CTCTACAGCA AGCTCACCGT GGACAAGAGC AGGTGGCAGC AGGGGAACGT CTTCTCATGC 
                 1260 
               
               
                   
               
               
                 TCCGTGATGC ATGAGGCTCT GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG 
                 1320 
               
               
                   
               
               
                 GGTAAA 
                 1326 
               
             
          
         
       
     
         [0039]    In certain aspects, the present invention provides for a recombinant mammalian expression plasmid for high expression of a polypeptide comprising the leading 215 amino acids of the human OPG followed by 227 amino acids of the Fc portion of the human Ig Gamma-1, optionally connected via a flexible linker. This plasmid comprises the cytomegalovirus (CMV) promoter to drive transcription of the gene coding for said polypeptide, followed by the bGH polyadenylation and transcription termination sequence. The plasmid also contains a pUC origin of replication and β-lactamase gene, which confers ampicillin resistance, for supporting plasmid propagation and selection in bacteria. The plasmid further contains a gene for Glutamine synthetase, a selectable marker widely used for establishing stable CHOK1 and NSO cell lines. 
         [0040]    In an example embodiment, the mammalian expression plasmid of the present invention comprises nucleotide sequence of SEQ ID NO. 3. 
         [0000]    
       
         
               
               
             
               
               
               
             
           
               
                 hOPG-hIgG1-Fc expression plasmid 
                   
               
               
                 (SEQ ID NO. 3) 
                   
               
             
          
           
               
                 GAATTCATTG ATCATAATCA GCCATACCAC ATTTGTAGAG GTTTTACTTG CTTTAAAAAA 
                 60 
                   
               
               
                   
               
               
                 CCTCCCACAC CTCCCCCTGA ACCTGAAACA TAAAATGAAT GCAATTGTTG TTGTTAACTT 
                 120 
               
               
                   
               
               
                 GTTTATTGCA GCTTATAATG GTTACAAATA AAGCAATAGC ATCACAAATT TCACAAATAA 
                 180 
               
               
                   
               
               
                 AGCATTTTTT TCACTGCATT CTAGTTGTGG TTTGTCCAAA CTCATCAATG TATCTTATCA 
                 240 
               
               
                   
               
               
                 TGTCTGGCGG CCGCGAGACG CCATCCACGC TGTTTTGACC TCCATAGAAG ACACCGGGAC 
                 300 
               
               
                   
               
               
                 CGATCCAGCC TCCGCGGCCG GGAACGGTGC ATTGGAACGC GGATTCCCCG TGCCAAGAGT 
                 360 
               
               
                   
               
               
                 GACGTAAGTA CCGCCTATAG AGTCTATAGG CCCACCCCCT TGGCTTCTTA TGCATGCTAT 
                 420 
               
               
                   
               
               
                 ACTGTTTTTG GCTTGGGGTC TATACACCCC CGCTTCCTCA TGTTATAGGT GATGGTATAG 
                 480 
               
               
                   
               
               
                 CTTAGCCTAT AGGTGTGGGT TATTGACCAT TATTGACCAC TCCCCTATTG GTGACGATAC 
                 540 
               
               
                   
               
               
                 TTTCCATTAC TAATCCATAA CATGGCTCTT TGCCACAACT CTCTTTATTG GCTATATGCC 
                 600 
               
               
                   
               
               
                 AATACACTGT CCTTCAGAGA CTGACACGGA CTCTGTATTT TTACAGGATG GGGTCTCATT 
                 660 
               
               
                   
               
               
                 TATTATTTAC AAATTCACAT ATACAACACC ACCGTCCCCA GTGCCCGCAG TTTTTATTAA 
                 720 
               
               
                   
               
               
                 ACATAACGTG CTCCACGCGA ATCTCGGGTA CGTGTTCCGG ACATGGGCTC TTCTCCGGTA 
                 780 
               
               
                   
               
               
                 GCGGCGGAGC TTCTACATCC GAGCCCTGCT CCCATGCCTC CAGCGACTCA TGGTCGCTCG 
                 840 
               
               
                   
               
               
                 GCAGCTAGTG GAGGCCAGAC TTAGGCACAG CACGATGCCC ACCACCACCA GTGTGCCGCA 
                 900 
               
               
                   
               
               
                 CAAGGCCGTG GCGGTAGGGT ATGTGTCTGA AAATGAGCTC GGGGAGCGGG CTTGCACCAA 
                 960 
               
               
                   
               
               
                 AAATTTTCGC GTCGACTATA CCGTCCACAT GTGAGCAAAA GGCCAGCAAA AGGCCAGGAA 
                 1020 
               
               
                   
               
               
                 CCGTAAAAAG GCCGCGTTGC TGGCGTTTTT CCATAGGCTC CGCCCCCCTG ACGAGCATCA 
                 1080 
               
               
                   
               
               
                 CAAAAATCGA CGCTCAAGTC AGAGGTGGCG AAACCCGACA GGACTATAAA GATACCAGGC 
                 1140 
               
               
                   
               
               
                 GTTTCCCCCT GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG ACCCTGCCGC TTACCGGATA 
                 1200 
               
               
                   
               
               
                 CCTGTCCGCC TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT CATAGCTCAC GCTGTAGGTA 
                 1260 
               
               
                   
               
               
                 TCTCAGTTCG GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT GTGCACGAAC CCCCCGTTCA 
                 1320 
               
               
                   
               
               
                 GCCCGACCGC TGCGCCTTAT CCGGTAACTA TCGTCTTGAG TCCAACCCGG TAAGACACGA 
                 1380 
               
               
                   
               
               
                 CTTATCGCCA CTGGCAGCAG CCACTGGTAA CAGGATTAGC AGAGCGAGGT ATGTAGGCGG 
                 1440 
               
               
                   
               
               
                 TGCTACAGAG TTCTTGAAGT GGTGGCCTAA CTACGGCTAC ACTAGAAGAA CAGTATTTGG 
                 1500 
               
               
                   
               
               
                 TATCTGCGCT CTGCTGAAGC CAGTTACCTT CGGAAAAAGA GTTGGTAGCT CTTGATCCGG 
                 1560 
               
               
                   
               
               
                 CAAACAAACC ACCGCTGGTA GCGGTTTTTT TGTTTGCAAG CAGCAGATTA CGCGCAGAAA 
                 1620 
               
               
                   
               
               
                 AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG TCTGACGCTC AGTGGAACGA 
                 1680 
               
               
                   
               
               
                 AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA AGGATCTTCA CCTAGATCCT 
                 1740 
               
               
                   
               
               
                 TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA TATGAGTAAA CTTGGTCTGA 
                 1800 
               
               
                   
               
               
                 CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG ATCTGTCTAT TTCGTTCATC 
                 1860 
               
               
                   
               
               
                 CATAGTTGCC TGACTCCCCG TCGTGTAGAT AACTACGATA CGGGAGGGCT TACCATCTGG 
                 1920 
               
               
                   
               
               
                 CCCCAGTGCT GCAATGATAC CGCGAGACCC ACGCTCACCG GCTCCAGATT TATCAGCAAT 
                 1980 
               
               
                   
               
               
                 AAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT GCAACTTTAT CCGCCTCCAT 
                 2040 
               
               
                   
               
               
                 CCAGTCTATT AATTGTTGCC GGGAAGCTAG AGTAAGTAGT TCGCCAGTTA ATAGTTTGCG 
                 2100 
               
               
                   
               
               
                 CAACGTTGTT GCCATTGCTA CAGGCATCGT GGTGTCACGC TCGTCGTTTG GTATGGCTTC 
                 2160 
               
               
                   
               
               
                 ATTCAGCTCC GGTTCCCAAC GATCAAGGCG AGTTACATGA TCCCCCATGT TGTGCAAAAA 
                 2220 
               
               
                   
               
               
                 AGCGGTTAGC TCCTTCGGTC CTCCGATCGT TGTCAGAAGT AAGTTGGCCG CAGTGTTATC 
                 2280 
               
               
                   
               
               
                 ACTCATGGTT ATGGCAGCAC TGCATAATTC TCTTACTGTC ATGCCATCCG TAAGATGCTT 
                 2340 
               
               
                   
               
               
                 TTCTGTGACT GGTGAGTACT CAACCAAGTC ATTCTGAGAA TAGTGTATGC GGCGACCGAG 
                 2400 
               
               
                   
               
               
                 TTGCTCTTGC CCGGCGTCAA TACGGGATAA TACCGCGCCA CATAGCAGAA CTTTAAAAGT 
                 2460 
               
               
                   
               
               
                 GCTCATCATT GGAAAACGTT CTTCGGGGCG AAAACTCTCA AGGATCTTAC CGCTGTTGAG 
                 2520 
               
               
                   
               
               
                 ATCCAGTTCG ATGTAACCCA CTCGTGCACC CAACTGATCT TCAGCATCTT TTACTTTCAC 
                 2580 
               
               
                   
               
               
                 CAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCC GCAAAAAAGG GAATAAGGGC 
                 2640 
               
               
                   
               
               
                 GACACGGAAA TGTTGAATAC TCATACTCTT CCTTTTTCAA TATTATTGAA GCATTTATCA 
                 2700 
               
               
                   
               
               
                 GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT TAGAAAAATA AACAAATAGG 
                 2760 
               
               
                   
               
               
                 GGTTCCGCGC ACATTTCCCC GAAAAGTGCC ACCTGACCTC GACGGATCGG GAGATCTCCC 
                 2820 
               
               
                   
               
               
                 GATCCCCTAT GGTGCACTCT CAGTACAATC TGCTCTGATG CCGCATAGTT AAGCCAGTAT 
                 2880 
               
               
                   
               
               
                 CTGCTCCCTG CTTGTGTGTT GGAGGTCGCT GAGTAGTGCG CGAGCAAAAT TTCTGTGGAA 
                 2940 
               
               
                   
               
               
                 TGTGTGTCAG TTAGGGTGTG GAAAGTCCCC AGGCTCCCCA GCAGGCAGAA GTATGCAAAG 
                 3000 
               
               
                   
               
               
                 CATGCATCTC AATTAGTCAG CAACCAGGTG TGGAAAGTCC CCAGGCTCCC CAGCAGGCAG 
                 3060 
               
               
                   
               
               
                 AAGTATGCAA AGCATGCATC TCAATTAGTC AGCAACCATA GTCCCGCCCC TAACTCCGCC 
                 3120 
               
               
                   
               
               
                 CATCCCGCCC CTAACTCCGC CCAGTTCCGC CCATTCTCCG CCCCATGGCT GACTAATTTT 
                 3180 
               
               
                   
               
               
                 TTTTATTTAT GCAGAGGCCG AGGCCGCCTC TGCCTCTGAG CTATTCCAGA AGTAGTGAGG 
                 3240 
               
               
                   
               
               
                 AGGCTTTTTT GGAGGCCTAG GCTTTTGCAA AAAGCTAAGC TACAACAAGG CTCTGGCTAA 
                 3300 
               
               
                   
               
               
                 CTAGAGAACC CACTGCTTAC TGGCTTATCG AAAGCTAGCT TAATACGACT CAATGAATCA 
                 3360 
               
               
                   
               
               
                 GGGTGCAAAC AAGACGGTAT TAGACCGATA TTTACGGTTA GATATCCCGG ACCAGAAATG 
                 3420 
               
               
                   
               
               
                 TCAAGCTATG TACATCTGGG TCGATGGAAC CGGCGAAAAC CTCCGCTCTA AGACCAGGAC 
                 3480 
               
               
                   
               
               
                 ACTCAACTTT ACTCCTAAAT CTCCCAGTGA GCTGCCAATA TGGAATTTCG ATGGGTCATC 
                 3540 
               
               
                   
               
               
                 AACGGGCCAG GCCGAACGGA GCAACAGTGA CGTGTACCTG TATCCAGTCG CTGTTTATCG 
                 3600 
               
               
                   
               
               
                 AGATCCATTC AGGCTGGGTA ACAATAAGCT GGTCCTCTGT GAAACCTACA AATACAACAA 
                 3660 
               
               
                   
               
               
                 GAAGCCTGCT GATACTAACC AGCGTTGGAA GTGTATGGAA GTAATGACAA GGGCAGCAGA 
                 3720 
               
               
                   
               
               
                 CCAGCACCCA TGGTTCGGCA TGGAACAAGA ATATACTCTT TTGGACATTG ACAAACATCC 
                 3780 
               
               
                   
               
               
                 CTTGGGTTGG CCCAAGAATG GCTATCCAGG CCCTCAGGGT CCCTATTACT GTGGTGTGGG 
                 3840 
               
               
                   
               
               
                 TGCTAATAGG GTGTATGGGC GCGATGTGGT CGAGGCTCAC TACAGGGCGT GTCTTTGCGC 
                 3900 
               
               
                   
               
               
                 TGGCATCAAC ATCTCTGGGG AGAACGCGAA AGTCATGCCG GCCCAATGGG AGTTCCAGGT 
                 3960 
               
               
                   
               
               
                 TGGTCCGTGT GAAGGCATAA CCATGGGCGA CGACCTCTGG ATGGCTCGCT ACCTTCTTCA 
                 4020 
               
               
                   
               
               
                 CAGGGTCGCT GAGGACTTTG ATGTTGTAGT AACACTCGAC CCCAAGCCAA TCCCTGGTGA 
                 4080 
               
               
                   
               
               
                 CTGGAACGGC GCTGGAATGC ACACTAATTT CTCTACTGAA GCCATGCGTG GTCCCAATGG 
                 4140 
               
               
                   
               
               
                 CATTCTGGAA ATTGAGAGTG CCATCGACAA ATTGTCGAAG GTTCATGAGA AACACATCAA 
                 4200 
               
               
                   
               
               
                 GGCATACGAC CCACACGCAG GCAAGGATAA CGAAAGGCGC TTGACTGGTC ATTATGAAAC 
                 4260 
               
               
                   
               
               
                 TTCCTCCATC CATGACTTTT CTGCAGGTGT GGCCAACCGT GGTGCCTCCA TCCGCATCCC 
                 4320 
               
               
                   
               
               
                 CAGAGGAGTG GCTGAGGAGA AAACCGGCTA CCTGGAGGAC CGTCGCCCTT CCTCCAACGC 
                 4380 
               
               
                   
               
               
                 TGACCCTTAT GTGGTGTCTG AGAGGCTTGT GCGTACCATC TGCCTGAACG AGCAGTGACT 
                 4440 
               
               
                   
               
               
                 ATAGGGAGAC CCAAGCTGAC GCGCCCTGTA GCGGCGCATT AAGCGCGCCC GGGCTGGTGG 
                 4500 
               
               
                   
               
               
                 TTACGCGCAG CGTGACCGCT ACACTTGCCA GCGCCCTAGC GCCCGCTCCT TTCGCTTTCT 
                 4560 
               
               
                   
               
               
                 TCCCTTCCTT TCTCGCCACG TTCGCCGGCT TTCCCCGTCA AGCTCTAAAT CGGGGGCTCC 
                 4620 
               
               
                   
               
               
                 CTTTAGGGTT CCGATTTAGT GCTTTACGGC ACCTCGACCC CAAAAAACTT GATTAGGGTG 
                 4680 
               
               
                   
               
               
                 ATGGTTCACG TAGTGGGCCA TCGCCCTGAT AGACGGTTTT TCGCCCTTTG ACGTTGGAGT 
                 4740 
               
               
                   
               
               
                 CCACGTTCTT TAATAGTGGA CTCTTGTTCC AAACTGGAAC AACACTCAAC CCTATCTCGG 
                 4800 
               
               
                   
               
               
                 TCTATTCTTT TGATTTATAA GGGATTTTCT CTAGCTAGAG CTTGGCGTAA TCATGGTCAT 
                 4860 
               
               
                   
               
               
                 AGCTGTTTCC TGTGTGAAAT TGTTATCCGC TCACAATTCC ACACAACATA CGAGCCGGAA 
                 4920 
               
               
                   
               
               
                 GCATAAAGTG TAAAGCCTGG GGTGCCTAAT GAGTGAGCTA ACTCACATTA ATTGCGTTGC 
                 4980 
               
               
                   
               
               
                 GCTCACTGCC CGCTTTCCAG TCGGGAAACC TGTCGTGCCA GCTGCATTAA TGAATCGGCC 
                 5040 
               
               
                   
               
               
                 AACGCGCGGG GAGAGGCGGT TTGCGTATTG GGCGCTCTTC CGCTTCCTCG CTGTCCACCT 
                 5100 
               
               
                   
               
               
                 CGCTGCGCTC GGTCGTTCGG CTGCGGCGAG CGGTATCAGC TCACTCAAAG GCGGTAATAC 
                 5160 
               
               
                   
               
               
                 GGTTAGAGAT TTCGATTCCA CCGCCGCCTT CTATGAAAGG TTGGGCTTCG GAATCGTTTT 
                 5220 
               
               
                   
               
               
                 CCGGGACGCC GGCTGGATGA TCCTCCAGCG CGGGGATCTC ATGCTGGAGT TCTTCGCCCA 
                 5280 
               
               
                   
               
               
                 CCCCAACTTG TTTATTGCAG CTTATAATGG TTACAAATAA AGCAATAGCA TCACAAATTT 
                 5340 
               
               
                   
               
               
                 CACAAATAAA GCATTTTTTT CACTGCATTC TAGTTGTGGT TTGTCCAAAC TCATCAATGT 
                 5400 
               
               
                   
               
               
                 ATCTTATCAT GTCTGTATAC CGTCGACCTC AAGGCTTGAC CGACAATTGC ATGAAGACGC 
                 5460 
               
               
                   
               
               
                 GTAATCTGCT TAGGGTTAGT TTTACAGGAT GGGGTCTCAT TTATTATTTA CAAATTCACA 
                 5520 
               
               
                   
               
               
                 TATACAACAC CACCAGATCG CCTGGAGACG CCATCCACGC TGTTTTGACC TCCATAGAAG 
                 5580 
               
               
                   
               
               
                 ACACCGGGAC CGATCCAGCC TCCGCGGCCG GGAACGGTGC ATTGGAACGC GGATTCCCCG 
                 5640 
               
               
                   
               
               
                 TGCCAAGAGT GACGTAAGTA CCGCCTATAG AGTCTATAGG CCCACCCCCT TGGCTTCTTA 
                 5700 
               
               
                   
               
               
                 TGCATGCTAT ACTGTTTTTG GCTTGGGGTC TATACACCCC CGCTTCCTCA TGTTATAGGT 
                 5760 
               
               
                   
               
               
                 GATGGTATAG CTTAGCCTAT AGGTGTGGGT TATTGACCAT TATTGACCAC TCCCCTATTG 
                 5820 
               
               
                   
               
               
                 GTGACGATAC TTTCCATTAC TAATCCATAA CATGGCTCTT TGCCACAACT CTCTTTATTG 
                 5880 
               
               
                   
               
               
                 GCTATATGCC AATACACTGT CCTTCAGAGA CTGACACGGA CGCGTTTTGC GCTGCTTCGC 
                 5940 
               
               
                   
               
               
                 GATGTACGGG CCAGATATAC GCGTTGACAT TGATTATTGA CTAGTTATTA ATAGTAATCA 
                 6000 
               
               
                   
               
               
                 ATTACGGGGT CATTAGTTCA TAGCCCATAT ATGGAGTTCC GCGTTACATA ACTTACGGTA 
                 6060 
               
               
                   
               
               
                 AATGGCCCGC CTGGCTGACC GCCCAACGAC CCCCGCCCAT TGACGTCAAT AATGACGTAT 
                 6120 
               
               
                   
               
               
                 GTTCCCATAG TAACGCCAAT AGGGACTTTC CATTGACGTC AATGGGTGGA GTATTTACGG 
                 6180 
               
               
                   
               
               
                 TAAACTGCCC ACTTGGCAGT ACATCAAGTG TATCATATGC CAAGTACGCC CCCTATTGAC 
                 6240 
               
               
                   
               
               
                 GTCAATGACG GTAAATGGCC CGCCTGGCAT TATGCCCAGT ACATGACCTT ATGGGACTTT 
                 6300 
               
               
                   
               
               
                 CCTACTTGGC AGTACATCTA CGTATTAGTC ATCGCTATTA CCATGGTGAT GCGGTTTTGG 
                 6360 
               
               
                   
               
               
                 CAGTACATCA ATGGGCGTGG ATAGCGGTTT GACTCACGGG GATTTCCAAG TCTCCACCCC 
                 6420 
               
               
                   
               
               
                 ATTGACGTCA ATGGGAGTTT GTTTTGGCAC CAAAATCAAC GGGACTTTCC AAAATGTCGT 
                 6480 
               
               
                   
               
               
                 AACAACTCCG CCCCATTGAC GCAAATGGGC GGTAGGCGTG TACGGTGGGA GGTCTATATA 
                 6540 
               
               
                   
               
               
                 AGCAGAGCTC GTTTAGTGAA CCGTCAGATC GCCTGGAGAC GCCATCCACG CTGTTTTGAC 
                 6600 
               
               
                   
               
               
                 CTCCATAGAA GACACCGGGA CCGATCCAGC CTCCGCGGCC GGGAACGGTG CATTGGAACG 
                 6660 
               
               
                   
               
               
                 CGGATTCCCC GTGCCAAGAG TGACGTAAGT ACCGCCTATA GAGTCTATAG GCCCACCCCC 
                 6720 
               
               
                   
               
               
                 TTGGCTTCTT ATGCATGCTA TACTGTTTTT GGCTTGGGGT CTATACACCC CCGCTTCCTC 
                 6780 
               
               
                   
               
               
                 ATGTTATAGG TGATGGTATA GCTTAGCCTA TAGGTGTGGG TTATTGACCA TTATTGACCA 
                 6840 
               
               
                   
               
               
                 CTCCCCTATT GGTGACGATA CTTTCCATTA CTAATCCATA ACATGGCTCT TTGCCACAAC 
                 6900 
               
               
                   
               
               
                 TCTCTTTATT GGCTATATGC CAATACACTG TCCTTCAGAG ACTGACACGG ACTCTGTATT 
                 6960 
               
               
                   
               
               
                 TTTACAGGAT GGGGTCTCAT TTATTATTTA CAAATTCACA TATACAACAC CACCGTCCCC 
                 7020 
               
               
                   
               
               
                 AGTGCCCGCA GTTTTTATTA AACATAACGT GGGATCTCCA CGCGAATCTC GGGTACGTGT 
                 7080 
               
               
                   
               
               
                 TCCGGACATG GGCTCTTCTC CGGTAGCGGC GGAGCTTCTA CATCCGAGCC CTGCTCCCAT 
                 7140 
               
               
                   
               
               
                 GCCTCCAGCG ACTCATGGTC GCTCGGCAGC TCCTTGCTCC TAACAGTGGA GGCCAGACTT 
                 7200 
               
               
                   
               
               
                 AGGCACAGCA CGATGCCCAC CACCACCAGT GTGCCGCACA AGGCCGTGGC GGTAGGGTAT 
                 7260 
               
               
                   
               
               
                 GTGTCTGAAA ATGAGCTCGG GGAGCGGGCT TGCACCGCTG ACGCATTTGG AAGACTTAAG 
                 7320 
               
               
                   
               
               
                 GCAGCGGCAG AAGAAGATGC AGGCAGCTGA GTTGTTGTGT TCTGATAAGA GTCAGAGGTA 
                 7380 
               
               
                   
               
               
                 ACTCCCGTTG CGGTGCTGTT AACGGTGGAG GGCAGTGTAG TCTGAGCAGT ACTCGTTGCT 
                 7440 
               
               
                   
               
               
                 GCCGCGCGCG CCACCAGACA TAATAGCTGA CAGACTAACA GACTGTTCCT TTCCATGGGT 
                 7500 
               
               
                   
               
               
                 CTTTTCTGCA GTCACCGTCC TTGACACGAA GCTTGCCACC ATGAATAAGC TGCTGTGCTG 
                 7560 
               
               
                   
               
               
                 TGCCCTCGTG TTTCTCGATA TAAGCATTAA GTGGACTACC CAGGAGACAT TCCCTCCTAA 
                 7620 
               
               
                   
               
               
                 GTATCTGCAC TATGACGAGG AGACAAGCCA TCAGCTGCTG TGCGATAAGT GTCCTCCTGG 
                 7680 
               
               
                   
               
               
                 GACCTATCTC AAACAACATT GTACAGCCAA ATGGAAGACA GTCTGCGCTC CATGTCCTGA 
                 7740 
               
               
                   
               
               
                 CCACTACTAC ACCGACTCTT GGCATACTAG CGACGAATGT CTGTATTGTT CACCCGTGTG 
                 7800 
               
               
                   
               
               
                 CAAGGAGCTG CAATACGTGA AACAGGAATG CAATAGGACA CATAACCGCG TGTGTGAATG 
                 7860 
               
               
                   
               
               
                 CAAAGAGGGC AGGTATCTGG AGATCGAATT TTGTCTGAAG CACCGGAGCT GCCCACCCGG 
                 7920 
               
               
                   
               
               
                 CTTTGGAGTG GTCCAGGCCG GGACTCCCGA GAGAAACACT GTGTGCAAAA GATGCCCAGA 
                 7980 
               
               
                   
               
               
                 CGGATTCTTT TCAAACGAGA CATCTTCTAA GGCACCATGT CGGAAGCACA CTAACTGTTC 
                 8040 
               
               
                   
               
               
                 CGTCTTTGGG CTGCTGCTCA CCCAGAAGGG CAATGCCACC CACGATAATA TTTGCTCCGG 
                 8100 
               
               
                   
               
               
                 AAACTCCGAA TCCACCCAAA AGTGCGGGAT AGATGTTACC CTCTGCGAAG AGGCATTCTT 
                 8160 
               
               
                   
               
               
                 CCGCTTCGCT GTTCCTACCA AGTTCGACAA AACTCACACA TGCCCACCGT GCCCAGCTCC 
                 8220 
               
               
                   
               
               
                 GGAACTCCTG GGCGGACCGT CAGTCTTCCT CTTCCCCCCA AAACCCAAGG ACACCCTCAT 
                 8280 
               
               
                   
               
               
                 GATCTCCCGG ACCCCTGAGG TCACATGCGT GGTGGTGGAC GTGAGCCACG AAGACCCTGA 
                 8340 
               
               
                   
               
               
                 GGTCAAGTTC AACTGGTACG TGGACGGCGT GGAGGTGCAT AATGCCAAGA CAAAGCCGCG 
                 8400 
               
               
                   
               
               
                 GGAGGAGCAG TACAACAGCA CGTACCGTGT GGTCAGCGTC CTCACCGTCC TGCACCAGGA 
                 8460 
               
               
                   
               
               
                 CTGGCTGAAT GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT 
                 8520 
               
               
                   
               
               
                 CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA CCACAGGTGT ACACCCTGCC 
                 8580 
               
               
                   
               
               
                 CCCATCCCGG GATGAGCTGA CCAAGAACCA GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT 
                 8640 
               
               
                   
               
               
                 CTATCCCAGC GACATCGCCG TGGAGTGGGA GAGCAATGGG CAGCCGGAGA ACAACTACAA 
                 8700 
               
               
                   
               
               
                 GACCACGCCT CCCGTGTTGG ACTCCGACGG CTCCTTCTTC CTCTACAGCA AGCTCACCGT 
                 8760 
               
               
                   
               
               
                 GGACAAGAGC AGGTGGCAGC AGGGGAACGT CTTCTCATGC TCCGTGATGC ATGAGGCTCT 
                 8820 
               
               
                   
               
               
                 GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG GGTAAATAAT A 
                 8871 
               
             
          
         
       
     
         [0041]    In certain aspects, the present invention provides for a mammalian expression system for production of a polypeptide comprising the leading 215 amino acids of the human OPG followed by 227 amino acids of the Fc portion of the human Ig Gamma-1, optionally connected via a flexible linker The expression system of the present invention comprises a mammalian cell harboring a recombinant mamalian expression plasmid for high expression of a polypeptide comprising the leading 215 amino acids of the human OPG followed by 227 amino acids of the Fc portion of the human Ig Gamma-1, optionally connected via a flexible linker 
         [0042]    In an example embodiment, the mammalian expression system of the present invention comprises Chinese hamster ovary cells (CHO-K1) harboring a plasmid comprising nucleotide sequence of SEQ ID NO. 3. 
         [0043]    In certain aspects, the present invention provides for a method of treatment of a mammal effected by a disorder associated with bone resorption or remodeling. 
       EXAMPLES 
       [0044]    The following Examples illustrate the forgoing aspects and other aspects of the present invention. These non-limiting Examples are put forth so as to provide those of ordinary skill in the art with illustrative embodiments as to how the compounds, compositions, articles, devices, and/or methods claimed herein are made and evaluated. The Examples are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventor regard as his invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. 
       Example 1 
     Preparation of Polypeptides of the Present Invention 
       [0045]    hOPG-hIgG1-Fc polypeptide of SEQ ID. 1 was expressed in CHO-K1 using molecular biology, cell culture and protein biochemistry techniques known in the art and described PCT Publication WO/2013/147899. Essentially, CHO-K1 cells expressing the polypeptide were harvested and lysed utilizing well established protocols. After cell lysate clarification, the supernatant containing expressed hOPG-hIgG1-Fc polypeptide was first applied to a Protein A affinity column. The pH adjusted Protein A column eluate was further purified by anion-exchange chromatography (AIEX) utilizing Q Sepharose resin. The AIEX flowthrough was analyzed by size-exclusion HPLC (SEC-HPLC), SDS-PAGE and other analytical techniques, as appropriate. 
         [0046]    For subsequent studies, a therapeutic composition comprising hOPG-hIgG1-Fc polypeptide was formulated to contain 40 mg hOPG-hIgG1-Fc polypeptide in 4 mL also containing 1% sucrose, 100 mM sodium chloride, 20 mM L-arginine hydrochloride and 25 mM sodium phosphate pH 6.3. A single vial contains about 40 mg hIgG1-Fc polypeptide in a volume of 4 mL. Thus the protein concentration in a vial is 10±1 mg/mL. 
       Example 2 
     Evaluation of Polypeptides of the Present Teachings Affinity Binding to RANKL Using Surface Plasmon Resonance (SPR) Assay 
       [0047]    The binding affinity of prepared hOPG-hIgG1-Fc polypeptide of SEQ ID NO. 1 to the recombinant soluble human Receptor Activator of Nuclear Factor Kappa-B Ligand (rshRANKL) was measured using a specially designed Surface Plasmon Resonance (SPR) assay. In the assay, hOPG-hIgG1-Fc polypeptide was captured on the SPR sensor surface by recombinant Protein A solutions of rshRANKL at different concentrations were dispensed over the sensor surface and kinetics of association and dissociation were monitored. Affinity is calculated by fitting a 1:1 Langmuir binding model to the data. 
         [0048]    Materials, Reagents and Equipment:
       Biacore CM5 Sensor Chip (GE Healthcare); Amine Coupling Kit (GE Healthcare); 50 mM sodium hydroxide (GE Healthcare); 10mM NaAcetate PH 4.5 (GE Healthcare); Surfactance P20 (GE Healthcare); Glycine (Sigma-Aldrich); 10×PBS Buffer (GE Healthcare)   Buffers:   Working Buffer: PBS+0.05% Surfactance P20 (pH7.26)   Regeneration Buffer: 5.4 ml of 10 mM Glycine buffer+4.6 ml of 10 mM Glycine buffer (pH 1.7)   Materials:   Denosumab (Prolia, commercial product, Amgen) concentration 60 mg/mL   hOPG-hIgG1-Fc polypeptide preparation per foregoing Example 1   rshRANKL concentration 0.76 mg/mL (13 μM)   Equipment:   Biacore X100 Instrument (GE Healthcare)   Biacore X100 Evaluation Software V2.0.1 (GE Healthcare)       
 
         [0060]    Procedures: 
         [0061]    Recombinant Protein A was diluted using pH4.5 10 mM NaAcetate buffer to a final concentration of 0.025 mg/ml. Protein A was immobilized on Flow Cells 1-2 using coupling kit and following parameters: a) 7 minute injection of 1:1 EDC: NHS; b) 5 minute injection of diluted Protein A in 10 mM NaAcetate pH4.5 at 10μL/min; c) 7 minute injection of 1M ethanolamine pH8.5. rshRANKL (MW 57.9 kDa) was dilute to desired concentrations with BIAcore working buffer. Five different rshRANKL dilutions were used for affinity measurements: 1.0415 nM, 2.083 nM, 4.166 nM, 8.333 nM, 16.666 nM. Six different rshRANKL dilutions were used for Denosumab affinity measurements: 0.7359 nM, 1.4719 nM, 2.94375 nM, 5.8875 nM, 11.775 nM, 23.55 nM. Denosumab was diluted using BIAcore working buffer to a final concentration of 0.8 μM. hOPG-hIgG1-Fc polypeptide sample was diluted using BIAcore working buffer to a final concentration of 37 nM 
         [0062]    Assay Protocol: 
         [0063]    The assay was performed according to the manufacturer&#39;s protocol Sample compartment temperature was 25° C.; data collection rate—1 Hz; flow rate—30μL/min; five different concentrations of rshRANKL were used for hOPG-hIgG1-Fc polypeptide evaluation (4.166 nM dilution was measured twice); six different concentrations of rshRANKL ligand were used for Denosumab evaluation. All measurements were performed as following: Denosumab/ hOPG-hIgG1-Fc capture with contact time of 180s; rshRANKL capture with contact time of 180s; dissociation using working buffer for 3600s; regeneration using regeneration buffer for 70s. 
         [0064]    The Biacore X100 Evaluation software is used to estimate the kinetic association (k a ) and dissociation (k d ) constants, the equilibrium dissociation constant (K D ) and the maximum RANKL binding level (R max ) for each sample. The model parameters are estimated for each sample individually by fitting a 1:1 Langmuir binding model to the data. 
         [0065]    Association and dissociation curves of Denosumab at different RANKL concentrations are showed in  FIG. 1 . Correlated data for each curve is listed in Table 1. The model parameters of Denosumab-RANKL binding which were estimated via Biacore X100 Evaluation software are listed in Table 2. 
         [0066]    The affinity (K D ) of anti-RANKL antibody Denosumab binding to RANKL is 2.6×10 −11 M, which is consistent with reported data from the manufacturer. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Correlated data for Denosumab-rshRANKL binding. 
               
             
          
           
               
                 Curve 
                 Conc (M) 
                 Flow (ul/min) 
                 kt (RU/Ms) 
                 RI (RU) 
               
               
                   
               
             
          
           
               
                 Cycle: 5  
                 0.7359 
                 nM 
                  7.359E−10 
                 30 
                 1.07346E+18 
                 2.729222012 
               
               
                 Cycle: 6  
                 1.4719  
                 nM 
                  1.4719E−09 
                 30 
                 1.07346E+18 
                 4.866918331 
               
               
                 Cycle: 7  
                 2.94375 
                 nM 
                 2.94375E−09 
                 30 
                 1.07346E+18 
                 6.830413213 
               
               
                 Cycle: 8  
                 5.8875  
                 nM 
                  5.8875E−09 
                 30 
                 1.07346E+18 
                 5.581231192 
               
               
                 Cycle: 9  
                 11.775  
                 nM 
                  1.1775E−08 
                 30 
                 1.07346E+18 
                 2.692233789 
               
               
                 Cycle: 10  
                 23.55 
                 nM 
                  2.355E−08 
                 30 
                 1.07346E+18 
                 −1.728466535 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Model parameters used for estimating Denosumab-RANKL binding. 
               
             
          
           
               
                 k a    
                 k d   
                 K D    
                 R max   
                   
                 Chi 2   
                   
               
               
                 (1/Ms) 
                 (1/s) 
                 (M) 
                 (RU) 
                 t c   
                 (RU 2 ) 
                 U-value 
               
               
                   
               
               
                 235360.1515 
                 6.21706E−06 
                 2.64151E−11 
                 562.403892 
                 3.45471E+17 
                 8.62705 
                 11.64904 
               
               
                   
               
             
          
         
       
     
         [0067]    Association and dissociation curves of hOPG-hIgG1-Fc polypeptide at different rshRANKL concentrations are showed in  FIG. 2 . Correlated data for each curve is listed in Table 3. The model parameters of hOPG-hIgG1-Fc-RANKL binding estimated by Biacore X100 Evaluation software are listed in the Table 4. 
         [0068]    The affinity (K D ) of hOPG-hIgG1-Fc binding to RANKL is 4.85×10 −13 M. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Correlated data for each curve of hOPG-hIgG1-Fc-RANKL binding. 
               
             
          
           
               
                 Curve 
                 Rmax (RU) 
                 Conc (M) 
                 Flow (ul/min) 
                 RI (RU) 
                 kt (RU/Ms) 
               
               
                   
               
             
          
           
               
                 Cycle: 5  
                 1.0415 
                 nM 
                 44.96516 
                 1.04E−09 
                 30 
                 −1.0584 
                 4.33E+08 
               
               
                 Cycle: 6  
                 2.083  
                 nM 
                 87.28024 
                 2.08E−09 
                 30 
                 −6.8384 
                 4.33E+08 
               
               
                 Cycle: 7  
                 4.166 
                 nM 
                 205.2188 
                 4.17E−09 
                 30 
                 −23.1182 
                 4.33E+08 
               
               
                 Cycle: 8  
                 8.333 
                 nM 
                 567.9522 
                 8.33E−09 
                 30 
                 −29.2618 
                 4.33E+08 
               
               
                 Cycle: 9  
                 16.666  
                 nM 
                 639.3375 
                 1.67E−08 
                 30 
                 −4.20587 
                 4.33E+08 
               
               
                 Cycle: 11  
                 4.166 
                 nM 
                 218.8427 
                 4.17E−09 
                 30 
                 −20.7052 
                 4.33E+08 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Model parameters of hOPG-hIgG1-Fc-RANKL binding estimated via Biacore X100. 
               
             
          
           
               
                 k a  (1/Ms) 
                 k d  (Vs) 
                 K D  (M) 
                 t c   
                 Chi 2  (RU 2 ) 
                 U-value 
               
               
                   
               
               
                 13476795.16 
                 6.54E−06 
                 4.85E−13 
                 1.39E+08 
                 5.541974 
                 5.30225 
               
               
                   
               
             
          
         
       
     
         [0069]    Thus, the affinity (K D ) of hOPG-hIgG1-Fc polypeptide of SEQ ID NO. 1 binding to rhsRANKL was estimated to be about 4.9×10 −13  M, which is approximately 50 times higher compared to that of commercially available Denosumab, which under substantially similar experimental conditions was estimated to be about 2.6×10 −11 M. 
       Example 3 
     Formulation Stability Study of hOPG-hIgG1-Fc Polypeptide 
       [0070]    hOPG-hIgG1-Fc polypeptide of SEQ ID NO. 1 was expressed and purified essentially as described in the forgoing. Long-term stability study of the hOPG-hIgG1-Fc polypeptide performed to estimate product stability at 2-8° C. hOPG-hIgG1-Fc polypeptide accelerated stability study at 40° C. was performed to evaluate product stability in formulation buffers of the different compositions. The polypeptide stability was analyzed by SEC HPLC. Integration of the SEC HPLC chromatograms was performed to evaluate hOPG-hIgG1-Fc polypeptide monomers, aggregates and degradation products and to monitor the changes in the protein composition. hOPG-hIgG1-Fc polypeptide was aliquoted into screw capped vials. The aliquots were stored in the dark at designated temperatures during required periods of time. 
         [0071]    Materials and Equipment: 
         [0072]    All reagents used were at least HPLC grade: Milli-Q Water (or equivalent); Sodium Chloride (J T Baker); Sodium Phosphate Dibasic, Heptahydrate (Na 2 HPO 4 .7H 2 O, J T Baker) or Sodium Phosphate Dibasic Anhydrous (Na 2 HPO 4 , J T Baker); 6 N Hydrochloric Acid (J T Baker); Sodium Hydroxide 6N NaOH (BDH); Sodium Azide (Sigma Aldrich); Methanol (J T Baker); rhsRANKL (Alphamab, Inc.); goat anti-human IgG:HRP conjugate, (Perkin-Elmer). 
         [0073]    pH Meter (Corning Pinnacle 542); Analytical Balance (Mettler Toledo XS603S); Waters HPLC System with PDA and Empower Software; YMC-Pack Diol 300, 6.0 mm ID×30 cm, (YMC Catalog Number DL06S053006WT); G2000 SW×1, 7.5 mm×300 mm (TOSOH Bioscience); TSK Guard SW, 7.5 mm×75 mm (TOSOH Bioscience); Inline Filter with 2 μm frit (VWR Catalog Number 21511-442); Replacement 2 μm Frit (VWR Catalog Number 21511-423); Filter, PES, 1000 mL (Nalgene, Catalog Number 567-0020); Total Recovery Vial, screw top 12×32 mm cap with PTFE/Silicone septa (Waters); Cap/Septa 12×32 screw neck with bonded pre-slit PTFE/Silicone septa (Waters). 
         [0074]    Buffers: 
         [0075]    Mobile Phase buffers:
       100 mM NaPhosphate, 200 mM NaCl pH 7.0 (PBS) for YMC-Pack Diol 300 column   20 mM NaPhosphate, 300 mM NaCl, pH 7.4 buffer. Filtered and degassed for G2000SW×1 column       
 
         [0078]    Following drug formulation buffers were prepared:
   1. 25 mM NaPhosphate, 100 mM NaCl, 25 mM L-Arginine HCl, 10 mg/mL Sucrose, pH 6.3   2. 25 mM NaPhosphate, 100 mM NaCl, 25 mM L-Arginine HCl, 10 mg/mL Sucrose, pH 6.8   3. 20 mM Histidine, 100 mM NaCl, 25 mM L-Arginine HCl, 10 mg/mL Mannitol, pH 6.8   4. 20 mM Histidine, 50 mM NaCl, 25 mM L-Arginine HCl, 25 mg/mL Mannitol, pH 6.8   5. 20 mM Histidine, 50 mM NaCl, 25 mM L-Arginine HCl, 25 mg/mL Mannitol, 0.1 mg/mL Methionine, pH 6.8   6. 20 mM Histidine, 100 mM NaCl, 25 mM L-Arginine HCl, 10 mg/mL Mannitol, pH 6.3   7. 20 mM Histidine, 50 mM NaCl, 25 mM L-Arginine HCl, 25 mg/mL Mannitol, pH 6.3   8. 20 mM Histidine, 50 mM NaCl, 25 mM L-Arginine HCl, 25 mg/mL Mannitol, 0.1 mg/mL Methionine, pH 6.3   
 
         [0087]    Procedures: 
         [0088]    Samples were diluted with mobile phase to reach protein concentration of 1 mg/ml or 2 mg/mL. 
         [0089]    Chromatography parameters: 
         [0090]    Flow Rate: 0.5 ml/min 
         [0091]    Column Temperature: 25±3° C. 
         [0092]    Autosampler Temperature: 5±3° C. 
         [0093]    Injection Volume: 15 μl for samples with 2 mg/mL polypeptide concentration
       25 μl for samples with 1 mg/mL polypeptide concentration       
 
         [0095]    Detector Wavelength: 280 nm for samples with 2 mg/mL polypeptide concentration
       214 nm for samples with 1 mg/mL polypeptide concentration       
 
         [0097]    Run Time: 35 min 
         [0098]    Two lots of hOPG-hIgG1-Fc polypeptide individual preparations were tested, both at about 10 mg/ml total protein concentration formulated in 25 mM NaPhosphate, 100 mM NaCl, 25 mM L-Arginine HCl, 10 mg/mL Sucrose, at pH 6.3. 
         [0099]    Results of the 2-8° C. stability studies for two different preparations lots of hOPG-hIgG1-Fc polypeptide are summarized in Table 5 and Table 6 below. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 hOPG-hIgG1-Fc polypeptide composition  
               
               
                 at the stability study time points 
               
             
          
           
               
                   
                   
                   
                 Time points (days) 
               
             
          
           
               
                   
                 Sample 
                 Composition 
                 0 
                 67 
                 176 
               
               
                   
                   
               
             
          
           
               
                   
                 Lot No 1 
                 Monomer (%) 
                 94.3 
                 94.4 
                 93.9 
               
               
                   
                   
                 Aggregates (%) 
                 0.6 
                 1.3 
                 1.3 
               
               
                   
                   
                 Degradation products (%) 
                 5.1 
                 4.3 
                 4.8 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 hOPG-hIgG1-Fc polypeptide composition  
               
               
                 at the stability study time points 
               
             
          
           
               
                   
                   
                 Time points 
               
             
          
           
               
                 Sample 
                 Composition 
                 1 day 
                 4 weeks 
                 9 weeks 
                 12 weeks 
               
               
                   
               
             
          
           
               
                 Lot No 2 
                 Monomer (%) 
                 98.1 
                 97.2 
                 97.2 
                 97.1 
               
               
                   
                 Aggregates (%) 
                 0.38 
                 0.43 
                 0.2 
                 0.56 
               
               
                   
                 Degradation  
                 1.5 
                 2.4 
                 2.6 
                 2.4 
               
               
                   
                 products (%) 
               
               
                   
               
             
          
         
       
     
         [0100]    Preparation Lot No 2 was subjected accelerated stability tests at 40° C. in formulation buffers of various compositions. Formulation buffer of Lot No 2 preparation (25 mM NaPhosphate, 100 mM NaCl, 25 mM L-Arginine HCl, 10 mg/mL Sucrose, pH 6.3) was exchanged with the drug formulation buffers listed in the forgoing disclosure. Results of the accelerated stability study are summarized in Table 7 below. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 hOPG-hIgG1-Fc polypeptide composition at the accelerated  
               
               
                 stability study time points (formulation buffers F1-F8) 
               
             
          
           
               
                   
                   
                 Time points (days) 
               
             
          
           
               
                 Sample 
                 Composition 
                 0 
                 7 
                 14 
                 21 
                 28 
               
               
                   
               
             
          
           
               
                 F1-25 mM NaPhosphate, 100 mM NaCl, 25 mM L-Arginine HCl, 10 mg/mL Sucrose, pH 6.3 
               
             
          
           
               
                 Lot No 2  
                 Monomer (%) 
                 96.6 
                 94.2 
                 91.4 
                 87.3 
                 83.7 
               
               
                   
                 Aggregates (%) 
                 0.8 
                 1.6 
                 3.2 
                 4.9 
                 6.3 
               
               
                   
                 Degradation products (%) 
                 2.6 
                 4.2 
                 5.4 
                 7.8 
                 10.0 
               
             
          
           
               
                 F2-25 mM NaPhosphate, 100 mM NaCl, 25 mM L-Arginine HCl, 10 mg/mL Sucrose, pH 6.8 
               
             
          
           
               
                 Lot No 2 
                 Monomer (%) 
                 96.8 
                 92.6 
                 89.1 
                 83.4 
                 83.7 
               
               
                   
                 Aggregates (%) 
                 0.6 
                 2.1 
                 4.1 
                 6.3 
                 6.3 
               
               
                   
                 Degradation products (%) 
                 2.6 
                 5.3 
                 6.8 
                 10.3 
                 10.0 
               
             
          
           
               
                 F3-20 mM Histidine, 100 mM NaCl, 25 mM L-Arginine HCl, 10 mg/mL Mannitol, pH 6.8 
               
             
          
           
               
                 Lot No 2 
                 Monomer (%) 
                 96.6 
                 94.2 
                 91.3 
                 88.3 
                 84.8 
               
               
                   
                 Aggregates (%) 
                 0.6 
                 1.5 
                 2.4 
                 3.2 
                 3.7 
               
               
                   
                 Degradation products (%) 
                 2.8 
                 4.3 
                 6.3 
                 8.5 
                 11.5 
               
             
          
           
               
                 F4-20 mM Histidine, 50 mM NaCl, 25 mM L-Arginine HCl, 10 mg/mL Mannitol, pH 6.8 
               
             
          
           
               
                 Lot No 2 
                 Monomer (%) 
                 96.6 
                 94.3 
                 91.2 
                 88.2 
                 86.4 
               
               
                   
                 Aggregates (%) 
                 0.6 
                 1.6 
                 2.4 
                 3.8 
                 3.8 
               
               
                   
                 Degradation products (%) 
                 2.8 
                 4.1 
                 6.4 
                 8.0 
                 9.8 
               
             
          
           
               
                 F5-20 mM Histidine, 50 mM NaCl, 25 mM L-Arginine HCl,  
               
               
                 25 mg/mL Mannitol, 0.1 mg/mL Methionine, pH 6.8 
               
             
          
           
               
                 Lot No 2 
                 Monomer (%) 
                 96.8 
                 94.3 
                 85.8 
                 87.7 
                 86.7 
               
               
                   
                 Aggregates (%) 
                 0.6 
                 1.5 
                 2.1 
                 2.3 
                 3.1 
               
               
                   
                 Degradation products (%) 
                 2.6 
                 4.2 
                 12.1 
                 10.0 
                 10.2 
               
             
          
           
               
                 F6-20 mM Histidine, 50 mM NaCl, 25 mM L-Arginine HCl, 10 mg/mLMannitol, pH 6.3 
               
             
          
           
               
                 Lot No 2 
                 Monomer (%) 
                 96.8 
                 94.4 
                 93.4 
                 88.7 
                 88.1 
               
               
                   
                 Aggregates (%) 
                 0.6 
                 1.4 
                 1.9 
                 2.3 
                 2.2 
               
               
                   
                 Degradation products (%) 
                 2.6 
                 4.2 
                 4.7 
                 9.0 
                 9.7 
               
             
          
           
               
                 F7-20 mM Histidine, 100 mM NaCl, 25 mM L-Arginine HCl, 25 mg/mL Mannitol, pH 6.3 
               
             
          
           
               
                 Lot No 2 
                 Monomer (%) 
                 96.8 
                 94.4 
                 93.0 
                 91.2 
                 87.9 
               
               
                   
                 Aggregates(%) 
                 0.6 
                 1.4 
                 1.8 
                 3.0 
                 2.9 
               
               
                   
                 Degradation products (%) 
                 2.6 
                 4.2 
                 5.2 
                 5.8 
                 7.2 
               
             
          
           
               
                 F8-20 mM Histidine, 50 mM NaCl, 25 mM L-Arginine HCl,  
               
               
                 25 mg/mL Mannitol, 0.1 mg/mL Methionine, pH 6.3 
               
             
          
           
               
                 Lot No 2 
                 Monomer (%) 
                 96.4 
                 95.0 
                 92.8 
                 91.0 
                 88.6 
               
               
                   
                 Aggregates (%) 
                 0.6 
                 1.3 
                 1.8 
                 2.1 
                 2.0 
               
               
                   
                 Degradation products (%) 
                 3.0 
                 3.7 
                 5.4 
                 6.9 
                 9.4 
               
               
                   
               
             
          
         
       
     
         [0101]    As is apparent from the results summarized in Table 7, addition of mannitol into formulation buffer up to 10 mg/mL improved hOPG-hIgG1-Fc polypeptide composition stability, however pH increase of the formulation buffer from 6.3 to 6.8 did not affect product stability, neither did changes in NaCl concentration. 
         [0102]    Stability of Lot No 2 preparation was also tested at room temperature (RT). The preparation was diluted with 0.9% NaCl into three samples having total protein concentrations of 0.6, 1.2, 1.8 mg/mL, respectively. The samples were stored at room temperature for 24 h, and analyzed at 0 hr time point and 24 hr time point. hOPG-hIgG1-Fc polypeptide composition stability was analyzed with SEC HPLC, which monitors integrity of protein composition and ELISA, to assess the binding of hOPG-hIgG1-Fc to RANKL with acceptance criteria of 70-130% of the reference standard binding to RANKL. The results of the study are summarized in Table 8. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 hOPG-hIgG1-Fc polypeptide composition at the RT stability study time points 
               
             
          
           
               
                   
                 Control 10 mg/mL 
                 0.6 mg/mL 
                 1.2 mg/mL 
                 1.8 mg/mL 
               
             
          
           
               
                   
                 0 hr 
                 24 hr 
                 0 hr 
                 24 hr 
                 0 hr 
                 24 hr 
                 0 hr 
                 24 hr 
               
               
                   
               
               
                 Monomer (SEC-HPLC) 
                 95.12% 
                 94.63% 
                 94.19% 
                 93.56% 
                 95.08% 
                 94.29% 
                 95.04% 
                  94.57% 
               
               
                 Binding Activity 
                  94.9% 
                 — 
                  88.8% 
                 94.10% 
                  89.3% 
                 79.40% 
                  88.6% 
                 115.60% 
               
               
                 (ELISA) 
               
               
                   
               
             
          
         
       
     
         [0103]    As is apparent from the results summarized in Table 8, hOPG-hIgG1-Fc polypeptide 10 mg/ml stock diluted to 0.6 mg/mL, 1.2 mg/mL and 1.8 mg/mL and stored at room temperature for 24 hours demonstrated integrity of composition and binding activity similar to the reference standard. Therefore, the data confirmed stability of the post-reconstituted hOPG-hIgG1-Fc polypeptide solutions during time sufficient for the drug preparation and intravenous administration. 
         [0104]    Long-term stability of Lot No 2 preparation was tested at 2-8° C. The results of the study are summarized in Table 9. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 hOPG-hIgG1-Fc polypeptide 2-8° C. long term stability study results 
               
             
          
           
               
                 Assays 
                 Test Method 
                 Specification 
                 T = 0 
                 T = 3 months 
                 T = 6 months 
               
               
                   
               
               
                 Appearance 
                 999-GMP-064 
                 Clear, colorless, free 
                 Clear, colorless, free 
                 Clear, colorless, free 
                 Clear, colorless, free of 
               
               
                   
                   
                 of visible particles 
                 visible particles 
                 of visible particles 
                 of visible particles 
               
               
                 pH 
                 999-GMP-017 
                 6.1-6.5 
                 6.4 
                 6.4 
                 6.4 
               
               
                 A280 
                 774-01-001 
                 9.0-11.0 mg/mL 
                 10.1 mg/mL 
                 10.8 mg/mL 
                 10.0 mg/mL 
               
               
                 Coomassie 
                 774-01-004 
                 Conforms to 
                 Conforms to reference 
                 Conforms to reference 
                 Conforms to 
               
               
                 Reduced 
                   
                 reference standard 
                 standard 
                 standard 
                 reference standard 
               
               
                 Coomassie  
                 774-01-004 
                 Conforms to 
                 Conforms to reference  
                 Conforms to reference 
                 Conforms to 
               
               
                 Non-Reduced 
                   
                 reference standard 
                 standard 
                 standard 
                 reference standard 
               
               
                 RankL ELISA 
                 774-01-009 
                 70-130% 
                 100% 
                 89% 
                 81% 
               
               
                 IEF 
                 774-01-007 
                 FIO, report pl range 
                 16 bands; pl range 6.0- 
                 11 bands; pl range 6.0- 
                 11 bands; pl range 
               
               
                   
                   
                 and number of bands  
                 8.0 
                 7.8 
                 6.0-7.8 
               
               
                 SEC-HPLC 
                 774-01-002 
                 ≧92% monomer 
                 96% monomer 
                 97% monomer 
                 97% monomer 
               
               
                 Osmolality 
                 999-GMP-024 
                 280-350 mOsm/kg 
                 298 mOsm/kg 
                 301 mOsm/kg 
                 300 mOsm/kg 
               
               
                 Endotoxin 
                 774-01-028 
                 ≦0.25 EU/mg 
                 &lt;0.04 EU/mg 
                 N/A 
                 N/A 
               
               
                 Bioburden 
                 999-GMP-484 
                 ≦3 CFU/10 mL with 
                 &lt;1 CFU/10 mL with no 
                 N/A 
                 N/A 
               
               
                   
                   
                 no objectionable 
                 objectionable 
                   
                   
               
               
                   
                   
                 organisms 
                 organisms 
               
               
                   
               
             
          
         
       
     
         [0105]    As is apparent from the results summarized in Table 8, hOPG-hIgG1-Fc polypeptide of SEQ ID NO. 1 at 10 mg/ml, formulated in 25 mM NaPhosphate, 100 mM NaCl, 25 mM L-Arginine HCl, 10 mg/mL Sucrose, at pH 6.3, maintains structural stability and specific activity at least for 6 months. 
       Example 4 
     Single Dose Pharmacokinetics Study of hOPG-hIgG1-Fc Polypeptide in Primates 
       [0106]    Pharmacokinetic profile and the maximum tolerated dose of hOPG-hIgG1-Fc polypeptide following subcutaneous and intravenous (bolus) administration were studied in the 12 Cynomolgus monkeys (6 males and 6 females) that received single dose of hOPG-hIgG1-Fc polypeptide via subcutaneous administration at dose levels of 0.3, 3, 10, 30 and 100 mg/kg or via intravenous administration at dose levels of 0.3, 3 and 10 mg/kg. Subcutaneous dosing was done at a dose volume of 1 mL/kg, whilst intravenous dosing was done at a dose volume of 2 mL/kg. The test substance concentration was 9.73 mg/mL, for higher dose levels the dose volume were adjusted (based on animals weight) to achieve the target dose level/s. 
         [0107]    The following vehicle was used for the preparation of the test substance to the required dose concentrations; formulation buffer (1% w/v Sucrose, 100 mM Sodium Chloride, 20 mM L-Arginine Hydrochloride, 25 mM Sodium Bicarbonate, final adjusted pH of 6.3). The vehicle was stored refrigerated and used within one week after preparation. 
         [0108]    The animals were approximately 2-4 years and weighed 2-4 kg at the time of study commencement. The animals were observed twice daily for clinical signs. Body weight was recorded prior to each dose escalation. Food consumption was visually assessed daily during the study. Blood samples for clinical pathology investigations of haematology and clinical chemistry were collected once pre-trial and 24 h after each dose level. 
         [0109]    The animals were initially allocated to 2 dose groups and treated as follows: 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                 Dose  
                 Dose  
                 Dose  
                   
               
               
                 Route of 
                 Level 
                 Concentration 
                 Volume 
                 No. of Animals 
               
             
          
           
               
                 Administration 
                 (mg/kg) 
                 (mg/mL) 
                 (mL/kg) 
                 Male 
                 Female 
               
               
                   
               
             
          
           
               
                 Subcutaneous 
                 0.3 
                 0.3 
                 1 
                 1 
                 1 
               
               
                 injection 
                 3 
                 3 
                 1 
                 1 
                 1 
               
               
                   
                 10 
                 9.73 
                 1 
                 1 
                 1 
               
               
                 Intravenous 
                 0.3 
                 0.15 
                 2 
                 1 
                 1 
               
               
                 (bolus) 
                   
                   
                   
                   
                   
               
               
                 injection 
               
               
                   
               
             
          
         
       
     
         [0110]    The animals were subsequently dosed as follows: 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                 Dose  
                 Dose  
                 Dose  
                   
               
               
                 Route of 
                 Level 
                 Concentration 
                 Volume 
                 No. of Animals 
               
             
          
           
               
                 Administration 
                 (mg/kg) 
                 (mg/mL) 
                 (mL/kg) 
                 Male 
                 Female 
               
               
                   
               
             
          
           
               
                 Subcutaneous 
                 10 
                 4.85 
                 2.06 
                 1* 
                 1* 
               
               
                 injection 
                 30 
                 9.73 
                 3.08 
                 1** 
                 1** 
               
               
                 Intravenous 
                 3 
                 1.5 
                 2 
                 1* 
                 1* 
               
               
                 (bolus) injection 
                 10 
                 5 
                 2 
                 1** 
                 1** 
               
               
                   
               
               
                 *Animals previously dosed with hOPG-hIgG1-Fc polypeptide at 0.3 mg/kg 
               
               
                 **Naïve animals 
               
             
          
         
       
     
         [0111]    Additional subcutaneous dosing for 2 animals was done to evaluate the liver enzyme activity (i.e. Aspartate Aminotransferase, Alanine Aminotransferase and Lactate Dehydrogenase), dosing was as follows: 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                 Dose  
                 Dose  
                 Dose  
                   
               
               
                 Route of 
                 Level 
                 Concentration 
                 Volume 
                 No. of Animals 
               
             
          
           
               
                 Administration 
                 (mg/kg) 
                 (mg/mL) 
                 (mL/kg) 
                 Male 
                 Female 
               
               
                   
               
               
                 Subcutaneous 
                 10 
                 4.85 
                 2.06 
                 1*** 
                 1*** 
               
               
                 injection 
               
               
                   
               
               
                 ***Animals previously dosed with hOPG-hIgG1-Fc polypeptide at 3.0 mg/kg via subcutaneous route 
               
             
          
         
       
     
         [0112]    Two animals received a single subcutaneous injection of 100 mg/kg followed by a 2 week post dose observation period. Blood samples were collected for liver enzyme activity. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                 Dose  
                 Dose  
                 Dose  
                   
               
               
                 Route of 
                 Level 
                 Concentration 
                 Volume 
                 No. of Animals 
               
             
          
           
               
                 Administration 
                 (mg/kg) 
                 (mg/mL) 
                 (mL/kg) 
                 Male 
                 Female 
               
               
                   
               
               
                 Subcutaneous 
                 100 
                 9.73 
                 10.3 
                 1 
                 1 
               
               
                 injection 
               
               
                   
               
             
          
         
       
     
         [0113]    Both animals were previously dosed with hOPG-hIgG1-Fc polypeptide at 0.3 and 10 mg/kg via subcutaneous route and at 0.3 and 3 mg/kg via intravenous route, respectively. 
         [0114]    Blood samples for pharmacokinetic investigations were obtained at designated time points at each dose escalation. 
         [0115]    A pharmacokinetic analysis on hOPG-hIgG1-Fc polypeptide plasma levels was undertaken using a non-compartmental method using Phoenix™ for WinNonlin® (version 6.1, from Pharsight Corporation). C 0  (extrapolated concentration at T=0 For IV administration), Cmax (maximal concentration for SC administration) were obtained from the observed individuals values. AUC last  (Area Under the Curve to the last data point) were determined by mixed logarithmic-linear regression. Results from the PK data showed the C max  of hOPG-hIgG1-Fc polypeptide to be approximately 6-8 h following subcutaneous administration at dose levels of 0.3, 3, 10 and 30 mg/kg, and approximately 1 h following intravenous injection at dose levels of 0.3 and 3 and 10 mg/kg. Clearance of hOPG-hIgG1-Fc polypeptide was approximately 168 h post dose for both intravenous and subcutaneous routes of administrations. For the SC route, hOPG-hIgG1-Fc polypeptide was not always immediately absorbed as showed by a T lag  of 1 h and 2 h observed at the lowest dose (0.3 mg/kg) and for animal 0590 (3 mg/kg), respectively. The T half  values indicated that the elimination of hOPG-hIgG1-Fc polypeptide was relatively slow with a T half  of about 55 h and 45 h for the IV and SC route, respectively, based on the 3 and 10 mg/kg doses values. For the IV route the T half  ranged from 27.5 h to 57.8 h and ranged from 35.5 h to 53.3 h for the SC route. The elimination rate showed no concomitant increase with dose rate. The elimination was similar for the both routes. For the IV and SC routes, the Cl and V d  were similar irrespective of the administered dose. For the IV route, the Cl and V d  ranged between 0.75 and 1.22 mL/h/kg and between 48.3 and 76.6 mL/kg, respectively. The corresponding ranges, for the SC route, were between 0.97 to 1.69 mL/h/kg and between 65.3 and 94.6 mL/kg. Cl and V d  did not increase with the increasing dose. These results showed that the T half , Cl and V d  were independent of the administered dose. The Linearity of exposure for hOPG-hIgG1-Fc was only determined graphically for the SC route, from Cmax and AUClast by linear regression and these are presented in  FIG. 4 . Exposure increased linearly, R 2 =0.96 and 0.98 for AUC last  and C max , respectively. 
         [0116]    For the SC route, based the C max  values, it appeared that the dose proportionality can be showed ( FIG. 5 ). The mean Cmax corrected by the dose were 6410, 11309, 10409 and 11739 (ng/mL)/(mg/kg) for 0.3, 3, 10 and 30 mg/kg doses, respectively. The dose proportionality was clearly demonstrated from 3 to 0.3 mg/kg doses. The exposure increased more than proportionality between 0.3 mg/kg dose in comparison to the 3 others. Actually, for a 10 fold increase in dose between 0.3 and 3 mg/kg, for a 33.3 fold increase between 0.3 and 10 mg/kg and for a100 fold increase in dose between 0.3 and 30 mg/kg, there were 17.6, 54.1 and 183.1 fold increase in C max , respectively. The corresponding values for AUC last  were 82.3, 249.5 and 1095.1, respectively which showed that the exposure increased for about 10 fold more than the dose ratios. 
         [0117]    Between 3 and 10 mg/kg, relative dose proportionality was showed based on the C max /D and AUC last  /D and was confirmed by the dose ratio calculation which was closed to the theoretical dose ratios. 
         [0118]    For the IV route, the exposure increased slightly more than the dose proportionality, for a 3.3 fold increase in dose there was a 4.3 and 4.1 increased in AUC last  and C max , respectively. 
         [0119]    Thus, AUC last  and C max  increased linearly (R 2 ≈1.0) with a relatively dose-proportional exposure only from 3 to 30 mg/kg. These results showed that the systemic exposure increased after SC administration almost proportionally with the increasing dose from 3 to 30 mg/kg. 
         [0120]    hOPG-hIgG1-Fc polypeptide bioavailability was estimated using the mean values obtained at 3 and 10 mg/kg and represented 88.6% and 59.2% at 3 and 10 mg/kg, respectively. It seemed that hOPG-hIgG1-Fc polypeptide had a better bioavialability at 3 mg/kg (88.6%) in comparison to 10 mg/kg (59.2%). 
         [0121]    Thus, as is apparent from the results of the study, all animals were exposed to hOPG-hIgG1-Fc polypeptide at both administration routes evaluated and at all administered doses. After SC administration, systemic exposure to hOPG-hIgG1-Fc polypeptide (AUC last  and C max ) increased linearly with the increasing dose with relatively dose-proportional exposure only from 3 to 30 mg/kg. The elimination rate was not affected by the increasing dose and was similar for the both routes. The Cl and V d  were similar irrespective of the administered dose whatever the administration route. The results showed that the T half , Cl and Vd were independent of the administered dose. The results suggest that hOPG-hIgG1-Fc polypeptide had a better bioavailability at 3 mg/kg (88.6%) in comparison to 10 mg/kg (59.2%). 
         [0122]    Mean plasma pharmacokinetic parameters for hOPG-hIgG1-Fc polypeptide after single subcutaneous and intravenous doses in primates are summarized in Table 9 below. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 Summary of mean plasma pharmacokinetic parameters for hOPG-hIgG1-Fc 
               
               
                 polypeptide after single subcutaneous and intravenous doses in primates 
               
             
          
           
               
                   
                   
                   
                 AUC 
                 Cmax 
                 T 1/2   
                 CL 
                 V 
               
               
                 Species 
                 Route 
                 Dose 
                 (ng.h/mL) 
                 (ng/mL) 
                 (h) 
                 (mL/h/kg) 
                 (mL/kg) 
               
               
                   
               
             
          
           
               
                 Cynomolgus 
                 SC 
                 0.3 
                 mg/kg 
                 27651 
                 1923 
                 NC* 
                 NC 
                 NC 
               
               
                 monkeys 
                   
                 3 
                 mg/kg 
                 2276384 
                 33928 
                 48.2 
                 1.25 
                 84.5 
               
               
                   
                   
                 10 
                 mg/kg 
                 6899231 
                 104091 
                 38.9 
                 1.38 
                 76.0 
               
               
                   
                   
                 30 
                 mg/kg 
                 30278949 
                 352175 
                 NC 
                 NC 
                 NC 
               
               
                 Cynomolgus 
                 IV 
                 3 
                 mg/kg 
                 2647530 
                 112132 
                 42.6 
                 1.07 
                 62.4 
               
               
                 monkeys 
                   
                 10 
                 mg/kg 
                 11256849 
                 460319 
                 54.9 
                 0.78 
                 61.5 
               
               
                   
               
               
                 *NC—Not calculated 
               
             
          
         
       
     
       Example 5 
     Repeat-Dose Pharmacokinetics Study of hOPG-hIgG1-Fc Polypeptide in Primates 
       [0123]    A repeat-dose pharmacokinetic analysis was completed during the toxicokinetic study in Cynomolgous monkeys, where four groups of 3 males and 3 females each from the main and the recovery group were treated twice weekly for 2 successive weeks by subcutaneous injection at concentrations of 0, 0.3, 3 and 10 mg/kg (n=3 animals per sex per group). Toxicokinetic analysis was undertaken only on animals administered the hOPG-hIgG1-Fc polypeptide. 
         [0124]    A toxicokinetic analysis on plasma levels was undertaken using a non-compartmental method (for each day of kinetics, on Day 1 and on Day 13) using Phoenix™ for WinNonlin® (version 6.1, from Pharsight Corporation). Thus, the pharmacokinetic parameters were compared after administrations done on Days 1 and 13, respectively. 
         [0125]    The study demonstrated the following. No quantifiable concentrations were detected in the control group (Group 1) even when hOPG-hIgG1-Fc polypeptide were measured in one male and one female of this group. All animals were exposed to hOPG-hIgG1-Fc polypeptide twice weekly for 2 weeks at the doses of 0.3, 3 and 10 mg/kg. Systemic exposure to hOPG-hIgG1-Fc polypeptide (mean AUC 72  and mean C max ) increased with the increasing dose in both males and females. Moderate accumulation of hOPG-hIgG1-Fc polypeptide was observed after a 2-week administration. The administration period did not depend on the administered dose levels. The C max  and AUC 72  increased following repeat administration at all dose levels in both males and females, as indicated by the ratio of AUC 72  between Day 13 and Day 1. This ratio ranged from 0.28 to 2.50 confirming a modest plasma accumulation of hOPG-hIgG1-Fc polypeptide. The accumulation increased with the administered dose level. No gender effect was showed higher than the intra-individual variability. No clear gender difference can be concluded and it can be considered that there was no difference between males and females. With respect to gender differences, the exposures were similar in both males and females across the different doses from 0.3 to 10 mg/kg. On day 1 and day 13, the systemic plasma exposure of hOPG-hIgG1-Fc polypeptide increased more than dose-proportionally between 0.3 and 10 mg/kg in both genders. No dose proportionality was observed, except on day 1, for females between the intermediate and the low doses in regards of the AUC 72  and for males between the high and low dose based on the C max . On day 13, the increase of plasma exposure was at least 5 fold higher than the targeted dose ratio at the high level. 
         [0126]    Mean plasma pharmacokinetic parameters for hOPG-hIgG1-Fc polypeptide after repeat-dose subcutaneous administration in primates are summarized in Table 10 below. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
             
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 10 
               
               
                   
               
               
                 Summary of mean plasma pharmacokinetic parameters in male  
               
               
                 and female Cynomolgous monkeys (N = 3 per sex per dose  
               
               
                 group) for hOPG-hIgG1-Fc polypeptide after twice weekly  
               
               
                 subcutaneous dosing for two weeks 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Dose (mg/kg) 
               
             
          
           
               
                   
                 0.3 
                 3 
                 10 
               
             
          
           
               
                   
                 Occasion 
               
               
                   
                 Day 1 
               
               
                   
                 Sex 
               
             
          
           
               
                   
                 Male 
                 Female 
                 Male 
                 Female 
                 Male 
                 Female 
               
               
                   
               
               
                 AUC 72   
                 452067 
                 235964 
                 2673970 
                 3203140 
                 14821856 
                 15224149 
               
               
                 (ng ·  
                   
                   
                   
                   
                   
                   
               
               
                 h/mL) 
                   
                   
                   
                   
                   
                   
               
               
                 C max    
                 14197 
                 5365 
                 45623 
                 52601 
                 233410 
                 250589 
               
               
                 (ng/mL) 
                   
                   
                   
                   
                   
                   
               
               
                 T max  (h) 
                 38.00 
                 8.00 
                 24.00 
                 24.00 
                 72.00 
                 24.00 
               
             
          
           
               
                 AUC 72    
                 1.92 
                 0.83 
                 0 97 
               
               
                 (M/F) 
                   
                   
                   
               
               
                 C max    
                 2.65 
                 0.87 
                 0.93 
               
               
                 (M/F) 
                   
                   
                   
               
               
                 Dose  
                 10 (I/L) 
                 3.33 (H/I) 
                 33.3 (H/L) 
               
               
                 Ratio 
                   
                   
                   
               
             
          
           
               
                 AUC 72    
                 5.91 
                 13.57 
                 5.54 
                 4.75 
                 32.8 
                 64.5 
               
               
                 Ratio 
                   
                   
                   
                   
                   
                   
               
               
                 C max    
                 3.21 
                 9.80 
                 5.12 
                 4.76 
                 16.44 
                 46.71 
               
               
                 Ratio 
               
               
                   
               
             
          
           
               
                   
                 Occasion 
               
               
                   
                 Week 2 (Day 13) 
               
               
                   
               
             
          
           
               
                 AUC 72   
                 189783 
                 210980 
                 4638633 
                 6785492 
                 33486023 
                 35935204 
               
               
                 (ng ·  
                   
                   
                   
                   
                   
                   
               
               
                 h/mL) 
                   
                   
                   
                   
                   
                   
               
               
                 C max    
                 4262 
                 3965 
                 74853 
                 110957 
                 547783 
                 606545 
               
               
                 (ng/mL) 
                   
                   
                   
                   
                   
                   
               
               
                 T max  (h) 
                 8.00 
                 8.00 
                 24.00 
                 8.00 
                 8.00 
                 24.00 
               
             
          
           
               
                 Dose  
                 10 (I/L) 
                 3.33 (H/I) 
                 33.3 (H/L) 
               
               
                 Ratio 
                   
                   
                   
               
             
          
           
               
                 AUC 72    
                 24.44 
                 32.16 
                 7.22 
                 5.30 
                 176.4 
                 170.3 
               
               
                 Ratio 
                   
                   
                   
                   
                   
                   
               
               
                 C max    
                 17.56 
                 27.98 
                 7.32 
                 5.47 
                 128.51 
                 152.96 
               
               
                 Ratio 
                   
                   
                   
                   
                   
                   
               
               
                 R ac  (day  
                 0.28 
                 1.07 
                 1.76 
                 2.11 
                 2.50 
                 2.39 
               
               
                 13/day 1) 
                   
                   
                   
                   
                   
                   
               
               
                   
               
             
          
         
       
     
       Example 6 
     Single Dose Pharmacokinetics Study of hOPG-hIgG1-Fc Polypeptide in Humans 
       [0127]    Pharmacokinetic profile of hOPG-hIgG1-Fc polypeptide of SEQ ID NO. 1 following subcutaneous administration were studied in healthy male volunteers (ages 19-39) who received single dose of hOPG-hIgG1-Fc polypeptide via subcutaneous administration at dose levels of 10 mg, 30 mg and 60 mg. The drug formulation of hOPG-hIgG1-Fc polypeptide used was at about 10mg/ml total protein concentration formulated in 25 mM NaPhosphate, 100 mM NaCl, 25 mM L-Arginine HCl, 10 mg/mL Sucrose, at pH 6.3. The blood levels hOPG-hIgG1-Fc polypeptide at various time points post administration for the three doses tested are summarized in Tables 11-13. The results indicate a substantial post-subcutaneous administration bioavailability of the drug substance under study in systemic circulation in human subjects. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 11 
               
             
             
               
                   
               
               
                 Blood levels of hOPG-hIgG1-Fc polypeptide in healthy volunteers  
               
               
                 after SC administration of a single 10 mg dose 
               
             
          
           
               
                 Time Point 
                 hOPG-hIgG1-Fc polypeptide (ng/mL) per subject ID 
               
             
          
           
               
                 (Hours) 
                 Ctrl 1 
                 Ctrl 2 
                 A** 
                 B 
                 C 
                 D 
                 E 
                 F 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 0.5 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 52.6 
               
               
                 4 
                 0 
                 0 
                 0 
                 18.1 
                 24.7 
                 0 
                 32.1 
                 98.2 
               
               
                 8 
                 0 
                 0 
                 0 
                 37.1 
                 69.3 
                 17.5 
                 196.4 
                 120.5 
               
               
                 9 
                 0 
                 0 
                 0 
                 32.5 
                 72.3 
                 17.8 
                 198.3 
                 116.6 
               
               
                 10 
                 0 
                 0 
                 0 
                 31.6 
                 86.3 
                 22.9 
                 215.2 
                 121.7 
               
               
                 11 
                 0 
                 0 
                 0 
                 28.7 
                 76.2 
                 18.4 
                 221.9 
                 113.7 
               
               
                 12 
                 0 
                 0 
                 0 
                 35.7 
                 78.4 
                 23.1 
                 234.4 
                 97.0 
               
               
                 16 
                 0 
                 0 
                 0 
                 29.5 
                 0 
                 64.9 
                 220.5 
                 91.6 
               
               
                 24 
                 0 
                 0 
                 0 
                 24.5 
                 105.5 
                 35.5 
                 215.9 
                 95.3 
               
               
                 48 
                 0 
                 0 
                 0 
                 52.5 
                 97.1 
                 48.2 
                 167.4 
                 123.2 
               
               
                 168 
                 0 
                 0 
                 0 
                 36.1 
                 53.4 
                 49.9 
                 67.0 
                 84.4 
               
               
                 288 
                 0 
                 0 
                 0 
                 26.6 
                 26.0 
                 28.6 
                 35.8 
                 49.1 
               
               
                 480 
                 0 
                 0 
                 0 
                 15.2 
                 0 
                 0 
                 0 
                 45.5 
               
               
                 648 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 40.0 
               
               
                   
               
               
                 * 0.0 denotes that value obtained is below Lowest Limit of Quantification (1.683 ng/mL) 
               
               
                 **Values were obtained for all time points, however were all below Lowest Limit of Quantification (1.683 ng/mL), thus cannot be reliably quantitated. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 12 
               
             
             
               
                   
               
               
                 Blood levels of hOPG-hIgG1-Fc polypeptide in healthy  
               
               
                 volunteers after SC administration of a single 30 mg dose 
               
             
          
           
               
                 Time Point 
                 hOPG-hIgG1-Fc polypeptide (ng/mL) per subject ID 
               
             
          
           
               
                 (Hours) 
                 Ctrl 1 
                 Ctrl 2 
                 A 
                 B 
                 C 
                 D 
                 E 
                 F 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 0.5 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 1 
                 0 
                 0 
                 0 
                 24.8 
                 17.7 
                 34.1 
                 0 
                 0 
               
               
                 2 
                 0 
                 0 
                 0 
                 67.2 
                 340.4 
                 37.7 
                 17.6 
                 0 
               
               
                 4 
                 0 
                 0 
                 44.6 
                 160.2 
                 1044.2 
                 116.7 
                 51.5 
                 74.2 
               
               
                 8 
                 0 
                 0 
                 123.2 
                 225.4 
                 1940.2 
                 288.4 
                 92.2 
                 187.0 
               
               
                 9 
                 0 
                 0 
                 143.3 
                 198.9 
                 2057.3 
                 279.2 
                 76.9 
                 175.1 
               
               
                 10 
                 0 
                 0 
                 163.1 
                 214.2 
                 2085.1 
                 352.7 
                 93.2 
                 221.6 
               
               
                 11 
                 0 
                 0 
                 167.6 
                 194.4 
                 2052.0 
                 303.8 
                 84.1 
                 205.2 
               
               
                 12 
                 0 
                 0 
                 173.1 
                 178.9 
                 2331.4 
                 353.6 
                 77.8 
                 205.7 
               
               
                 16 
                 0 
                 0 
                 196.6 
                 156.9 
                 2466.4 
                 298.3 
                 81.1 
                 181.4 
               
               
                 24 
                 0 
                 0 
                 103.9 
                 165.4 
                 2321.4 
                 262.6 
                 109.4 
                 171.3 
               
               
                 48 
                 0 
                 0 
                 168.1 
                 164.3 
                 1883.5 
                 224.1 
                 176.6 
                 164.1 
               
               
                 168 
                 0 
                 0 
                 69.9 
                 61.9 
                 264.9 
                 36.6 
                 100.5 
                 116.0 
               
               
                 288 
                 0 
                 0 
                 40.2 
                 0 
                 54.6 
                 30.3 
                 44.0 
                 59.1 
               
               
                 480 
                 0 
                 0 
                 0 
                 0 
                 24.5 
                 0 
                 22.6 
                 33.1 
               
               
                 648 
                 0 
                 0 
                 0 
                 0 
                 17.0 
                 45.5 
                 0 
                 19.1 
               
               
                   
               
               
                 * 0.0 denotes that value obtained is below Lowest Limit of Quantification (1.683 ng/mL) 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 13 
               
             
             
               
                   
               
               
                 Blood levels of hOPG-hIgG1-Fc polypeptide in healthy volunteers after SC administrsingle 60 mg dose 
               
             
          
           
               
                 Time Point 
                 hOPG-hIgG1-Fc polypeptide (ng/mL) per subject ID 
               
             
          
           
               
                 (Hours) 
                 Ctrl 1 
                 Ctrl 2 
                 A 
                 B 
                 C 
                 D 
                 E 
                 F 
               
               
                   
               
             
          
           
               
                   0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   0.5 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   1 
                 0 
                 0 
                 0 
                 83.6 
                 0 
                 0 
                 0 
                 0 
               
               
                   2 
                 0  
                 0 
                 191.5 
                 836.0 
                 56.5 
                 98.7 
                 0 
                 66.7 
               
               
                   4 
                 0 
                 0 
                 879.5 
                 1818.6 
                 222.9 
                 370.8 
                 276.3 
                 275.7 
               
               
                   8 
                 0 
                 0 
                 1505.1 
                 2963.0 
                 376.7 
                 567.1 
                 579.3 
                 686.7 
               
               
                   9 
                 0 
                 0 
                 1392.3 
                 2744.2 
                 365.4 
                 632.7 
                 719.7 
                 739.3 
               
               
                  10 
                 0 
                 0 
                 1605.2 
                 3054.6 
                 450.6 
                 720.5 
                 732.5 
                 1048.0 
               
               
                  11 
                 0 
                 0 
                 1672.7 
                 3231.3 
                 453.9 
                 683.7 
                 717.3 
                 1028.3 
               
               
                  12 
                 0 
                 0 
                 1987.3 
                 3293.0 
                 434.6 
                 708.4 
                 700.6 
                 1026.4 
               
               
                  16 
                 0 
                 0 
                 1860.1 
                 3226.6 
                 368.8 
                 903.4 
                 749.4 
                 1180.0 
               
               
                  24 
                 0 
                 0 
                 1749.6 
                 3252.6 
                 547.9 
                 834.8 
                 718.1 
                 1508.0 
               
               
                  48 
                 0 
                 0 
                 1849.4 
                 3041.6 
                 971.8 
                 1119.4 
                 1026.1 
                 1766.0 
               
               
                 168 
                 0 
                 0 
                 523.7 
                 1015.2 
                 409.6 
                 753.5 
                 444.6 
                 1804.0 
               
               
                 288 
                 0 
                 0 
                 251.1 
                 339.6 
                 113.0 
                 174.7 
                 182.3 
                 575.5 
               
               
                 480 
                 0 
                 0 
                 81.5 
                 0 
                 41.7 
                 62.7 
                 84.1 
                 321.5 
               
               
                 648 
                 0 
                 0 
                 43.5 
                 0 
                 37.6 
                 0 
                 71.1 
                 180.6 
               
               
                   
               
               
                 *0.0 denotes that value obtained is below Lowest Limit of Quantification (1.683 ng/mL) 
               
             
          
         
       
     
       Example 7 
     Human Tissue Binding Characteristics Study of hOPG-hIgG1-Fc Polypeptide 
       [0128]    The purpose of this immunohistochemistry (IHC) study is to determine tissue binding characteristics for hOPG-hIgG1-Fc polypeptide of SEQ ID NO. 1, and to compare the binding pattern to a commercially available therapeutic (Prolia). A commercially available TNF-alpha binding therapeutic etanercept (Enbrel) was used as an isotype control. 
         [0129]    Methods 
         [0130]    Titration experiments were conducted with 3 protein therapeutics, hOPG-hIgG1-Fc-FITC, PROLIA, and ENBREL (FITC (fluorescein isothiocyanate) labeling performed by Covance) to establish concentrations that would result in minimal background and maximal detection of signal. 
         [0131]    Serial dilutions were performed at 20 μg/ml, 10 μg/ml, 5 μg/ml, and 2.5 μg/ml on fresh frozen human tissues supplied by LifeSpan. In addition ENBREL was further titered at 1.25 μg/ml, 0.6 μg/ml, and 0.3 μg/ml. hOPG-hIgG1-Fc-FITC, PROLIA-FITC, and the isotype control therapeutic ENBREL-FITC were used as the primary binding reagents, and the principal detection system consisted of an anti-FITC mouse secondary antibody (Sigma-Aldrich, catalog# F5636), followed by an anti-mouse secondary antibody (Vector, BA-2000), and a ABC-AP kit (AP=alkaline phosphatase secondary, Vector, AK-5000) with a Red substrate kit (Vector, SK-5100), which was used to produce a fuchsia-colored deposit. Tissues were also stained with positive control antibodies (CD31 and vimentin) to ensure that the tissue antigens were preserved and accessible for immunohistochemical analysis. Only tissues that were positive for CD31 and vimentin staining were selected for the remainder of the study. The negative controls consisted of performing the entire immunohistochemistry procedure on adjacent sections in the absence of primary reagents, or in the absence of both the primary reagent and the anti-FITC secondary antibody (using the anti-mouse tertiary and all other downstream reagents in all cases). The slides were interpreted by a pathologist and each reagent was evaluated for the presence of specific signal, level of background, and concordance with expression results reported in the literature. Staining intensity was recorded on a 0-4 scale (0=negative, 1=blush, 2=faint, 3=moderate, 4=strong). Slides were imaged with a DVC 1310C digital camera coupled to a Nikon microscope. Experimental results are summarized in Table 14 below. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 14 
               
             
             
               
                   
               
               
                 Results of the IHC study in human tissue 
               
             
          
           
               
                   
                   
                   
                   
                   
                   
                 CD31/ 
                   
                 hOPG-hIgG1  
                 PROLIA 
                 ENBREL 
                 No Primary 
               
               
                 Sam- 
                   
                   
                   
                 Path- 
                   
                 Vimentin 
                   
                 5 ug/ml 
                 2.5 ug/ml 
                 1.25 ug/ml 
                 With 
               
               
                 ple 
                 Sex 
                 Age 
                 Tissue 
                 ology 
                 Type 
                 Validation 
                 Cell Type 
                 IHC Score 
                 IHC Score 
                 IHC Score 
                 Secondary 
               
               
                   
               
             
          
           
               
                 1 
                 M 
                 70 
                 Bone  
                 Normal 
                 Normal 
                 P 
                 Erythroid precursor 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                   
                   
                 Marrow 
                   
                   
                   
                 Myeloid precursor 
                 2 
                 2-3 
                 0-1 
                 0 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Megakaryocytes 
                 2-3 
                 3 
                 2-3 
                 1 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Macrophages 
                 1-2 
                 2-3 
                 2 
                 0 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Granulocytes 
                 3 
                 3-4 
                 0-1 
                 0 
               
               
                 2 
                 M 
                 58 
                 Bone  
                 Normal 
                 Normal 
                 P 
                 Erythroid precursor 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                   
                   
                 Marrow 
                   
                   
                   
                 Myeloid precursor 
                 2 
                 2 
                 0 
                 0 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Megakaryocytes 
                 2-3 
                 2-3 
                 1 
                 0 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Macrophages 
                 1-2 
                 2 
                 0 
                 0 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Granulocytes 
                 3 
                 3 
                 0 
                 occ 2 
               
               
                 3 
                 M 
                 76 
                 Bone  
                 Normal 
                 Normal 
                 P 
                 Erythroid precursor 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                   
                   
                 Marrow 
                   
                   
                   
                 Myeloid precursor 
                 2 
                 2 
                 0-1 
                 0 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Megakaryocytes 
                 2-3 
                 2-3 
                 1 
                 0 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Macrophages 
                 2 
                 2 
                 1 
                 0 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Adipocytes 
                 2 
                 2 
                 2 
                 2 
               
               
                 4 
                 M 
                 16 
                 Liver 
                 Normal 
                 Normal 
                 P 
                 Hepatocytes 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Bile Duct Epithelium 
                 0-1 
                 0 
                 0 
                 0 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Kupffer Cells 
                 1-2 
                 0-1 
                 0 
                 0 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Other 
                 0 
                 0 
                 0 
                 0 
               
               
                 5 
                 M 
                 55 
                 Small 
                 Normal 
                 Normal 
                 P 
                 Epithelium 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                   
                   
                 Intestine 
                   
                   
                   
                 Smooth Muscle 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Inflammatory 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Goblet Mucin,  
                 3 
                 3 
                 3 
                 3 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Brush B              
               
               
                   
               
               
                             indicates data missing or illegible when filed 
               
             
          
         
       
     
         [0132]    Results With hOPG-hIgG1-Fc-FITC 
         [0133]    hOPG-hIgG1-Fc-FITC, at a concentration of 5-10 μg/ml, showed faint to occasional moderate staining within lymphocytes in the tonsil, including the mantle zone, and within thymocytes in the thymus. Moderate staining was also seen in Hassall&#39;s corpuscles. The prostate was largely negative or showed occasional blush staining of smooth muscle. Within the testis, spermatogonia and occasional spermatocytes showed rare blush staining and Leydig cells were negative. The uterus showed blush staining of myometrial smooth muscle, and the ovary showed blush staining of stromal cells. The placenta showed moderate staining of syncytiotrophoblasts, and faint to moderate staining of endothelium and occasional stromal cells. The small intestine showed blush staining of epithelium with moderate to strong staining of the brush border and goblet cell mucin. Vessels, fibroblasts, ganglia, and smooth muscle of the submucosa and muscularis were negative. The liver section showed faint to moderate staining of hepatocytes, and faint to occasional moderate staining of sinusoidal lining cells. 
         [0134]    Results with PROLIA-FITC: 
         [0135]    PROLIA-FITC, at a concentration of 2.5-5 Vector g/ml, showed moderate to occasional strong staining within subsets of lymphocytes in the tonsil, particularly within the mantle zone of lymphoid follicles and faint to moderate staining of thymocytes, with occasional strong staining of medullary lymphocytes in the thymus. The prostate was negative for staining The testis showed rare blush staining of spermatogonia, but was largely negative in spermatocytes and Leydig cells. The uterus showed faint staining of myometrial smooth muscle, and the ovary section showed faint staining of vascular smooth muscle. The placenta showed moderate staining of syncytiotrophoblasts and faint to moderate staining of endothelium and occasional stromal cells. The small intestine showed faint staining of epithelium with moderate to strong staining of the brush border. Vessels, fibroblasts, ganglia, and smooth muscle of the submucosa and muscularis were negative. The liver section showed faint staining of hepatocytes. 
         [0136]    Results with ENBREL-FITC: 
         [0137]    ENBREL-FITC, at a concentration of 1.25 μg/ml, showed moderate to occasionally strong membranous staining of lymphocytes in the thymus and tonsil. The prostate showed faint to moderate staining of epithelial cells and faint staining of smooth muscle. The placenta showed moderate to occasionally strong staining of subsets of syncytiotrophoblasts faint to moderate staining of stromal cells and occasional cytotrophoblasts, and faint staining of endothelium. The ovary showed faint staining of stromal cells. The uterus sample showed faint staining of myometrial smooth muscle, and largely negative staining of vascular endothelium and vascular smooth muscle. Within the testis, spermatogonia and occasional spermatocytes showed moderate staining and Leydig cells were negative. The small intestine showed moderate staining of epithelium, strong staining of the brush border, and negative staining of submucosal vessels and fibroblasts. 
         [0138]    In summary, hOPG-hIgG1-Fc-FITC at 5 μg/ml and PROLIA-FITC at 2.5 μg/ml showed positive staining within lymphocytes of the tonsil and thymus, with positive staining of sinusoidal endothelium of the splenic red pulp. Both reagents also showed positive staining of placental trophoblasts and endothelium, faint staining of uterine myometrial smooth muscle, and were largely negative in the prostate. Hepatocytes were also positive with both reagents. Cell types that showed positive staining were very similar between PROLIA-FITC and hOPG-hIgG1-Fc-FITC. ENBREL-FITC also showed positive staining of lymphocytes, placental trophoblasts and endothelium, and myometrial smooth muscle, but also showed positive staining in prostate epithelium and seminiferous tubules of the testis, in contrast to the other two reagents. The pattern of staining of ENBREL-FITC showed differences compared to PROLIA-FITC and hOPG-hIgG1-Fc-FITC, which were more similar to one another. 
         [0139]    “Prolia”, “Xgeva” and “Enbrel” are registered trademarks of Amgen Inc., a Delaware Corporation. 
         [0140]    All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. 
         [0141]    While specific embodiments of the subject matter have been discussed, the above specification is illustrative and not restrictive. Many variations will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.