JP6550413B2 - Compositions derived from osteoprotegerin and uses thereof - Google Patents

Description

TECHNICAL FIELD In general, the invention relates to the field of biological medicine as well as its use in conditions associated with bone resorption, such as tumors. More specifically, the present invention relates to osteoprotegerin derived compositions that bind to receptor activators of the NF-kappa B ligand (RANKL).

BACKGROUND The approaches described in this section may be pursued, but not necessarily approaches that have been previously conceived or pursued. Thus, unless stated otherwise, it should not be assumed that any approach described in this section should be considered as prior art simply because it is included in this section.

  Human osteoprotegerin (OPG; GenBank: U94332.1) is a 401 amino acid protein that contains a 21 amino acid signal peptide, which is cleaved in front of glutamate 22, resulting in the maturation of 380 amino acids. It produces soluble proteins. OPG is a member of the tumor necrosis factor receptor (TNFR) family, which contains a TNFR-like domain rich in four cysteines in its N-terminal part. OPG has been shown to have a role in bone development, and mice lacking the OPG gene had an osteoporosis phenotype and gross skeletal abnormalities.

  OPG, produced by osteoblasts and bone marrow stromal cells, acts as a secreted decoy receptor that has no overt direct signaling function. OPG works by binding to the natural ligand-osteoprotegerin ligand (OPGL), also known as RANKL (receptor activator of NF-kappa B ligands). The binding between OPG and RANKL prevents RANKL from activating the cognate receptor RANK, an osteoclast receptor which is crucial for osteoclast differentiation, activation and survival.

  Recombinant OPG is present in monomeric and dimeric forms of apparent molecular weights of about 55 kDa and about 110 kDa, respectively. Cleavage of the N-terminal domain to residue cysteine 185 causes OPG inactivation, presumably by breaking the disulfide bond of the TNFR-like domain, whereas cleavage of the C-terminal part of the protein to residue 194 is biologically active Not change.

  Overexpression of OPG in transgenic mice causes severe osteopetrosis characterized by near complete absence of osteoclasts in mice. Conversely, ablation of the OPG gene causes severe osteoporosis in mice, indicating an important physiological role of OPG in regulating bone resorption. Secretion of OPG and RANKL from osteoblasts and stromal cells is regulated by a number of hormones and cytokines. The relative levels of OPG and RANKL production are thought to control the degree of bone resorption: expression of RANKL increases bone resorption while excess OPG has an adverse effect. Recombinant OPG blocks the effects of the vast majority of factors that stimulate osteoclasts in vitro and in vivo. OPG also inhibits bone resorption in various animal disease models, including ovariectomy, induced osteoporosis, malignant humoral hypercalcemia and experimental bone metastases. Thus, OPG may represent an effective therapeutic option for diseases associated with excessive osteoclast activity (Kosteneik PJ, Shalhoub V., Curr Pharm Des. 2001 May; 7 (8): 613-35).

  Denosumab is a high affinity fully human monoclonal antibody that binds human RANKL and inhibits its interaction with RANK and thus has a similar mechanism of action to OPG. Denosumab under the trade name Prolia has been approved by the US Food and Drug Administration (FDA) for the prevention and treatment of osteoporosis in postmenopausal women. Denosumab under the tradename Xgeva has been approved by the US 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 are currently underway for other bone remodeling related conditions, ie for bone metastases from other cancer forms.

  Thus, it is desirable to have a therapeutic composition that can bind to RANKL, have an acceptable pharmacological profile, and have broader therapeutic potential based on natural OPG molecules.

SUMMARY OF THE INVENTION 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 important 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.

  In one aspect, the invention provides a polypeptide that binds human RANKL. The polypeptide comprises a first amino acid sequence comprising amino acids 1 to 215 of human osteoprotegerin (GenBank: U94332.1). The polypeptide further comprises a second amino acid sequence comprising amino acids 103 to 329 of human immunoglobulin gamma-1 Fc (GenBank: J00228.1). The polypeptide may be capable of inhibiting RANKL-induced osteoclast differentiation in an in vitro cell-based assay with an EC 50 of about 50 ng / ml, or up to 10% at a concentration of 100 ng / ml. The polypeptide may comprise the amino acid sequence of SEQ ID NO: 1.

  In one aspect, the invention provides a therapeutic composition. The composition comprises a polypeptide that binds human RANKL. The polypeptide comprises a biologically active portion of human osteoprotegerin and an Fc portion of human immunoglobulin gamma-1. The polypeptide is substantially purified to homodimeric form in solution. The polypeptide may exhibit a half-life in systemic circulation of mice of at least 80 hours after subcutaneous administration at a dose of 5 mg / kg. The polypeptide substantially inhibits human breast cancer-induced osteolysis in a subcutaneous administration course of the composition at a dose of up to 1 mg / kg three times weekly at a dose of up to 1 mg / kg in a mouse osteolysis model It can be done.

  In one aspect, the invention provides a therapeutic composition. The composition comprises a first amino acid sequence comprising amino acids 1 to 215 of human osteoprotegerin. The polypeptide further comprises a second amino acid sequence comprising amino acids 103 to 329 of human immunoglobulin gamma-1 Fc. The polypeptide can be substantially purified to homodimeric form in solution. The polypeptide may exhibit a half-life in systemic circulation of mice of at least 80 hours after subcutaneous administration at a dose of 5 mg / kg. The polypeptide substantially inhibits human breast cancer-induced osteolysis in a subcutaneous administration course of the composition at a dose of up to 1 mg / kg three times weekly at a dose of up to 1 mg / kg in a mouse osteolysis model It can be done. The polypeptide may comprise the amino acid sequence of SEQ ID NO: 1.

  In one aspect, the invention provides an isolated nucleic acid encoding a polypeptide that binds human RANKL. The polypeptide comprises a first amino acid sequence comprising amino acids 1 to 215 of human osteoprotegerin. The polypeptide further comprises a second amino acid sequence comprising amino acids 103 to 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 the amino acid sequence of SEQ ID NO: 1. The nucleic acid can have a codon usage of the nucleic acid that is optimized for high expression of the polypeptide in mammalian cells. The nucleic acid may comprise the sequence of SEQ ID NO: 2. The nucleic acid may comprise an expression vector.

  In one aspect, the invention provides a heterologous expression system. The expression system comprises an expression vector comprising a nucleic acid sequence encoding a polypeptide that binds human RANKL. The polypeptide comprises a first amino acid sequence comprising amino acids 1 to 215 of human osteoprotegerin. The polypeptide further comprises a second amino acid sequence comprising amino acids 103 to 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 the amino acid sequence of SEQ ID NO: 1. The expression vector of the expression system can be contained in mammalian cells. The mammalian cells can be Chinese hamster ovary (CHO) cells. The expression level of the polypeptide in the expression system may be at least 125 mg per liter of cell culture.

  In one aspect, the invention provides the use of a substance for the 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 to 215 of human osteoprotegerin. The polypeptide further comprises a second amino acid sequence comprising amino acids 103 to 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 the amino acid sequence of SEQ ID NO: 1. The disease associated with bone resorption or remodeling may be carcinoma, breast cancer, prostate cancer, multiple myeloma, osteosarcoma, bone metastases from solid tumors, osteoporosis, rheumatoid arthritis, or psoriatic arthritis.

  In one aspect, the invention provides a method of treating or preventing a disease associated with bone resorption or remodeling. The method comprises administering a pharmaceutical composition comprising a polypeptide that binds a therapeutically effective amount of human RANKL to a patient in need of treating or preventing a disease associated with bone resorption or remodeling. The polypeptide comprises a first amino acid sequence comprising amino acids 1 to 215 of human osteoprotegerin. The polypeptide further comprises a second amino acid sequence comprising amino acids 103 to 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 the amino acid sequence of SEQ ID NO: 1. The disease associated with bone resorption or remodeling may be carcinoma, breast cancer, prostate cancer, multiple myeloma, osteosarcoma, bone metastases from solid tumors, osteoporosis, rheumatoid arthritis, or psoriatic arthritis.

  These and other aspects and advantages of the invention described herein will become apparent in view of the following drawings and detailed description.

  The following drawings and a brief description of the drawings are provided to aid the understanding of the present invention.

FIG. 1 schematically shows the map and annotated sequences of the pCDNA4-Fc plasmid used in cloning the polypeptide of the invention.

Figure 2 schematically shows the map and annotated sequences of the pKN002 plasmid used in cloning of the polypeptide of the invention.

FIG. 3 shows a typical transfection growth curve obtained in the process of producing stable cell lines for expressing the polypeptide of the invention.

FIG. 4 shows a size exclusion HPLC analysis chromatogram of a sample containing the polypeptide of SEQ ID NO: 1 after an anion exchange chromatography purification step.

FIG. 5 shows SDS-PAGE analysis (under reducing conditions) of a sample containing the polypeptide of SEQ ID NO: 1 after an anion exchange chromatography purification step.

FIG. 6 shows a hydroxylapatite chromatography elution profile showing excellent separation of samples containing the polypeptide of SEQ ID NO: 1.

FIG. 7 shows a size exclusion HPLC analysis chromatogram of a sample of the main peak of the eluate containing the polypeptide of SEQ ID NO: 1 from hydroxylapatite chromatography.

FIG. 8 shows X-ray images of typical mouse tibia from group 1 (subcutaneous administration of formulation buffer) 8 weeks after inoculation used for evaluation of osteolysis (the assigned score is each Shown below the image).

FIG. 9 is an X-ray image of a typical mouse tibia from group 2 (subcutaneous administration of polypeptide of SEQ ID NO: 1 at 1 mg / kg) 8 weeks after inoculation used for evaluation of osteolysis (Assigned scores are shown below each image).

FIG. 10 shows two-dimensional micro CT scan images for one representative mouse in each of groups 1, 2 and 5.

FIG. 11 shows three dimensional micro CT scan images for one exemplary mouse in each of groups 1, 2 and 5.

DETAILED DESCRIPTION OF THE INVENTION The teachings described herein are based, in part, on engineering a protein molecule comprising a biologically active N-terminal portion of OPG fused to the Fc portion of human IgG. In order to allow the recombinant production of such OPG-derived protein molecules, a DNA expression vector has been constructed to overproduce the protein molecule in a heterologous protein expression system, and mammalian cells It is prepared to stably express at high expression levels. Unexpectedly, protein molecules from recombinant sources formed homodimers and homotetramers in solution. Protein purification procedures are designed to make it possible to obtain physiologically relevant substantially pure homodimeric preparations of the protein molecule. Thus, purified protein molecules demonstrate a high degree of inhibition of RANKL induced osteoclast differentiation in related in vitro cell based assays. Unexpectedly, the protein molecule exhibits an acceptable pharmacokinetic profile upon subcutaneous animal administration and does not exhibit any adverse toxicity or immunogenicity. The protein molecule also demonstrates high efficacy in predictive animal models of bone metastases induced by human breast cancer.

  The terms used herein generally have their ordinary meaning in the art within the context of the present invention and in the specific context in which each term is used. Specific terms are discussed below or elsewhere in the specification, and provide additional guidance to those skilled in the art in describing the compositions and methods of the invention and the manner of making them and their manner of use. Do. The scope or meaning of any use of a term is apparent from the specific context in which the term is used. "About" and "approximately" generally mean an acceptable degree of error for the quantity measured given the nature or accuracy of the measurement. Generally, a typical degree of error is within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, particularly in biological systems, the terms "about" and "approximately" may mean values which are within one order of magnitude of the predetermined value, preferably within 5 times, and more preferably within 2 times. A given quantity herein is, unless otherwise stated, an approximate meaning by which the term "about" or "approximately" can be inferred when not expressly stated.

  The method of the invention may comprise the step of comparing the sequence comprising the wild type sequence and one or more mutants (sequence variants) to one another. Such comparisons generally involve alignments of polymer sequences, such as using sequence alignment programs and / or algorithms well known in the art (eg, BLAST, FASTA and MEGALIGN to name but a few examples). ). In such alignments, where the mutation involves insertion or deletion of residues, the sequence alignment does not include inserted or deleted residues and "gaps" in the polymer sequence (generally dashes, A person skilled in the art can easily understand to introduce or "represented by" A ".

  Methods of the invention may include statistical calculations, such as determination of IC50 or EC50 values. Those skilled in the art will readily understand that such can be done using various commercially available software, such as PRISM (GraphPad Softqare Inc, La Jolla, Calif., USA) or the like. Can.

  "Homology" has, in all its grammatical forms and spelling variations, a "common evolutionary origin" that includes proteins from the superfamily in the same species of organism, and homologous proteins from different species of organism. The relationship between two proteins is shown. Such proteins (and the nucleic acids encoding them) have sequence homology as reflected in their sequence similarity, percent identity, or the presence of particular residues or motifs and conserved positions. However, in general usage and in the present application, the term "homologous" may mean sequence similarity when modified with adverbs such as "highly", even with respect to a common source of evolution. It does not have to be related.

  The term "sequence similarity" in all its grammatical forms indicates the degree of identity or correspondence between nucleic acid or amino acid sequences which may or may not share a common source of evolution.

  The terms "protein" and "polypeptide" are used interchangeably. Generally, OPG-derived proteins of the present teachings for use in mammals are expressed in mammalian cells that allow for appropriate post-translational modifications, such as CHO or HEK 293 cell lines, but other mammals Expression cell lines are also expected to be useful. Thus, it is expected that OPG-derived proteins can be post-translationally modified without substantially affecting their biological function.

  In one aspect, functional variants of OPG-derived protein molecules of the present teachings include fusion proteins having at least a biologically active portion of human OPG and one or more fusion domains. Well known examples of such fusion domains are polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, immunoglobulin heavy chain constant region (eg Fc), maltose binding protein (MBP), Or including but not limited to human serum albumin. Fusion domains can be selected to confer desired properties. For example, the OPG polypeptide moiety can be fused to a domain ("stabilizer" domain) that stabilizes the OPG polypeptide in vivo, optionally via a suitable peptide linker. The term "stabilization" refers to an increase in the half-life of a polypeptide in any systemic circulation, whether due to a decrease in destruction, a decrease in clearance, or other pharmacokinetic effects. Fusion with immunoglobulin Fc portions is known to confer desirable pharmacokinetic properties on certain proteins. Similarly, fusion to human serum albumin can provide desirable properties. Other types of fusion domains that may be selected are multimerized (eg, dimerization, tetramerization) to provide additional biological functions, such as the function of promoting accumulation at the site of action targeted in vivo. Including functional and functional domains.

In one aspect, the present invention provides a polypeptide comprising the first 215 amino acids of human OPG (GenBank: U94332.1) and then the 227 amino acids of the Fc portion of human Ig gamma-1 (GenBank: J00228.1). . In an exemplary embodiment, a protein molecule of the invention comprises the amino acid sequence of SEQ ID NO: 1.
hOPG-hIgG1-Fc polypeptide (SEQ ID NO: 1)

In one aspect, the invention comprises a polypeptide comprising the first 215 amino acids of human OPG, then 227 amino acids of the Fc portion of human Ig gamma-1, optionally linked via a flexible linker. Provided is a recombinant DNA molecule having an open reading frame that encodes. In an example embodiment, the recombinant DNA molecule of the invention comprises the nucleotide sequence of SEQ ID NO: 2.
hOPG-hIgG1-Fc DNA (SEQ ID NO: 2)

  In one aspect, the present invention provides high expression of a polypeptide comprising the first 215 amino acids of human OPG and then the 227 amino acids of the Fc portion of human Ig gamma-1 and optionally linked via a flexible linker Provides a recombinant mammalian expression plasmid for This plasmid contains the cytomegalovirus (CMV) promoter driving transcription of the gene encoding the polypeptide, followed by bGH polyadenylation and transcription termination sequences. The plasmid also contains the pUC origin of replication and the β-lactamase gene that confers ampicillin resistance to support plasmid growth and selection in bacteria. The plasmid further contains the glutamine synthetase gene, which is a selectable marker that is widely used to establish stable CHOK1 and NSO cell lines. The plasmid of the present invention is illustratively shown in FIG.

In an example embodiment, the mammalian expression plasmid of the invention comprises the nucleotide sequence of SEQ ID NO: 3.
hOPG-hIgG1-Fc expression plasmid (SEQ ID NO: 3)

  In one aspect, the invention provides for the production of a polypeptide comprising the top 215 amino acids of human OPG and then the 227 amino acids of the Fc portion of human Ig gamma-1 and optionally linked via a flexible linker. The present invention provides a mammalian expression system for The expression system of the present invention comprises the top 215 amino acids of human OPG and then the 227 amino acids of the Fc portion of human Ig gamma-1 for high expression of polypeptides optionally linked via a flexible linker. And mammalian cells having a recombinant mammalian expression plasmid.

  In an example embodiment, the mammalian expression system of the invention comprises Chinese hamster ovary cells (CHO-K1) comprising a plasmid comprising the nucleotide sequence of SEQ ID NO: 3.

  In one aspect, the invention provides a method of treating a mammal achieved by a disorder associated with bone resorption or remodeling.

EXAMPLES The following examples illustrate the above aspects and other aspects of the present invention. These non-limiting examples are set forth to provide the skilled artisan with an illustrative manner in the manner in which the claimed compounds, compositions, articles, devices and / or methods are made and evaluated. The examples are intended as pure illustrations of the present invention, and do not limit the scope considered by the present inventors as the invention. Efforts are made to ensure accuracy with respect to numbers (eg, amounts, temperatures, etc.), but some errors and deviations should be considered.

Example 1 : Construction of a plasmid for expression of a polypeptide of the present invention First, a DNA encoding hOPG amino acid residues 1 to 215, immediately followed by a DNA encoding a leading fragment of hIgG1-Fc amino acid DNA fragments were chemically synthesized. At the 5 'end of the first DNA fragment, the Hind III restriction enzyme recognition site, Kozak sequence (GCCACC) and its signal sequence preceded the hOPG coding sequence. The hIgG1-Fc fragment-encoding DNA of the first DNA fragment contained at the 3 ', a Sac II restriction site.

  The synthetic gene was then subcloned into a pcDNA4-Fc vector containing DNA encoding the remaining hIgG1-Fc amino acids using previously engineered Hind III and Sac II restriction sites. The pcDNA4-Fc vector utilizes the cytomegalovirus (CMV) promoter to drive transcription of the gene of interest and has been shown to produce high level expression in a wide variety of mammalian cell lines. The vector contains bGH polyadenylation and transcription termination sequences. The vector also contains a pUC origin of replication and a β-lactamase gene that confers ampicillin resistance to support growth and selection in bacteria. FIG. 1 shows the map and annotated sequences of the pCDNA4-Fc plasmid.

  Finally, a second DNA fragment comprising DNA encoding hOPG residues 1 to 215 and residues 103 to 329 of hIgG1-Fc is utilized using the pCDNA4-Fc plasmid schematically shown in FIG. , Was subcloned into the pKN002 mammalian expression vector for high expression of hOPG-hIgG1-Fc polypeptide of SEQ ID NO: 1. The pKH002 vector utilizes the cytomegalovirus (CMV) promoter followed by bGH polyadenylation and transcription termination sequences to drive transcription of the gene of interest. The vector also contains a pUC origin of replication and a β-lactamase gene that confers ampicillin resistance to support growth and selection in bacteria. Finally, the pKN002 vector contains the glutamine synthetase gene, a selectable marker that is widely used to establish stable CHOK1 and NSO cell lines. FIG. 2 exemplarily shows the map and annotated sequences of the resulting high expression pKH002-hOPG-hIgG1-Fc plasmid of SEQ ID NO: 3.

Example 2 Generation of Stable Cell Lines for Expressing the Polypeptide of the Present Invention Stable clones of CHO-K1 cells overexpressing hOPG-hIgG1-Fc polypeptide of SEQ ID NO: 1 through standard protocol Made. The expression plasmid pKH002-hOPG-hIgG1-Fc described in Example 1 was used to generate stable cell lines for high expression of the polypeptide. The expression level of the polypeptide in multiple clones was 125 mg / L or more in 7-day batch culture. One clonal cell line showed expression levels of about 125 mg / L or 125 mg / L or more in shake flask batch cultures.

Materials Chinese hamster ovary cells (CHO-K1) were obtained as frozen stocks from ATCC (CCL- ^61TM ). The cells were adapted to in-house CD CHO medium. Media and reagents were obtained from commercial sources. After a complete fit, cells were grown at high density for a few passages. The resulting cells were subcloned. One of the resulting clones had a doubling time of 20 hours and the good form was selected as the parent cell line.

Method A vial of CHO-K1 cells (7.5 × 10 ⁶ viable cells in a volume of 1.5 ml) is thawed in 20 ml CD CHO medium supplemented with 4 mM L-glutamine and orbitally at 80 to 100 rpm A cell density of 3.75 × 10 ⁵ cells / ml was obtained in an Erlenmeyer flask at 37 ° C./10% CO ₂ on a shaker platform. Cells were subcultured every 3 to 4 days.

Each of the 19 cryovials of the research cell bank containing 7.5 × 10 ⁶ cells (passage 1) in 1.5 ml 50% fresh growth medium / 50% conditioned medium containing 7.5% DMSO, liquid nitrogen I saved it inside. After further expansion, 50 cryovials, each containing 7.5 × 10 ⁶ cells (passage 3) in 1.5 ml, were frozen in liquid nitrogen (as described above) and used as working cell banks.

Transfection of CHO-K1 cells with the pKH002-hOPG-hIgG1-Fc plasmid described in Example 1 was electroporated using a Gene Pulse Xcell device (BIO-RAD) using standard protocols. went. One to two days prior to electroporation, low concentrations of dilute CHO-K1 cells are reached to reach mid-log phase (generally about 2-3 x 10 ⁶ cells / ml) on the day of the experiment. Transfer to fresh growth media (CD CHO media). After transfection, cell lines were adapted to suspension culture using a standard protocol and then 50 ml culture growth curves were measured. Cell number and viability were determined daily for each cell line tested. The level of production of the desired polypeptide was determined by two methods: ELISA; and test purification with a Protein A column.

  A number of transfections were performed in CHO-K1 cells in the process of production of potential hOPG-hIgG1-Fc expressing cell lines. A typical transfection growth curve is shown in FIG. 3 and the data is shown in Table 1. Supercoiled DNA was transfected into CHO-K1 cells under electroporation and selected with MSX. Cells were selected as minipools in 96 well plates and productivity was assessed using SDS-PAGE and ELISA. Based on these analyses, two transfections were identified as producing the highest levels of hOPG-hIgG1-Fc. These transfections are characterized in more detail.

  After initial selection in 96 well plates, 24 cell lines were scaled up to 24 well plates and then T25 flasks. Media was harvested from 24-well plates or T25 flasks and subjected to SDS-PAGE and ELISA. Four lines that demonstrated good hOPG-hIgG1-Fc expression were transferred to suspension culture. The expression levels of two of the four cell lines were 125 mg / L or more in a 7 day batch culture, and the two lines were selected as high expression parent cell lines.

  Two high expressing parental cell lines were seeded in 5x96 well plates for single cloning. In both parental cell lines, multiple clones were picked from 96 well plates and transferred to 24 well plates. Media was harvested from confluent wells and assessed as before by SDS-PAGE and ELISA tests. Clones that did not produce the desired result were discarded. Subsets of multiple clones were grown well in serum free suspension culture and 50 mL growth curves were evaluated. All clones were further reanalyzed by SDS-PAGE and ELISA. From the subset of clones, select one clone with the highest productivity to be evaluated in an additional set of 1 L growth curves, which clone has the highest productivity and is compared with the parent cell line. It was confirmed that they showed a significant improvement.

The selected highest producing cell line was selected and thawed into CD-CHO. Growth curves for cell lines were evaluated and samples were collected daily for cell number, cell viability and hOPG-hIgG1-Fc productivity. Based on these studies, it was determined that the highest productivity cell line selected expressed hOPG-hIgG1-Fc polypeptide in the amount necessary to support commercial production. The yield of polypeptide after purification was at least 125 mg / L (without any production optimization).

Example 3 Purification of the Polypeptide of the Invention The hOPG-hIgG1-Fc polypeptide of SEQ ID NO: 1 was expressed in CHO-K1 essentially as described in Example 2 above. Cells were harvested and lysed using a well established protocol. After clearing of the cell lysate, the supernatant containing the expressed hOPG-hIgG1-Fc polypeptide was first applied to a protein A affinity column. The affinity purification step was performed according to the process outlined in Table 2. The protein A eluate containing hOPG-hIgG1-Fc was pH adjusted to about pH 7.5 using 1 M Tris-HCl, pH 9.0. The conductivity was adjusted with deionized water (dH2O) as needed.

  The pH adjusted protein A column eluate was further purified by anion exchange chromatography (AIEX) utilizing Q Sepharose resin to reduce the content of DNA, HCP, endotoxin and possible viral contamination. The AIEX process is operated in flow through mode according to the process outlined in Table 3.

  The AIEX flow through was analyzed by size exclusion HPLC (SEC-HPLC) and SDS-PAGE (reduction). The results of the analysis are shown in FIGS. 4 and 5, respectively.

The SEC-HPLC procedure is outlined in Table 4. A biologically relevant form of hOPG-hIgG1-Fc polypeptide is a homodimer linked via a C-terminal disulfide. Based on tests performed on protein size markers, the molecular weight of the pre-shoulder peak (retention time RT = 13.5 minutes) is approximately 378 KDa, presumably indicating that it represents a tetramer of hOPG-hIgG1-Fc The

  In order to determine whether the low pH elution conditions used in the Protein A affinity purification step can cause higher oligomer formation, tests were conducted for correlation of eluate concentration and oligomer content. An analytical protein A column (2 mL) with MabSelect Sure resin was loaded and used according to the operating procedure outlined in Table 1. The column loading volume was varied, ie four experiments of 20, 50, 100 and 150 mL respectively were performed. The collected eluate was analyzed by SEC-HPLC to estimate the oligomer content. The results show that although oligomers were formed in low pH elution, the oligomer content was not correlated with pool concentration.

  Therefore, the following further purification methods were performed to reduce undesired oligomer formation. Hydrophobic interaction chromatography (HIC), cation exchange chromatography (CIEX), and hydroxylapatite (HA) chromatography.

In HIC, 2 one resin, phenyl - and butyl -, and three salt mobile phase, NaCl, _{over Na} 2 SO ₄ and _{(NH 4)} 2 SO ₄ were tested. Utilizing a phenyl based resin and a NaCl based mobile phase, good results have been demonstrated that allow achieving> 97% purity and about 50% yield. The two CIEX resins were screened without showing better results than those obtained from HIC.

Further studies utilizing hydroxylapatite chromatography (HAC) have yielded excellent results. The HA column was packed with Macro-Prep Ceramic HA Type II 40 μm (Bio-Rad) to a column volume (CV) of 10.4 mL (11 cm bed height). Screening experiments were performed to identify the best elution conditions (see Table 6). The starting material used was the AIEX flow through after the first two step chromatography step. First, two batches of protein were prepared. Total protein expression levels for the first and second batches were about 588 mg / L and 50 mg / mL, respectively. The purity and concentration in AIEX flow through from the two batches were comparable, about 92% and 2.5 mg / mL, respectively. The AIEX flow through was dialyzed against HAC loading buffer and loaded onto the HA column. All experiments were performed at a flow rate of 2 mL / min.

  Of the experiments summarized in Table 5, Experiment No. 6 gave excellent results. The HAC chromatogram from Experiment No. 6 is shown in FIG. The pool of major peak fractions from Experiment No. 6 had a purity of greater than 99% (an example of an SEC-HPLC analytical chromatogram of the major peak fractions of HAC is shown in FIG. 7) The protein yield was 63%. Of note, Experiment No. 5 also produced relatively good separation, but with a low protein yield of about 55%.

  In the in vitro cell-based assay, expressed and purified polypeptide of SEQ ID NO: 1, as essentially described in this example, at a concentration of about 100 ng / ml, 50 ng / ml of RANKL Inducible osteoclast differentiation was completely blocked (RAW 264.7 cells, EC 50 at approximately 50 ng / ml).

Example 4 : Toxicity test of hOPG-hIgG1-Fc polypeptide of SEQ ID NO: 1 for subcutaneous administration to mice The polypeptide of SEQ ID NO: 1 is expressed and purified essentially as described in the previous example I did. For administration to animals, the polypeptides were formulated in the following buffer: 1% w / v sucrose, 100 mM sodium chloride, 25 mM L-arginine hydrochloride, 25 mM sodium bicarbonate, pH 6.3. The administered stock concentration used was 0.5 mg / mL of polypeptide.

  The SPF mice used in the study were randomly divided into three groups: formulation buffer control group, low dose group and high dose group, each group had 5 mice. The mice were anesthetized by intraperitoneal injection of pentobarbital sodium and the dose of anesthetic was 30 mg / kg. The back of the mouse was shaved to monitor the injection site. 20 μL formulation buffer was administered to the control group by subcutaneous injection. The 16 mg / kg polypeptide is administered to the low dose group, the 32 mg / kg polypeptide is administered to the high dose group, and the response at the injection site is observed, 15 minutes, 6 hours, 24 hours, 72 hours and 7 hours. I took a photo on the day.

  In the tests performed, no acute response was observed in mice after subcutaneous administration of formulation buffer alone or the polypeptide of SEQ ID NO: 1 at doses of 16 and 32 mg / kg. No skin reactions or other abnormalities were observed during up to 7 days of observation.

Example 5 : Pharmacokinetics (PK) of hOPG-hIgG1-Fc polypeptide of SEQ ID NO: 1 after subcutaneous administration in mice
The polypeptide of SEQ ID NO: 1 was expressed and purified essentially as described in the previous example. For administration to animals, the polypeptides were formulated in the following buffer: 1% w / v sucrose, 100 mM sodium chloride, 20 mM L-arginine hydrochloride, 25 mM sodium bicarbonate, pH 6.3. The administered stock concentration used was 0.5 mg / mL of polypeptide.

  On day 0 of the study, 14 female Balb / c nu / nu mice were randomized based on body weight into 2 groups of 7 animals. A single treatment with polypeptide (5 mg / kg) was administered subcutaneously on day 0 to all groups except mouse in group 1 which was bled via cardiac puncture for plasma preparation on day 0 of the study Given). Blood samples were collected from mice via the orbital sinus in the remaining groups at various time points, except in the study for plasma preparation.

  Body weights were recorded for all animals on the day of treatment (day 0) and then three times a week, including the end day of each group.

  Groups of mice were selected at specific time points for plasma preparation. Body weight change was not measured in the group selected for sample collection at time 0 and within 36 hours of administration. Most groups gained weight during the study period. Only group 5 ended 7 days after treatment, which failed to achieve an overall increase in weight. Neither excessive weight loss nor adverse clinical signs of mice were reported during the study period.

  Following the survival phase of the test, plasma samples were analyzed by ELISA for Hu-Fc protein. Quantification of Hu-Fc in mouse plasma samples by ELISA was used as a read-out for circulating levels of polypeptide. The assay was performed on samples from all mice in the test.

One hour after administration, the polypeptide was detected in the plasma of the animal. Then, as detected by Hu-Fc ELISA using the One Phase Decay Model equation using Prism 5.0c (GraphPad Software Inc, La Jolla, Calif., USA), Pharmacokinetics were determined. The peak circulating level of Hu-Fc (Cmax) was determined to be 26.49 μg / mL, and the peak time of circulating levels (Tmax) was 10 hours after treatment. Polypeptide levels decreased to nearly undetectable levels by 3 weeks after administration, which is the final score. The half lives (T1 / 2) were 83.52 h and K 0.0083 h- ¹ . Hu-Fc was not detected in the plasma of untreated group 1 animals. The results of the study are summarized in Table 6.
The SEM that can not be calculated as the level of Hu-Fc was below the detectable limit of the ELISA for one of the samples in the group.
Human-Fc protein concentrations were determined by Prism Software based.
^{^} Blood collection via the orbital venous plexus
^# Blood collection via terminal cardiac puncture

Example 6 : Determination of the efficacy of hOPG-hIgG1-Fc polypeptide of SEQ ID NO: 1 against human breast cancer as a mouse osteolysis model The mouse osteolysis model used in this example is well known in the art (See, eg, Arrington, SA, et al., Bone. 2006 Mar; 38 (3): 359-67). The polypeptide of SEQ ID NO: 1 was expressed and purified essentially as described in the previous example. For administration to animals, the polypeptides were formulated in the following buffer: 1% w / v sucrose, 100 mM sodium chloride, 20 mM L-arginine hydrochloride, 25 mM sodium bicarbonate, pH 6.3. The administered stock concentration used was 0.5 mg / mL of polypeptide.

Sixty female athymic Nude-Foxn1 ^nu mice were each inoculated with 5 × 10 ⁵ MDA-MB-468 human breast cancer cells (in 5 μL) in the right tibia. Fourteen days after inoculation, mice were randomized by weight into five groups of 12 (day 0).

  Mice in each group were treated subcutaneously with vehicle (formulation buffer) or polypeptide of SEQ ID NO: 1 (1, 5 or 10 mg / kg) or intravenously with Zometa (zoledronic acid) (0.1 mg / kg) I gave treatment. Treatment started on day 0 and was administered 3 times weekly for 6 weeks.

  Body weight was recorded for all animals three times a week, including the first treatment day (day 0) and then the end of the study (48 hours after the last treatment, 42 days after the first treatment). There was a significant (p <0.05) mean weight gain in all groups during the study course. No adverse clinical signs, including excessive weight loss, were observed in mice treated with the polypeptide of SEQ ID NO: 1 or Zometa.

  Treatment with the polypeptide of SEQ ID NO: 1 and Zometa at all doses, compared to vehicle, was significant for osteolysis evaluated in tibial radiographs obtained both after 6 and 8 weeks of inoculation (P <0.05) inhibition. With little or no evidence of osteolysis, the inoculated leg muscles were inoculated 6 mg / kg from one mouse receiving 10 mg / kg of the polypeptide of SEQ ID NO: 1 and 2 mice receiving Zometa. An increase appeared in the x-ray images obtained after a week. Eight weeks after inoculation, the increased leg muscle incidence increased in all groups. All mice with increased leg muscles that received the polypeptide of SEQ ID NO: 1 or Zometa had mild or no osteolysis 8 weeks after inoculation. Present x-ray images from all mice.

  Treatment with the polypeptide of SEQ ID NO: 1 at 1 mg / kg and Zometa compared to vehicle showed osteolysis as indicated by bone density parameters assessed by microCT scan of inoculated tibia excised at the end of the study Resulted in a significant (p <0.05) inhibition. Typical 2D and 3D microCT scan images from one mouse in each group analyzed for microCT are presented.

Materials and Methods All materials for testing were obtained from commercial sources. MDA-MB-468 human breast tumor cells were supplied by the American Type Culture Collection (ATCC) (Rockville, MD, USA). MDA-MB-468 human breast tumor cells (passage 5 from working stock) were cultured in RPMI 1640 cell culture medium supplemented with 10% FBS, 1% Glutamax and 1% penicillin-streptomycin. Cells were harvested by trypsinization, washed twice with HBSS and counted. The cells were then resuspended in HBSS to a final concentration of 1 × 10 ⁸ cells / mL.

The injection site was thoroughly disinfected with alcohol prior to tumor inoculation. A 27G needle was inserted through the skin to tunnel a 5-7 mm long (sufficient to provide 5 μL cell suspension) from the right hind tibial plateau to the bone marrow channel. Using a 50 μL Hamilton-type syringe equipped with a 27 G needle, 5 μL of MDA-MB-468 cell suspension (5 × 10 ⁵ cells) was flushed through the pre-formed tunnel. Treatment of mice was initiated 14 days after MDA-MB-468 cell inoculation.

  In total, 65 female mice (Mus musculus) were inoculated for the test, of which 60 were actually used for the test (5 in group, 12 groups in total). At the beginning of the treatment, the animal weight ranged from about 20.3 to about 27.8 g (average 24. 1 g) at inoculation. The animals were placed 4 individually into individually ventilated cages (Alternative Design Max, USA), with 3 cages per group. The animals were maintained in a controlled environment under barrier (quarantine) conditions with a 12 hour light / dark cycle (target range: temperature 21 ± 3 ° C., humidity 30-70%, ventilation 15 times per hour). Temperature and relative humidity were monitored continuously. All animals were subjected to the same environmental conditions.

  Standard certified commercial rodent diets (Teklad Global 18% Protein Rodent Diet, Harlan Laboratories, Inc, IN, USA) and tap water were provided ad libitum to the animals. Mortality check was performed once a day in the morning during the trial. Clinical signs (e.g. health problems and behavioral changes) were also recorded for all animals once daily. Body weight was recorded for all animals on the first treatment day (day 0) and then three times a week, including the end of the study.

  The following vehicle formulations were used in the test: 1% sucrose, 100 mM sodium chloride, 20 mM 1-arginine hydrochloride, 25 mM sodium phosphate, pH 6.3. The solution was stored at 4 ° C. The reference substance Zometa (zoledronic acid) was used in a clinical preparation (0.8 mg / mL) and stored at room temperature. The test substance (polypeptide of SEQ ID NO: 1) (45 mg / mL) was formulated with formulation buffer (1% sucrose, 100 mM sodium chloride, 20 mM l-arginine hydrochloride, 25 mM sodium phosphate) to achieve the concentration necessary for administration. , PH 6.3). Zometa® Clinical Formulation (0.8 mg / mL) was diluted with sterile saline to achieve the required concentration for administration. The solution to be administered was prepared fresh on each day of the administration.

The dosing regimens used in this study are summarized in Table 7. Sixty mice were randomized into five groups of twelve by weight on day 0 of the study, 14 days after inoculation.

  Vehicle control (formulation buffer; group 1) and polypeptide of SEQ ID NO: 1 (1, 5 and 10 mg / kg; groups 2, 3 and 4 respectively) were administered by subcutaneous injection (sc) (Table 7) . Zometa (0.1 mg / kg; group 5) was administered by intravenous injection (iv) (Table 6). Treatment was administered 3 times a week starting on day 0 and continued for 6 weeks.

  Vehicle control and test substances were dosed at a dosing volume of 10 mL / kg. The weight of each animal was measured immediately before dosing. The volume of dosing solution administered to each animal was calculated and adjusted based on individual weight.

  All mice underwent X-ray examination of both tibiae at 6 and 8 weeks after inoculation (4 and 6 weeks after the first treatment). A visual assessment of the osteolysis of the inoculated right tibia was made from each X-ray image and scores were given to indicate the severity of lysis and the presence of increased leg muscles. The degree of lysis was given on a scale of 0 (no lysis) to 4 (very severe osteolysis). The presence of increased leg muscles as indicated by the swelling of the muscle tissue gave an additional score of 0.5.

  At the end, the right tibia inoculated from all mice in all groups was excised. The extracted tibia is analyzed by microCT analysis (Adelaide Microscopy, Adelaide, SA, including total bone mass (TBV), trabecular bone volume (Tb. BV), trabecular pattern factor (Tb. Pf) and structural model index (SMI). Stored in 10% neutral buffered formalin for Australia).

Total bone mass (TBV) (mm ³ ) is a measure of total cortex and trabecular volume (Tb. BV) (mm ³ ) within the volume of interest (VOI), and VOI is a cross-sectional area consisting of cortex and trabeculae Including. Trabecular pattern factor (Tb.Pf) (mm ⁻¹ ) is a reverse collapse index, which indicates structural connectivity that can be specifically applied to trabecular bone. Lower Tb. Pf values indicate better coupled column lattices, higher Tb. Pf values indicate more severed pillar structures (ie greater disintegration / osteolysis). Structural model index (SMI) is an indicator of the relative prevalence of rods and plates in 3D structures like trabecular bone. This parameter is important in bone osteolysis and is characterized by the transition from plate-like (normal) to rod-like (degraded) structures. The ideal plate, cylinder and sphere have SMI values of 0, 3 and 4 respectively. The higher the value, the more damage there is. All statistical calculations were performed using Prism 5.0c (GraphPad Software Inc, La Jolla, Calif., USA).

  A paired t-test was used to determine if body weight significantly changed within the treatment group on day 0 and the end of the study. If the data did not pass the Normality Test, the Wilcoxon Matched Pairs Test was performed.

  Comparisons of osteolytic scores assessed in x-ray images at 6 and 8 weeks after inoculation were made between all groups. Kruskal-Wallis One Way Analysis of Variance (ANOVA) was performed in ranking, as both data sets failed the normality test. Multiple Comparison vs. Control Procedure (Dunn's method) was performed to compare each treatment with vehicle.

  Comparison of osteolytic parameters assessed by microCT scan of excised inoculated tibiae was performed between mice in groups 1, 2 and 5. One-way ANOVA was performed on normal variance data and comparisons between all groups were made using Tukey's Multiple Comparison Test. If the data did not pass the normality test, Kruskal-Wallis one-way analysis of variance (ANOVA) was performed in the ranking. In this case, comparisons between all groups were made using the All Pairwise Multiple Comparison Procedure (Dunn's method). A p-value less than 0.05 was considered significant.

Results and Observations No adverse clinical signs were observed in mice treated with the polypeptide of SEQ ID NO: 1 or Zometa (groups 2-5). The mice treated with the polypeptide of SEQ ID NO: 1 or Zometa (groups 2-5) did not lose more than 15% of their original weight during the test period. There was a significant (p <0.05) mean weight gain in all groups (Table 8).

On days 28 and 42 (6 and 8 weeks after inoculation), x-rays of both tibiae were obtained from all mice. Visual assessment of osteolysis of the inoculated right tibia was made from each X-ray image and scores were given to indicate the severity of lysis and the presence of increased muscle in the inoculated leg (Table 9, and 8 and 9). Treatment with the polypeptides of SEQ ID NO: 1 (1, 5 and 10 mg / kg; groups 2, 3 and 4 respectively) and Zometa (group 5) at all doses compared to vehicle control (group 1) This resulted in significant (p <0.05) inhibition of osteolysis assessed in X-ray images obtained both at 6 and 8 weeks after inoculation.
^a : significantly different from vehicle (group 1) (p <0.05, in ranking, Kruskal-Wallis one-way ANOVA)
Score Description:
0 no osteolysis 1 onset / mild osteolysis 2 moderate osteolysis 3 severe osteolysis 4 very severe osteolysis increased muscle-score + 0.5

  MicroCT scan parameters for assessment of osteolysis include three-dimensional morphometry including total bone mass (TBV), trabecular bone volume (Tb. BV), trabecular pattern factor (Tb. Pf) and structural model index (SMI) Analysis included. The data is shown in Table 10. Typical images of microCT scans from one animal in each of the analyzed groups are shown in FIG. 10 and FIG. Treatment with both SEQ ID NO: 1 polypeptide (group 2) and Zometa (group 5) at 1 mg / kg showed osteolytic lysis as indicated by all microCT scan parameters compared to vehicle control (group 1) Resulted in significant (p <0.05) inhibition (significantly higher TBV and Tb. BV, and significantly lower Tb. Pf and SMI).

Thus, the polypeptide of SEQ ID NO: 1 (1, 5 and 10 mg / kg, sc three times a week, 6 weeks) was inoculated in the tibia of female athymic Nude-Foxn1 ^nu mice MDA-MB-468 It resulted in being effective in preventing the osteolysis induced by human breast cancer cells. This efficacy, as measured by x-ray images at 6 and 8 weeks after inoculation and at the end of the procedure (8 weeks after inoculation), as measured by microCT scan of the tibia removed, did not appear to be dose dependent. Similarly, the reference compound for these studies, Zometa (0.1 mg / kg, iv 3 times weekly, 6 weeks) also proved effective.

  "Zometa" is a registered trademark of Novartis AG Corporation, Switzerland. "Prolia" and "Xgeva" are registered trademarks of Amgen Inc., a Delaware Corporation.

  All documents and patent documents mentioned in this specification are as if each individual document or patent document was specifically and individually indicated to be incorporated by reference in its entirety. It is hereby incorporated by reference.

  Although particular aspects of the subject are discussed, the above specification is illustrative and not restrictive. Many modifications will be apparent to one of ordinary skill in the art upon review of the specification and claims. 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.

Claims (1)

A polypeptide comprising the amino acid sequence of SEQ ID NO: 1, wherein said polypeptide is bound to human RANKL and said polypeptide is in the purified homodimeric form .