Parathyroid hormone-anti-RANKL antibody fusion compounds

Abstract

Fusion compounds and methods of using same are provided which bind and neutralize human receptor activator of nuclear factor kappa-B ligand and are agonistic to parathyroid hormone receptor 1 signaling, said compounds are useful as agents for bone healing or treating conditions associated with bone mass loss or degeneration including treating osteoporosis.

Claims

1. A compound comprising a first polypeptide and a second polypeptide, wherein a) said first polypeptide comprises a parathyroid hormone (PTH) peptide and a monoclonal antibody (mAb) IgG heavy chain (HC), the PTH peptide having the amino acid sequence given by SEQ ID NO: 13, and the HC having a heavy chain variable region (HCVR) comprising heavy chain complementary determining regions (HCDR) 1-3, HCDR1 having the amino acid sequence given by SEQ ID NO: 7, HCDR2 having the amino acid sequence given by SEQ ID NO: 8, and HCDR3 having the amino acid sequence given by SEQ ID NO: 9; and b) said second polypeptide comprises a mAb light chain (LC) comprising a light chain variable region (LCVR) comprising light chain complementary determining regions (LCDR) 1-3, LCDR1 having the amino acid sequence given by SEQ ID NO: 10, LCDR2 having the amino acid sequence given by SEQ ID NO: 11, and LCDR3 having the amino acid sequence given by SEQ ID NO: 12, wherein the PTH peptide is linked to the HC via a polypeptide linker (L1), L1 being covalently attached to the N-terminus of HC and the C-terminus of the PTH peptide.

2. The compound of claim 1, wherein the HCVR has the amino acid sequence given by SEQ ID NO: 5 and the LCVR has the amino acid sequence given by SEQ ID NO: 6.

3. The compound of claim 1, wherein L1 has the amino acid sequence given by SEQ ID NO: 14.

4. The compound of claim 3, wherein the first polypeptide has the amino acid sequence given by SEQ ID NO: 1 and the second polypeptide has the amino acid sequence given by SEQ ID NO: 2.

5. The compound of claim 4 comprising two first polypeptides and two second polypeptides.

6. A pharmaceutical composition comprising a compound of claim 1 and one or more pharmaceutically acceptable carriers, diluents, or excipients.

7. The compound of claim 2, wherein L1 has the amino acid sequence given by SEQ ID NO: 14.

8. A DNA molecule comprising a polynucleotide sequence encoding the first polypeptide and the second polypeptide of the compound of claim 1, the first polypeptide having the amino acid sequence of SEQ ID NO: 1 and the second polypeptide having the amino acid sequence of SEQ ID NO: 2.

9. An isolated mammalian cell comprising the DNA molecule of SEQ ID NO: 3 and the DNA molecule of SEQ ID NO:4, which cell is capable of expressing polypeptides comprising the first polypeptide and the second polypeptide of the compound of claim 1, the first polypeptide having the amino acid sequence of SEQ ID NO: 1 and the second polypeptide having the amino acid sequence of SEQ ID NO: 2.

10. The mammalian cell of claim 9, wherein the mammalian cell is a Chinese Hamster Ovary cell.

11. An isolated mammalian cell comprising the DNA molecule of claim 8, which cell is capable of expressing the first polypeptide and the second polypeptide.

12. The mammalian cell of claim 11, wherein the mammalian cell is a Chinese Hamster Ovary cell.

13. A process for producing a compound comprising a first polypeptide having the amino acid sequence given by SEQ ID NO: 1 and a second polypeptide having the amino acid sequence given by SEQ ID NO: 2, comprising cultivating the mammalian cell of claim 11 under conditions such that the compound is expressed, and recovering the expressed compound.

14. A process for producing a compound comprising a first polypeptide having the amino acid sequence given by SEQ ID NO: 1 and a second polypeptide having the amino acid sequence given by SEQ ID NO: 2, the process comprising cultivating the mammalian cell of claim 9 under conditions such that the compound is expressed, and recovering the expressed compound.

15. A compound produced by the process of claim 13.

16. A compound produced by the process of claim 14.

17. A method of treating a bone disorder, comprising administering to a patient in need thereof a therapeutically effective amount of a compound of claim 1.

18. The method of claim 17, wherein the bone disorder is osteoporosis.

19. The method of claim 17, wherein the bone disorder is selected from the group consisting of: osteopenia, osteogenesis imperfecta, transplant-associated bone loss, autoimmune-induced bone loss, disuse-induced bone loss, degenerative lumbar spondylolisthesis, and degenerative disk disease.

Description

EXAMPLES

(1) Fusion Compound Expression and Purification

(2) An exemplified fusion compound of the present invention is expressed and purified essentially as follows. A glutamine synthetase (GS) expression vector containing the polynucleotide sequences given by SEQ ID NO: 3 (encoding an exemplified first polypeptide of SEQ ID NO: 1 and a post-translationally cleaved signal peptide) and SEQ ID NO: 4 (encoding an exemplified second polypeptide of SEQ ID NO: 2 and a post-translationally cleaved signal peptide) is used to transfect a Chinese hamster cell line (CHO, GS knockout), by electroporation. The expression vector encodes a SV Early (Simian Virus 40E) promoter and the gene for GS. Expression of GS allows for the biochemical synthesis of glutamine, an amino acid required by the CHO cells. Post-transfection, cells undergo bulk selection with 50 μM L-methionine sulfoximine (MSX). The inhibition of GS by MSX is utilized to increase the stringency of selection. Cells with integration of the expression vector cDNA into transcriptionally active regions of the host cell genome can be selected against CHO wild type cells. Transfected pools are plated at low density to allow for close-to-clonal outgrowth of stable expressing cells. The master-wells are screened for fusion compound expression and then scaled up in serum-free, suspension cultures to be used for production.

(3) Clarified medium, into which the exemplified compound has been secreted, is applied to a Protein A affinity column that has been equilibrated with a compatible buffer such as phosphate buffered saline (pH 7.4). The column is washed to remove nonspecific binding components. The bound fusion compound is eluted, for example, by pH gradient and neutralized for example with Tris, pH 8 buffer. Fusion compound fractions are detected, such as by SDS-PAGE or analytical size-exclusion, and then are pooled. Soluble aggregate and multimers may be effectively removed by common techniques including size exclusion, hydrophobic interaction, CAPTO™ multimodal chromatography, ion exchange, or hydroxyapatite chromatography. The fusion compound is concentrated and/or sterile filtered using common techniques. The purity of the exemplified fusion compound after these chromatography steps is greater than 98% (monomer). The fusion compound may be immediately frozen at −70° C. or stored at 4° C. for several months.

(4) The relationship of the various regions and linkers comprising an exemplified fusion compound of the present invention, expressed and purified following procedures essentially as described above, is presented in Table 1 (numbering of amino acids applies linear numbering; assignment of amino acids to variable domains is based on the International Immunogenetics Information System® available at www.imgt.org; assignment of amino acids to CDR domains is based on the well-known Kabat and North numbering conventions as reflected at the end of Table 1):

(5) TABLE-US-00001 TABLE 1 Amino acid regions of an exemplified fusion compound of the present invention. SEQ ID NO: 1 SEQ ID NO: 2 A.A. A.A. Portion Region Pos. Portion Region Pos. Exemplified PTH Peptide  1-34 PTH Peptide Exemplified L1 35-49 Linker Exemplified FRH1 50-74 Exemplified FRL1-1  1-23 RANKL HCDR1 75-84 RANKL LCDR1 24-34 HCVR FRH2 85-98 LCVR FRL1-2 35-49 HCDR2  99-115 LCDR2 50-56 FRH3 116-147 FRL1-3 57-88 HCDR3 148-159 LCDR3 89-97 FRH4 160-170 FRL1-4  98-107 HC CH 171-496 LC CL 108-214 Constant Constant Region Region Starting Amino Acid Ending Amino Acid CDR Residue Defined By: Residue Defined By: HCDR1 North Kabat HCDR2 Kabat Kabat HCDR3 North Kabat LCDR1 Kabat Kabat LCDR2 North Kabat LCDR3 Kabat Kabat

(6) The exemplified compound presented in Table 1 comprises two first polypeptides having amino acid sequences of SEQ ID NO: 1 and two second polypeptides having amino acid sequences of SEQ ID NO: 2. According to the exemplified fusion compound, each of the first polypeptides forms an inter-chain disulfide bond with each of the second polypeptides between cysteine residue 184 of SEQ ID NO: 1 and cysteine residue 214 of SEQ ID NO: 2; at least two inter-chain disulfide bonds with the other first polypeptide, the first inter-chain disulfide bond forming between cysteine residue 276 (of SEQ ID NO: 1) of the first polypeptide and cysteine residue 276 (of SEQ ID NO: 1) of the other first polypeptide, the second inter-chain disulfide bond forming between cysteine residue 279 (of SEQ ID NO: 1) of the first polypeptide and cysteine residue 279 (of SEQ ID NO: 1) of the other first polypeptide. Further, the exemplified compound presented in Table 1 is glycosylated at asparagine residue 347 of SEQ ID NO: 1 of both first polypeptides.

(7) Except as noted otherwise herein, the exemplified fusion compound referred to throughout the Examples refers to the exemplified compound of the present invention presented in Table 1.

(8) Fusion Compound Binding Affinity to RANKL

(9) Binding affinity and binding stoichiometry of the exemplified fusion compound to human and murine RANKL is determined using a surface plasmon resonance assay on a BIACORE 2000™ instrument primed with HBS-EP+ (10 mM Hepes, pH7.4+150 mM NaCl+3 mM EDTA+0.05% (w/v) surfactant P20) running buffer and analysis temperature set at 25° C. A CM5 chip (Biacore, p/n.BR-100530) containing immobilized protein A (generated using standard NHS-EDC amine coupling) on all four flow cells (Fc) is used to employ a capture methodology. Fusion compound samples are prepared at 2 μg/mL by dilution into running buffer. Human and murine RANKL samples, respectively, are prepared at final concentrations starting at 5 nM and using two-fold serial dilutions (in running buffer) for each cycle.

(10) Each analysis cycle consists of (1) capturing antibody samples on separate flow cells (Fc2 and Fc3); (2) injection of each human and murine RANKL concentration, respectively, over all Fc at 100 μL/min for 150 seconds followed by return to buffer flow for 1800 seconds to monitor dissociation phase; (3) regeneration of chip surfaces with injection of 10 mM glycine, pH 1.5, for 120 seconds at 5 μL/min over all cells; and (5) equilibration of chip surfaces with a 10 μL (60-sec) injection of HBS-EP+. Data are processed using standard double-referencing and fit to a 1:1 binding model using BIACORE 2000™ Evaluation software, version 2.0.3, to determine the association rate (k.sub.on, M.sup.−1s.sup.−1 units), dissociation rate (k.sub.off, s.sup.−1 units), and R.sub.max (RU units). The equilibrium dissociation constant (K.sub.D) is calculated from the relationship K.sub.D=k.sub.off/k.sub.on, and is in molar units. Results are provided in Table 2.

(11) TABLE-US-00002 TABLE 2 Binding affinity to human and murine RANKL by the exemplified fusion compound. k.sub.on k.sub.off Avg. Avg. K.sub.D (10.sup.7 s.sup.−1 Avg. Antigen M.sup.−1s.sup.−1) (10.sup.−5) pM n Human RANKL 0.35 1.69 4.82 4 Murine RANKL 1.16 9.69 8.37 1

(12) The results provided in Table 2 demonstrate that the exemplified fusion compound of the present invention binds human and murine RANKL with high affinity at 25° C.

(13) Neutralization of RANKL-Induced NF-kB-Driven Luciferase Activity In Vitro

(14) HEK293 cells, which stably co-express human RANK and a NF-kB driven luciferase reporter, are used to assess the ability of the exemplified fusion compound presented in Table 1 to neutralize RANKL activity. In the above-described HEK293 cell model, RANK, when bound by human RANKL, induces NF-kB signaling resulting in luciferase luminescence. Neutralization of RANKL binding to RANK, by the exemplified fusion compound, is measured by a reduction of luciferase luminescence.

(15) HEK293 cells are routinely cultured under selective pressure of 700 μg/mL Geneticin (HyClone, p/n.SV30069.01). 25,000 cells/well are added to the wells of 96 well tissue culture plates (Benton Dickinson, p/n.354620) in assay media (504, DMEM/F12 (1:3) media (Gibco, p/n.930152DK) containing 0.5% FBS (Gibco, p/n.10082-147), 20 nM Hepes (HyClone, p/n.SH30237.01), 1×GlutaMax GLUTAMAX™ media supplement (Gibco, p/n.35050-61) and 1× penicillin/streptomycin (Hyclone, p/n.SV30010)). Cells are incubated at 37° C. (with 5% CO.sub.2 and 95% humidity) overnight.

(16) Assay media including 1 nM and 10 nM concentrations of human RANKL are used to prepare dose ranges of 10 nM to 0.005 nM (with 1:3 serial dilutions) for each of: a.) the exemplified fusion compound; and b.) a RANKL neutralizing antibody (an IgG4 RANKL mAb having the same HC and LC amino acid sequences as the mAb portion of the exemplified fusion compound). Assay medium is used for a “media only” control. All treatment groups are incubated for 15 minutes at room temperature. Thereafter, 50 μl of each treatment group is added to 50 μl of media containing cultured cells. The mixture of treatment group and cultured cells are incubated overnight at 37° C.

(17) Following overnight incubation existing growth media is removed and cells are suspended in 504, of BUGLITE™ solution (2.296 g DTT (Sigma, p/n.D0632), 1.152 g Coenzyme A (Sigma, p/n. C-3019), 0.248 g ATP (Sigma, p/n.A7699) in 1 L 1% TRITON X-100™ Lysis Buffer (30 mL TRITON X-100™ (Fisher, p/n.BP151-500), 3 mL MgCl (Sigma, p/n.M9272), 108.15 mL 1M TRIZMA™ HCL (Sigma, p/n.T-3253), 41.85 mL 1M TRIZMA™ Base (Sigma, p/n.T-1503) and 817 mL H2O)). Cells are then lysed with gentle agitation on a plate shaker for between 5 to 10 minutes. Following cell lysis, luminescence is measured on a plate reader (Envision Plate Reader). IC.sub.50 values for all treatment groups are calculated using a three-parameter logistic regression model with GraphPad Prism 6 and are presented in Table 3.

(18) TABLE-US-00003 TABLE 3 Neutralization of human RANKL by the exemplified fusion compound. IC50 (nM) Molecule hRANKL n Exemplified 0.067 11 Fusion Compound RANKL mAb 0.069 3

(19) The results presented in Table 3 demonstrate that the exemplified fusion compound of the present invention neutralizes human RANKL induced NF-kB driven luciferase luminescence. The inhibition was comparable to that observed with the positive control RANKL antibody. Media controls did not neutralize human RANKL induced NF-kB driven luciferase luminescence in the HEK293 cell model at any concentration tested. These results demonstrate the exemplified fusion compound of the present invention effectively neutralizes RANKL.

(20) Activation of PTHR1 Receptor-Induced Luciferase Activity In Vitro

(21) Rat osteosarcoma UMR-106 cells (ATCC, p/n.CRL-1661) which endogenously express PTH receptor and which have been stably co-transfected (using Roche Fugene6 reagent) with a pTranslucent CRE(1) Luciferase Reporter Vector (Panomics, p/n. LR0093) and pEGFP-N1 (Clontech), are used to assess the ability of the exemplified fusion compound presented in Table 1 to activate the PTHR1 receptor. PTH binding of the PTH receptor (expressed by the UMR-106 cells) induces CRE-regulated luciferase luminescence. Activation of the PTHR1 receptor by the exemplified fusion compound is measured through quantification of luciferase luminescence.

(22) The co-transfected UMR-106 cells are grown at 37° C. and 10% CO.sub.2 in DMEM/HEPES, 10% FBS, 1× Penicillin, Streptomycin, and Glutamine, and 2 mg/ml G418. UMR-106 cells (at a concentration of 50,000 cells/well) are added to an opaque white plate and incubated overnight at 37° C. (under 10% CO.sub.2). Following incubation, a dose range of 0 nM to 1250 nM of one of a.) the exemplified fusion compound; b.) a RANKL neutralizing antibody (an IgG4 RANKL mAb having the same HC and LC amino acid sequences as the mAb portion of the exemplified fusion compound) and c.) a PTH peptide (a 38 amino acid parathyroid hormone peptide) is added to the seeded plates and plates are incubated at 37° C. (under 10% CO.sub.2) for four to six hours. Thereafter, 50 μL of BugLite is added to each plate and luminescence is measured on a plate reader (Envision Plate Reader). EC.sub.50 values for all treatment groups are calculated using a three-parameter logistic regression model with GraphPad Prism or JMP and are presented in Table 4.

(23) TABLE-US-00004 TABLE 4 PTHR1 Activation by the exemplified fusion compound. Molecule EC50 (nM) n Exemplified 6.6 10 Fusion Compound RANKL mAb 0.0 10 PTH Peptide 5.6 5

(24) The results presented in Table 4 demonstrate that the exemplified fusion compound of the present invention both binds PTHR1 receptor and activates the downstream PTHR1 signaling cascade in vitro. As the results demonstrate, the ability of the exemplified fusion compound to activate PTHR1 signaling compares favorably with the PTH peptide alone.

(25) In Vivo Efficacy Analysis in Intact Murine Model

(26) Effects on bone mass density, in vivo, are assessed using an intact female murine model. C57/B6 intact female mice, aged twenty to twenty-two weeks (Charles River) are maintained on a 12 hour light/dark cycle at 22° C. with ad lib access to food (TD 2014 with 0.72% Ca and 0.61% P, Vit. D 0.99 IU/g, Teklad, Madison, Wis.) and water.

(27) The mice are divided into treatment groups or a PBS vehicle control group. Each treatment group of mice receives a weekly subcutaneous injection of one of: a.) 3 mg/kg or 10 mg/kg exemplified fusion compound; b.) 10 mg/kg RANKL neutralizing antibody (an IgG4 RANKL mAb having the same HC and LC amino acid sequences as the mAb portion of the exemplified fusion compound); or c.) co-administration of 10 mg/kg of the RANKL neutralizing antibody and 3 mg/kg PTH peptide (a 38 amino acid parathyroid hormone peptide). Mice are sacrificed at four weeks.

(28) Bone mass density (BMD) of distal and mid-femur is monitored by quantitative computed tomography (qCT) using Aloka LaTheta LTC-100 model CT scanner. Results are provided in Table 5 (data presented as mean % difference compared to vehicle control using Dunnett's Method with a significance level of P<0.05).

(29) TABLE-US-00005 TABLE 5 Skeletal BMD Analysis. % BMD Increase Over Vehicle Control Mice Distal-Femur Middle-Femur n Exemplified fusion compound 31 12 6 (3 mg/kg) Exemplified fusion compound 55 14 6 (10 mg/kg) Rank mAb Alone 23 4 6 (10 mg/kg) Rank mAb (10 mg/kg) + PTH 17 9 6 peptide (3 μg/kg)

(30) The results presented in Table 5 demonstrate that, dosed weekly, the exemplified fusion compound of the present invention demonstrates a dose-dependent increase of BMD at both the distal and middle femur over RANKL antibody only treated mice and co-administration of RANKL antibody and PTH peptide treated mice.

(31) In Vivo Efficacy Analysis in Ovariectomized Murine Model

(32) Effects on bone mass density, in vivo, are assessed using an ovariectomized murine model. Twenty week old female C57/B6 mice (Harlan, Indianapolis, Ind.) are ovariectomized (or sham operated control group) and maintained on a 12 hour light/dark cycle at 22° C. with ad lib access to food (TD 2014 with 0.72% Ca and 0.61% P, Vit. D 0.99 IU/g, Teklad, Madison, Wis.) and water. Osteopenia is established in the mice by allowing ovariectomized mice to lose bone mass for a six-week period.

(33) Following a six-week osteopenia-establishing period, mice are divided into treatment groups or a vehicle PBS control group. Each treatment group of mice receives a weekly subcutaneous injection of one of: a.) 1 mg/kg or 3 mg/kg exemplified fusion compound; b.) 3 mg/kg or 10 mg/kg RANKL neutralizing antibody (an IgG4 RANKL mAb having the same HC and LC amino acid sequences as the mAb portion of the exemplified fusion compound); or c.) co-administration of 10 mg/kg of the RANKL neutralizing antibody and 10 mg/kg PTH peptide (a 38 amino acid parathyroid hormone peptide). Mice are sacrificed at four weeks.

(34) Skeletal bone mass density (BMD) of vertebrae 5 is assessed by quantitative computed tomography (qCT), using Aloka LaTheta LTC-100 model CT scanner, following sacrifice. Results are provided in Table 6 (data presented as mean % difference compared to sham-operated control group using Dunnett's Method with a significance level of P<0.05).

(35) TABLE-US-00006 TABLE 6 Skeletal BMD Analysis. % BMD Increase Over Molecule OVX vehicle control Mice n Exemplified fusion 11 6 compound (1 mg/kg) Exemplified fusion 19 6 compound (3 mg/kg) Rank mAb Alone 2 6 (3 mg/kg) Rank mAb Alone 11 6 (10 mg/kg) Rank mAb (10 mg/kg) + 13 6 PTH peptide (10 μg/kg)

(36) The results presented in Table 6 demonstrate that, dosed weekly, the exemplified fusion compound of the present invention demonstrates a dose-dependent increase of bone mass density of vertebrae over RANKL antibody only treated mice and co-administration of RANKL antibody and PTH peptide treated mice.

(37) In Vivo Efficacy Analysis in Orchidectomized Murine Model

(38) Effects on bone mass density and bone mineral content, in vivo, are assessed using an orchidectomized murine model. Sixteen week old female C57/B6 mice (Harlan, Indianapolis, Ind.) are orchidectomized (or vehicle control group, n=6) and maintained on a 12 hour light/dark cycle at 22° C. with ad lib access to food (TD 2014 with 0.72% Ca and 0.61% P, Vit. D 0.99 IU/g, Teklad, Madison, Wis.) and water. Osteopenia is established in the orchidectomized mice by allowing mice to lose bone mass for a six-week period.

(39) Following a six-week osteopenia-establishing period, mice are divided into treatment groups and a vehicle PBS control group. Each treatment group of mice receives, either weekly or twice-per week (as outlined in Table 7 below), a subcutaneous injection of one of: a.) 0.5 mg/kg or 2.0 mg/kg exemplified fusion compound per week; b.) 0.5 mg/kg or 2.0 mg/kg exemplified fusion compound twice-per week; c.) 2 mg/kg RANKL neutralizing antibody (an IgG4 RANKL mAb having the same HC and LC amino acid sequences as the mAb portion of the exemplified fusion compound) twice-per week; d.) 5 μg/kg PTH peptide (a 38 amino acid parathyroid hormone peptide) daily; or e.) co-administration of 2 mg/kg of the RANKL neutralizing antibody twice-per week and 5 mg/kg PTH peptide daily. Mice are sacrificed at two weeks.

(40) Bone mass density (BMD) of distal femur and bone mineral content (BMC) of lumbar vertebra are assessed by quantitative computed tomography (qCT) using Aloka LaTheta LTC-100 model CT scanner. Results are provided in Table 7 (data presented as mean % difference compared to vehicle control mice using Dunnett's Method with a significance level of P<0.05).

(41) TABLE-US-00007 TABLE 7 Skeletal BMD and BMC Analysis (results presented as percent change from vehicle control mice). Distal-Femur Lumbar Vertebra Dosing (% BMD (% BMC Molecule n Regimen change) change) Exemplified 9 weekly 0 6 fusion compound (0.5 mg/kg) Exemplified 10 weekly 24 26 fusion compound (2 mg/kg) Exemplified 9 twice/week 17 12 fusion compound (0.5 mg/kg) Exemplified 10 twice/week 46 38 fusion compound (2 mg/kg) Rank mAb 9 twice/week 19 13 Alone (2 mg/kg) PTH peptide 9 daily 16 1 (5 μg/kg) Rank mAb (2 9 twice/week + 39 24 mg/kg) + daily PTH peptide (5 μg/kg)

(42) The results presented in Table 7 demonstrate that the exemplified fusion compound of the present invention demonstrates a dose-dependent increase of both BMD (of distal femur) and BMC (of lumbar vertebra) over RANKL antibody only and PTH peptide only treated mice, and compares favorably to co-administration of RANKL antibody and PTH peptide treated mice.

(43) Pharmacodynamic Effects, In Vivo, in Cynomolgus Monkey Model

(44) In vivo pharmacodynamic effects on serum calcium, bone formation biomarker P1NP, bone resorption biomarker CTx, and arterial pressure are assessed using cynomolgus monkey model as set forth in detail below. The data presented below demonstrates the exemplified fusion compound, unlike RANKL mAb-only treated animals, stimulates an increase (relative to baseline) in serum bone formation biomarker P1NP; stimulates a decrease (relative to baseline) of bone resorption biomarker CTx at a level similar to that seen in RANKL mAb-only treated animals; does not impact serum calcium concentrations relative to control-treated groups; and does not stimulate a mean difference in arterial pressure between treated and untreated groups of greater than 10 mm Hg for at least the first 8 hours post-treatment.

(45) Serum Calcium Effects in Cynomolgus Monkey Model

(46) Effect on serum calcium is assessed using a cynomolgus monkey model. Female cynomolgus monkeys aged five to six years receive a single subcutaneous injection of either 0.1 mg/kg of the exemplified fusion compound or a PBS vehicle control, or 1.0 mg/kg of the exemplified fusion compound or a PBS vehicle control. Additionally, male cynomolgus monkeys aged two to three years receive a single subcutaneous injection of either 0.1 mg/kg or 1.0 mg/kg of the exemplified fusion compound or PBS vehicle control.

(47) Blood samples are collected from the femoral vein of each monkey prior to dosing and at twenty-four hour intervals thereafter for one week. Serum calcium concentrations of each collected sample are analyzed using a Roche P800 Modular Chemistry Analyzer (Roche Diagnostics Corp., Indianapolis Ind.). Results are presented in Table 8.

(48) TABLE-US-00008 TABLE 8 Total Serum Calcium in Cynomolgus Monkeys (NC = sample not collected; ND = not determined). Total Mean (±SD where applicable) Serum Calcium (mg/dL) Days pre/post-dosing Molecule N Sex −1 0 0.5 1 2 3 Exemplified 3 F 10.6 ± NC 10.0 ± 10.6 ± 10.0 ± 10.1 ± fusion 0.8 0.4 0.6 0.7 0.3 compound (0.1 mg/kg) Vehicle 4 F 11.2 ± NC 10.2 ± 10.7 ± 10.6 ± 10.4 ± control 0.6 0.5 0.5 0.8 0.7 Exemplified 3 F 10.6 NC 9.8 ± 11.1 ± 10.6 ± 10.7 ± fusion (except (N = 2) 0.7 0.6 0.9 0.3 compound where (1.0 mg/kg) noted) Vehicle 3 F 10.4 ± NC 8.8 ± 10.0 ± 10.5 ± 10.4 ± control 0.3 0.2 0.3 1.0 0.5 Exemplified 2 M 10.0 10.0 NC NC 9.7 9.6 fusion compound (0.1 mg/kg) Vehicle 3 M 10.3 ± 10.3 ± NC NC 10.0 ± 10.1 ± control 0.2 0.2 0.1 0.5 Exemplified 3 M 10.3 ± NC 11.7 ± 9.0 ± 9.1 ± 8.3 ± fusion 0.4 0.4 0.5 0.5 0.8 compound (1.0 mg/kg) Vehicle 3 M 10. ± NC 11.0 ± 9.4 ± 9.5 ± 9.2 ± control 0.4 1.1 0.4 0.2 0.4 Total Mean (±SD where applicable) Serum Calcium (mg/dL) Days pre/post-dosing Molecule 4 5 6 7 8 9 10 Exemplified 10.3 ± 10.4 ± 10.4 ± 9.8 ± 10.1 ± NC NC fusion 1.0 0.4 0.7 0.4 0.6 compound (0.1 mg/kg) Vehicle 10.3 ± 10.1 ± 10.4 ± 10.2 ± 10.2 ± NC NC control 0.7 0.4 0.7 0.9 0.4 Exemplified 9.2 ± 10.2 ± 10.1 ± 9.7 ± 10.2 ± 10.0 ± 10.1 ± fusion 0.8 0.6 0.6 0.2 0.6 0.5 1.1 compound (1.0 mg/kg) Vehicle 9.5 ± 9.8 ± 9.9 ± 9.7 ± 9.8 ± 9.6 ± 9.5 ± control 1.1 0.5 0.8 0.5 0.3 0.4 0.1 Exemplified 9.0 9.8 9.8 10.1 10.0 10.4 NC fusion compound (0.1 mg/kg) Vehicle 10.5 ± 10.3 ± 10.9 ± 10.3 ± 10.6 ± 10.2 ± 10.2 ± control 0.5 0.3 0.6 0.3 0.3 0.2 0.2 Exemplified 8.6 ± 9.5 ± NC 6.8 ± 9.7 ± 8.7 ± 8.7 ± fusion 0.7 0.2 0.2 0.8 1.3 0.7 compound (1.0 mg/kg) Vehicle 9.4 ± 10.5 ± NC 10.1 ± 10.2 ± 10.1 ± 10.4 ± control 0.3 0.3 0.2 0.5 0.2 0.6

(49) The results presented in Table 8 demonstrate the exemplified fusion compound has no impact, relative to control dosed animals, on serum calcium levels in female cynomolgus monkeys at either dose concentration. The results presented in Table 8 also demonstrate the exemplified fusion compound has no impact, relative to control dosed animals, on serum calcium levels in male cynomolgus monkeys at 0.1 mg/kg. Monkeys dosed at a concentration of exemplified fusion compound at 1.0 mg/kg show a decrease, relative to control dosed animals, in serum calcium only at day 7 (on day 8, however, serum calcium levels had returned to levels equivalent that of control dosed animals).

(50) Bone Formation Biomarker P1NP Effects in Cynomolgus Monkey Model

(51) Effects on serum bone formation biomarker P1NP are assessed using a cynomolgus monkey model. Female cynomolgus monkeys aged five to six years receive a single subcutaneous injection of either: 0.1 mg/kg of the exemplified fusion compound or a PBS vehicle control; or 1.0 mg/kg of the exemplified fusion compound or a PBS vehicle control. Additionally, male cynomolgus monkeys aged two to three years receive a single subcutaneous injection of either 0.1 mg/kg or 1.0 mg/kg of the exemplified fusion compound; 0.1 mg/kg or 1.0 mg/kg of RANKL neutralizing antibody (an IgG4 RANKL mAb having the same HC and LC amino acid sequences as the mAb portion of the exemplified fusion compound); or a PBS vehicle control.

(52) Blood samples are collected from the femoral vein of each monkey prior to dosing and at twenty-four hour intervals thereafter for one week. Serum P1NP concentrations of each collected sample are analyzed using a UNIQ™ P1NP RIA assay (Orion Diagnostica, Espoo, Finland). Results are presented in Table 9 as a mean % change from baseline P1NP concentrations (e.g., P1NP concentration at day 0).

(53) TABLE-US-00009 TABLE 9 % Change in Serum P1NP Levels in Cynomolgus Monkeys. Mean Percent Change (±SD where applicable) from Baseline in Serum P1NP Levels Days pre/post-dosing Molecule N Sex 0 1 2 3 4 5 Exemplified 3 F NC −2.92 ± −1.04 ± −29.91 ± −32.77 ± −15.05 ± fusion 6.62 4.86 7.82 3.45 10.33 compound (0.1 mg/kg) Vehicle 3 F NC 13.16 ± 20.73 ± 18.73 ± 18.00 ± 27.81 ± control 25.17 36.75 22.19 28.25 20.53 Exemplified 3 F NC −21.86 ± −55.09 ± −45.66 ± −25.26 ± −7.95 ± fusion 3.33 7.16 12.22 13.57 9.22 compound (1.0 mg/kg) Vehicle 3 F NC −14.03 ± −9.60 ± −12.16 ± −15.05 ± −11.84 ± control 11.76 11.91 20.96 13.30 12.46 Exemplified 2 M 2.00 −26.01 −8.36 −7.00 32.82 7.19 fusion compound (0.1 mg/kg) Vehicle 3 M −5.69 + 2.7647 + −4.44 + −8.81 + −0.93 + 8.06 + control 6.61 28.98 21.82 18.77 23.74 21.16 Exemplified 3 M −6.16 ± −45.09 ± −27.07 + 8.50 + 79.24 + 96.12 + fusion 8.45 28.85 31.06 23.35 17.65 25.69 compound (1.0 mg/kg) Vehicle 3 M −1.54 ± −8.27 ± −7.32 ± −6.72 ± 22.61 ± 5.28 ± control 4.50 3.81 12.43 9.41 22.89 30.43 RANKL 3 M 0 ± −26.56 ± −21.92 ± −29.43 ± −20.77 ± −34.27 ± mAb 0 11.63 12.70 4.01 5.82 3.71 (0.1 mg/kg) RANKL 3 M 0 ± −31.10 + −41.68 ± −42.40 ± −39.48 ± −48.68 ± mAb 0 17.74 7.58 3.69 10.09 10.03 (1.0 mg/kg) Mean Percent Change (±SD where applicable) from Baseline in Serum P1NP Levels Days pre/post-dosing Molecule 6 7 8 9 10 16 Exemplified −6.43 ± −2.80 ± 3.53 ± 6.47 ± 19.16 ± 31.92 ± fusion 9.04 13.31 3.11 12.44 3.85 24.52 compound (0.1 mg/kg) Vehicle 14.99 ± 27.54 ± 25.91 ± 22.34 ± 23.01 ± 25.22 ± control 14.98 12.88 13.66 18.95 16.02 28.95 Exemplified 15.71 ± 15.63 ± 19.62 ± 30.25 ± 24.62 ± 28.69 ± fusion 2.72 7.20 7.79 21.86 8.84 12.36 compound (1.0 mg/kg) Vehicle −7.16 ± −17.08 ± −17.53 ± −22.57 ± −12.06 ± −13.85 ± control 13.90 12.09 10.76 16.91 11.73 21.88 Exemplified −10.55 −16.58 15.93 13.77 NC −16.98 fusion compound (0.1 mg/kg) Vehicle −1.65 + −6.02 + 0.21 + −3.94 + NC −7.64 + control 19.84 18.42 26.52 25.91 19.22 Exemplified NC 21.34 ± NC 43.80 + NC 47.04 + fusion 9.52 18.82 11.04 compound (1.0 mg/kg) Vehicle NC −19.59 ± NC −13.47 ± NC 3.51 ± control 16.35 16.26 9.86 RANKL NC −32.45 ± NC NC −32.08 ± NC mAb 7.74 12.29 (0.1 mg/kg) RANKL NC −50.50 + NC NC −57.22 ± NC mAb 6.72 5.32 (1.0 mg/kg)

(54) The results presented in Table 9 demonstrate that, after a single dose of either 0.1 or 1.0 mg/kg, the exemplified fusion compound (unlike RANKL mAb-only treated animals), stimulates a dose-dependent increase in serum P1NP levels in both male and female monkeys relative to control treated animals. RANKL mAb-only treated animals demonstrate a decrease in serum P1NP levels in both male and female monkeys relative to control treated animals.

(55) Bone Resorption Biomarker CTx Effects in Cynomolgus Monkey Model

(56) Effects on serum bone resorption biomarker CTx are assessed using a cynomolgus monkey model. Male cynomolgus monkeys aged two to three years receive a single subcutaneous injection of either: 0.1 mg/kg or 1 mg/kg of the exemplified fusion compound; 0.1 mg/kg or 1.0 mg/kg of RANKL neutralizing antibody (an IgG4 RANKL mAb having the same HC and LC amino acid sequences as the mAb portion of the exemplified fusion compound); or a PBS vehicle control. Additionally, female cynomolgus monkeys aged five to six years receive a single subcutaneous injection of either 0.1 mg/kg or 1.0 mg/kg of the exemplified fusion compound.

(57) Blood samples are collected from the femoral vein of each monkey prior to dosing and at twenty-four hour intervals thereafter for one week. Serum CTx concentrations of each collected sample are analyzed using ELISA method per manufacture's instruction. (Immunodiagnostic Systems Inc.). Results are presented in Table 10 as mean % change from baseline in serum CTx concentrations (e.g., CTx concentration at day 0).

(58) TABLE-US-00010 TABLE 10 Serum CTx Levels in Cynomolgus Monkeys. Mean Percent Change (±SD where applicable) from Baseline in Serum CTx Days post-dosing Molecule N Sex 0 1 2 3 4 Exemplified 3 F NC 26.03 ± −49.06 ± −44.08 ± −52.34 ± fusion 55.00 32.81 22.07 10.94 compound (0.1 mg/kg) Vehicle 3 F NC −13.66 ± 0.76 ± −23.52 ± −16.12 ± control 40.37 25.07 21.09 35.68 Exemplified 3 F NC −20.50 ± −52.02 ± −68.82 ± −66.73 ± fusion (except 27.29 17.11 5.17 9.61 compound where (1.0 mg/kg) noted) Vehicle 3 F NC 26.70 ± 47.79 ± 43.29 ± 46.39 ± control 12.66 7.32 25.16 42.97 Exemplified 2 M 3.29 −23.14 −56.08 −50.52 −45.72 fusion compound (0.1 mg/kg) Vehicle 3 M −5.68 ± −33.02 ± −2.75 ± 2.48 ± −2.51 ± control 10.28 11.30 21.19 32.61 27.10 Exemplified 3 M −1.45 ± −32.50 + −63.03 + −65.67 + −67.21 + fusion 13.62 11.15 6.09 11.75 14.11 compound (1.0 mg/kg) Vehicle 3 M 17.05 ± −12.04 ± −39.88 ± −15.25 ± −15.25 ± control 9.97 30.16 4.83 15.01 9.95 RANKEL 6 M 0 ± 0 −49.32 ± −29.83 ± −41.11 ± −60.32 ± mAb 6.93 22.69 11.58 8.75 (0.1 mg/kg) RANKEL 3 M 0 ± 0 −61.37 + −58.89 ± −64.36 ± −77.70 ± mAb 1.32 8.28 8.41 0.38 (1.0 mg/kg) Mean Percent Change (±SD where applicable) from Baseline in Serum CTx Days post-dosing Molecule 5 6 7 8 9 10 Exemplified −43.25 ± −31.24 ± −22.77 ± −22.89 ± −20.65 ± −29.29 ± fusion 6.95 10.12 6.47 10.39 11.22 24.20 compound (0.1 mg/kg) Vehicle −13.64 ± −15.22 ± −15.43 ± −26.97 ± −21.27 ± 7.2213 ± control 39.17 26.67 22.75 8.18 26.03 35.54 Exemplified −67.65 ± −70.07 ± −69.20 ± −67.24 ± −68.19 ± −62.46 ± fusion 13.75 7.15 15.01 9.46 17.41 11.29 compound (1.0 mg/kg) Vehicle 55.54 ± 63.37 ± 68.26 ± 73.74 ± 79.19 ± 92.41 ± control 57.18 71.66 86.77 102.74 119.35 128.03 Exemplified −47.30 −13.26 −25.46 −18.61 −27.29 NC fusion compound (0.1 mg/kg) Vehicle −10.43 ± −4.15 ± −1.80 ± 3.537707 15.19 ± NC control 22.82 34.72 29.84 11.56 Exemplified −66.17 + NC −69.30 + NC −64.36 + NC fusion 13.95 8.27 14.50 compound (1.0 mg/kg) Vehicle −2.22 ± NC 26.93 ± NC −28.87 ± NC control 19.06 10.66 5.85 RANKEL −64.90 ± NC −28.04 ± NC NC −16.63 ± mAb 3.43 12.42 8.43 (0.1 mg/kg) RANKEL −76.0411 ± NC −75.22 + NC NC −71.80 + mAb 2.44 4.81 7.89 (1.0 mg/kg)

(59) The results presented in Table 10 demonstrate that after a single dose of either 0.1 or 1.0 mg/kg of the exemplified fusion compound, in both male and female monkeys, serum CTx levels are reduced relative to control-treated monkeys (and reduced to levels similar to the RANKL mAb-only treated groups).

(60) Arterial Pressure Effects in Cynomolgus Monkey Model

(61) Effects on arterial pressure are assessed using a female cynomolgus monkey model. Female cynomolgus monkeys, aged five to six years, receive a single subcutaneous injection of either: 0.1 mg/kg or 1.0 mg/kg of the exemplified fusion compound; or of PBS vehicle control. Mean arterial pressure is measured for each animal at one-hour intervals for the first eight hours post-injection. A baseline arterial pressure for each animal is measured at time point 0. Results are presented in Table 11, including the mean difference in mmHg between treated and control groups (adjusted for baseline correction).

(62) TABLE-US-00011 TABLE 11 Mean Arterial Pressure in Cynomolgus Monkeys. Mean Arterial Pressure (mm Hg) ± SD from Baseline hours post-dosing Molecule N 0 1 2 3 4 5 6 7 8 Exemp. 3 77.71 85.15 64.79 65.08 64.50 62.42 66.23 64.17 68.61 fusion (0.93) (3.00) (6.90) (5.59) (2.50) (7.21) (10.75) (3.96) (1.74) cmpd 0.1 mg/kg Vehicle 4 84.72 85.81 79.98 78.84 75.40 72.81 74.10 76.90 80.18 Control (6.06) (9.28) (7.83) (3.74) (2.43) (4.39) (3.84) (2.80) (5.68) Mean +6.35 −8.18 −6.75 −3.89 −3.38 −0.86 −5.63 −4.56 +6.35 difference Exemp. 3 84.72 82.13 77.34 77.38 74.73 75.91 74.14 73.18 73.59 fusion (6.06) (11.92) (6.68) (6.18) (7.79) (10.27) (10.13) (8.27) (12.58) cmpd 1.0 mg/kg Vehicle 6 77.71 75.24 59.04 61.54 62.98 67.52 66.71 67.86 66.44 Control (0.93) (6.02) (9.18) (3.64) (5.90) (9.42) (7.24) (3.74) (2.95) Mean +0.12 +11.29 +8.83 +4.74 +1.38 +0.42 −1.69 +0.14 +0.12 difference

(63) The results presented in Table 11 demonstrate that a single dose of 0.1 or 1.0 mg/kg of the exemplified fusion compound does not stimulate a mean difference in arterial pressure between treated and untreated groups of greater than 10 mm Hg for the first 8 hours post treatment (subsequent testing, data not shown, demonstrates the mean arterial pressure between treated and untreated groups does not exceed 10 mm Hg for at least 69 hours post-dosing).

(64) Fusion Compound Physical and Chemical Property Analysis

(65) Fusion Compound Solubility Analysis

(66) Solubility of the exemplified fusion compound is analyzed at 4° C. after a 4 week incubation period. Solubility is assessed with fusion compound concentrated to between 100 and 150 mg/mL using a Millipore centrifugal filter device (p/n. #UFC803024). Samples are formulated in three buffers: (a) 10 mM citrate at pH 6.0; (b) 10 nM citrate at pH6.0 plus 150 mM NaCl; and (c) PBS at pH 7.4. The exemplified fusion compound exhibited a solubility of greater than 110 mg/mL for all formulations. Formulation (b), 10 nM citrate at pH6.0 plus 150 mM NaCl, exhibited a solubility of greater than 150 mg/mL.

(67) Additionally, formulated samples (a-c, as described above) are analyzed for percent high molecular weight (% HMW) soluble aggregate using size exclusion chromatography (SEC) with a TSKGEL Super SW3000™ (Tosoh Bioscience product #18675) column. Samples are assayed both at 100 mg/mL and 1 mg/mL. Chromatograms are analyzed using Chem Station and % high molecular weight (HMW) is calculated using the ratio of AUC of the peaks eluted before the monomer peak to total AUC. Both formulations (a) and (b), at both 100 and lmg/mL concentrations, exhibited less than a 5% increase in HMW soluble aggregate formation.

(68) Low Concentration Freeze/Thaw Analysis

(69) Low concentration freeze/thaw analysis of the exemplified fusion compound is assessed with fusion compound concentrated at 1 mg/ml and formulated in 10 mM citrate, pH 6.0, with and without 150 mM NaCl and with and without 0.02% TWEEN-80™ detergent (pH 5.5 and pH6.0, respectively). Three freeze/thaw cycles (a single cycle including incubation at −70° C. for at least four hours, followed by thawing at ambient temperature, then gentle mixing) are performed and particle growth for each sample is assessed using a HIAC Particle Counter (Pacific Scientific, p/n. 9703). Percent high molecular weight (% HMW) soluble aggregate using SEC is also assessed. Particle counts for both formulations (with and without 0.02% TWEEN-80™ detergent) are less than 400. Formulations with 0.02% TWEEN-80™ detergent demonstrated a reduction in particle counts. Additionally, all formulations exhibit less than a 5% increase in HMW soluble aggregate formation. These results demonstrate the exemplified fusion compound of the present invention, under low concentration conditions, is stable following multiple freeze/thaw cycles.

(70) High Concentration Physical Stability Analysis

(71) High concentration freeze/thaw analysis of the exemplified bispecific antibody is assessed with bispecific antibody concentrated at 50 mg/ml and formulated in either 10 mM citrate, pH 6.0 with 150 mM NaCl or 10 mM citrate, pH 6.0, 0.02% TWEEN-80™ detergent, with 150 mM NaCl. Samples are either incubated for four weeks at 4° C., 25° C., or subjected to three freeze/thaw cycles (a single cycle including incubation at −70° C. for at least four hours, followed by thawing at ambient temperature, then gentle mixing). Following the respective incubation or freeze thaw period, samples are analyzed for particle growth using HIAC Particle Counter or % HMW soluble aggregate using SEC. Particle counts for both formulations under all treatment conditions are less than 1000. Additionally, all formulations exhibited less than a 6.5% increase in HMW soluble aggregate formation. These results demonstrate the exemplified fusion compound of the present invention, under high concentration conditions, is stable following incubation under various conditions and multiple freeze/thaw cycles.

(72) TABLE-US-00012 Sequences SEQ ID NO: 1 - Exemplified First Polypeptide (of exemplified fusion compound of Table 1) SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFGGGGSGGGGSGGGGSQ VQLVQSGAEVKKPGSSNIKVSCKASGYAFTNYYIEWVRQAPGQGLEWMGV INPGWGDTNYNEKFKGRVTITADKSTSTAYMELSSLASEDTAVYYCARRD TAHGYYALDPWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK TYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG SEQ ID NO: 2 - Exemplified Second Polypeptide (of the exemplified fusion compound of Table 1) DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKLLIYS ASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYWDYPLTFGG GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC SEQ ID NO: 3 - DNA Seq. Encoding the Exemplified First Polypeptide (SEQ ID NO. 1) and a Signal Peptide AGCGTGTCCGAGATCCAGCTGATGCACAACCTCGGCAAGCACCTGAATAG CATGGAGCGCGTCGAGTGGCTGCGGAAGAAACTGCAGGACGTGCACAACT TCGGCGGCGGCGGCAGCGGCGGTGGCGGCTCCGGTGGCGGCGGAAGCCAG GTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCAGT GAAGGTTTCCTGCAAGGCATCTGGCTACGCCTTCACCAACTACTATATCG AGTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGTGATC AACCCCGGCTGGGGCGACACGAACTACAACGAGAAGTTCAAGGGCAGAGT CACCATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCA GCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGACGCGATACG GCTCACGGCTACTACGCCCTTGATCCGTGGGGCCAAGGAACCACGGTCAC CGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCGCTAGCGCCCT GCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAG GACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGAC CAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT CCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACC TACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAG AGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGG CCGCCGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACT CTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAG CCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGG TGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTAC CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAA GGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGA AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACC CTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTG CCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAAAGCA ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCC GACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTG GCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACA ACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTTGA SEQ ID NO: 4 - DNA Seq. Encoding the Exemplified Second Polypeptide (SEQ ID NO. 2) and a Signal Peptide GGCGGCGGCGGCAGCGGCGGTGGCGGCTCCGGTGGCGGCGGAAGCGACAT CCAGATGACCCAGTCTCCATCCTCTCTGTCTGCATCTGTAGGAGACAGAG TCACCATCACTTGCAAGGCCAGCCAGAATGTGGGCACCAACGTGGCCTGG TATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGCGCCAG CTACAGATACAGCGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGA CAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACT TACTACTGTCAGCAGTACTGGGACTACCCCCTGACCTTCGGCGGAGGGAC CAAGGTGGAGATCAAACGGACTGTGGCTGCACCATCTGTCTTCATCTTCC CGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTG CTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAA CGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCA AGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGAC TACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAG CTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGCTAA SEQ ID NO: 5 - Exemplified HCVR (of exemplified fusion compound of Table 1) QVQLVQSGAEVKKPGSSVKVSCKASGYAFTNYYIEWVRQAPGQGLEWMGV INPGWGDTNYNEKFKGRVTITADKSTSTAYMELSSLASEDTAVYYCARRD TAHGYYALDPWGQGTTVTVSS SEQ ID NO: 6 - Exemplified LCVR (of exemplified fusion compound of Table 1) DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKLLIYS ASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYWDYPLTFGG GTKVEIK SEQ ID NO: 7 - Exemplified HCDR1 (of exemplified fusion compound of Table 1) GYAFTNYYIE SEQ ID NO: 8 - Exemplified HCDR2 (of exemplified fusion compound of Table 1) VINPGWGDTNYNEKFKG SEQ ID NO: 9 - Exemplified HCDR3 (of exemplified fusion compound of Table 1) RDTAHGYYALDP SEQ ID NO: 10 - Exemplified LCDR1 (of exemplified fusion compound of Table 1) KASQNVGTNVA SEQ ID NO: 11 - Exemplified LCDR2 (of exemplified fusion compound of Table 1) SASYRYS SEQ ID NO: 12 - Exemplified LCDR3 (of exemplified fusion compound of Table 1) QQYWDYPLT SEQ ID NO: 13 - Exemplified PTH Peptide of exemplified fusion compound of Table 1 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF SEQ ID NO: 14 - Exemplified Linker of exemplified fusion compound of Table 1 GGGGSGGGGSGGGGS SEQ ID NO: 15 - Full length Human Parathyroid Hormone SVSEIQLMHN LGKHLNSMER VEWLRKKLQD VHNFVALGAP LAPRDAGSQR PRKKEDNVLV ESHEKSLGEA DKADVNVLTK AKSQ