Bispecific Antibody Binding To RANKL and Osteonectin with

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Brief Article

A bispecific antibody binding to RANKL and osteonectin with enhanced localization to the bone Jou-Han Chen, Chun Yu Lin, Yi-Chun Maria Chen, Wei-Ting Tian, Hsing-Mao Chu, and Tse Wen Chang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00501 • Publication Date (Web): 27 Sep 2017 Downloaded from http://pubs.acs.org on September 29, 2017

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Molecular Pharmaceutics

A bispecific antibody binding to RANKL and osteonectin with enhanced localization to the bone Jou-Han Chen,† Chun Yu Lin,† Yi-Chun Maria Chen,† Wei-Ting Tian,† Hsing-Mao Chu,† Tse Wen Chang†‡ †

‡

Immunwork, Inc., Taipei 115, Taiwan

Genomics Research Center, Academia Sinica, Taipei 115, Taiwan

KEYWORDS Anti-RANKL, denosumab, osteonectin, SPARC, bispecific antibody, bone-targeting, disease site delivery

ABSTRACT

Therapeutics reducing bone turnover, such as denosumab (Dmab), an anti-RANKL antibody, can provide treatments for patients with bone destruction. However, some

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patients with osteoporosis or localized primary bone tumors and many patients with various types of bone-metastatic cancer display unsatisfactory responses to Dmab. For achieving greater efficiency of RANKL neutralization in the bone microenvironment by enhancing the distribution of Dmab to the bone, we reengineered Dmab by fusing with single-chain variable fragments of an antibody specific for osteonectin (On), which is abundantly expressed in osseous tissues. The bispecific antibody, Dmab-FvOn, showed a similar activity as Dmab in inhibiting RANKL as examined in an osteoclast differentiation assay. When administered to mice, Dmab-FvOn was found to localize in increased proportions at the endosteum of the bone where osteonectin is abundant. Our study suggests that by linking anti-RANKL with an osteonectin-targeting moiety, a greater proportion of the therapeutic effector can be distributed in the bone. Future studies are needed to investigate whether the bispecific antibody can achieve higher therapeutic efficacy and lower toxicity. INTRODUCTION Bone remodeling is a continuous process that renews the skeleton through highly regulated osteoblast-mediated bone formation and osteoclast-mediated bone resorption. Receptor activator of nuclear factor kappa-B ligand (RANKL), a cytokine of the tumor necrosis factor superfamily produced by osteoblasts, is an essential regulator of bone remodeling due to its role as a resorptive signal1-3. Upon binding to its cell surface receptor (RANK) on osteoclasts and their precursors, RANKL promotes the formation and activity of osteoclasts, resulting in enhanced bone resorption. Osteoprotegerin, the soluble “decoy receptor” of RANKL, prevents the binding of RANKL to its receptor, thereby reducing osteoclastic activity4, 5. This RANK-RANKL-osteoprotegerin pathway


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thus constitutes the principal regulating mechanism balancing bone formation and resorption. RANKL is also suspected to be a chemotactic factor influencing the metastatic migration of tumor cells into bones6, 7. Furthermore, abnormally increased RANKL expression by stromal cells promotes the neoplastic transformation of these cells to giant cell tumor of bone8-10. Similar to osteoprotegerin, the anti-RANKL monoclonal antibody denosumab (Dmab) binds to RANKL and inhibits its interaction with RANK, yielding clinical benefits in patients with unbalanced RANK-RANKL-osteoprotegerin system11-14. Dmab has been approved for treating bone loss and bone destruction in patients with osteoporosis, advanced cancer with bone metastasis, and giant cell tumor of bone15, 16. In a clinical trial on postmenopausal women with low bone mineral density, Dmab showed 68% and 20% reduction in relative risks of vertebral and non-vertebral fractures, respectively, over placebo11. When compared to the conventional dosing of zoledronic acid in treating bone metastasis from breast and prostate tumors, Dmab has been demonstrated to be more effective in delaying skeletal-related events13, 14, although no superiority was shown in patients having other types of advanced cancer with bone metastasis, such as non-small cell lung cancer and multiple myeloma17. The overall response rate to Dmab in patients with giant cell tumor of bone was 25% by RECIST 1.1 analysis18. As for safety concerns, cancer patients receiving Dmab were reported to have increased incidences of hypocalcemia13, 14, and osteoporotic subjects treated with Dmab were more likely to develop skin-related adverse events11, 19. In an earlier study, it was found that upon injection in a mouse, Dmab was distributed like a regular IgG antibody without increased concentrations in bone matrix or bone



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surfaces12. With a purpose of raising response rates and reducing side effects of Dmab, it is desirable to channel increased proportions of the injected Dmab to the bone and decreased amounts to other sites of the body. We therefore adopt an approach of combining Dmab with a component bearing bone-targeting function in the same pharmaceutical molecule. The chosen component is a fragment of an antibody specific for osteonectin (also referred to as “secreted protein acidic and rich in cysteine or SPARC”), which is a matrix-associated protein abundant in osseous tissues20,

21

. We

rationalize that an anti-RANKL effector imparted with an osteonectin-targeting ability can neutralize RANKL in the bone microenvironment more effectively than an antiRANKL antibody alone and hence can result in better therapeutic effects and safety profiles.

EXPERIMENTAL SECTION Preparation of antibodies The hybridoma line producing an anti-osteonectin monoclonal antibody (mAb 175) was obtained from the Developmental Studies Hybridoma Bank, Iowa University and maintained in Iscove’s DMEM supplemented with 20% FBS (Gibco, USA) and 50 µg/mL gentamicin at a density of 0.2 - 1.0 x 106 cells/mL. The secreted mAb 175 was purified using rProtein A Sepharose column (GE Healthcare, USA). The purified mAb 175 was buffer-exchanged to PBS and stored at -20°C. The anti-RANKL mAb, denosumab (ProliaTM; Amgen, USA) was a gift of Dr. Wen-Hung Chung, Chang Gung Medical Foundation, Taiwan. The bispecific antibody, Dmab-FvOn, was prepared by combining denosumab and the single chain variable fragments (scFv) of mAb 175. The cDNA of VH and VL of mAb 175 were


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obtained using pairs of mouse γ and κ primer sets (Merck, Germany). Amino acid sequences of γ2a and κ chains of denosumab were retrieved from DrugBank database (accession number DB06643). Respective codon-optimized DNA encoding the light chain of denosumab and DNA encoding the heavy chain of denosumab fused with the scFv of mAb 175 were synthesized by Protech Technology Enterprise (Taiwan). The light chain gene was cloned into pIgG1κ expression vector with DraIII and EcoRI sites and the heavy chain gene into the same vector with MluI and BamHI sites. For the recombinant heavy chain, a (GGGGS)3 linker was inserted between the CH3 domain of denosumab and the VH from mAb 175, and a linker peptide, GSTSGSGKPGSGEGSTKG, between the VH and VL of mAb 175 scFv. Expi293F cells were transfected with the expression plasmid and incubated for 7 days. The expressed Dmab-FvOn in cell culture supernatant was purified by rProtein A Sepharose (GE Healthcare, USA). The protein concentration was measured by A280 and its identity verified by mass spectrometry. It was then stored in PBS at -20°C. Production and purification of RANKL and osteonectin Human RANKL (hRANKL), which was used in ELISA and osteoclastogenesis assays, was generated by cloning the fragment of amino acid residues #143-317 (the TNF-α-like domain) from

human

PBMCs

by

using

primers

5′-cacgatgtgagaaagcgatggtggatg-3′

and

5′-

ggatcccgatcaatctatatctcgaactttaaaagcccca-3′, placing DraIII and BamHI sites at the N- and Ctermini, respectively. The fragment, with sequence confirmed with the NCBI database, was incorporated into pIgG1κ expression vector for transfecting Expi293F cells (Thermo Fisher Scientific, USA). Sepharose medium (GE Healthcare, USA) conjugated with Dmab was prepared to affinity-purify the expressed RANKL. Protein concentration of purified RANKL was



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measured by A280 and its identity verified by mass spectrometry. The purified RANKL was buffer-exchanged to PBS and stored at -20°C. The gene of full-length human osteonectin was synthesized according to the NCBI database (accession number CAG33080) (Protech Technology Enterprise, Taiwan) and subcloned into pIgG1κ vector using BglII and BamHI. The protein was expressed in Expi293F cells and purified with mAb 175-conjugated affinity column. Protein identity was verified by mass spectrometry. Biotin and DyLight 680 labeling of proteins and antibodies Human RANKL and human osteonectin were labeled with biotin using an EZ-Link SulfoNHS-Biotin kit (Thermo Fisher Scientific, USA) for use in ELISA. To study the bio-distribution of antibodies in mice, Dmab, mAb 175, and Dmab-FvOn were labeled with DyLight 680 (Thermo Fisher Scientific, USA) and the conjugates purified according to the manufacturer’s instruction. An average 2-2.5 molecules of DyLight 680 were conjugated per IgG molecule. In vitro osteoclastogenesis assay All experiments with mice were conducted under the guidelines of the Institutional Animal Care and Use Committee of Academia Sinica for the care and use of animals. C57BL/6 mice aged 8-10 weeks were purchased from BioLASCO, Taiwan. Isolation and culturing of primary cells were performed according to Hsu et al.22 Bone marrow cells from the femurs were incubated in α-MEM (Gibco, USA) containing 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin, for 12 hrs at 37°C in 5% CO2. Subsequently, non-adherent cells were collected and seeded in 24-well plates at 2 x 106 cells/well supplemented with 30 ng/mL of recombinant mouse macrophage colony stimulating factor (R&D Systems, USA). After 48 hrs, 40 ng/mL of mouse macrophage colony stimulating factor and 100 ng/mL hRANKL were applied to trigger the differentiation of osteoclasts, with or without denosumab, DmabON, or mAb 175. During the


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osteoclast induction, fresh medium was replenished on day 3, and the experiment was terminated on day 5. To reveal tartrate-resistant acid phosphatase (TRAP) activity, cells were washed with PBS and fixed with 4% paraformaldehyde before staining with a TRAP assay kit (Sigma Aldrich, USA). TRAP-positive cells with three or more nuclei were regarded as mature osteoclasts and their numbers in each wells enumerated. ELISA To test the binding specificity of Dmab-FvOn, 50 ng/well of human RANKL, human osteonectin or mouse osteonectin (R&D Systems, USA) was coated on wells of 96-well plates (Greiner Bio-One, Austria) at 4°C overnight. The wells were blocked with PBS containing 0.5% BSA, 0.05% Tween 20 and 0.01 % Thimerosal, at room temperature for 1 hr, and then washed, and incubated with 1 g/mL Dmab-FvOn, Dmab or mAb 175 for 1 hr. After washing, bound antibodies were detected with HRP-labeled goat anti-human or anti-mouse antibodies (Jackson ImmunoResearch, USA) and 3,3′,5,5′-tetramethylbenzidine substrate (BioVision, USA). Absorbance at 450 nm was measured by a SpectraMax M2 reader (Molecular Devices, USA). The dual binding activity of Dmab-FvOn to osteonectin and to RANKL was also examined in an ELISA. Human osteonectin was coated at 50 g/well on an ELISA plate, followed by blocking and incubation with 1 µg/mL Dmab-FvOn. Biotin-labeled hRANKL was subsequently added at 1 µg/mL. The assay was performed in comparison with Dmab and mAb175. Bound antibodies were detected by HRP-labeled goat anti-human or goat anti-mouse antibodies (Jackson ImmunoResearch, USA), and biotin-labeled hRANKL was detected by HRP-labeled streptavidin (Sigma Aldrich, USA). To compare the binding strength of Dmab-FvOn and Dmab to hRANKL, various concentrations (10-fold serial dilutions from 10 µg/mL to 10-5 µg/mL) of respective antibodies



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were added to RANKL-coated plates. Bound antibodies were measured with HRP-labeled goat anti-human antibodies (Jackson ImmunoResearch, USA). Tissue distribution and serum levels of antibodies in mice Female BALB/c mice of 8-10 weeks old were purchased from BioLASCO Taiwan. Mice were given single intravenous injections of vehicle (PBS), DyLight 680-labeled denosumab (100 µg/mouse), DyLight 680-labeled mAb 175 (100 µg/mouse) or DyLight 680-labeled Dmab-FvOn (130 µg/mouse, based on its 1.3x molecular weight of regular antibodies). After the antibody injection, mice were shaved, excluding the head and neck areas, for subsequent imaging procedures. At 3, 24, 48 and 72 hrs after injection, mice were anesthetized with isofurane and placed in an IVIS Spectrum Imager (PerkinElmer, USA) to observe the distribution of injected antibodies at Ex/Em = 675/720 nm. One mouse was used for each time point (3, 24, 48, and 72 hrs) for each of four groups (PBS, Dmab, mAb 175 and Dmab-FvOn); a total of 16 mice were used in each distribution assay. Three separate assays were performed and a total of 48 mice were used. Data from one of the three assays were presented. After image acquisition, mice were sacrificed for sampling blood and tissues. Blood samples were allowed to clot for 30 min at room temperature and subjected to centrifugation at 10000 rcf for 5 minutes to obtain sera. Aliquots of 50 µL of sera were placed in wells of a black 96-well plate (Greiner Bio-One, Austria) for measuring fluorescence using an IVIS Spectrum Imager. Tissue preparation and immunohistochemistry After biofluorescence imaging, the femoral bones of mice were excised and fixed in 10% formaldehyde in PBS for 1 day. After washing, the bone tissues were decalcified with 10% EDTA for 7 days, with daily renewal of the decalcifying solution. Bone samples were dehydrated and paraffinized using Shandon Excelsier tissue processor (Thermo Fisher Scientific, USA).


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Sections of 3 µm were deparaffinized and hydrated using Leica Autostainer XL. Recovery of antigenicity of proteins in the bone tissues was performed with 20 µg/mL of proteinase K and 0.5% solution of hyaluronidase, followed by quenching the activity of endogenous peroxidase in 3% H2O2. The blocking and staining procedures were performed according to the instruction provided in the Tyramide Signal Amplification - Biotin Kit (PerkinElmer, USA) and visualized with 3,3'-diaminobenzidine substrate (Enzo Life Sciences, USA). In brief, HRP-labeled goat antihuman IgG (Jackson ImmunoResearch, USA) was used at 25 µg/mL to bind to Dmab or DmabFvOn in the tissue sections, which were subsequently reacted with Tyramide amplification reagent to generate biotin conjugation onto the goat anti-human IgG. After further incubation with streptavidin-HRP, 3,3'-diaminobenzidine substrate (Enzo Life Sciences, USA) was used to form brown precipitate. Sections were further counterstained with hematoxylin. Histological images were acquired using a ScanScope XT slide scanner (Leica Biosystems, Germany). The images were further analyzed semi-quantitatively using the “Positive Pixel Count Algorithm” in the ImageScope software (Leica Biosystems, Germany) to enumerate diaminobenzidine-positive pixels in four randomly selected fields in cortical area and trabecular areas in each stained bone sections. Statistical Analysis Prism 6.0 (GraphPad Software, USA) was used for statistical analysis. One-way ANOVA and Tukey’s multiple comparison test was used to compare data between groups in TRAP staining and IHC experiments. Results were expressed as means ± s.d. and P values < 0.05 were considered as having significant differences.

RESULTS



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Preparation and Characterization of Dmab-FvOn The molecular configuration of Dmab-FvOn is a tetravalent bispecific antibody, which consists of a Dmab IgG and two scFv from an osteonectin-targeting mAb 175 fused to the C-termini of Dmab CH3 domains (Figure 1A). The fused heavy chain of Dmab-FvOn had a size of 70 kDa and the light chain, 25 kDa, as shown in the SDS-PAGE analysis (Figure 1B). Dmab-FvOn maintained the same binding properties of Dmab in binding to hRANKL and of mAb 175 in binding to human and mouse osteonectin (Figure 1C). The ability of Dmab-FvOn in binding to mouse osteonectin allowed its in vivo experiments in mice. The ELISA in Figure 1D showed that Dmab-FvOn could bind to both human osteonectin and hRANKL simultaneously, whereas Dmab did not bind to human osteonectin, and mAb 175 not to hRANKL. Furthermore, Dmab-FvOn and Dmab exhibited very similar binding to hRANKL over a broad range of concentrations (Figure 1E). The above results indicate that in Dmab-FvOn, the scFvs at the C-termini can bind to osteonectin like parental mAb 175 and that the IgG moiety can bind to RANKL as well as denosumab. To examine whether Dmab-FvOn and Dmab had similar activities in neutralizing RANKL’s biological effects, their inhibitory activity on RANKL-induced osteoclast formation was determined. Osteoclast precursor cells were prepared from the bone marrow of excised femurs of C57BL/6 mice, and stimulated with mouse macrophage colony stimulation factor and hRANKL to drive osteoclast differentiation for 5 days, with or without Dmab-FvOn, Dmab or mAb 175. Figure 2 shows that 100 ng/mL of hRANKL markedly induced the formation of osteoclasts and that Dmab-FvOn and Dmab both nearly abolished osteoclast formation. The osteonectin-specific mAb 175 was observed to partially inhibit osteoblast formation for unknown reasons. Evaluation of in vivo bone-targeting effect of Dmab-FvOn


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To evaluate the bone-targeting effect of Dmab-FvOn, we studied its relative distribution in blood and bones, and compared it with those of Dmab and mAb 175. Following single intravenous injections in BALB/c mice, the blood clearance of DyLight 680-labeled antibodies in the circulation was investigated by measuring fluorescence in the serum at different time points (Figure 3). Linearity was observed between fluorescence intensity (Y) and the concentration of antibodies (X) in the range of 1.56-100 µg/mL (Y = 4x10-7 X + 8x10-7), and R2 = 0.9991, indicating a strong linear relationship. Thus, the antibody concentration in the blood can be calculated from the measured fluorescence. Dmab-FvOn and mAb 175 displayed similar kinetic properties and their serum concentrations dropped quickly and leveled off after 48 hrs. Dmab in serum remained at levels higher than those of mAb 175 and Dmab-FvOn throughout the 72-hr period. To study how the additional osteonectin-targeting feature would alter the tissue distribution of antibodies, biofluorescence imaging of mice was performed at 3 hrs, 24 hrs, 48 hrs, and 72 hrs after the administration of DyLight 680-labeled antibodies (Figure 4A). Dmab was visualized evenly throughout the mouse body, which is in accordance with previous studies that Dmab is primarily detected in the circulation12, 23. In comparison with Dmab, both mAb 175 and DmabFvOn appeared to distribute disproportionately in the spine area, as revealed by using the IRDye 680RD BoneTag reagent, which accumulates in mineralized bones24 (Figure 4B). To further examine the skeletal disposition of antibodies, immunohistochemistry was performed on tissue sections of mouse femoral bone to detect the presence of bound Dmab and Dmab-FvOn, after the antibodies were injected into the mice (Figure 5A). In PBS-treated mice, there was very faint, spotty staining in the bone matrix, which was possibly background signal produced by the HRP-labeled goat anti-human IgG antibody, since such staining was absent in



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control sections without this antibody. In tissue sections from Dmab-treated mice, only similar residual background staining was found in the bone matrix (Figure 5B). On the contrary, in tissue sections from Dmab-FvOn-treated mice, conspicuous stains were observed along the cortical and trabecular bone surfaces facing the marrow cavities. It is known that these regions are aligned by osteoblasts and osteoclasts. It is also consistent with the report that osteonectin is mainly produced by osteoblasts25-27. The staining of Dmab-FvOn in cortical and trabecular bones was significantly more intense than that of Dmab (Figure 5B). Dmab-FvOn was also present in blood vessels, possibly due to the expression of osteonectin by vascular endothelial cells28, 29. The results that Dmab-FvOn is cleared much faster than Dmab in the circulation (Figure 3) and that Dmab-FvOn is present at much higher levels than Dmab in the bone tissues indicate that osteonectin can serve as a target for increased bone targeting of therapeutic agents, such as Dmab.

DISCUSSION In this study, we explore a therapeutic approach of delivering proportionally larger amounts of the administered Dmab to the bone and less to other parts of the body, so that more effective inhibition of RANKL in the bone and fewer side effects elsewhere can be achieved. Towards this aim, we generated Dmab-FvOn, a bispecific antibody, which is capable of both binding to osteonectin in bone tissues and neutralizing RANKL, and characterized its biological properties in in vitro and in vivo studies. In skeletal tissues, bone remodeling takes place in the cortical and trabecular endosteum. In this region, osteoblasts express RANKL on the cell surface30. Since the membrane-bound RANKL is more potent than the shed and soluble RANKL in driving the formation and activity of


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osteoclasts31, 32, it is the primary target for a RANKL inhibitor drug. Osteonectin, a major protein of the extracellular matrix in the endosteum, is also produced by osteoblasts33-35. Thus, DmabFvOn, by targeting osteonectin, should bring a RANKL inhibitor to the vicinity of the membranebound RANKL and blocks its activity to activate osteoclasts. The fact that osteonectin is not expressed on the cell surface helps alleviate the concern of possible direct adverse effects of antibody binding on cells. The experiments on whole body imaging of mice and histological examination comparing the distribution of the injected Dmab-FvOn and Dmab showed that the staining of Dmab-FvOn in the bone tissue sections was more abundant in the endosteum. This indicates that osteonectin-targeting increased the localization of Dmab-FvOn to where membrane-bound RANKL is located. The molecular mechanism, which causes Dmab-FvOn to be cleared much faster than Dmab from blood circulation, is not understood. One possibility is that osteonectin is present in other tissues in addition to bones and thus helps exhaust Dmab-FvOn. Nonetheless, while Dmab-FvOn existed at lower levels than Dmab in the blood at various time points, Dmab-FvOn was found at abundant amounts in the bone endosteum. The fact that Dmab was detected at high levels in serum, and at scarce levels in the bone endosteum is consistent with the earlier report that the administered Dmab in mice was readily observable in the vasculatures in the bones, but not in the endosteum12. These findings relating to the kinetics and tissue distribution of Dmab and DmabFvOn support the value of osteonectin-targeting in channeling Dmab-FvOn to the bone. In summary, the present study shows that Dmab-FvOn, a bispecific antibody binding to both RANKL and osteonectin, can potentially provide an improved anti-RANKL therapy. Further studies investigating long-term effects of Dmab-FvOn on bone mineral density and comparing therapeutic effects of Dmab-FvOn and Dmab in animal models of osteoporosis are warranted.



FIGURES

A

C

B

Dmab

kDa

D

ab m

D

m

vO -F b a

n

2.0

hRANKL hOn mOn HSA

1.5 OD450

130 100 70

scFv anti-On

55

1.0 0.5

40 35

0.0

Dmab-FvOn

Dmab

mAb 175

25

D

E 2.0

Detection with goat anti-human Ig or goat antii-mouse Ig

1.5

Detection with biotin-hRANKL and streptavidin-HRP

Dmab-FvOn Dmab

OD450

OD450

1.5 1.0

1.0

0.5 0.5 0.0 5 A

b

17

ab m

vO -F ab

10-5 10-4 10-3 10-2 10-1 100 101 10 Antibody concentration (µg/mL)

D

m

D m

n

5 17 b A

D m

ab m

m

ab

-F

vO

n

0.0

D

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 27

Figure 1. Characterization of the bispecific antibody Dmab-FvOn. (A) Schematic representation of the bispecific construct containing denosumab, denoted “Dmab” (with variable regions in green) and two scFvs derived from osteonectin (On)-specific mAb 175 (orange) at the C-termini of the heavy chains. (B) SDS-PAGE analysis of Dmab and Dmab-FvOn, showing their respective heavy and light chains. (C) ELISA showing binding of Dmab-FvOn, Dmab, and mAb 175 to solid-phase antigens: human osteonectin, mouse osteonectin, human RANKL, and human serum albumin. (D) ELISA examining simultaneous binding of Dmab-FvOn to RANKL and osteonectin; the binding of antibodies to coated human osteonectin was revealed by secondary


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antibodies (left part), while the binding activity to human RANKL was detected by biotin-human RANKL followed by streptavidin-HRP (right part). The bars represent the mean of three replicates ± s.d. (E) ELISA showing the binding of different concentrations of Dmab and DmabFvOn to immobilized RANKL.

A 60 TRAP+ multinucleated cells/well

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40

*

20 **

**

+

+

+

+ -

-

-

0 hRANKL

-

+

Dmab

-

-

Dmab-FvOn mAb 175

+ -

+

B RANKL

RANKL + Dmab-FvOn

RANKL + Dmab

RANKL + mAb 175

Figure 2. Inhibition of osteoclastogenesis by Dmab-FvOn in vitro. Bone marrow cells from C57BL/6 mice were incubated with RANKL, with or without the addition of Dmab, DmabFvOn, or mAb 175. After 5 days the transformed osteoclasts were TRAP-stained and counted. TRAP-positive cells with more than three nuclei were considered as mature osteolclasts. (A)



Numbers of TRAP+ osteoclasts per well. Data were plotted as group mean ± s.d. (n = 3 wells/group). Statistical analysis was made comparing the effects of antibody-treated groups with the group treated with human RANKL alone. *P < 0.05, **P < 0.01. (B) Representative TRAPstaining of osteoclasts. Scale bar: 200 µm.

Serum levels of injected antibodies (µg/mL)

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80

Dmab mAb 175 Dmab-FvOn PBS

60 40 20 0 0

20

40

60

80

Time post injection (hr)

Figure 3. Kinetics of injected Dmab, mAb 175, and Dmab-FvOn in mice. Equal molar amounts of DyLight 680-labeled Dmab (100 µg), mAb 175 (100 µg) or Dmab-FvOn (130 µg) were administered intravenously. At 3, 24, 48, 72 hrs post injection, blood was drawn by orbital sinus sampling and let to clot, and 50 µL of sera were placed in wells of a black ELISA plate and the concentrations of the labeled antibodies were measured against a DyLight 680-labeled antibody standard using an IVIS Spectrum imager.


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A

B 1

2

3

4

BoneTag

3 hr

3 hr

Efficiency Color Scale Min = 9.35e-5 Max = 9.72e-4

24 hr

Efficiency

48 hr Color Scale Min = 5.13e-5 Max = 5.45e-4

72 hr

Figure 4. Live imaging of DyLight 680-labeled antibodies in vivo. (A) Mice were injected i.v. with vehicle PBS (set 1), 100 µg Dmab (set 2), 100 µg mAb 175 (set 3), or 130 µg of DmabFvOn (set 4). The images were captured for each set of 4 mice at 3, 24, 48, and 72 hrs post injections. (B) A mouse was injected with IRDye 680RD-labeled “BoneTag”, a Ca2+-chelating agent, and imaged at 3 hr after i.v. injection.



A 24 hr, Dmab

24 hr, Dmab-FvOn

72 hr, Dmab

72 hr, Dmab-FvOn

trabecular bone

cortical bone

PBS

B 24 hr, trabecular bone

24 hr, cortical bone Intensity of positive pixels/µm 2

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40

40

250

***

***

*** 30

30

20

20

72 hr, trabecular bone

72 hr, cortical bone 600

***

500

200

400 150 300 100 10

10

0

0 PBS

Dmab

Dmab-FvOn

200

50

100 0

0 PBS

Dmab

Dmab-FvOn

PBS

Dmab

Dmab-FvOn

PBS

Dmab

Dmab-FvOn

Figure 5. Immunohistochemical analysis of Dmab and Dmab-FvOn in the femoral bone of mice. Tissue specimens were sampled from mice at indicated time points after antibody injection. (A) Sections were stained with HRP-labeled goat anti-human IgG Fc, the signal was amplified using a Tyramide Signal Amplification kit. Counterstaining was performed with hematoxylin. Scale bar: 25 µm. (B) Semi-quantitation of DAB signals in IHC results. Four randomly selected areas of cortical and trabecular bone in each stained section were analyzed for positive brown pixels and plotted as group mean ± s.d. Statistical analysis was made comparing the signals of Dmabwith Dmab-FvOn-treated groups. ***P < 0.001.


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AUTHOR INFORMATION Corresponding Author Tse Wen Chang [email protected] Immunwork, Inc., Taipei, Taiwan 1F., No. 47 Sec. 2, Academia Rd. Nangang Dist., Taipei City 115 Author Contributions Jou-Han Chen designed the overall study and individual experiments, conducted the experiments, and wrote manuscript, Chun Yu Lin and Wei-Ting Tian conducted experiments, Yi-Chun Maria Chen and Hsing-Mao Chu designed the overall study, Tse Wen Chang designed the study and experiments and wrote manuscript.

ACKNOWLEDGMENT We thank Dr. Wen-Hung Chung of Chang Gung Medical Foundation for the gift of a vial of Prolia.

ABBREVIATIONS RANKL, receptor activator of nuclear factor kappa-B ligand; Dmab, donosumab; Dmab-FvOn, denosumab fused with scFv anti-osteonectin; TRAP, tartrate-resistant acid phosphatase.



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REFERENCES 1.

Wong, B. R.; Josien, R.; Lee, S. Y.; Sauter, B.; Li, H. L.; Steinman, R. M.; Choi, Y.

TRANCE (tumor necrosis factor [TNF]-related activation-induced cytokine), a new TNF family member predominantly expressed in T cells, is a dendritic cell-specific survival factor. The Journal of experimental medicine 1997, 186, (12), 2075-80. 2.

Yasuda, H.; Shima, N.; Nakagawa, N.; Yamaguchi, K.; Kinosaki, M.; Mochizuki, S.;

Tomoyasu, A.; Yano, K.; Goto, M.; Murakami, A.; Tsuda, E.; Morinaga, T.; Higashio, K.; Udagawa, N.; Takahashi, N.; Suda, T.

Osteoclast differentiation factor is a ligand for

osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proceedings of the National Academy of Sciences of the United States of America 1998, 95, (7), 3597-602. 3.

Lacey, D. L.; Timms, E.; Tan, H. L.; Kelley, M. J.; Dunstan, C. R.; Burgess, T.; Elliott,

R.; Colombero, A.; Elliott, G.; Scully, S.; Hsu, H.; Sullivan, J.; Hawkins, N.; Davy, E.; Capparelli, C.; Eli, A.; Qian, Y. X.; Kaufman, S.; Sarosi, I.; Shalhoub, V.; Senaldi, G.; Guo, J.; Delaney, J.; Boyle, W. J.

Osteoprotegerin ligand is a cytokine that regulates osteoclast

differentiation and activation. Cell 1998, 93, (2), 165-76. 4.

Mizuno, A.; Amizuka, N.; Irie, K.; Murakami, A.; Fujise, N.; Kanno, T.; Sato, Y.;

Nakagawa, N.; Yasuda, H.; Mochizuki, S.; Gomibuchi, T.; Yano, K.; Shima, N.; Washida, N.; Tsuda, E.; Morinaga, T.; Higashio, K.; Ozawa, H.

Severe osteoporosis in mice lacking

osteoclastogenesis inhibitory factor/osteoprotegerin. Biochemical and biophysical research communications 1998, 247, (3), 610-5.


Page 21 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


5.

Yasuda, H.; Shima, N.; Nakagawa, N.; Mochizuki, S. I.; Yano, K.; Fujise, N.; Sato, Y.;

Goto, M.; Yamaguchi, K.; Kuriyama, M.; Kanno, T.; Murakami, A.; Tsuda, E.; Morinaga, T.; Higashio, K. Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): a mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro. Endocrinology 1998, 139, (3), 1329-37. 6.

Saad, F.; Markus, R.; Goessl, C. Targeting the receptor activator of nuclear factor-

kappaB (RANK) ligand in prostate cancer bone metastases. BJU international 2007, 101, (9), 1071-5. 7.

Jones, D. H.; Nakashima, T.; Sanchez, O. H.; Kozieradzki, I.; Komarova, S. V.; Sarosi, I.;

Morony, S.; Rubin, E.; Sarao, R.; Hojilla, C. V.; Komnenovic, V.; Kong, Y. Y.; Schreiber, M.; Dixon, S. J.; Sims, S. M.; Khokha, R.; Wada, T.; Penninger, J. M. Regulation of cancer cell migration and bone metastasis by RANKL. Nature 2006, 440, (7084), 692-6. 8.

Atkins, G. J.; Haynes, D. R.; Graves, S. E.; Evdokiou, A.; Hay, S.; Bouralexis, S.;

Findlay, D. M. Expression of osteoclast differentiation signals by stromal elements of giant cell tumors. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 2000, 15, (4), 640-9. 9.

Atkins, G. J.; Kostakis, P.; Vincent, C.; Farrugia, A. N.; Houchins, J. P.; Findlay, D. M.;

Evdokiou, A.; Zannettino, A. C. RANK Expression as a cell surface marker of human osteoclast precursors in peripheral blood, bone marrow, and giant cell tumors of bone. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 2006, 21, (9), 1339-49.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 27

10. Cowan, R. W.; Singh, G.; Ghert, M. PTHrP increases RANKL expression by stromal cells from giant cell tumor of bone. Journal of orthopaedic research : official publication of the Orthopaedic Research Society 2012, 30, (6), 877-84. 11. Cummings, S. R.; San Martin, J.; McClung, M. R.; Siris, E. S.; Eastell, R.; Reid, I. R.; Delmas, P.; Zoog, H. B.; Austin, M.; Wang, A.; Kutilek, S.; Adami, S.; Zanchetta, J.; Libanati, C.; Siddhanti, S.; Christiansen, C. Denosumab for prevention of fractures in postmenopausal women with osteoporosis. The New England journal of medicine 2009, 361, (8), 756-65. 12. Kostenuik, P. J.; Nguyen, H. Q.; McCabe, J.; Warmington, K. S.; Kurahara, C.; Sun, N.; Chen, C.; Li, L.; Cattley, R. C.; Van, G.; Scully, S.; Elliott, R.; Grisanti, M.; Morony, S.; Tan, H. L.; Asuncion, F.; Li, X.; Ominsky, M. S.; Stolina, M.; Dwyer, D.; Dougall, W. C.; Hawkins, N.; Boyle, W. J.; Simonet, W. S.; Sullivan, J. K. Denosumab, a fully human monoclonal antibody to RANKL, inhibits bone resorption and increases BMD in knock-in mice that express chimeric (murine/human) RANKL. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 2009, 24, (2), 182-95. 13. Stopeck, A. T.; Lipton, A.; Body, J. J.; Steger, G. G.; Tonkin, K.; de Boer, R. H.; Lichinitser, M.; Fujiwara, Y.; Yardley, D. A.; Viniegra, M.; Fan, M.; Jiang, Q.; Dansey, R.; Jun, S.; Braun, A. Denosumab compared with zoledronic acid for the treatment of bone metastases in patients with advanced breast cancer: a randomized, double-blind study. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2010, 28, (35), 5132-9. 14. Fizazi, K.; Carducci, M.; Smith, M.; Damiao, R.; Brown, J.; Karsh, L.; Milecki, P.; Shore, N.; Rader, M.; Wang, H.; Jiang, Q.; Tadros, S.; Dansey, R.; Goessl, C. Denosumab versus


Page 23 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


zoledronic acid for treatment of bone metastases in men with castration-resistant prostate cancer: a randomised, double-blind study. Lancet (London, England) 2011, 377, (9768), 813-22. 15.

XGEVA® (denosumab) prescribing information, Amgen. http://www.xgeva.com.

16.

Prolia® (denosumab) prescribing information, Amgen. https://www.prolia.com.

17. Henry, D. H.; Costa, L.; Goldwasser, F.; Hirsh, V.; Hungria, V.; Prausova, J.; Scagliotti, G. V.; Sleeboom, H.; Spencer, A.; Vadhan-Raj, S.; von Moos, R.; Willenbacher, W.; Woll, P. J.; Wang, J.; Jiang, Q.; Jun, S.; Dansey, R.; Yeh, H. Randomized, double-blind study of denosumab versus zoledronic acid in the treatment of bone metastases in patients with advanced cancer (excluding breast and prostate cancer) or multiple myeloma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2011, 29, (9), 1125-32. 18. Chawla, S.; Henshaw, R.; Seeger, L.; Choy, E.; Blay, J. Y.; Ferrari, S.; Kroep, J.; Grimer, R.; Reichardt, P.; Rutkowski, P.; Schuetze, S.; Skubitz, K.; Staddon, A.; Thomas, D.; Qian, Y.; Jacobs, I. Safety and efficacy of denosumab for adults and skeletally mature adolescents with giant cell tumour of bone: interim analysis of an open-label, parallel-group, phase 2 study. The Lancet. Oncology 2013, 14, (9), 901-8. 19. Bone, H. G.; Bolognese, M. A.; Yuen, C. K.; Kendler, D. L.; Wang, H.; Liu, Y.; San Martin, J. Effects of denosumab on bone mineral density and bone turnover in postmenopausal women. The Journal of clinical endocrinology and metabolism 2008, 93, (6), 2149-57. 20. Termine, J. D.; Kleinman, H. K.; Whitson, S. W.; Conn, K. M.; McGarvey, M. L.; Martin, G. R. Osteonectin, a bone-specific protein linking mineral to collagen. Cell 1981, 26, (1 Pt 1), 99-105.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 27

21. Jundt, G.; Berghauser, K. H.; Termine, J. D.; Schulz, A. Osteonectin--a differentiation marker of bone cells. Cell and tissue research 1987, 248, (2), 409-15. 22. Hsu, Y. H.; Chen, W. Y.; Chan, C. H.; Wu, C. H.; Sun, Z. J.; Chang, M. S. Anti-IL-20 monoclonal antibody inhibits the differentiation of osteoclasts and protects against osteoporotic bone loss. The Journal of experimental medicine 2011, 208, (9), 1849-61. 23. Baron, R.; Ferrari, S.; Russell, R. G.

Denosumab and bisphosphonates: different

mechanisms of action and effects. Bone 2011, 48, (4), 677-92. 24. Kovar, J. L.; Xu, X.; Draney, D.; Cupp, A.; Simpson, M. A.; Olive, D. M. Near-infraredlabeled tetracycline derivative is an effective marker of bone deposition in mice. Analytical biochemistry 2011, 416, (2), 167-73. 25. Yang, R.; Davies, C. M.; Archer, C. W.; Richards, R. G. Immunohistochemistry of matrix markers in Technovit 9100 New-embedded undecalcified bone sections. European cells & materials 2003, 6, 57-71; discussion 71. 26. Wittenburg, G.; Volkel, C.; Mai, R.; Lauer, G. Immunohistochemical comparison of differentiation markers on paraffin and plastic embedded human bone samples. Journal of physiology and pharmacology : an official journal of the Polish Physiological Society 2009, 60 Suppl 8, 43-9. 27. Bianco, P.; Silvestrini, G.; Termine, J. D.; Bonucci, E. Immunohistochemical localization of osteonectin in developing human and calf bone using monoclonal antibodies. Calcified tissue international 1988, 43, (3), 155-61.


Page 25 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


28. Kupprion, C.; Motamed, K.; Sage, E. H. SPARC (BM-40, osteonectin) inhibits the mitogenic effect of vascular endothelial growth factor on microvascular endothelial cells. The Journal of biological chemistry 1998, 273, (45), 29635-40. 29. Kato, Y.; Lewalle, J. M.; Baba, Y.; Tsukuda, M.; Sakai, N.; Baba, M.; Kobayashi, K.; Koshika, S.; Nagashima, Y.; Frankenne, F.; Noel, A.; Foidart, J. M.; Hata, R. I. Induction of SPARC by VEGF in human vascular endothelial cells. Biochemical and biophysical research communications 2001, 287, (2), 422-6. 30. Kartsogiannis, V.; Zhou, H.; Horwood, N. J.; Thomas, R. J.; Hards, D. K.; Quinn, J. M.; Niforas, P.; Ng, K. W.; Martin, T. J.; Gillespie, M. T.

Localization of RANKL (receptor

activator of NF kappa B ligand) mRNA and protein in skeletal and extraskeletal tissues. Bone 1999, 25, (5), 525-34. 31. Nakashima, T.; Kobayashi, Y.; Yamasaki, S.; Kawakami, A.; Eguchi, K.; Sasaki, H.; Sakai, H. Protein expression and functional difference of membrane-bound and soluble receptor activator of NF-kappaB ligand: modulation of the expression by osteotropic factors and cytokines. Biochemical and biophysical research communications 2000, 275, (3), 768-75. 32. Udagawa, N.; Takahashi, N.; Yasuda, H.; Mizuno, A.; Itoh, K.; Ueno, Y.; Shinki, T.; Gillespie, M. T.; Martin, T. J.; Higashio, K.; Suda, T. Osteoprotegerin produced by osteoblasts is an important regulator in osteoclast development and function. Endocrinology 2000, 141, (9), 3478-84. 33. Sage, H.; Vernon, R. B.; Decker, J.; Funk, S.; Iruela-Arispe, M. L. Distribution of the calcium-binding protein SPARC in tissues of embryonic and adult mice. The journal of



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 27

histochemistry and cytochemistry : official journal of the Histochemistry Society 1989, 37, (6), 819-29. 34. Rosset, E. M.; Bradshaw, A. D. SPARC/osteonectin in mineralized tissue. Matrix biology : journal of the International Society for Matrix Biology 2016, 52-54, 78-87. 35. Tera Tde, M.; Nascimento, R. D.; Prado, R. F.; Santamaria, M. P.; Jardini, M. A. Immunolocalization of markers for bone formation during guided bone regeneration in osteopenic rats. Journal of applied oral science : revista FOB 2014, 22, (6), 541-53.


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For Table of Contents Only


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Bispecific Antibody Binding To RANKL and Osteonectin with

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