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Oct 30, 2013 - Madhuri Newa, Michael Lam, Krishna Hari Bhandari, Biwen Xu, and Michael R. Doschak*. Faculty of Pharmacy & Pharmaceutical Sciences, ...
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Expression, Characterization, and Evaluation of a RANK-Binding Single Chain Fraction Variable: An Osteoclast Targeting Drug Delivery Strategy Madhuri Newa, Michael Lam, Krishna Hari Bhandari, Biwen Xu, and Michael R. Doschak* Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2N8, Canada ABSTRACT: A single chain Fraction variable (scFv) employs antibody-like target recognition specificity. Osteoclasts, responsible for bone resorption, express Receptor Activator of Nuclear factor Kappa B (RANK) receptors. This study aimed to express, characterize, and evaluate scFv against RANK receptors that may serve as a platform to target osteoclasts. Using phage display technology, scFv against RANK receptor was expressed and characterized by DNA sequencing, sodium dodecyl sulfate− polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption−ionization time-of-flight (MALDI TOF), enzyme-linked immunosorbent assay (ELISA), Western blot, and immunocytochemistry. The potential for cytotoxicity was evaluated using an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay, and its cross reactivity was evaluated using ELISA. Osteoclast-like cells were generated from RAW 264.7 cells, and the osteoclast targeting ability of scFv was evaluated using immunocytochemistry. ScFv’s antiresorptive efficacy was studied using a tartrate-resistant acid phosphatase (TRAP) assay and resorption assay. Anti-RANK scFv was successfully expressed and characterized. No cross reactivity with other tumor necrosis factor receptor (TNFR) members and no cytotoxic effect on a non-RANK bearing cell line were observed. It showed specificity toward a RANK receptor and an inhibitory effect on osteoclast activity. With the increase in development trends for biologics as therapeutics and growing knowledge on the importance of osteoclast targeted therapy, this study may provide a drug delivery strategy to target osteoclasts, thereby leading to a promising therapy for resorptive bone diseases. KEYWORDS: osteoclast, RANK receptor, single chain fraction variable, phage display, drug delivery system

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INTRODUCTION Osteoclasts (OC) are multinucleated giant cells associated with bone resorption1 and involved with the pathogenesis of osteoporosis and osteoarthritis. Osteoclast cells express the RANK receptor (Receptor Activator of Nuclear factor Kappa B), which from a drug delivery perspective may serve as useful molecular targets. RANK is the essential signaling receptor for osteoclast differentiation during the process of osteoclastogenesis, as triggered by the osteoclast differentiation factor known as RANK-ligand (RANKL).2 RANK is a member of the tumor necrosis factor (TNF) receptor superfamily of proteins,3 designated as member 11a (TNFRSF11a). The only known ligand for RANK is RANKL,4 the TNF ligand superfamily member 11 (TNFS11). The probability that TNFRSF member RANK is the only functional receptor for RANK-L is also supported by the fact that RANK −/− and RANK-L −/− mice display similar phenotypes.5,6 Interactions between RANKL and RANK are essential for osteoclastogenesis. According to Boyle et al. (2003), RANK is now recognized as an important receptor for RANKL in the context of osteoclast differentiation, the cell type responsible for the resorption of bone during bone remodeling.7 RANK signaling, with additional signaling through c-Fms, the receptor for the macrophage-colony stimulating factor (M-CSF), triggers the proliferation and fusion of mononuclear cells and the formation of multinucleated, mature osteoclasts.7 Antibodies represent an important category of proteins for their ability to recognize epitopes with high affinity and specificity. © 2013 American Chemical Society

Despite the widespread application of monoclonal antibodies produced by hybridoma technology, immunotherapy is limited by the immunogenicity of murine-derived antibodies. Furthermore, production of antibody by hybridoma technology with immortalization and propagation of antigen-specific B-cell clones is timeconsuming, laborious, and a very expensive process. An antibodylike phage display has been developed as an alternative bioreactor technology to circumvent such problems. The phage display technique was discovered in 1985 by Smith, who developed libraries of peptides on the surface of phages. It has become a widely used and well-established technique for the selection and production of antibody-like binding fragment expressed in host libraries.8,9 Molecular engineering technologies made it possible to clone specific domains from antibodies resulting in “miniaturized antibodies”.10 Thus, only the antigen-binding sequences can subsequently be selected for “humanization”, and those sequences can be expressed as single-chain variable fragment (scFv). Using this technique, it is possible to mimic the strategy used by the adaptive humoral immune response to produce humanized antibodies or antibody fragments, while further averting the need for the lengthy immunization process and the subsequent construction of hybridomas.11 Received: Revised: Accepted: Published: 81

March 30, 2013 August 12, 2013 October 30, 2013 October 30, 2013 dx.doi.org/10.1021/mp400188r | Mol. Pharmaceutics 2014, 11, 81−89

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A single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulin and, hence, retains the binding specificity of the original immunoglobulin. These antibody-like fragments have shown advantages over conventional and monoclonal antibodies.12,13 They show good penetration of tissues, low immunogenicity in theory, low retention in the kidneys and other nontarget organs, and better penetration in target tumors, are relatively easy to construct, and have low commercial cost in large-scale production, and moreover, it is possible to restructure them in order to improve their activity and production. The phage display process is simpler, less time-consuming, and more efficient than conventional methods of antibody production as B-cell immortalization is not required and the selected antibodies carry with them the genetic material necessary for their replication. It is a powerful technology for antibody production outside the immune system, thus avoiding the use of experimental animals. In prior work, we developed a drug delivery system targeting bone mineral surfaces using bone seeking bisphosphonate drugs.14−16 We subsequently developed an osteoclast-targeting drug delivery strategy using monoclonal antibodies generated by the hybridoma technique.17 Our current work evaluates the use of the phage display technique for the generation of antibody-like humanized osteoclast-targeting fragments which may prove useful as an effective osteoclast targeting platform.

Figure 1. Overall concept of phage display. A phage display library was constructed in pIT2 construct containing myc and 6xHis tag. A human scFv-phage library was incubated with recombinant human soluble RANK protein. Unbound phage were rinsed away, while RANK-binding phage were eluted, amplified in Escherichia coli, and screened for two additional rounds, and RANK-binding scFv phage clones were identified to express soluble antigen-binding scFv.

TG1 (provided along with the purchase of the Tomlinson library). The Tomlinson scFv phage display library requires the use of two E. coli strainsTG1 and HB2151, where TG1 was used entirely for phage screening and HB2151 was used entirely for scFv expression. TG1 is an amber codon suppressing strain where a glutamine residue is produced instead of stopping translation when the amber stop codon (TAG) was encountered. This genotype is taken as an advantage where the scFv was fused with the pIII phage protein separated by an amber codon. Therefore, TG1 was used as a phage display strain due to its ability to produce scFv displaying phage. The amplified phages were screened for two additional rounds with same procedure, and RANK-binding phage was used to infect E. coli HB2151 for scFv expression. As antigen-binding scFv phage clones were identified, the pIII protein was no longer needed. By infecting the nonamber suppressing strain HB2151 with the scFv phagemid, translation would be halted at the amber stop codon, and only the soluble antigen-binding scFv were expressed. An enzymelinked immunosorbent assay (ELISA) was performed to identify individual RANK-binding scFv clones. DNA Sequencing. DNA template from individual antigenbinding clone was isolated using QIAprep Spin Miniprep Kit. A sample of 3 mL of overnight culture was pelleted by centrifugation, and the pellet was resuspended in the resuspension buffer provided. Subsequently, lysis buffer was added to the suspension, followed by addition of the provided neutralization buffer. The resulting mixture was centrifuged to remove all precipitate. The supernatant was then applied to the provided spin column to allow binding of DNA to column. Wash buffer 1 was added to the column and spun to remove endonucleases, and wash buffer 2 was added to the column and spun to remove all salts. Finally, 50 μL of the provided elution buffer was added to the column to isolate DNA. For DNA sequencing, 2 μL of the BigDye, 3 μL of the BigDye buffer, 4 μL of the isolated DNA, 1 μL of 5 μM primer, and 10 μL DNase and RNase free water were mixed to create a 20 μL reaction mixture. The reaction mixture was left to react in a thermocycler for 25 cycles with the following settings: (i) denaturing temperature: 96 °C, 30 s; annealing temperature: 50 °C, 15 s; extension temperature: 60 °C, 2 min. All reactions were carried out using primer 5′-CAG GAA ACA GCT ATG AC-3′.

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MATERIALS AND METHODS Materials. The phage display library used for this work was the Tomlinson Human scFv Library purchased from Source BioScience, UK. Recombinant human soluble RANK protein was from Peprotech, USA. Trypsin, 50 mM NaH2PO4 buffer, 300 mM NaCl, 10 mM imidazole, 3,3′,5,5′-tetramethylbenzidine (TMB), and 4−6-diamidino-2 phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). QIAprep Spin Miniprep Kit was from Qiagen Sciences (MD, USA). The dye used for DNA sequencing was BigDye v3.1 purchased from the MBSU facility at the University of Alberta. A micro BCA protein assay kit and isopropyl β-D-1-thiogalactopyranoside (IPTG) was from Thermo Fisher Scientific (Canada). BugBuster MasterMix extraction buffer was from EMD Chemicals (NJ, USA). The Bradford assay was from Bio-Rad (CA, USA). Anti-M13 HRP was kindly provided by Dr. Suresh’s Lab, University of Alberta. TNF I receptor, TNF II receptor, osteoprotegerin, macrophage colony stimulating factor (M-CSF), and RANKL were from PeproTech (NJ, USA). Antimyc HRP antibody was from Invitrogen (CA, USA). Western blotting reagent and blotting film were from GE Healthcare (UK). Light seal cassette and developing and fixing solution from Kodak (NY, USA) were used. RAW 264.7 cells (transformed murine monocytic cell line) were purchased from the American type culture collection (ATCC, VA, USA). Alexa Fluor 488 conjugate was from Millipore (USA) and acid phosphatase Assay Kit was from Cayman Chemical (USA). Generation of Anti-RANK scFv Using the Phage Display Technique. The overall concept of phage display is shown in Figure 1. The phage display library used for this work was constructed in the pIT2 construct, which contained both myc and 6xHis tag. This commercial human scFv-phage library was incubated in wells containing 1 mcg of recombinant human soluble RANK protein. Unbound phage were rinsed away, while RANK-binding phage were eluted with trypsin and amplified in Escherichia coli 82

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After reaction was completed, 20 μL of the reaction mixture was added to 2 μL of the provided ethyl acetate and 80 μL of 95% ethanol. The resulting mixture was mixed by vortex and incubated on ice for 15 min for DNA precipitation. After incubation, DNA was pelleted by centrifugation, and the supernatant was removed by vacuum and resuspended in 70% ethanol. The mixture was again mixed by vortex and pelleted by centrifugation at 10 800 rcf, 4 °C for 5 min. After removing the supernatant by vacuum, the dried product was submitted to the MBSU DNA sequencing facility at the University of Alberta. Large-Scale scFv Expression and Purification. Once phage selection was completed and antigen-binding clones were identified and cross infected to the expression strain HB2151, large-scale amplification was conducted by isopropyl β-D-1thiogalactopyranoside (IPTG)-induced overnight expression. After centrifugation to pellet the cell bodies, the periplasmic fraction of scFv was extracted by adding Bug Buster Master Mix extraction buffer, followed by scFv purification with Ni-NTA column. The extract was passed through the column entirely at least once to ensure maximum binding. The fraction collected was labeled as “flow through (FT)”. After all flow through fraction was collected, wash buffer 1(W1), which consisted of 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, and pH 8.0, was added to the column until minimal protein was detected using a Bradford assay. Subsequently, wash buffer 2 (W2), which consisted of identical NaH2PO4 and NaCl concentration and pH except with 50 mM imidazole, was added to wash the column until minimal protein was detected using a Bradford assay. For eluting the expressed scFv, the column was loaded with 10 mL of elution buffer, which consisted of identical NaH2PO4 and NaCl concentration and pH as the wash buffers except with 250 mM Imidazole, and the sample was collected in two fractions (E1 and E2). The resin was resuspended with the remaining elution buffer. The eluted samples were analyzed by SDS-PAGE (Figure 3) and subsequent dialysis (MWCO = 13 000) against PBS pH 7.2 overnight at 4 °C. The protein concentration was then determined by a micro BCA protein assay. Characterization of Anti-RANK scFv Using Sodium Dodecyl Sulfate−Polyacrylamide Gel Electrophoresis (SDS-PAGE). SDS-PAGE analysis was performed for the preliminary characterization of anti-RANK scFv. The various fractions collected during purification process and labeled as “flow through (FT)”, wash 1(W1), wash 2 (W2), Elution1 (E1), Elution2 (E2), and resin were mixed with loading buffer and run in 10% polyacrylamide gel under nonreducing condition. The gel was stained for 1 h with Coomassie brilliant blue R-250 (0.008% in 10% acetic acid), followed by destaining. Destaining solution composed of 10% (v/v) glacial acetic acid and 25% methanol in water. The destained gel was then visualized and stored digitally using the Alpha Imager gel documentation system. Characterization of Anti-RANK scFv Using an EnzymeLinked Immunosorbent Assay. For phage ELISA, indirect ELISA was performed in flat bottomed 96-well plates. A portion of 100 μL of antigen (human sRANK receptor) was used for coating at a concentration of 1 μg/100 μL overnight at 4 °C. The wells were washed three times with PBS (pH 7.2), and to avoid nonspecific binding, incubated with 2% bovine serum albumin (BSA) in PBS for 2 h at room temperature with agitation. The blocking solution was then discarded, and the wells were further washed three times with 200 μL of PBS. At the same time, the overnight culture plate was pelleted by centrifugation at 2000 rpm for 10 min at room temperature, and 50 μL of the phage-containing supernatant was used for phage ELISA.

The supernatant was incubated at room temperature with agitation for 1 h, at which time the wells were washed 3 times with 200 μL of 0.1% PBST. Once the wells were thoroughly washed, 100 μL of 1:10 000 dilution of anti-M13 HRP in PBS was added to each well and left to incubate 1 h at room temperature with agitation. The wells were then washed three times again with 200 μL of 0.1% PBST, at which time 100 μL of substrate solution 3,3′,5,5′-tetramethylbenzidine, or TMB, was added to each well and left to incubate at room temperature for 10−15 min. After incubation, the plate was read at 650 nm by a plate reader (BioTek EL808) and analyzed using the software KC Junior. To confirm that the scFv function has been retained after purification, a 96-well plate was coated with 100 μL/well of 10 μg/mL of antigen overnight at 4 °C. After blocking with BSA, the wells were incubated with different concentrations of scFv for 1 h, followed by the incubation with 100 μL of antimyc HRP antibody at room temperature for 1 h. After final washing, 100 μL of TMB substrate was added to each well and incubated for 15 min. The optical density (OD) was measured at 650 nm. Characterization of scFv Using Western Blot. The sample of purified scFv was electrophoresed on SDS-PAGE using 10% acrylamide gel and transferred onto nitrocellulose membrane using mini trans-blot apparatus. The membrane was blocked with 5% skim milk in PBST overnight at 4 °C. After washing with PBST, it was incubated with 1:5000 antimyc HRP for 1 h at room temperature, followed by detection of protein band using enhanced chemiluminescence. Characterization of scFv Using MALDI-TOF. Purified scFv was also characterized in positive mode. Samples were diluted 20-fold in 50% acetonitrile/water. A portion of 1 μL of each sample was mixed with 1 μL of sinapic acid (10 mg/mL in 50% acetonitrile/water + 0.1% trifluoroacetic acid). A sample of 1 μL of the sample/matrix solution was then spotted onto a steel target plate and allowed to air-dry. All MALDI-MS experiments were carried out using a Bruker Ultraflex MALDI-ToF/ToF (Bruker Daltonic GmbH) in positive mode. Data analysis was carried out using the flexAnalysis software (Bruker Daltonic GmbH) at the Institute for Biomolecular design. Effect of scFv on the Viability of Different Cell Lines. To ensure that the scFv did not have any adverse effect on the proliferation of other cells, we used UMR-106 (Rat osteosarcoma cell line: ATCC) cells for the cytotoxicity test. Cell culture was treated with 1 nM and 100 nM of scFv every 48 h over a period of 6 days. An observation was made to check if there is any difference in the proliferation of cells treated with testing agent and the cells treated with media alone. For quantitative analysis, cell viability was assayed by using thiazolyl blue tetrazolium blue (MTT; Sigma). UMR-106 cells were seeded on well plates in DMEM medium supplemented with 10% FBS and 1% penicillin−streptomycin.The culture was treated with media, 100 nM and 1 nM scFv. Media and factors were replaced every 48 h. On day 6, the media was aspirated, and MTT was added to each well and incubated further for four hours at 37 °C. The medium was then discarded; the formazan crystals were dissolved in 200 μL of solubilization solution using an in vitro toxicology assay kit (TOX-1, Sigma), and the absorbance was measured at 570 nm using a microplate reader. Evaluation of the Cross Reactivity of scFv with Other Members of TNFR Superfamily. ELISA was conducted to check for the cross reactivity of scFv with other members of TNFR superfamily. Wells were coated with 1 mcg of sRANK receptor, 1 mcg of TNF I receptor, 1 μg of TNF II receptor, and 83

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1 μg of osteoprotegerin. After blocking with BSA, the wells were incubated with 2 μg of scFv for 1 h, followed by the incubation with 100 μL of 1:5000 antimyc HRP antibody at room temperature for 1 h. After final washing, 100 μL of TMB substrate was added to each well and incubated for 15 min. The optical density (OD) was measured at 650 nm. Confirmation of the RANK Receptor Binding Ability of the Generated scFv by Western Blot. RANK receptor (19.3 kDa) was electrophoresed on SDS-PAGE gel and transferred onto nitrocellulose membrane as mentioned above. After blocking with 5% skim milk in PBST, the membrane was incubated with anti-RANK scFv followed by the incubation with antimyc HRP. The binding of scFv to RANK receptor was detected using enhanced chemiluminescence. Osteoclast Generation and TRAP Staining. RAW264.7 cells were seeded in culture plates and incubated for 24 h. Peptide factors 50 ng/mL RANKL and MCSF 25 ng/mL were added to the culture, and the medium was replaced every 48 h. With tartrate-resistant acid phosphatase (TRAP) being an osteoclast marker, the cultured cells were stained for the enzyme TRAP using the leukocyte acid phosphatase (TRAP) kit according to the manufacturer’s instructions. TRAP-positive multinucleated osteoclasts were visualized by light microscopy. Osteoclast Targeting Ability of Anti-RANK scFv. Immunocytochemistry was conducted to check for the osteoclast targeting potential of the generated scFv. Osteoclast-like cells were generated in Lab Tek II chamber slide system (Nunc) from RAW 264.7 cells (transformed murine monocytic cell line) by dosing them with 25 ng/mL macrophage colony stimulating factor (M-CSF) and 50 ng/mL RANKL every 48 h for 7 days. An osteoblast-like cell MG-63 cell line was used as a negative control. Likewise, an osteoclast cell culture omitting primary mAb was also used as a negative control. Cell cultures were washed with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde in PBS (pH 7.4) for 5 min, and rinsed thoroughly. After blocking with 3% BSA in PBS for 1 h, the cells were incubated with our anti-RANK scFv (4 μg/mL) for 2 h at 4 °C, followed by incubation with anti-Myc Tag, clone 9E10, Alexa Fluor 488 conjugate for 60 min at 4 °C. To visualize cell nuclei, slides were counterstained with 1 μg/mL 4−6-diamidino-2 phenylindole (DAPI) diluted in PBS. Culture slides were separated from their chambers, mounted, and photographed using confocal microscopy (Zeiss LSM 710 with ZEN software and the microscope the Observer.Z1). Spectrophotometric Assay of in Vitro TRAP Activity. To examine the effect of expressed scFv osteoclast culture and TRAP activity, RAW264.7 cells were seeded in culture plates and incubated for 24 h. Peptide factors 50 ng/mL RANKL and MCSF 25 ng/mL were added to the culture, with or without the addition of 1nM scFv or 100nM scFv. The medium and factors were replaced every 48 h. Osteoclastogenesis was assessed by the spectrophotometric measurement of TRAP activity on day 7. TRAP activity in the cell lysate was determined using an acid phosphatase assay kit. The absorbance was measured at 405 nm using a microplate reader. Resorption Assay. Resorptive ability is one of the most important assays for osteoclast functionality. Accordingly, RAW264.7 cells were suspended in cell culture medium and seeded in Corning Osteo Assay Surface Stripwells (which have calcium phosphate coating) and incubated for 24 h. Cells were treated with 50 ng/mL RANKL and 25 ng/mL M-CSF, with or without the addition of 1nM scFv or 100nM scFv every 48 h over the course of 7 days. After 7 days of culture, the medium was

aspirated and washed with double-distilled water. To remove the adherent cells, 100 mcl of 10% bleach solution was added for 5 min at room temperature. Bleach solution was aspirated, and the wells were washed twice with 150 mcl/well of doubledistilled water. The wells were allowed to air-dry completely at room temperature. To quantify the amount of calcium phosphate remained after the culture, 50 mcl of 1N HCl was added to each well, and QuantiChromTM Calcium Assay Kit (DICA-500) was used. The absorbance was measured at 630 nm using a microplate reader. All experiments were performed in triplicate. Descriptive statistical values are means ± standard deviation. Statistical Analysis. All experiments were performed in triplicate. Statistical analysis was conducted using Microsoft excel 2007. Results are presented as the mean ± standard deviation. Unpaired t-tests of two samples assuming equal variances were used to assign significance between groups, with a P-value of less than 0.05 as the threshold for significance.

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RESULTS DNA Sequencing. The DNA template from individual antigen-binding clone was isolated using a QIAprep Spin Miniprep Kit and was submitted to the Molecular Biology Service Unit DNA sequencing facility of University of Alberta. The DNA sequence result was obtained as a notepad file via e-mail. The DNA sequence was then analyzed by an online translation tool ExPASy Translate tool (http://expasy.org/ tools/dna.html). Full details of sequencing are shown in Figure 2.

Figure 2. Amino acid sequence of RANK-binding scFv. The amino acid sequence of anti-RANK scFv clone was deduced from obtained DNA sequencing result using primer 5′-CAG GAA ACA GCT ATG AC-3′. The DNA sequence was translated to amino acid sequence using an online translation tool ExPASy Translation Tool (http://expasy.org/ tools/dna.html). Each letter represents the letter abbreviation of individual amino acid sequence. Bold letters denote the heavy chain of the scFv. Underlined letters denote the light chain of the scFv. Italicized letters denote the polyglycine serine linker. 6xHis and c-myc tag are highlighted in black. Asterisk (*) represents translation termination due to the amber (TAG) stop codon.

SDS-PAGE. Taking advantage of the 6xHis tag within the scFv construct, the desired RANK-binding scFv was purified from the overnight culture using the Ni-NTA column. Figure 3 shows the SDS-PAGE image of the purification process. While some impurities were found present in the elution fractions, the percentage was deemed lower than 5% judging at the intensity of the desired scFv band in comparison. An intense protein band at 28 kDa was seen for the anti-RANK scFv. Enzyme-Linked Immunosorbent Assay. After purification of RANK-binding scFv and determination of protein concentration by BCA assay, a serial dilution scFv ELISA was performed. The optical density was seen to linearly increase with the concentration of scFv (Figure 4). No cross reactivity was 84

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Figure 5. ELISA to check for the cross reactivity of scFv with other members of TNFR superfamily. Wells were coated with sRANK receptor, TNF I receptor, TNF II receptor, and osteoprotegerin. After blocking with BSA, the wells were incubated with scFv, followed by the incubation with antimyc HRP antibody. After final washing, TMB substrate was added and incubated for 15 min. The optical density (OD) was measured at 650 nm.

Figure 3. SDS-PAGE of Ni-NTA column purification of anti-RANK scFv. Each lane was labeled as shown. The purification process was competitive affinity chromatography. Wash 1 (W1), wash 2 (W2), and elutions (E1 and E2) were performed using I with concentration 10 mM, 50 mM, and 250 mM, respectively. 10% SDS gel was used, and the gel was run at 100 V for approximately 1 h. An intense protein band was observed above 25 KDa for the expressed anti-RANK scFv, and elution fraction 1 contained significantly more scFv than elution fraction 2.

is the molecular weight of scFv, was detected by antimyc antibody. For the more sensitive method to confirm the binding of scFv with RANK receptor, recombinant human sRANK receptor, a 19.3 kDa polypeptide, electrophoresed on a 10% polyacrylamide gel and transferred to nitrocellulose membrane. On incubation with generated scFv and followed by incubation with antimyc antibody, a band was observed between 15 and 20 kDa. Osteoclast Targeting Ability of Anti-RANK scFv. Immunocytochemistry was conducted to check for the osteoclast targeting potential of the generated scFv. Confocal images confirmed the osteoclast receptor targeting potential of generated scFv (Figure 9). Antimyc Alexa Fluor 488 detected the binding of scfv with osteoclast receptor. Counterstaining with DAPI (blue color) confirmed the multinucleated phenotype of osteoclast-like giant cells. There was no demonstrable fluorescent Alexa 488 staining seen in the negative control slides. Spectrophotometric Assay of in Vitro TRAP Activity. The effect of scFv on TRAP release from osteoclast cell culture was determined using a spectrophotometric assay of In vitro TRAP activity (Figure 10). TRAP has long been used as a histochemical marker of the osteoclast.18 The enzyme has been shown to be a specific and sensitive indicator of bone resorption19 and contributes to the intracellular processing of primary bone matrix degradation products prior to its final release through the basolateral membrane of resorbing osteoclast cells.20,21 The effect of scFv on osteoclast TRAP enzyme was quantified by enzyme assay kit on the cell lysate. Osteoclast cell culture treated with scFv showed decrease in TRAP release. Resorption Assay. Antiresorptive effect of scFv on the osteo assay surface is shown in Figure 11. Osteoclasts, as the boneresorbing cells of the body, show the ability to dissolve the mineralized inorganic phase of bone matrix known as hydroxyapatite.21 To evaluate if this antibody-like fragment has any effect on the ability of osteoclasts to resorb bone, we cultured osteoclast-like cells on calcium phosphate coated culture wells. The wells treated with RANKL and MCSF showed a decrease in the calcium remains when measured by calcium assay kit. scFv treated osteo assay surface showed more of the calcium amount as compared to that of osteoclast culture. The greater absorbance was observed for 100 nM scFv treatment.

Figure 4. ELISA of the generated anti-RANK scFv using commercially available recombinant soluble human RANK receptor. To confirm that the scFv function has been retained after purification, indirect ELISA was performed. Human sRANK receptor was used as an antigen. 96 well plates were coated with Human sRANK receptor. After blocking with BSA, the wells were incubated with different concentrations of scFv, followed by the incubation with the antimyc HRP antibody. After final washing, TMB substrate was added to each well and incubated for 15 min. The optical density (OD) was measured at 650 nm.

observed for the anti-RANK scFv with other TNFR members when ELISA was conducted with wells coated with TNFR I, TNFR II, and OPG receptors (Figure 5). MALDI-TOF. Molecular weight of the generated scFv was confirmed by MALDI-TOF analysis. MALDI-TOF result of scFv showed a sharp peak at 28011.008 m/z. Other peaks represent either impurities or degradation products during analysis (Figure 6). Effect of scFv on Proliferation of Other Cells. As shown in Figure 7, no noticeable difference was observed in the growth of UMR-106 cells on treatment with scFv every 48 h over a period of 6 days. When observed under microscope, the same degree of cell confluency was observed for all of the groups. The MTT assay further confirmed that this antibody-like fragment did not have any adverse effect on the proliferation of UMR-106. Western Blot. RANK receptor binding ability of the generated scFv was confirmed by Western blot analysis (Figure 8). ScFv has an myc epitope, and when it was electrophoresed on SDS gel and transferred to nitrocellulose, a band at 28 kDa, which

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DISCUSSION The phage display technique was used to screen and express RANK binding scFv clone from the Tomlinson human scFv library. SDS PAGE analysis and MALDI TOF analysis 85

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Figure 6. Confirmation of molecular weight of the generated scFv by MALDI-TOF: Purified scFv was also characterized in positive mode. Samples were diluted in acetonitrile/water, mixed with sinapic acid solution in acetonitrile/water containing 0.1% trifluoroacetic acid, and spotted onto a steel target plate. MALDI-MS experiments were carried out using a Bruker Ultraflex MALDI-TOF/TOF (Bruker Daltonic GmbH), and data analysis was carried out using the flexAnalysis software (Bruker Daltonic GmbH).

Figure 7. Cell viability test using MTT assay on UMR-106 cells. No demonstrable cytotoxicity was seen as measured by the absorbance of formazan solution formed after incubation with compounds, compared to that seen with untreated media.

Figure 9. Osteoclast targeting ability of anti-RANK scFv. Immunocytochemistry was conducted to check for the osteoclast targeting potential of the generated scFv. (A) Confocal images of immunocytochemistry performed with anti-RANK scFv which detected RANK receptors on the osteoclasts. (B) Confocal image of immunocytochemistry for osteoclast cell culture omitting primary antibody, as a negative control. (C) Confocal image of immunocytochemistry for osteoblast MG-63 cell line as a negative control. Figure 8. Confirmation of RANK receptor binding ability of the generated scFv by Western blot analysis. Lane 1: A band seen at 28 kDa as a result of chemiluminescent detection of epitope c-myc of antiRANK scFv using anti myc HRP antibody. Lane 2: RANK receptor (19.3 kDa) was electrophoresed on 10% SDS-PAGE. Protein was transferred to nitrocellulose membrane which was blocked with 5% skim milk, followed by the incubation with anti-RANK scFv. Reaction was detected by using the antimyc HRP antibody, and the membrane was developed using ECL reagent and exposure to X-ray film.

density with the increase in concentration of scFv when ELISA was performed on RANK receptor coated wells also confirmed the generated scFv to have RANK binding specificity. It did not show cross reactivity with other members of TNFR superfamily. Western blot also confirmed the specificity of scFv for the RANK receptor. Immunocytochemistry further confirmed the osteoclast targeting potential of scFv. The fluorescent staining of AntiMyc Tag, clone 9E10, Alexa Fluor 488 conjugate detected the myc epitope of scFv and hence the binding of scFv on osteoclast receptor. The effect of scFv on inhibition in osteoclast enzyme TRAP and conservation of calcium phosphate coated osteo assay

characterized scFv, showing the isolated protein band at 28 KD and a peak at 28011.008 m/z, respectively. The increase in optical 86

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Molecular Pharmaceutics

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The therapeutic targeting of the RANK−RANKL interaction may be of concern in itself, as it not only plays a pivotal role in osteoclast formation and function but is also involved in other biological processes such as lymph node development,5 mammary gland development,22 and dendritic cell survival and activation.23,24 Systemically perturbing RANK−RANKL interactions may result in adverse side effects of the patient immune response.25 RANKL−RANK signaling is not entirely restricted to osteoclasts and their precursors, as it is also known to be involved in the regulation of other cell types. RANK is also known to be expressed by macrophages and dendritic cells which are specialized immune cells programmed to capture and process antigens in the body and present them to naı̈ve T cells. That may yet prove to be one potential limitation of this targeting strategy. However, therapeutics targeting RANK−RANKL interaction are unlikely to affect mammary gland development since that biological process is not a pressing issue for most patients with bone disorders such as postmenopausal women22 and may not affect lymph node organogenesis since that process is complete in adults.26 RANK signaling is indispensable for lymph node formation at the developmental stage but not for adult lymph node function.27 Denosumab, a new drug for treating osteoporosis, targets RANK−RANKL interaction and shows the great potential to serve as a therapeutic target for various bone disorders without significant immune system perturbation.28 With growing appreciation for the importance of the RANK receptor in the differentiation and activity of osteoclast, the scFv targeting RANK receptor may find a meaningful role as a viable strategy in the treatment of various bone diseases. Future studies involving animal models will further address the potency and systemic specificity of these antibody-like fragments. Osteoclasts have drawn attention as a therapeutic target for various bone disorders.29 The osteoclast is the sole cell responsible for the resorption of bone and is central in pathologic situations, where bone destruction is highly involved. The increase in development trends for therapeutic fragments30 and numerous publications on the importance of RANK in osteoclast formation and activity is the prime impetus for this research work. Osteoclast targeted therapy has great potential in the management of various bone diseases in which excess resorption of bone is a key pathological process.31 The increased bone resorption in osteoporosis is due both to increased osteoclastogenesis and to decreased osteoclast apoptosis.32,33 Many researchers have shown that subchondral bone modifications occur early in the development of osteoarthritis. At an early stage of osteoarthritis pathogenesis, there is a phase of increased bone resorption and an increased number of osteoclasts.34 Increased bone resorption results in increased articular damage.35,36 Since osteoclasts are the sole bone cells responsible for resorption, targeting osteoclasts may have a role in osteoarthritis therapy as well. Paget’s disease of bone and multiple myelomas are characterized by increased numbers of osteoclasts and markedly increased bone resorption at the sites of the disease.37 As with all pharmaceutical products, development of this antibody formulation requires continuous comparison of the cellular and in vitro studies with in vivo studies. The anti-RANK antibody/fragment has shown good efficacy in inhibiting osteoclast activity in in vitro assays. Future studies involving animal models can further address the potency and specificity of these antibody fragments. Biodistribution of any therapeutic agent is a key consideration for getting the desirable in vivo pharmacological effect. Selective localization of such agent in desired site of action is important in achieving the efficacy.

Figure 10. In vitro TRAP activity assay to determine the effect of scFv on TRAP release from osteoclast cell culture. RAW264.7 cells were incubated with RANKL and MCSF with or without the addition of scFv. The medium and factors were replaced every 48 h. Osteoclastogenesis was assessed by the spectrophotometric measurement of TRAP activity on day 7. TRAP activity in the cell lysate was determined using an acid phosphatase assay kit. The absorbance was measured at 405 nm using a microplate reader. *P < 0.05 versus OC. **P < 0.05 versus 1 nM scFv.

Figure 11. Antiresorptive effect of scFv on the osteo assay surface. RAW 264.7 cells were cultured on osteo assay surface Stripwells and were treated with RANKL and M-CSF, with or without the addition of scFv. After 7 days of culture, the medium was aspirated, cells were removed, and the amount of calcium phosphate remained was quantified by a calcium assay kit. *P < 0.05 versus OC. **P < 0.05 versus 1 nM scFv.

surface supports the possibility of use of this generated antibodylike fragment as an antiresorptive agent rather than just the targeting agent. One possible explanation for the inhibitory effect of antiRANK scFv on osteoclast activity could be due to interference (antagonism) in binding of RANKL on RANK receptoran indispensible step for osteoclast formation and activity. Another possibility is that the binding of scFv to monomeric receptor subunits is disrupting the RANK receptor trimer assembly around the ligand and interrupting the intracellular signaling necessary for osteoclastogenesis. Thus, understanding the mechanisms underlying the interaction of the scFv and osteoclast receptor are key for the subsequent development of novel antiresorptive approaches capable of modulating metabolic bone disease. 87

dx.doi.org/10.1021/mp400188r | Mol. Pharmaceutics 2014, 11, 81−89

Molecular Pharmaceutics

Article

(4) Hsu, H.; Lacey, D. L.; Dunstan, C. R.; Solovyev, I.; Colombero, A.; Timms, E.; Tan, H. L.; Elliott, G.; Kelley, M. J.; Sarosi, I.; Wang, L.; Xia, X. Z.; Elliott, R.; Chiu, L.; Black, T.; Scully, S.; Capparelli, C.; Morony, S.; Shimamoto, G.; Bass, M. B.; Boyle, W. J. Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 3540−5. (5) Dougall, W. C.; Glaccum, M.; Charrier, K.; Rohrbach, K.; Brasel, K.; De Smedt, T.; Daro, E.; Smith, J.; Tometsko, M. E.; Maliszewski, C. R.; Armstrong, A.; Shen, V.; Bain, S.; Cosman, D.; Anderson, D.; Morrissey, P. J.; Peschon, J. J.; Schuh, J. RANK is essential for osteoclast and lymph node development. Genes Dev. 1999, 13, 2412−24. (6) Li, J.; Sarosi, I.; Yan, X. Q.; Morony, S.; Capparelli, C.; Tan, H. L.; McCabe, S.; Elliott, R.; Scully, S.; Van, G.; Kaufman, S.; Juan, S. C.; Sun, Y.; Tarpley, J.; Martin, L.; Christensen, K.; McCabe, J.; Kostenuik, P.; Hsu, H.; Fletcher, F.; Dunstan, C. R.; Lacey, D. L.; Boyle, W. J. RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 1566−71. (7) Boyle, W. J.; Simonet, W. S.; Lacey, D. L. Osteoclast differentiation and activation. Nature 2003, 423, 337−42. (8) Maynard, J.; Georgiou, G. Antibody engineering. Annu. Rev. Biomed. Eng. 2000, 2, 339−76. (9) Smith, G. P. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 1985, 228, 1315−7. (10) Holliger, P.; Hudson, P. J. Engineered antibody fragments and the rise of single domains. Nat. Biotechnol. 2005, 23, 1126−36. (11) Roque, A. C.; Lowe, C. R.; Taipa, M. A. Antibodies and genetically engineered related molecules: production and purification. Biotechnol. Prog. 2004, 20, 639−54. (12) Chester, K. A.; Hawkins, R. E. Clinical issues in antibody design. Trends Biotechnol. 1995, 13, 294−300. (13) Verhaar, M. J.; Chester, K. A.; Keep, P. A.; Robson, L.; Pedley, R. B.; Boden, J. A.; Hawkins, R. E.; Begent, R. H. A single chain Fv derived from a filamentous phage library has distinct tumor targeting advantages over one derived from a hybridoma. Int. J. Cancer 1995, 61, 497−501. (14) Bhandari, K. H.; Newa, M.; Uludag, H.; Doschak, M. R. Synthesis, characterization and in vitro evaluation of a bone targeting delivery system for salmon calcitonin. Int. J. Pharmaceutics 2010, 394, 26−34. (15) Bhandari, K. H.; Newa, M.; Chapman, J.; Doschak, M. R. Synthesis, characterization and evaluation of bone targeting salmon calcitonin analogs in normal and osteoporotic rats. J. Controlled Release 2012, 158, 44−52. (16) Doschak, M. R.; Kucharski, C. M.; Wright, J. E.; Zernicke, R. F.; Uludag, H. Improved bone delivery of osteoprotegerin by bisphosphonate conjugation in a rat model of osteoarthritis. Mol. Pharmaceutics 2009, 6, 634−40. (17) Newa, M.; Bhandari, K. H.; Tang, L.; Kalvapalle, R.; Suresh, M.; Doschak, M. R. Antibody-mediated “universal” osteoclast targeting platform using calcitonin as a model drug. Pharm. Res. 2011, 28, 1131− 43. (18) Burstone, M. S. Histochemical localization of oxidase activity in the mitochondria of the human heart. Nature 1959, 184 (Suppl 7), 476− 7. (19) Halleen, J. M.; Ranta, R. Tartrate-resistant acid phosphatase as a serum marker of bone resorption. Am. Clin. Lab. 2001, 20, 29−30. (20) Vaananen, H. K.; Zhao, H.; Mulari, M.; Halleen, J. M. The cell biology of osteoclast function. J. Cell Sci. 2000, 113 (Pt 3), 377−81. (21) Vaaraniemi, J.; Halleen, J. M.; Kaarlonen, K.; Ylipahkala, H.; Alatalo, S. L.; Andersson, G.; Kaija, H.; Vihko, P.; Vaananen, H. K. Intracellular machinery for matrix degradation in bone-resorbing osteoclasts. J. Bone Miner. Res. 2004, 19, 1432−40. (22) Fata, J. E.; Kong, Y. Y.; Li, J.; Sasaki, T.; Irie-Sasaki, J.; Moorehead, R. A.; Elliott, R.; Scully, S.; Voura, E. B.; Lacey, D. L.; Boyle, W. J.; Khokha, R.; Penninger, J. M. The osteoclast differentiation factor osteoprotegerin-ligand is essential for mammary gland development. Cell 2000, 103, 41−50.

The RANK receptor is highly expressed on osteoclasts. However, they are expressed in dendritic cells, liver, colon, and small intestine. Hence, the biodistribution study for the RANK targeting antibody should be conducted to compare the level of localization at the desired site of action with other RANK bearing cells other than osteoclasts. Furthermore, it is important to study if it leads to adverse effects by binding of this antibody to those cells. The effective targeting of therapeutic agents to skeletal tissues may greatly improve the efficacy of a variety of drug molecules while potentially greatly reducing undesired side effects. Thus, although still at the developmental stage, antibodymediated osteoclast-targeting strategies show tremendous potential as novel management options for bone disease. Despite that optimism, a cautious approach is required in determining their beneficial effects on bone biology, while identifying potential systemic side effects of this novel antiresorptive strategy.

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CONCLUSION Since the pharmacological arrest of the osteoclast is the mainstay of treating various bone disorders, drug delivery research targeting the osteoclast remains a viable option for the development of novel therapeutics. With the increase in developmental trends for biologic therapeutic fragments and the importance of targeting therapeutic compounds to the desired site of biological action, this research may provide a meaningful strategy in terms of osteoclast targeting and drug delivery leading to a novel targeted therapy for bone disease with the aim of controlling/reducing bone resorption.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +1 780 492-8758. E-mail: [email protected]. Author Contributions

M.N. and M.L. contributed equally. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank Dr. Greg Winter’s lab at the MRC Laboratory of Molecular Biology and the MRC Centre for Protein Engineering (Cambridge, U.K.) for providing the Tomlinson human scFv library. This research was funded by the Alberta Osteoarthritis Team grant from Alberta Innovates Health Solutions (AIHS). M.N. was supported by the Queen Elizabeth II Graduate Scholarship (Doctoral) from the Government of the Province of Alberta. M.L. was supported by the CIHR Frederick Banting and Charles Best Canada Graduate ScholarshipsMaster’s Award.

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REFERENCES

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