TocilizumabâAlendronate Conjugate for Treatment of Rheumatoid

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Tocilizumab – Alendronate Conjugate for Treatment of Rheumatoid Arthritis Hwiwon Lee, Suk Ho Bhang, Jeong Ho Lee, Hyemin Kim, and Sei Kwang Hahn Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00008 • Publication Date (Web): 20 Jan 2017 Downloaded from http://pubs.acs.org on January 21, 2017

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Bioconjugate Chemistry

Tocilizumab – Alendronate Conjugate for Treatment of Rheumatoid Arthritis

Hwiwon Lee,† Suk Ho Bhang,‡ Jeong Ho Lee,† Hyemin Kim,† Sei Kwang Hahn†,*

† Department of Materials Science and Engineering, Pohang University of Science and Technology (POSTECH), 77 Cheongam-ro, Nam-gu, Pohang 790-784, Korea. ‡ School of Chemical Engineering, Department of Energy Science, Sungkyunkwan University, Suwon 440-746, Korea.

*Corresponding Author Tel.: +82 54 279 2159. Fax: +82 54 279 2399. E-mail: [email protected] (S.K. Hahn)

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ABSTRACT An autoimmune disease of rheumatoid arthritis (RA) causes severe inflammation on the synovial membrane, which results in the destruction of articular cartilage and bone. Here, Tocilizumab (TCZ) – Alendronate (ALD) conjugate is synthesized for the early intervention of RA. A humanized monoclonal antibody of TCZ shows an immunosuppressive effect, targeting interleukin-6 (IL-6) receptor in the RA pathogenesis. ALD is an anti-inflammatory bisphosphonate drug which can bind to the exposed bone surface. ALD is conjugated selectively to N-glycan on Fc region of TCZ using a chemical linker of 3-(2pyridyldithio) propionyl hydrazide (PDPH) – poly(ethylene glycol) – N-hydroxysuccinimide (PDPHPEG-NHS). The successful synthesis of TCZ-ALD conjugate is corroborated by 1H NMR, the purpald assay, mass spectrometry (MS), and high performance liquid chromatography (HPLC). In vitro binding affinity and cell viability tests confirmed the biological activity of TCZ-ALD conjugate. Furthermore, in vivo efficacy of TCZ-ALD conjugate is confirmed by micro computed tomography (CT), histological, and western blot analyses for the treatment of RA. [KEYWORDS] Tocilizumab; Alendronate; Antibody-drug conjugate; Rheumatoid arthritis


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INTRODUCTION Rheumatoid arthritis (RA) is one of the serious chronic autoimmune diseases which results in the synovial membrane inflammation and bone destruction.1 RA accompanies persistent cellular activation, and triggers autoimmunity and tissue injury by the overexpressed pro-inflammatory cytokines such as interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α in the serum and synovial fluid.2-5 Among them, IL-6 is one of the most important cytokine targets for the treatment of RA. IL-6 is an important pleiotropic cytokine which affects biological activities of various cells including T cells, B cells, neutrophils, monocytes, and osteoclasts.6 Tocilizumab (TCZ) is a kind of humanized monoclonal antibody and inhibits IL-6 signaling by competitively binding to IL-6R, ameliorating the severe symptoms of RA.7,8 Alendronate (ALD) is an anti-inflammatory bisphosphonate drug commonly used for treating osteoporosis and other bone related diseases. ALD can bind to bone surface, induce antiinflammatory effect, and inhibit the growth of macrophage and the osteoclast-mediated bone resorption by interrupting cellular enzymes related with the biosynthetic pathway.9 Antibody-drug conjugate (ADC) is a highly potent combinational biopharmaceutical drug taking advantages of a monoclonal antibody and a small molecular chemical drug.10-14 More than 30 ADCs have been investigated in various stages of clinical trials,15,16 Recently, FDA approved two ADCs, brentuximab vedotin in 201117 and trastuzumab emtansine in 2013.18 ADC has a high sensitivity to target cells by the monoclonal antibody, binding to a specific antigen in the body. After that, ADC internalizes into the cell via receptor mediated endocytosis and releases the small molecule drug, killing the target cells.19 The well-designed combination of an antibody and a cytotoxic drug can greatly improve the therapeutic efficacy. Furthermore, site-specific conjugation of chemical linkers to antibody can improve the therapeutic efficacy, maintaining the antigen binding site without steric hindrance.20 The chemical linker should be also stable in the extracellular space before ADC metabolism to reduce the side effect of cytotoxic drugs in the body. In this work, a new ADC system of TCZ-ALD conjugate was developed to treat RA. Since many types of pro-inflammatory cytokines and cells are involved in the pathogenesis of RA, there have been ACS Paragon Plus Environment

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extensive investigations to improve the therapeutic efficacy of RA by the combination therapy.6,21 TCZ in the TCZ-ALD conjugate is well known to block IL-6R for the alleviation of RA symptom, and ALD in the conjugate shows anti-inflammatory effect and inhibitory effect on macrophages.22-24 Macrophage is involved in osteoclastogenesis and the major source of pro-inflammatory cytokines.25 ALD was sitespecifically conjugated to N-glycan on Fc region of TCZ without hindering the antigen binding site.26,27 As a specific chemical linker, 3-(2-pyridyldithio)propionyl hydrazide (PDPH) – poly(ethylene glycol) – N-hydroxysuccinimide (PDPH-PEG-NHS) with a cleavable disulfide bond28 was used to release ALD inside the target cells, reducing its side effect to normal cells. The highly potent TCZ-ALD conjugate was assessed by in vitro characterization and in vivo therapeutic evaluation.


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RESULTS AND DISCUSSION

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Figure 1. (a) Schematic illustration for the therapeutic mechanism of TCZ-ALD conjugate by IL-6 signal inhibition and anti-inflammation on macrophage within the joint. (b) Schematic representation for the synthetic strategy of TCZ-ALD conjugate.

Preparation and characterization of TCZ-ALD conjugate. The pathogenesis of RA is so complicated that the exact mechanism of RA is not fully understood yet. Nevertheless, IL-6 signaling is considered to be one of the main factors to aggravate the progression of inflammation and RA symptom.5,6 Many pro-


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inflammatory immunocytes including macrophage, osteoclast, B cell, and T cell express IL-6R and respond to the IL-6 signaling.6 Because these pro-inflammatory immunocytes work together leading to the highly complicated pathological environment, it is difficult to significantly alleviate the symptom of RA with only one interfering agent. For further therapeutic effect, small chemical drugs can be used, but they indiscriminately affect both target cells and normal cells, requiring the target-specific delivery and dose reduction of cytotoxic drugs with the minimal side-effect.13 Recently, ADC systems have been widely investigated to selectively deliver cytotoxic small chemical drugs to target cells with greatly reduced side-effects.12,14 As schematically shown in Figure 1a, we developed a highly potent biopharmaceutical drug of TCZ-ALD conjugate, which is able to simultaneously block IL-6R and inhibit macrophage pro-inflammatory activities for the treatment of RA. TCZ is a humanized monoclonal antibody against IL-6R and used as an immunosuppressive drug. ALD, a bisphosphonate drug, inhibits osteoclast activities and macrophage activities. These two molecules were chemically connected together using a PDPH-PEG-NHS linker which was synthesized by conjugating PDPH to thiol-PEG-NHS (Figure 1b). The NHS group of PEG was conjugated to amine group of ALD for the preparation of PDPH-PEGALD, which was confirmed by 1H NMR and

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C NMR (Figure 2a, Figures S1 and S2). The hydrazide

group of PDPH was conjugated to aldehyde group of oxidized TCZ. After that, the successful conjugation of ALD to TCZ was confirmed by the purpald assay (Figure 2b) and mass spectrometry (MS) (Figure 2c). The purpald assay, a kind of colorimetric assay, can detect aldehyde groups on oxidized TCZ by the color change from clear to purple. If aldehyde groups on TCZ were blocked by linker-ALD, the purpald agent does not show the color change indicating no aldehyde group. Accordingly, the conjugation was indirectly confirmed by the purpald assay. In addition, MS was also performed to identify whether ALD was conjugated on TCZ or not. After reducing dithiol groups on PDPH-PEG-ALD with dithiothreitol (DTT), we attained a peak corresponding to the molecular weight (MW) of about 2,400, which is equal to the MW of SH-PEG-ALD (Figure 2c). Furthermore, the number of PDPH-PEG-ALD attached to TCZ was analyzed by high performance liquid chromatography (HPLC) ACS Paragon Plus Environment

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with DTT treatment (Figure 2d). According to the peak area analysis, the average number of ALD conjugated to TCZ was ca. 1.14. From the results, we could clearly confirm the successful conjugation of PDPH-PEG-ALD to TCZ. The serum stability of PDPH-PEG linker between TCZ and ALD was assessed in bovine serum albumin (BSA) solution without DTT, which showed only 6.5 % linker cleavage even after incubation at 37 °C for 48 h (Figure 2d).

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Figure 2. (a) 1H NMR of PDPH-PEG-ALD. (b) The purpald assay for the analysis of oxidized TCZ (***P < 0.0001 in comparison with oxidized TCZ). (c) MS and (d) HPLC analysis of purified SH-PEGALD from TCZ-ALD conjugate after reduction by DTT treatment (104 µg/mL) or after incubation with bovine serum albumin (BSA) (6.76 µg/mL) as a model serum condition. PEG-ALD was used as a control (333 µg/mL).


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In vitro biological activity of TCZ-ALD conjugate. The binding affinity of TCZ-ALD conjugate to IL6R was assessed by enzyme-linked immunosorbent assay (ELISA) (Figure 3). Anti-IL-6R antibody coated plate was incubated with IL-6R, and various concentrations of TCZ and TCZ-ALD conjugate. After washing the plate, anti-human immunoglobulin G (IgG)- horseradish peroxidase (HRP) was added to the plate for detecting TCZ attached to IL-6R. As expected, there was no difference on the binding affinity between TCZ alone and TCZ-ALD conjugate, because TCZ was site-specifically modified with ALD on N-glycan regions without hindrance of antigen binding sites. Besides, the anti-inflammatory effect of TCZ-ALD conjugate was confirmed by the MTT assay to analyze the viability of Raw 264.7 macrophages (Figure 4). They have pivotal roles on the RA pathogenesis. When treated with ALD, ALD-linker, TCZ+ALD mixture, and TCZ-ALD conjugate, the survival rate of Raw 264.7 macrophages, which is affected by ALD, was dramatically reduced in comparison with that of phosphate buffered saline (PBS), IgG-ALD conjugate, and TCZ treated groups. There was no statistically significant difference between TCZ+ALD mixture and TCZ-ALD conjugate. The results corroborated the inhibitory effect of ALD on the growth of macrophages even after chemical conjugation to the linker and TCZ. Besides, in comparison to the control group of IgG-ALD conjugate which has few binding sites on the macrophage, we clearly confirmed the receptor mediated endocytosis29 of TCZ-ALD conjugate via IL-6R on the macrophage.

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TCZ TCZ-ALD

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Figure 3. (a) Schematic illustration for the binding test of TCZ and TCZ-ALD conjugate to IL-6R by ELISA. (b) The binding affinity of TCZ and TCZ-ALD conjugate with increasing concentration of TCZ (n = 3). ACS Paragon Plus Environment

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120

Cell Viability (%)

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Figure 4. The viability of Raw 264.7 macrophages after treatment with PBS, ALD, ALD-linker, IgGALD, TCZ, TCZ+ALD mixture, and TCZ-ALD conjugate for 48 h. The value of ***P < 0.0001 in comparison to the PBS treated group was considered to be statistically significant (n = 4).

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Figure 5. Severity scores for the paws of CIA model mice in comparison to the normal mice (score = 0) after treatment with PBS, ALD, TCZ or TCZ-ALD conjugate. The CIA model mice were monitored once a week for 2 months (n = 5). ACS Paragon Plus Environment

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In vivo efficacy of TCZ-ALD conjugate in RA model mice. The efficacy of TCZ-ALD conjugate was assessed in collagen induced arthritis (CIA) model mice (n = 5). In comparison with the normal control (group 1), four groups of CIA model mice were treated with PBS (group 2), ALD (group 3), TCZ (group 4), and TCZ-ALD conjugate (group 5), respectively. The severity of RA was monitored and scored every week for 2 months (Figure 5). Group 3 treated with ALD alone showed a better severity score than the negative control of group 2. Group 4 treated with TCZ alone showed a better score than group 3. However, in case of group 5 treated with TCZ-ALD conjugate, there was almost no symptom of RA in the mice after 8 weeks, reflecting the excellent efficacy of TCZ-ALD conjugate for the treatment of RA. In addition, the level of bone degradation was evaluated by micro computed tomography (CT) (Figure 6). In case of group 2 (Figure 6b) and group 3 (Figure 6c), the surface of bone was significantly degraded. Group 4 showed almost clear bone surface, but one toe was harshly degraded (Figure 6d). In contrast, there was no bone degradation for the case of group 5 (Figure 6e). All these results confirmed the synergistic effect of TCZ-ALD conjugate on inhibiting the bone resorption of osteoclast, despite the low injected dose of ALD far below the therapeutic range. As well as the therapeutic effect of TCZ, the conjugation of ALD to TCZ might facilitate the intracellular delivery of ALD into IL-6R expressed cells strongly related with the pathogenesis of RA.

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Figure 6. Representative micro CT images of the bones in (a) normal control, (b) disease control, treated groups with (c) ALD, (d) TCZ, and (e) TCZ-ALD conjugate, respectively. ACS Paragon Plus Environment

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Histological and western blot analysis. The efficacy of TCZ-ALD conjugate was evaluated by histological analysis of dissected joint tissues with hematoxylin and eosin (H&E) staining in CIA model mice. The level of inflammation, and degeneration of cartilage and bone in the treated tissues were investigated as shown in Figure 7. The normal mice without RA induction in the control group showed no sign of inflammation, and the destruction of cartilage and bone (Figure 7a). The area of cartilage and bone in the tissues of the control group had a clear boundary without collapse. However, the interface between bone and cartilage in group 2 of the negative control was hard to distinguish because of infiltration of inflammatory cells, and degeneration of cartilage and bone (Figure 7b). Histological analysis of group 3 treated with ALD also showed inflammatory cell infiltration, and unclear boundary between cartilage and bone (Figure 7c). The treatment of ALD alone showed an insignificant therapeutic effect. In contrast to group 2 and group 3, group 4 treated with TCZ showed a significantly reduced level of inflammation, and degeneration of cartilage and bone (Figure 7d). In the case of group 5 treated with TCZ-ALD conjugate, the interface between cartilage and bone was very clear without inflammatory cell infiltration, which was almost comparable to that of the normal control group. Histological analysis clearly visualized the combinational therapeutic effect of TCZ-ALD conjugate on RA. For further efficacy evaluation of TCZ-ALD conjugate on RA, we performed the western blot analysis of IL-6 (Figure 8b), cluster of differentiation 68 (CD68) (Figure 8c), Type II collagen (CII) (Figure 8d), and osteocalcin (Figure 8e) in the dissected joint tissues. The relative amount of IL-6 indicating the severity of inflammation was significantly up-regulated in PBS treated group and ALD treated group comparing to the normal control group. However, the treatment of TCZ and TCZ-ALD conjugate, especially TCZ-ALD conjugate treatment, led to the considerably reduced level of IL-6. The relative amount of CD68, which is a biomarker for the macrophage lineage, showed the similar trend with that of IL-6. The combined delivery of TCZ and ALD in the conjugate into the cells might block IL-6R and suppress the macrophage activity simultaneously, inhibiting autocrine IL-6 signaling loop for the treatment of RA. In contrast, both CII in the cartilage and osteocalcin in the bone were more highly detected in the normal control and TCZ-ALD conjugate treated groups than PBS and ALD treated 11 ACS Paragon Plus Environment


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groups. TCZ-ALD conjugate alleviated the cartilage destruction and osteoclast activity for bone resorption. The combinational therapeutic effect of TCZ-ALD conjugate was clearly confirmed by statistical analysis between TCZ and TCZ-ALD conjugate treated groups (Figure 8). The pathogenesis of RA is highly complicated involving many pathological factors like cytokines in vivo. TCZ is able to block IL-6 signaling, one of pathological factors of RA, showing significant therapeutic effect on RA. ALD also has therapeutic effect on RA by inhibiting macrophage activities (Fig. 4). However, because a small chemical drug of ALD has a short residence time within the joint fluid,30 ALD without conjugation has minor therapeutic effect on RA as shown in Fig. 7 and 8. By conjugating a small drug of ALD to TCZ (MW of 150 kDa) which binds to overexpressed IL-6R within the joint, the residence time of ALD in TCZ-ALD conjugate might be significantly extended,31 resulting in drastically enhanced therapeutic effect.


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Figure 7. Histological analysis with H&E staining of dissected joint tissues of (a) normal control, (b) disease control, treated groups with (c) ALD, (d) TCZ, and (e) TCZ-ALD conjugate, respectively (B = bone, C = cartilage, I = inflammation).


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Figure 8. (a) Western blot analysis of IL-6, CD68, collagen type II, and osteocalcin in the dissected joint tissues. Densitometric analysis of the Western blot bands of (b) IL-6, (c) CD68, (d) Collagen type II, and (e) Osteocalcin. The values of **P < 0.01 and ***P < 0.0001 in comparison with PBS treated group and between TCZ and TCZ-ALD conjugate treated groups were considered to be statistically significant (n = 4). ACS Paragon Plus Environment

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CONCLUSION Highly potent TCZ-ALD conjugate was successfully developed for treating RA. The formation of TCZ-ALD conjugate was corroborated by 1H NMR, the purpald assay, MS, and HPLC analysis. In vitro binding affinity of TCZ-ALD conjugate to IL-6R was confirmed by ELISA and in vitro antiinflammatory effect was confirmed by measuring macrophage viability after treatment with TCZ-ALD conjugate. In vivo therapeutic effect of TCZ-ALD conjugate on RA was verified by micro CT, histological, and Western blot analysis in CIA model mice. Taken together, TCZ-ALD conjugate would be successfully developed as a highly potent drug candidate to treat RA. Furthermore, the antibody-drug conjugate strategy can be used to design a combination therapy for various autoimmune diseases.

EXPERIMENTAL PROCEDURES

Materials. Thiol-PEG-NHS was obtained from Nanocs (MW = 2 kDa, New York, NY) and PDPH was supplied from Thermo Scientific (Waltham, MA). Alendronate sodium trihydrate, sodium periodate, purpald, dithiothreitol (DTT), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and histological decalcifying solution were purchased from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), antibiotics, and phosphate buffered saline (PBS, pH 7.4) were obtained from Invitrogen (Carlsbad, CA). Raw 264.7 cells were provided from Korean Cell Line Bank (Seoul, Korea). Recombinant human IL-6, human IL-6Rα antibody, and recombinant human IL-6Rα were obtained from R&D systems (Minneapolis, MN). Anti-IL-6 antibody containing horseradish peroxidase (HRP) and primary antibodies detecting IL-6, CD68, collagen type II, and osteocalcin were purchased from Abcam (Cambridge, MA). Goat anti-human IgG-HRP was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Tocilizumab (TCZ) was purchased from JW Pharmaceutical (Seoul, Korea).


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Synthesis and characterization of TCZ-ALD conjugate. First, SH-PEG-ALD was synthesized by the conjugation reaction between amine group of ALD (5 mg) and SH-PEG-NHS (MW = 2 kDa, 10 mg) in distilled water at pH 8 overnight, and then purified by dialysis (MWCO 2,000) in distilled water for 3 days. After that, SH-PEG-ALD was treated with DTT to reduce the disulfide linkage overnight and dialyzed in distilled water for a day. Finally, SH-PEG-ALD was reacted with 5 mg of PDPH at pH 7 overnight, which was purified by dialysis in distilled water for 3 days and then freeze dried. The prepared PDPH-PEG-ALD was analyzed by 1H NMR (400 MHz). ALD was site-specifically conjugated to N-glycan on Fc region of TCZ. As a first step, 100 µL of TCZ (1 mg/mL) was oxidized with 10 µL of sodium periodate (21.39 mg/mL) in PBS (pH 7.4) at 4 ˚C in dark for 30 min and diluted with PBS. The excess amount of sodium periodate was filtered out by centrifugation with a syringe filter (MWCO 10 kDa, 5,000×g, 10 min). The oxidation of TCZ was analyzed by the purpald assay.27 Then, 50 molar excess of PDPH-PEG-ALD was added to the oxidized TCZ, which was incubated overnight to chemically attach PDPH to aldehyde group of oxidized TCZ. The unbound PDPH-PEG-ALD was dialyzed with a centrifugal filter (MWCO 10 kDa, 5,000×g, 10 min) five times. The conjugation of PDPH-PEG-ALD to TCZ was analyzed by MS (Applied Biosystems 4700 Proteomics Analyzer, AB Sciex, MA). The number of ALD attached to TCZ and the serum stability of the linker between TCZ and ALD in BSA solution were determined by HPLC (Waters, MA) using the Superdex peptide 10/300 GL column. At a PBS flow rate of 0.4 mL/min, 100 µL of the sample in PBS was injected and detected at 267 nm. For MS and HPLC analysis, SH-PEG-ALD was detached from TCZ-ALD conjugate by incubation with DTT (100 mM) to reduce the disulfide linkage. After centrifugation (MWCO 10k) to remove TCZ and dialysis (MWCO 500) to remove DTT, the purified SH-PEG-ALD was analyzed by MS and HPLC. The HPLC peak area of SH-PEG-ALD was compared with that of SH-PEG-ALD standard solutions (1 mg/mL ~ 0.037 mg/mL) to determine the ratio of ALD to TCZ. The serum stability of the linker was assessed in BSA solution (10 mg/mL) at 37 °C for 48 h by the HPLC analysis of the detached SH-PEG-ALD from TCZ-ALD conjugate.


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In vitro biological activity of TCZ-ALD conjugate. The biological activity of TCZ-ALD conjugate was evaluated in vitro to specifically block IL-6R by ELISA.32 First, anti-IL-6R antibody (4 µg/mL) in sodium carbonate buffer (pH 9) was added to a 96-well plate, which was incubated with a plate sealer at 4 ˚C overnight to coat anti-IL-6R antibody on the plate. After washing the plate with TTBS buffer four times, the wells were incubated with a blocking buffer (1% skim milk in coating buffer) for 2 h and then with 100 µL of recombinant human IL-6Rα (2 µg/mL) with mild shaking for 2 h. After washing with TTBS buffer four times, TCZ or TCZ-ALD conjugate ranging from 0.0005 to 10 µg/mL were added to the wells, which were incubated with mild shaking for 2 h and washed with TTBS buffer four times. Goat anti-human IgG-HRP specific to TCZ was added and incubated with mild shaking for 2 h. Finally, 50 µL of TMB solution made by mixing 100 µL of TMB (6 mg/mL in DMSO) and 0.6 µL of H2O2 (30 %) in 6 mL of acetic acid (100 mM, pH 5.5) was added into each well to activate HRP. After incubation in dark for 20 min, the stop solution prepared by diluting 533 µL of sulfuric acid (1 M) in 10 mL of DI water was added in each well. The optical absorbance was analyzed at 450 nm with a microplate reader (EMax, Molecular Devices, CA). Effect of TCZ-ALD conjugate on macrophage viability. The anti-inflammatory effect of TCZ-ALD conjugate was assessed on the viability of Raw 264.7 cells, macrophages derived from an ascites of a tumor by intraperitoneal injection of Abselon Leukaemia Virus to BALB/c mice.33,34 Raw 264.7 macrophages at a density of 2 × 104 cells/well were seeded on 96-well plates and cultured in DMEM containing 10 vol% FBS and 1 vol% antibiotics at 37°C and 5% CO2 for a day. After that, PBS, ALD, ALD-Linker, IgG-ALD, TCZ, TCZ+ALD mixture or TCZ-ALD conjugate (1 µM of ALD, IgG, TCZ) were incubated with Raw 264.7 macrophages in 100 µL of serum free DMEM for 48 h, respectively. Goat anti-human IgG was used as a non-specific control antibody. Then, the medium in wells was removed by gentle suction and 100 µL of fresh serum free DMEM containing 10 µL of MTT solution at a concentration of 5 mg/mL was added to Raw 264.7 macrophages. After incubation for 2 h, the plate was cleaned by suction and filled with 50 µL of DMSO in each well. The optical density was analyzed with a microplate reader at 540 nm. ACS Paragon Plus Environment

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In vivo therapeutic assessment of TCZ-ALD conjugate. DBA/1j mice were used to prepare a CIA animal model. CII emulsified in Complete Freund’s Adjuvant (CFA) was injected to DBA/1j mice for first immunization, followed by the second immunization with CII emulsified in Incomplete Freund's Adjuvant (IFA) 3 weeks after the first immunization.35 The prepared mice were observed once-a-week after the first immunization. The level of severity on each paw was blindly scored at a scale from 0 (normal) to 4 (severe) depending on the degree of swelling and erythema.35 The RA model mice (n = 5) were locally injected on the arthritic ankle joints with four types of samples (10 µL) at a dose of 2 µg/kg of ALD and 0.8 mg/kg of TCZ 4 weeks after the first immunization with CFA: group 1 - PBS, group 2 ALD, group 3 - TCZ, and group 4 - TCZ-ALD conjugate. The therapeutic effect of samples was assessed by measuring severity scores of RA for two months.35 The level of bone degradation on arthritic ankle joints was evaluated by micro CT (Quantum FX micro CT, PerkinElmer, Waltham, MA). In vivo tests were performed according to the institutional ethical protocols of POSTECH for animals. Histological analysis. After sacrifice, the treated ankle joints of the mice were harvested for histological analysis. All dissected tissues were fixed in 10 vol% formalin and then decalcified with a histological decalcifying solution for a week. Each sample was washed with DI water, dehydrated by simple immersion in a graded ethanol series several times, and embedded in paraffin. The samples were sliced into 4 µm-thick for H&E staining. The images were taken using a light microscope (Model IX71 Olympus, Tokyo, Japan). Western blot analysis. Western blot analysis was performed to separate and quantify proteins in the harvested tissues. Dissected tissues were washed with PBS and lysed with sodium dodecyl sulphate (SDS) buffer containing 2% SDS, 62.5 mM Tris–HCl (pH 6.8), 50 mM DTT, 10% glycerol, and 0.1% bromophenol blue. The extracted samples were separated on SDS polyacrylamide gels (4~10%) by electrophoresis and transferred to membranes, which were incubated with primary antibodies detecting IL-6, CD68, CII, or osteocalcin at 4 ˚C overnight. After washing, samples were incubated with secondary antibodies containing HRP with mild shaking for 1 h. The luminescence of Western blots was


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represented on X-ray film (Fuji super RX, Fujifilm Medical Systems, Tokyo, Japan) with quantification by the image densitometry (Bio-Rad, Hercules, CA). Statistical analysis. The data are shown as means ± standard deviation after several separate experiments. Statistical analysis was performed via the t-test using SigmaPlot12.0. The value *P < 0.05 was considered to be statistically meaningful.

ACKNOWLEDGEMENT This research was supported by the Bio & Medical Technology Development Program (No. 2012M3A9C6049791) of the National Research Foundation (NRF). This research was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health and Welfare, Korea (HI14C1658).

SUPPORTING INFORMATION The Supporting Information of 1H NMR (Figures S1) and

13

C NMR (Figures S2) for PDPH-PEG-

ALD, SH-PEG-ALD, SH-PEG-NHS, ALD and PDPH is available free of charge on the ACS Publications website.

ABBREVIATIONS USED TCZ, tocilizumab; ALD, alendronate; ADC, antibody-drug conjugate; CIA, collagen induced arthritis; CFA, complete freund’s adjuvant; IFA, incomplete freund’s adjuvant; CII , type II collagen


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– For Table of Contents Use Only –

Tocilizumab – Alendronate Conjugate for Treatment of Rheumatoid Arthritis

Hwiwon Lee,† Suk Ho Bhang,‡ Jeong Ho Lee,† Hyemin Kim,† Sei Kwang Hahn†,*

IL-6 Bone resorption & Cartilage destruction

× IL-6R Anti-inflammation IL-6 signal inhibition

Macrophage


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TocilizumabâAlendronate Conjugate for Treatment of Rheumatoid

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