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Hyaluronate - Death Receptor 5 Antibody Conjugates for Targeted Treatment of Liver Metastasis Hwiwon Lee, Beom-Ju Hong, Jeong Ho Lee, Sujin Yeo, HoeYune Jung, Junho Chung, G-one Ahn, and Sei Kwang Hahn Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 12 Aug 2016 Downloaded from http://pubs.acs.org on August 12, 2016

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Hyaluronate - Death Receptor 5 Antibody Conjugates for Targeted Treatment of Liver Metastasis

Hwiwon Lee,† Beom-Ju Hong,‡ Jeong Ho Lee,† Sujin Yeo,‡ Hoe-Yune Jung,§ Junho Chung,¶ G-One Ahn,‡,* 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. ‡ Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), 77 Cheongam-ro, Nam-gu, Pohang 790-784, Korea. § R&D Center, NovMetaPharma Co., Ltd., Jigokdong, Nam-gu, Pohang, 790-834, Korea. ¶ Department of Biochemistry and Molecular Biology and Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea.

*Corresponding authors Tel.: +82 54 279 2159. Fax: +82 54 279 2399. E-mail: [email protected] (S.K. Hahn) Tel.: +82 54 279 2353. Fax: +82 54 279 8379. E-mail: [email protected] (G.O. Ahn) ACS Paragon Plus Environment

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ABSTRACT Liver is the most frequent site of metastasis with a 5-year survival rate of only 20~40 %. In this work, hyaluronate (HA) - death receptor 5 antibody (DR5 Ab) conjugate was synthesized as a dual targeting therapeutic agent to treat the liver metastasis. Dual targeting is achieved by DR5 Ab, a humanized agonistic monoclonal antibody binding to DR5 frequently overexpressed in many kinds of cancer cells, and by HA, a natural polysaccharide binding to HA receptors highly expressed in both the liver and cancer cells. Thiol end-modified HA was site-specifically conjugated to N-glycan on Fc region of oxidized DR5 Ab using a hetero-bifunctional linker of 3-(2-pyridyldithio)propionyl hydrazide (PDPH). The successful synthesis of HA-DR5 Ab conjugate was confirmed by 1H NMR, purpald assay, dynamic light scattering (DLS), and high-performance liquid chromatography (HPLC). In vitro analysis of HA-DR5 Ab conjugate revealed that the conjugation of HA to DR5 Ab did not affect the binding affinity and anti-cancer efficacy of DR5 Ab. Remarkably, according to in vivo bioimaging study, HADR5 Ab conjugate appeared to be highly accumulated in the liver and dramatically effective in inhibiting the tumor growth in liver metastasis model mice. [KEYWORDS] Death receptor 5; Antibody; Hyaluronate; Targeted delivery; Liver metastasis

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INTRODUCTION Liver metastasis frequently occurs in colorectal cancer patients with a median survival period of approximately 5 ~ 10 months.1-5 Liver is a highly susceptible organ for metastasis because of plentiful dual blood supply from hepatic artery and portal vein. Given the fact that only a small proportion of patients can be benefited from surgical resection and that other treatment modalities such as chemotherapy or radiofrequency ablation are often associated with the risk of recurrence,6 it is crucial to develop new therapeutic strategies. One of such strategies is to utilize tumor necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL), a member of the TNF ligand superfamily.7,8 TRAIL can bind to four transmembrane receptors: TRAIL-R1 also known as death receptor (DR) 4, TRAIL-R2 (DR5), TRAIL-R3 (decoy receptor 1), and TRAIL-R4 (decoy receptor 2), as well as to the soluble receptor osteoprotegerin. It is known that only DR4 and DR5 can trigger apoptosis as TRAIL-R3/R4 and osteoprotegerin lack the functional cytoplasmic death domain required for inducing apoptosis.9,10 The expression of DR4 and, even more so, DR5 is often being high in a number of malignancies, leaving normal cells being hardly affected.11-16 Although TRAIL binding to DR4 and DR5 triggers both intrinsic and extrinsic pathways of apoptosis,17 the results of recent clinical trials have been rather disappointing.18,19 Suggested limitations are such that many human tumors are partially or completely resistant to TRAIL monotherapy and that TRAIL agonistic capacity is not potent enough.20 In order to avoid the TRAIL resistance induced by TRAIL binding to the antagonistic receptors of TRAIL-R3 and TRAIL-R4, an alternative therapeutic approach has been developed utilizing agonistic monoclonal antibodies targeting DR4 or DR5.21-23 A number of these agents are now in clinical trials against various types of advanced cancers including colorectal carcinomas, hepatocellular carcinomas, non-small cell lung cancers, and breast cancers demonstrating good tolerability and safety profiles in these patients.24 A strategy to further improve the therapeutic efficacy of agonistic antibodies against DR4 or DR5 is chemical modifications to enhance the targeting efficiency towards cancers or particular organ types. In this aspect, we have exploited hyaluronate (HA), a biodegradable, non-toxic, non-immunogenic, and natural polysaccharide,25 as a target-specific drug delivery carrier. HA binds to its receptors, namely ACS Paragon Plus Environment

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RHAMM and CD44, which are significantly overexpressed on many cancers.26-28 Furthermore, there are a number of HA receptors and enzymes for HA degradation (e.g. hyaluronidase) in the liver, allowing the selective HA accumulation to the liver.29-31 Therefore, we hypothesized that the conjugation of HA to DR5 antibody (Ab) might improve the targeting effect of such an antibody in the liver, which may in turn increase the therapeutic efficacy for cancers in the liver. In this work, we investigated the hypothesis by conjugating HA to humanized IgG1 monoclonal DR5 Ab and examined its therapeutic efficacy in a mouse model of the liver metastasis induced by the splenic injection of colorectal human cancer cells. We hereby report a novel dual-targeting strategy for both HA receptors and DR5 in the liver cancer, evaluating in vitro and in vivo characteristics to demonstrate that this HA-modified antibody therapeutics can be applied to various other antibody therapeutics for primary or metastatic liver cancers.


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EXPERIMENTAL SECTION Materials. Hyaluronate (HA, MW = 5 kDa) was purchased from Lifecore Biomedical (Chaska, MN). Sodium periodate, purpald, tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), dithiothreitol (DTT), N-hydroxysulfosuccinimide sodium salt and MTT formazan were purchased from SigmaAldrich (St.Louis, MO). 3-(2-Pyridyldithio)propionyl hydrazide (PDPH), fetal bovine serum (FBS), antibiotics and phosphate buffered saline (PBS, pH 7.4) were obtained from Thermo Fisher Scientific (Waltham, MA). HiLyte FluorTM 647 amine and HiLyte FluorTM 647 acid were purchased from AnaSpec (Fremont, CA). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Roswell Park Memorial Institute (RPMI) 1640 medium was purchased from ATCC and human colon carcinoma cell line HCT 116 expressing luciferase was obtained from Perkin Elmer (Waltham, MA). Mounting medium with 4',6-diamidino-2-phenylindole (DAPI) was obtained from Vector Laboratories (Burlingame, CA). Goat anti-human IgG-HRP was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). DR5 was obtained from ProSpec (East Brunswick, NJ) and DR5 human ELISA kit was purchased from Abcam (Cambridge, MA). Centrifugal filter (MWCO = 10 kDa) was obtained from Merck Millipore (Darmstadt, Germany) and NAP-5 column was purchased from GE Healthcare (Buckinghamshire, UK). Death receptor 5 antibody (DR5 Ab) was kindly donated from iBio (Seoul, Korea). Synthesis of thiol end-modified HA. Thiol end-modified HA (HA-SH) was synthesized by reductive amination as described elsewhere.32 HA (MW = 5 kDa, 10 mg) and cystamine dihydrochloride (6 mg) were dissolved in 2 mL of borate buffer (0.1 M, pH 8.5) with 0.4 M sodium chloride (NaCl), which was reacted for 2 h. Sodium cyanoborohydride (NaBH3CN) was added to the solution at a final concentration of 200 mM and incubated at 40 °C for 5 days. The reaction mixture stirred with 100 mM of DTT for 12 h to reduce disulfide bonds (S-S) of cystamine. The solution was dialyzed using a dialysis tube (MWCO = 3500 Da) against 5 L of NaCl solution (100 mM) for 2 days, 25% ethanol for a day, and distilled water for a day to remove unreacted chemicals and freeze-dried for 3 days. Before use, HA-SH was mixed


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with TCEP to completely reduce disulfide bonds of cystamine and purified through a PD-10 desalting column to remove TCEP. The introduction of thiol groups at the end of HA dissolved in deuterium oxide (D2O) was analyzed by 1H NMR. Synthesis of target-specific HA-DR5 Ab conjugate. As a first step, 100 µL of DR5 Ab (1 mg/mL) was oxidized with 10 µL of sodium periodate (21.39 mg/mL) in phosphate buffer (pH 7.4) at 4 °C for 30 min in dark and then diluted with 1 mL of PBS. The excess amount of sodium periodate was filtered out with a centrifugal filter (MWCO = 10 kDa, 5000 g, 10 min). Then, 50 molar excess of PDPH, a heterobifunctional linker, was added to the oxidized DR5 Ab, which was incubated overnight for chemical conjugation between the hydrazide group of PDPH and the aldehyde group of oxidized DR5 Ab.33 The unbound PDPH was dialyzed against PBS with a centrifugal filter five times. The conjugation of PDPH to DR5 Ab was analyzed by the purpald assay, which detects free aldehyde groups on DR5 Ab by measuring the absorbance at 550 nm.33 After that, HA-SH was incubated with PDPH-DR5 Ab at room temperature for 6 h for conjugation reaction between thiol group of HA-SH and pyridyldisulfide group of PDPH-DR5 Ab. The conjugation of HA-DR5 Ab was assessed by 1H NMR, dynamic light scattering (DLS) and high-performance liquid chromatography (HPLC) using the ultrahydrogel 500 and 1000 columns (Waters, MA). With a PBS flow rate of 0.4 mL/min, 100 µL of the sample in PBS was injected to the column and analyzed by detecting the absorbance at 210 nm. In vitro biological activity of HA-DR5 Ab conjugate. In vitro biological activity of HA-DR5 Ab conjugate was evaluated for specific binding to DR5 by enzyme-linked immunosorbent assay (ELISA). First, DR5 (2 ng/mL) in sodium carbonate buffer (pH 9) was coated on a 96-well plate with a plate sealer by incubating at 4˚C overnight. After washing the plate with a TTBS buffer four times, the wells were incubated with a blocking buffer (1% skim milk in pH 9 carbonate buffer) at room temperature for 2 h. Then, DR5 Ab and HA-DR5 Ab conjugate ranging from 0.0137 to 10 µg/mL were incubated in the DR5 coated wells at room temperature with mild shaking for 2 h and washed with a TTBS buffer four times. Goat anti-human IgG-horseradish peroxidase (HRP) specifically binding to DR5 Ab was


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incubated with mild shaking for 2 h and washed again. Finally, 50 µL of TMB solution prepared by mixing 100 µL of TMB at 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 to each well to activate HRP in dark for 20 min. Then, the stop solution prepared by diluting 533 µL of sulfuric acid (1 M) in 10 mL of DI water was added into each well to stop the reaction. The absorbance of each well was measured at 450 nm with a microplate reader (EMax, Molecular Devices, Sunnyvale, CA). Anti-tumor effect of HA-DR5 Ab conjugate. Anti-tumor effect of HA-DR5 Ab conjugate was assessed in human colon cancer cells of HCT116 and mouse hepatocytes of FL83B. HCT116 and FL83B at a density of 2 × 104 cells/well were seeded on 96-well plates and cultured in RPMI 1640 and DMEM, containing 10 vol% FBS and 1 vol% antibiotics at 37°C and 5% CO2 atmosphere for a day. After that, FL83B mouse hepatocyte was treated with DR5 Ab (20 µg/mL) as a control and HCT116 was treated with PBS, IgG1, HA, HA + DR5 Ab mixture and HA-DR5 Ab conjugate in 100 µL of serum free media for 24 h, respectively. The concentration of antibody for the case of HCT116 was fixed at 0.2 µg/mL, 2 µg/mL, and 20 µg/mL. HA was tested for comparison at concentrations of 0.02 µg/mL, 0.2 µg/mL, and 2 µg/mL. Then, samples containing media in each well were removed by gentle suction and 100 µL of fresh serum free media containing 10 µL of MTT solution (5 mg/mL) was added to the well. After incubation at 37°C and 5% CO2 atmosphere for 2 h, the plate was cleaned by gently suction and treated with 50 µL of DMSO in each well. The optical density of each well was measured with a microplate reader at 540 nm to determine the cell viability. In vitro assessment of HA-DR5 Ab conjugate for dual receptor binding. The dual receptor binding of HA-DR5 Ab conjugate was evaluated in mouse hepatocytes of FL83B with HA receptors and HCT116 cancer cells with both HA receptors and DR5. FL83B and HCT116 cells were cultured in DMEM and RPMI, respectively, containing 10 vol% FBS and 1 vol% antibiotics at 37°C and 5% CO2 atmosphere. Both cells were incubated in a confocal microplate with 200 µL of media for a day and 20 mg/mL of free HA was incubated with the cells for 1 h to block HA receptors before the sample


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treatment. HA was labeled with HiLyte FluorTM 647 amine (red) by EDC chemistry and DR5 Ab was labeled with FITC (green) for fluorescent observation. Cells were incubated with HA or HA-DR5 Ab conjugate overnight, washed with PBS thrice and fixed with 4 % of paraformaldehyde (100 µL) at 37°C for 5 min. After gentle suction of paraformaldehyde, DAPI containing mounting gel which stains cell nuclei in blue was dropped on the cells and the cell seeded plate was covered with a cover slip. Cells treated with HA and HA-DR5 Ab conjugate were observed with a confocal microscope (Leica TCS SP5, Wetzlar, Germany). In vivo bioimaging of HA-DR5 Ab conjugate for liver targeting. In vivo liver targeting of HA-DR5 Ab conjugate was evaluated in comparison with DR5 Ab after tail vein injection to mice in quadruplicate. HiLyte FluorTM 647 acid containing succinimidyl ester, an amino-reactive form, was labeled to amine groups of DR5 Ab. In detail, 10 molar ratio of the dye was reacted with DR5 Ab and HA-DR5 Ab conjugate at room temperature in dark overnight and then purified with a centrifugal filter (MWCO = 10 kDa, 10000 g, 10 min) and NAP-5 desalting column (GE Healthcare, UK) to remove unbound HiLyte FluorTM 647. Four types of samples (100 µL) including PBS, HiLyte FluorTM 647, DR5 Ab and HA-DR5 Ab conjugate with the same fluorescent intensity were injected via the tail vein of mice. After 24 h, the mice were sacrificed, whole blood was perfused out and their livers were harvested to measure the fluorescence intensity via in vivo imaging systems (IVIS) (ex/em = 640/680 nm) (Perkin Elmer, Waltham, MA) for the assessment of liver targeting of HA-DR5 Ab conjugate. In vivo therapeutic assessment of HA-DR5 Ab conjugate. To assess in vivo therapeutic effect of HADR5 Ab conjugate, metastatic liver cancer model mice were prepared by splenic injection of cancer cells.1-3 The direct inoculation of cancer cells into the livers and the chemical treatment to prepare liver cancer model mice are considered as an artificial approach. For liver metastasis model mice, HCT116 expressing luciferase tumor cells (5 × 106 cells/40 µL/mouse) were injected into the spleen of 7-week old male BALB/c (nu/nu) mice (Orient Bio, Seongnam, Korea) using a 26-gauge 1/2 syringe. Eleven days after splenic injection, the spleen was removed surgically and liver metastasis was identified by


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observing the intensity of luciferase after peritoneal injection of luciferin (100 µL, 10 mg/mL) using the IVIS. To investigate the therapeutic efficacy of HA-DR5 Ab conjugate on the liver metastasis, the liver cancer model mice were treated with four types of samples at a dose of 0.1 mg/mouse of DR5 Ab: PBS for group 1, HA (5 kDa, 5 µg/mouse) for group 2, DR5 Ab for group 3, and HA-DR5 Ab conjugate for group 4 (n = 4). The therapeutic effect of each sample was determined by measuring the level of luciferase expressed by HCT116 which invaded into the liver from the spleen. Sample treated liver metastasis model mice were monitored for a week by measuring luciferase intensity with an IVIS system. Animal experiments were conducted under the guiding principle approved by the Institutional Animal Care and Use Committee of POSTECH (approval # POSTECH-2015-0035). Histological analysis. After one week monitoring for the progress of liver metastasis using an IVIS system, livers of sample treated mice were harvested for histological analysis. All harvested liver tissues were washed with PBS and fixed in 10 vol% buffered formalin for storage. The fixed liver tissues were washed with DI water, dehydrated by simple immersion in a graded ethanol series (70%, 80%, 95%, 100%) several times, and embedded in paraffin. The paraffin embedded liver tissues were sliced into 4 µm-thick and stained with hematoxylin and eosin (H&E). The sample images were observed with a light optical microscope (Model IX71 Olympus, Topkyo, Japan). Statistical analysis. The data are expressed as means ± standard deviation from several separate experiments. Statistical analysis was carried out via the t-test using the software of SigmaPlot12.0. A value for *P < 0.05 was considered statistically significant.


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RESULTS AND DISCUSSION Synthesis and characterization of HA-DR5 Ab conjugate. Fig. 1 shows a schematic representation of HA-DR5 Ab conjugate for dual targeting both DR5 and HA receptors. To synthesize HA-DR5 Ab conjugate, HA was chemically tethered to DR5 Ab using a hetero-bifunctional linker of PDPH containing pyridyldisulfide and hydrazide reactive groups. The hydrazide group of PDPH was reacted with aldehyde group on carbohydrate region of oxidized DR5 Ab and the pyridyldisulfide group of PDPH was reacted with thiol group of HA. 1H NMR analysis confirmed the successful synthesis of HADR5 Ab conjugate (Fig. 2a and b). The peak around 2.9 ppm in Fig. 2a confirmed that thiol groups were introduced at the end of HA chains and the peak around 2.7 ppm in Fig. 2b showed that PDPH was successfully conjugated to HA chains. The successful conjugation of HA to DR5 Ab was also confirmed by the purpald assay, DLS and HPLC. The purpald assay can detect aldehyde groups on oxidized DR5 Ab via the colorimetric change from clear to purple. The absorbance at 550 nm greatly increased after oxidization of DR5 Ab and decreased after PDPH and HA conjugation on DR5 Ab (Fig. 2c), which revealed the site-specific conjugation of HA to DR5 Ab. DLS showed the much bigger hydrodynamic size of HA-DR5 Ab conjugate (around 170 nm) than that of DR5 Ab (around 10 nm) (Fig. 2d). Moreover, HPLC analysis showed a peak with an increased molecular weight for HA-DR5 Ab conjugate compared to PDPH-DR5 Ab (Fig. 2e), further confirming the successful conjugation of HA on PDPHDR5 Ab. In vitro biological activity of HA-DR5 Ab conjugate. We examined the binding affinity of HA-DR5 Ab conjugate to DR5 by ELISA (Fig. 2f). DR 5-coated plate was incubated with various concentrations of DR5 Ab or HA-DR5 Ab conjugate ranging from 0.0137 to 10 µg/mL. After washing the plate, antihuman IgG-HRP was added to the plate for detecting DR5 Ab bound to DR5. As expected, there was no significant difference in the binding affinity between DR5 Ab alone and HA-DR5 Ab conjugate, because HA was site-specifically tethered to N-glycan of Fc region of DR5 Ab without hindrance of DR5 binding sites. In general, amine groups on the antibody were randomly modified with amine reactive


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functional groups, which frequently blocks antigen binding sites and reduces biological activity of antibody. However, in this work, the site-specific modification of antibody resulted in the well preserved biological activity of antigen binding site. Besides, as shown in Figure 3, the cytotoxic effect of HA-DR5 Ab conjugate on cancer cells was confirmed from the viability of HCT116, a human colon cancer cell line, which was exploited to prepare liver metastasis model mice for the following in vivo experiments. In vitro cytotoxicity of HA-DR5 Ab conjugate was dose-dependent and comparable to that of DR5 Ab alone or HA + DR5 Ab mixture against the human colon cancer cell line of HCT116. On the other hand, we observed no significant cell death caused by HA alone. In addition, DR5 Ab alone exerted no cytotoxicity towards the normal mouse hepatocyte of FL83B, showing the human tumor-specific cytotoxic effect. From the results, we clearly confirmed in vitro biological activity of HA-DR5 Ab conjugate as a promising anti-cancer therapeutics. In vitro dual receptor binding of HA-DR5 Ab conjugate. The dual receptor binding of HA-DR5 Ab conjugate to DR5 and HA receptors was evaluated by confocal microscopy after labeling HA and DR5 Ab with red and green fluorescence, respectively (Fig. 4). For the competitive binding test, we blocked HA receptors by incubating with free HA (not labeled) prior to incubation of HA-DR5 Ab conjugate with either FL83B normal mouse hepatocytes or HCT116 cancer cells which were stained with DAPI in blue. We observed that red labeled HA bound well to FL83B cells (Fig. 4a) with HA receptors but not DR5. As expected, we did not observe any significant red signal from HA in FL83B cells pre-incubated with free HA (Fig. 4b). We observed yellowish color arising from co-localization of red HA and green DR5 Ab after treatment with HA-DR5 Ab conjugate (Fig. 4c). There was no signal from cells preincubated with free HA (Fig. 4d). These results suggest that HA-DR5 Ab conjugate effectively targets HA receptors in hepatocytes. We further examined HA and HA-DR5 Ab conjugates in HCT116 cells, which expresses both HA receptors and DR5. Consistent with FL83B cells, HA and HA-DR5 Ab conjugate were well bound to HCT116 cells (Fig. 4e, 4g). However, when cells were pre-incubated with free HA, while red colored HA could not bind (Fig. 4f), HA-DR5 Ab conjugate labeled in red and green


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bound to HCT116 cells (Fig. 4h). These results indicate that HA-DR5 Ab conjugate has the dual targetspecificity, namely towards HA receptors and DR 5. In vivo bioimaging of HA-DR5 Ab conjugate for liver targeting. The effect of HA conjugation to DR5 Ab on its liver targeting was examined in comparison to DR5 Ab alone by monitoring their biodistribution in mice. After labeling a fluorescent dye of HiLyte FluorTM 647 acid on DR5 Ab, the accumulation of DR5 Ab, HA + DR5 Ab mixture and HA-DR5 Ab conjugate was visualized by IVIS imaging at 24 h after intravenous injection (Fig. 5). We observed that the highest level of fluorescence was detected in the liver of mice after injection of HA-DR5 Ab conjugate, which was significantly higher than that of DR5 Ab alone (***P < 0.0001) and HA + DR5 Ab mixture (***P < 0.0001), confirming the significantly enhanced liver accumulation by the dual targeting effect. HiLyte FluorTM 647 alone showed a fluorescence level lower than that of DR5 Ab alone (**P < 0.01), HA + DR5 Ab mixture (**P < 0.01) or HA-DR5 Ab conjugate (***P < 0.0001), reflecting the rapid urinary clearance of HiLyte FluorTM 647 and the long half-life of DR5 Ab antibodies. In vivo efficacy of HA-DR5 Ab conjugate in liver metastasis model mice. The antitumor efficacy of HA-DR5 Ab conjugate was evaluated in liver metastasis model mice, set up by splenic implantation of luciferase-expressing HCT116 cancer cells, by bioluminescence imaging (Fig. 6). We observed that the liver metastasis progressed very fast, resulting in the lethal level of cancer cell spreading by 7 days postimplantation. The treatment with PBS or HA alone had no inhibitory effect on the progression of liver metastasis, resulting in the fast invasion and growth of HCT116 cancer cells in the liver, and the broad spreading through the whole body. On the other hand, the metastatic progression was significantly reduced after treatment with DR5 Ab alone compared to that of PBS or HA treated mice, but cancer cells were slowly becoming bigger in the liver. Remarkably, the cancer regression in size was definitely observed in the mice treated with HA-DR5 Ab conjugate, which supported the dual targeting efficacy of HA-DR5 Ab conjugate on liver metastasis.


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For further in-depth evaluation, histological analysis of dissected liver tissues was performed with H&E staining. After sacrifice of mice, the size of harvested livers treated with PBS or HA was much bigger than that with DR5 Ab or HA-DR5 Ab conjugate. The abnormal cancer tissues were evidently observed with naked eyes. Furthermore, histological analysis with H&E staining of dissected liver tissues definitely showed that cancer cells deeply and frequently invaded into liver tissues after treatment with PBS or HA (Fig. 7a, 7b). Although DR5 Ab alone significantly reduced the progression of liver metastasis, bioluminescence signal still remained and histological examination by H&E staining revealed macroscopic regions of cancer cells in the liver (Fig. 7c). Noticeably, there were few regions for cancer cell invasion into the livers after treatment with HA-DR5 Ab conjugate (Fig. 7d) in consistent with the significantly reduced bioluminescence signal. These results overall demonstrated that HA-DR5 Ab conjugate achieved a significantly improved antitumor efficacy compared to DR5 Ab alone via the dual targeting to DR5 and HA receptors, and the selective accumulation of the conjugate in the liver.


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CONCLUSION We have successfully developed HA-DR5 Ab conjugate for targeted treatment of liver metastasis. After characterization by 1H NMR, purpald assay, DLS and HPLC analysis, we could confirm the comparable cytotoxic effect of HA-DR5 Ab conjugate to that of DR5 Ab alone in cancer cells. According to in vitro confocal microscopy, HA-DR5 Ab conjugate showed the dual targeting effect to both DR5 and HA receptors. Furthermore, in vivo bioimaging revealed that HA-DR5 Ab conjugate accumulated much more significantly in the liver than DR5 Ab alone, resulting in the drastically enhanced antitumor efficacy in liver metastasis model mice. Histological analysis also confirmed the anti-tumor effect of HA-DR5 Ab conjugate. Taken together, HA-DR5 Ab conjugate might have a great potential as a dual targeting anticancer drug for the treatment of liver metastasis. This HA-based dual targeting strategy to the liver can be also applied to many other antibody therapeutics for treating various liver diseases.

ACKNOWLEDGEMENT This research was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health and Welfare, Korea (HI14C1658), and the Bio & Medical Technology Development Program (No. 2012M3A9C6049791) and Mid-career Researcher Program (No. 2015R1A2A1A15053779) of the National Research Foundation (NRF) funded by the Korean government (MEST).


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10. Weichhaus, M.; Segaran, P.; Renaud, A.; Geerts, D.; Connelly, L. Osteoprotegerin expression in triple-negative breast cancer cells promotes metastasis. Cancer Med. 2014, 3, 1112-1125. 11. Yada, A.; Yazawa, M.; Ishida, S.; Yoshida, H.; Ichikawa, K.; Kurakata, S.; Fujiwara, K. A novel humanized anti-human death receptor 5 antibody CS-1008 induces apoptosis in tumor cells without toxicity in hepatocytes. Ann. Oncol. 2008, 19, 1060-1067. 12. Wilson, N. S.; Yang, A.; Yang, B.; Couto, S.; Stern, H.; Gogineni, A.; Pitti, R.; Marsters, S.; Weimer, R. M.; Singh, M.; Ashkenazi, A. Proapoptotic activation of death receptor 5 on tumor endothelial cells disrupts the vasculature and reduces tumor growth. Cancer Cell 2012, 22, 80-90. 13. Yagita, H.; Takeda, K.; Hayakawa, Y.; Smyth, M. J.; Okumura, K. TRAIL and its receptors as targets for cancer therapy. Cancer Sci. 2004, 95, 777-783. 14. Ichikawa, K.; Liu, W.; Zhao, L.; Wang, Z.; Liu, D.; Ohtsuka, T.; Zhang, H.; Mountz, J. D.; Koopman, W. J.; Kimberly, R. P.; Zhou, T. Tumoricidal activity of a novel anti-human DR5 monoclonal antibody without hepatocyte cytotoxicity. Nat. Med. 2001, 7, 954-960. 15. Huet, H. A.; Growney, J. D.; Johnson, J. A.; Li, J.; Bilic, S.; Ostrom, L.; Zafari, M.; Kowal, C.; Yang, G.; Royo, A.; Jensen, M.; Dombrecht, B.; Meerschaert, K. R.; Kolkman, J. A.; Cromie, K. D.; Mosher, R.; Gao, H.; Schuller, A.; Isaacs, R.; Sellers, W. R.; Ettenberg, S. A. Multivalent nanobodies targeting death receptor 5 elicit superior tumor cell killing through efficient caspase induction. mAbs 2014, 6, 1560-1570. 16. Wang, G.; Wang, X.; Yu, H.; Wei, S.; Williams, N.; Holmes, D. L.; Halfman, R.; Naidoo, J.; Wang, L.; Li, L.; Chen, S.; Harran, P.; Lei, X.; Wang, X. Small-molecule activation of the TRAIL receptor DR5 in human cancer cells. Nat. Chem. Biol. 2013, 9, 84-89.


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17. Wiezorek, J.; Holland, P.; Graves, J. Death receptor agonists as a targeted therapy for cancer. Clin. Cancer Res. 2010, 16, 1701-1708. 18. Dimberg, L. Y.; Anderson, C. K.; Camidge, R.; Behbakht, K.; Thorburn, A.; Ford, H. L. On the TRAIL to successful cancer therapy? Predicting and counteracting resistance against TRAIL-based therapeutics. Oncogene 2013, 32, 1341-1350. 19. Holland, P. M. Death receptor agonist therapies for cancer, which is the right TRAIL? Cytokine Growth Factor Rev. 2014, 25, 185-193. 20. Zhang, L.; Fang, B. Mechanisms of resistance to TRAIL-induced apoptosis in cancer. Cancer Gene Ther. 2005, 12, 228-237. 21. Camidge, D. R.; Herbst, R. S.; Gordon, M. S.; Eckhardt, S. G.; Kurzrock, R.; Durbin, B.; Ing, J.; Tohnya, T. M.; Sager, J.; Ashkenazi, A.; Bray, G.; Mendelson, D. A phase I safety and pharmacokinetic study of the death receptor 5 agonistic antibody PRO95780 in patients with advanced malignancies. Clin. Cancer Res. 2010, 16, 1256-1263. 22. Graves, J. D.; Kordich, J. J.; Huang, T. H.; Piasecki, J.; Bush, T. L.; Sullivan, T.; Foltz, I. N.; Chang, W.; Douangpanya, H.; Dang, T.; O’Neil, J. W.; Mallari, R.; Zhao, X.; Branstetter, D. G.; Rossi, J. M.; Long, A. M.; Huang, X.; Holland, P. M. Apo2L/TRAIL and the death receptor 5 agonist antibody AMG 655 cooperate to promote receptor clustering and antitumor activity. Cancer Cell 2014, 26, 177-189. 23. Sung, E. S.; Park, K. J.; Lee, S. H.; Jang, Y. S.; Park, S. K.; Park, Y. H.; Kwag, W. J.; Kwon, M. H.; Kim, Y. S. A novel agonistic antibody to human death receptor 4 induces apoptotic cell death in various tumor cells without cytotoxicity in hepatocytes. Mol. Cancer Ther. 2009, 8, 2276-2285. 24. Dai, X.; Zhang, J.; Arfuso, F.; Chinnathambi, A.; Zayed, M. E.; Alharbi, S. A.; Kumar, A. P.; Ahn, K. S.; Sethi, G. Targeting TNF-related apoptosis-inducing ligand (TRAIL) receptor by natural products as a potential therapeutic approach for cancer therapy. Exp. Biol. Med. 2015, 240, 760-773.


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25. Choi, K. Y.; Chung, H.; Min, K. H.; Yoon, H. Y.; Kim, K.; Park, J. H.; Kwon, I. C.; Jeong, S. Y. Self-assembled hyaluronic acid nanoparticles for active tumor targeting. Biomaterials 2010, 31, 106-114. 26. Jiang, T.; Zhang, Z.; Zhang, Y.; Lv, H.; Zhou, J.; Li, C.; Hou, L.; Zhang, Q. Dual-functional liposomes based on pH-responsive cell-penetrating peptide and hyaluronic acid for tumor-targeted anticancer drug delivery. Biomaterials 2012, 33, 9246-9258. 27. Ganesh, S.; Iyer, A. K.; Morrissey, D. V.; Amiji, M. M. Hyaluronic acid based self-assembling nanosystems for CD44 target mediated siRNA delivery to solid tumors. Biomaterials 2013, 34, 3489-3502. 28. Qhattal, H. S.; Hye, T.; Alali, A.; Liu, X. Hyaluronan polymer length, grafting density, and surface poly(ethylene glycol) coating influence in vivo circulation and tumor targeting of hyaluronangrafted liposomes. ACS Nano 2014, 8, 5423-5440. 29. Lee, M. Y.; Yang, J. A.; Jung, H. S.; Beack, S.; Choi, J. E.; Hur, W.; Koo, H.; Kim, K.; Yoon, S. K.; Hahn, S. K. Hyaluronic acid-gold nanoparticle/interferon alpha complex for targeted treatment of hepatitis C virus infection. ACS Nano 2012, 6, 9522-9531. 30. Yang, J. A.; Park, K.; Jung, H.; Kim, H.; Hong, S. W.; Yoon, S. K.; Hahn, S. K. Target specific hyaluronic acid-interferon alpha conjugate for the treatment of hepatitis C virus infection. Biomaterials 2011, 32, 8722-8729. 31. Zhou, Q.; Stefano, J. E.; Manning, C.; Kyazike, J.; Chen, B.; Gianolio, D. A.; Park, A.; Busch, M.; Bird, J.; Zheng, X.; Simonds-Mannes, H.; Kim, J.; Gregory, R. C.; Miller, R. J.; Brondyk, W. H.; Dhal, P. K.; Pan, C. Q. Site-specific antibody-drug conjugation through glycoengineering. Bioconjugate Chem. 2014, 25, 510-520. 32. Lee, H.; Lee, M. Y.; Bhang, S. H.; Kim, B. S.; Kim, Y. S.; Ju, J. H.; Kim, K. S; Hahn, S. K.. Hyaluronate-gold nanoparticle/tocilizumab complex for the treatment of rheumatoid arthritis. ACS Nano 2014, 8, 4790-4798.


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33. Kumar, S.; Aaron, J.; Sokolov, K. Directional conjugation of antibodies to nanoparticles for synthesis of multiplexed optical contrast agents with both delivery and targeting moieties. Nat. Protoc. 2008, 3, 314-320.


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FIGURES Fig. 1

a

Dual-targeting system

HA-DR5 Ab conjugate

Death receptor mediated apoptosis Liver

HA receptor mediated targeting

DR5

HA receptor

Cancer Cancer cell

Hepatocyte

b NaIO4 --OH

PDPH

HA-SH

--CH=O


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Fig. 2

a

5.0

4.0

3.0

2.0

1.0

ppm

4.0

3.0

2.0

1.0

ppm

b

5.0

c

d

0.6

18 DR5 Ab HA-DR5 Ab

16

0.5

Intensity (%)

14

Absorbance

0.4 0.3 0.2 ***

0.1

12 10 8 6 4

***

2 0.0

Ab

1000

10000

R

R5

5

Ab

100

-D

Size (nm )

HA

-D

10

PD

PH

1

O

xi

di

ze

d

D

DR

R5

5

Ab

Ab

S

0

PB

e

f

1.6 DR5 Ab HA-DR5 Ab

1.4

Absorbance

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

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1.2 1.0 0.8 0.6 0.01

0.1

1

10

Concentration (µ µ g/m L)


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Biomacromolecules

Fig. 3

120

FL83B

HCT116

100

Cell viability (%)

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

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80 60 40

***

***

***

20 0.02 0.2 2 0.2 2

20 0.2 2

20

20 0 20

0 0.2 2

20 0.2 2

Concentration (µ µ g/mL) DR5 Ab PBS

IgG1 DR5 Ab

HA

HA-DR5 Ab conjugate HA+DR5 Ab mixture


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Fig. 4

a

b

c

HA (FL83B)

HA + free HA (FL83B)

HA-DR5 Ab (FL83B)

d

e

f

HA-DR5 Ab + free HA (FL83B)

HA (HCT116)

HA + free HA (HCT116)

g

h

HA-DR5 Ab (HCT116)

HA-DR5 Ab + free HA (HCT116)


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Biomacromolecules

Fig. 5

a

5.0

Dye

PBS

4.0 3.0

Mixture

DR5 Ab

× 10

Conjugate

8

2.0 1.0

b

18 ***

16

***

14 12 10 **

8 6 4 2

ug nj

R5 -D HA

HA

+D

R5

Ab

Ab

co

m

D

ix

R5

tu

at

e

re

Ab

ye D

S

0 PB

ROI (x10 7)

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

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Fig. 6

a

PBS

HA

DR5 Ab HA-DR5 Ab

0d

2d

7d

b

*


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Fig. 7

b

a

C

L

C

L

c

d

C

L


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FIGURE CAPTIONS

Fig. 1. Schematic illustration (a) for dual-targeting mechanism of hyaluronate (HA) - death receptor 5 antibody (DR5 Ab) conjugate by targeting DR5 and HA receptors, and (b) for the synthesis of HA-DR5 Ab conjugate. Fig. 2. 1H NMR of (a) thiol-end modified HA and (b) PDPH conjugated HA. (c) The purpald assay for PBS, DR5 Ab, oxidized DR5 Ab, PDPH-DR5 Ab and HA-DR5 Ab conjugate (***P < 0.0001 in comparison with oxidized DR5 Ab). (d) DLS and (e) HPLC analysis of DR5 Ab and HA-DR5 Ab conjugate. (f) DR5 binding affinity of DR5 Ab and HA-DR5 Ab conjugate with increasing concentration of DR5 Ab from 0.0137 to 10 µg/mL by ELISA (n = 3). Fig. 3. Cell viability of FL83B mouse hepatocyte treated with DR5 Ab (20 µg/mL) as a control and HCT116 treated with PBS, IgG1, HA, HA + DR5 Ab mixture and HA-DR5 Ab conjugate. The concentration of antibody for the case of HCT116 was fixed at 0.2 µg/mL, 2 µg/mL, and 20 µg/mL. For comparison, we used HA at concentrations of 0.02 µg/mL, 0.2 µg/mL, 2 µg/mL. The value of ***P < 0.0001 in comparison with PBS treated group was considered to be statistically significant (n = 4). Fig. 4. The receptor mediated endocytosis of HA and HA-DR5 Ab conjugate to FL83B mouse hepatocytes and HCT116 cancer cells. FL83B was incubated with HA labeled in red (a) in the absence or (b) in the presence of free HA. Also, FL83B was incubated with HA (red) - DR5 Ab (green) conjugate (c) in the absence or (d) in the presence of free HA. In addition, HCT116 was incubated with HA (e) in the absence or (f) in the presence of free HA. Furthermore, HCT116 was incubated with HA (red) – DR5 Ab (green) conjugate (g) in the absence or (h) in the presence of free HA (scale bar = 50 µm).


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Fig. 5. (a) In vivo bioimaging of the liver after treatment with HiLyte FluorTM 647 dye, fluorescently labeled DR5 Ab, HA + DR5 Ab mixture and HA-DR5 Ab conjugate, and (b) the average level of fluorescent ROI values using the IVIS system 24 h after tail vein injection to mice (n = 4). The values of **P < 0.01 and ***P < 0.0001 in comparison with HA-DR5 Ab conjugate treated group were considered to be statistically significant (n = 4). Fig. 6. In vivo bioimaging for the luciferase levels of liver metastasis model mice reflecting anti-tumor effect for 7 days after treatment with PBS, HA, DR5 Ab and HA-DR5 Ab conjugate: (a) IVIS images and (b) the average level of luciferase ROI values. The value of *P < 0.05 was considered to be statistically significant (n = 4). Fig. 7. Histological analysis with H&E staining of dissected liver tissues after treatment with (a) PBS, (b) HA, (c) DR5 Ab and (d) HA-DR5 Ab conjugate, respectively. The invasion of cancer cells into liver tissues is indicated with white dotted lines (C: cancer cell part, L: liver cell part, scale bar = 200 µm).


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– For Table of Contents Use Only –

Hyaluronate - Death Receptor 5 Antibody Conjugates for Targeted Treatment of Liver Metastasis Hwiwon Lee,1 Beom-Ju Hong,2 Jeong Ho Lee,1 Sujin Yeo,2 Hoe-Yune Jung,3 Junho Chung,4 G-One Ahn,2,* Sei Kwang Hahn1,*

Dual-targeting system HA receptor mediated targeting

Death receptor mediated apoptosis

DR5

HA receptor

Cancer cell

Hepatocyte


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HyaluronateâDeath Receptor 5 Antibody ... - ACS Publications

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