Conjugation to 10 kDa Linear PEG Extends Serum Half-Life and

Publication Date (Web): December 28, 2016. Copyright © 2016 American Chemical Society. *E-mail: [email protected]. Cite this:Mol. Pharmaceutics 14...
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Conjugation to 10 kDa Linear PEG Extends Serum Half-Life and Preserves the Receptor-Binding Ability of mmTRAIL with Minimal Stimulation of PEG-Specific Antibodies Qianxue Nie,# Dianlong Jia,# Hao Yang, Yanru Feng, Qing Fan, Qiuxiao Shi, Lin Wan, and Xiaofeng Lu* Key Lab of Transplant Engineering and Immunology, MOH, West China Hospital, Sichuan University, Chengdu 610041, China S Supporting Information *

ABSTRACT: The poor in vivo potencies of most therapeutic proteins might be attributed to their short serum half-lives. PEGylation is a well-established method and has been clinically proven to improve pharmacokinetics. mmTRAIL exhibited supercytotoxicity in a variety of tumor cells, but its serum half-life was less than 10 min in mice. Here, mmTRAIL5K, mmTRAIL-10K, and mmTRAIL-20K were produced by N-terminus-specific PEGylation of mmTRAIL with 5, 10, or 20 kDa mPEG, respectively. The particle sizes of mmTRAIL5K, mmTRAIL-10K, and mmTRAIL-20K were 9.09 ± 2.76, 12.62 ± 4.05, and 15.68 ± 4.95 nm, which were higher than the threshold (∼7 nm) of renal clearance. Accordingly, mmTRAIL5K exhibited a serum half-life of 30 min only 3 times longer than that of mmTRAIL. However, both mmTRAIL-10K and mmTRAIL-20K exhibited similar serum half-lives ranging from 350 to 400 min, indicating that PEGylation with 10 or 20 kDa mPEG significantly improved the pharmacokinetics of mmTRAIL. However, death receptor binding of mmTRAIL-20K was reduced 5- to 8-fold, resulting in a 3-fold reduction of cytotoxicity. Additionally, repeated administration of mmTRAIL-20K elicited both mPEG-specific IgG and IgM antibody responses in rats. In contrast, the receptor binding and cytotoxicity of mmTRAIL-10K were similar to those of mmTRAIL. Repeated administration of mmTRAIL-10K did not obviously stimulate mPEG-specific antibody responses in rats and rhesus monkeys. Of the three PEGylated mmTRAIL analogues, mmTRAIL-10K exerted the greatest tumor suppression in mice bearing human tumor xenografts. These results demonstrated that conjugation of mmTRAIL to 10 kDa mPEG was better than that to 5 or 20 kDa mPEG for enhancing antitumor effects. KEYWORDS: serum half-life, PEGylation, anti-PEG antibody, cancer targeted therapy, tumor necrosis factor-related apoptosis-inducing ligand

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INTRODUCTION Recombinant proteins are considered to be an important class of medicine that might be used for the treatment of a wide range of diseases. Most small protein drugs exhibit short halflives, which significantly lower their in vivo potencies. In fact, clinical application of these short-acting protein drugs is hampered by frequent dosing requirements, particularly for chronic diseases.1 Extending the circulation time is essential for the development of proteins as therapeutic drugs. Three strategies, including PEGylation, fusion to the IgG1 Fc fragment, and conjugation/fusion/binding to albumin, have been widely used to improve the pharmacokinetics of biological drugs.2−4 Of these strategies, PEGylation is the most established method for half-life extension, and it is the first approach that was proven to be clinically applicable. To date, 12 PEGylated biopharmaceuticals have been approved by the FDA for clinical treatment. Clinical trials for over 20 PEGylated biodrugs are currently ongoing.1,2,5 Each ethylene glycol subunit of polyethylene glycol (PEG) is surrounded by a minimum of 2−3 water molecules, making it exceptionally hydrophilic.6 Consequently, the conjugation to © XXXX American Chemical Society

PEG increases the hydrodynamic diameter, thus extending the circulation time of the protein by reducing the renal elimination rate. PEG might be synthesized as polymers with a wide range of molecular weights. Usually, the serum half-life of PEGylated protein is proportional to the size of the conjugated PEG.7 Nevertheless, the biological activity of the PEGylated protein was usually reduced by the PEG-related steric hindrance that is proportional to the molecular weight of conjugated PEG.7−9 Thus, a balance between the extension of the serum half-life and the reduction in biological activity must be considered for the PEGylation of proteins. In this case, it is necessary to screen PEGs with different molecular weights and identify the one that is optimal for a given protein. In past decades, PEG was widely believed to be inert and nonimmunogenic.10 However, recent works revealed PEGspecific antibodies in animal models and humans that were Received: Revised: Accepted: Published: A

October 25, 2016 December 20, 2016 December 28, 2016 December 28, 2016 DOI: 10.1021/acs.molpharmaceut.6b00964 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

mmTRAIL (∼5.0 mg/mL) was dialyzed against acetate buffer (50 mM, pH 5.0) overnight. Subsequently, sodium cyanoborohydride was added to the mmTRAIL protein solution to obtain a final concentration of 20 mM. mPEG (mPEGbutyraldehyde, Kaizheng Biotech Development Co., Ltd., Beijing, China) of different molecular weights (5, 10, or 20 kDa) was mixed with mmTRAIL followed by incubation at 4 °C with gentle shaking for the appropriate time. PEGylation of mmTRAIL was monitored with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of β-mercaptoethanol (2-ME) or by size exclusion chromatography in the absence of 2-ME according to our previous description.19 The PEGylation conditions, including the molar ratio of mPEG to mmTRAIL (2.5:1, 5:1, or 10:1) and the incubation time (6−24 h), were optimized. To remove the unconjugated mPEG contaminant, PEGylated mmTRAIL was adsorbed by SP-sepharose Fast Flow equilibrated with phosphate buffer (20 mM, pH 6.8), followed by elution with a linear gradient (0−1 M) of NaCl. After verification by sizeexclusion chromatography and SDS-PAGE, the recovered PEGylated mmTRAIL was dialyzed against phosphate-buffered saline (PBS; 137 mM NaCl, 2.68 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4, 20 μM ZnSO4) and stored at −70 °C for further use. mmTRAIL conjugated to 5, 10, or 20 kDa mPEG was designated mmTRAIL-5K, mmTRAIL-10K, and mmTRAIL-20K, respectively. The particle sizes of these PEGylated mmTRAIL analogues were determined using a Zetasizer Nano-ZS (Malvern Instruments, U.K.) and transmission electron microscopy (TEM) according to methods described by Li et al.21 Death Receptor-Binding Assays. The binding kinetics of PEGylated mmTRAIL to death receptors was measured with biolayer interferometry carried out on a BlItz System (Pall ForteBio LLC, CA) according to methods described by Wheatly et al. with some modifications.22 Briefly, all assays were performed at room temperature in PBS. IgG1 Fc-fusion death receptor proteins, including DR4-Fc and DR5-Fc (Sinobiological, Beijing, China), were first immobilized on a protein A-coated biosensor. After a quick wash with PBS, the biosensor tip was inserted into the solution containing PEGylated mmTRAIL at different concentrations for binding assessment prior to disassociation in PBS. The kinetic constants, such as the association constant (ka), dissociation constant (kd) and affinity (KD, KD = kd/ka), were calculated using software according to a 1:1 binding model. In Vitro Cytotoxicity Assays. Colorectal cancer COLO205 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a 5% CO2 humidified atmosphere. Cells (∼2 × 104 cells/well in 100 μL of medium) were seeded onto 96-well plates and cultured overnight, followed by the addition of PEGylated mmTRAIL at different concentrations. PBS was used as a negative control. After treatment at 37 °C overnight, the surviving cells were measured by the addition of CCK-8 solution (5 μL/well) according to methods described by Yang et al.23 The viability of cells treated with PEGylated mmTRAIL analogues was expressed as the percentage relative to the viable PBS-treated cells, which was considered to be 100%. The IC50 values of these proteins were calculated using SPSS 13.0 software according to their respective cell viability curves. To determine the receptor-dependence of the cytotoxicity of PEGylated mmTRAIL, before addition into the cells, the

treated with PEGylated substances, suggesting that PEG is immunogenic. The anti-PEG antibodies might induce accelerated blood clearance (ABC) phenomena that might drastically reduce the in vivo potency of repeatedly administered PEGylated substances.11−14 It was found that the immunogenic character of PEG depends on its molecular structure and molecular weight.10 Interestingly, free PEG only elicited a weak and transitory immune response. However, conjugation to immunogenic proteins enhanced the PEG-specific antibody responses.14,15 In contrast, conjugation to PEG usually reduced the immunogenicity of the protein core.16 These results suggest that the immune response for PEGylated proteins could not be simply predicted by the immunogenicity of the unconjugated PEG and the protein. For a given protein, it is necessary to screen PEGs with different immunogenicities to identify the one that would not stimulate obvious immune responses after conjugation to it. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) plays an important role in antitumor surveillance under physiological conditions. Recombinant human TRAIL (hTRAIL) was considered an attractive antitumor drug candidate due to its ability to induce rapid, potent, and selective apoptosis in a variety of tumor cells at low concentrations (nM). However, hTRAIL is limited by its intrinsic characteristics, such as poor solubility, instability, and short half-life.17 Consequently, a series of hTRAIL variants have been developed in the past decades.18 In our previous work, we found that mmTRAIL derived from rhesus monkey Macaca mulatta differed from hTRAIL at only four positions.19 However, mmTRAIL was more soluble and stable than hTRAIL, resulting in yields of recombinant mmTRAIL that were 2-times higher than those of hTRAIL. Moreover, mmTRAIL and hTRAIL showed similar cytotoxicity in sensitive tumor cells, whereas mmTRAIL induced apoptosis in many resistant tumor cells more strongly than hTRAIL.19 These results suggest that mmTRAIL might be a novel candidate for anticancer drugs. Similar to hTRAIL, mmTRAIL also exhibited a short serum half-life that was less than 10 min in mice. An essential work for increasing the therapeutic potency of mmTRAIL is to improve its pharmacokinetics. In this study, we first modified mmTRAIL by conjugating methoxyPEG (mPEG) with different molecular weights to the N-terminus of mmTRAIL. Subsequently, we investigated the impact of PEGylation on the receptor binding, cytotoxicity, stability, serum half-life, and antitumor effects of mmTRAIL. In particular, we evaluated mPEG-specific IgG and IgM antibody responses elicited by PEGylated mmTRAIL analogues in rats as well as in rhesus monkeys. Finally, we identified the optimal mPEG that preserved the biological activity and significantly prolonged the serum half-life of mmTRAIL but did not significantly stimulate mPEG-specific antibody responses when conjugated to mmTRAIL.

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EXPERIMENTAL SECTION Recombinant Expression and PEGylation of mmTRAIL. The recombinant mmTRAIL was produced using E. coli M15 containing plasmid pQE30 mmTRAIL according to our previous work.19 The endotoxins in protein solutions were removed by using ToxinErase endotoxin removal kit combined with ToxinSensor chromogenic LAL Endotoxin Assay Kit (Genscript, Nanjing, China). N-Terminal site-specific PEGylation of mmTRAIL was performed according to methods described by Chae et al.20 with some modification. Briefly, B

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Figure 1. Preparation and PEGylation of mmTRAIL. (A) SDS-PAGE of mmTRAIL and its PEGylated analogues in the presence of 2-ME. Purified mmTRAIL was conjugated to 5, 10, or 20 kDa mPEG to produce its PEGylated analogues including mmTRAIL-5K, mmTRAIL-10K, and mmTRAIL-20K. The SDS-PAGE gel was stained with Coomassie brilliant blue. M: protein markers. (B) Size-exclusion chromatography of mmTRAIL and its PEGylated analogues. (C) Particle sizes of mmTRAIL and its PEGylated analogues measured using a Zetasizer Nano-ZS. (D) TEM of mmTRAIL-10K counterstained with phosphotungstic acid.

viability was measured by adding CCK8 solution. Moreover, the activation of caspase in cells was determined using caspasespecific substrates provided by the caspase 3, 8, and 9 colorimetric assay kit (Genscript, NJ). In Vivo Pharmacokinetics. For the pharmacokinetics assay, BALB/c mice (4−6 weeks old, n = 3 for each time point) were intravenously injected with PEGylated mmTRAIL analogues at a single dose of 10 mg/kg. The blood samples were collected from periorbital venous sinus at different time points (0.16, 0.5, 1, 2, 4, 8, 16, 24, and 48 h) postinjection. The residual PEGylated mmTRAIL in the plasma was measured using an enzyme-linked immunosorbent assay (ELISA) kit (Boster, Wuhan, China) for human TRAIL. The circulating half-life (t1/2) and area under the curve (AUC) were calculated according to the perspective curve of plasma concentration using DAS software version 2.11.24 To verify the timedependent elimination of PEGylated mmTRAIL, the in vitro cytotoxicity of the residual protein in plasma was detected at different time points after the plasma was diluted 25−1000 times. The plasma derived from mice that did not receive injections of protein was used as a negative control. Immunogenicity Assays. To evaluate the immunogenicity of proteins, Wistar rats (8 weeks old, male, n = 3 in each group) were injected via the tail vein with 10 mg/kg PEGylated mmTRAIL 3−5 times with a week interval. The blood samples were collected via the tail vein at 0.16, 1, 3, 6, 24, 48, 72, 120, and 168 h postinjection. The specific antibodies against PEGylated mmTRAIL or mPEG were detected according to

soluble death receptor protein (DR4-Fc or DR5-Fc, Sino Biological Inc., Beijing, China) was mixed with PEGylated mmTRAIL (10 nM) at different molar ratios (0−5.0) and incubated at room temperature for 30 min. A receptordependent increase in the viability rate of cells treated with the mixture reflects the receptor-dependent cytotoxicity of PEGylated mmTRAIL. Moreover, to investigate the thermal stability, PEGylated mmTRAIL (100 nM in PBS) was preincubated at 37 °C for different times (0−24 h), followed by the addition into cells at a final concentration of 10 nM. A time-dependent increase in cell viability reflects the instability of PEGylated mmTRAIL. Apoptosis Assays. Approximately 5 × 105 COLO205 cells (in 600 μL of medium) were treated with 2.5 nM PEGylated mmTRAIL in a 6-well plate at 37 °C for 2 h, followed by FITCAnnexin V/propidium iodide (PI) dual staining according to the kit manual (Invitrogen, CA). Annexin V+/PI− cells revealed by flow cytometry were considered as the early apoptotic cells. To detect the nucleus condensation and fragmentation in apoptotic cells, 2 × 104 cells (in 100 μL of medium) were treated with 5 nM PEGylated mmTRAIL in a 96-well plate at 37 °C for 1.5−2 h. Subsequently, the cells were stained with Hoechst 33342 followed by observation under a fluorescent microscope. To verify the involvement of caspase pathways in apoptosis, COLO205 cells (2 × 104 cells/well in 50 μL of medium) were preincubated with caspase inhibitors (40 μM) at 37 °C for 2 h, followed by the addition of PEGylated mmTRAIL (5 nM, 50 μL). After treatment overnight, the cell C

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Molecular Pharmaceutics methods described by Shimizu et al.25 with some modification. Briefly, the 96-well immunosorbent plates were coated with PEGylated mmTRAIL or mPEG conjugated to BSA (100 nM, in 100 μL of PBS) at 4 °C overnight. Subsequently, the plate was washed three times with PBS, followed by incubation with 1% BSA at 37 °C for 1 h. The plasma was diluted 100−2500 times with PBS plus 500 mM NaCl added into the wells and incubated at 37 °C for 1 h. Finally, the specific antibodies were measured using horseradish peroxidase (HRP)-labeled secondary antibody against rat IgG (Earthox, CA) or IgM (Abcam, Cambridge, U.K.) combined with tetramethylbenzidine (TMB) as the substrate. The reaction was stopped by the addition of 2 M H2SO4. The absorbance at 450 nm was measured with a microplate reader (Biorad, CA). Moreover, PEGylated mmTRAIL was also intravenously injected into rhesus monkeys (4−5 years old, n = 1 in each group) three times with a week interval. The specific antibodies against PEGylated mmTRAIL or mPEG were detected using antimonkey IgG (Sigma, MO) or antihuman IgM (Zen BioScience, Chengdu, China). The plasma samples derived from the rats and monkeys without injection were used as a negative control. The efficacies of all secondary antibodies were verified using either rat or monkeyderived plasma containing IgG and IgM antibodies. To investigate whether repeated injections of PEGylated mmTRAIL would induce accelerated blood clearance (ABC) phenomena, the plasma samples collected at 0.16, 1, 3, 6, 24, and 48 h after each injection were diluted 50 or 100 times for in vitro cytotoxicity assay. Repeat injection-dependent reductions of cytotoxicity indicate ABC phenomena. Assay of in Vivo Antitumor Effects. BALB/c nude mice (4−6 weeks old) were subcutaneously injected with COLO205 cells (5 × 105 cells/mouse) in the right thigh. Once the tumor grafts were palpable, the mice were randomly divided into different groups (n = 7). To compare the tumor growth suppression of PEGylated mmTRAIL analogues, these mice were intravenously injected with mmTRAIL and mmTRAIL5K, -10K, and -20K at 10 mg/kg on day 6 postinjection. The mice in the control group were injected with the same volume of PBS. Moreover, the tumor growth suppression mediated by 2 or 5 mg/kg mmTRAIL-10K was further compared with those mediated by 10 mg/kg mmTRAIL. Tumor growth was monitored every day by measuring the longitudinal (L) and transverse (W) diameters of the tumor graft. The tumor volume (V) was calculated using the following formula: V = L × W2/2. All mice were sacrificed at the end of the experiment or once the tumor volume exceeded 800 mm3 during the experiment. To verify the in vivo antitumor activity of mmTRAIL and mmTRAIL-10K, tumor grafts were collected 24 h postinjection and immediately sectioned under frozen conditions. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) mix (Promega, WI) combined with DAPI staining was used to identify DNA fragmentation in apoptotic cells in tumor tissues. Statistical Analysis. The one-way analysis of variance (ANOVA) test for multiple comparisons was performed using SPSS software version 13.0. The significance level was defined as P < 0.05. The results were expressed as the mean ± standard deviation (SD).

Figure 2. Death receptor-binding assays for mmTRAIL (A), mmTRAIL-5K (B), mmTRAIL-10K (C), and mmTRAIL-20K (D). The death receptor fusion protein (DR4-Fc or DR5-Fc) was immobilized onto a protein A-coated biosensor followed by incubation with increasing mmTRAIL or its PEGylated analogues. The KD values of each protein for DR4 and DR5 are indicated.

SDS-PAGE in the presence of 2-ME indicated that the apparent molecular weight (MW) of mmTRAIL was approximately 20 kDa, corresponding to the MW of the monomer (Figure 1A). Size-exclusion chromatography in the absence of 2-ME indicated that the apparent MW of mmTRAIL was between 44 and 67 kDa (Figure 1B). Similar to our previous results,19 these results suggested that mmTRAIL formed a trimer in solution. Due to the differences in the pKa values between the α-amino group of the N-terminal residue and ε-amino group of internal lysine residues, the N-terminal site-specific PEGylation of mmTRAIL was achieved under acidic aqueous conditions (pH 5.0). Under monitoring by SDS-PAGE combined with size-exclusion chromatography, the molar ratio of mPEG to mmTRAIL and the reaction time were optimized. Finally, PEGylated mmTRAIL analogues, including mmTRAIL-5K, mmTRAIL-10K, and mmTRAIL-20K, were prepared under the optimized condition (mPEG/mmTRAIL molar ratio of 5:1, reaction time of 12 h at 4 °C) using 5, 10, and 20 kDa mPEG, respectively. As shown in Figure 1A, on the SDS-PAGE gel, PEGylated mmTRAIL analogues produced two bands corre-

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RESULTS Preparation of PEGylated mmTRAIL Analogues. Histagged mmTRAIL produced by E. coli was purified to homogeneity by Ni-NTA agarose affinity chromatography. D

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Figure 3. Impact of PEGylation on the biological characteristics of mmTRAIL. (A,B) In vitro cytotoxicity (A) and stability (B). For the stability assay, the proteins (100 nM) were incubated at 37 °C for different lengths of time followed by cytotoxicity examination. (C,D) In vivo pharmacokinetics. Proteins were intravenously injected into mice at a single dose of 10 mg/kg. Subsequently, the plasma samples were collected at different time points and used for the quantitative assay (C) or cytotoxicity measurements (D) for residual proteins.

IC50s of mmTRAIL-5K (0.53 ± 0.12 nM) and mmTRAIL-10K (0.69 ± 0.06 nM) were comparable to those of mmTRAIL (0.72 ± 0.05 nM). However, the IC50 of mmTRAIL-20K was 1.83 ± 0.01 nM, which was approximately 3 times higher than that of mmTRAIL. These results demonstrate that conjugation to mPEG with an MW of 10 kDa or less did not reduce the cytotoxicity of mmTRAIL. When incubated at 37 °C, mmTRAIL-5K, mmTRAIL-10K, and mmTRAIL-20K slowly lost their cytotoxicity. In particular, the cytotoxicity of mmTRAIL-10K and mmTRAIL-20K was only reduced by approximately 8% and 40%, respectively, within 24 h. However, mmTRAIL lost over 60% of its cytotoxicity within 6 h (Figure 3B). These results demonstrated that PEGylation especially with 10 and 20 kDa mPEG significantly improved the thermal stability of mmTRAIL. In addition, both mmTRAIL-10K and mmTRAIL induced caspase-dependent apoptosis in tumor cells (Figure S1), indicating that PEGylation did not change the action mode of mmTRAIL. Serum Half-Life of PEGylated mmTRAIL Analogues. As shown in Figure 3C, mmTRAIL was eliminated rapidly from the blood with a serum half-life of less than 10 min; however, all PEGylated mmTRAIL analogues were cleared more slowly than mmTRAIL. Although the serum half-life of mmTRAIL-5K was only approximately 30 min, the serum half-lives of mmTRAIL-10K and mmTRAIL-20K were approximately 350−400 min. In addition, the AUC(0‑t) values (μg/L·h) of mmTRAIL-10K and mmTRAIL-20K were 275333 and 300816, compared to 50356 and 11299 measured for mmTRAIL-5K and mmTRAIL. These results demonstrated that conjugation to 10 or 20 kDa mPEG extended the circulation time of mmTRAIL (35−40 times longer). PEGylation-mediated

sponding to the mmTRAIL monomer and the PEGylated mmTRAIL monomer in the presence of 2-ME. However, only a single peak corresponding to the PEGylated mmTRAIL trimer was detected by size-exclusion chromatography in the absence of 2-ME. These results suggested that all the trimers, but not monomers, of mmTRAIL were PEGylated under this condition. Dynamic light scattering demonstrated that the particle sizes of mmTRAIL-5K, mmTRAIL-10K, and mmTRAIL-20K were 9.09 ± 2.76, 12.62 ± 4.05, and 15.68 ± 4.95 nm, compared to 5.85 ± 1.49 nm of that of mmTRAIL (Figure 1C). The particle size of mmTRAIL-10K was verified with TEM (Figure 1D). Death Receptor-Binding Ability of PEGylated mmTRAIL Analogues. As shown in Figure 2, biolayer interferometry analysis demonstrated that all PEGylated mmTRAIL analogues were able to bind to the DR4 and DR5 death receptors. The KD values of mmTRAIL-5K, mmTRAIL10K, and mmTRAIL-20K for DR4 were 3.75 ± 1.58, 2.56 ± 0.59, and 15.7 ± 6.1 nM and for DR5 were 2.61 ± 0.91, 1.12 ± 0.58, and 8.3 ± 2.88 nM, respectively. Compared to the death receptor affinities of mmTRAIL (2.17 ± 0.53 nM for DR4 and 1.77 ± 0.39 nM for DR5), the affinities for DR4 and DR5 were well preserved in mmTRAIL-5K and mmTRAIL-10K. However, the affinities of mmTRAIL-20K for both DR4 (15.7 ± 6.1 nM) and DR5 (8.3 ± 2.88 nM) exhibited approximately a 5- to 8-fold reduction. These results demonstrated that conjugation to mPEG with an MW of 10 kDa or less did not significantly reduce the death receptor-binding ability of mmTRAIL. In Vitro Cytotoxicity and the Stability of PEGylated mmTRAIL Analogues. Figure 3A shows that all PEGylated mmTRAIL analogues were cytotoxic in COLO205 cells. The E

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Figure 4. Detection of IgG antibody in the plasma of rats. (A) Antibody against mmTRAIL elicited by the injection of mmTRAIL. (B) The antibody against mmTRAIL-20K and 20 kDa mPEG elicited by the injection of mmTRAIL-20K. (C) The antibody against mmTRAIL-10K and 10 kDa mPEG elicited by the injection of mmTRAIL-10K. Proteins were repeatedly (5 times for mmTRAIL and 3 times for its PEGylated analogues) injected into rats (n = 3) with a week interval. The IgG antibody against the injected protein or conjugated mPEG in the plasma samples was examined using ELISA. The results are from one of three similar experiments.

prolongation of the serum half-life was further verified by timedependent loss of cytotoxicity assays. As shown in Figure 3D and Figure S2, the cytotoxicity of plasma derived from mice injected with mmTRAIL and mmTRAIL-5K drastically decreased (from over 90% to 10% or below) within several hours postinjection. However, the cytotoxicity of plasma derived from mice injected with mmTRAIL-10K or mmTRAIL-20K was detectable even at 48 h postinjection. These results indicated that conjugation to 10 or 20 kDa, but not 5 kDa, mPEG significantly prolonged the circulation time of mmTRAIL. Immunogenicity of PEGylated mmTRAIL Analogues. As shown in Figure 4A, mmTRAIL was immunogenic in rats. After the initial injection of mmTRAIL, little IgG antibody against mmTRAIL was detected in the plasma. However, after the second injection, the level of mmTRAIL-specific IgG antibody in the plasma drastically increased on day 3, peaked on day 5 and then gradually decreased on day 7 postinjection. The subsequent injections of mmTRAIL steadily elicited high levels of IgG antibody responses. Repeated injection of mmTRAIL only stimulated low levels of IgM antibody production (Figure 5A). Both IgG (Figure 4B) and IgM (Figure 5B) antibodies against mmTRAIL-20K were detectable

in plasma derived from rats with 2 or 3 injections of mmTRAIL-20K. The levels of IgG and IgM antibodies against 20 kDa mPEG and mmTRAIL-20K were comparable, suggesting that antibodies against mmTRAIL-20K were predominantly induced by the conjugated mPEG. Surprisingly, repeated injections of mmTRAIL-10K did not elicit a significant antibody response. Although low levels of IgG (Figure 4C) and IgM (Figure 5C) antibodies against mmTRAIL-10K were detected in the plasma of rats with three injections of mmTRAIL-10K, the IgG and IgM antibodies against 10 kDa mPEG were undetectable during the whole experiment. These results demonstrated that mmTRAIL, but not conjugated 10 kDa mPEG, contributed to the low level antibody response to mmTRAIL-10K in rats. Nevertheless, neither IgG nor IgM antibody against mmTRAIL-10K or 10 kDa mPEG was detected in the plasma of rhesus monkeys repeatedly injected with mmTRAIL-10K (Figure 6A,B). No obvious ABC phenomena for mmTRAIL-10K in the rats (Figure S3) or rhesus monkeys (Figure 6C) repeatedly treated with mmTRAIL-10K were detected by time-dependent loss of cytotoxicity assays. These results suggested that 10 kDa mPEG conjugated to mmTRAIL did not stimulate an obvious antibody response in monkeys. Surprisingly, although F

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Figure 5. Detection of IgM antibody in rats. (A) The antibody against mmTRAIL elicited by the injection of mmTRAIL. (B) The antibody against mmTRAIL-20K and 20 kDa mPEG elicited by the injection of mmTRAIL-20K. (C) The antibody against mmTRAIL-10K and 10 kDa mPEG elicited by the injection of mmTRAIL-10K. Proteins were repeatedly (5 times for mmTRAIL and 3 times for its PEGylated analogues) injected into rats (n = 3) with a week interval. The IgM antibody against the injected protein or conjugated mPEG in the plasma samples was examined using ELISA. The results are from one of three similar experiments.

measuring the apoptotic cells using TUNEL staining. Numerous apoptotic cells were observed in tumor grafts of mice administrated with a single dose of 10 mg/kg mmTRAIL, which was in agreement with the efficacy of mmTRAIL tested in our previous work.19 Definitely, the apoptosis induced by the same amount of mmTRAIL-10K was greater than that induced by mmTRAIL (Figure 7C). These results demonstrated that conjugation to 10 kDa mPEG was more effective than that to 5 or 20 kDa mPEG in enhancing the in vivo antitumor effects of mmTRAIL.

mmTRAIL and mmTRAIL-20K elicited obvious antibody responses in rats, repeated injection did not accelerate the elimination of these proteins (Figure S3), suggesting that the antibodies decreased to a low level when the protein was injected again. In Vivo Antitumor Effects of PEGylated mmTRAIL Analogues. As shown in Figure 7A, tumor grafts in mice treated with 10 mg/kg mmTRAIL or its PEGylated analogues grew much more slowly than those in PBS-treated mice. The tumor growth curve for mice treated with a 10 mg/kg dose of mmTRAIL-5K was similar to that of mmTRAIL-treated mice, indicating that conjugation to 5 kDa mPEG did not enhance the in vivo antitumor effect of mmTRAIL. Compared to mmTRAIL and mmTRAIL-5K, mmTRAIL-20K exerted greater tumor suppression, but the difference was not significant. However, the tumor growth rate in mice treated with 10 mg/kg mmTRAIL-10K was significantly (P < 0.01) lower than that in mice injected with the same amount of mmTRAIL or mmTRAIL-5K. Further comparison revealed that tumor suppression mediated by 5 mg/kg mmTRAIL-10K was greater than that mediated by 10 mg/kg mmTRAIL (Figure 7B). In addition, the difference between mmTRAIL-10K and mmTRAIL in tumor suppression was further verified by

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DISCUSSION mmTRAIL, a variant of hTRAIL, induced apoptosis in different types of tumor cells at nanomolar concentrations in vitro. However, its in vivo antitumor effects are not consistent with its cytotoxicity detected in vitro. We found that the serum half-life of mmTRAIL is less than 10 min in mice, which might contribute to its poor in vivo potency. To enhance the antitumor effects of mmTRAIL, we attempted to extend the serum half-life of mmTRAIL by site-specific PEGylation. Here, we produced mmTRAIL-5K, mmTRAIL-10K, and mmTRAIL20K by conjugating mmTRAIL to 5, 10, and 20 kDa mPEG, respectively. We found that all PEGylated mmTRAIL analogues G

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Figure 6. Detection of antibody against mmTRAIL-10K and mPEG in rhesus monkeys. (A,B) IgG (A) and IgM (B) antibody measurement; 10 mg/ kg mmTRAIL-10K was repeatedly (3 times) injected into rhesus monkeys with a week interval. After each injection, the plasma samples were collected on days 1, 2, 3, 5, and 7. mmTRAIL-10K or 10 kDa mPEG-specific IgG and IgM antibodies in the plasma samples were examined using ELISA. (C) Time-dependent loss of cytotoxicity of mmTRAIL-10K repeatedly injected into rhesus monkeys. After each injection of mmTRAIL-10K at 10 mg/kg, plasma samples of rhesus monkeys were collected at 0.16, 1, 3, 6, 24, and 48 h postinjection. Subsequently, the cytotoxicity of residual mmTRAIL-10K in the plasma samples diluted 50 and 100 times was determined using CCK-8.

achieved by conducting the reductive alkylation of proteins with PEG−aldehydes in a cold acidic environment.26 In fact, the protein amino groups included α-amino group (NH2terminal amine) and ε-amino group (internal lysine residue). The pKa value of the α-amino group (7.6−8.0) is relatively lower than that of the ε-amino group (10.0−10.2). Consequently, the reductive amination reactions under cold acidic conditions exhibit excellent selectivity for NH2-terminalspecific PEGylation. In this experiment, PEGylation of mmTRAIL with a mPEG/mmTRAIL molar ratio of 5:1 was performed at pH 5.0 and 4 °C for 12 h. PEGylation was monitored by SDS-PAGE and size exclusion chromatography. As shown in Figure 1B, size-exclusion chromatography only showed the protein peak corresponding to PEG-conjugated mmTRAIL, indicating that the PEGylation of mmTRAIL was highly efficient under the optimized conditions. In contrast, SDS-PAGE revealed both conjugated and unconjugated mmTRAIL in the PEGylated products (Figure 1B). It is known that mmTRAIL exists as a trimer in solution.19 It is possible that at least one mPEG was conjugated to each mmTRAIL trimer, but not to every individual monomeric

were more stable than mmTRAIL. Conjugation to 5 kDa mPEG did not interfere with the binding of mmTRAIL to the death receptors but had little effect on serum half-life extension. Conjugation to 20 kDa mPEG extended the serum half-life of mmTRAIL 35- to 40-fold but reduced the death receptor binding ability 5- to 8-fold. Interestingly, conjugation to 10 kDa mPEG preserved the death receptor binding ability of mmTRAIL. Its ability to prolong the serum half-life was similar to that of 20 kDa mPEG. In addition, unlike 20 kDa mPEG, conjugation to 10 kDa mPEG did not elicit obvious mPEGspecific IgG and IgM antibody responses in either the rats or monkeys. Of the three PEGylated mmTRAIL analogues, mmTRAIL-10K exerted the greatest tumor growth suppression in mice bearing human tumor xenografts. These results demonstrated that 10 kDa mPEG is more effective than 5 and 20 kDa mPEG for mmTRAIL PEGylation. Monosite directed PEGylation is required for preparing homogeneous mPEG−protein derivatives. Since the fusion of hTRAIL at the N-terminus does not interfere with its receptor binding and receptor activation,21 we decided to modify mmTRAIL by N-terminal amine-specific PEGylation. This was H

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Figure 7. Impact of PEGylation on in vivo antitumor effects of mmTRAIL in mice. (A) Comparison of the antitumor effects of mmTRAIL and its PEGylated analogues. BALB/c nude mice (n = 7) bearing subcutaneous COLO205 xenografts were intravenously injected with 10 mg/kg mmTRAIL, mmTRAIL-5K, mmTRAIL-10K, or mmTRAIL-20K on day 6 postinoculation. The tumor volume was measured every day. (B) Dosedependent antitumor effects of mmTRAIL-10K. BALB/c nude mice (n = 7) with subcutaneous COLO205 xenografts were intravenously injected with 10 mg/kg mmTRAIL or 2 or 5 mg/kg mmTRAIL-10K on day 6 postinoculation. (C) Apoptosis of tumor cells in xenografts. BALB/c nude mice (n = 3) with subcutaneous COLO205 xenografts (200−300 mm3) were injected with a single dose of 10 mg/kg mmTRAIL or mmTRAIL-10K. The same volume of PBS was used as the control. On the second day, the tumor grafts were removed and sectioned under frozen conditions. The apoptotic cells were detected by TUNEL staining. The nuclei of the cells were visualized using DAPI.

higher than the threshold of renal clearance. Although mmTRAIL-10K was smaller than mmTRAIL-20K, both PEGylated mmTRAIL analogues showed comparable serum half-lives of approximately 350−400 min (Figure 3C). These results demonstrated that the degree of serum half-life extension is related to the MW of the conjugated mPEG. However, the steric hindrance is also related to the MW of conjugated mPEG and may reduce the biological activity of the anchoring protein. Actually, receptor binding assays demonstrated that conjugation to 20 kDa mPEG reduced the DR4 and DR5 binding ability of mmTRAIL 5- to 8-fold (Figure 2). Thus, the cytotoxicity of mmTRAIL-20K is approximately 3 times lower than that of mmTRAIL (Figure 3A). However, conjugation to 5 or 10 kDa mPEG did not interfere with the receptor binding (Figure 2) and cytotoxicity (Figure 3A) of mmTRAIL. Considering the balance between serum half-life extension and biological activity reduction, 10 kDa mPEG is better than 5 and 20 kDa mPEG for the modification of mmTRAIL.

mmTRAIL within each trimer. The same phenomenon was observed during PEGylation of hTRAIL.7 It is well-known that rapid renal clearance contributes to the short serum half-life of a protein that is smaller than the kidney filtration cutoff of approximately 70 kDa in MW3 or 7 nm in size.27 PEGylation could increase both the size and hydrodynamic diameter of proteins in a PEG MW-dependent manner. In fact, the MW (∼50 kDa) and size (∼6 nm) of the mmTRAIL trimer is below the threshold of renal clearance. However, the conjugation to 5, 10, and 20 kDa mPEG increased the apparent protein-equivalent MW of PEGylated mmTRAIL to approximately 90, 450, and 580 kDa, respectively, corresponding to 9.09 ± 2.76, 12.62 ± 4.05, and 15.68 ± 4.95 nm in size (Figure 1B,C). Conjugation to 5 kDa mPEG slightly increased the MW and size of mmTRAIL. Consequently, the serum half-life of mmTRAIL was only extended from less than 10 to 30 min in mice (Figure 3C). In contrast, conjugation to either 10 kDa or 20 kDa mPEG increased the MW and size of mmTRAIL to a level that was far I

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from hTRAIL at four positions, it may also exhibit low immunogenicity in humans. Consequently, attention must also be paid to the antibody responses if mmTRAIL-10K is to be repeatedly administered to patients.

In addition to the impact of PEGylation on the pharmacokinetics and biological activity of mmTRAIL, the immunogenicity of conjugated mPEG should also be of concern. Early studies considered PEG to be a biologically inert material with no immunogenicity. Recently, increasingly studies have correlated the PEG-specific antibody responses with the loss of therapeutic efficacy of PEGylated proteins.28,29 It is known that the repeated administration of PEGylated protein might induce anti-PEG IgM or IgG antibodies, which might reduce the therapeutic efficacy of PEGylated proteins by mediating accelerated blood clearance or by neutralizing the activity.29 Only conjugation to 10 or 20 kDa mPEG significantly prolonged the serum half-life of mmTRAIL; therefore, we evaluated the IgM and IgG antibody responses elicited by mmTRAIL-10K and mmTRAIL-20K. Repeated administration of mmTRAIL in rats induced high levels of IgG antibody against mmTRAIL (Figure 4A), indicating that mmTRAIL was immunogenic in rats. Obvious IgG and IgM antibody responses were detected in rats after repeated injections of mmTRAIL-20K. The levels of IgG (Figure 4B) and IgM (Figure 5B) antibody against 20 kDa mPEG were comparable to those against mmTRAIL-20K. In contrast, repeated administration of mmTRAIL-10K stimulated weak IgG and IgM antibody responses for mmTRAIL-10K but not for conjugated mPEG. The mPEG-specific IgG antibody was not detectable. The strength of the IgM antibody response for 10 kDa mPEG was much weaker than that for mmTRAIL-10K. These results demonstrated that the immunogenicity of 10 kDa mPEG in rats was lower than that of 20 kDa mPEG when conjugated to mmTRAIL. Although mmTRAIL, mmTRAIL20K, and mmTRAIL-10K stimulated antibody responses in rats, there was no obvious ABC phenomena or biological activity neutralization according to the time-dependent loss of cytotoxicity assays for proteins that were administered repeatedly (Figure S3). This may have been because the protein was injected on the eighth day after the previous injection, when the antibody had decreased to a low level. It was known that free PEG elicited very weak or no antibody responses, whereas repeated administration of PEG conjugated to immunogenic protein usually induced PEG-specific antibody production in animal.14,15 Consequently, PEG was considered to be a hapten whose immunogenicity was dependent on the immunogenicity of the anchoring protein. Repeated injection of mmTRAIL-10K induced a weak mPEG-specific IgM antibody response that might be attributed to the immunogenicity of mmTRAIL in rats. Because mmTRAIL is theoretically nonimmunogenic in rhesus monkeys, we further evaluated the antibody responses for mmTRAIL-10K in rhesus monkeys. We found that repeated injection of mmTRAIL-10K did not stimulate IgG (Figure 6A) or IgM antibodies (Figure 6B) against either mmTRAIL-10K or 10 kDa mPEG. Accordingly, ABC phenomena were not observed with repeated injections of mmTRAIL-10K in monkeys by the time-dependent loss of cytotoxicity assay (Figure 6C). These results demonstrate that 10 kDa mPEG conjugated to mmTRAIL was not very immunogenic in monkeys. In mice bearing human tumor xenografts, tumor suppression mediated by mmTRAIL-10K was much greater than that mediated by mmTRAIL, mmTRAIL-5K, and mmTRAIL-20K (Figure 7A,B), suggesting that conjugation to 10 kDa mPEG was most effective in enhancing the antitumor effects of mmTRAIL. These results suggest that mmTRAIL-10K might be a novel candidate for cancer therapy. Nevertheless, although mmTRAIL only differs

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CONCLUSIONS Although receptor-binding activity of mmTRAIL was preserved well, serum half-life of mmTRAIL was prolonged slightly by conjugation to 5 kDa mPEG. Conjugation to 20 kDa mPEG significantly prolonged the serum half-life of mmTRAIL but reduced its receptor-binding ability. Conjugation to 10 kDa mPEG significantly extended the serum half-life and preserved the receptor-binding of mmTRAIL. Unlike 20 kDa mPEG, 10 kDa mPEG conjugated to mmTRAIL did not elicit mPEGspecific antibody responses. Finally, 10 kDa mPEG-mmTRAIL conjugate exerted the greatest tumor suppression. For PEGylation of a given protein, screening on PEGs with different MWs might identify the one that prolongs serum halflife and preserves biological activity of protein, but elicits little PEG-specific antibody responses.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.6b00964. Apoptosis induction and time-dependent loss of cytotoxicity of mmTRAIL and its PEGylated analogues (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions #

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Guangleng Liao (Animal center of West China Hospital, Sichuan University) for his assistance in animal administration. This work was supported by the Natural Science Fund of China (81273419, to X.L.) and the National Key Clinical Program (to X.L.).

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REFERENCES

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