Anti-PEG IgM Is a Major Contributor to the Accelerated Blood

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Anti-PEG IgM is a major contributor to the accelerated blood clearance of polyethylene glycol-conjugated protein Yu Mima, Yosuke Hashimoto, Taro Shimizu, Hiroshi Kiwada, and Tatsuhiro Ishida Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00144 • Publication Date (Web): 12 Jun 2015 Downloaded from http://pubs.acs.org on June 22, 2015

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Molecular Pharmaceutics

Anti-PEG IgM is a major contributor to the accelerated blood clearance of polyethylene glycol-conjugated protein

Yu Mima,1 Yosuke Hashimoto,1 Taro Shimizu,1 Hiroshi Kiwada,1 and Tatsuhiro Ishida,1,* 1, Department of Pharmacokinetics and Biopharmaceutics, Institute of Health Biosciences, Tokushima University, 1-78-1 Sho-machi, Tokushima 770-8505, Japan

*Corresponding author: Tatsuhiro Ishida, Ph.D. Department of Pharmacokinetics and Biopharmaceutics, Institute of Health Biosciences, Tokushima University, 1-78-1, Sho-machi, Tokushima 770-8505 Japan Tel: +81-88-633-7260, Fax: +81-88-633-7259 e-mail: [email protected]

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Table of Contents Graphic

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Abstract

Limited therapeutic efficacy of polyethylene glycol-conjugated (PEGylated) protein drugs has been recently reported in animals and human following repeat injections. Since there are reports that an accelerated blood clearance (ABC)

phenomenon is caused by repeated injection of PEGylated liposome, there is an assumption that PEGylated proteins lose their long circulating property when they are injected repeatedly due to the induction of anti-PEG antibody. Although induction of anti-PEG antibody by PEGylated protein has been reported, there is little evidence of accelerated blood clearance of PEGylated protein upon repeated injection. Herein, we investigated the blood concentration of PEGylated ovalbumin (PEG-OVA), a model PEGylated protein, upon its repeated injection. A single intravenous administration of PEG-OVA elicited an anti-PEG IgM response but not anti-PEG IgG response, while the administration did not elicit antibody against OVA. At 24 hr post-injection of test PEG-OVA, although control mice showed 41.6 %dose of PEG-OVA in blood, the mice pretreated with PEG-OVA showed rapid clearance of test PEG-OVA from blood and undetectable level of PEG-OVA. Interestingly, the anti-PEG IgM induced by PEGylated liposome did not affect the blood concentration of subsequent dose of PEG-OVA. Our result suggests that anti-PEG IgM is a major contributor to the accelerated blood clearance of PEG-conjugated protein, but the presence of anti-PEG IgM in blood circulation does not necessarily affect circulating property of entire PEGylated materials. Keywords

Polyethylene glycol (PEG); PEGylated protein; ABC phenomenon; PEGylated liposome; anti-PEG IgM 3



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Introduction

The conjugation of proteins and nanocarriers with polyethylene glycol (PEG), so-called PEGylation, is currently considered to be one of the most successful techniques that can be used to prolong their half-life in the body 1-4. By modifying proteins with PEG, solubility is increased due to the PEG hydrophilicity, recognition by protease and the immune system is decreased due to PEG-steric hindrance, and glomerular filtration is decreased due to an increased molecular weight 5-7. Several PEGylated protein drugs such as Oncaspar® (PEG-asparaginase), Pegintron® (PEG-interferon α2b), Pegasys® (PEG-interferon α2a), and Neulasta® (PEG-G-CSF) have been approved by the FDA 8, 9. In a similar manner, the PEGylation of nanocarrier systems such as liposomes and micelles attracts a water shell, which results in a reduced adsorption of opsonins and a resultant reduction in the recognition of the nanocarrier by the cells of the mononuclear phagocyte system (MPS) 10, 11. The PEGylated nanocarriers thus obtain long circulation properties, and therefore can accumulate efficiently in solid tumors because of the enhanced permeability and retention (EPR) effect in growing tumors 12, 13. Despite the preceding argument, we and other groups have reported that PEGylated liposome loses its long-circulating properties upon repeat injections (the so-called accelerated blood clearance (ABC) phenomenon) 14-17. In earlier work 18, 19, we determined that anti-PEG IgM, secreted from the spleen in response to a first dose of PEGylated liposome, was responsible for this phenomenon. Following the anti-PEG IgM production, antibody selectively binds to PEG on the second dose of liposome and subsequently activates a complement system, whereby the liposome is taken up by the Kupffer cells in the liver. 4


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In recent studies, PEGylated proteins elicited anti-PEG antibody response including both IgG and IgM following administration 20-23. In addition, repeated injections of PEGylated proteins are reported to have caused a rapid loss of enzymatic activity in rats and humans 24-26. These reports might suggest that PEGylated proteins lose their long-circulating properties as a result of the induction of anti-PEG antibody upon repeat injections. In the present study, therefore, we investigated the blood concentrations of PEG-OVA, a model PEGylated protein, upon repeated injections. First, we evaluated anti-PEG IgM production after a single injection of PEG-OVA. Next, we examined whether the anti-PEG IgM, induced by PEGylated protein, affects the biodistribution of subsequent doses. We found that PEGylated protein induces anti-PEG IgM production, which is responsible for the accelerated blood clearance of subsequent doses of PEGylated protein.

Experimental Section Materials

α-Succinimidyl carbonyl-ω-methoxy, polyoxyethylene (SUNBRIGHT ME-400TS), hydrogenated egg phosphatidylcholine (HEPC), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy (polyethylene glycol)-2000] (mPEG2000-DSPE) were generously donated by NOF (Tokyo, Japan). The cholesterol (Chol) was of analytical grade (Wako Pure Chemical, Osaka, Japan). All lipids were used without further purification. 3H-Cholesterylhexadecyl ether (3H-CHE) was purchased from Perkin Elmer Japan (Yokohama, Japan). PEG2000-OH and PEG20000-OH were purchased from Sigma (MO, USA). The egg-white albumin (OVA) was purchased from Sigma (MO, USA). All other reagents were of analytical grade. 5



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Animals

BALB/c and BALB/c (nu/nu) male mice, 5 weeks old, were purchased from Japan SLC (Shizuoka, Japan). All animal experiments were evaluated and approved by the Animal and Ethics Review Committee of the University of Tokushima. Preparation of PEG-OVA

PEG40000-OVA was prepared by adding a 20 molar excess of SUNBRIGHT ME-400TS to OVA with gentle stirring for 4 hr at room temperature according to a previously reported method with minor modifications 7, 9. To eliminate unreacted PEG, the reaction mixture was applied to cation exchange chromatography using a MacroCap SP (GE Healthcare, Japan). After the column was washed with phosphate buffer (pH 3.1) to eliminate unreacted PEG, 2M NaCl in the phosphate buffer was applied to elute the PEG-OVA. Then, the fraction was dialyzed against PBS (pH 7.4) and concentrated using VIVASPIN-20 (Sartorius Stedim Biotech GmbH, Goettingen, Germany). Preparation of PEGylated liposome

PEGylated liposome, composed of HEPC:Chol:mPEG2000-DSPE=1.85:1.00:0.15 (molar ratio), was prepared as previously described 18. A NICOMP 370 HPL submicron particle analyzer (Particle Sizing System, CA, USA) was used to determine that the mean diameter of the prepared liposome was 96.2 ± 7.6 nm. The concentration of phospholipids was determined via colorimetric assay 27. To follow the biodistribution of a second dose of PEGylated liposome, it was labeled with a trace amount of 3H-CHE (40 µCi/µmol of phospholipids) as a non-exchangeable lipid phase marker. Generation of a monoclonal anti-PEG IgG

A previously demonstrated method 28 was used to generate a monoclonal 6


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anti-PEG IgG (HIK-G11, mouse IgG1κ). The HIK-G11 recognized the backbone of PEG (Supplemental Figure 1). Detection of serum anti-PEG IgM, anti-PEG IgG or anti-OVA IgM

Mice received intravenous injections of either free PEG diol (either PEG2000 or PEG20000 (15, 150 or 1500 mg/kg) or PEGylated products (PEG-OVA (5, 50, 500, 2500 or 5000 µg/kg) and PEGylated liposome (0.1 µmol phospholipids/kg)). Serum samples were collected on day 5 following the injections. Either anti-PEG IgM or anti-OVA IgM in the serum was determined using the ELISA described earlier18. Briefly, each well of a 96-well plate was coated with either 10 nmol of PEG-DSPE (50 µl of ethanol), 1 µg of PEG-OVA, or 1 µg of OVA (100 µl of PBS (pH 7.4)). The coated plate was then washed three times with 250 µl of wash solution (50 mM Tris, 0.14 M NaCl, 0.05% CHAPS). Then, 200 µl of blocking buffer (50 mM Tris, 0.14 M NaCl, 1% BSA) was added followed by incubation for 1 hr at room temperature. After washing three times, 100 µl of serum was added after a 200-fold dilution with blocking buffer. After incubation for 1 hr at room temperature, the plate was washed five times. Then, 100 µl of horseradish peroxidase (HRP)-conjugated antibody (Goat anti-mouse IgM-HRP conjugate or Goat anti-mouse IgM-HRP conjugate 25 ng/ml; Bethyl Laboratories, TX, USA) was added followed by incubation for 1 hr at room temperature. The plate was washed five times, and then incubated for 15-30 min at room temperature followed by the addition of 100 µl of o-phenylene diamine (1 mg/ml, Sigma, MO, USA). The reaction was stopped by adding 100 µl of 2 M H2SO4. Absorbance at 490 nm was measured using a Microplate reader (Sunrise Rainbow RC-R, Tecan, Japan). Quantitation of PEG-OVA concentration in serum

Mice received intravenous injections of PEG-OVA (500 µg/kg) or PEGylated 7



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liposome (0.1 µmol phospholipids/kg). On day 5, PEG-OVA (500 µg protein/kg) was intravenously injected into the mice. Then, serum was collected first at 2 min, and then at 1, 4, 12, and 24 hr following injection. The concentration of PEG-OVA in the serum was measured via our previously developed sandwich ELISA (Supplemental Figure 2) referred to reported method with minor modifications 29. The sandwich ELISA was performed using a 96-well plate coated with 1 µg of monoclonal anti-PEG IgG (HIK-G11) in Tris-buffered saline (pH 8.0). The coated plate was washed three times with 250 µl of wash solution (50 mM Tris, 0.14 M NaCl, 0.5% CHAPS). Then, 200 µl of blocking buffer (50 mM Tris, 0.14 M NaCl, 1% BSA) was added followed by incubation for 1 hr at 37 ℃. After three washes, 100 µl of serum diluted 1,500-fold with blocking buffer was added. After incubation for 1 hr at room temperature, the plate was washed five times. Then, HRP-conjugated HIK-G11 was added and incubated for 1 hr at room temperature. The plate was washed five times, and then it was incubated for 10-20 min at room temperature followed by the addition of 1 mg/ml of o-phenylene diamine. The reaction was stopped by adding 2 M H2SO4. Absorbance at 490 nm was measured using a Microplate reader. Biodistribution of PEGylated liposome

The mice received intravenous injections of either PEG-OVA (500 µg/kg) or PEGylated liposome (0.1 µmol phospholipids/kg). On day 5, 3H-CHE-labeled test PEGylated liposome (5 µmol phospholipids/kg) was intravenously injected into the mice. Then, samples (blood, liver and spleen) were collected at 1 hr post-injection. Radioactivity in the samples was assayed as described previously 30. Mice receiving only 3H-CHE-labeled test PEGylated liposome with no pre-treatment served as the control. 8


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Statistics

All values are expressed as the mean ± S.D. Statistical analysis was performed with a two-tailed unpaired t-test. The level of significance was set at p

Anti-PEG IgM Is a Major Contributor to the Accelerated Blood

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