Effect of protein immunogenicity and PEG size and branching on the anti-PEG immune response to PEGylated proteins
Graphical abstract
Introduction
Polyethylene glycol (PEG) has been successfully used to improve the pharmacological and biological properties of therapeutic proteins [1]. PEGylation prolongs the circulation time of proteins by enhancing their hydrodynamic radii and decreasing the kidney filtration [2]. PEGylation also increases the stability of proteins and decreases the immunogenicity of proteins [3]. Recently, several PEGylated proteins have been approved for clinical use, including Oncaspar (PEG-asparaginase, Ovation, USA) and Krystexxa (PEG–uricase, Savient, USA) [4]. These PEGylated proteins show higher therapeutic efficacy than non-PEGylated counterparts. Thus, PEGylation of proteins has been considered as a milestone breakthrough in the field of therapeutic proteins [5].
PEG has very low toxicity and its simple structure is assumed to be weakly immunogenic. However, the anti-PEG immune response has burdened the development of PEGylated proteins [6], [7]. For instance, Oncaspar and Krystexxa can induce high level of anti-PEG antibodies and lead to unexpected immune-mediated side effects [8], [9]. Anti-PEG antibodies have also been reported to accelerate the clearance and reduce the efficacy of PEGylated proteins [10], [11].
The immune response against the PEG portion of a PEGylated protein was first reported in 1983 [12]. In brief, anti-PEG antibodies in rabbits were raised by immunization with PEGylated ovalbumin (OVA), PEGylated bovine superoxide dismutase, and PEGylated ragweed pollen extract in Freund’s complete adjuvant (FCA). In contrast, the PEGylated proteins elicited weak or undetectable level of anti-PEG antibodies in the absence of FCA [12]. The authors concluded that the anti-PEG immune response depended on the nature of proteins and the degree of modification. however, this conclusion may be interfered by the presence of FCA and needs further demonstration. Sherman et al. assessed the role of methoxy group in the immune responses to methoxy PEG (mPEG) conjugates and the potential advantages of replacing mPEG with hydroxyl–PEG [13]. Saifer et al. found that the clinical use of hydroxyl–PEG–protein conjugates could induce less intense anti-PEG immune responses than the use of mPEG–protein conjugates [14]. Cheng et al. reported that anti-PEG IgM accelerated the clearance of PEGylated proteins [10]. Mima et al. reported that anti-PEG IgM was a major contributor to the accelerated blood clearance of PEGylated proteins [11].
Anti-PEG antibodies are also implicated in the increased clearance of PEGylated liposomes after repeated administration in animal studies [15]. Several studies have shown that anti-PEG antibodies (predominant IgM) are associated with the rapid clearance of subsequent doses of PEGylated liposomes [16]. PEGylated liposomes can activate the complement system and cause hypersensitivity reactions [15], [17]. Extensive studies suggest that anti-PEG IgM is secreted by splenic B cells without the stimulation of T helper cells and the immune memory is thus not induced [18]. This is only the case for PEGylated liposomes, but not for PEGylated proteins.
Anti-PEG immune response to the PEGylated proteins possibly depends on the nature of proteins and the conjugated mPEG. Thus, it is necessary to investigate the effects of protein immunogenicity, the extent of PEGylation, the molecular weight (Mw), and the branching of mPEG on the anti-PEG immune response. In addition, PEGylated proteins with strong anti-PEG immunogenicity are highly preferred to immunize the animals in the absence of adjuvants.
In the present study, OVA, tetanus toxoid (TT), TT–TT conjugate (TT–TT), and TT–bovine serum albumin conjugate (TT–BSA) were of different immunogenicities and acted as target proteins. The PEGylated proteins with different PEGylation extents were obtained by fractionation of the PEGylated TT, using size exclusion chromatography (SEC). The PEGylated proteins with different Mw and branching of mPEG were obtained by modification of TT with linear mPEG (5 kDa and 20 kDa) and branched mPEG (20 kDa). The PEGylated proteins were used to immunize the BALB/c mice in the absence of adjuvants. The immunological characteristics of the PEGylated proteins were then investigated.
Section snippets
Materials
Horseradish peroxidase-conjugated goat anti-mouse IgG Fc antibody (HRP-anti-IgG), IgG1 Fc antibody (HRP-anti-IgG1), IgG2a Fc antibody (HRP-anti-IgG2a), IgG2b Fc antibody (HRP-anti-IgG2b), IgG3 Fc antibody (HRP-anti-IgG3), and IgM Fc antibody (HRP-anti-IgM) were purchased from Abcam (USA). Bovine serum albumin (BSA), OVA, 3-maleimidohexanoic acid N-hydroxysuccinimide ester (EMCS), N-ethylmaleimide (NEM), 3,3′,5,5′-tetramethylbenzidine (TMB), 5,5′-dithio-bis-(2-nitrobenzoic acid), and
Purification of TT–BSA, TT–TT, and the PEGylated proteins
TT–BSA, TT–TT, and TT–L–P5K were fractionated from the reaction mixtures, using a Superdex 200 column (2.6 cm × 60 cm). As shown in Fig. 1a, two partially resolved elution peaks were observed. TT–BSA was fractionated as indicated by the arrow. As shown in Fig. 1b, a major asymmetric elution peak was observed for TT–TT. TT–TT was fractionated as indicated by the arrows. As shown in Fig. 1c, the PEGylated TT (TT–L–P5K) was eluted as a single and broad peak on the column. The broad elution peak
Discussion
The main objective of the present study was to investigate the effect of protein immunogenicity, PEG size and PEG branching on the anti-PEG immune response to the PEGylated proteins. Here, the PEGylated proteins (TT, TT–BSA, and OVA) were used to investigate their anti-PEG immunological properties.
The amines of TT were converted to thiols for preparation of TT–BSA. The thiolated TT was conjugated with maleimide groups of the EMCS–modified BSA. Subsequently, the maleimide mPEG reacted with the
Conclusion
In summary, our result suggested that the anti-PEG immune response to the PEGylated proteins depended on the immunogenicity of proteins, the extent of PEGylation, and the Mw of mPEG. In contrast, the branching of mPEG had an insignificant effect on the anti-PEG immune response to the PEGylated proteins.
Acknowledgments
This study was financially supported by the Beijing Natural Science Foundation (Grant No. 7142104), the National Natural Science Foundation of China (Grant Nos. 20906095 and 81402861), and the STS Project of Chinese Academy of Sciences (Grant Nos. KFJ-EW-STS-027 and KFJ-EW-STS-098).
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