Role of the Methoxy Group in Immune Responses to mPEG-Protein Conjugates

作者：Merry R. Sherman, L. David Williams, Monika Sobczyk, Shawnya Michaels, Mark G.P. Saifer · 发表于：Bioconjugate Chemistry · 年份：2012 · DOI：10.1021/bc200551b · 被引用次数：184 · 研究领域：Monoclonal and Polyclonal Antibodies Research、Glycosylation and Glycoproteins Research、Nanoparticle-Based Drug Delivery

Anti-PEG antibodies have been reported to mediate the accelerated clearance of PEG-conjugated proteins and liposomes, all of which contain methoxyPEG (mPEG). The goal of this research was to assess the role of the methoxy group in the immune responses to mPEG conjugates and the potential advantages of replacing mPEG with hydroxyPEG (HO-PEG). Rabbits were immunized with mPEG, HO-PEG, or t-butoxyPEG (t-BuO-PEG) conjugates of human serum albumin, human interferon-α, or porcine uricase as adjuvant emulsions. Assay plates for enzyme-linked immunosorbent assays (ELISAs) were coated with mPEG, HO-PEG, or t-BuO-PEG conjugates of the non-cross-reacting protein, porcine superoxide dismutase (SOD). In sera from rabbits immunized with HO-PEG conjugates of interferon-α or uricase, the ratio of titers of anti-PEG antibodies detected on mPEG-SOD over HO-PEG-SOD ("relative titer") had a median of 1.1 (range 0.9-1.5). In contrast, sera from rabbits immunized with mPEG conjugates of three proteins had relative titers with a median of 3.0 (range 1.1-20). Analyses of sera from rabbits immunized with t-BuO-PEG-albumin showed that t-butoxy groups are more immunogenic than methoxy groups. Adding Tween 20 or Tween 80 to buffers used to wash the assay plates, as is often done in ELISAs, greatly reduced the sensitivity of detection of anti-PEG antibodies. Competitive ELISAs revealed that the affinities of antibodies raised against mPEG-uricase were c. 70 times higher for 10 kDa mPEG than for 10 kDa PE...