Selectivity of binding of PEGs and PEG-like oligomers to anti-PEG antibodies induced by methoxyPEG-proteins.

The use of methoxypoly(ethylene glycol) (mPEG) in PEG conjugates of proteins and non-protein therapeutic agents has led to the recognition that the polymer components of such conjugates can induce anti-PEG antibodies (anti-PEGs) that may accelerate the clearance and reduce the efficacy of the conjugates. Others have classified anti-PEGs as "methoxy-specific" or "backbone-specific". The results of our previous research on anti-PEGs in the sera of rabbits immunized with mPEG or hydroxyPEG (HO-PEG) conjugates of three unrelated proteins were consistent with that classification (Sherman, M.R., et al., 2012. Bioconjug. Chem. 23, 485-499). Enzyme-linked immunosorbent assays (ELISAs) were performed on rabbit antisera and rabbit monoclonal anti-PEGs with competitors including 10 kDa mPEG, 10 kDa PEG diol and six linear or cyclic oligomers of oxyethylene (CH2CH2O), with molecular weights of ca. 150-264 Da. Our results demonstrate that (1) the binding affinities of anti-mPEGs depend more on the backbone lengths of the polymers and the hydrophobicities of their end-groups than on their resemblance to the methoxy terminus of the immunogenic polymer; (2) anti-PEGs raised against HO-PEG-proteins are not directed against the terminal hydroxy group, but against the backbone; (3) rabbit anti-PEGs bind to and distinguish among PEG-like oligomers with as few as three oxyethylene groups; and (4) none of the monoclonal or polyclonal anti-PEGs was absolutely "methoxy-specific" or "backbone-specific", but displayed distinct relative selectivities. If these results are relevant to human immune responses, the clinical use of stable conjugates of HO-PEG with proteins and non-protein therapeutic agents would be expected to produce fewer and less intense immune responses than those induced by conjugates with mPEG or PEGs with larger alkoxy groups.

Role of the methoxy group in immune responses to mPEG-protein conjugates.

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 PEG diol and that anti-PEG antibodies raised against mPEG conjugates of three proteins had >1000 times higher affinities for albumin conjugates with c. 20 mPEGs than for analogous HO-PEG-albumin conjugates. Overall, these results are consistent with the hypothesis that antibodies with high affinity for methoxy groups contribute to the loss of efficacy of mPEG conjugates, especially if multiply-PEGylated. Using monofunctionally activated HO-PEG instead of mPEG in preparing conjugates for clinical use might decrease this undesirable effect.