bc200551b_si_001.pdf (282.03 kB)
Role of the Methoxy Group in Immune Responses to mPEG-Protein Conjugates
journal contribution
posted on 2015-12-16, 21:11 authored by Merry R. Sherman, L. David Williams, Monika A. Sobczyk, Shawnya
J. Michaels, Mark G. P. SaiferAnti-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.