electivity of binding of PEGs and PEG-like oligomers to anti-PEG ntibodies induced by methoxyPEG-proteins ark

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 (CH2 CH2 O), 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. Abbreviations: AAALAC, Association for the Assessment and Accreditation of aboratory Animal Care; Albumin, human serum albumin; Anti-PEGs, anti-PEG antiodies; D50, dilution of serum that corresponds to 50% of maximal binding in a irect ELISA; ELISA, enzyme-linked immunosorbent assay; H and L chains, heavy nd light immunoglobulin chains; HO-PEG, hydroxyPEG; IACUC, Institutional Anial Care and Use Committee; IC50, inhibitor concentration that reduces binding to 0% of maximal binding in a competitive ELISA; IFN, interferon; KLH, keyhole impet hemocyanin; mAb, monoclonal antibody; mAU/min, milli-absorbance units er minute; mPEG, monomethoxypoly(ethylene glycol); mPEG-KLH, 5 kDa mPEGLH; NPC, p-nitrophenylcarbonate; PEG, poly(ethylene glycol); PEG20K-KLH, 20 kDa PEG-KLH; pNPCOCl, p-nitrophenylchloroformate; RI, refractive index; RSD, reltive standard deviation = s.d./mean; SOD, porcine Cu–Zn superoxide dismutase; V, ultraviolet absorbance. Crown ether; EtO-TEG, mTEG; n-BuO-TEG, TEG diol and etraEG are defined in Table 1. This is an open-access article distributed under the terms of the Creative Comons Attribution-NonCommercial-No Derivative Works License, which permits on-commercial use, distribution, and reproduction in any medium, provided the riginal author and source are credited. ∗ Corresponding author. Tel.: +1 650 365 5515x224; fax: +1 650 365 5525. E-mail address: sherman@mvpharm.com (M.R. Sherman). 161-5890/$ – see front matter © 2013 The Authors. Published by Elsevier Ltd. All rights ttp://dx.doi.org/10.1016/j.molimm.2013.07.014 © 2013 The Authors. Published by Elsevier Ltd. All rights reserved.