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

• Published in: Molecular immunology Vol. 57; no. 2; pp. 236 - 246
• Main Authors: Saifer, Mark G.P., Williams, L. David, Sobczyk, Monika A., Michaels, Shawnya J., Sherman, Merry R.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.02.2014
• Subjects: Animals; Anti-PEG; Antibodies, Anti-Idiotypic - blood; Antibodies, Anti-Idiotypic - immunology; Antibodies, Monoclonal - analysis; Antibodies, Monoclonal - immunology; Antibody Affinity - immunology; Backbone-specific; Humans; Methoxy-specific; Monoclonal; Poly(ethylene glycol); Polyclonal; Polyethylene Glycols - chemistry; Polyethylene Glycols - metabolism; Proteins - immunology; Rabbits; NPC; Backbone-specific; KLH; Anti-PEG; IFN-α; mPEG-KLH; mAU/min; IC50; mPEG; Poly(ethylene glycol); Methoxy-specific; Polyclonal; IACUC; Monoclonal; D50; UV; RSD; EtO-TEG; HO-PEG; Albumin; SOD; n-BuO-TEG; Anti-PEGs; H and L chains; mAb; AAALAC; PEG; RI; PEG20K-KLH; pNPCOCl; ELISA; Institutional Animal Care and Use Committee; hydroxyPEG; ultraviolet absorbance. Crown ether; inhibitor concentration that reduces binding to 50% of maximal binding in a competitive ELISA; anti-PEG antibodies; IC; relative standard deviation=s.d./mean; p-nitrophenylchloroformate; p-nitrophenylcarbonate; milli-absorbance units per minute; TEG diol and TetraEG are defined in Table 1; Association for the Assessment and Accreditation of Laboratory Animal Care; 5kDa mPEG-KLH; human serum albumin; keyhole limpet hemocyanin; D; interferon-α; heavy and light immunoglobulin chains; mTEG; porcine Cu–Zn superoxide dismutase; 20kDa mPEG-KLH; monomethoxypoly(ethylene glycol); enzyme-linked immunosorbent assay; refractive index; monoclonal antibody; dilution of serum that corresponds to 50% of maximal binding in a direct ELISA
• ISSN: 0161-5890; 1872-9142
• DOI: 10.1016/j.molimm.2013.07.014

•Monoclonal and polyclonal anti-PEG antibody affinities were measured by ELISAs.•Anti-PEGs induced by mPEG-proteins can be “methoxy-specific” or “backbone-specific”.•For “backbone-specific” anti-PEGs, backbone length dominates the binding affinities.•For “methoxy-specific” anti-PEGs, end-group hydrophobicity dominates the affinities.•HydroxyPEG-proteins are less immunoreactive than analogous 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 10kDa mPEG, 10kDa PEG diol and six linear or cyclic oligomers of oxyethylene (CH2CH2O), with molecular weights of ca. 150–264Da. 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.