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

Abstract

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.

The Mystery of Antibodies Against Polyethylene Glycol (PEG) - What do we Know?

“…The competition efficiency of the different PEG species on a molar basis increased with polymer length and with the presence of terminal methoxy groups. Saifer (40) recently tested the specificity of various monoclonal and polyclonal anti-PEG antibodies raised against PEG polymers with or without terminal methoxy groups. The authors demonstrated that antibodies against PEG polymers were never truly specific for methoxy PEGs or ethoxy PEGs, but recognized PEG polymers of different sizes with and without terminal Example of a subject who showed persistent positive results for anti-PEG IgG antibodies which were high enough in titer to be confirmed for specificity.…”

“…The anti-PEG-B-47 antibody recognizes PEG polymers of different lengths with and without terminal methoxy groups (40). Details of the monoclonal antibody and the study design are outlined in Online Supplement 3.…”

“…To obtain further information on the safety of anti-PEG antibodies, we asked if anti-PEG antibodies bind to any human tissue. For this purpose, we used a high-affinity anti-PEG antibody which recognizes PEG polymers of different lengths with and without terminal methoxy groups (40). We tested this antibody against a panel of 36 different human tissues (Table V) using an optimized immunohistochemistry protocol and included tissue spiked with a PEGylated protein as positive control.…”

Anti-PEG IgM Is a Major Contributor to the Accelerated Blood Clearance of Polyethylene Glycol-Conjugated Protein

“…This suggests that an induced anti-PEG IgM will recognize only a PEG moiety. Recently, Sherman et al proposed a methodology to classify anti-PEG 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 13 antibodies into the following categories: "terminal methoxy-specific" or "backbone-specific" 21, 38 . They showed that methoxy-specific anti-PEG antibodies bind preferentially to methoxy-PEG rather than to the PEG diol, but backbone-specific anti-PEG antibodies show no apparent differences in the affinity for either methoxy-PEG or the PEG diol.…”

Development of a Generic Anti-PEG Antibody Assay Using BioScale’s Acoustic Membrane MicroParticle Technology

“…For this particular format, Casein in PBS from Thermo Fisher Scientific provided the lowest signal for the (14,15) previously reported on the detrimental effects of such surfactants on the sensitivity of detection for anti-PEG antibodies. Since our positive control antibody is PEG backbone unit specific, the interference was expected.…”