Engineering a High-Affinity Anti-Methoxy Poly(ethylene glycol) (mPEG) Antibody for Sensitive Immunosensing of mPEGylated Therapeutics and mPEG Molecules.

Sensitive quantification of methoxy poly(ethylene glycol) (mPEG)-conjugated therapeutics for pharmacokinetic determination is critical for mPEGylated drug development. However, sensitive measurement of low-molecular-weight (lmw) mPEG compounds remains challenging due to epitope competition between backbone-specific anti-PEG antibodies. Here, we engineered a high-affinity methoxy-specific anti-mPEG antibody for sensitive quantification of free mPEG molecules and mPEGylated therapeutics. The affinity-enhanced h15-2Y antibody variant shows a 10.3-fold increase in mPEG-binding activity compared to parental h15-2b. h15-2Y-based sandwich ELISA can effectively quantify lmw mPEG5K and high-molecular-weight (hmw) mPEG20K at concentrations as low as 3.4 and 5.1 ng mL-1, respectively. Moreover, lmw mPEG compounds (560, 750, 1000, and 2000 Da) can be efficiently quantified via h15-2Y-based competitive ELISA with detection limits at nanomolar levels. This study provides a promising approach for application in the quantitative analysis of the various sizes of mPEG molecules to accelerate the timeline of mPEG-conjugated drug development.

Structural determination of an antibody that specifically recognizes polyethylene glycol with a terminal methoxy group.

Covalent attachment of methoxy poly(ethylene) glycol (mPEG) to therapeutic molecules is widely employed to improve their systemic circulation time and therapeutic efficacy. mPEG, however, can induce anti-PEG antibodies that negatively impact drug therapeutic effects. However, the underlying mechanism for specific binding of antibodies to mPEG remains unclear. Here, we determined the first co-crystal structure of the humanized 15-2b anti-mPEG antibody in complex with mPEG, which possesses a deep pocket in the antigen-binding site to accommodate the mPEG polymer. Structural and mutational analyses revealed that mPEG binds to h15-2b via Van der Waals and hydrogen bond interactions, whereas the methoxy group of mPEG is stabilized in a hydrophobic environment between the VH:VL interface. Replacement of the heavy chain hydrophobic V37 residue with a neutral polar serine or threonine residue offers additional hydrogen bond interactions with methoxyl and hydroxyl groups, resulting in cross-reactivity to mPEG and OH-PEG. Our findings provide insights into understanding mPEG-binding specificity and antigenicity of anti-mPEG antibodies.