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

Abstract

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.

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

“…We have described the cocrystal structure of h15-2b Fab in complex with mPEG . We performed structure-guided discovery of high-affinity 15-2b variants based on the structural analysis of the h15-2b Fab mPEG crystal structure.…”

“…We have described the cocrystal structure of h15-2b Fab in complex with mPEG. 18 We performed structure-guided discovery of high-affinity 15-2b variants based on the structural analysis of the h15-2b Fab mPEG crystal structure. W104 H Y (h15-2Y) Fab exhibited mild improvement of mPEG-binding activity compared to parental h15-2b when produced by bacterial expression systems (Figure 1A).…”

“…The structural analysis reveals that h15-2Y Fab exhibits a similar mPEGbinding mode with h15-2b Fab. 18 Moreover, Y104 H forms an additional hydrogen bond with the methoxy group of mPEG compared with parental W104 H , leading to the enhanced mPEG-binding activity of h15-2Y Fab (Figure 1C,D).…”

“…On the other hand, mutations in the framework regions (FR) of an antibody may also play a crucial role in the affinity improvement of hapten-binding antibodies . In our previous study, we determined the binding mechanism of mPEG and the h15-2b antibody, which provided valuable insights to assist the rational design of high-affinity h15-2b antibodies …”

“…17 In our previous study, we determined the binding mechanism of mPEG and the h15-2b antibody, which provided valuable insights to assist the rational design of high-affinity h15-2b antibodies. 18 In this study, we engineer an affinity-improved h15-2b variant for quantitative analysis of lmw and hmw mPEG molecules by anti-mPEG sandwich ELISA and competitive ELISA. Our results suggest that the affinity-enhanced anti-mPEG antibody might provide a useful tool to be easily adopted in laboratories and industries for quantification of a wide range of mPEG reagents.…”