Synthesis of disulfide-bridging trehalose polymers for antibody and Fab conjugation using a bis-sulfone ATRP initiator

Abstract

Antibodies and antigen binding fragments (Fabs) are widely used as therapeutics and conjugated polymers can enhance the properties of these important biomolecules. However, limitations to the selectivity and stability of current conjugation methodologies can inhibit the exploration of new antibody–polymer conjugates. Herein, we describe a new strategy for the synthesis of these conjugates that forms a stable thioether bond and can be directly incorporated into an atom transfer radical polymerization (ATRP) initiator. Specifically, a bis-sulfone alkyl bromide initiator was synthesized and utilized in the activators generated by electron transfer (AGET) ATRP of ethylene glycol methacrylate and trehalose methacrylate to form the respective polymers. The trehalose polymer was then irreversibly inserted into the disulfide bonds of Herceptin and Herceptin Fab after mild reduction to form the conjugates with quantitative conversions as verified by Western Blot and mass spectrometry after cleavage of the polymer. The binding of the Herceptin and Herceptin Fab conjugates to the receptor was investigated by indirect ELISA (enzyme-linked immunosorbent assay) and the EC50s were 0.90 and 2.74 nM, respectively, compared to Herceptin (0.26 nM) and the Fab (0.56 nM). The conjugates were subjected to heating studies at a constant 75 °C, the temperature determined in a heat ramp to be the threshold of stability for the antibody and FAB; the trehalose polymer was found to considerably increase the thermal stability of both Herceptin and Herceptin Fab. This work provides a new way to prepare polymer–antibody/Fab conjugates utilizing bis-sulfone end groups installed by atom transfer radical polymerization of the functionalized initiators and a way to stabilize these important molecules by conjugation to trehalose polymers.