Multi-domain unfolding of the Fab fragment of a humanized anti-cocaine mAb characterized by non-reducing SDS-PAGE.

Monoclonal antibodies and their fragments are widely used for research and therapy. Fab fragments are useful since they retain antigen binding specificity, but being smaller proteins, are better able to penetrate biological compartments and tumors, and do not induce Fc-dependent immunological system activation. Our laboratory developed an anti-cocaine mAb (named h2E2) for the treatment of cocaine use disorders, which is currently in advanced pre-clinical development. We are also interested in the Fab fragment of this mAb for potential therapy of acute cocaine overdose. Previously, we showed that this mAb and its F(ab')2 and Fab fragments undergo discrete domain unfolding, as detected by non-reducing SDS-PAGE, and that ligand-induced protein thermal stabilization can be quantitated utilizing differential scanning fluorimetry in the absence of SDS. Here, we demonstrate that multiple Fab protein gel bands observed using non-reducing SDS-PAGE in the presence and absence of cocaine and its metabolites can be explained and interpreted based on the differential stabilization of two unfolding domains in the Fab fragment. The variable domain is stabilized by ligands against SDS unfolding, while the constant domain is not. This data and its interpretation are also supported by differential scanning fluorimetry data for the Fab fragment in SDS. It is likely that these non-reducing SDS-PAGE results and the gel band domain unfolding model will be applicable to other small molecule binding antibodies. Thus, non-reducing SDS-PAGE is a widely available and simple method for assessing domain stability and multi-step unfolding of Fab fragments.