Folding and aggregation of a multi-domain engineered immunotoxin

Engineered immunotoxins with specific targeting mechanisms have potential applications for the treatment of cancer and other diseases; however, their folding behavior is often poorly understood and this presents challenges during process development, manufacturing, and formulation. Folding thermodynamics of an antibody variable domain (V_H/V_L) genetically fused to a biological toxin payload were characterized at pH 6.0 and pH 8.0 in order to assess the relative domain stabilities, along with time scales on which they fold, and the competition between aggregation and folding. The toxin and V_H/V_L domains had considerably different unfolding free energies (ΔG_UNF), leading to a thermodynamically-distinct intermediate species, with the toxin domain unfolded and the V_H/V_L folded. The intermediate is the majority species over a range of denaturant concentrations (∼4–6 M urea; ∼2–4 M guanidine HCl). Thermal unfolding resulted in reversible unfolding of the toxin domain at pH 8, but at pH 6 thermal unfolding was convoluted with aggregation due to irreversible unfolding and aggregation for the V_H/V_L domain. Chemical unfolding of both domains was more easily reversible, provided that the refold was done stepwise, allowing the antibody domain to fold first at intermediate denaturant concentration, as folding of the V_H/V_L domain played a key role in aggregation of this antibody fusion protein.