Efficient spontaneous site-selective cysteine-mediated toxin attachment within a structural loop of antibodies.

Site-specific coupling of toxin entities to antibodies has become a popular method of synthesis of antibody-drug conjugates (ADCs), as it leads to a homogenous product and allows a free choice of a convenient site for conjugation. We introduced a short motif, containing a single cysteine surrounded by aromatic residues, into the N-terminal FG-loop of the C H 2 domain of two model antibodies, cetuximab and trastuzumab. The extent of conjugation with toxic payload was examined with hydrophobic interaction chromatography and mass spectrometry and the activity of resulting conjugates was tested on antigen-overexpressing cell lines. Antibody mutants were amenable for rapid coupling with maleimide-based linker endowed toxin payload and the modifications did not impair their reactivity with target cell lines or negatively impact their biophysical properties. Without any previous reduction, up to 50% of the antibody preparation was found to be coupled with two toxins per molecule. After the isolation of this fraction with preparative hydrophobic interaction chromatography, the ADC could elicit a potent cytotoxic effect on the target cell lines. By fine-tuning the microenvironment of the reactive cysteine residue, this strategy offers a simplified protocol for production of site-selectively coupled ADCs. Our unique approach allows the generation of therapeutic ADCs with controlled chemical composition, which facilitates the optimization of their pharmacological activity. This strategy for directional coupling could in the future simplify the construction of ADCs with double payloads ("dual warheads") introduced with orthogonal techniques. [Display omitted] • A structural loop in C H 2 antibody domain is used for site-selective toxin coupling. • Grafted pi-clamp motif enables rapid efficient conjugation of maleimide linker-MMAE. • Resulting antibody-drug conjugates are of good biophysical properties. • Such derivatives of cetuximab and trastuzumab are highly cytotoxic for target cells. [ABSTRACT FROM AUTHOR]