Expanding the Scope of Antibody Rebridging with New Pyridazinedione–TCO Constructs

Abstract

Disulfide rebridging methods have recently emerged as a route to hinge region-specific antibody modification, and there exist numerous examples of successful rebridging chemistry applied to clinically relevant human IgG1 antibodies. Here, dibromopyridazinedione disulfide rebridging is adapted to fast trans-cyclooctene/tetrazine (TCO/Tz) bioorthogonal ligations and extended beyond therapeutic human IgG1 antibodies for the first time to include mouse and rat monoclonal antibodies integral to multiplexed analytical diagnostics. In spite of a common architecture, only a subset of antibody host species and IgG isotype subclasses can be rebridged, highlighting the intricate relationship between hinge region sequence, structure, biological activity, and the conjugation chemistry of IgG antibodies.

Leucine-rich alpha-2-glycoprotein 1 (LRG1) as a novel ADC target

“…This method was selected in view of their favourable properties in terms of reproducibility, homogeneity, serum stability and exemplification in vitro and in vivo. 32,[36][37][38][39] We demonstrate that this ADC retains binding post-modification, is stable in serum and effective in in vitro cell studies. We developed a human LRG1 (hLRG1)-expressing B16F0 mouse melanoma cell line and demonstrate that Magacizumab binds to LRG1 and localises to the tumour site.…”

Site-selective modification strategies in antibody–drug conjugates

“…This PD-TCO reagent was successfully conjugated to three therapeutically relevant IgG1 antibodies (trastuzumab, cetuximab and rituximab) and were further functionalised with tetrazine-containing fluorophores. 143 All three conjugates were highly homogenous and could be recovered in high yields. Notably even rituximab, which is known to exhibit 490% protein loss after TCO modification via traditional NHS-ester chemistry, could be recovered in 83% yield after PD-TCO modification.…”

A General Strategy to Design Highly Fluorogenic Far‐Red and Near‐Infrared Tetrazine Bioorthogonal Probes

“…[1] It is being used to activate drugs in as pecific manner, [2] noninvasively image immune responses [3] and guide the design of antibody-drug conjugates. [4] Te trazine-based fluorogenic probes have been developed to visualize specific biomolecules with high spatio-temporal resolution in live cells, [5] which may make them capable of delineating key structures during image-guided surgery. [6] Ther ationale behind using tetrazine bioorthogonal chemistry to generate fluorogenic probes is that the probe is prepared as an on-or weakly fluorescent precursor that undergoes ab ioorthogonal reaction to become strongly fluorescent.…”