Exatecan, DXd, Enhertu and trastuzumab-LP5 induce S phase and G2/M arrest. Cell cycle arrest induced by exatecan, DXd, trastuzumab-LP5 DAR 8 and Enhertu. HER2-positive cells were treated for indicated time points and concentrations of respective free payloads and ADCs. Afterwards, permeabilized cells were stained with propidium iodide (PI) and analyzed by flow cytometry. (A) Flow cytometric histogram of PI-stained untreated SKBR-3 cells or cells treated for 48 hours with 20 nM or 0.2 nM trastuzumab-LP5 DAR 8 ADC, showing concentration-dependent induction of S phase or G2/M arrest. (B) SKBR-3 cells were treated for 48 hours with indicated concentrations of free exatecan or trastuzumab-LP5 DAR 8 ADC. (C) HER2-positive SKBR-3 and N-87 cells were treated for indicated time points with 20 nM of by exatecan, DXd, trastuzumab-LP5 DAR 8 and Enhertu. Bar graphs show % of cells in S, G2/M G0/G1 and sub G0 (apoptotic) phase. Graphs show means of n = 2 ± SEM.
ARTICLE ABSTRACTTopoisomerase I (TOP1) Inhibitors constitute an emerging payload class to engineer antibody–drug conjugates (ADC) as next-generation biopharmaceutical for cancer treatment. Existing ADCs are using camptothecin payloads with lower potency and suffer from limited stability in circulation. With this study, we introduce a novel camptothecin-based linker–payload platform based on the highly potent camptothecin derivative exatecan. First, we describe general challenges that arise from the hydrophobic combination of exatecan and established dipeptidyl p-aminobenzyl-carbamate (PAB) cleavage sites such as reduced antibody conjugation yields and ADC aggregation. After evaluating several linker–payload structures, we identified ethynyl-phosphonamidates in combination with a discrete PEG24 chain to compensate for the hydrophobic PAB–exatecan moiety. Furthermore, we demonstrate that the identified linker–payload structure enables the construction of highly loaded DAR8 ADCs with excellent solubility properties. Head-to-head comparison with Enhertu, an approved camptothecin-based ADC, revealed improved target-mediated killing of tumor cells, excellent bystander killing, drastically improved linker stability in vitro and in vivo and superior in vivo efficacy over four tested dose levels in a xenograft model. Moreover, we show that ADCs based on the novel exatecan linker–payload platform exhibit antibody-like pharmacokinetic properties, even when the ADCs are highly loaded with eight drug molecules per antibody. This ADC platform constitutes a new and general solution to deliver TOP1 inhibitors with highest efficiency to the site of the tumor, independent of the antibody and its target, and is thereby broadly applicable to various cancer indications.