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Antibody-drug conjugate (ADC) is a class of targeted cancer therapies that combine the advantages of monoclonal antibody (mAb)'s specific targeting and chemotherapy's potent cytotoxicity. The therapeutic effect of ADC is significantly affected by its bioproduction process. This study aims to develop an effective ADC production process using anti-HER2 mAb-drug as a model therapeutic. First, a high titer (>2 g/L) of mAb was produced by Chinese hamster ovary cells from fed-batch cell culture. Both live-cell confocal microscopy imaging and flow cytometry analysis demonstrated that the produced mAb and ADC had strong and specific binding to HER2+ cell line BT474. Second, various conjugation conditions of mAb and drug, including linker selection, ratio of drug and mAb, and conjugation approaches, were investigated to improve the production yield and product quality. Finally, the ADC structure and biological quality were evaluated by SDS-PAGE and anti-breast cancer toxicity study, respectively. The ADC with integral molecular structure and high cytotoxicity (IC50 of 1.95 nM) was produced using the optimized production process. The robust bioproduction process could guide the development of ADC-based biopharmaceuticals.

Cardiac glycosides have been used for the treatment of heart failure because of their capabilities of inhibiting Na+/K+ ATPase (NKA), which raises [Na+]i and attenuates Ca2+ extrusion via the Na+/Ca2+ exchanger (NCX), causing [Ca2+]i elevation. The resulting [Ca2+]i accumulation further enhances Ca2+-induced Ca2+ release, generating the positive inotropic effect. However, cardiac glycosides have some toxic and side effects such as arrhythmogenesis, confining their extensive clinical applications. The mechanisms underlying the proarrhythmic effect of glycosides are not fully understood. Here we investigated the mechanisms by which glycosides could cause cardiac arrhythmias via impairing mitochondrial energetics using an integrative computational cardiomyocyte model. In the simulations, the effect of glycosides was mimicked by blocking NKA activity. Results showed that inhibiting NKA not only impaired mitochondrial Ca2+ retention (thus suppressed reactive oxygen species (ROS) scavenging) but also enhanced oxidative phosphorylation (thus increased ROS production) during the transition of increasing workload, causing oxidative stress. Moreover, concurrent blocking of mitochondrial Na+/Ca2+ exchanger, but not enhancing of Ca2+ uniporter, alleviated the adverse effects of NKA inhibition. Intriguingly, NKA inhibition elicited Ca2+ transient and action potential alternans under more stressed conditions such as severe ATP depletion, augmenting its proarrhythmic effect. This computational study provides new insights into the mechanisms underlying cardiac glycoside-induced arrhythmogenesis. The findings suggest that targeting both ion handling and mitochondria could be a very promising strategy to develop new glycoside-based therapies in the treatment of heart failure.