Anti-HER2 monoclonal antibody-MCC-DM1 conjugate(Shanghai Jiaolian Drug Development Co. Ltd.)

Antibody drug conjugates are emerging as a powerful class of antitumor agents with efficacy across a range of cancers; therefore, understanding the disposition of this class of therapeutic is crucial. Reported here is a method of enriching a specific organelle (lysosome) to understand the catabolism of an anti-CD70 Ab-MCC-DM1, an antibody drug conjugate with a noncleavable linker. With such techniques a higher degree of concentration-activity relationship can be established for in vitro cell lines; this can aid in understanding the resultant catabolite concentrations necessary to exert activity.

Transamidation of α-synuclein by the Ca(2+)-dependent enzyme tissue transglutaminase (tTG, EC 2.3.2.13) is implicated in Parkinson's disease (PD). tTG may therefore offer a novel therapeutic target to intervene in PD. Here we first evaluated the potency and efficacy of three recently developed irreversible active-site inhibitors of tTG (B003, Z006 and KCC009) to inhibit tTG activity in vitro and in living cells. In vitro, all compounds were found to be full inhibitors of tTG activity showing a rank order of potency (defined by IC-50 values) of Z006>B003>KCC009. Upon Ca(2+) ionophore (A23187) induced activation of cellular tTG (measured by incorporation of the tTG-specific amine substrate 5-(biotinamido)pentylamine (BAP) into cellular proteins) in neuroblastoma SH-SY5Y cells, only Z006 (0.3-30 μM) retained the capacity to completely inhibit tTG activity. Under these conditions B003 (3-300 μM) only partially blocked tTG activity whereas KCC009 (3-100 μM) failed to affect tTG activity at any of the concentrations used. Z006 (30 μM) also blocked the tTG mediated incorporation of BAP into α-synuclein monomers and SDS-resistant multimers in vitro and in α-synuclein overexpressing SHSY5Y cells exposed to A23187 or the PD mimetic 1-methyl-4-phenylpyridine (MPP(+)). Moreover, Z006 (30 μM) substantially reduced formation of SDS-resistant α-synuclein multimers in SH-SY5Y cells exposed to A23187 or MPP(+) in the absence of BAP. We conclude that α-synuclein is a cellular substrate for tTG under conditions mimicking PD and blockade of tTG activity counteracts α-synuclein transamidation and aggregation in vitro and in living cells. Moreover, our cell model appears an excellent readout to identify candidate inhibitors of intracellular tTG.