Antibody-drug conjugates (ADC) are emerging as powerful cancer treatments that combine antibody-mediated tumor targeting with the potent cytotoxic activity of toxins. We recently reported the development of a novel ADC that delivers the cytotoxic payload monomethyl auristatin E (MMAE) to tumor cells expressing tissue factor (TF). By carefully selecting a TFspecific antibody that interferes with TF:FVIIa-dependent intracellular signaling, but not with the procoagulant activity of TF, an ADC was developed (TF-011-MMAE/HuMax-TF-ADC) that efficiently kills tumor cells, with an acceptable toxicology profile. To gain more insight in the efficacy of TF-directed ADC treatment, we compared the internalization characteristics and intracellular routing of TF with the EGFR and HER2. Both in absence and presence of antibody, TF demonstrated more efficient internalization, lysosomal targeting, and degradation than EGFR and HER2. By conjugating TF, EGFR, and HER2-specific antibodies with duostatin-3, a toxin that induces potent cytotoxicity upon antibody-mediated internalization but lacks the ability to induce bystander killing, we were able to compare cytotoxicity of ADCs with different tumor specificities. TF-ADC demonstrated effective killing against tumor cell lines with variable levels of target expression. In xenograft models, TF-ADC was relatively potent in reducing tumor growth compared with EGFR-and HER2-ADCs. We hypothesize that the constant turnover of TF on tumor cells makes this protein specifically suitable for an ADC approach.

The high reaction rate of the 'click-to-release' reaction between allylic substituted trans-cyclooctene and tetrazine has enabled exceptional control over chemical and biological processes. Here we report the development of a new bioorthogonal cleavage reaction based on trans-cyclooctene and tetrazine with up to 3 orders of magnitude higher reactivity compared to the parent reaction, and 4 to 6 orders higher than other cleavage reactions. In this new pyridazine elimination mechanism, wherein the roles a reversed, a trans-cyclooctene activator reacts with a tetrazine that is substituted with a methylene-linked carbamate, leading to an 1,4-elimination of the carbamate and liberation of an amine. Through a series of mechanistic studies, we identified the 2,5-dihydropyridazine tautomer as the releasing species and found factors that govern its formation and subsequent fragmentation. The bioorthogonal utility was demonstrated by the selective cleavage of a tetrazine-linked antibody-drug conjugate by trans-cyclooctenes, affording efficient drug liberation in plasma and cell culture. Finally, the parent and the new reaction were compared at low concentration, showing that the use of a highly reactive trans-cyclooctene as activator leads to a complete reaction with antibody-drug conjugate in seconds vs. hours for the parent system. We believe that this new reaction may allow markedly reduced click-to-release reagent doses in vitro and in vivo and could expand the application scope to conditions wherein the trans-cyclooctene has limited stability. File list (2) download file view on ChemRxiv 20191217_Click-to-Release from Tetrazine.pdf (1.66 MiB) download file view on ChemRxiv 20191217_Click-to-Release from Tetrazine_SI.pdf (12.72 MiB)

The emanations of electronic and mechanical devices have raised serious privacy concerns. It proves possible for an attacker to recover the keystrokes by acoustic signal emanations. Most existing malicious applications adopt contextbased approaches, which assume that the typed texts are potentially correlated. Those approaches often incur a high cost during the context learning stage, and can be limited by randomly typed contents (e.g., passwords). Also, context correlations can increase the risk of successive false recognition. We present a context-free and geometry-based approach to recover keystrokes. Using off-the-shelf smartphones to record acoustic emanations from keystrokes, this design estimates keystrokes' physical positions based on the Time Difference of Arrival (TDoA) method. We conduct extensive experiments and the results show that more than 72.2% of keystrokes can be successfully recovered.

Inner ear cochlear spiral ganglion neurons (SGNs) transmit sound information to the brainstem. Recent single cell RNA-Seq studies have revealed heterogeneities within SGNs. Nonetheless, much remains unknown about the transcriptome of SGNs, especially which genes are specifically expressed in SGNs. To address these questions, we needed a deeper and broader gene coverage than that in previous studies. We performed bulk RNA-Seq on mouse SGNs at five ages, and on two reference cell types (hair cells and glia). Their transcriptome comparison identified genes previously unknown to be specifically expressed in SGNs. To validate our dataset and provide useful genetic tools for this research field, we generated two knockin mouse strains: Scrt2-P2A-tdTomato and Celf4-3xHA-P2A-iCreER-T2A-EGFP. Our comprehensive analysis confirmed the SGN-selective expression of the candidate genes, testifying to the quality of our transcriptome data. These two mouse strains can be used to temporally label SGNs or to sort them.