Abstract 211: A novel FRET assay for the intracellular activation of ADC linkers

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Evaluating the intracellular activation of ADC by a novel FRET assay. Despite the recent success of ADCs as cancer therapeutics, their mechanisms of action are not fully understood. In this study, we developed ADCs using a novel fluorescence resonance energy transfer (FRET) linker in order to facilitate monitoring the details of intracellular uptake, vesicular trafficking and payload release. In the FRET linker, the cathepsin-cleavable dipeptide of val-cit was inserted between a fluorescence donor Alexa Fluor 488 (or later, fluorescein) and an acceptor tetramethylrhodamine (TAMRA). Upon cleavage of the val-cit linker, a fluorescence signal from Alexa Fluor 488 or fluorescein is expected to appear as a probe for monitoring intracellular activation of ADC. We used two in-vitro human cancer cell lines, SKBR3 and PC3. SKBR3 is a Her2-positive breast cancer cell line and PC3 is a prostate cancer cell line that has been engineered to express a prostate cancer-specific growth factor receptor, tomoregulin-2 (TenB2). We assessed the intracellular processing of the FRET conjugates using flow cytometry. We found that 74% and 41% of the linker was cleaved in PC3 and SKBR3 cells after 20 hours of treatment, respectively. In the live-celling imaging of the FRET probe in SKBR3 and PC3 cells, Anti-Her2 and TenB2 FRET ADCs showed a remarkable difference of internalization pathways and cleavage following receptor-mediated endocytosis. In PC3 cells, FRET ADCs were internalized and congregated into a certain localized area during the early stage of uptake in 2 to 3 hours. The ADC-containing vesicles were spread over the cytosol and near the plasma membrane in a subset of cells. A similar directed localization at the early stage of uptake was not observed for the SKBR3 cells. The endosomal vesicles in SKBR3s were spread over the cytosol during the course of ADC internalization. The amount of the release payload in the cytosol of SKBR3s was 3-fold greater than that of PC3 cells, suggesting that the endosomal/lysosomal membrane of SKBR3 may be more permeable than that of PC3. In summary, our ADC FRET probes allowed for the facile, robust and non-invasive evaluation of intracellular processing for ADC linkers. Quantitative measurements for the rate of antigen-mediated intracellular cleavage of the FRET linker as well as cytosolic release could be evaluated by our FRET probes. Citation Format: Byoung-Chul Lee, Cecile Chalouni, Sam Nalle, Sophia Doll, Martine Darwish, Ira Mellman, Richard Vandlen. A novel FRET assay for the intracellular activation of ADC linkers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 211. doi:10.1158/1538-7445.AM2015-211