Abstract 4837: Extracellular proteolytic cleavage of peptide-linked antibody-drug conjugates promotes bystander killing of cancer cells
Authors: Lam My-Hanh , Lucas Judy , Maderna Andreas , Wald Hallie , Wojciechowicz Megan , Dushin Russell , Peano Bryan , Wang Fang , Myers Jeremy , Tan Xingzhi , Musto Sylvia , Charati Manoj , Gerber Hans-Peter , Loganzo Frank 
Published: May 2015

Abstract
Antibody drug conjugates (ADCs) are designed to deliver cytotoxics to tumor cells via binding to surface antigen followed by internalization and intracellular drug release. ADC linkers are typically categorized as non-cleavable or cleavable; a cleavable linker example is Y_mcValCitPABC_X, with antibody Y, a dipeptide sequence with self-immolative PABC spacer, and payload X. This linker is known to be cleaved by endosomal/lysosomal proteases such as cathepsins, releasing attached payload. In addition to intracellular processing of this linker, we report that conditioned media of cultured tumor cell lines is sufficient to promote extracellular cleavage of ADCs with peptide-linked payloads. Cultured cell lines N87 (gastric) and U87 (glioblastoma), and patient-derived xenograft PA0165 (pancreatic) adapted to in vitro culture, were plated either in standard 2D culture or in 3D Cultrex embedded culture. After 3 - 7 days, conditioned media from cells was transferred onto MDA-MB-468 or HT29 cells, and then ADCs (Y_mcValCitPABC_Aur) were added to cultures. ADCs were non-targeting IgG conjugated via cleavable dipeptide-PABC linker to auristatin tubulin inhibitor. Minimal cytotoxicity was observed with ADC alone on 468 or HT29 cells. However, in the presence of conditioned media from N87, U87, or PA0165 cells plus the ADC, cytotoxicity was observed in the recipient cells (up to 31, 22, 56% growth inhibition respectively at 100 nM ADC). Moreover, in all cases, the magnitude of the response was greatest when cells providing conditioned media were grown in 3D culture (up to 56, 48, 70%, respectively). In contrast, minimal response was observed using conditioned media from other cancer cell lines (ie HCC2429, 1 - 17%). Additional analyses were conducted by incubating conditioned media from these cells with a dipeptide-based cleavable substrate with fluorescent probe and measuring released product in a plate-based assay. Conditioned media promoted fluorescence, suggesting proteolytic enzymes secreted by cells. An ELISA confirmed the presence of cathepsins in conditioned media. Complementing these studies, proteolytic activity was detected in the interstitial fluid derived from tumors grown in athymic mice. Fluid extracted from xenograft tumors (cultured cancer lines and patient-derived tumors) was analyzed for proteolytic activity using cleavable-fluorescent linker-probe in a plate assay. The majority of samples demonstrated proteolytic activity. These data are consistent with reported secretion of cathepsins by cancer cells and we now show that these proteases may mediate extracellular release of cytotoxic payloads from ADCs containing peptide-based cleavable linkers. This activity is magnified when cells are grown in 3D culture and is observed in tumor xenografts grown in vivo. This response may provide a beneficial bystander effect of ADCs on antigen negative cells in a heterogenous tumor population.
Citation Format: My-Hanh Lam, Judy Lucas, Andreas Maderna, Hallie Wald, Megan Wojciechowicz, Russell Dushin, Bryan Peano, Fang Wang, Jeremy Myers, Xingzhi Tan, Sylvia Musto, Manoj Charati, Hans-Peter Gerber, Frank Loganzo. Extracellular proteolytic cleavage of peptide-linked antibody-drug conjugates promotes bystander killing of cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4837. doi:10.1158/1538-7445.AM2014-4837