MMAE Payload Responsible for EV Resistance in Bladder Cancer

While antibody-drug conjugates (ADCs) can act as strong therapeutic treatments for various forms of cancer, primary or secondary resistance is a common occurrence alongside ADC treatment. For patients with non-muscle invasive bladder cancer (NMIBC), enfortumab vedotin (EV) has proven to be a beneficial ADC that targets the NECTIN4 surface protein, which is highly expressed in bladder cancer tumors.

Previous research has suggested that the loss of NECTIN4 expression can potentially lead to EV resistance. To further researchers’ understanding of the mechanisms behind EV resistance, a team led by Kevin Chang and Drs. Carissa Chu and Jonathan Chou, of the University of California, San Francisco, presented new research on an in vitro preclinical bladder cancer model of EV resistance at the 24th Annual Meeting of the Society of Urologic Oncology. The model was investigated to further the current understanding of mechanisms and strategies to overcome EV resistance.

The RT112 bladder cancer cell line, which expresses high levels of NECTIN4, was used to develop the resistance model. RT112 cells underwent treatment cycles, which consisted of 5 to 7 days of EV administration that escalated with each cycle, with subsequent recovery and passaging of the surviving cells. EV dosage began at 0.5 µg/ml and culminated at 30 µg/ml, yielding a generation of cells that exhibited a 4- to 5-fold increase in IC50 for EV. The cells were cultured using flow cytometry and Western blotting, and the parental RT112 cells were cultured in parallel.

Drug dose-response and chimeric antigen receptor (CAR) T-cell killing assays were carried out using parental RT112 cells and EV-exposed cells; after 24 hours, the drug or CAR T cells were added at indicated concentrations or effector-to-target ratios. Cell count and growth were also monitored.

The EV-resistant RT112 cells showed similar surface NECTIN4 levels and increased surface TROP2 levels compared with the parental cells. The EV-resistant cells were also less sensitive to the microtubule inhibitor MMAE, which serves as the payload of EV. EV-resistant cells were more sensitive to treatment with sacituzumab govitecan (SG) and were equally susceptible to killing by NECTIN4-directed CAR T cells compared with the parental RT112 cells.

The in vitro model of EV-resistant bladder cancer showed that EV resistance was mainly due to resistance of the payload MMAE and not because of NECTIN4 downregulation. EV-resistant bladder cancer cells remained sensitive to NECTIN4-directed CAR T cells, demonstrating that NECTIN4 is still a relevant target even after cells develop EV resistance.

Treatment with SG can serve as an alternate strategy after EV resistance in patients.