Identification of antibody-drug conjugate payloads that are substrates of ATP-binding cassette drug efflux transporters

<b>Aim:</b> Antibody-drug conjugates (ADCs) feature an antibody recognizing a specific protein joined to a potent toxic payload. Numerous ADCs have received U.S. Food and Drug Administration (FDA) approval; however, clinical resistance arises. Resistance mechanisms include decreased expression or mutation of the antibody target, impaired payload release, or increased expression of adenosine triphosphate (ATP)-binding cassette (ABC) efflux transporters associated with multidrug resistance. We therefore sought to characterize the interactions of ABC multidrug transporters with ADC payloads. <b>Methods:</b> We performed a high-throughput screen with 27 common ADC payloads using cell lines expressing ABC transporters P-glycoprotein [P-gp, encoded by ABC subfamily B member 1 (<i>ABCB1</i>)] or ABC subfamily B member G2 (ABCG2, encoded by <i>ABCG2</i>). Confirmatory assays were also performed using cells transfected to express P-gp, ABCG2, or multidrug resistance-associated protein 1 (MRP1, encoded by <i>ABCC1</i>). <b>Results:</b> Several commonly used ADC payloads were substrates of P-gp, including calicheamicin γ1, monomethyl auristatin E, mertansine (DM1), and ravtansine (DM4). All the pyrrolobenzodiazepines tested - SJG136, SGD-1882, SG2057, and SG3199 - were substrates of P-gp, ABCG2, and MRP1. The modified anthracyclines nemorubicin and its metabolite PNU-159682 were poorly transported by both ABCB1 and ABCG2 and displayed nanomolar to picomolar toxicity. Further, we found that the efficacy of the FDA-approved ADC mirvetuximab soravtansine, with DM4 as the toxic payload, was decreased in cell lines expressing P-gp. In contrast, Duocarmycin DM and PNU-159682 were exquisitely toxic to a panel of 99 cancer cell lines of varying origins. <b>Conclusion:</b> Several commonly used ADC payloads can be transported by ABC transporters, potentially leading to transporter-mediated drug resistance in patients. Future ADCs should be developed using payloads that are not ABC transporter substrates.