SLC46A3 Mediates Lysosomal Egress of Auristatin Derivatives

The lysosomal transporter SLC46A3 has been implicated in the pharmacology of antibody−drug conjugates (ADCs) by facilitating the transport of payloads from lysosomes to the cytosol. To date, two payload classes, maytansinoid catabolites (e.g., DM1 and Lys-SMCC-DM1) and the pyrrolobenzodiazepine (PBD) dimer SG3199, have been identified as SLC46A3 substrates; however, the transport mechanisms for other payloads remain to be elucidated. Auristatin derivatives (auristatins) are among the most widely deployed payloads in approved ADCs, yet the mechanism by which these highly potent peptides egress from the lysosome remains unclear. Here, we demonstrate that SLC46A3 directly transports auristatins. At pH 5.0, mimicking the lysosomal environment, SLC46A3 significantly facilitated the membrane transport of all tested auristatins, whereas transport via passive diffusion was negligible. Among the auristatins, monomethyl auristatin F (MMAF), which bears a negative charge, was a superior substrate for SLC46A3 compared to monomethyl auristatin E (MMAE), a finding consistent with the preference of SLC46A3 for organic anions. Interestingly, MMAF conjugated with a non-cleavable maleimidocaproyl (mc) linker, was also recognized as an SLC46A3 substrate, exhibiting higher transport activity than unmodified MMAF. Furthermore, depletion of SLC46A3 in HER2-positive cancer cells attenuated the cytotoxicity of trastuzumab-mcMMAF, suggesting that SLC46A3 mediates the lysosomal egress of its active metabolites. Taken together, our findings highlight lysosomal transporters including SLC46A3, as critical determinants of the efficient cytoplasmic release of auristatins, providing an opportunity to optimize ADC efficacy.