Obinutuzumab induces lysosomal destabilization via sphingomyelin-dependent inhibition of TRPML2.

Obinutuzumab (OBI), a type II glycoengineered anti-CD20 antibody, induces direct cell death (DCD) in B-cell lymphomas more effectively than rituximab, yet the upstream mechanisms underlying this activity remain unclear. Here, we identify a lipid-ion channel axis linking antibody internalization to lysosomal destabilization. Using imaging, genetic, and biochemical approaches, we show that OBI is rapidly internalized into acidic compartments where it colocalizes with sphingomyelin (SM). SM-dependent inhibition of TRPML2-mediated lysosomal Ca(2)⁺ release sensitizes lysosomes to OBI-induced stress, lowering the threshold for LMP and direct cell death. Restoration of TRPML2 function by SMase treatment, or blockade of OBI internalization, attenuates LMP and DCD, underscoring the critical role of the SM-TRPML2 pathway. These findings reveal a previously uncharacterized mechanism by which OBI exerts cytotoxicity, highlighting lipid remodeling and ion channel regulation as potential targets to enhance the efficacy of antibody-based therapies in B-cell malignancies.