Inhibiting α2,6-sialylation and its association with nectin-4 stability and macropinocytic uptake in bladder cancer.

792 Background: Enfortumab vedotin (EV), a Nectin-4–directed ADC, improves outcomes in advanced urothelial carcinoma; however, determinants of EV resistance remain unclear. Tumour cell surface sialylation and complex N-glycans are dysregulated in cancer and may modulate antigen abundance and ADC internalisation. To test whether α2,6-sialylation drives EV resistance by downregulating Nectin-4 and limiting macropinocytic uptake, and whether pharmacologic desialylation restores EV efficacy and potentiates EV–PD-1 combination therapy. Methods: We profiled sialylation/N-glycan states, Nectin-4 levels and EV sensitivity across bladder cancer (BCa) cell lines, patient-derived organoids and EV-resistant derivatives. Mechanistic studies included ST6GAL1 knockdown, ubiquitination/stability assays, FITC-EV tracking with endocytosis inhibitors, and FITC-dextran uptake. The sialyltransferase inhibitor P-3Fax-Neu5Ac was used to deplete α2,6-sialylation. In vivo efficacy was tested in subcutaneous models and in EV±anti-PD-1 ±P-3Fax-Neu5Ac settings. Immunogenic cell death (ICD) readouts included p-eIF2α, ATP release, HMGB1, and calreticulin exposure; T-cell phenotypes were analysed in tumour–PBMC co-culture and tumours. Statistics used t-tests/ANOVA with p < 0.05 considered significant. Results: High α2,6-sialylation/complex-type N-glycans correlated with low Nectin-4 and EV resistance, whereas high-mannose signatures associated with higher Nectin-4. Pharmacologic or genetic desialylation increased total and membranous Nectin-4 by reducing its ubiquitin-dependent degradation and restored EV sensitivity in vitro, including EV-resistant cells/organoids. EV internalisation in BCa cells occurred via macropinocytosis; α2,6-desialylation enhanced macropinocytic uptake and EV cytotoxicity. In mice, P-3Fax-Neu5Ac augmented EV antitumour activity and prolonged survival without added toxicity. Desialylation amplified EV-induced ICD and improved CD8^+ T-cell proliferation and function, thereby further potentiating EV+anti-PD-1 in vivo. Conclusions: α2,6-Sialylation is a tractable driver of EV resistance through Nectin-4 destabilisation and reduced macropinocytosis. Targeting sialylation reinstates antigen density, increases EV internalisation, heightens ICD, and strengthens EV±PD-1 efficacy. These data nominate tumour desialylation as a rational co-strategy to expand and deepen clinical benefit from EV in bladder cancer.