PSCA-directed nanosized bio-immune conjugates (NANO:BICs) enable selective uptake of TLR9 agonists in bladder cancer cells.

Introduction: Bladder cancer (BCa), particularly non-muscle invasive bladder cancer (NMIBC), remains a significant healthcare challenge due to high recurrence rates and limited non-surgical treatment options. Methods: Prostate stem cell antigen (PSCA)-transduced HEK-BlueTMhTLR9 and PSCA-positive SW780 bladder cancer cells were stimulated with PSCA-targeting NANO:BICs, which were assembled from a scFv(AM1)-KiBAP, NeutrAvidin, and the Toll-like Receptor 9 (TLR9) agonist ODN2006. Functional analyses included a secreted embryonic alkaline phosphatase (SEAP) reporter assay to measure TLR9 activation, a Cytometric Bead Array for cytokine quantification, and confocal microscopy to assess cellular uptake. Results: PSCA-targeting NANO:BICs demonstrated significantly enhanced uptake into PSCA-positive cancer cells compared to non-targeting controls, with the PSCA receptor increasing uptake by a factor of 3.63. This targeted delivery led to potent activation of the TLR9 signaling pathway, evidenced by a robust reporter gene response and secretion of key antiviral cytokines, including type I and III interferons and the chemokine IP-10. Discussion: These findings highlight the potential of this approach not only for reinvigorating anti-tumor immune responses in BCa but also for broader applications in other PSCA-expressing malignancies.