biostudies-literatureYan JNatural Science Foundation of China grantJiangsu Province Key Research and Development Program grantJiangsu Provincial Medical Key Discipline grant1323https://www.ebi.ac.uk/biostudies/studies/S-EPMC12632142biostudies-literatureUnknown23(1)<h4>Background</h4>Although antibody-conjugated drugs have achieved success in clinical practice for cancer treatment, challenges remain in developing a highly efficient drug delivery system with specific accumulation in tumors and reduction in side effects. With improved pharmacokinetics, strong covalent bonding and quick binding reactions, a pre-targeting approach via molecular pairs represents an attractive platform for two-step delivery system construction.<h4>Methods</h4>Bioinformatics and immunohistochemistry assays were performed to assess Claudin-6 (CLDN6) as a highly specific tumor target in solid tumors. A phage-displayed library was used to screen and optimize anti-CLDN6 designed ankyrin repeat proteins (DARPins), which were incorporated into a two-step delivery system based on SpyTag/SpyCatcher. Fluorescent staining, flow cytometry and near-infrared imaging were performed to assess the tumor-targeting ability and biodistribution of this delivery system. The cytotoxic drug, Monomethyl auristatin E (MMAE), was conjugated with the delivery system to evaluate its anti-tumor efficacy and safety profile.<h4>Results</h4>Anti-CLDN6 DARPins exhibited specific binding to CLDN6⁺ cancer cells with high affinity instead of negative cells in vitro, ex vivo and in vivo. The DARPins-based two-step delivery system improved background clearance with a high signal-to-noise ratio, enhancing the specific accumulation of payloads in tumors. The cytotoxic drug delivered via the two-step system appeared superior to the one-step approach in IC50, biodistribution, and tumor growth inhibition.<h4>Conclusions</h4>Our study presented the de novo design of a two-step drug delivery system targeting Claudin-6 with enhanced anti-tumor efficacy and improved biosafety. These findings highlighted the potential of this approach to enhance the efficacy of tumor-targeting therapies and reduce adverse effects, paving the way for more effective cancer treatments.Journal of translational medicineDe novo design of a two-step approach targeting Claudin-6 for enhanced drug delivery to solid tumors.PMC12632142ZDXK202233BE202365482272811Lei LYan JShao JAn MLiu QWei XLi RLi LZhao YGuo JLiu BChen XWang YZhong LLiu FYu XChen TfalseDe novo design of a two-step approach targeting Claudin-6 for enhanced drug delivery to solid tumors.<h4>Background</h4>Although antibody-conjugated drugs have achieved success in clinical practice for cancer treatment, challenges remain in developing a highly efficient drug delivery system with specific accumulation in tumors and reduction in side effects. With improved pharmacokinetics, strong covalent bonding and quick binding reactions, a pre-targeting approach via molecular pairs represents an attractive platform for two-step delivery system construction.<h4>Methods</h4>Bioinformatics and immunohistochemistry assays were performed to assess Claudin-6 (CLDN6) as a highly specific tumor target in solid tumors. A phage-displayed library was used to screen and optimize anti-CLDN6 designed ankyrin repeat proteins (DARPins), which were incorporated into a two-step delivery system based on SpyTag/SpyCatcher. Fluorescent staining, flow cytometry and near-infrared imaging were performed to assess the tumor-targeting ability and biodistribution of this delivery system. The cytotoxic drug, Monomethyl auristatin E (MMAE), was conjugated with the delivery system to evaluate its anti-tumor efficacy and safety profile.<h4>Results</h4>Anti-CLDN6 DARPins exhibited specific binding to CLDN6⁺ cancer cells with high affinity instead of negative cells in vitro, ex vivo and in vivo. The DARPins-based two-step delivery system improved background clearance with a high signal-to-noise ratio, enhancing the specific accumulation of payloads in tumors. The cytotoxic drug delivered via the two-step system appeared superior to the one-step approach in IC50, biodistribution, and tumor growth inhibition.<h4>Conclusions</h4>Our study presented the de novo design of a two-step drug delivery system targeting Claudin-6 with enhanced anti-tumor efficacy and improved biosafety. These findings highlighted the potential of this approach to enhance the efficacy of tumor-targeting therapies and reduce adverse effects, paving the way for more effective cancer treatments.2025-01-01T00:00:00Z2025 Nov2026-06-05T17:14:08.828Z2026-05-19T03:11:44.744ZS-EPMC126321424126707410.1186/s12967-025-07316-2