A bifunctional CPG2-anti PD-L1 fusion protein combining specific tumor targeting and enzyme-mediated prodrug...

The specific targeting of therapeutic agents to tumor cells remains a pivotal challenge in cancer treatment. While antibody-directed enzyme prodrug therapy (ADEPT) offers a promising approach by delivering enzymes such as carboxypeptidase G2 (CPG2) to the tumor microenvironment, achieving efficient targeting and multifunctionality remains a significant hurdle. This study developed a novel bifunctional fusion protein by integrating an anti-PD-L1 nanobody with CPG2, enabling specific immune checkpoint targeting and localized prodrug activation. The fusion protein was computationally engineered using in silico analyses to optimize structural stability and linker flexibility. Molecular modeling and docking simulations were performed to evaluate binding to the PD-L1 receptor, and the construct exhibiting the most favorable binding energy and strongest intramolecular interactions was selected for experimental validation. The optimized fusion protein was expressed in E. coli BL21 (DE3) and purified. Cell-ELISA revealed values for the Nb and fusion protein against MDA-MB-231 (12.97 vs. 25.86 nM) and RKO (11.75 vs. 24.36 nM) cells, respectively. Despite a moderate shift in K_d values, the fusion protein maintained nanomolar binding affinity, with negligible binding to PD-L1-low HEK-293 cells. Enzymatic activity assays demonstrated that the fusion protein preserved significant catalytic efficiency (24.83 vs. 31.82 μM^-1·min^-1 for native CPG2). In addition, cell-based viability analyses indicated minimal intrinsic cytotoxicity, supporting the functional integrity of the bifunctional construct. Overall, these findings demonstrate that the engineered CPG2-anti-PD-L1 fusion protein preserves both antigen-binding and enzymatic activities, highlighting its potential as a dual-function platform that integrates immune checkpoint targeting with site-specific prodrug activation for targeted cancer therapy.