<p>Pancreatic cancer is a highly aggressive malignancy with a dismal prognosis, characterized by a complex tumor microenvironment that promotes immunosuppression and limits the efficacy of immune checkpoint blockade (ICB) therapy. Fibroblast activation protein (FAP) is overexpressed in the tumor stroma and represents a promising target for therapeutic intervention. Here, we developed a novel antibody-drug conjugate (ADC) targeting FAP, and investigated its anti-tumor activity and ability to enhance ICB efficacy in pancreatic cancer. We conjugated a humanized anti-FAP antibody with the potent topoisomerase I inhibitor exatecan to generate a novel FAP-targeting ADC (FAP-ADC) with a drug-to-antibody ratio of eight. The cytotoxicity and internalization of FAP-ADC were evaluated in vitro using FAP-expressing cell lines, and its anti-tumor activity was assessed in vivo using cell-derived xenograft models. Mechanistic studies revealed that FAP-ADC synergistically improved the efficacy of anti-PD-L1 antibody in vivo by leading to an increased level of M1-polarized macrophages and reduced abundance of myeloid-derived suppressor cells and regulatory T cells in the tumor microenvironment. Furthermore, FAP-ADC treatment enhanced the infiltration of CD8⁺ T cells into the tumor and upregulated the expression of pro-inflammatory cytokines. Combination therapy with FAP-ADC and anti-PD-L1 antibodies resulted in superior anti-tumor efficacy compared to either monotherapy. Collectively, our novel FAP-targeting ADC exerts potent anti-tumor activity in pancreatic cancer by selectively depleting FAP-expressing cells and reversing the immunosuppressive tumor microenvironment. The combination of FAP-ADC with ICB therapy represents a promising therapeutic strategy to improve the treatment outcomes for patients with this fatal cancer.</p><p></p>