FAP-targeted radioligand therapy with 68Ga/177Lu-DOTA-2P(FAPI)2 enhance immunogenicity and synergize with PD-L1 inhibitors for improved antitumor efficacy

Background Fibroblast activation protein (FAP)-targeted radioligand therapy, with immunomodulatory effects, has shown efficacy in both preclinical and clinical studies. We recently reported on a novel dimeric FAP-targeting radiopharmaceutical, 68 Ga/ 177 Lu-DOTA-2P(FAPI) 2 , which demonstrated increased tumor uptake and prolonged retention in various cancers. However, further exploration is required to understand the therapeutic efficacy and underlying mechanisms of combining 68 Ga/ 177 Lu-DOTA-2P(FAPI) 2 radioligand therapy with PD-1/PD-L1 immunotherapy. Methods Regarding the change in PD-L1 expression and DNA double-strand breaks induced by radiopharmaceuticals, CT26-FAP tumor cells were incubated with 68 Ga and 177 Lu labeled DOTA-2P(FAPI) 2 , respectively. Monotherapy with 68 Ga-DOTA-2P(FAPI) 2 , 177 Lu-DOTA-2P(FAPI) 2 , and PD-L1 immunotherapy as well as combination therapy ( 68 Ga/ 177 Lu-DOTA-2P(FAPI) 2 and PD-L1 immunotherapy) were tested and evaluated to evaluate in vivo antitumor efficacy. Furthermore, immunohistochemical staining and single-cell RNA sequencing were used to analyze changes in the tumor microenvironment (TME) and elucidate the underlying mechanisms of action of this combination therapy. Results Our findings indicated that FAP-targeting radiopharmaceuticals can induce DNA double-strand breaks and upregulate PD-L1 expression, with 177 Lu-DOTA-2P(FAPI) 2 proving to be more effective than 68 Ga-DOTA-2P(FAPI) 2 . Both 68 Ga-DOTA-2P(FAPI) 2 and 177 Lu-DOTA-2P(FAPI) 2 radiopharmaceuticals significantly improved therapeutic outcomes when combined with anti-PD-L1 monoclonal antibody (αPD-L1 mAb). Notably, the combination of 177 Lu-DOTA-2P(FAPI) 2 with αPD-L1 mAb immunotherapy eliminated tumors in mouse models. Mice treated with this regimen not only exhibited exceptional responses to the initial immune checkpoint inhibitor therapy but also showed 100% tumor rejection on subsequent tumor cell re-inoculation. Further mechanistic studies have shown that 177 Lu-DOTA-2P(FAPI) 2 combined with αPD-L1 mAb can reprogram the TME, enhancing antitumor intercellular communication, which activates antitumor-related intercellular contacts such as FasL-Fas interactions between T cells and NK cells with tumor cells and increasing the proportion of infiltrating CD8+ T-cells while reducing regulatory T cells and inhibiting tumor progression. Our research also demonstrates that mature neutrophils play a role in enhancing the efficacy of the combined therapy, as shown in neutrophil-blocking experiments. Conclusions Our study robustly advocates for use of FAP-targeting radiopharmaceuticals, particularly 177 Lu-DOTA-2P(FAPI) 2 , alongside immunotherapy in treating FAP-positive tumors. This combination therapy transforms the TME and enables a translatable approach to increasing the sensitivity to PD-1/PD-L1 immunotherapy, leading to improved complete remission rates and extended overall survival.