PurposeFAP is a membrane-bound protease under investigation as a pan-cancer target, given its high levels in tumors but limited expression in normal tissues. FAP-2286 is a radiopharmaceutical in clinical development for solid tumors that consists of two functional elements: a FAP-targeting peptide and a chelator used to attach radioisotopes. Preclinically, we evaluated the immune modulation and anti-tumor efficacy of FAP-2287, a murine surrogate for FAP-2286, conjugated to the radionuclide lutetium-177 (177Lu) as a monotherapy and in combination with a PD-1 targeting antibody.MethodsC57BL/6 mice bearing MCA205 mouse FAP-expressing tumors (MCA205-mFAP) were treated with177Lu-FAP-2287, anti-PD-1, or both. Tumor uptake of177Lu- FAP-2287 was assessed by SPECT/CT scanning, while therapeutic efficacy was measured by tumor volume and survival. Immune profiling of tumor infiltrates was evaluated through flow cytometry, RNA expression, and immunohistochemistry analyses.Results177Lu-FAP-2287 rapidly accumulated in MCA205-mFAP tumors leading to significant tumor growth inhibition (TGI) and longer survival time. Significant TGI was also observed from anti-PD-1 and the combination. In flow cytometry analysis of tumors,177Lu-FAP-2287 increased CD8+T cell infiltration which was maintained in the combination with anti-PD-1. The increase in CD8+T cells was accompanied by an induction of STING-mediated type I interferon response and higher levels of co-stimulatory molecules such as CD86.ConclusionIn a preclinical model, FAP-targeted radiotherapy enhanced anti-PD-1-mediated TGI by modulating the TME and increasing the recruitment of tumor-infiltrating CD8+T cells. These findings provide a rationale for clinical studies of combined177Lu-FAP-2286 radiotherapy and immune checkpoint inhibition in FAP-positive tumors.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00259-023-06211-6.