Rheumatoid arthritis is a chronic autoimmune disorder resulting in synovial inflammation. Fibroblast activation protein (FAP) is overexpressed by fibroblastlike synoviocytes in arthritic joints. Radioimmunoimaging with an anti-FAP antibody might be used to monitor the response to therapy, thus enabling tailored therapy strategies and therapeutic outcomes. The aim of this study was to assess whether a radiolabeled anti-FAP antibody could be used to monitor the efficacy of treatment with long-circulating liposomes (LCL) containing prednisolone phosphate (PLP-LCL) in a mouse model of arthritis. METHODS: Collagen-induced arthritis (CIA) was induced in male DBA/1J mice. Mice were treated with a single injection (10 mg/kg) of PLP-LCL or empty LCL as a control. SPECT and CT images were acquired 24 h after injection of 99mTc-labeled succinimidyl-hydrazinonicotinamide (99mTc-S-HYNIC)-conjugated anti-FAP antibody 28H1 at 2, 5, and 9 d after treatment. The uptake of 99mTc-S-HYNIC-28H1 in all joints was quantified and correlated with macroscopic arthritis scores. RESULTS: Treatment of CIA with PLP-LCL significantly suppressed joint swelling. At just 1 d after treatment, the macroscopic arthritis scores had decreased by 50%. Scores decreased further, to only 10% of the initial scores, at 5 and 9 d after treatment. In contrast, macroscopic arthritis scores had increased up to 600% in untreated mice at 9 d after the injection of empty LCL. 99mTc-S-HYNIC-28H1 uptake ranged from 1.5 percentage injected dose per gram in noninflamed joints to 22.6 percentage injected dose per gram in severely inflamed joints. The uptake of radiolabeled 28H1 in inflamed joints (percentage injected dose) correlated with the arthritis score (Spearman ρ, 0.77; P < 0.0001). Moreover, the uptake of 99mTc-S-HYNIC-28H1 was slightly increased at 9 d after therapy but was not seen macroscopically, indicating that SPECT/CT imaging might be more sensitive than the macroscopic arthritis scoring method. CONCLUSION: SPECT/CT imaging with 99mTc-S-HYNIC-28H1 specifically monitored the response to therapy, and tracer accumulation correlated with the severity of inflammation. In addition, SPECT/CT imaging was potentially more sensitive than the macroscopic arthritis scoring method. This study showed that SPECT/CT with 99mTc-S-HYNIC-28H1 could be used to noninvasively monitor the course of CIA in mice.