Abstract Rationale Zampilimab is a humanized monoclonal antibody that selectively inhibits transamidation activity of human tissue transglutaminase-2 (hTG2)1. The enzymatic function of hTG2 within the extracellular matrix has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF) and other fibrotic diseases2,3. Zampilimab has recently completed a Phase Ib clinical trial in IPF patients4. However, non-invasive quantification of target occupancy (TO) in the lungs remains a major challenge. In this study, we investigated the feasibility of radiolabeling zampilimab and longitudinally assessing its TO in a bleomycin (BLM)-induced lung fibrosis model in hTG2-humanized mice using single photon computed tomography (SPECT) imaging. Methods BLM was administered on days 0 and 4 via oropharyngeal aspiration to hTG2 male mice, while saline was instilled in the control group. On day 14, all mice received an intravenous injection of 111InIn-DOTAGA-Zampilimab (90% radiochemical purity, 25 µg/mouse). A subset of BLM-treated mice also received a 100-fold excess of unlabeled antibody (blocking group). SPECT imaging was performed at 24, 48, 72, and 96 hours post-injection. CT-derived mean lung attenuation (MLA) and SPECT signal were quantified using VivoQuant software. Ex-vivo gamma counting of the lungs was conducted to validate the in vivo results. Results On day 14, CT imaging revealed a significant increase in MLA in both BLM-treated groups compared to controls, confirming the establishment of fibrosis. At all timepoints, SPECT imaging showed a significantly higher pulmonary uptake of radiolabeled zampilimab in BLM-treated mice compared to control group. Specific binding of zampilimab to hTG2 was confirmed in the blocking group, where an excess of unlabeled antibody reduced the uptake of radiolabeled antibody by approximately 80%. Ex vivo gamma counting corroborated the persistence of 111InIn-DOTAGA-Zampilimab in fibrotic lungs up to 96 hours post-injection and confirmed its specificity for hTG2. A good positive correlation (R2=0.83, p 0.0001) was also observed between the degree of fibrosis measured by MLA and the pulmonary uptake of 111InIn-DOTAGA-Zampilimab, suggesting its potential utility as a quantitative imaging tool for monitoring disease progression and therapeutic response in pulmonary fibrosis models. Conclusion Zampilimab can be effectively radiolabeled for SPECT imaging, enabling non-invasive and quantitative assessment of lung TO in a BLM-induced fibrosis model in hTG2 humanized mice. These findings support the role of hTG2 transamidation activity in fibrosis progression and demonstrate that zampilimab can reach the lung and selectively bind the active enzyme, paving the way for its theragnostic application in clinical settings. This abstract is funded by: Chiesi Farmaceutici
Capelli et al. (Fri,) studied this question.