Spect as a Translational Non-invasive Method to Assess Zampilimab Lung Target Occupancy in Mice

Abstract RATIONALE Zampilimab is a humanized monoclonal antibody that selectively inhibits transamidation of human tissue transglutaminase-2 (hTG2). The enzymatic activity of hTG2 in the extracellular matrix has been associated with the development of Idiopathic Pulmonary Fibrosis (IPF) and other fibrotic disorders. Recently Zampilimab has completed Phase Ib in IPF patients. Since measuring target occupancy (TO) non-invasively in the lung is challenging, in this study, we evaluated the feasibility to radiolabel Zampilimab and longitudinally measure its TO in the murine lung with nuclear imaging (single photon computed tomography – SPECT). METHODS Zampilimab was bioconjugated with DOTAGA and subsequently radiolabeled with indium-111 (90% radiochemical purity), then administered intravenously (i.v., 25 µg/mouse) to nude female mice receiving hTG2+ A375 cells either subcutaneously or intratracheally. In blocking groups, [111 In]In-DOTAGA-Zampilimab was simultaneously injected with a 100X excess of unlabeled antibody. SPECT imaging was performed on mice at 48, 72, and 96 hours following [111 In]In-DOTAGA-Zampilimab administration. RESULTS For all the timepoints, SPECT imaging showed a significant uptake of radiolabeled Zampilimab in xenograft tumors where hTG2+ cells were subcutaneously implanted. Furthermore, mice receiving hTG2+ cells intratracheally displayed a greater pulmonary uptake of [111 In]In-DOTAGA-Zampilimab compared to control group, which did not receive cells. The binding specificity of Zampilimab to human TG2 was confirmed with blocking experiments where the administration of the excess of unlabeled antibody significantly prevented the uptake of the radiolabeled one. CONCLUSION The administration of hTG2+ cells has proven to be a straightforward, rapid, and cost-effective animal model for specifically evaluating the TO of Zampilimab. Since hTG2+ cells intratracheally administered are more uniformly distributed within the lung compared to xenograft tumors, where hTG2 is overexpressed in a localized and vascularized area, results from intratracheal model consistently confirmed that Zampilimab can reach the lung and specifically bind the hTG2. Additionally, our study demonstrated that Zampilimab can be effectively radiolabeled for SPECT imaging, allowing for a non-invasive and quantitative assessment of lung TO, paving the way for its clinical application.