A110-02 Cryo-fluorescence Tomography to Assess Zampilimab Lung Target Occupancy in Humanized Fibrotic Mouse Lung

Rationale Transglutaminase-2 (TG2) is a multifunctional enzyme whose extracellular transamidation activity has been implicated in the pathogenesis of Idiopathic Pulmonary Fibrosis (IPF) and other fibrotic disorders1,2. Zampilimab is a therapeutic monoclonal antibody that selectively inhibits the catalytic crosslinking function of human TG2, representing a promising candidate for IPF treatment3. This proof-of-concept study aimed to confirm the precize lung localization of hTG2 expression and assess zampilimab biodistribution and target occupancy (TO) in fibrotic lung regions using Cryo-Fluorescence Tomography (CFT) in bleomycin-induced fibrosis model in humanized hTG2 mice. Methods Pulmonary fibrosis was induced in male humanized hTG2 mice via oropharyngeal aspiration of bleomycin on days 0 and 4.Zampilimab was administered intravenously at 100 mg/kg on days 7 and 14. On day 20, saline, fibrotic and zampilimab-treated mice received an intravenous injection of DyLight800-labeled zampilimab (100 μg/mouse). After 48 hours, in-vivo micro-Computed Tomography (µCT) was performed to quantify lung fibrosis based on mean lung attenuation (HU)4.Lungs were then removed, inflated with Dark D31 OCT, snap frozen in dry ice and embedded blocks of Dark D31 OCT.Lungs positioned for top-to-bottom were imaged at 780/840nm (DyLight800) and 470/511nm (autofluorescence) at 20μm resolution and from serial 20μm sections.Volumetric datasets were reconstructed from 2D image stacks, preserving both anatomical and molecular information. Results Micro-CT imaging revealed increased lung density in the apical regions of bleomycin (BLM)-treated mice, consistent with fibrotic tissue deposition.This increase was attenuated in zampilimab-treated animals, indicating a partial reduction in fibrosis.CFT-derived autofluorescence further confirmed these structural changes, with fibrotic regions displaying dense tissue architecture and elevated pixel intensities. The same lungs were scanned at 800 nm to detect DyLight800-labeled zampilimab.A strong fluorescent signal was observed in fibrotic areas of BLM-treated mice, whereas low signal was detected in saline- or zampilimab-treated animals. The high signal in BLM lungs confirmed that hTG2 is enzymatically active in fibrotic lesions.Conversely, the strong decrease in signal in zampilimab-treated mice indicates target occupancy by the therapeutic antibody, preventing binding of the labeled probe and confirming its specificity for hTG2. Conclusion CFT enables high-resolution spatial mapping of hTG2 in lung fibrosis, showing its localization within fibrotic lesions and role in disease progression.Zampilimab effectively reaches fibrotic lung tissue and selectively binds the active form of hTG2, supporting its further clinical development as antifibrotic therapy. Moreover, labeled zampilimab shows potential as an imaging tool to assess drug biodistribution and TO in clinical settings. REFERENCES1. Tatsukawa H. SciRep.2018. 2. Olsen Ks. AJRCCM2011. 3. Vitiello G. AJRCCM2025. 4.Ferrini E.Methods.inMolecularBiology.2025 This abstract is funded by: Chiesi Farmaceutici S.p.A.