Purpose <p>The triggering receptor expressed on myeloid cells 2 (TREM2) has become a promising target for biologics in both monitoring and treating neuroinflammation in Alzheimer’s disease (AD). This study aimed to develop and compare bispecific anti-TREM2 antibodies featuring different transferrin receptor (TfR) binders to enhance brain delivery, identifying the most suitable format for in vivo PET imaging of TREM2 in transgenic AD mice.</p> Methods <p>Three bispecific TREM2-antibody formats were produced and evaluated for their ability to cross the blood-brain barrier (BBB) via TfR-mediated transcytosis and bind TREM2. Blood concentration profiles up to 72&#xa0;h post-injection (p.i.), and ex vivo brain uptake of iodine-125-labeled antibody constructs were quantified in App^NL−G−F and age-matched wild type (WT) mice using a γ-counter. The best-performing bispecific TREM2-antibody was radiolabeled with iodine-124 and used for in vivo PET imaging of brain TREM2 levels in App^NL−G−F mice at 72&#xa0;h p.i. Brain TREM2 concentrations were subsequently quantified using ELISA.</p> Results <p>The antibody format carrying two scFv8D3 TfR-binders (IgG-scFv₂), demonstrated the highest brain concentrations of all tested bispecific constructs. This antibody also exhibited significantly higher brain concentrations in App^NL−G−F mice compared to WT mice at both 48 and 72&#xa0;h p.i. This difference was further visualized and quantified through in vivo PET imaging. Moreover, brain concentrations of the antibody ligand correlated with elevated TREM2 levels in brain homogenates.</p> Conclusion <p>These findings highlight IgG-scFv₂ as a promising radioligand for in vivo PET imaging of TREM2, advancing non-invasive neuroinflammation studies and supporting drug development for AD and other neurodegenerative diseases.</p>