PET imaging of microglia in Alzheimer's disease using copper-64 labeled TREM2 antibodies

Abstract

Triggering receptor expressed on myeloid cells 2 (TREM2) plays an essential role in microglia activation and is being investigated as a potential therapeutic target for modulation of microglia in several neurological diseases. In this study, we present the development and preclinical evaluation of ⁶⁴Cu-labeled antibody-based PET radiotracers as tools for non-invasive assessment of TREM2 expression. Furthermore, we tested the potential of an antibody transport vehicle (ATV) that binds human transferrin receptor to facilitate transcytosis of TREM2 antibody-based radiotracers to the CNS and improve target engagement. <b>Methods:</b> A TREM2 antibody with an engineered transport vehicle (ATV:4D9) and without (4D9) were covalently modified with <i>p</i>NCS-benzyl-NODAGA and labeled with copper-64. Potency, stability, and specificity were assessed <i>in vitro</i> followed by <i>in vivo</i> PET imaging at the early 2 h, intermediate 20 h, and late imaging time points 40 h post-injection using a human transferrin receptor (hTfR) expressing model for amyloidogenesis (5xFAD;TfR^mu/hu) or wild-type mice (WT;TfR^mu/hu), and hTfR negative controls. Organs of interest were isolated to determine biodistribution by <i>ex vivo</i> autoradiography. Cell sorting after <i>in vivo</i> tracer injection was used to demonstrate cellular specificity for microglia and to validate TREM2 PET results in an independent mouse model for amyloidogenesis (App^SAA;TfR^mu/hu). For translation to human imaging, a human TREM2 antibody (14D3) was radiolabeled and used for <i>in vitro</i> autoradiography on human brain sections. <b>Results:</b> The ⁶⁴Cu-labeled antibodies were obtained in high radiochemical purity (RCP), radiochemical yield (RCY), and specific activity. Antibody modification did not impact TREM2 binding. ATV:4D9 binding proved to be specific, and the tracer stability was maintained over 48 h. The uptake of [⁶⁴Cu]Cu-NODAGA-ATV:4D9 in the brains of hTfR expressing mice was up to 4.6-fold higher than [⁶⁴Cu]Cu-NODAGA-4D9 in mice without hTfR. TREM2 PET revealed elevated uptake in the cortex of 5xFAD mice compared to wild-type, which was validated by autoradiography. PET-to-biodistribution correlation revealed that elevated radiotracer uptake in brains of 5xFAD;TfR^mu/hu mice was driven by microglia-rich cortical and hippocampal brain regions. Radiolabeled ATV:4D9 was selectively enriched in microglia and cellular uptake explained PET signal enhancement in App^SAA;TfR^mu/hu mice. Human autoradiography showed elevated TREM2 tracer binding in the cortex of patients with Alzheimer's disease. <b>Conclusion:</b> [⁶⁴Cu]Cu-NODAGA-ATV:4D9 has potential for non-invasive assessment of TREM2 as a surrogate marker for microglia activation <i>in vivo</i>. ATV engineering for hTfR binding and transcytosis overcomes the blood-brain barrier restriction for antibody-based PET radiotracers. TREM2 PET might be a versatile tool for many applications beyond Alzheimer's disease, such as glioma and chronic inflammatory diseases.