Evaluating the cellular binding specificity of a copper-64 labelled antibody transport vehicle enabled TREM2 PET tracer using scRadiotracing in mice

Evaluating the cellular binding specificity of a copper-64 labelled antibody transport vehicle enabled TREM2 PET tracer using scRadiotracing in mice

Rebecca Schaefer¹, Marlies Haertel¹, Lea H Kunze¹, Laura M Bartos¹, Giovanna Palumbo¹, Kai Schlepckow², Christian Haass²³⁴, Joseph W Lewcock⁵, Kathryn M Monroe⁵, Matthias Brendel¹²⁴, Simon Lindner¹⁴

(1)Department of Nuclear Medicine, University Hospital, LMU Munich, Munich, Germany (2)German Center for Neurodegenerative Diseases (DZNE) Munich, Munich, Germany (3)Biomedical Center (BMC), Division of Metabolic Biochemistry, Faculty of Medicine, LMU Munich, Munich, Germany (4)Munich Cluster for Systems Neurology (SyNergy), Munich, Germany (5)Denali Therapeutics Inc., South San Francisco, CA, United States of America

Objective

TREM2 (Triggering receptor expressed on myeloid cells 2) is expressed on myeloid cells, and plays a pivotal role in the activation of microglia¹. Thus, it is a potential target for non-invasive imaging of neuroinflammatory and neurodegenerative diseases by positron emission tomography (PET). Currently available radiotracers (i.e., translocator protein (TSPO) PET tracers) lack specificity for microglia, as their target is expressed in various other cell types². The aim of this project was to develop a ⁶⁴Cu-labelled antibody-based PET tracer targeting TREM2 and to examine its cellular binding specificity after radiotracer injection in vivo via single-cell (sc)Radiotracing³ in mice.

Methods

For this study, the Fc domain of a TREM2 antibody (clone 4D9) was engineered with an antibody transport vehicle (ATV), such that it binds human transferrin receptor to generate ATV:4D9 which was further modified with pNCS-benzyl-NODAGA and labelled with copper-64. Cellular specificity of ATV:4D9 for microglia was assessed in vivo using scRadiotracing 20 h post tracer injection, comparing APP-SAA-KI x hTfR-KI (an amyloid Alzheimer's disease model), WT and WTxTREM2-/- mice (n = 4 per group). Furthermore, PET and autoradiography signals of WT mice implanted with SB28 glioblastoma cells (week 2), as well as scRadiotracing data, were compared to a TSPO tracer.

Results

The ⁶⁴Cu-labelled antibody exhibited high radiochemical purity (RCP) and radiochemical yield (RCY). scRadiotracing revealed that the tracer was selectively enriched in microglia, demonstrating two-fold increase in microglial cell activity in APP-SAA-KI x hTfR-KI vs WT, and a 20-fold increase in comparison to WTxTREM2-/-. Depleted fractions exhibited minimal activity per cell. In glioblastoma mice the tracer demonstrated high tumour-lesion-to-background-ratios (TBR) in PET and autoradiography (PET TBR: 8.8 ± 0.4, autoradiography TBR: 14.9 ± 0.3), and scRadiotracing revealed a 94% cell specificity of tumour-associated microglia/macrophages (TAMs) vs tumour cells.

Conclusions

Preliminary data from scRadiotracing indicated that the tracer binds with high specificity to TREM2 on microglia. Further studies are required to evaluate the potential of [⁶⁴Cu]Cu-NODAGA-ATV:4D9 as a TREM2 PET tracer in patients with neuroinflammatory conditions.

1. Cantoni C, Bollman B, Licastro D, et al. TREM2 regulates microglial cell activation in response to demyelination in vivo. Acta Neuropathologica. 2015;129(3):429-447. doi:10.1007/s00401-015-1388-1 2. Nutma E, Ceyzériat K, Amor S, et al. Cellular sources of TSPO expression in healthy and diseased brain. European Journal of Nuclear Medicine and Molecular Imaging. 2021;49(1):146-163. doi:10.1007/s00259-020-05166-2 3. Bartos LM, Kunte ST, Beumers P, et al. Single-Cell radiotracer allocation via immunomagnetic sorting to disentangle PET signals at cellular resolution. Journal of Nuclear Medicine. 2022;63(10):1459-1462. doi:10.2967/jnumed.122.264171