Structural and functional characterisation of Alzheimer's disease risk factor TREM2

Single point mutations in TREM2, a receptor expressed by microglia in the brain, are associated with an increased risk of neurodegeneration, including Alzheimer’s disease. Numerous studies support a role for TREM2 in sensing damaging stimuli and triggering signalling cascades necessary for neuroprotection. TREM2 contains an immunoglobulin-like domain followed by a flexible stalk region, a transmembrane domain and a cytoplasmic tail. Despite its significant role, ligands and regulators of TREM2 activation, and the mechanisms governing TREM2-dependent responses and its cleavage from the membrane, remain poorly characterised. Here, I characterise phage display generated single-chain variable antibody fragments (scFvs) to human TREM2 ectodomain. The highest published affinity to human TREM2 was measured for one of the produced antibodies. Co-crystal structures revealed the binding of two scFvs to an epitope on the TREM2 Ig-like domain distal to the putative ligand-binding site. Functional activity was observed for oligomeric scFv species, which inhibited the accumulation of soluble TREM2 shed by transfected HEK293 or THP-1 cells. Treatment of HEK293 cells with fluorochrome-conjugated scFvs and quantification of surface TREM2 revealed scFv-induced receptor endocytosis as the likely mechanism of action in these cells. Furthermore, the multivalent scFv led to the activation of a TREM2 signalling cascade measured by phosphorylation of spleen tyrosine kinase in human iPSC-derived macrophages (iMac) and THP-1 cells. Similar activation in iMac cells was achieved upon conversion to monoclonal antibodies, with agonist values exceeding those in the literature. Further studies in combination with physiological ligands, such as apoptotic neurons, need to be performed to probe the overall effect on the TREM2 pathway and microglial activity in models of AD. The detailed characterisation of the epitopes and properties of these antibodies should facilitate the use of these renewable binders as structural and functional biology tools for TREM2 research.