Single domain antibody‐based non‐invasive in vivo imaging of tau pathology

Abstract

BackgroundNumerous studies have shown that the extent of pathological tau protein in the brain correlates strongly with the severity of Alzheimer’s dementia at the time of death, and similar association with functional phenotypes have been seen in other tauopathies. In recent years, several dye‐based imaging probes with selectivity for tau aggregates have been developed. However, all are β‐sheet binders with varying affinity for different amyloids, and thereby lack specificity for tau lesions. In contrast, single domain antibodies (sdAbs) are specific for their target and because of their small size have much greater access into the brain than standard whole antibodies.MethodTo examine sdAbs’ suitability as non‐invasive in vivo imaging probes for tauopathies, we immunized a llama with a full‐length recombinant tau protein followed by boosters with human brain‐derived pathological tau proteins. Phage display libraries were generated from its peripheral blood mononuclear cells and screened for binding to tau proteins using various assays, resulting in a prototype sdAb deemed promising for in vivo imaging. Subsequently, this anti‐tau sdAb was labeled with a near‐infrared tag (680 nm), and injected intravenously in tauopathy and control mice, followed by imaging using an In Vivo Imaging System (IVIS). After the imaging, brains were extracted for tissue analysis.ResultIntravenous injection of labeled sdAb resulted in a strong in vivo brain signal detected through the intact head in two different models of transgenic tauopathy mice, but not in wild‐type or transgenic α‐synucleinopathy mice. Importantly, the in vivo brain signal correlated strongly with insoluble (r = 0.9839, p < 0.0001) and soluble (human tau (CP27) and phospho‐tau (PHF1): r = 0.9603 and 0.9459, p < 0.0001) tau protein within the brain. Furthermore, postmortem analysis revealed extensive co‐localization of the sdAb imaging probe with tau aggregates within neurons in the endosomal‐lysosomal system, indicating their interaction in these degradation pathways.ConclusionOverall, this specific sdAb imaging ligand has great potential as an in vivo diagnostic marker for Alzheimer's disease and related tauopathies, and sdAb‐based imaging may be applicable to a variety of protein conformational disorders.

Single-Domain Antibody-Based Protein Degrader for Synucleinopathies

“…In the next phase of our study, we assessed the in vivo effectiveness of the unmodified and modified sdAb in an M83 mouse model of synucleinopathy. Previous findings from our diagnostic imaging study indicated that the anti-α-syn sdAbs 2D8 and 2D10 traversed the blood-brain barrier and colocalized with intraneuronal α-syn aggregates after an intravenous injection in this mouse model ( 37 ). The intensity of the sdAb brain signal correlated strongly with the levels of pathological α-syn in the brain, with 2D10 showing a clear superiority over 2D8 in signal intensity, and a slight advantage in degree of correlation and specificity for α-syn lesions.…”

“…Therefore, the half-life of sdAbs in blood is relatively short, typically less than 2 h, rendering them potentially less suitable as therapeutic candidates compared to whole antibodies that usually have blood half-life of 1-3 weeks ( 68 ). However, during our imaging study, we observed that at the time point of 24 and 96 hours following a single intravenous injection of near-infrared-labeled 2D8, its brain signal registered at 47% and 22% of peak signal, respectively ( Supplemental Figure 7 , ( 37 )). This relatively slow brain clearance is likely explained by sdAb binding to α-syn aggregates within neurons.…”

“…Our previous work showed that unmodified sdAb 2D8 readily entered the brain of M83 synucleinopathy mice and bound to pathological α-syn protein intracellularly after a single intravenous (i.v.) injection ( 37 ). To determine whether 2D8-PEG4-T could bind to α-syn protein intracellularly in a similar manner as its unmodified version, primary M83 transgenic neurons were incubated with 10 µg/ml of LBD brain-derived soluble S1 α-syn fraction for 24 h. The culture was then washed and treated with 5 µg/ml of either unmodified sdAb 2D8 or modified sdAb 2D8-PEG4-T for 2 h, followed by immunohistochemistry and analysis by confocal microscopy ( Figure 4E ).…”

“…Furthermore, our previous findings demonstrated that following its i.v. injection, near-infrared-labeled sdAb 2D8 enters the brain and neuronal cells, where it colocalizes with pathological α-syn within the endosome-lysosome system ( 37 ). In addition, the in vivo studies show that acute treatment with unmodified 2D8 clears insoluble α-syn but not soluble α-syn, which supports its efficacy via lysosomal clearance ( Figure 8 ).…”

Anti-tau single domain antibodies clear pathological tau and attenuate its toxicity and related functional defects

“…They are also highly amenable to engineering to increase their half-life and enhance their degradation capabilities. We have described in detail the development of our anti-tau sdAbs by immunizing a llama with recombinant tau and pathological tau derived from a human tauopathy brain (21,22). We have also reported on the e cacy of two of them in tauopathy culture models and by in vivo microdialysis in a mouse tauopathy model, as well as the suitability of one of them as a diagnostic imaging agent in two different tauopathy mouse models (21,22).…”