In Vivo Detection of Amyloid-β Deposits Using Heavy Chain Antibody Fragments in a Transgenic Mouse Model for Alzheimer's Disease

Abstract

This study investigated the in vivo properties of two heavy chain antibody fragments (VHH), ni3A and pa2H, to differentially detect vascular or parenchymal amyloid-β deposits characteristic for Alzheimer's disease and cerebral amyloid angiopathy. Blood clearance and biodistribution including brain uptake were assessed by bolus injection of radiolabeled VHH in APP/PS1 mice or wildtype littermates. In addition, in vivo specificity for Aβ was examined in more detail with fluorescently labeled VHH by circumventing the blood-brain barrier via direct application or intracarotid co-injection with mannitol. All VHH showed rapid renal clearance (10–20 min). Twenty-four hours post-injection 99mTc-pa2H resulted in a small yet significant higher cerebral uptake in the APP/PS1 animals. No difference in brain uptake were observed for 99mTc-ni3A or DTPA(111In)-pa2H, which lacked additional peptide tags to investigate further clinical applicability. In vivo specificity for Aβ was confirmed for both fluorescently labeled VHH, where pa2H remained readily detectable for 24 hours or more after injection. Furthermore, both VHH showed affinity for parenchymal and vascular deposits, this in contrast to human tissue, where ni3A specifically targeted only vascular Aβ. Despite a brain uptake that is as yet too low for in vivo imaging, this study provides evidence that VHH detect Aβ deposits in vivo, with high selectivity and favorable in vivo characteristics, making them promising tools for further development as diagnostic agents for the distinctive detection of different Aβ deposits.

Fusion of hIgG1-Fc to 111In-anti-amyloid single domain antibody fragment VHH-pa2H prolongs blood residential time in APP/PS1 mice but does not increase brain uptake

“…The immunoreactivity is highly similar to conventional monoclonal anti-Aβ antibodies (e.g. clone 4G8) and to the specificity previously shown by unmodified VHH-pa2H [7,8,10,30]. Addition of the chelator DTPA to VHH-pa2H-Fc did not impair the ability to recognize and bind Aβ.…”

“…All animal studies have been approved by the Leiden University Medical Center institutional Animal Ethics Committee (DEC permits 10097 and 12065). Besides standard genotyping, Aβ pathology was confirmed on brain sections by standard Thioflavin T staining [10].…”

“…Monovalent VHH may not have enough time to interact with hard-to-reach epitopes or to cross regulate endothelial barriers -such as the blood brain barrier (BBB) -in sufficient amounts [8]. They may be rapidly filtered out of the system, unless they are injected at very high doses, continuously via infusion or the BBB is locally impaired [9,10]. Even though for imaging applications short half-lives are interesting [11], for systemic immunotherapeutic applications, elongated serum half-lives of VHH are preferred.…”

New Applications of Nanotechnology for Neuroimaging

“…[60][61][62][63][64] Nanobodies can detect vascular and parenchymal amyloid beta (A␤) deposits in vivo to differentiate cerebral ␤-amyloid indicative of Alzheimer disease and vascular A␤ plaques associated with cerebral amyloid angiopathy. 65 With the transgenic Alzheimer Disease Cerebral Amyloid Angiopathy (AD/CAA) mouse model, 2 distinct A␤ targeting nanobodies, ni3A and pa2H, were administered to evaluate their ability in vivo to differentially detect vascular or parenchymal amyloid-␤ deposits and their ability to cross the blood-brain barrier. In vivo specificity for A␤ was evaluated in this mouse model by direct topical application or intracarotid coinjection with mannitol with a fluorescently labeled nanobody.…”

“…Limited in vivo BBB passage was postulated by the authors to be due to low concentrations of nanobodies, low circulation time, or possibly a paucity of active transport receptors involved in BBB transport in this mouse model. 65 Future methods, such as nanobodies encapsulated in polysorbate 80 -coated PBCA nanoparticles, could be used to increase in vivo BBB passage.…”