1. New tools for the diagnosis of Alzheimer's disease by MRI:
Llama antibody conjugates targeting amyloid plaques
M. Vandesquille1-2, T. Li2-3, C. Po1-2, F. Koukouli2, C. Ganneau2, C. Czech4, C. Duyckaerts3,U. Maskos2, B. Delatour3, S. Bay2, P. Lafaye2, M. Dhenain1
1MIRCen, CEA, Fontenay-aux-Roses, France, 2Institut Pasteur, Paris, France, 3ICM, Hôpital de la Pitié Salpêtrière, Paris, France, 4Hoffman LaRoche, Basel, Suisse
Contacts:
Specificity against amyloid plaques
We have obtained a VHH targeting specifically the Aβ peptide, and able to recognize amyloid plaques in human and mouse tissues.
Site specific conjugation
This approach is a versatile strategy to produce well defined compounds without major modification of the VHH properties.
Blood brain barrier crossing
This VHH, once linked to an AlexaFluor488 could be visualized in vivo after iv injection by two-photon microscopy. Our results demonstrate that our VHHs are able to cross the BBB rapidly and to specificallly label amyloid plaques.
MRI visualisation of amyloid plaques
Brains of PS2APP mice incubated with the VHH linked to DOTA/Gd show hypointense spots colocalized with amyloid plaques on MR images.
Moreover, after iv injection, our agent is able to cross the BBB and to induce local hypointensity colocalized with amyloid plaques.
Conclusion
Altogether, our results highlight the potential of VHH to cross the BBB and to detect amyloid plaques in vivo.
Context: In Alzheimer’s disease, amyloid (Aβ) plaques occur up to 20 years prior to dementia [1]. Even if few magnetic resonance imaging (MRI) contrast agents have been developed to detect these plaques, none of them are transposable to human because of their inability to cross the blood brain barrier (BBB) [2, 3, 4].
Rationale: Heavy chain only antibody fragments (VHH) are nanobodies that can cross the BBB [5]. Thus, VHH targeting amyloid plaques could be used for in vivo MR imaging of these lesions.
Aim: The present work describes the production of a VHH (and its conjugates) specifically targeting amyloid plaques, the description of their ability to cross the BBB, and their use to detect amyloid plaques by MRI.
Context & Aim
Results
Discussion
Validation of amyloid plaques specificity by IHC on human and mouse tissue
Proof of concept of MRI labelling after in vitro incubation
Site specific conjugaison
Patent: EP
Patent: EP
VHH
LCMS monitoring
Animals: PS2APP mice (20-22 month-old) and their control littermates.
Two-photon experiments: a 1mm2 skull window was realized and protected with a cover glass. Mice (n=4) were injected in the tail vein with VHH-S-AF488 (20mg/kg), imaged for 4 hours and then sacrificed for correlative IHC.
MRI experiments:
In vitro: brains (n=2) incubation with VHH-S-(DOTA/Gd)3 (0.1 mg/ml in PBS + Triton 0.2%) overnight, followed by 6x30min wash-out before MR images (7tesla, 25*25*100µm, TR/TE=40/15ms, α=20°, Acq time=11h39min).
Ex vivo: mice (n=2) were iv injected with VHH-S-(DOTA/Gd)3 (20mg/kg) and sacrificed after 4 hours. Brains were then extracted prior to MR acquisition (11.7tesla, 50µm isotropic, TR/TE=40/12ms, α=20°, Acq time=1h29min).
Each acquisitions were compared to the same brain imaged after a Gd-staining procedure to reveal amyloid plaques (positive control) [3]. Negative controls were PS2APP mice injected with PBS.
Immunohistochemistry: paraffin sections were performed (4µm). The VHH was revealed by a secondary anti-His antibody, and positive controls were performed on adjacent brain sections with a reference antibody (4G8).
Materials & Methods
MWth =
Δ=31ppm
Evaluation of BBB crossing after iv injection by two-photon microscopy
MR visualisation of amyloid plaques on ex vivo images after iv injection
anti-Aβ VHH with Cter Cysteine
+ Maleimide - AlexaFluor488
+ Maleimide - (DOTA/Gd)3
MWth =
Δ=38ppm
MWth =
Δ=33ppm
References: [1] Villemagne et al. Lancet Neurol 12(4): (2013); [2] Ramakrishnan et al. Phar Res 25(8): (2008); [3] Petiet et al. Neurobiol Aging 33(8): (2012); [4] Wadghiri et al, Plos One 8(2):e57097 (2013); [5] Li et al. FASEB 26(10): (2012).
=> Two-photons imaging
=> MR imaging