Targeting tau neuronal internalization using anti-tau nanobodies

TARGETING TAU NEURONAL INTERNALIZATION USING ANTI-TAU NANOBODIES® Clément Danis1,2,3*, Elian Dupré1,2, Thomas Bouillet3, Marine Denéchaud3, Camille Lefebvre3, Marine Nguyen1,2, Justine Mortelecque1,2, François-Xavier Cantrelle1,2, Jean-Christophe Rain4, Xavier Hanoulle1,2, Morvane Colin3, Luc Buée3*, Isabelle Landrieu1,2* In Alzheimer's disease, tau pathology spreads across brain regions as the disease progresses. Intracellular tau can be released and taken up by nearby neurons. Here, we evaluated anti-tau nanobodies®, also called VHHs, as inhibitors of neuronal tau internalization. We identified two potent VHH inhibitors of tau uptake: A31 and H3-2. These VHHs compete with the membrane protein LRP1, a major receptor mediating neuronal uptake of monomeric tau. A31 bind to microtubule binding domain repeat 2, which is involved in the interaction with LRP1. VHH H3-2 is the only VHH from our library that reduces the internalization of both monomeric tau and tau fibrils. VHH H3-2 binds a C-terminal tau epitope with high affinity. Its three-dimensional structure in complex with a tau peptide reveals a unique binding mode as a VHH-swapped dimer. These anti-tau VHHs are interesting tools to study tau prion-like propagation in tauopathies and potentially develop novel biotherapies. 1Univ. Lille, Inserm, CHU Lille, U1172 - LilNCog -Lille Neuroscience & Cognition, F-59000 Lille, France, 2CNRS EMR9002 BSI Integrative Structural Biology F-59000 Lille, France, 3Univ. Lille, Inserm, CHU Lille, Institut Pasteur de Lille, U1167 - RID-AGE - Risk Factors and Molecular Determinants of Aging-Related Diseases, F-59000 Lille, France, 4Hybrigenics Services SAS 1 rue Pierre Fontaine, 91000 Evry-Courcouronnes, France Summary ⑤H3-2 dimerizes in interaction with tau in crystals and in solution in vitro ②VHHs A31 and H3-2 block tau cellular uptake in mouse primary neuronal culture Overview of the biochemical characterization (NMR, SPR) of the VHHtau interactions (a, b). Schematic representation of the tau neuronal uptake assays using mouse primary neuronal culture and recombinant monomeric tau1N4R (tau_M) or tau1N4R fibrils (tau_F) (c). X-ray crystal structure of H3-2 with its corresponding Ctertau peptide epitope revealed the formation of H3-2 swapped dimer involving exchange of their CDR3 that adopt a β-strand conformation.Each C-terminus domain framework of the VHH is binding to the other VHH through a β-augmentation mechanism. We have identified by NMR the tau region recognized by H3-2 which is located in the C-terminus domain of tau (a). Interaction of H3-2 with tau fibrils have been validated using nanobodies linked to gold particles and TEM (b). KD and specificity of the interaction were confirmed using SPR with monomeric and fragment of tau containing only the repeat domains (tauRD) (c). SPR associated to steady state analysis confirmed the specific and stong binding between VHH H3-2 and the C-terminal tau peptide (Cter peptide) that corresponds to the binding region determined by NMR (d). We investigated the mechanisms of monomeric tau neuronal uptake inhibition driven by the VHHs, using SPR. We first confirmed the binding of LRP1 cluster domains to tau (a) and the inhibition of this interaction using the RAP protein (b). We then performed competition experiments using tau in absence (black curve) or in presence of increasing concentrations of VHH (yellow orange and red curve). We observed a inhibition effect using VHHs A31 and H32 but not E2-2, suggesting that inhibition of tau internalization by the VHH could be mediated by competition for the interaction between LRP1 and tau (c). ③VHHs A31 and H3-2 compete for the interaction of tau with LRP1 ④VHH H3-2 binds both monomericand tau fibrils and interacts with high afffinity to its tau C-terminal epitope Response (RU) Literature TONIC / NanoTarget H3-2 binding site tau NMR 15N, 1H, HSQC 2D Danis, C. et al. Inhibition of Tau seeding by targeting Tau nucleation core within neurons with a single domain antibody fragment. Mol. Ther. 30, 1484–1499 (2022). Dupré, E. et al. Single Domain Antibody Fragments as New Tools for the Detection of Neuronal Tau Protein in Cells and in Mice Studies. ACS Chem. Neurosci. 10, 3997–4006 (2019). Sensorgram Steady state KD = 0,34µM tau1N4R Cter peptide Cter peptide H3-2 H3-2 Cter peptide CDR3 We also performed size-exclusion chromatography in order to confirm the dimer formation of H3-2 in solution. We identified a major peak corresponding in size to a dimer (34kDa), without or in complex with the peptide, suggesting that H3-2 spontaneously self-dimerize in solution. Finally we also investigated by HTRF the dimerization of VHH H3-2 in the presence of the peptide. Interestingly, HTRF data confirmed that VHH H3-2 dimerization can be induced by the presence of the peptide, which confirmed that H3-2 interaction with tau peptide may enhance and stabilize its dimerization. * Correspondence : [email protected] ; [email protected] ; [email protected] VHH tau epitope kon (M-1.s-1)*10+2 koff (s-1)*10-3 KD(nM) B1-1 PRD 608 ± 18 27.9 ± 0.40 460 ± 15 C3-2 R1 29 ± 0.5 2.5 ± 0.04 857 ± 20 A31 R2 3311 ± 24 12.1 ± 0.06 37 ± 0.1 Z70mut1 R3 1523 ± 30 71.4 ±0.65 468 ± 10 A5-2 R4 618 ± 25 32.4 ±0.62 525 ± 24 F8-2 C-terminal 575 ± 11 42.5 ± 0.39 740 ± 16 E2-2 467 ± 3 4.5 ± 0.04 96 ± 1 H3-2 5544 ± 25 6.0 ± 0.03 11 ± 0.1 ® Nanobody isa trademark of Ablynx/Sanofi tauRD Conclusion From this screen, we identified VHHs A31 and H3-2 as the most potent inhibitors of neuronal internalization of monomeric tau in primary mouse neuronal cultures. VHHs A31 compete with the binding between tau and a LRP1 membrane receptor domain, supporting the importance of the tauRD for binding to LRP1 and in theinternalization process of monomeric tau. VHH H3-2, a C-terminal tau-binding VHH, was the strongest inhibitor of tau uptake in both monomeric and fibrillar forms. We showed that VHH H3-2 forms a swapped dimer through the formation of an intermolecular β-sheet between two subunits, each binding to its C-terminal tau epitope. This binding mode may explain its potent activity in inhibiting tau cellular uptake. These VHHs thus represent alternative immunological tools that can be used to better explore the epitopes and mechanisms of interest for targeting tau propagation, which are critical parametersfor designing the best approach to tau immunotherapy. Danis, C. et al. Inhibition of tau neuronal internalization using anti-tau single domain antibodies. Nat. Commun. 16, 3162 (2025). Percentage of tau neuronal uptake based on fluorescence signal coming from monomeric tau1N4R (tau_M) or tau 1N4R fibrils (tau_F) coincubated with RAP, heparin and the anti-tau VHHs. RAP, VHHs A31 and H3-2 significantly reduced cellular uptake of monomeric tau, while heparin and H3-2 significantly reduced cellular uptake of tau fibrils. These data were confirmed by confocal microscopy analysis. Distinct inhibition effect from RAP and heparin suggest that monomeric tau neuronal entry are principally taken in charge by the LRP1 membrane receptor, while tau fibrils is internalized through their interaction with Heparan Sulfates ProteoGlycans(HSPGs) at the cell membrane surface. ①Materials and assays design a b c tau a b c a b c d Tau_M heparinRAP A31 Z70mut1 H3-2 Tau_F heparinRAP A31 Z70mut1 H3-2 Anti-tau VHHs screening