Crystal structure of a conformational antibody that binds tau oligomers and inhibits pathological seeding by extracts from donors with Alzheimer's disease

Abstract

Soluble oligomers of aggregated tau accompany the accumulation of insoluble amyloid fibrils, a histological hallmark of Alzheimer disease (AD) and two dozen related neurodegenerative diseases. Both oligomers and fibrils seed the spread of Tau pathology, and by virtue of their low molecular weight and relative solubility, oligomers may be particularly pernicious seeds. Here, we report the formation of in vitro tau oligomers formed by an ionic liquid (IL15). Using IL15-induced recombinant tau oligomers and a dot blot assay, we discovered a mAb (M204) that binds oligomeric tau, but not tau monomers or fibrils. M204 and an engineered single-chain variable fragment (scFv) inhibited seeding by IL15-induced tau oligomers and pathological extracts from donors with AD and chronic traumatic encephalopathy. This finding suggests that M204-scFv targets pathological structures that are formed by tau in neurodegenerative diseases. We found that M204-scFv itself partitions into oligomeric forms that inhibit seeding differently, and crystal structures of the M204-scFv monomer, dimer, and trimer revealed conformational differences that explain differences among these forms in binding and inhibition. The efficiency of M204-scFv antibodies to inhibit the seeding by brain tissue extracts from different donors with tauopathies varied among individuals, indicating the possible existence of distinct amyloid polymorphs. We propose that by binding to oligomers, which are hypothesized to be the earliest seeding-competent species, M204-scFv may have potential as an early-stage diagnostic for AD and tauopathies, and also could guide the development of promising therapeutic antibodies.

Keywords: Alzheimer disease; amyloid; antibody; antibody engineering; fibril; inhibitor; neurodegeneration; neurodegenerative disease; oligomerization; prion; protein aggregation; protein crystallization; protein structure; tau; tauopathy.

Figure 1.

IL15 induces formation of tau-K18 oligomers and amyloid fibrils. A, aggregation kinetics of tau-K18 induced with 0.225 mg/ml of heparin (blue), 2% (w/v) IL15 (green), or 2% (w/v) IL23 (magenta), measured by fluorescence of ThT dye at 480 nm. B, separation of tau-K18 monomer and oligomer after 16-18 h of incubation and shaking with IL15, as in A, by SEC using a HiLoad 16/600 Superdex 75 pg column. C, immunoblot analysis of tau-K18 oligomer and monomer fractions from the SEC peaks using anti-oligomer A11 polyclonal antibody. D–F, negative stain electron micrographs of tau-K18 (D) monomer, (E) oligomer, and (F) fibril prepared with IL15. G, X-ray fibril diffraction from tau-K18 fibrils prepared with IL15. H, HEK293 tau biosensor cells expressing YFP-tagged tau-K18 and seeded with 1.5 nm tau-K18 oligomer. I, as in H, except seeded with 1.5 nm tau-K18 monomer. J, HEK293 tau biosensor cells without addition of tau seed. Representative cells that contain aggregates are marked by red arrows, and cells without by white arrows. K, quantification of seeding from representative images shown in H–J. Statistical analysis was performed using a one-way ANOVA (**, p < 0.005, n.s., no significance) and Tukey's multiple comparison in GraphPad Prism. Error bars show the S.D. of three replicates.

Figure 2.

Tau oligomer purification and antibody binding. A, schematic representation of full-length tau (tau40) and the tau-K18 construct containing the microtubule binding domains R1–R4. B, S75 16/600 SEC purification of tau-K18 oligomer prepared as described in the legend to Fig. 1 with IL15. C, dot blot of chromatographed tau-K18 oligomer and monomer fractions (left set of four), and of purified tau-K18 monomer, tau40 monomer, and tau40 fibril (right set of 3). Only the oligomer peak shows immunoreactivity with M204. D, ELISA showing immunoreactivity of monoclonal M204 antibody with chromatographed S75 fractions containing tau-K18 oligomer (fractions 44-49, colored red), but not tau monomer (fractions 58-61, colored black). A volume of 20 µl of the indicated SEC fraction was diluted into 100 µl of coating buffer and applied to ELISA plate. In fractions 46 and 61 (the highest peaks), 20 µl = 100 ng of protein/well. E, S200 10/300 SEC purification of oligomeric tau40 prepared with IL15. F, ELISA showing immunoreactivity of monoclonal M204 with chromatographed S200 fractions containing tau40 oligomer (fractions 10-15, colored red) but not tau monomer (fractions 17-19, colored black). A volume of 50 µl of the indicated SEC fraction containing tau40 oligomer (fractions 8-15) were diluted into 100 µl of coating buffer. For tau40 monomer fractions 17 and 19, 10 µl was used to coat ELISA plates rather than 50 µl because the peak monomer fraction was about 5 times the intensity of the oligomer peak. For reference, the amount of protein used to coat the ELISA plate in fractions 13 and 19 (the highest peaks) was 100 ng of protein. Error bars show the S.D. of triplicate measurements.

Figure 3.

M204 binds to aggregation-driving segments in synthetic, recombinant tau and AD brain-derived tau. A, epitope mapping using a peptide array with overlapping synthetic tau peptides shows M204 binding most strongly to peptides containing the sequences KVQIINK and SVQIVY (in red). B, seeding by IL15-mediated tau-K18 purified oligomers in HEK293 biosensor cells expressing YFP-tagged tau-K18. C, as in B, except following treatment of tau-K18 oligomer with 10 μm M204 antibody. Seeding inhibition by M204 is interpreted based on the appearance of fewer puncta compared with B. Representative cells containing aggregated tau are marked by red arrows, and cells without by white arrows. D, quantification of seeding from representative images shown in B and C. Tau oligomer seeding and inhibition by M204 antibody were determined by calculating the number of normalized puncta per well of a 96-well–plate. Statistical analysis was performed using one-way ANOVA (****, p < 0.0001; ***, p < 0.0006; **, p < 0.003) and Tukey's multiple comparison in GraphPad Prism. Error bars show the S.D. of three replicates. E, Western blotting of tissue from the brain of a donor with AD, fractionated into: crude brain homogenate, Sarkosyl-insoluble, and Sarkosyl-soluble fractions, each probed with M204 or A11 anti-oligomer antibodies.

Figure 4.

M204-scFv purifies as a mixture of monomer and oligomers. A, schematic comparing a full-length mAb and the scFv fragment. B, sequence of M204-scFv. The VH and VL variable domains are colored blue and green, respectively, and the sequences of the CDRs loops are bolded and underlined. The VH and VL fragments are connected by a flexible linker with sequence (Gly₄Ser)₃. C, S75 SEC of M204-scFv overlaid with gel filtration standards (in red) showing apparent monomer, dimer, and trimer M204-scFv species, as judged by comparison to the SEC protein standards. D, SDS-PAGE analysis of monomeric, dimeric, and trimeric M204-scFv fractions, run under reducing and nonreducing conditions, as indicated, showing high molecular weight bands that correspond to the dimeric and trimeric species (marked by black arrows) under nonreducing conditions.

Figure 5.

M204-scFv antibody delays in vitro aggregation. A, SEC purification of tau-K18 oligomer using a S200 10/300 column. The protein elution fractions were also monitored by absorption at 280 nm and presented in Fig. S7. B–D, ELISA showing immunoreactivity of the corresponding tau-K18 oligomer fractions from A with M204-scFv (B) monomer, (C) dimer, and (D) trimer. E–G, M204-scFv mediated inhibition of tau-K18 aggregation. Aggregation was measured by ThT fluorescence with M204-scFv added at molar ratios of 1:1 and 1:0.5 (tau-K18: M204-scFv), as indicated. Inhibition of IL15-induced tau aggregation by M204-scFv is shown for the M204-scFv monomer (E), dimer (F), and trimer (G). Tau-k18 is a positive control that was run in parallel but without the addition of M204-scFv, and is the same in aggregation experiments shown in E–G, colored black. Error bars represent the mean ± S.D. of three replicates.

Figure 6.

M204-scFv antibodies inhibit the seeding of tau aggregation by autopsied brain extracts from three human AD brain patients. A, negative stain electron micrograph of fibrils purified from human AD brain tissue. Scale bar, 200 nm. Zoom view shows a single fibril with a twist of two protofilaments, with the appearance of a paired helical filament. B–E, quantification of the inhibitory effect of M204-scFv antibodies on inhibition of seeding by Sarkosyl-insoluble fractions from AD brain tissue, measured in HEK293 biosensor cells expressing YFP-tagged tau-K18. Statistical analysis was performed using one-way ANOVA (****, p < 0.0001; ***, p < 0.0002; **, p < 0.001; *, p < 0.01, n.s., no significance) followed by a Tukey's multiple comparison test in GraphPad Prism. Error bars show the S.D. of three replicates. F, representative images of seeding and inhibition in HEK293 biosensor cells expressing YFP-tagged tau-K18. Cells seeded with the Sarkosyl-insoluble fraction from AD Donor 1 without pre-treatment with M204-scFv (left panel) or no inhibitor (No I), and following overnight incubation with M204-scFv trimer, dimer, or monomer (as indicated). The seeding can vary dramatically from different regions of a given brain tissue section, and this reflects a naturally occurring nonuniform distribution of tau pathology in the brain. Representative cells that contain aggregates are marked by red arrows, and cells without by white arrows.

Figure 7.

A–D, tests of the effectiveness of M204-scFv for inhibition of tau fibril seeding by diseased brain extracts in our HEK293 biosensor cell-based assay. M204-scFv dimer and trimer inhibit seeding by extract from (A) CTE Donor 1, but not (B) CTE Donor 2, (C) CBD donor, or (D) a donor with a P301L familial mutation. Statistical analysis was performed using one-way ANOVA (****, p < 0.0001; ***, p < 0.0008; **, p < 0.005; *, p < 0.01, n.s., no significance) followed by a Tukey's multiple comparison test in GraphPad Prism. Error bars show the S.D. of three replicates.

Figure 8.

Crystal structures of M204-scFv monomer, dimer, and trimer antibodies. A–C, structures of M204-scFv monomer (A), dimer (B), and trimer (C), showing the positions of the complementarity-determining region (CDR) loops from the heavy (H) and light (L) chains. The structure of the M204-scFv monomer is colored pink, and in B, the dimer is formed between protomers colored pink and cyan. In C, the interaction of the protomers that form the dimer (colored in pink and cyan) is conserved, and a third protomer that interacts to form a trimer is colored in green. For each species in A–C, the CDR loops are solvent exposed, and oligomerization of the antibody could thus, in principle, enhance its interaction with the tau oligomer by polyvalent interactions. D, ribbon diagram of M204-scFv showing the electron density and proximity of cysteine 201 from two adjacent protomers, with the potential to form a disulfide bond (yellow). E, comparison of CDR loops from the heavy chains (H1, H2, and H3) of the M204-scFv monomer (pink), dimer (cyan) and trimer (green) species, and (F) CDR loops of the light chains (L1, L2, and L3) with the same coloring scheme.