NeuroMab™ Anti-Tau Oligomer BBB Shuttle Antibody(NRZP-1022-ZP3131)

Figure 1 depicts the characteristics of the TOC1 monoclonal antibody.

FIG. A shows dot blot demonstrating TOC1 immunoreactivity. It shows that TOC1 preferentially (i.e., selectively) labels uncross-linked Tau oligomeric and filamentous aggregates prepared with AA as opposed to unaggregated Tau (−AA). Moreover, it shows that TOC1 did not react with either α-synuclein (αS) or Aβ in the monomeric, oligomeric, or filamentous states. 45 ng/spot was applied to the nitrocellulose. Total Tau was determined using Tau12. FIG. B shows quantification of TOC1 and Tau12 immunoreactivity at varying Tau concentrations. Whereas, Tau12 (gray dashed line) demonstrates a high affinity for both unaggregated (0) and aggregated (●) Tau, TOC1 (black solid line) reacts exclusively with Tau aggregates. Each point represents a minimum of three independent measurements. FIG. C shows EMs of Tau oligomers and filaments (Tau Olig+Fil), α-synuclein oligomers and filaments (αS Olig+Fil), Aβ oligomers (Aβ Olig), and Aβ filaments (Aβ Fil) confirm the generation of aggregates of the appropriate morphology for each protein. Scale bar is 200 nm. FIG. D shows that TOC1 preferentially reacts with Tau dimers on dot blots. Monomeric and dimeric Tau were isolated from the same aggregation reaction. Also included is an electroeluted monomeric sample that was never exposed to AA (−AA). 12 ng/spot was applied to the nitrocellulose.

Figure 2 depicts TOC1 preferentially labeling Tau oligomers (i.e., distinct 180-kDa Tau oligomers).

FIG. A shows TOC1 immune-gold labeling which revealed preferential labeling of oligomeric structures. Labeling of the ends of a few filaments was observed as well (arrow). FIG. 6B shows higher magnification of TOC1 immunogold labeling. FIG. C is immunogold labeling of the poly-His tag which revealed abundant labeling of both Tau oligomers and filaments. FIG. D is quantification of TOC1 immunogold labeling of filaments (>50 nm) and oligomers (<50 nm) relative to His tag immune-gold labeling of structures of the same category.*, p

Figure 3 depicts epitope mapping of TOC1. Deletion mutants of hTau40 were assembled with AA, spotted on nitrocellulose, and probed with TOC1.

FIG. A is schematic representations of the deletion mutants utilized. FIG. B is EM of wild type (WD, A9-155, A273-305, A144-273, A291-349, and A321-441). hTau40 was used to confirm the presence of aggregates. The morphological characteristics of the other deletion mutants were described elsewhere (30, 36, 37, 53). Scale bar is 500 nm. FIG. C is immunoreactivity of TOC1 to deletion mutants of hTau40 expressed as the ratio of TOC1:total Tau. Total Tau was measured with eitherTau12 or a H is probe antibody. Results were normalized to WT hTau40. FIG. D is the discontinuous epitope of TOC1, its preferential binding (e.g., selective binding) to dimeric over monomeric Tau are consistent with an antiparallel dimer conformation. The gray shaded box represents the MTBR, and the proposed TOC1 epitope is circled.

Figure 4 depicts elevated TOC1 immunoreactivity in AD.

FIG. A shows TOC1 preferentially (i.e., selectively) labeling Tau in AD brains, compared with that from controls in non-denaturing conditions on dot blots (580 ng/spot), indicating that Tau in the TOC1 conformation is more abundant in AD. FIG. B is a quantification of TOC1 labeling in extracts from the frontal cortex of control and AD brains in A represented as TOC1:Tau12 ratios.*, p<0.01, unpaired t test. FIG. 8C depicts that the superior temporal gyrus (STG) does not contain TOC1 immunoreactive Tau pathology in control cases (Braak I and II). FIG. D depicts that, in the entorhinal cortex (EC) of control cases, TOC1 labels early pretangle neurons with staining extending into both apical and basal dendritic processes. FIG. E is higher magnification of TOC1-labeled pretangle neuron in the EC from a control case and reveals that these neurons do not contain mature compact NFTs. FIG. F shows that TOC1 labels abundant pathology in the STG of AD cases (Braak V. and VI), including neuropil threads, neuritic plaques, as well as pretangle and tangle-bearing neurons. FIG. G shows that, in the EC of AD cases, TOC1 labels similar pathology in addition to neurons that have lost their dendritic processes indicating that they are further along in the process of tangle evolution. FIG. H is a higher magnification of TOC1 staining of the EC in AD. FIG. I is a low magnification TOC1 immunostaining of the hippocampus of an AD case. FIG. J shows that TOC1 immunostaining in the CA1 region of the hippocampus in AD reveals flame-shaped inclusions within pyridimal neurons that are characteristic of this region. FIG. K is a higher magnification of TOC1 immunolabeling of a neuritic plaque and pyramidal neurons in the CA1 region of an AD case. Scale bars are 50 μm.

Figure 5 depicts TOC1 immunoreactivity co-localized with early markers of Tau pathology.

FIGS. A and B show laser scanning confocal microscopy used to determine the degree of colocalization between TOC1 (green) and pS422 (red) in the entorhinal cortex. Colocalization between the two antibodies was almost complete in both control (A) and AD (B) cases, indicating that formation of the TOC1 epitope correlates with phosphorylation of Ser422, an early event in AD pathogenesis. FIG. C shows immunofluorescence which was performed using TOC1 (green) and MN423 (red) in severe AD cases. Very little colocalization was observed. FIG. D shows that, in severe AD cases, TOC1 and TR did not colocalize, indicating that the TOC1 epitope precedes β-sheet formation characteristic of NFTs. Although occasional inclusions with both TOC1 and TR exist (arrow), different portions of the cell are labeled by each. Scale bar is 50 μm.

Figure 6 depicts TOC1 preferentially labeling tau oligomers purified by sucrose gradient density fractionation (fraction 3).

Figure 7 depicts TOC1 preferentially labels tau oligomeric tau rather than filamentous or unaggregated tau.

FIGS. A-C are negatively stained EM images of tau in the absence of arachidonic acid (−AA) or in its presence after 15 minutes or 6 hours of aggregation. FIGS. D-F show TOC1 Immunogold labeling with negatively stained EM on the same aggregate preparations. Scale bar=200 nm.