Anti-Human Abeta Recombinant Antibody (Gantenerumab) (CAT#: TAB-229)

Figure 1 Immunohistochemical localization of gantenerumab.

Electron micrographs demonstrate binding of gantenerumab to A fibrils within amyloid- plaques from an AD patient (a) a PS2APP mouse (b) and synthetic A 1-42 fibrils (c). Gantenerumab was detected by a secondary goat anti-human antibody conjugated to 10 nm colloidal gold. Immunofluorescence staining of gantenerumab at 7 nM (1g/mL) revealed specifically diffuse and dense-cored amyloid- plaques in the frontal cortex of an AD patient (d) and PS2APP mouse (e). Staining of a human AD brain section with gantenerumab at 0.07 nM (10 ng/mL) demonstrated sensitive reactivity with a dense-core A plaque (f). Controls with an unrelated human IgG1 antibody were negative for human amyloid- plaques (g), A 1-42 fibrils (h) and immunofluorescence staining of human (left) and PS2APP (right) brain tissue (i). Bars on electron microgaphs in a, b, c, g and h represent 200 nm. Bars in fluorescence micrographs, d, e and i represent 200 m except 20 m in f.
g

Bohrmann, B., Baumann, K., Benz, J., Gerber, F., Huber, W., Knoflach, F.,... & Rothe, C. (2012). Gantenerumab: a novel human anti-Aβ antibody demonstrates sustained cerebral amyloid-β binding and elicits cell-mediated removal of human amyloid-β. Journal of Alzheimer's Disease, 28(1), 49-69.

Figure 2 Cellular phagocytosis of human amyloid- plaques.

Human AD brain sections were preincubated with gantenerumab followed by cell culturing in presence of primary human macrophages as effector cells. Plaques were labeled afterwards with an anti-A antibody (BAP2) conjugated to Alexa488. Brain section without gantenerumab shows the distribution of numerous amyloid- plaques in the grey matter of the frontal cortical lobe after culturing with human macrophages (a) and a concentration-dependent decrease of amyloid- plaques when gantenerumab was added (b and c). Plaques were slightly reduced after preincubation with gantenerumab at 0.07 nM (10 ng/mL) (b) and substantially at 7 nM (1,000 ng/mL) (c). Control section preincubated with gantenerumab 35 nM (5,000 ng/mL) in the absence of cells indicated ability of BAP2 to label amyloid- plaques (d). Quantitative analysis of the experiment shown in (a–c) revealed a significant concentrationdependent reduction of amyloid- plaques, while control with unrelated human IgG1 antibody is inactive (e). Determination of EC50 showed a consistent plaque clearance by macrophages from two different human donors in additional independent experiments (f). Double-staining of human macrophages for A and gantenerumab (g–j) and A together with the lysosomal marker LAMP2 (k–n) indicated internalization of AD brain derived amyloid- into vesicles. Differential interference contrast (DIC) images are shown for comparison to depict individual cells. The arrow in j points to a macrophage cell with numerous internalized complexes of colocalized A and gantenerumab. Bars represent 300 m in a–d, 5 m in g–j and 3 m in k–n. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Bohrmann, B., Baumann, K., Benz, J., Gerber, F., Huber, W., Knoflach, F.,... & Rothe, C. (2012). Gantenerumab: a novel human anti-Aβ antibody demonstrates sustained cerebral amyloid-β binding and elicits cell-mediated removal of human amyloid-β. Journal of Alzheimer's Disease, 28(1), 49-69.

Figure 3 Cerebral amyloid- plaque binding of gantenerumab in PS2APP mice.

High resolution confocal microscopy image of gantenerumab binding at amyloid- plaque after a single iv injection of 20 mg/kg and imaging 3 days postdose (a-c). Gantenerumab was detected as described in Fig. 2. Structural details are shown after image deconvolution with strong reactivity of gantenerumab bound to an amyloid- plaque (a) counterstained with anti-A antibody BAP2 (b) demonstrating the amyloid- specificity by the high degree of colocalization shown in the merged image (c). Gantenerumab binds dose-dependent to plaques as shown after one week iv bolus at indicated doses (d–f). Triple labeling of gantenerumab, A and anti- I-A/I-E-antibody revealed microglia cells adjacent to gantenerumab bound to A deposits (g). At higher magnification the close proximity of a microglia cell to a gantenerumab-reactive plaque (h) can be appreciated and is depicted in a volumetric visualization (i) of the microglia cell shown in h. Evidence for internalized gantenerumab that colocalized with A in the cytoplasm of a microglia cell is shown in three different planes of view (j). The pharmacokinetic profile of gantenerumab bound to cerebral plaques showed sustained binding in PS2APP mice (k). Values represent mean fluorescence intensity at plaques in the occipital cortex over 9 weeks after a single iv bolus dose at 20 mg/kg. Error bars are ± SD (n = 3 animals per time point). The sustained binding of gantenerumab to plaques is markedly different from its peripheral mean plasma levels (dotted line). Bars represent 25 m in a-i and 5 m in j.

Bohrmann, B., Baumann, K., Benz, J., Gerber, F., Huber, W., Knoflach, F.,... & Rothe, C. (2012). Gantenerumab: a novel human anti-Aβ antibody demonstrates sustained cerebral amyloid-β binding and elicits cell-mediated removal of human amyloid-β. Journal of Alzheimer's Disease, 28(1), 49-69.

Figure 4 PS2APP mice treated with gantenerumab for 5 months.

Plaque bearing PS2APP mice were treated by weekly iv injections of gantenerumab at 20 mg/kg. Reduction of A plaques was seen after treatment with gantenerumab (a) compared with vehicle (b). Quantitative morphometry after immunohistochemical staining of A plaques is shown for the hippocampus (c), neocortex (d) and thalamus (e). Plaque load of untreated animals sacrificed at an age of 5 months is shown for comparison as baseline level of amyloidosis at study begin (day 0, n = 15). A significant
reduction in plaque number is evident after treatment with gantenerumab (n = 12), compared to the progressive plaque formation seen in vehicle treated animals (n = 14). A significant clearance activity of gantenerumab was seen specifically for smaller amyloid deposits up to a size of 400 m2 as shown for the hippocampal region (f). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Bohrmann, B., Baumann, K., Benz, J., Gerber, F., Huber, W., Knoflach, F.,... & Rothe, C. (2012). Gantenerumab: a novel human anti-Aβ antibody demonstrates sustained cerebral amyloid-β binding and elicits cell-mediated removal of human amyloid-β. Journal of Alzheimer's Disease, 28(1), 49-69.

Figure 5 Gantenerumab attenuates the inhibition of long-term potentiation (LTP) by A 42 in rat CA1 in vivo.

After stable baseline recordings were obtained, rats were injected icv with 5 L of vehicle or 10 nM A 42 oligomers in the absence or presence of gantenerumab or control
anti-A antibody 6E10 at 1 g/L. One hour later, high frequency stimulation (HFS) was issued that induced LTP in vehicle treated animals and in animals injected with soluble A 42 oligomers co-injected with gantenerumab or 6E10. Histograms represent normalized, mean fEPSP slopes ± SEM of 5-6 animals normalized to the 10-min period before the HFS. For comparison, mean fEPSP values are shown 5 min (a) and 150 min (b) after HFS with a significant blocking of LTP induction seen after injecting A 42 oligomers after 150 min. Co-injection of gantenerumab or 6E10, effectively attenuated the A 42 oligomers induced inhibition of LTP induction. *p ≤ 0.05.

Bohrmann, B., Baumann, K., Benz, J., Gerber, F., Huber, W., Knoflach, F.,... & Rothe, C. (2012). Gantenerumab: a novel human anti-Aβ antibody demonstrates sustained cerebral amyloid-β binding and elicits cell-mediated removal of human amyloid-β. Journal of Alzheimer's Disease, 28(1), 49-69.

Figure 6 Efficacy of mono- and combination treatments on brain amyloid burden and plaque number.

Animals were treated with BACE inhibitor RO5508887 at either 30 or 90 mg/kg daily (BI30, BI90), with anti-Aβ antibody gantenerumab (Gant) at 20 mg/kg weekly or with both compounds (BI30+Gant, BI90+Gant). A–D, Fluorescence images of entire sagittal brain sections after staining of amyloid plaques at study end. Representative images are shown after vehicle-treatment (A), treatment with BI90 (B), gantenerumab (C), and gantenerumab combined with BI90 (D). Notably, plaque reduction is most substantial in the animals that received both drugs. E, The quantification and statistical evaluation of the area surface occupied by plaques across all treatment groups for cortex and hippocampus. Decrease of plaque surface is significant while variable in treatment groups compared with vehicle-treated mice. The combination treatment significantly enhances the amyloid-plaque lowering for both the low- and the high-dose of RO5508887 in the cortex. F, Effect of mono- and combination treatments on total plaque number. Significantly enhanced efficacy by combination treatment is seen in both brain regions examined. Bars represent the mean of 10–4 animals (+SD).

Jacobsen, H., Ozmen, L., Caruso, A., Narquizian, R., Hilpert, H., Jacobsen, B.,... & Bohrmann, B. (2014). Combined treatment with a BACE inhibitor and anti-Aβ antibody gantenerumab enhances amyloid reduction in APPLondon mice. Journal of Neuroscience, 34(35), 11621-11630.

Figure 1 Anti-Human Abeta Antibody (TAB-229) in ELISA

ELISA analysis of TAB-229 was performed by coating with recombinant human Beta-amyloid 42 with His & GST Tag (10703-H20E2) (1 μg/mL). Then blocked with BSA and incubated with Anti-Human Abeta Antibody (TAB-229) at a starting concentration of 2.5 ng/mL. The HRP-conjugated goat anti-human IgG as a secondary antibody. Detection was performed using TMB substrate and stopped with sulfuric acid. The absorbances were read on a spectrophotometer at 450 nm.

Figure 2 Anti-Human Abeta Antibody (TAB-229) in WB

Western blot analysis was performed by loading 1 µg (lane 1, Non-Reduced) and 1 µg (lane 2, Reduced) recombinant human Beta-amyloid 42 with His & GST Tag (10703-H20E2) onto a 12% Tris-HCl polyacrylamide gel. Proteins were transferred to a CN membrane and blocked with 5% skim milk for at least 1 hour. Membranes were probed with Anti-Human Abeta Antibody (TAB-229) at a concentration of 1 μg/mL overnight at 4°C on a rocking platform. Then probed with Goat anti-Human IgG HRP secondary antibody at a dilution of 1:8,000 for one hour. Chemiluminescent was detected.

Figure 3 Anti-Human Abeta Antibody in DB

Dot Blot analysis of TAB-229 was performed by coating with Human Abeta Protein (His Tag).