Anti-Abeta42- and anti-Abeta40-specific mAbs attenuate amyloid deposition in an Alzheimer disease mouse model

Abstract

Accumulation and aggregation of amyloid beta peptide 1-42 (Abeta42) in the brain has been hypothesized as triggering a pathological cascade that causes Alzheimer disease (AD). To determine whether selective targeting of Abeta42 versus Abeta40 or total Abeta is an effective way to prevent or treat AD, we compared the effects of passive immunization with an anti-Abeta42 mAb, an anti-Abeta40 mAb, and multiple Abeta(1-16) mAbs. We established in vivo binding selectivity of the anti-Abeta42 and anti-Abeta40 mAbs using novel TgBRI-Abeta mice. We then conducted a prevention study in which the anti-Abeta mAbs were administered to young Tg2576 mice, which have no significant Abeta deposition, and therapeutic studies in which mAbs were administered to Tg2576 or CRND8 mice with modest levels of preexisting Abeta deposits. Anti-Abeta42, anti-Abeta40, and anti-Abeta(1-16) mAbs attenuated plaque deposition in the prevention study. In contrast, anti-Abeta42 and anti-Abeta40 mAbs were less effective in attenuating Abeta deposition in the therapeutic studies and were not effective in clearing diffuse plaques following direct injection into the cortex. These data suggest that selective targeting of Abeta42 or Abeta40 may be an effective strategy to prevent amyloid deposition, but may have limited benefit in a therapeutic setting.

Figure 1

Specificity of Ab40.1 and Ab42.2. (A) Serial dilutions of Aβ₄₀, Aβ₄₂, and Aβ₃₈ were used to determine the crossreactivity of Ab42.2 by capture ELISA. Ab42.2 was used as capture and Ab9-HRP as detection. (B) Serial dilutions of Aβ₄₀, Aβ₄₂, and Aβ₃₈ were used to determine the crossreactivity of Ab40.1 by capture ELISA. Ab9 was used as capture and Ab40.1 as detection. (C) Schematic depicting the method for capture ELISA of biotinylated mAb–Aβ complexes in plasma. (D) Specificity of Ab42.2 and Ab40.1 in vivo. Biotinylated Ab42.2, Ab40.1, and Ab9 (500 μg) were injected i.p. into TgBRI-Aβ₄₀ and TgBRI-Aβ₄₂ mice (n = 3 per group). Seventy-two hours after injection, levels of biotinylated mAb–Aβ complexes in plasma were determined using capture ELISA as illustrated in C. Plasma levels of Aβ₄₀ were ∼1000 pM in TgBRI-Aβ₄₀ mice, and plasma Aβ₄₂ levels were ∼1000 pM in TgBRI-Aβ₄₂ mice. No Aβ₄₂ was detected in the plasma of TgBRI-Aβ₄₀ mice, and no Aβ₄₀ was detected in the plasma of TgBRI-Aβ₄₂ mice.

Figure 2

Effect of immunization with C-terminal–specific mAbs on Aβ levels in brains of Tg2576 mice. (A and B) Seven-month-old Tg2576 mice (n = 6 per group) were immunized with 500 μg of Ab40.1 and Ab42.2 biweekly for 4 months, and Aβ levels were compared with those following immunization with Ab9. Control mice received PBS. Mice were killed following treatment, and both SDS Aβ (A) and FA Aβ (B) were analyzed by capture ELISA. SDS Aβ₄₀ and FA Aβ₄₀ in control mice were 123 ± 27 and 3,613 ± 610 pmol/g, respectively; SDS Aβ₄₂ and FA Aβ₄₂ in control mice were 44 ± 4 and 840 ± 180 pmol/g, respectively. (C) Representative immunostained sections for amyloid plaques from brains of mAb-immunized 7-month-old Tg2576 mice. Magnification, ×100. (D) Quantitative image analysis of amyloid plaque burden in the neocortices of immunized 7-month-old Tg2576 mice. (E and F) Eleven-month-old Tg2576 mice (n = 6 per group) were immunized with Ab40.1, Ab42.2, and Ab9 biweekly for 4 months. SDS Aβ (E) and FA Aβ (F) were analyzed by capture ELISA. SDS Aβ₄₀ and FA Aβ₄₀ in control mice were 1,115 ± 72 and 4,675 ± 430 pmol/g, respectively; SDS Aβ₄₂ and FA Aβ₄₂ in control mice were 348 ± 34 and 737 ± 62 pmol/g, respectively. *P < 0.05, **P < 0.01 vs. control.

Figure 3

Effect of immunization with anti-Aβ mAbs on Aβ levels in brains of CRND8 mice. (A and B) Three-month-old CRND8 mice (n = 6 per group) were immunized with 500 μg of Ab9 or Ab42.2 weekly for 8 weeks. Control mice received PBS. Mice were killed following treatment, and both SDS Aβ (A) and FA Aβ (B) fractions were analyzed by capture ELISA. SDS Aβ₄₀ and FA Aβ₄₀ in control mice were 217 ± 40 and 563 ± 95 pmol/g, respectively; SDS Aβ₄₂ and FA Aβ₄₂ in control mice were 189 ± 12 and 636 ± 51 pmol/g, respectively. (C) Representative immunostained sections for amyloid plaques from brains of mAb-immunized CRND8 mice. Magnification, ×40. (D) Quantitative image analysis of amyloid plaque burden in the neocortices of immunized CRND8 mice. *P < 0.05, **P < 0.01 vs. control.

Figure 4

Effect of direct cortical injections with anti-Aβ mAbs on Aβ plaque burdens in 18-month-old Tg2576 mice. Mice were injected in the frontal cortex with 1 μg each the following mAbs: control mouse IgG, Ab2, Ab3, Ab5, Ab9, Ab40.1, and Ab42.2. (A) Representative images of immunostained Aβ plaques taken from injection sites in cortex following injection with Ab9, control IgG, Ab42.2, and Ab40.1. (B) Representative images of thioflavin-S–positive Aβ plaques taken from injection sites in cortex following injection with Ab9, control IgG, Ab42.2, and Ab40.1. Magnification, ×100. (C) Quantitative analysis of immunostained amyloid plaque burdens in mice following mAb injections. *P < 0.01 vs. mouse IgG. (D) Quantitative analysis of thioflavin-S–positive amyloid plaque burdens in mice following mAb injections.

Figure 5

Effect of immunization with N-terminal–specific mAbs on Aβ levels in brains of 10-month-old Tg2576 mice. (A) Unfixed, frozen cryostat serial sections of human AD tissue (hippocampus) were stained with Ab9, Ab5, Ab3, Ab2, Ab40.1, and Ab42.2. Representative plaque staining is shown. Magnification, ×400. (B) Quantitative image analysis of the average fluorescence intensity level per plaque following mAb binding. ^#P < 0.001 vs. Ab40.1; ^†P < 0.05 vs. Ab2. (C and D) Aβ levels in brains of Ab2-, Ab5-, Ab9-, and Ab3-immunized Tg2576 mice. Ten-month-old Tg2576 mice (n = 6 per group) were immunized biweekly with 500 μg N-terminal mAbs for 4 months. Mice were sacrificed following treatment, and brain tissue was subject to a 2-step SDS or FA extraction. Both SDS Aβ (C) and FA Aβ (D) were analyzed by capture ELISA. SDS Aβ₄₀ and FA Aβ₄₀ in control mice were 1,115 ± 72 and 4,675 ± 430 pmol/g, respectively; SDS Aβ₄₂ and FA Aβ₄₂ in control mice were 348 ± 54 and 737 ± 62 pmol/g, respectively. *P < 0.05, **P < 0.01 vs. control.