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Effector Function of anti-pyroglutamate-3 Aβ Antibodies Affects Cognitive Benefit, Glial Activation and Amyloid Clearance in Alzheimer's-like Mice

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Effector Function of anti-pyroglutamate-3 Aβ Antibodies Affects Cognitive Benefit, Glial Activation and Amyloid Clearance in Alzheimer's-like Mice

Helen Crehan et al. Alzheimers Res Ther.

Abstract

Background: Pyroglutamate-3 Aβ (pGlu-3 Aβ) is an N-terminally truncated and post-translationally modified Aβ species found in Alzheimer's disease (AD) brain. Its increased peptide aggregation propensity and toxicity make it an attractive emerging treatment strategy for AD. We address the question of how the effector function of an anti-pGlu-3 Aβ antibody influences the efficacy of immunotherapy in mouse models with AD-like pathology.

Methods: We compared two different immunoglobulin (Ig) isotypes of the same murine anti-pGlu-3 Aβ mAb (07/1 IgG1 and 07/2a IgG2a) and a general N-terminal Aβ mAb (3A1 IgG1) for their ability to clear Aβ and protect cognition in a therapeutic passive immunotherapy study in aged, plaque-rich APPSWE/PS1ΔE9 transgenic (Tg) mice. We also compared the ability of these antibodies and a CDC-mutant form of 07/2a (07/2a-k), engineered to avoid complement activation, to clear Aβ in an ex vivo phagocytosis assay and following treatment in APPSLxhQC double Tg mice, and to activate microglia using longitudinal microPET imaging with TSPO-specific 18F-GE180 tracer following a single bolus antibody injection in young and old Tg mice.

Results: We demonstrated significant cognitive improvement, better plaque clearance, and more plaque-associated microglia in the absence of microhemorrhage in aged APPSWE/PS1ΔE9 Tg mice treated with 07/2a, but not 07/1 or 3A1, compared to PBS in our first in vivo study. All mAbs cleared plaques in an ex vivo assay, although 07/2a promoted the highest phagocytic activity. Compared with 07/2a, 07/2a-k showed slightly reduced affinity to Fcγ receptors CD32 and CD64, although the two antibodies had similar binding affinities to pGlu-3 Aβ. Treatment of APPSLxhQC mice with 07/2a and 07/2a-k mAbs in our second in vivo study showed significant plaque-lowering with both mAbs. Longitudinal 18F-GE180 microPET imaging revealed different temporal patterns of microglial activation for 3A1, 07/1, and 07/2a mAbs and no difference between 07/2a-k and PBS-treated Tg mice.

Conclusion: Our results suggest that attenuation of behavioral deficits and clearance of amyloid is associated with strong effector function of the anti-pGlu-3 Aβ mAb in a therapeutic treatment paradigm. We present evidence that antibody engineering to reduce CDC-mediated complement binding facilitates phagocytosis of plaques without inducing neuroinflammation in vivo. Hence, the results provide implications for tailoring effector function of humanized antibodies for clinical development.

Keywords: APPSWE/PS1ΔE9; Immunotherapy; Microhemorrhage; Phagocytosis; Pyroglutamate-3 amyloid-β; microPET.

Conflict of interest statement

According to the journal’s requirements, we hereby declare that Martin Kleinschmidt, Jens-Ulrich Rahfeld, Stephan Schilling, and Inge Lues are former and Thore Hettmann, present, employees of Vivoryon Therapeutics AG, Germany and hold stock options of the company. Jens-Ulrich Rahfeld and Stephan Schilling are advisors to Vivoryon Therapeutics AG. Senior author, Cynthia A. Lemere, was an unpaid scientific advisory board member for Vivoryon Therapeutics AG, receives antibodies, and has previously received unrestricted funding from Vivoryon Therapeutics AG for some of her past work on pGlu-3 Aβ immunotherapy. She has also served as a consultant to Biogen.

Figures

Fig. 1
Fig. 1
Anti-pGlu-3 Aβ IgG2a isotype mAb shows significant cognitive improvement in 15-mo-old Tg mice. No effect was observed on locomotor activity in the APPSWE/PS1ΔE9 Tg mice, as measured by total distance traveled in the open field (OF) (a). Less distance was spent in the center of the OF by PBS-treated Tg versus Wt mice (p < 0.05), and 07/1 treatment showed a trend (p < 0.0836) to reverse this (b). Day by day analysis of the acquisition phase of the water T-maze (WTM) shows there were fewer (p < 0.05) correct responses to find the platform in PBS-treated Tg versus Wt mice on days 3–8 (c). 07/2-treated Tg mice displayed a significant increase (p < 0.05) in the percent of correct responses on days 3 and 7 compared to Tg PBS-treated mice (c). Day by day analysis of the reversal phase of the WTM (c) demonstrated a reduction (p < 0.05) in percent of correct responses on days 2–4 by the PBS-injected Tg vs. Wt mice, indicating that the Tg mice had impaired cognitive flexibility. A significant increase in correct responses was observed in Tg mice treated with 07/2a (p < 0.05) on days 2 and 3 (c). WTM results averaged across all days confirmed that Tg mice made significantly fewer (p < 0.05) correct responses to find the platform compared to Wt PBS-injected mice in both the acquisition and reversal phases, which appeared to be partially rescued following 07/2a (p < 0.05) treatment compared to PBS Tg mice (d, e). n = 11–13 per group. All data are expressed as the mean ± SEM. Fisher’s PLSD: a *p < 0.05 Wt PBS versus Tg 3A1, Tg 07/1, and Tg 07/2a at 10 and 20 min, p < 0.05 Wt PBS versus Tg 07/1, Tg 07/2a at 30 min, #p < 0.05 Wt PBS versus Tg PBS. b, d, and e *p < 0.05 versus Tg PBS, #p < 0.05 Wt PBS versus all groups. c *p < 0.05 Tg PBS versus 07/2a, #p < 0.05 Wt PBS versus Tg PBS
Fig. 2
Fig. 2
Lowering of multiple Aβ species in aged APPSWE/PS1ΔE9 Tg brains following 07/2a IgG2a mAb immunotherapy. Representative photomicrographs of hippocampus (HC) or cortex (CTX) from each treatment group of serial sections immunolabeled with a novel pGlu-3 Aβ IgG2b monoclonal antibody (ad; HC), a general Aβ polyclonal antibody, R1282 (eh; HC), stained with a marker for fibrillar amyloid, Thioflavin S (il; HC), and with a marker that specifically recognizes Aβ starting at Asp1, 82E1 (mp; CTX). Quantitative image analysis on six stained sections at thee equidistant planes per marker demonstrated that there was a significant reduction of pGlu-3 Aβ (p < 0.001), general Aβ (p < 0.05), and fibrillar amyloid (p < 0.05) in the hippocampus of Tg mice immunized with 07/2a IgG2a mAb compared to Tg mice treated with PBS (qt). Similarly, there was a significant reduction of pGlu-3 Aβ (p < 0.05), general Aβ, fibrillar amyloid, and Aβ1−x (p < 0.05) in the cortex of the Tg mice immunized with 07/2a IgG2a mAb compared to Tg mice treated with PBS (ux). n = 13–15 per group. All data, excluding baseline, are expressed as the mean ± SEM. ANOVA with Neuman-Keuls post test: ***p < 0.001 and *p < 0.05 versus Tg PBS
Fig. 3
Fig. 3
07/2a IgG2a and 3A1 IgG1 mAb’s lowered guanidine-HCl-extracted Aβ in aged APPSWE/PS1ΔE9 mice. Significant decreases in guanidine-HCl-extracted pGlu-3 Aβ (p < 0.05), Aβx-42 (p < 0.01), Aβx-40 (p < 0.05), and Aβx-38 (p < 0.05) were observed in Tg mice treated with 07/2a IgG2a mAb compared to PBS-treated Tg mice (ad). Similarly, treatment with 3A1 mAb reduced guanidine-HCl-extracted Aβx-42 (p < 0.01), Aβx-40 (p < 0.05, t-test), and Aβx-38 (p < 0.05, t-test) in Tg mice compared to PBS controls (bd). Treatment of the Tg mice with 3A1 IgG1 mAb resulted in a significant increase in plasma levels of Aβx-42 (0.01), Aβx-40 (p < 0.001), and Aβx-38 (p < 0.001) with no differences observed in Tg mice treated with anti-pGlu-3 Aβ mAbs, 07/1 IgG1 or 07/2a IgG2a, compared to PBS-treated Tg mice (eg). No significant (N.S) differences in hemosiderin-positive staining were observed between antibody-treated Tg mice versus PBS-injected Tg mice or WT mice (h). n = 13–15 per group. All data, excluding baseline, are expressed as the mean ± SEM. ANOVA with Neuman-Keuls post test: ***p < 0.001, **p < 0.01, and *p < 0.05 versus Tg PBS. Student’s t-test: #p < 0.05
Fig. 4
Fig. 4
Immunohistochemical analysis of microglia and macrophage markers in the hippocampus and cortex of APPSWE/PS1ΔE9 Tg mice. Passive immunization, in aged APPSWE/PS1ΔE9 mice, resulted in a significant increase in plaque-associated microglia in mice treated with 07/2a IgG2a mAb. Representative photomicrographs of the hippocampus from each treatment group immunolabeled with microglial and macrophage marker, Iba1 (ae), and activated microglial and macrophage marker, CD68 (fj), showed an increased immunolabeling in immunized mice compared to PBS-treated Tg mice. Image analysis demonstrated that there was a significant increase in the percent area of Iba1-positive staining in the hippocampus (p < 0.05; k) and cortex (p < 0.05; l) of 07/2a IgG2a mAb-immunized mice compared to PBS-treated Tg mice. Analysis of the cortex demonstrated that there was a significant increase in CD68-positive immunolabeling in the hippocampus (p < 0.01; m) and cortex (p < 0.05; n) of 07/2a IgG2a mAb-immunized mice compared to PBS-treated Tg mice. A significant increase in plaque-associated CD68-positive cells was observed in Tg mice immunized with 07/2a IgG2a mAb (o, s). n = 13–15 per group. All data are expressed as the mean ± SEM. ANOVA with Neuman-Keuls post test: **p < 0.01 and *p < 0.05 versus Tg PBS
Fig. 5
Fig. 5
Ex vivo antibody-mediated phagocytosis assay demonstrates differences in plaque clearance based on antibody isotype. Unfixed, frozen plaque-rich tissue sections (20 μm) from 20-mo-old APPSWE/PS1ΔE9 Tg mice were pre-incubated with the following antibodies for 1 h: 07/1 IgG1, isotype control IgG1, 07/2a IgG2a, 07/2a-k IgG2a, or isotype control IgG2a. Following washing, the tissue was incubated with primary murine microglia (PMG) for 24 h and Aβ levels were determined. Representative photomicrographs of the hippocampus from each treatment group co-immunolabeled with a general Aβ marker, R1282, an activated microglial and macrophage marker, CD68 and a nucleic acid dye, DAPI (a-g) and showed decreased R1282 immunolabeling in the hippocampus of tissue pre-incubated with 07/2a IgG2a compared to isotype control (ah). pGlu-3 Aβ, Aβx-40, and Aβx-42 levels were also determined by ELISA (ik). Significant reductions in pGlu-3 Aβ were observed in tissue pre-incubated with 07/1 IgG1 and 07/2 IgG2a mAbs compared to their respective isotype controls (i). Tissue incubation with 07/2a IgG2a and 07/2a-k IgG2a resulted in decreased Aβx-40 levels compared to isotype control (j). There were no differences in Aβx-42 levels observed between groups (k). n = 3 per group. All data are expressed as the mean ± SEM. ANOVA with Neuman-Keuls post test: ***p < 0.001, **p < 0.01, and *p < 0.05 versus isotype control
Fig. 6
Fig. 6
07/2a and 07/2a-k immunotherapy lowers Aβ in hAPPSL:hQC mice. Quantitative image analysis of the hippocampus on six stained sections at three equidistant planes per marker demonstrated that there was a significant reduction of pGlu-3 Aβ following 4 months of treatment with 07/2a at 150 μg (p < 0.05), 07/2a at 500 μg (p < 0.01), and 07/2a-k at 500 μg (p < 0.05) (a). General Aβ as measured by R1282 IHC demonstrated a reduction after dosing with 07/2a at 150 μg (p < 0.05), 07/2a at 500 μg (p < 0.01), and 07/2a-k at 500 μg (p < 0.01) (b). Aβx-42 levels measured in the TBS soluble fraction were reduced in the brains of animals treated with 07/2a at 150 μg (p < 0.05), 07/2a at 500 μg (p < 0.01), and 07/2a-k at 500 μg (p < 0.05) (c). n = 7–8 mice per group. All data are expressed as the mean ± SEM. ANOVA with Bartlett’s post test: ***p < 0.001, **p < 0.01, and *p < 0.05 versus isotype control
Fig. 7
Fig. 7
Uptake of 18F-GE180 tracer in mouse hippocampus at day 0, day 3, and day 30. One-hour microPET imaging was carried out using a novel TSPO radioligand, 18F-GE180, to visualize the microglia activation in mouse brain pre- and post-antibody immunization. The tracer uptake was quantitatively estimated as the percentage of injected dose per unit volume of hippocampus. ae Time-activity curve (TAC) of hippocampal 18F-GE180 uptake in 4-mo-old mice with a single injection of 0.25 ml PBS (a) or 500 μg of antibody including 3A1-IgG1 (b), 07/1-IgG1 (c), 07/2a-IgG2a (d), or 07/2a-k (e). eh TAC of hippocampal 18F-GE180 uptake in 16-mo-old mice with a single injection of 0.25 ml PBS (f) or 500 μg of antibody including 3A1-IgG1 (g), 07/1-IgG1 (h), 07/2a-IgG2a (i), or 07/2a-k IgG2a (j). n = 3–4 mice per treatment group per age. Two-way ANOVA with Bonferroni post hoc test was applied for statistical analysis

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