Chronic Intranasal Treatment with an Anti-Aβ30-42 scFv Antibody Ameliorates Amyloid Pathology in a Transgenic Mouse Model of Alzheimer's Disease

Amyloid-beta peptide (Aβ)-directed active and passive immunization therapeutic strategies reduce brain levels of Aβ, decrease the severity of beta-amyloid plaque pathology and reverse cognitive deficits in mouse models of Alzheimer's disease (AD). As an alternative approach to passive immunization with full IgG molecules, single-chain variable fragment (scFv) antibodies can modulate or neutralize Aβ-related neurotoxicity and inhibit its aggregation in vitro. In this study, we characterized a scFv derived from a full IgG antibody raised against the C-terminus of Aβ, and studied its passage into the brains of APP transgenic mice, as well as its potential to reduce Aβ-related pathology. We found that the scFv entered the brain after intranasal application, and that it bound to beta-amyloid plaques in the cortex and hippocampus of APP transgenic mice. Moreover, the scFv inhibited Aβ fibril formation and Aβ-mediated neurotoxicity in vitro. In a preventative therapeutic approach chronic intranasal treatment with scFv reduced congophilic amyloid angiopathy (CAA) and beta-amyloid plaque numbers in the cortex of APPswe/PS1dE9 mice. This reduction of CAA and plaque pathology was associated with a redistribution of brain Aβ from the insoluble fraction to the soluble peptide pool. Due to their lack of the effector domain of full IgG, scFv may represent an alternative tool for the treatment of Aβ-related pathology without triggering Fc-mediated effector functions. Additionally, our observations support the possibility that Aβ-directed immunotherapy can reduce Aβ deposition in brain vessels in transgenic mice

Effect of IVIG Formulation on IgG Binding to Self- and Exo- Antigens In Vitro and In Vivo.

In relation to the recent trials of Intravenous Immunoglobulin (IVIG) in Alzheimer's Disease (AD) it was demonstrated that different IgG preparations contain varying amounts of natural anti-amyloid β (Aβ) antibodies as measured by ELISA. We therefore investigated the relevance of ELISA data for measuring low-affinity antibodies, such as anti-Aβ. We analysed the binding of different commercial Immunoglobulin G (IgG) preparations to Aβ, actin and tetanus toxoid in different binding assays to further investigate the possible cause for observed differences in binding to Aβ and actin between different IgG preparations. We show that the differences of commercial IgG preparations in binding to Aβ and actin in ELISA assays are artefactual and only evident in in vitro binding assays. In functional assays and in vivo animal studies the different IVIG preparations exhibited very similar potency. ELISA data alone are not appropriate to analyse and rank the binding capacity of low-affinity antibodies to Aβ or other endogenous self-antigens contained in IgG preparations. Additional analytical methods should be adopted to complement ELISA data

<i>In vitro</i> Comparison of reformulated IgG preparations activity to Aβ, actin and tetanus toxoid by ELISA and Octet.

<p>Unformulated Privigen (in H₂O) was formulated in Proline pH 4.8, Glycine pH 4.7 or Glycine pH 4.2. (A) ELISA measurements on plate-bound Aβ oligomers. The panel shows combined results from repeated measurements (n = 3). (B) Octet binding measurements to immobilized Aβ and <b>C</b> to bound tetanus toxoid. Privigen Fc fragments were used as negative controls.</p

<i>In vivo</i> pharmacokinetics of different IgG preparations, proline and glycine in rats.

<p>Clr:CD(SD) rats (n = 10 in groups 0 min and 2 min; n = 5 in groups 6h and 24h) were intravenously injected with 500 mg/kg of Privigen 10%, Gammagard 10%, Octagam 10% or Gamunex 10%, repectively. Blood was taken at baseline (0 min) as well as 2 min, 6h and 24h after injection. Plasma (10% citrate) was prepared and analyzed for total IgG by Nephelometry and Proline and Glycine concentration by HPLC. The elimination profile for IgG is shown in (A) and for proline and glycine in (B).</p

<i>In vivo</i> comparison of the binding to Aβ, Actin, tetanus toxoid and Varicella Zoster Virus by ELISA.

<p>Clr:CD(SD) rats (n = 10 in groups 0 min and 2 min; n = 5 in groups 6h and 24h) were intravenously injected with 500 mg/kg of Privigen 10%, Gammagard 10%, Octagam 10% or Gamunex 10%, respectively. Blood was taken at baseline (0 min) as well as 2 min, 6h and 24h after injection. Plasma (10% citrate) was prepared and the binding capacity to oligomeric Aβ (A and B), actin (C and D), Tetanus Toxoid (E and F), and VZV (G and H) was analyzed by ELISA. (*p<0.05, **p<0.01). Each ELISA was performed 3 times n = 3 (with all animals n = 30) except VZV t = 2 min n = 2 (with all animals n = 30) due to limited sample volume.</p

Amyloid-β peptide-specific DARPins as a novel class of potential therapeutics for Alzheimer disease

Passive immunization with anti-amyloid-β peptide (Aβ) antibodies is effective in animal models of Alzheimer disease. With the advent of efficient in vitro selection technologies, the novel class of designed ankyrin repeat proteins (DARPins) presents an attractive alternative to the immunoglobulin scaffold. DARPins are small and highly stable proteins with a compact modular architecture ideal for high affinity protein-protein interactions. In this report, we describe the selection, binding profile, and epitope analysis of Aβ-specific DARPins. We further showed their ability to delay Aβ aggregation and prevent Aβ-mediated neurotoxicity in vitro. To demonstrate their therapeutic potential in vivo, mono- and trivalent Aβ-specific DARPins (D23 and 3×D23) were infused intracerebroventricularly into the brains of 11-month-old Tg2576 mice over 4 weeks. Both D23 and 3×D23 treatments were shown to result in improved cognitive performance and reduced soluble Aβ levels. These findings demonstrate the therapeutic potential of Aβ-specific DARPins for the treatment of Alzheimer disease

Intravenous Immunglobulin Binds Beta Amyloid and Modifies Its Aggregation, Neurotoxicity and Microglial Phagocytosis <i>In Vitro</i>

<div><p>Intravenous Immunoglobulin (IVIG) has been proposed as a potential therapeutic for Alzheimer's disease (AD) and its efficacy is currently being tested in mild-to-moderate AD. Earlier studies reported the presence of anti-amyloid beta (Aβ) antibodies in IVIG. These observations led to clinical studies investigating the potential role of IVIG as a therapeutic agent in AD. Also, IVIG is known to mediate beneficial effects in chronic inflammatory and autoimmune conditions by interfering with various pathological processes. Therefore, we investigated the effects of IVIG and purified polyclonal Aβ -specific antibodies (pAbs-Aβ) on aggregation, toxicity and phagocytosis of Aβ <i>in vitro</i>, thus elucidating some of the potential mechanisms of action of IVIG in AD patients. We report that both IVIG and pAbs-Aβ specifically bound to Aβ and inhibited its aggregation in a dose-dependent manner as measured by Thioflavin T assay. Additionally, IVIG and the purified pAbs-Aβ inhibited Aβ-induced neurotoxicity in the SH-SY5Y human neuroblastoma cell line and prevented Aβ binding to rat primary cortical neurons. Interestingly, IVIG and pAbs-Aβ also increased the number of phagocytosing cells as well as the amount of phagocytosed fibrillar Aβ by BV-2 microglia. Phagocytosis of Aβ depended on receptor-mediated endocytosis and was accompanied by upregulation of CD11b expression. Importantly, we could also show that Privigen dose-dependently reversed Aβ-mediated LTP inhibition in mouse hippocampal slices. Therefore, our <i>in vitro</i> results suggest that IVIG may have an impact on different processes involved in AD pathogenesis, thereby promoting further understanding of the effects of IVIG observed in clinical studies.</p></div