Acute targeting of pre-amyloid seeds in transgenic mice reduces Alzheimer-like pathology later in life.

Amyloid-ß (Aß) deposits are a relatively late consequence of Aß aggregation in Alzheimer's disease. When pathogenic Aß seeds begin to form, propagate and spread is not known, nor are they biochemically defined. We tested various antibodies for their ability to neutralize Aß seeds before Aß deposition becomes detectable in Aß precursor protein-transgenic mice. We also characterized the different antibody recognition profiles using immunoprecipitation of size-fractionated, native, mouse and human brain-derived Aß assemblies. At least one antibody, aducanumab, after acute administration at the pre-amyloid stage, led to a significant reduction of Aß deposition and downstream pathologies 6 months later. This demonstrates that therapeutically targetable pathogenic Aß seeds already exist during the lag phase of protein aggregation in the brain. Thus, the preclinical phase of Alzheimer's disease-currently defined as Aß deposition without clinical symptoms-may be a relatively late manifestation of a much earlier pathogenic seed formation and propagation that currently escapes detection in vivo.

Antibody-mediated clearance of tau in primary mouse microglial cultures requires Fcγ-receptor binding and functional lysosomes.

Neurodegenerative diseases such as Alzheimer's disease are characterized by the progressive spreading and accumulation of hyper-phosphorylated tau protein in the brain. Anti-tau antibodies have been shown to reduce tau pathology in in vivo models and antibody-mediated clearance of tau exerted by microglia has been proposed as a contributing factor. By subjecting primary microglia cultured in vitro to anti-phospho-tau antibodies in complex with pathological tau, we show that microglia internalise and degrade tau in a manner that is dependent on FcγR interaction and functional lysosomes. It has recently been discussed if anti-tau antibody effector-functions are required for induction of tau clearance. Using antibodies with compromised FcγR binding and non-compromised control antibodies we show that antibody effector functions are required for induction of microglial clearance of tau. Understanding the inflammatory consequences of targeting microglia using therapeutic antibodies is important when developing these molecules for clinical use. Using RNA sequencing, we show that treatment with anti-tau antibodies increases transcription of mRNA encoding pro-inflammatory markers, but that the mRNA expression profile of antibody-treated cells differ from the profile of LPS activated microglia. We further demonstrate that microglia activation alone is not sufficient to induce significant tau clearance.

Highly specific and selective anti-pS396-tau antibody C10.2 targets seeding-competent tau.

INTRODUCTION: The abnormal hyperphosphorylation of the microtubule-associated protein tau plays a crucial role in neurodegeneration in Alzheimer's disease (AD) and other tauopathies. METHODS: Highly specific and selective anti-pS396-tau antibodies have been generated using peptide immunization with screening against pathologic hyperphosphorylated tau from rTg4510 mouse and AD brains and selection in in vitro and in vivo tau seeding assays. RESULTS: The antibody C10.2 bound specifically to pS396-tau with an IC50 of 104 pM and detected preferentially hyperphosphorylated tau aggregates from AD brain with an IC50 of 1.2 nM. C10.2 significantly reduced tau seeding of P301L human tau in HEK293 cells, murine cortical neurons, and mice. AD brain extracts depleted with C10.2 were not able to seed tau in vitro and in vivo, demonstrating that C10.2 specifically recognized pathologic seeding-competent tau. DISCUSSION: Targeting pS396-tau with an antibody like C10.2 may provide therapeutic benefit in AD and other tauopathies.

Fibrin-targeting immunotherapy protects against neuroinflammation and neurodegeneration.

Activation of innate immunity and deposition of blood-derived fibrin in the central nervous system (CNS) occur in autoimmune and neurodegenerative diseases, including multiple sclerosis (MS) and Alzheimer's disease (AD). However, the mechanisms that link disruption of the blood-brain barrier (BBB) to neurodegeneration are poorly understood, and exploration of fibrin as a therapeutic target has been limited by its beneficial clotting functions. Here we report the generation of monoclonal antibody 5B8, targeted against the cryptic fibrin epitope γ377-395, to selectively inhibit fibrin-induced inflammation and oxidative stress without interfering with clotting. 5B8 suppressed fibrin-induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and the expression of proinflammatory genes. In animal models of MS and AD, 5B8 entered the CNS and bound to parenchymal fibrin, and its therapeutic administration reduced the activation of innate immunity and neurodegeneration. Thus, fibrin-targeting immunotherapy inhibited autoimmunity- and amyloid-driven neurotoxicity and might have clinical benefit without globally suppressing innate immunity or interfering with coagulation in diverse neurological diseases.

Development of a Cell-Based Assay to Assess Binding of the proNGF Prodomain to Sortilin.

Sortilin was first identified based on its activity as part of intracellular protein sorting machinery. Recently, it was discovered that sortilin also acts as a cell surface receptor for the propeptide form of nerve growth factor (proNGF), progranulin, and neurotensin. The interaction of sortilin to these neurotrophic ligands is linked to diseases of the nervous system that lead to neurodegeneration and neuropathic pain. Blocking of the interaction of sortilin to these ligands may prevent or slow the progress of these nervous system disorders. In vitro screening assays for blocking compounds or peptides are part of the standard set of tools for drug discovery. However, assays for sortilin biology are not readily available to determine if the selected blocking agent inhibits sortilin activity on the surface of cells. We have developed a sortilin specific cell based assay to identify compounds that specifically block interaction between sortilin and proNGF prodomain. The assay system records both the presence of sortilin on the cell surface and the interaction with the pro domain of NGF. Fluorescent images of the sortilin expressing cells are analyzed for the presence of pro domain of NGF. Sortilin-positive and sortilin-negative cells within one well are concomitantly and automatically analyzed. Sortilin-pro domain interaction can be blocked dose dependently by neurotensin and synthetic compounds. The assay will facilitate the discovery of entities interfering with the binding of sortilin to the NGF pro domain. This assay can be modified to screen for inhibitors of the binding of ligands to other complex cell surface receptors.

Comparing the efficacy and neuroinflammatory potential of three anti-abeta antibodies.

Immunotherapy is a promising strategy for the treatment of Alzheimer's disease (AD). Antibodies directed against Amyloid Beta (Aß) are able to successfully clear plaques and reverse cognitive deficits in mouse models. Excitement towards this approach has been tempered by high profile failures in the clinic, one key issue has been the development of inflammatory side effects in the brain (ARIAs). New antibodies are entering the clinic for Alzheimer's disease; therefore, it is important to learn all we can from the current generation. In this study, we directly compared 3 clinical candidates in the same pre-clinical model, with the same effector function, for their ability to clear plaques and induce inflammation in the brain. We produced murine versions of the antibodies: Bapineuzumab (3D6), Crenezumab (mC2) and Gantenerumab (chGantenerumab) with an IgG2a constant region. 18-month transgenic APP mice (Tg2576) were injected bilaterally into the hippocampus with 2 µg of each antibody or control. After 7 days, the mice tissue was analysed for clearance of plaques and neuroinflammation by histology and biochemical analysis. 3D6 was the best binder to plaques and in vitro, whilst mC2 bound the least strongly. This translated into 3D6 effectively clearing plaques and reducing the levels of insoluble Aß, whilst chGantenerumab and mC2 did not. 3D6 caused a significant increase in the levels of pro-inflammatory cytokines IL-1ß and TNFα, and an associated increase in microglial expression of CD11B and CD68. chGantenerumab increased pro-inflammatory cytokines and microglial activation, but minimal changes in CD68, as an indicator of phagocytosis. Injection of mC2 did not cause any significant inflammatory changes. Our results demonstrate that the ability of an antibody to clear plaques and induce inflammation is dependent on the epitope and affinity of the antibody.

New roles for Fc receptors in neurodegeneration-the impact on Immunotherapy for Alzheimer's Disease.

There are an estimated 18 million Alzheimer's disease (AD) sufferers worldwide and with no disease modifying treatment currently available, development of new therapies represents an enormous unmet clinical need. AD is characterized by episodic memory loss followed by severe cognitive decline and is associated with many neuropathological changes. AD is characterized by deposits of amyloid beta (Aß), neurofibrillary tangles, and neuroinflammation. Active immunization or passive immunization against Aß leads to the clearance of deposits in transgenic mice expressing human Aß. This clearance is associated with reversal of associated cognitive deficits, but these results have not translated to humans, with both active and passive immunotherapy failing to improve memory loss. One explanation for these observations is that certain anti-Aß antibodies mediate damage to the cerebral vasculature limiting the top dose and potentially reducing efficacy. Fc gamma receptors (FcγR) are a family of immunoglobulin-like receptors which bind to the Fc portion of IgG, and mediate the response of effector cells to immune complexes. Data from both mouse and human studies suggest that cross-linking FcγR by therapeutic antibodies and the subsequent pro-inflammatory response mediates the vascular side effects seen following immunotherapy. Increasing evidence is emerging that FcγR expression on CNS resident cells, including microglia and neurons, is increased during aging and functionally involved in the pathogenesis of age-related neurodegenerative diseases. Therefore, we propose that increased expression and ligation of FcγR in the CNS, either by endogenous IgG or therapeutic antibodies, has the potential to induce vascular damage and exacerbate neurodegeneration. To produce safe and effective immunotherapies for AD and other neurodegenerative diseases it will be vital to understand the role of FcγR in the healthy and diseased brain. Here we review the literature on FcγR expression, function and proposed roles in multiple age-related neurological diseases. Lessons can be learnt from therapeutic antibodies used for the treatment of cancer where antibodies have been engineered for optimal efficacy.

The identification of AF38469: an orally bioavailable inhibitor of the VPS10P family sorting receptor Sortilin.

The identification of the novel, selective, orally bioavailable Sortilin inhibitor AF38469 is described. Structure-activity relationships and syntheses are reported, along with an X-ray crystal structure of the sortilin-AF38469 protein-inhibitor complex.

Fc optimization of therapeutic antibodies enhances their ability to kill tumor cells in vitro and controls tumor expansion in vivo via low-affinity activating Fcgamma receptors.

Monoclonal antibodies (mAb) are widely used in the treatment of non-Hodgkin's lymphoma and autoimmune diseases. Although the mechanism of action in vivo is not always known, the therapeutic activity of several approved mAbs depends on the binding of the Fcgamma regions to low-affinity Fcgamma receptors (FcgammaR) expressed on effector cells. We did functional genetic screens to identify IgG1 Fc domains with improved binding to the low-affinity activating Fc receptor CD16A (FcgammaRIIIA) and reduced binding to the low-affinity inhibitory Fc receptor, CD32B (FcgammaRIIB). Identification of new amino acid residues important for FcgammaR binding guided the construction of an Fc domain that showed a dramatically enhanced CD16A binding and greater than a 100-fold improvement in antibody-dependent cell-mediated cytotoxicity. In a xenograft murine model of B-cell malignancy, the greatest enhancement of an Fc-optimized anti-human B-cell mAb was accounted for by improved binding to FcgammaRIV, a unique mouse activating FcgammaR that is expressed by monocytes and macrophages but not natural killer (NK) cells, consistent with experimental and clinical data suggesting that mononuclear phagocytes, effector cells expressing both activating and inhibitory FcgammaR, are critical mediators of B-cell depletion in vivo. By using mice transgenic for human CD16A, enhanced survival was observed due to expression of CD16A-158(phe) on monocytes and macrophages as well as on NK cells in these mice. The design of new generations of improved antibodies for immunotherapy should aim at Fc optimization to increase the engagement of activating FcgammaR present on the surface of tumor-infiltrating effector cell populations.