Oskar Adolfsson

An Effector-Reduced Anti-β-Amyloid (Aβ) Antibody with Unique Aβ Binding Properties Promotes Neuroprotection and Glial Engulfment of Aβ

Passive immunization against β-amyloid (Aβ) has become an increasingly desirable strategy as a therapeutic treatment for Alzheimers disease (AD). However, traditional passive immunization approaches carry the risk of Fcγ receptor-mediated overactivation of microglial cells, which may contribute to an inappropriate proinflammatory response leading to vasogenic edema and cerebral microhemorrhage. Here, we describe the generation of a humanized anti-Aβ monoclonal antibody of an IgG4 isotype, known as MABT5102A (MABT). An IgG4 subclass was selected to reduce the risk of Fcγ receptor-mediated overactivation of microglia. MABT bound with high affinity to multiple forms of Aβ, protected against Aβ1–42 oligomer-induced cytotoxicity, and increased uptake of neurotoxic Aβ oligomers by microglia. Furthermore, MABT-mediated amyloid plaque removal was demonstrated using in vivo live imaging in hAPP(V717I)/PS1 transgenic mice. When compared with a human IgG1 wild-type subclass, containing the same antigen-binding variable domains and with equal binding to Aβ, MABT showed reduced activation of stress-activated p38MAPK (p38 mitogen-activated protein kinase) in microglia and induced less release of the proinflammatory cytokine TNFα. We propose that a humanized IgG4 anti-Aβ antibody that takes advantage of a unique Aβ binding profile, while also possessing reduced effector function, may provide a safer therapeutic alternative for passive immunotherapy for AD. Data from a phase I clinical trial testing MABT is consistent with this hypothesis, showing no signs of vasogenic edema, even in ApoE4 carriers.

Aβ42 Neurotoxicity Is Mediated by Ongoing Nucleated Polymerization Process Rather than by Discrete Aβ42 Species

The identification of toxic Aβ species and/or the process of their formation is crucial for understanding the mechanism(s) of Aβ neurotoxicity in Alzheimer disease and also for the development of effective diagnostic and therapeutic interventions. To elucidate the structural basis of Aβ toxicity, we developed different procedures to isolate Aβ species of defined size and morphology distribution, and we investigated their toxicity in different cell lines and primary neurons. We observed that crude Aβ42 preparations, containing a monomeric and heterogeneous mixture of Aβ42 oligomers, were more toxic than purified monomeric, protofibrillar fractions, or fibrils. The toxicity of protofibrils was directly linked to their interactions with monomeric Aβ42 and strongly dependent on their ability to convert into amyloid fibrils. Subfractionation of protofibrils diminished their fibrillization and toxicity, whereas reintroduction of monomeric Aβ42 into purified protofibril fractions restored amyloid formation and enhanced their toxicity. Selective removal of monomeric Aβ42 from these preparations, using insulin-degrading enzyme, reversed the toxicity of Aβ42 protofibrils. Together, our findings demonstrate that Aβ42 toxicity is not linked to specific prefibrillar aggregate(s) but rather to the ability of these species to grow and undergo fibril formation, which depends on the presence of monomeric Aβ42. These findings contribute significantly to the understanding of amyloid formation and toxicity in Alzheimer disease, provide novel insight into mechanisms of Aβ protofibril toxicity, and important implications for designing anti-amyloid therapies.

Efficacy and Safety of A Liposome-Based Vaccine against Protein Tau, Assessed in Tau.P301L Mice That Model Tauopathy

Progressive aggregation of protein Tau into oligomers and fibrils correlates with cognitive decline and synaptic dysfunction, leading to neurodegeneration in vulnerable brain regions in Alzheimers disease. The unmet need of effective therapy for Alzheimers disease, combined with problematic pharmacological approaches, led the field to explore immunotherapy, first against amyloid peptides and recently against protein Tau. Here we adapted the liposome-based amyloid vaccine that proved safe and efficacious, and incorporated a synthetic phosphorylated peptide to mimic the important phospho-epitope of protein Tau at residues pS396/pS404. We demonstrate that the liposome-based vaccine elicited, rapidly and robustly, specific antisera in wild-type mice and in Tau.P301L mice. Long-term vaccination proved to be safe, because it improved the clinical condition and reduced indices of tauopathy in the brain of the Tau.P301L mice, while no signs of neuro-inflammation or other adverse neurological effects were observed. The data corroborate the hypothesis that liposomes carrying phosphorylated peptides of protein Tau have considerable potential as safe and effective treatment against tauopathies, including Alzheimers disease.

Discovery and structure activity relationship of small molecule inhibitors of toxic β-amyloid-42 fibril formation

Background: Self-aggregation of β-amyloid plays an important role in the pathogenesis of Alzheimer disease. Results: Small molecule inhibitors of β-amyloid fibril formation reduce β-amyloid mediated cell toxicity. Conclusion: Rational design led to the successful development of small molecule inhibitors of β-amyloid oligomerization and toxicity. Significance: Small molecules targeting β-amyloid misfolding may provide new treatments for Alzheimer disease. Increasing evidence implicates Aβ peptides self-assembly and fibril formation as crucial events in the pathogenesis of Alzheimer disease. Thus, inhibiting Aβ aggregation, among others, has emerged as a potential therapeutic intervention for this disorder. Herein, we employed 3-aminopyrazole as a key fragment in our design of non-dye compounds capable of interacting with Aβ42 via a donor-acceptor-donor hydrogen bond pattern complementary to that of the β-sheet conformation of Aβ42. The initial design of the compounds was based on connecting two 3-aminopyrazole moieties via a linker to identify suitable scaffold molecules. Additional aryl substitutions on the two 3-aminopyrazole moieties were also explored to enhance π-π stacking/hydrophobic interactions with amino acids of Aβ42. The efficacy of these compounds on inhibiting Aβ fibril formation and toxicity in vitro was assessed using a combination of biophysical techniques and viability assays. Using structure activity relationship data from the in vitro assays, we identified compounds capable of preventing pathological self-assembly of Aβ42 leading to decreased cell toxicity.

Structure of Crenezumab Complex with Aβ Shows Loss of β-Hairpin

Accumulation of amyloid-β (Aβ) peptides and amyloid plaque deposition in brain is postulated as a cause of Alzheimer’s disease (AD). The precise pathological species of Aβ remains elusive although evidence suggests soluble oligomers may be primarily responsible for neurotoxicity. Crenezumab is a humanized anti-Aβ monoclonal IgG4 that binds multiple forms of Aβ, with higher affinity for aggregated forms, and that blocks Aβ aggregation, and promotes disaggregation. To understand the structural basis for this binding profile and activity, we determined the crystal structure of crenezumab in complex with Aβ. The structure reveals a sequential epitope and conformational requirements for epitope recognition, which include a subtle but critical element that is likely the basis for crenezumab’s versatile binding profile. We find interactions consistent with high affinity for multiple forms of Aβ, particularly oligomers. Of note, crenezumab also sequesters the hydrophobic core of Aβ and breaks an essential salt-bridge characteristic of the β-hairpin conformation, eliminating features characteristic of the basic organization in Aβ oligomers and fibrils, and explains crenezumab’s inhibition of aggregation and promotion of disaggregation. These insights highlight crenezumab’s unique mechanism of action, particularly regarding Aβ oligomers, and provide a strong rationale for the evaluation of crenezumab as a potential AD therapy.

Increased Efflux of Amyloid-β Peptides through the Blood-Brain Barrier by Muscarinic Acetylcholine Receptor Inhibition Reduces Pathological Phenotypes in Mouse Models of Brain Amyloidosis

The formation and accumulation of toxic amyloid-β peptides (Aβ) in the brain may drive the pathogenesis of Alzheimers disease. Accordingly, disease-modifying therapies for Alzheimers disease and related disorders could result from treatments regulating Aβ homeostasis. Examples are the inhibition of production, misfolding, and accumulation of Aβ or the enhancement of its clearance. Here we show that oral treatment with ACI-91 (Pirenzepine) dose-dependently reduced brain Aβ burden in AβPPPS1, hAβPPSL, and AβPP/PS1 transgenic mice. A possible mechanism of action of ACI-91 may occur through selective inhibition of muscarinic acetylcholine receptors (AChR) on endothelial cells of brain microvessels and enhanced Aβ peptide clearance across the blood-brain barrier. One month treatment with ACI-91 increased the clearance of intrathecally-injected Aβ in plaque-bearing mice. ACI-91 also accelerated the clearance of brain-injected Aβ in blood and peripheral tissues by favoring its urinal excretion. A single oral dose of ACI-91 reduced the half-life of interstitial Aβ peptide in pre-plaque mhAβPP/PS1d mice. By extending our studies to an in vitro model, we showed that muscarinic AChR inhibition by ACI-91 and Darifenacin augmented the capacity of differentiated endothelial monolayers for active transport of Aβ peptide. Finally, ACI-91 was found to consistently affect, in vitro and in vivo, the expression of endothelial cell genes involved in Aβ transport across the Blood Brain Brain (BBB). Thus increased Aβ clearance through the BBB may contribute to reduced Aβ burden and associated phenotypes. Inhibition of muscarinic AChR restricted to the periphery may present a therapeutic advantage as it avoids adverse central cholinergic effects.

Novel Phospho-Tau Monoclonal Antibody Generated Using a Liposomal Vaccine, with Enhanced Recognition of a Conformational Tauopathy Epitope

The microtubule-associated protein Tau is an intrinsically unfolded, very soluble neuronal protein. Under still unknown circumstances, Tau protein forms soluble oligomers and insoluble aggregates that are closely linked to the cause and progression of various brain pathologies, including Alzheimer’s disease. Previously we reported the development of liposome-based vaccines and their efficacy and safety in preclinical mouse models for tauopathy. Here we report the use of a liposomal vaccine for the generation of a monoclonal antibody with particular characteristics that makes it a valuable tool for fundamental studies as well as a candidate antibody for diagnostic and therapeutic applications. The specificity and affinity of antibody ACI-5400 were characterized by a panel of methods: (i) measuring the selectivity for a specific phospho-Tau epitope known to be associated with tauopathy, (ii) performing a combination of peptide and protein binding assays, (iii) staining of brain sections from mouse preclinical tauopathy models and from human subjects representing six different tauopathies, and (iv) evaluating the selective binding to pathological epitopes on extracts from tauopathy brains in non-denaturing sandwich assays. We conclude that the ACI-5400 antibody binds to protein Tau phosphorylated at S396 and favors a conformation that is typically present in the brain of tauopathy patients, including Alzheimer’s disease.

CHANGES IN TAU PROTEIN LANDSCAPE DURING ALZHEIMER’S DISEASE PROGRESSION

P4-099 CHANGES IN TAU PROTEIN LANDSCAPE DURING ALZHEIMER’S DISEASE PROGRESSION Hsu-Hsin Chen, Paul Auger, Peter Liu, Julie Lee, Oskar Adolfsson, Maria Pihlgren, Andreas Muhs, Andrea Pfeifer, Stephen Schauer, Dana Baker, Akash Datwani, Kristin R. Wildsmith, Thomas G. Beach, Marcel P. van der Brug, Genentech, South San Francisco, CA, USA; AC Immune SA, Lausanne, Switzerland; Banner Sun Health Research Institute, Sun City, AZ, USA. Contact e-mail: chen.hsuhsin@gene.com

Tau antibodies lacking effector function minimize inflammatory responses while effectively blocking spread of tau pathology

P2-054 TAU ANTIBODIES LACKING EFFECTOR FUNCTION MINIMIZE INFLAMMATORY RESPONSES WHILE EFFECTIVELY BLOCKING SPREAD OF TAU PATHOLOGY Seung-Hye Lee, Claire Le Pichon, Oskar Adolfsson, Val erie Gafner, Maria Pihlgren, Han Lin, Hilda Solanoy, Robert Brendza, Hai Ngu, Oded Foreman, Ruby Chan, James Ernst, Danielle Dicara, Isidro Hotzel, Karpagam Srinivasan, David Hansen, Jasvinder Atwal, Andrea Pfeifer, Ryan Watts, Andreas Muhs, Kimberly Scearce-Levie, Gai Ayalon, Genentech, Inc., South San Francisco, CA, USA; National Institute of Neurological Disorders and Stroke, Bethesda, MD, USA; AC Immune SA, Lausanne, Switzerland. Contact e-mail: lee.seunghye@gene.com

A liposomal anti-phospho-tau vaccine demonstrates higher immunogenicity and specificity than a classical peptide vaccine approach

However, the relationship between these two proteins and neuronal loss lacks amechanistic explanation. Furthermore, evidence from animal models suggests that amyloid beta toxicity is mediated by tau [1-6]. We hypothesize that tau oligomers formation plays curial role in driving AD pathogenesis. Thus, tau oligomers represent an ideal therapeutic target for the treatment of AD. In order to study the removal of toxic tau assemblies in an animal model of AD (Tg2576), we generated a tau oligomer specific antibody (TOMA). This antibody does not recognize the functional monomeric tau or oligomers from other amyloidogenic proteins. Methods: Here we used the Tg2576 mouse model which overexpress the human APP with the Swedish double mutations (K670N, M671L) under the control of a hamster prion protein promoter .14-month old Tg2576 mice, received a single iv injection of 30 mg of the TOMA antibody. Control group received 30 mg of non-specific IgG. Cognitive function was assessed by novel object recognition test, 15 days after injection. In addition, western blot, ELISA and Immunostaining were performed to evaluate the response to treatment. Results: Our results indicate that single iv-injection of the TOMA antibody, reduce endogenous tau oligomers and improve cognition in the Tg2576 mouse. Interestingly, removal of tau oligomers by immunotherapy decreases beta-amyloid-56* and increases deposition of plaques in immunized mice. Conclusions: Our results support the findings that tau oligomers mediate beta-amyloid toxicity in vivo. Moreover, removal of tau oligomers by immunotherapy may induce beta-amyloid aggregates to assembly into inert and perhaps protective plaques. Thus, targeting tau oligomers by immunotherapymay represent a novel strategy for the treatment of AD and other neurodegenerative tauopathies.